Activity Intrinsic

Vol. (Suppl. ) Intrinsic 201 Activity

www.IntrinsicActivity.org Published by th ISSN 2309-8503 Austrian Pharmacological Society

Dopamine 2016 Vienna, 5–8 September 2016

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Correspondence Intrinsic Activity c/o Institute for Experimental and Clinical Pharmacology Medical University of Graz Universitätsplatz 4 8010 Graz, Austria Tel.: +43 (316) 380-4305 Fax: +43 (316) 380-9645 E-mail: [email protected] Website: www.IntrinsicActivity.org ISSN: 2309-8503 Austrian Pharmacological Society c/o Institute of Pharmacology Centre for Physiology and Pharmacology Medical University of Vienna Währinger Straße 13a 1090 Wien, Austria E-mail: [email protected]

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Dopamine 2016 Vienna, 5 – 8 September 2016

Plenary Lectures & Oleh Hornykiewicz Symposium combined lines of evidence suggest that DA dysfunction may be an early event leading to additional consequences on the rest of the circuitry and behavior. We will review the evidence critically and A1.1 develop a model as well as future directions to move this under- Dopamine in schizophrenia: where does it stand in the cascade standing further. of pathological events? References Anissa ABI-DARGHAM* 1. Weinstein JJ, Chohan MO, Slifstein M, Kegeles LS, Moore H, Abi- Department of Psychiatry, Columbia University, New York, NY, Dargham A: Pathway-specific dopamine abnormalities in United States of America schizophrenia. Biol Psychiatry, 2016; in press. *E-mail: [email protected] doi:10.1016/j.biopsych.2016.03.2104 Intrinsic Activity, 2016; 4(Suppl. 2): A1.1 2. Slifstein M, van de Giessen E, Van Snellenberg J, Thompson JL, http://www.intrinsicactivity.org/2016/4/S2/A1.1 Narendran R, Gil R, Hackett E, Girgis R, Ojeil N, Moore H, D’Souza D, The dopamine (DA) dysfunction in schizophrenia has recently moved Malison RT, Huang Y, Lim K, Nabulsi N, Carson RE, Lieberman JA, from being a hypothesis based on clinical observations, to a stage of Abi-Dargham A: Deficits in prefrontal cortical and extrastriatal refined and topographically precise sets of alterations documented dopamine release in schizophrenia: a positron emission across multiple labs with positron emission tomography (PET) tomographic functional magnetic resonance imaging study. imaging studies, that showed excess striatal dopamine synthesis, JAMA Psychiatry, 2015; 72(4):316–324. doi:10.1001/jamapsychiatry.2014.2414 release, and D2 supersensitivity [1]. Recent data suggest also profound extrastriatal deficits in DA release [2]. The presence of 3. Kellendonk C, Simpson EH, Kandel ER: Modeling cognitive opposing findings of striatal excess and extrastriatal deficit including endophenotypes of schizophrenia in mice. Trends Neurosci, 2009; midbrain deficit is puzzling as it suggests that striatal excess may not 32(6):347–358. doi:10.1016/j.tins.2009.02.003 be a consequence of midbrain DA cells overactivity. A mechanistic 4. Schizophrenia Working Group of the Psychiatric Genomics understanding of the DA dysfunction is missing. Animal models may Consortium: Biological insights from 108 schizophrenia-associated shed some light on the pathogenic mechanisms involved. In genetic loci. Nature, 2014; 511(7510):421–427. doi:10.1038/nature13595 particular, the D2 overexpressing (D2OE) [3] mouse has shown that cortical-dependent cognitive deficit and abnormal cortical DA signaling can be a consequence of developmental abnormalities in striatal D2 stimulation. Since many developmental factors, both Edited by: Thomas Griesbacher (Austrian Pharmacological Society APHAR, genetic [4] and environmental, have been shown to be at play in and Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria; [email protected]) schizophrenia, and shown to affect dopaminergic indices, these

A1.2 studies have shown that dopamine transients are an important Allosteric mechanisms of the dopamine signaling machines: component of monoamine transmission. New properties of transients The moving stories of GPCRs and transporters are still being described, such as previously unappreciated cross- Harel WEINSTEIN*, George KHELASHVILI, Michael V. LEVINE and hemispheric dopamine signaling. Remarkably, we find that a majority Michel CUENDET of dopamine transients are synchronous between hemispheres, Physiology and Biophysics, Weill Cornell Medical College, Cornell concurring with the physiological synchrony that is observed in many University, New York, NY, United States of America parts of the brain.To explore the specific actions of dopamine at its *E-mail: [email protected] receptors we developed a method to chemotype dopamine receptors Intrinsic Activity, 2016; 4(Suppl. 2): A1.2 in situ. We use a modified form of iontophoresis enabling controlled http://www.intrinsicactivity.org/2016/4/S2/A1.2 delivery of dopamine receptor agonists and antagonists locally to the site of the recording electrode. We monitor their effects at dopamine The molecular machines that enable dopamine (DA) signaling belong autoreceptors by examining how they alter dopamine release. By to the large category of allosteric proteins that perform complex pairing electrochemical measurements with electrophysiology, we functions triggered and / or enabled by the binding of the neurotrans- can also monitor effects on postsynaptic receptors and cell-firing mitter in binding sites located distally from the intracellular regions of without disturbing animal behavior. These methods are sufficiently the proteins. The mechanisms of these “actions at a distance” are of quantitative to generate dose response curves in multiple dopamine- great interest not only because of the essential role of dopaminergic rich microenvironments. Chemotyping enables real-time observa- signaling in normal physiology and in disease, but also because the tions of dopamine interacting with different receptor subtypes to current assays of dopaminergic function, and the need to intervene mediate animal behavior. New tools also enable us to explore mechanistically and therapeutically can no longer ignore the role of unexpected facets of neurochemical communication in disease interactions of the specific DA targets with their membrane and models and monitor previously undescribed dopamine release during protein environments. But a suitable understanding of allostery spreading depression. As these examples indicate, our tools have requires the clear definitions of the states of the DA target proteins enabled exploration of novel properties of dopamine neurotrans- (both the DA GPCRs and the transporter, DAT) under various mission, and sets a firm foundation for understanding the role of conditions in structural terms, as well as the definition of the relation dopamine in behavior. among those states in specific kinetic models (not qualitative hand- waving about conformational changes inferred from inspection of A1.4 single structures). The development of such definitions, and of Modeling three variants of Parkinson’s in mice based on structure-informed allosteric models that can be interrogated for complex aetiologies in humans specific and quantitative inferences, is a multidisciplinary endeavor in Michael G. SCHLOSSMACHER1,*, Julianna J. TOMLINSON1, which computational approaches are central to (i) the rigorous Earl G. BROWN1 and David S. PARK2 exploration of the properties of the molecular systems, (ii) of their 1 dynamic transitions between states, and (iii) the evolution of physics- Neuroscience Program, The Ottawa Hospital, Univ Ottawa Brain 2 based testable and predictive models of function. We will describe and Mind Research Institute, Ottawa, ON, Canada; Department of the ongoing development of such quantitative models of allostery for Cellular and Molecular Medicine; University of Ottawa Brain and DA receptors and the DAT, and illustrate physics-based analyses of Mind Research Institute; Ottawa, ON, Canada extensive computational simulations that reveal with increasing *E-mail: [email protected] accuracy the dynamic mechanisms by which these machines achieve Intrinsic Activity, 2016; 4(Suppl. 2): A1.4 well-regulated and modulated functions. In both the DA GPCRs and http://www.intrinsicactivity.org/2016/4/S2/A1.4 in DAT these functional mechanisms include ligand-determined, and Parkinsonism in humans encompasses several variants. We allosteric modulation by protein–protein interaction, as well as speci- postulate that the faithful modeling of different subtypes of fic effects of disease-causing mutations. parkinsonism in mice — based on individual, complex aetiologies observed in humans — is essential in facilitating breakthroughs for A1.3 cause-directed treatment of our patients. Such breakthroughs will be Analytical tools propel discoveries about dopamine based on the better understanding of disease processes, related neurotransmission advances in target validation, and the successful development of R. Mark WIGHTMAN* objective laboratory markers. Department of Chemistry, University of North Carolina at Chapel Past efforts in replicating Parkinson’s in animals have been Hill, United States of America disappointing; published models were largely based on either a single *E-mail: [email protected] genomic change or single neurotoxin exposure. Our team has Intrinsic Activity, 2016; 4(Suppl. 2): A1.3 modeled the complex aetiology for three variants of human http://www.intrinsicactivity.org/2016/4/S2/A1.3 parkinsonism in mice. Specifically, we have rebuilt: (1) a complex variant of dominantly inherited disease with motoric and cognitive The goal of my laboratory has been to discover how dopamine decline (“synergy mouse”); (2) a multifactorial variant for causing regulation mechanisms exert their effects during real-time neuronal sporadic disease (“reovirus mouse”); and (3) a complex variant to communication deep within the brain, especially during behavior. To recreate recessively inherited disease (“DJ-1 mouse”). approach this problem, we developed very small, carbon-based In pursuing this work, we have incorporated allelic changes at the chemical sensors designed to measure physiological fluctuations in following genetic loci in our models: In the “synergy mouse”, four dopamine concentrations. At the time, changes in extracellular copies of a mutant human SNCA allele [1] were combined with knock- dopamine had only been measured as slow fluctuations in basal in mutations at gba1 [2]; in the “reovirus mouse”, mutant lrrk2 alleles concentrations. When we first used these sensors in freely moving [3] were coupled with exposure to a neurotropic virus [4]; and in the animals we were surprised to observe rapid, transient dopamine “DJ-1 mouse”, two park7-null alleles were combined with a second concentration changes. Introducing novel stimuli or psychostimulants hit in the nuclear genome [5]. In carrying out work on all three altered the frequency of transients. In behaving animals, dopamine variants, we have made substantive progress in the modeling of transients also became time-locked to reward retrieval, or the clinically relevant behavioral deficits, biochemical abnormalities, and presentation of a reward-predicting cue. Over the last 15 years,

histological changes; there, we also recorded disease worsening with disease in population-based studies. Together with pathological progression in age. Concrete examples for pathological changes will observations of a stagewise progression of PD-specific synuclein be reviewed. pathology with involvement of olfactory or lower brain stem We are optimistic that 55 years after Oleh Hornykiewicz and Walther structures, as well as the peripheral autonomic nervous system prior Birkmayer introduced L-DOPA replacement therapy, the better to any change in the nigrostriatal dopamine projection, these modeling of Parkinson’s variants in laboratory animals, as informed observations have begun to challenge the classical diagnostic and by the complex aetiologies seen in patients, will advance our goal of clinical concept of Parkinson’s disease. In particular, there is reason causal treatment. to hypothesize that individuals without any distinctive motor features Our work has been supported by the Bhargava Family Research but the combined presence of hyposmia, RBD and constipation might Chair, the Canadian Institutes of Health Research, the Michael J. Fox in reality represent patients in the earliest “extranigral” stages of Foundation, the Weston Brain Institute, the Parkinson Society Parkinson’s disease. A new definition and concept of PD needs to be Canada, and the Parkinson Research Consortium. developed that would take into account the prominent non-motor References facet of the disease as well as the distinction of a preclinical and 1. Kuo YM, Li Z, Jiao Y, Gaborit N, Pani AK, Orrison BM, Bruneau BG, premotor phase of the illness. What is needed for such a new concept Giasson BI, Smeyne RJ, Gershon MD, Nussbaum RL: Extensive to be successfully established is a set of biomarkers with high enteric nervous system abnormalities in mice transgenic for sensitivity and specificity for the future development of classical artificial chromosomes containing Parkinson disease-associated motor PD. Recent progress has been made in the field of genomics — α-synuclein gene mutations precede central nervous system imaging, tissue biopsies and proteomic / metabolism marker are an changes. Hum Mol Genet, 2010; 19(9):1633–1650. active field of study. Such concepts are needed not least to open up doi:10.1093/hmg/ddq038 new opportunities for defining at-risk subjects as the future targets for 2. Xu YH, Quinn B, Witte D, Grabowski GA: Viable mouse models of neuropreventive strategies that might ultimately lead to a reduced acid β-glucosidase deficiency: the defect in Gaucher disease. prevalence of and morbidity from Parkinson’s disease. Am J Pathol, 2003; 163(5):2093–2101. doi:10.1016/S0002-9440(10)63566-3 A1.6 3. Herzig MC, Kolly C, Persohn E, Theil D, Schweizer T, Hafner T, Deconstructing dopamine’s role in reward learning and seeking Stemmelen C, Troxler TJ, Schmid P, Danner S, Schnell CR, Mueller M, Patricia H. JANAK1,2,* Kinzel B, Grevot A, Bolognani F, Stirn M, Kuhn RR, Kaupmann K, van 1Department of Psychological and Brain Sciences, Johns Hopkins der Putten PH, Rovelli G, Shimshek DR: LRRK2 protein levels are University, Baltimore, MD, United States of America; 2Department determined by kinase function and are crucial for kidney and lung of Neuroscience, Johns Hopkins University, Baltimore, MD, homeostasis in mice. Hum Mol Genet, 2011; 20(21):4209–4223. United States of America doi:10.1093/hmg/ddr348 *E-mail: [email protected] 4. Gauvin L, Bennett S, Liu H, Hakimi M, Schlossmacher M, Majithia J, Intrinsic Activity, 2016; 4(Suppl. 2): A1.6 Brown EG: Respiratory infection of mice with mammalian http://www.intrinsicactivity.org/2016/4/S2/A1.6 reoviruses causes systemic infection with age and strain dependent pneumonia and encephalitis. Virol J, 2013; 10:67. Dopamine neuron activity has long been implicated in reward doi:10.1186/1743-422X-10-67 processes, both in the acquisition of reward-related associations and 5. Rousseaux MW, Marcogliese PC, Qu D, Hewitt SJ, Seang S, Kim RH, in performance based on those associations. Recent studies have Slack RS, Schlossmacher MG, Lagace DC, Mak TW, Park DS: focused on defining specific behavioral functions of this fascinating Progressive dopaminergic cell loss with unilateral-to-bilateral neuromodulator by manipulating dopamine neuron activity during progression in a genetic model of Parkinson disease. Proc Natl discrete behavioral events, such as during actions made to obtain Acad Sci U S A, 2012; 109(39):15918–15923. reward, during reward-predictive sensory cues, and during receipt of doi:10.1073/pnas.1205102109 reward itself. These studies reveal specificity in dopamine’s behavioral effect that depends on anatomy and depends on when in A1.5 relation to ongoing behavior dopaminergic activity is manipulated. The changing concept of Parkinson’s disease Werner POEWE* A1.7 Department of Neurology, Medical University of Innsbruck, Austria Multidimensional roles for dopamine in cognition and *E-mail: [email protected] behaviour: a translational perspective Intrinsic Activity, 2016; 4(Suppl. 2): A1.5 Trevor W. ROBBINS* http://www.intrinsicactivity.org/2016/4/S2/A1.5 Behavioural and Clinical Neuroscience Institute and Department of Psychology, University of Cambridge, United Kingdom Ever since the seminal description by James Parkinson almost 200 *E-mail: [email protected] years ago, the clinical definition of Parkinson’s disease (PD) is Intrinsic Activity, 2016; 4(Suppl. 2): A1.7 anchored on the presence of motor symptoms. The internationally http://www.intrinsicactivity.org/2016/4/S2/A1.7 most widely used UK Brain Bank Criteria require the presence of bradykinesia plus at least one additional feature of tremor, rigidity or The major forebrain dopamine systems have been implicated in deficient postural reflexes for a clinical diagnosis of PD. On the other motor, motivational and cognitive functions. These functions will be hand, it is now widely acknowledged that a variety of non-motor reviewed from the point of view of roles for dopamine in fronto–striatal symptoms form an integral part of the clinical spectrum of this systems in the control of cognition as well as of impulsive and disorder and that their first occurrence may antedate the manifesta- compulsive behaviour. Studies using a variety of techniques will be tion of classical motor signs. This has been particularly well studied surveyed of rodents, non-human primates and human volunteers for REM sleep behavior disorder (RBD), where up to 60 % of affected relevant to the treatment of patients with schizophrenia, Parkinson’s individuals may convert to clinical PD within 10 years or more. disease, addiction, attention deficit hyperactivity disorder (ADHD) Likewise, healthy subjects with hyposmia, constipation of depression and obsessive–compulsive disorder (OCD). We will consider roles of have been found to carry a two- to four-fold risk for Parkinson’s dopamine D1, and especially D2, receptors in animal behavioural

paradigms that are particularly well-suited to translational applica- Laboratories, United Kingdom; 3Department of Translational tions, using tests of attention, spatial working memory, response Neuroscience, Brain Center, Rudolf Magnus University Medical control and cognitive flexibility, but which also potentially throw light Center Utrecht, The Netherlands on the normal functions of the DA-modulated direct and indirect *E-mail: [email protected] output pathways from the striatum. Drugs affecting the dopamine Intrinsic Activity, 2016; 4(Suppl. 2): A2.2 systems appear to be especially sensitive to individual differences in http://www.intrinsicactivity.org/2016/4/S2/A2.2 their baseline states: several examples of this baseline-dependency Mesodiencephalic dopamine neurons (mDA) play crucial roles in the will be provided and the clinical and theoretical implications consi- control of a variety of brain functions, including voluntary movement dered. and behavioural processes such as mood, reward and attention. Two

main subgroups of mDA neurons, the subtantia nigra pars compacta Developmental biology and the dopamine system (SNpc) and the ventral tegmental area (VTA), form the well-known nigrostriatal and mesolimbic pathways innervating the dorsal and A2.1 ventral striatal regions respectively. Until now, the mechanisms Molecular heterogeneity of midbrain dopamine neurons involved in the segregation of these two important axonal pathways Siew-Lan ANG1,*, Shabana KHAN1, Anna TRUCKENBRODT1, remained unknown. Here we show that two transcription factors, Simon STOTT1, Audrey CHABRAT2 and Martin LÉVESQUE2 Lmx1a and Lmx1b, are required for the appropriate topographical axon innervation of dopamine neurons from the VTA and the SNpc. 1Developmental Neurobiology, Francis Crick Institute, Mill Hill In absence of Lmx1a/b, axon projections of SNpc neurons are Laboratories, United Kingdom; 2Centre de recherche de l’Institut misguided toward the ventral striatal region (VTA target site). Gene universitaire en santé mentale de Québec, Québec, Canada expression profiling experiments comparing Lmx1a/b double *E-mail: [email protected] conditional mutants to control mice identified PlexinC1 as Lmx1a/b Intrinsic Activity, 2016; 4(Suppl. 2): A2.1 target gene. PlexinC1 expression is normally restricted to VTA http://www.intrinsicactivity.org/2016/4/S2/A2.1 neurons while in Lmx1a/b mutants, PlexinC1 expression is expanded Midbrain dopamine (mDA) neurons can be subdivided into distinct to mDA neurons of the SNpc. In vitro examination of dopaminergic functional subsets but very little is known about the molecular mecha- growth cones exposed to Sema7a, a PlexinC1 ligand, indicates that nisms involved in this functional diversity. Recent transcriptome Sema7a acts as a chemorepellent for VTA neurons. The dorsal analysis of different subpopulations of adult mDA neurons, specifical- striatal region shows strong expression of Sema7a, and knockout of ly ventral tegmental area (VTA) and substantia nigra pars compacta Sema7a results in an inappropriate innervation of VTA neurons in the neurons, has improved our understanding of the molecular hetero- dorsal striatal region. Forced expression of PlexinC1 in mDA neurons geneity of these neurons. For example, the homeodomain-containing of transgenic mice results in the same axon guidance defect observed transcription factor Otx2 is expressed in subsets of VTA neurons in Lmx1a/b mutant animals. Our results revealed that Lmx1a and where it regulates the expression of the Girk2 and DAT genes and Lmx1b act as a repressor of PlexinC1 in SNpc neurons and that provides resistance to toxic insults in cell culture assays as well as in Sema7a / PlexinC1 are responsible of the segregation of nigrostriatal mice. One of the VTA-enriched molecules in one of the transcriptomic and mesolimbic dopaminergic pathways. studies is the basic helix-loop-helix transcripton factor Neurod6. Neurod6 is specifically expressed in a subset of VTA mDA neurons A2.3 at embryonic and adult stages. By combining a Cre knock-in strategy Understanding decisions by dopamine axons in adolescence and a loss-of-function approach in mice, we have characterised this Cecilia FLORES* mDA neuronal subset at embryonic, postnatal and adult stages Department of Psychiatry, McGill University, Montreal, Quebec, showing that they form a subset of the Calbindin+ and Raldh1a+ mDA Canada subpopulation of the VTA. Neurod6+ mDA neurons are localised *E-mail: [email protected] specifically in the interfasicular nucleus (IFN), dorsal and ventral Intrinsic Activity, 2016; 4(Suppl. 2): A2.3 paranigral nucleus (PN) and the lateral parabrachial nucleus (PBP) http://www.intrinsicactivity.org/2016/4/S2/A2.3 of the medial VTA. In the absence of Neurod6, 32 % of the total Neurod6-expressing mDA cells are lost within the medial VTA in Adolescence is an age of heightened vulnerability to develop Neurod6 mutants due to cell death as shown by TUNNEL analysis. psychiatric disorders that involve alterations in medial prefrontal Furthermore, retrograde fluorogold tracing experiments in the septum cortex (mPFC) circuitry and cognitive dysfunction. The maturation of have demonstrated that Neurod6-expressing mDA neurons specifi- mPFC function is linked to the establishment of dopamine connec- cally project to two distinct septal region, the dorsolateral (DL) and tivity within this region. The organization of mesocortical dopamine dorsointermediate (DI) septum. Specific loss of mDA axonal pro- circuitry however is a protracted process, which peaks in adoles- jecttions within the DI septum occurred, while projections of mDA cence and ends only in early adulthood. Because of its extended neurons to the DL septum remain unchanged in Neurod6 mutants. developmental course, the shaping of this dopamine projection is Altogether, our results demonstrate that Neurod6 labels a novel particularly susceptible to life experience. Yet, there is a significant population of mDA neurons projecting to the DL and DI septum, but gap in our knowledge about mechanisms of adolescent mPFC is only required for the survival of the latter neurons. dopamine development and about how they are influenced by experience. Via multidisciplinary and translational studies my group A2.2 has identified the guidance cue receptor gene, DCC, to control target Mechanisms controlling the diversity of dopaminergic axon recognition decisions by dopamine axons specifically in adolescence. projections In doing so, DCC determines the extent and organization of the Martin LÉVESQUE1,*, Audrey CHABRAT1, Guillaume BRISSON1, dopamine innervation to the mPFC, influencing significantly (1) the Emmanouil METZAKOPIAN2, Hélène DOUCET-BEAUPRÉ1, structure and function of mPFC pyramidal neurons and (2) cognitive Julien CHAREST1, Siew-Lan ANG2 and R. Jeroen PASTERKAMP3 processing, including behavioral inhibition. Altered function of DCC and its related genes determine differential vulnerability to develop 1Department of Psychiatry and Neuroscience, Université Laval, psychopathology-like phenotypes in mice and is associated with Québec, Québec, Canada; 2The Francis Crick Institute, Mill Hill schizophrenia and depression in humans. Pharmacological and

prophylactic interventions earlier in life may alter the function of this cells in the thyroid gland. These findings are of significance, consider- signaling pathway in the dopamine system and in turn may have an ing the increasing interest in the role of cilia in harboring functional impact on disease outcome. GPCR. We hypothesize that TAAR1 ligands like thyronamines can reach and activate this GPCR in thyroid follicular epithelia by acting A2.4 from within the thyroid follicle lumen, their potential site of synthesis, Unique encoding of reward-related learning and extinction as part of a non-classical mechanism of thyroid autoregulation. by adolescent dopamine neurons Experiments currently ongoing aim at further identifying the vesicles Bita MOGHADDAM* of the secretory pathway which harbor TAAR1. To decipher ligand- triggered actions of TAAR1 in a variety of secretory cells including Center for Neuroscience, University of Pittsburgh, PA, dopaminergic neurons, TAAR1-deficient mice were crossed with United States of America cathepsin-K-deficient animals featuring an overall induction of the *E-mail: [email protected] dopaminergic system. Double-deficient animals are characterized Intrinsic Activity, 2016; 4(Suppl. 2):A2.4 regarding the phenotypes of the thyroid gland and the central nervous http://www.intrinsicactivity.org/2016/4/S2/A2.4 system at the cellular and molecular levels. Background: Elucidating the neurobiology of the adolescent brain is fundamental to our understanding of the etiology of psychiatric A3.2 disorders such as schizophrenia and addiction, the symptoms of Activation of TAAR1 reduces cocaine addiction: from behavior which often manifest during this developmental period. Dopamine to neurobiology neurons in the ventral tegmental area (VTA) are strongly implicated Jun-Xu LI* in adolescent behavioral and psychiatric vulnerabilities, but little is Department of Pharmacology and Toxicology, University at Buffalo, known about how these neurons encode information during motivated NY, United States of America behavior. *E-mail: [email protected] Methods: We recorded daily from dopamine and non-dopamine VTA Intrinsic Activity, 2016; 4(Suppl. 2): A3.2 neurons in adolescent and adult rats during learning and http://www.intrinsicactivity.org/2016/4/S2/A3.2 maintenance of a cued, reward-motivated instrumental task, and extinction from this task. Background: A novel G protein-coupled receptor, trace amine- Results: Both age groups similarly learned the task and quickly associated receptor 1 (TAAR1), has been shown to be a promising adapted their behavior to the absence of reward during extinction. target to prevent stimulant relapse. Our recent studies showed that a During learning, putative dopamine neurons developed similar phasic systemic TAAR1 agonist decreased abuse-related effects of cocaine responses to cue onset in both age groups but showed larger and methamphetamine. However, the neural mechanisms underlying responses before and after reward delivery in adults compared the effect of TAAR1 in drug addiction are largely unknown. to adolescents. Simultaneously recorded putative non-dopamine Methods: Here, we assessed the effects of microinjections of neurons were activated by instrumental actions in adolescents, the selective TAAR1 full agonist RO 5166017 into the mesolimbic but not adults. During extinction, adult putative dopamine neurons regions, i. e. the ventral tegmental area (VTA), the medial prefrontal rapidly diminished their response to the cue as expected, whereas cortex (mPFC), and the nucleus accumbens (NAc), on cue- and drug adolescent neurons remained activated by the cue. priming-induced cocaine seeking in rats with a history of cocaine self- Conclusions: These results suggest that, in adolescent VTA, administration. Rats underwent extinction after cocaine self-adminis- (i) dopamine neurons have a muted response to reward anticipation, tration training. RO 5166017 (5 μg / side) or vehicle (0.5 μl / side) was (ii) non-dopamine neurons have a unique capacity to encode actions, microinjected into each brain region immediately before cue- and and (iii) dopamine neurons retain representations of reward opportu- drug priming-induced cocaine seeking test. nity for longer timescales than adults. Thus, during performance of Results: Microinjection of RO 5166017 into the VTA and the prelimbic the same motivated behavior, identical events are encoded differently cortex of mPFC decreased both cue- and drug priming-induced by adult and adolescent VTA neurons. These data may provide a new cocaine seeking. However, cocaine-seeking behaviors were not framework for understanding dopamine-related psychiatric vulnera- affected by RO 5166017 when microinjected into the infralimbic bilities in adolescents. cortex of mPFC. Furthermore, microinjection of RO 5166017 into the NAc core only inhibited cue-induced drug seeking, but had no effect Dopamine and trace amines on drug priming-induced cocaine seeking. RO 5166017 microinjected into all above brain regions did not affect locomotor activity. Discussion: Taken together, these results indicated that TAAR1 in A3.1 different mesolimbic regions distinctly contributes to cue- and drug Trace amine-associated receptor 1 localization: implications priming-induced cocaine-seeking behavior. for ligand-triggered action Klaudia BRIX1,*, Joanna SZUMSKA1, Maria QATATO1, E. Melisa A3.3 ULLRICH GAVILANES1, Zaina BATOOL1, Uillred DALLTO1, Maya Exploring effects of TAAR1 activation on real-time dopamine GUTEWORT1, Maren REHDERS1 and David K. GRANDY2 neurotransmission using optogenetics and voltammetry 1 Department of Life Sciences and Chemistry, Jacobs University Maria A. MIKHAILOVA1, Marius C. HOENER2 and 2 Bremen, Germany; Department of Physiology and Pharmacology, Evgeny A. BUDYGIN3,* Oregon Health and Science University, School of Medicine, 1Institute of Translational Biomedicine, St. Petersburg State Portland, OR, USA University, St. Petersburg, Russia; 2Neuroscience Discovery, *E-mail: [email protected] Roche Innovation Center Basel, F. Hoffmann-La Roche, Basel, Intrinsic Activity, 2016; 4(Suppl. 2): A3.1 Switzerland; 3Department of Neurobiology and Anatomy, Wake http://www.intrinsicactivity.org/2016/4/S2/A3.1 Forest School of Medicine, Winston-Salem, NC, United States Trace amine-associated receptor 1 (TAAR1) is transported along the of America secretory pathway and it localizes to the primary cilium of epithelial *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A3.3

http://www.intrinsicactivity.org/2016/4/S2/A3.3 in the PFC, as well as altered subunit composition and deficient functionality of the glutamate N-methyl-D-aspartate (NMDA) recep- Using animal models, it has been suggested that the activation tors in the pyramidal neurons of layer V of PFC in mice lacking of trace amine-associated receptor 1 (TAAR1) may represent a TAAR1. The dysregulated cortical glutamate transmission in TAAR1- novel therapeutic option for treatment of several neuropsychiatric dis- KO mice was associated with aberrant behaviors in several tests, orders, including schizophrenia and addiction. Several studies indicating a perseverative and impulsive phenotype of mutants. demonstrated the ability of TAAR1 to modulate dopamine (DA) auto- Conversely, pharmacological activation of TAAR1 with selective receptor function. However, the exact mechanism and real-time agonists reduced premature impulsive responses observed in the consequences of this regulatory action on different patterns of DA fixed-interval conditioning schedule in normal mice. These studies transmission are unknown. The present study explored the effects of indicate that TAAR1 plays an important role in the modulation of a partial TAAR1 agonist, RO 5263397, on presynaptic DA trans- dopamine-related processes in the basal ganglia and NMDA mission, using optogenetics and voltammetry. First of all, we estab- receptor-mediated glutamate transmission in the PFC. Furthermore, lished that high-frequency stimulation (50 Hz) of the VTA could these data suggest that the development of TAAR1-based drugs induce phasic patterns of DA release in terminals which were could provide a novel therapeutic approach for the treatment of characterized by a large, transient rise of extracellular DA levels neuropsychiatric disorders related to aberrant frontostriatal circuitry. (~ 100–600 nM). Applying lower frequencies (5 or 10 Hz) produced a relatively low (~ 40–100 nM), but longer-lasting increase in accumbal DA. Secondly, the effects of RO 5263397 (10 mg/kg, i.p.) on tonic and Dopamine transport phasic DA signaling in vivo were evaluated. The experiments indicated a significant drug effect on the amplitude of the optogenetically- A4.1 induced DA signal (p < 0.0001). Importantly, the drug decreased Dopamine transporter deficiency syndrome: clinical phasic DA more powerfully than tonic DA (p < 0.05). Finally, since characteristics, functional insights and novel therapeutic there are marked regional variations in DA dynamics between striatal strategies and accumbal terminals, we explored local effects of RO 5263397 on Manju A. KURIAN* electrically-evoked DA efflux in the nucleus accumbens core, shell Developmental Neurosciences, Institute of Child Health, and dorsal striatum, using brain slice preparations. High concen- University College London, United Kingdom trations of the compound decreased DA release in the nucleus *E-mail: [email protected] accumbens core (p < 0.05), as well as between the nucleus PPP Intrinsic Activity, 2016; 4(Suppl. 2): A4.1 accumbens shell and dorsal striatum (p < 0.05). In addition, a http://www.intrinsicactivity.org/2016/4/S2/A4.1 significant difference between the last two regions was observed with the drug application at 100 µM (p < 0.01). Therefore, DA release in Over the last few years, recessive bi-allelic mutations in the gene the shell region was less responsive to RO 5263397 application in encoding the dopamine transporter (DAT) have been identified as a comparison with the two other regions. In conclusion, the results of cause of infantile parkinsonism-dystonia and juvenile-onset parkin- this study provide valuable information on the modulation of different sonism. In the classical form of dopamine transporter deficiency DA release patterns through TAAR1 in vivo, and revealed region syndrome (DTDS), infants present with an early-onset hyperkinetic specificity in the direct action of the drug on DA terminals. disorder with dystonia, chorea and orolingual dyskinesia. During childhood, disease progression leads to a hypokinetic phenotype with A3.4 parkinsonism. DTDS is associated with significant morbidity and a Functional consequences of modulating dopamine and high risk of mortality in childhood. A wide range of mutations have glutamate transmission by TAAR1 been reported in DTDS which lead to multifaceted loss of DAT Raul GAINETDINOV1,* and Stefano ESPINOZA2 function, including reduced DAT surface membrane expression, abnormal DAT trafficking, impaired protein glycosylation and reduced 1Institute of Translational Biomedicine, St. Petersburg State substrate recognition and transport. We have utilized dopaminergic University, St. Petersburg, Russian Federation; 2Neuroscience and neurons differentiated from patient-derived pluripotent stem cells to Brain Technologies, Istituto Italiano di Tecnologia, Genova, Italy further understand the pathophysiology processes underpinning *E-mail: [email protected] DTDS, and plan to use this model as a platform for future high Intrinsic Activity, 2016; 4(Suppl. 2): A3.4 throughput drug screening. Furthermore, the use of animal models to http://www.intrinsicactivity.org/2016/4/S2/A3.4 elucidate disease mechanisms and assess novel treatment G protein-coupled trace amine-associated receptor 1 (TAAR1) is strategies, such as gene therapy, will be discussed. emerging as a promising new target for psychiatric disorders. Recent progress in identifying selective ligands for TAAR1 led to the A4.2 possibility of evaluation of the functional consequences of stimulation Pharmacochaperoning of the dopamine transporter / blockade of TAAR1. By using these compounds in an experimental Sonja SUCIC1,*, Ameya KASTURE1, Ali EL-KASABY1, Daniel paradigm developed in our laboratory that involves mouse models of SZÖLLŐSI1, Thomas STOCKNER1, Thomas HUMMEL2 and hyperdopaminergia and dopamine deficiency, as well as mice lacking Michael FREISSMUTH1 TAAR1 (TAAR1-KO mice), we explored the role of TAAR1 in 1Institute of Pharmacology, Medical University of Vienna, Austria; modulation of dopaminergic and glutamatergic transmission. Phar- 2Department of Neurobiology, University of Vienna, Austria macological or genetic targeting of TAAR1 revealed that stimulation *E-mail: [email protected] of TAAR1 suppressed dopamine-dependent behaviors, while TAAR1 Intrinsic Activity, 2016; 4(Suppl. 2): A4.2 deficiency potentiated them. Apparantely, this modulation involves http://www.intrinsicactivity.org/2016/4/S2/A4.2 regulation of striatal D2 dopamine receptor function via D2R–TAAR1 heterodimerization. TAAR1-selective ligands have shown potential The dopamine transporter (DAT) is a member of the solute carrier 6 antipsychotic, antidepressant, and pro-cognitive effects in several gene (SLC6) family. DAT has twelve membrane-spanning helices experimental animal models suggesting that TAAR1 may affect and cytosolic amino and carboxy termini. It is localised at the pre- prefrontal cortex (PFC)-related processes and functions as well. synaptic specialisation, where it rapidly retrieves previously released Recently, we documented a distinct pattern of expression of TAAR1 dopamine from the synaptic cleft, and operates in relay with the

vesicular monoamine transporter to replenish vesicular stores. identification of yet other DAT variants. This includes a variant, Folding-deficient mutants of the human dopamine transporter (DAT) located in the C-terminal PDZ binding sequence, which is also are known to cause a syndrome of infantile / juvenile dystonia / associated with neuropsychiatric disorder and early-onset neuro- parkinsonism. We here provide a proof-of-principle that the folding degenerative parkinsonism. In addition, sequencing of 155 patients deficit is amenable to correction both in vitro and in vivo by two with severe affective disorder has revealed new variants that current- means, the cognate DAT ligand noribogaine and the HSP70 inhibitor, ly are subject to phenotypic characterization in vitro. Our data provide pifithrin-µ. We examined the Drosophila melanogaster mutant dDAT- strong evidence that missense mutations in DAT can cause or G108Q, which leads to a sleepless phenotype in flies harboring this contribute to both neuropsychiatric diseases and movement dis- mutation. Molecular dynamics simulations suggested an unstable orders, and that the resulting disease phenotype depends on the structure of dDAT-G108Q consistent with a folding defect. This nature of the functional perturbations caused by the mutations. conjecture was verified: heterologously expressed dDAT-G108Q and Moreover, the results suggest a yet unappreciated commonality the human equivalent hDAT-G140Q were retained in the endo- between neurodegenerative and neuropsychiatric diseases and plasmic reticulum in a complex with endogenous folding sensors accordingly that the study of DAT missense variants can lead to novel (calnexin and HSP70-1A). Incubation of the cells with noribogaine understanding that pertain to dopamine pathologies in general. (a DAT ligand selective for the inward-facing state) and / or pifithrin-µ (an HSP70 inhibtor) restored the folding of, and hence dopamine A4.4 transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions Sigma-1 receptor modulates methamphetamine regulation of of DAT were confined to the cell bodies of the dopaminergic neurons dopamine transporter activity in the fly brain and failed to reach the axonal compartments. Axonal Habibeh KHOSHBOUEI* delivery was restored and sleep time increased to normal length (from Department of Neuroscience, University of Florida, Gainesville, FL, 300 to 1000 min/d), when dDAT-G108Q-expressing flies were treated United States of America with noribogaine and / or pifithrin-µ. Importantly, we found that a *E-mail: [email protected] number of clinically relevant human DAT (hDAT) mutations causing Intrinsic Activity, 2016; 4(Suppl. 2): A4.4 infantile dystonia / parkinsonism can also be functionally rescued by http://www.intrinsicactivity.org/2016/4/S2/A4.4 pharmacochaperoning. Rescuing misfolded versions of DAT is of therapeutic interest: it may provide opportunities to remedy disorders The dopamine (DA) transporter (DAT) is a membrane protein arising from folding defects, of not only human DAT, but also of implicated in multiple physiological and pathological conditions, related SLC6 transporters, e. g. of the human creatine transporter-1, including movement, reward, and psychostimulant addiction. DAT which gives rise to mental retardation in children, when a glycine functions to translocate DA across the plasma membrane and to residue equivalent to G108 of dDAT is mutated to valine. modulate the excitability of dopaminergic neurons, making it a crucial regulator of DA homeostasis. DAT is also the primary target for A4.3 methamphetamine (METH), a highly addictive and widely abused Missense mutations in the dopamine transporter gene: psychostimulant. What is not often recognized, however, is that commonality between neuropsychiatric and neurodegenerative methamphetamine is both a substrate for DAT as well as a ligand for diseases? the sigma-1 receptor (σ1R), an intracellular chaperone protein. Here, Ulrik GETHER* we identified the σ1R as a novel protein partner of DAT at the plasma membrane. We show that treatment with both methamphetamine and Department of Neuroscience and Pharmacology, Faculty of Health the selective σ R agonist PRE-084 increased the association of σ R and Medical Sciences, University of Copenhagen, Denmark 1 1 with DAT. Interestingly, we found that increased expression of σ R or *E-mail: [email protected] 1 agonist treatment prevented methamphetamine-induced, DAT- Intrinsic Activity, 2016; 4(Suppl. 2): A4.3 mediated increases in firing rates in dopaminergic neurons. We also http://www.intrinsicactivity.org/2016/4/S2/A4.3 found that administration of σ1R agonist also decreased methamphet- Dopamine dysfunction is of central importance in neuropsychiatric amine-mediated DA efflux, without affecting DA uptake or DAT diseases, such as schizophrenia, affective disorder, ADHD and trafficking. These data are the first to provide evidence on how an autism, as well as in neurodegenerative parkinsonism. The pre- intracellular target of methamphetamine can regulate dopamine synaptic dopamine transporter (DAT) mediates reuptake of dopamine neurotransmission. and thereby plays a key role in regulating dopamine homeostasis by terminating dopamine signaling and ensuring maintenance of re- Dopaminergic neurotransmission usable pools of transmitter. There is accumulating evidence that rare genetic variants in the DAT gene, including de novo mutations, can play a hitherto unknown key role in the pathophysiology of both A5.1 neuropsychiatric and neurodegenerative disorders. We recently iden- Regulated trafficking of dopamine and glutamate transporters: tified two novel DAT coding variants in an adult male diagnosed with a mechanism for signal integration by dopamine neurons both neuropsychiatric disorder and early-onset neurodegenerative Suzanne UNDERHILL* parkinsonism. The variants included Ile312Phe in transmembrane National Institute of Mental Health, National Institutes of Health, segment 6 and a presumed de novo mutant Asp421Asn in the second United States of America sodium site. In heterologous cells, both mutants exhibited markedly *E-mail: [email protected] reduced dopamine uptake capacity but preserved membrane Intrinsic Activity, 2016; 4(Suppl. 2): A5.1 targeting, consistent with impaired catalytic activity. For Asp421Asn, http://www.intrinsicactivity.org/2016/4/S2/A5.1 substrate efflux experiments revealed a constitutive, anomalous The actions of psychostimulants involve modulation of both efflux of dopamine, and electrophysiological analyses identified a dopaminergic and glutamatergic neurotransmission. We focus on the cation leak that might contribute to perturbed dopaminergic neuro- mechanisms of action of amphetamine (AMPH) in particular, since transmission. To assess causality and investigate disease mecha- they are widely used therapeutically, but also have a high potential nisms, knock-in mice expressing Asp421Asn and / or Ile312Phe have for addiction and abuse. Plasma membrane neurotransmitter trans- been generated. Importantly, our genetic screening has led to porters are important components of both dopamine and glutamate

neurotransmission and modulation of these carriers play important A5.3 physiological roles in the brain. The dopamine transporter, DAT, and Activity-dependence of dopamine release from the somato- the neuronal glutamate transporter, EAAT3, are both sensitive to dendritic region of nigral dopaminergic neurons AMPH through trafficking mechanisms. Using biochemistry and Andrew Gregory YEE1,*, Mark Henry BURRELL2, Peter Stuart microscopy on cultured cell lines, primary midbrain cultures and acute FREESTONE1 and Janusz LIPSKI1 brain slices, we found that AMPH stimulates internalization of DAT 1Department of Physiology, University of Auckand, New Zealand; and EAAT3. In particular, AMPH-induced EAAT3 internalization leads 2Department of Physiology, Development and Neuroscience, to potentiation of glutamatergic responses in dopamine neurons. We University of Cambridge, United Kingdom designed a small interfering peptide based on the structure of EAAT3 *E-mail: [email protected] that prevents AMPH-induced EAAT3 internalization, but not DAT Intrinsic Activity, 2016; 4(Suppl. 2): A5.3 trafficking. With the use of this peptide as well as traditional EAAT http://www.intrinsicactivity.org/2016/4/S2/A5.3 antagonists, we were able to determine that the potentiation of glutamatergic responses was attributable to EAAT3 internalization in The release of dopamine (DA) from the somato-dendritic region of dopamine neurons. We further identified that AMPH-mediated nigral DAergic neurons has been well established, however despite internalization of the carriers is clathrin-independent but dependent the importance of this process in modulating the excitability of these upon activation of the small GTPase RhoA, Rho kinase (ROCK), the and adjacent non-DAergic neurons, the mechanism of this release GTPase dynamin and release of calcium from intracellular stores. remains unclear. Our recent development of fast-scan controlled Interestingly, we also found that AMPH-induced RhoA activation is absorption voltammetry (FSCAV; [1]) has provided a novel way to transient. The small GTPase is inactivated by protein kinase A (PKA) investigate the activity-dependence of DA release. Using a combina- phosphorylation, which is also stimulated by AMPH; AMPH causes tion of FSCAV and single-unit extracellular electrophysiological Rho activation and then, effectively, Rho inactivation. In agreement recordings in horizontal midbrain slices (300 µm), we directly with this model, we found that PKA activation prior to AMPH correlated absolute levels of extracellular DA concentration ([DA]o) in application blocked Rho activation and subsequent internalization of the substantia nigra pars compacta (SNc) with pharmacologically

DAT and EAAT3. We have found that several other molecules are induced changes in the firing of DAergic neurons. Elevation of [DA]o capable of stimulating this pathway of EAAT3 and DAT internaliza- by pargyline (10 µM; MAO inhibitor) and either cocaine (10 µM; DAT tion, including dopamine itself; a high concentration of dopamine blocker) or L-DOPA (50 µM) decreased the frequency of regular

(10 µM) is sufficient to stimulate internalization of DAT and EAAT3. pacemaker activity by activating D2 auto-receptors. Blocking these The convergence of these two neurotransmitter systems in dopamine receptors with raclopride (1 µM) evoked a large increase of firing, but neurons and the mechanisms by which dopaminergic and glutamate- did not affect [DA]o. Furthermore, abrupt silencing of spontaneous ergic signaling is coordinated is an important avenue to explore in activity with TTX (0.5 µM) had no effect on [DA]o. Induction of burst order to further understand what shapes dopamine and psycho- firing with apamin (100 nM) and NMDA (20 µM) caused a small stimulant-mediated behaviors. elevation of [DA]o, but this rise was also unaffected by TTX. These findings indicate that changes in either regular or burst firing patterns A5.2 of activity do not significantly affect [DA]o, suggesting that somato- Phasic and tonic dopamine neurotransmission may be a dendritic DA release from nigral DAergic neurons is by an action continuum potential-independent mechanism. Further experiments are under- Sweyta LOHANI1,*, Adria MARTIG1, Suzanne UNDERHILL2,3, Alicia way to investigate how [DA]o is affected by optogenetic stimulation or DEFRANCESCO-LISOWITZ1, Susan AMARA2,3 and Bita MOGHADDAM1 inhibition of neuronal firing. Reference 1Center for Neuroscience, University of Pittsburgh, PA, United 1. Burrell MH, Atcherley CW, Heien ML, Lipski J: A novel States of America; 2Department of Neurobiology, University of electrochemical approach for prolonged measurement of absolute Pittsburgh, PA, United States of America; 3National Institute of levels of extracellular dopamine in brain slices. ACM Chem Mental Health, Bethesda, MD, United States of America Neurosci, 2015; 6(11):1802–1812. *E-mail: [email protected] doi:10.1021/acschemneuro.5b00120 Intrinsic Activity, 2016; 4(Suppl. 2): A5.2 http://www.intrinsicactivity.org/2016/4/S2/A5.2 A5.4 Both phasic and tonic modes of neurotransmission are implicated in Co-transmission diversity among dopamine and glutamate critical functions assigned to dopamine, but they are generally treated neurons of the ventral tegmental area as separate entities. Our findings bridge the multiple timescales of Marisela MORALES*, David H. ROOT, Jia QI, Shiliang ZHANG and dopamine neurotransmission by demonstrating that phasic burst Huiling WANG stimulation of VTA dopamine neurons produces a prolonged post- Neuronal Networks Section, Integrative Neuroscience Research burst increase of extracellular dopamine in nucleus accumbens and Branch, National Institutes of Health/National Institute on Drug prefrontal cortex. This prolonged elevation is not due to spillover from Abuse, Baltimore, MD, United States of America the stimulation surge but depends on impulse flow-mediated *E-mail: [email protected] dopamine release. We identified Rho-mediated internalization of Intrinsic Activity, 2016; 4(Suppl. 2): A5.4 dopamine transporter as a mechanism responsible for prolonged http://www.intrinsicactivity.org/2016/4/S2/A5.4 availability of actively released dopamine. These results demonstrate that phasic and tonic dopamine neurotransmission can be a The ventral tegmental area (VTA) has dopamine (DA) neurons continuum and may explain why both modes of signaling are critical expressing tyrosine hydroxylase (TH), GABA neurons expressing for motivational and cognitive functions associated with dopamine. vesicular GABA transporter (VGAT), and glutamatergic neurons expressing vesicular glutamate transporter (VGluT2) [1]. We have found that VTA VGluT2 neurons establish both local and long-range connections that participate in different behaviors. While most of VTA VGluT2 neurons lack both dopaminergic and GABAergic markers, some VGluT2 neurons co-express molecules for

the synthesis and vesicular accumulation of either dopamine [2–4] or A5.5 GABA [5]. At the local level, we had found that VTA VGluT2 neurons Mapping dopamine neuron transmission and co-transmission establish multiple asymmetric (associated with excitation) synapses across striatal subregions on single neighboring dopamine neurons. These synapses mediate Stephen RAYPORT1,2,* and Nao CHUHMA1,2 glutamate-receptor-dependent firing of some dopaminergic neurons 1Department of Psychiatry, Columbia University, New York, NY, that innervate the nucleus accumbens (NAcc), and participate in United States of America; 2Department of Molecular Therapeutics, reward. Regarding long-range outputs of VTA VGluT2 neurons, we NYS Psychiatric Institute, New York, NY, United States of America found that fibers from VTA TH-VGluT2 neurons establish asymmetric *E-mail: [email protected] excitatory synapses on dendritic spines of medium spiny neurons Intrinsic Activity, 2016; 4(Suppl. 2): A5.5 (MSNs) within the NAcc. We had demonstrated that in the nAcc, http://www.intrinsicactivity.org/2016/4/S2/A5.5 individual axons from TH-VGluT2 neurons release DA and glutamate, each from mutually exclusive axonal microdomains [6]. We had also We are conducting a functional connectome analysis of dopamine found that the subpopulation of VTA VGluT2-GAT neurons provides (DA) neuron synaptic actions. By expressing ChR2 in a genetically the major input from VTA to lateral habenula (LHb). Individual axon defined neuronal population — such as DA neurons — and then terminals from fibers of these mesohabenular VGluT2-GAT neurons recording in brain slices from identified target neurons — here in the establish both asymmetric and symmetric synapse, co-release striatum — , wide-field photostimulation activates all ChR2-expressing glutamate and GABA within the LHb, and produce conditional place terminals impinging on recorded postsynaptic neurons. Maps of aversion. Regarding VTA VGluT2-only neurons, a population of nAcc functional connectivity can then be established in systematic parvalbumin GABAergic-interneurons is a major target of fibers from recordings across the striatum, which in concert define the DA neuron the VGluT2-only neurons. These fibers establish multiple asymmetric functional connectome. In DAT-IREScre::Ai32 mice, we see that all synapses on single parvalbumin-GABAergic interneurons, releases DA neurons express ChR2, their terminals produce a uniform striatal GABA onto MSNs, and promotes aversion [7]. fluorescence, and there is complete overlap in ChR2 and immuno- These findings suggest that the midbrain glutamatergic neurons staining for the DA synthetic enzyme tyrosine hydroxylase (TH). participate in different aspects of reward or aversion. In addition, Postsynaptic striatal neurons are functionally identified by their some of these glutamatergic neurons have the capability to use both activity patterns or transgenically by conditional ChAT-, D1R- or D2R- glutamate and dopamine or glutamate and GABA as neurotrans- GFP expression. In the three major populations of striatal neurons, mitters, and they target brain structures implicated in several brain the D1R-expressing, direct pathway (dSPNs) and D2R-expressing, disorders. indirect pathway (iSPNs) spiny projection neurons and cholinergic This work was supported by the Intramural Research Program of the interneurons (ChIs), we have recorded DA neuron synaptic inputs, National Institute on Drug Abuse. and thereby mapped the effects of concerted DA terminal activation References on firing and amplitudes of post-synaptic currents (PSCs) in multiple 1. Morales M, Root DH: Glutamate neurons within the midbrain striatal subregions. With optical stimulation in trains mimicking DA dopamine regions. Neuroscience, 2014; 282:60–68. neuron phasic firing, DA neuron terminal activation reduced ChI firing doi:10.1016/j.neuroscience.2014.05.032 in all striatal regions, except the nucleus accumbens medial shell, 2. Yamaguchi T, Wang HL, Li X, Ng TH, Morales M: Mesocorticolimbic where it drives ChI burst firing. In the lateral dorsal striatum, ChIs glutamatergic pathway. J Neurosci, 2011; 31(23):8476–8490. show a delayed burst following the initial pause. In SPNs, activated doi:10.1523/JNEUROSCI.1598-11.2011 by current injection, we recorded a pause, in the striatum but little 3. Yamaguchi T, Wang HL, Morales M: Glutamate neurons in the effect in the nucleus accumbens. When pharmacologically isolated substantia nigra compacta and retrorubral field. Eur J Neurosci, PSCs were recorded, glutamate PSCs were prominent in the nucleus 2013; 38(11):3602–3610. doi:10.1111/ejn.12359 accumbens medial shell, in both ChIs and SPNs, but weak in most 4. Li X, Qi J, Yamaguchi T, Wang HL, Morales M: Heterogeneous other subregions. However, in the lateral dorsal striatum, large composition of dopamine neurons of the rat A10 region: glutamate PSCs were observed in SPNs, more prominently in molecular evidence for diverse signaling properties. Brain Struct dSPNs, and small glutamate PSCs were observed in ChIs. These Funct, 2013; 218(5):1159–1176. doi:10.1007/s00429-012-0452-z observations reveal new dimensions in DA neuron signaling and 5. Root DH, Mejias-Aponte CA, Zhang S, Wang HL, Hoffman AF, Lupica identify the medial shell of nucleus accumbens and lateral dorsal CR, Morales M: Single rodent mesohabenular axons release striatum as regions where DA neurons have prominent excitatory glutamate and GABA. Nat Neurosci, 2014; 17(11):1543–1551. actions. Despite apparently invariant cytoarchitecture across the doi:10.1038/nn.3823 striatum, considerable heterogeneity is revealed at the functional 6. Zhang S, Qi J, Li X, Wang HL, Britt JP, Hoffman AF, Bonci A, Lupica level, in particular in the synaptic signals conveyed by DA neurons. CR, Morales M: Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons. Nat Neurosci, 2015; A5.6 18(3):386–392. doi:10.1038/nn.3945 Inhibitory neurotransmission by midbrain dopamine neurons 7. Qi J, Zhang S, Wang HL, Barker DJ, Miranda-Barrientos J, Morales M: Nicolas TRITSCH* VTA glutamatergic inputs to nucleus accumbens drive aversion Department of Neuroscience and Physiology, NYU School of by acting on GABAergic interneurons. Nat Neurosci, 2016; Medicine, New York, NY, United States of America 19(5):725–733. doi:10.1038/nn.4281 *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A5.6 http://www.intrinsicactivity.org/2016/4/S2/A5.6

Midbrain dopamine-producing neurons (DANs) play crucial roles in the generation and reinforcement of purposive behaviors, and perturbations in the function of this small group of cells have been implicated in a plethora of neuropsychiatric conditions, including Parkinson’s disease, addiction, schizophrenia and depression. DANs have consequently been studied extensively: the anatomy of their

synaptic inputs and axonal projections, their electrophysiological as “fluorescent false neurotransmitters” (FFNs) that act as synaptic properties and their firing patterns to environmental stimuli are well vesicle transporter substrates, as well as endocytic probes and documented. However, how they synaptically influence the activity of calcium reporters that resolve activity simultaneously at hundreds of target cells in the striatum remains comparatively less well under- synapses both in the brain slice and, very recently, in vivo. These stood. We recently uncovered that DANs co-release the major optical approaches indicate not only presynaptic release probability inhibitory neurotransmitter GABA along with dopamine in dorsal and and reuptake kinetics but also new forms of presynaptic plasticity, ventral striatum, and showed that DANs utilize non-conventional including the rapid movement of presynaptic release sites, and molecular mechanisms to acquire GABA and package it into synaptic transfer between silent and active states, with the surprising vesicles. I will review these findings and discuss ongoing experiments observation that most dopamine “synapses” may be functionally aimed at revealing the functional implications of inhibitory signaling silent. by dopaminergic neurons for synaptic transmission, plasticity and We will also report the first in vivo optical observation of nor- behavior. adrenergic synapses and amphetamine’s action at single-synapse resolution, using a novel norepinephrine (NE)-specific FFN, FFN 270, A5.7 in mice surgically implanted with a cranial window. We find that in Dopamine neurons in vivo controls in the absence of sensory stimuli, only a small subset of Carlos A. PALADINI1,*, Kauê M. COSTA2 and Jochen ROEPER2 noradrenergic axonal varicosities actively released FFN. However, at 1 mg/kg D-amphetamine (i.p.), a dosage thought to be equivalent to 1Department of Biology, UTSA Neurosciences Institute, University treatment for ADHD, the FFN was emptied from all synapses within of Texas at San Antonio, San Antonio, TX, United States of 10 minutes (p = 0.005). This is to our knowledge a first optical in vivo America; 2Institute of Neurophysiology, Neuroscience Center, confirmation of amphetamine’s role in vesicular release of NE at Goethe University Frankfurt, Frankfurt am Main, Germany therapeutically relevant doses and at the level of resolution of *E-mail: [email protected] individual synapses. Intrinsic Activity, 2016; 4(Suppl. 2): A5.7

http://www.intrinsicactivity.org/2016/4/S2/A5.7 Dopamine receptors Intracellular in vivo recording in awake behaving animals remains the ultimate tool to understand which receptors and ionic conductances underlie reward-related activity of identified dopamine neurons. Up to A6.1 now, studies investigating the mechanisms underlying dopamine Modulation of D2 autoreceptors function in substantia nigra neuron activity have been limited to ex vivo intracellular recordings, dopaminergic neurons: an in vitro electrophysiological which lack ongoing behavior; or in vivo extracellular recordings, approach 1 1,2 1,2, which cannot measure subthreshold ionic conductances. We have Daniela AVERSA , Nicola BERRETTA and Nicola B. MERCURI * developed a method to routinely obtain in vivo whole-cell recordings 1Department of System Medicine, Università Tor Vergata, Rome, from identified dopamine neurons in mice. In the awake, behaving Italy; 2Fondazione Santa Lucia, Italy brain everything is active and dopamine neurons receive a sustained *E-mail: [email protected] combination of excitatory and inhibitory inputs. A reward-related burst Intrinsic Activity, 2016; 4(Suppl. 2): A6.1 of action potentials could just as likely result from a phasic increase http://www.intrinsicactivity.org/2016/4/S2/A6.1 in excitation or decrease in inhibition. A reward-related pause in firing One of the main hallmarks of the dopamine (DA)-releasing neuronal could result from a phasic decrease in excitation or increase in population of the substantia nigra pars compacta (SNpc) is the inhibition. We are determining whether reward-related bursts are presence of somato-dendritic D autoreceptors, whose activation caused by dynamic changes in the balance between excitation and 2 leads to opening of G protein-coupled inward rectifier K+ (GIRK) inhibition that result in a relative increase in excitatory synaptic channels and hence membrane hyperpolarization. The physiological transmission or a decrease in inhibitory synaptic transmission. importance of this auto-inhibition is implicitly understood by the fact Conversely, we are determining whether reward-related pauses are that DA neurons firing is constantly controlled by tonic DA acting mediated by a relative decrease in excitatory synaptic transmission locally. Interestingly, the membrane hyperpolarization induced by or an increase in inhibitory synaptic transmission. prolonged DA exposure is subject to a time-dependent desensitization, such that neuronal firing may recover almost to control levels, in A5.8 spite of persistent D2 stimulation. This phenomenon, often referred as Optical means to measure presynaptic regulation of dopamine reversal of DA inhibition (RDI), is mostly observed in young animals. release However, evidence exists that in Parkinson’s disease (PD) surviving 1, 2 1 3 David SULZER *, Daniela PEREIRA , Yvonne SCHMITZ , Matt DUNN , adult DA neurons respond with RDI under prolonged DA. This may 1 3 3 Sam CLARK , Adam HENKIE and Dalibor SAMES suggest a higher metabolic demand on the SNpc DA neurons 1Department of Psychiatry, Columbia University, United States of population, due to the reduced auto-inhibition, that could exacerbate America; 2University of Lisbon, Portugal; 3Department of Chemistry, their progressive loss in PD. Moreover, RDI may dramatically blunt Columbia University, United States of America the efficacy of common pharmacological therapies aimed at *E-mail: [email protected] increasing the DA tone in PD patients. With the aim to understand the Intrinsic Activity, 2016; 4(Suppl. 2): A5.8 cellular mechanisms underlying RDI, we have investigated DA http://www.intrinsicactivity.org/2016/4/S2/A5.8 neurons’ response to prolonged (> 15 min) perfusion of DA 100 µM in midbrain slices from young (p12–15) mice. While a clear recovery of Electrophysiological and electrochemical recordings provide time firing was observed with DA, the cells remained inhibited when the D resolution sufficient to measure quantal neurotransmission at CNS 2 receptor was continuously stimulated with its agonist quinpirole synapses, but do not resolve activity of individual synapses within (100 nM). Notably, if 100 µM DA was then added under quinpirole, populations. Modulation at the level of individual synapses, however, their firing soon recovered as with DA alone. This strongly suggests may be required in habit learning and decision-making. We will that (1) DA as such is required for RDI, and (2) RDI is not due to the discuss means to optically resolve the modulation of subgroups of sole D receptors stimulation. We then activated the GIRK channel individual synapses using a class of catecholamine derivatives known 2 through the converging GABAB receptor pathway by baclofen

(300 nM), under D2 receptor block with sulpiride (30 µM). No recovery signal in behaving animals is unknown. We therefore extended our from firing inhibition was observed under long exposure to baclofen previous mathematical models to infer the real time DA signal in freely (> 15 min), however, if we then added 100 µM DA to baclofen and moving animals from experimental data obtained using fast-scan sulpiride, the cells resumed their activity. We thus propose the GIRK cyclic voltammetry [4]. The models were tailored to match DA cell channel, rather than the D2 receptor, as the main actor of RDI, firing with recorded spontaneous fluctuations in extracellular DA whereby closure of the GIRK channel causes recovery from firing concentrations in individual animals. The output of the model includes inhibition in spite of a continuous D2 receptor stimulation. Implications direct estimates of the pre-synaptic D2 autoreceptor action and a for PD etiology and therapy will be discussed. measure of the individual efficacy of the presynaptic autoreceptor system in each animal. A6.2 We applied the model to recordings from 36 freely moving rats, 19 hereof were recorded in nucleus accumbens shell and 17 in nucleus Calcium mmodulation of somatodendritic dopamine D2 autoreceptor responses accumbens core. We tested the ability of the model to estimate the Birgit LISS* spontaneous real-time DA signal in freely moving conditions and tested predicted individual differences in DA release under raclopride Institute of Applied Physiology, University of Ulm, Germany and intravenous cocaine. We found that the DA level was negatively *E-mail: [email protected] correlated with autoreceptor efficacy. In other words, low average DA Intrinsic Activity, 2016; 4(Suppl. 2): A6.2 levels observed in some animals were the result of strong auto- http://www.intrinsicactivity.org/2016/4/S2/A6.2 receptor control, while other animals had high DA levels and corre-

Somatodendritic dopamine autoreceptors of the D2 type (D2ARs) spondingly low autoreceptor feedback. Our analysis suggests that modulate the spontaneous electrical activity of substantia nigra individual differences in efficacy of presynaptic autoreceptor account dopaminergic midbrain (SN DA) neurons in a negative feedback loop, for most of the between subject variability in DA signaling. due to direct βγ-subunit activation of G protein-coupled inwardly References + rectifying K channels (GIRKs). However, D2 receptor signaling in 1. Dreyer JK, Hounsgaard J: Mathematical model of dopamine general is modulated by a variety of — partly Ca2+-dependent — autoreceptors and uptake inhibitors and their influence on tonic signaling mechanisms. Ca2+ and voltage-gated Ca2+ channel (VGCC) and phasic dopamine signaling. J Neurophysiol, 2013; 109(1): activity seem to contribute to the high vulnerability of SN DA neurons 171–182. doi:10.1152/jn.00502.2012 to degeneration in Parkinson’s disease (PD); epidemiological studies 2. Marinelli M, White FJ: Enhanced vulnerability to cocaine self- indicate that specific blood–brain barrier-permeable VGCC blockers administration is associated with elevated impulse activity of (i. e. L-type VGCC inhibitors of the dihydropyridine type) reduce the midbrain dopamine neurons. J Neurosci, 2000; 20(23):8876–8885. risk to develop PD by about 30 %. However, the underlying molecular 3. Zald DH, Cowan RL, Riccardi P, Baldwin RM, Ansari MS, Li R, Shelby mechanisms as well as the physiological roles of VGCCs in SN DA ES, Smith CE, McHugo M, Kessler RM: Midbrain dopamine receptor neurons are still unclear. We identified a novel role of VGCCs for a availability is inversely associated with novelty-seeking traits in calcium- and age-dependent sensitization of inhibitory somato- humans. J Neurosci, 2008; 28(53):14372–14378. dendritic D2AR responses: a homeostatic interplay of VGCCs seems doi:10.1523/JNEUROSCI.2423-08.2008 to mediate context-dependent, flexible dopamine D2AR responses, 4. Dreyer JK, Vander Weele CM, Lovic V, Aragona BJ: Functionally selectively in highly vulnerable SN DA neurons, and thus modulate distinct dopamine signals in nucleus accumbens core and shell in their activity pattern and dopamine release. We currently investigate the freely moving rat. J Neurosci, 2016; 36(1):98–112. 2+ the possibility that Ca and VGCC-mediated sensitization of D2AR doi:10.1523/JNEUROSCI.2326-15.2016 responses might modulate SN DA vulnerability to degeneration. Dopaminergic signal transduction A6.3 Presynaptic dopamine autoreceptors account for individual A7.1 differences in the dopamine baseline: theory meets experiment Regulation of ribosomal S6 phosphorylation in D R- and D R- in freely moving rats 1 2 expressing striatal neurons Jakob Kisbye DREYER1,*, Caitlin VANDER WEELE2, Vedran LOVIC3 and Emmanuel VALJENT*, Anne BIEVER and Emma PUIGHERMANAL Brandon ARAGONA4 Department of Neurobiology, Inserm U 1191, CNRS UMR 5203, 1Department of Neuroscience and Pharmacology, University of Institut de génomique fonctionnelle (IGF), University of Montpellier, Copenhagen, Denmark; 2Department of Brain and Cognitive France Sciences, Massachusetts Institute of Technology, Cambridge, MA, *E-mail: [email protected] United States of America; 3Department of Psychology, University of Intrinsic Activity, 2016; 4(Suppl. 2): A7.1 Calgary, Alberta, Canada; 4Department of Psychology, University of http://www.intrinsicactivity.org/2016/4/S2/A7.1 Michigan, Ann Arbor, MI, United States of America *E-mail: [email protected] The striatum and its ventral part, the nucleus accumbens (NAc) are Intrinsic Activity, 2016; 4(Suppl. 2): A6.3 critical input structures of the basal ganglia, an ensemble of http://www.intrinsicactivity.org/2016/4/S2/A6.3 subcortical nuclei enabling adaptive control of behavior. The output neurons of these structures, the GABAergic medium-sized spiny Dopamine (DA) release is maintained within a desired working range neurons (MSNs), comprising the striatonigral (D R-MSNs) and by pre-synaptic and somatodendritic dendritic D -like autoreceptors 1 2 striatopallidal MSNs (D R-MSNs) are tightly modulated by dopamine- that provide negative feedback on DA release, synthesis and firing 2 ergic inputs. Dysfunction of these circuits participates in various rate. The complex concerted action of these somatodendritic and neurological and psychiatric disorders including Parkinson’s disease, presynaptic feedback systems has previously been studied obsessive–compulsive disorder, schizophrenia and addiction. computationally [1]. However, individual differences DA autoregula- Regulation of gene transcription and mRNA translation are key tion have been related to numerable psychiatric traits, including mechanisms by which dopamine can control physiologogical and novelty seeking and propensity for drug abuse [2, 3], but the pathological striatal plasticity. We will present data showing that functional effect of such differences on the real time functional DA modulation of DA tone can induce a rapid regulation of the state of

phosphorylation of the ribosomal protein S6 (rpS6), a component of between D2 receptors and other receptors would alter responses to the 40S ribosomal subunit. We also discuss recent findings D2 agonists, which we measured with a sensitive radioisotopic suggesting that neither basal nor drug-induced transient rpS6 methodology assessing D2 receptor inhibition of rat brain dopamine phosphorylation correlate with changes in global mRNA translation of synthesis. The inhibitory effect of D2 receptor full agonist quinpirole global or 5′-terminal oligopyrimidine tract mRNAs. Finally, we will could not be modified with mGlu5 negative allosteric modulators provide evidences that despite the lack of causal relationship MTEP (10 µM) or fenobam (1 µM) or with the GABAB antagonist between both events, rpS6 phosphorylation plays an important role CGP 55845 (100 nM). In contrast, MTEP completely blocked the in striatal plasticity. inhibition elicited by the D2 partial agonists (−)-PPP (1 µM) and

aripiprazole (10 nM). Additionally, fenobam, but not the GABAB A7.2 antagonist CGP 55845, blocked the inhibition elicited by (−)-PPP. The Regulation of mood related behavior by GSK3-mediated simplest explanation for this crosstalk between D2 and mGlu5 dopamine receptor signaling receptors in the regulation of dopamine synthesis is a membrane Jean-Martin BEAULIEU* interaction or heteromerization that has already been described in cultured cells. Such membrane interaction appears to affect only Department of Psychiatry and Neuroscience, Université Laval, responses to D partial agonists, but not full agonists. These results Québec, QC, Canada 2 could be relevant for the treatment of neuropsychiatric disorders, *E-mail: [email protected] where D receptors are important targets of pharmacotherapy. Intrinsic Activity, 2016; 4(Suppl. 2): A7.2 2 http://www.intrinsicactivity.org/2016/4/S2/A7.2 A7.4 Despite wide clinical applications, the mechanisms underlying the Dopamine mediates neuroinflammation and HIV infection of the shared actions and therapeutic specificity of mood stabilizers are CNS: critical roles in neuroAIDS unknown. Investigations conducted in animal models have shown Tina CALDERON1, Dionna WILLIAMS1, Lillie LOPEZ1, Eliseo EUGENIN2, that mood stabilizers can exert their effect partially by acting on Peter GASKILL1, Mike VEENSTRA1, Kathryn ANASTOS3, Susan Akt / GSK3 pathway downstream of dopamine D2 receptors. Few MORGELLO4 and Joan BERMAN1,* studies have show that regulation of distinct behaviors by the 1Department of Pathology, Albert Einstein College of Medicine, Akt / GSK3 pathway may be brain-region-dependent. However, cell- Bronx, NY, United States of America; 2Department of Microbiology, type-specific mechanisms by which Akt / GSK3 signaling regulates Biocemistry and Molecular Genetics, Rutgers State University of mood-related behaviors remains elusive. We identified fragile X New Jersey, Newark, NJ, United States of America; 3Department of mental retardation protein 1 (FXR1P), an RNA-binding protein Medicine, Albert Einstein College of Medicine, Bronx, NY, United associated to genetic risk for schizophrenia, as a substrate for States of America; 4Department of Neurology, Icahn School of GSK3β. Phosphorylation of FXR1P by GSK3β is facilitated by prior Medicine at Mount Sinai, New York, NY, United States of America phosphorylation by ERK2 and leads to its down-regulation. In *E-mail: [email protected] contrast, behaviorally effective chronic mood stabilizer treatments in Intrinsic Activity, 2016; 4(Suppl. 2): A7.4 mice inhibit GSK3β and increase FXR1P levels. In line with this, http://www.intrinsicactivity.org/2016/4/S2/A7.4 overexpression of FXR1P in the mouse prefrontal cortex also leads to comparable mood-related responses. Furthermore, functional Substance abuse may accelerate the development and / or increase genetic polymorphisms affecting either FXR1P or GSK3β gene the severity of HIV-associated neurocognitive disorders (HAND). HIV expression interact to regulate emotional brain responsiveness and neuropathogenesis in the drug-abusing infected population is not stability in humans. These observations uncovered a GSK3β / FXR1P completely characterized. The percentage of peripheral blood signaling pathway that contributes to regulating mood and emotion CD14+ CD16+ monocytes is increased with HIV infection. The trans- processing. Regulation of FXR1P by GSK3β also provides a migration of uninfected and infected CD14+ CD16+ monocytes mechanistic framework that may explain how inhibition of GSK3β can across the blood–brain barrier (BBB) contributes to HIV entry into the contribute to the regulation of mood by psychoactive drugs in mental CNS and to the establishment and propagation of chronic neuro- illnesses such as bipolar disorder. Moreover, this pathway could inflammation, which both mediate HAND. We demonstrate that the potentially be implicated in other biological functions, such as frequency of CD14+ CD16+ monocytes in the periphery of HIV- inflammation and cell proliferation, in which FXR1P and GSK3 are infected substance abusers is increased when compared to those known to play a role. without active substance use. Dopamine, elevated in the brain in response to drug use, enhances the transmigration of CD14+ CD16+ A7.3 monocytes, but not T cells, across our in vitro model of the human BBB. Therefore, elevated extracellular CNS dopamine may be a D2 receptor crosstalk with metabotropic glutamate receptors, novel mechanism by which active substance use increases but not with GABAB receptors, in the regulation of dopamine synthesis in rat brain CD14+ CD16+ monocyte entry into the CNS of HIV-infected individ- Doaa ABASS, Guofen MA, Marta GONZÁLEZ-SEPÚLVEDA, Carles GIL, uals. Transmigration is mediated by D1-like dopamine receptors as Jesús GIRALDO, Josefa SABRIA and Jordi ORTIZ* indicated by greater surface D1 and D5 dopamine receptor expression on CD14+ CD16+ monocytes as compared to T cells, and the ability Neuroscience Institute, Universitat Autònoma de Barcelona, Spain of the D -like dopamine receptor agonist, SKF 38393, to increase *E-mail: [email protected] 1 CD14+ CD16+ monocyte transmigration across the BBB. Pseudopod Intrinsic Activity, 2016; 4(Suppl. 2): A7.3 formation and active ADAM17 expression by CD14+ CD16+ mono- http://www.intrinsicactivity.org/2016/4/S2/A7.3 cytes are increased by dopamine and may contribute to the ability of Dopamine receptors play a key role in motor activity and goal- these cells to transmigrate. Thus, uninfected and HIV-infected directed behaviors and are relevant for the treatment of diverse dis- CD14+ CD16+ monocyte entry into the CNS may increase with active orders, including schizophrenia and Parkinson’s disease. G protein- substance use and subsequent increased dopamine, contributing to coupled receptors are known to form homo- and heterodimers at the CNS viral seeding, neuroinflammation, and HIV neuropathogenesis. plasma membrane, but the function of such receptor oligomers is relatively unknown. Here, we hypothesized that heteromerization

Dopamine and neuroplasticity Intrinsic Activity, 2016; 4(Suppl. 2): A8.2 http://www.intrinsicactivity.org/2016/4/S2/A8.2

A8.1 Childhood adversity increases the risk of psychosis in adulthood. Dopaminergic modulation of hippocampal spike timing- Theoretical and animal models suggest that this effect may be dependent plasticity mediated by increased striatal dopamine neurotransmission. Elke EDELMANN1,2,*, Efrain CEPEDA PRADO1 and Volkmar The primary objective of this study was to examine the relationship LESSMANN1,2 between adversity in childhood and striatal dopamine function in early adulthood. Secondary objectives were to compare exposure to child- 1Institute of Physiology, Otto-von-Guericke University Magdeburg, hood adversity and striatal dopamine function in young people at Germany; 2Center for Behavioral Brain Sciences, Magdeburg, ultra-high risk (UHR) of psychosis and healthy volunteers. Sixty- Germany seven young adults, comprising 47 individuals at UHR for psychosis *E-mail: [email protected] and 20 healthy volunteers were recruited from the same geographic Intrinsic Activity, 2016; 4(Suppl. 2): A8.1 area and were matched for age, gender and substance use. Pre- http://www.intrinsicactivity.org/2016/4/S2/A8.1 synaptic dopamine function in the associative striatum was assessed Long-term potentiation (LTP) is an accepted model for cellular using [18F]DOPA positron emission tomography. memory engrams. Strengthening of synaptic transmission can be Childhood adversity was assessed using the Childhood Experience induced with various stimulation patterns. A reliable tool to mimic of Care and Abuse questionnaire. Within the sample as a whole, both synaptic plasticity in a physiological relevant form is the spike timing- severe physical or sexual abuse (T63 = 2.92; p = 0.005), and unstable dependent plasticity (STDP) paradigm, which consists of repetitive family arrangements (T57 = 2.80; p = 0.007) in childhood were presynaptic stimulations paired with 1–4 action potentials (AP) fired associated with elevated dopamine function in the associative in the postsynaptic neuron. Applying different STDP paradigms at striatum in adulthood. Comparison of the UHR and volunteer sub- CA3–CA1 synapses, we can induce robust timing-dependent (t-)LTP. groups revealed similar incidence of childhood adverse experiences, Interestingly subtle changes in the number of postsynaptic APs and and there was no significant group difference in dopamine function. in the number of overall repetition of pairings lead to t-LTP forms This study provides evidence that childhood adversity is linked to which markedly differ in the recruited signaling cascades. Using patch elevated striatal dopamine function in adulthood. These findings clamp techniques in acute hippocampal slices, we show that a suggest that psychosocial factors may influence the risk of psychosis canonical 1 EPSP / 1 AP pairing (70 x) leads to a presynaptic t-LTP, through effects on neurobiological processes implicated in the patho- which is induced in a dopamine (DA)-dependent manner. In DA- physiology of the disorder. depleted slices, STDP can be restored by bath-applied DA. Non- canonical forms of STDP, like our 1EPSP / 4AP paradigm (30 x) leads A8.3 to postsynaptic t-LTP which is mediated by BDNF. We further show The role of prefrontal dopamine in stress reactivity in that, this 1 : 4 t-LTP is mediated via BDNF action at postsynaptic TrkB psychosis receptors. In order to investigate synaptic plasticity mechanisms Inez MYIN-GERMEYS1,*, Zuzana KASANOVA1, Jens PRUESSNER2, approaching in vivo-like conditions we tested threshold protocols with Johan LATASTER3, Dina COLLIP3 and Dennis HERNAUS3 low numbers of repeats. A decrease in number of repeats in both 1Department of Neuroscience, KU Leuven, Belgium; 2Department of paradigms to only 6 repeats still elicited t-LTP. Interestingly this low Psychiatry, McGill University, Montreal, Quebec, Canada; repeat t-LTP shows a dramatic change in the expression mechanism. 3Department of Psychiatry and Neuropsychology, Maastricht While we observe a clear DA dependency for the 1 : 1 70 x paradigm, University, Maastricht, The Netherlands the 6 x 1 : 1 variant is independent of D receptor signaling and most 1 *E-mail: [email protected] likely mediated by BDNF signaling. Similarly, decreasing the repeat Intrinsic Activity, 2016; 4(Suppl. 2): A8.3 number to 6 x leads to a loss of BDNF-mediated expression of t-LTP http://www.intrinsicactivity.org/2016/4/S2/A8.3 in case of the 1 : 4 paradigm and additionally to a shift from NMDAR dependency towards NMDAR-independent induction. Currently, we Background: Stress is a risk factor in the etiology of psychosis. investigate whether a change to lower repeats for STDP induction Stress is hypothesized to be associated with prefrontal and meso- leads to recruitment of other DA receptors. Taken together our data limbic dopamine neurotransmission. Similarly, whereas aberrant show, that similarly effective STDP can be induced with various striatal dopamine processing is associated with psychosis, the role of patterns and different numbers of repeats at hippocampal CA3–CA1 the prefrontal cortex remains under-researched. This study aims to synapses. These t-LTP forms are mediated by activation of distinct investigate prefrontal dopamine in a stress paradigm in subjects at signaling cascades and expression mechanisms. Hence, our data risk for psychosis. indicate a multitude of potential forms of memory engrams, which can Methods: The Montreal Imaging Stress Task was used in a be expressed simultaneously or independently at a single type of [18F]fallypride PET paradigm in healthy controls (n = 12), siblings synapse in the hippocampus. (n = 14), and unmedicated patients with psychotic disorder (n = 12). Supported by the SFB779 / B06. Data were acquired using a modified version of the simplified reference tissue model. Furthermore, the siblings and controls A8.2 participated in an experience sampling study to examine real-life Adversity in childhood linked to elevated striatum dopamine stress reactivity. function in adulthood Results: The stress challenge induced detectable amounts of Alice EGERTON*, Lucia VALMAGGIA, Oliver HOWES, Fern DAY, dopamine release throughout the prefrontal cortex of controls. Christopher CHADDOCK, Paul ALLEN, Toby WINTON-BROWN, Furthermore, higher levels of subjective stress were associated with Michael BLOOMFIELD, Sagnik BHATTACHARYYA, Jack CHILCOTT, decreased prefrontal DA activity under stress. However, we found no Julia LAPPIN, Robin MURRAY and Philip MCGUIRE absolute mean differences in stress-induced prefrontal DA release between the three groups. Still, psychotic reactivity to daily life stress Psychosis Studies, Institute of Psychiatry, Psychology and was found to be associated with decreased stress-related prefrontal Neuroscience, Kings College London, United Kingdom DA function. *E-mail: [email protected]

Discussion: The findings underscore the functional relevance of A8.5 prefrontal DA in the stress-response. However, the data do not offer Plasticity of the dopaminergic system: drug–stress cross- evidence for altered stress-induced extrastriatal DA release in sensitization and fear conditioning 18 vulnerability to psychosis. Still, decreased [ F]fallypride displace- Jennifer I. LISSEMORE1,*, Atsuko NAGANO-SAITO1, Linda BOOIJ1, ment to stress in VMPFC was associated with increased psychotic Paul GRAVEL2, Marco LEYTON1 and Chawki BENKELFAT1 reactivity to daily life stress. The implications of these findings for the 1Department of Psychiatry, McGill University, Montreal, QC, role of DA and other neurotransmitter systems in the context of Canada; 2McConnell Brain Imaging Center, Montreal Neurological psychosis and the stress network are discussed. Institute, McGill University, Montreal, QC, Canada *E-mail: [email protected] A8.4 Intrinsic Activity, 2016; 4(Suppl. 2): A8.5 Inverse incentive learning: dopamine and decreased http://www.intrinsicactivity.org/2016/4/S2/A8.5 responding to stimuli Richard J. BENINGER* Studies in laboratory animals indicate that pharmacological and environmental activations of mesocorticolimbic dopamine (DA) Department of Psychology, Queen’s University, Kingston, ON, pathways can lead to long-lasting changes in their reactivity. To test Canada for these effects in humans, we conducted positron emission *E-mail: [email protected] tomography (PET) studies of (i) drug–stress cross-sensitization, and Intrinsic Activity, 2016; 4(Suppl. 2): A8.4 (ii) fear conditioning. http://www.intrinsicactivity.org/2016/4/S2/A8.4 Study 1 used PET with [11C]raclopride to test whether a repeated Dopamine (DA)-mediated incentive learning refers to the acquisition intermittent psychostimulant regimen can sensitize the striatal DA by neutral stimuli of an increased ability to elicit approach and other response to stress. Seventeen healthy volunteers initially underwent responses. Inverse incentive learning (IIL) is the loss by stimuli of two baseline PET scans during a low stress motor control task and their ability to elicit approach and other responses and also may the Montreal Imaging Stress Task (MIST). During the following week, involve DA. When DA neurons signal negative prediction error, IIL they received three administrations of either D-amphetamine (0.3 may take place. IIL is observed as increased immobility across once- mg/kg, p.o.) or placebo. Two weeks after the last drug administration daily sessions using the bar test (like the apparatus often used to test (0 or 0.3 mg/kg), participants had a 2nd PET scan with the MIST. In catalepsy). Schmidt et al. [1] showed that paired rats treated with low- subjects who had previously received D-amphetamine, the stress- dose haloperidol (HAL, 0.25 mg/kg) 1 hr before testing descended induced striatal DA response was increased (p < 0.001), suggesting immediately on the first test but increased immobility over days; the development of drug–stress cross-sensitization. unpaired rats receiving the same dose of HAL each day 1 hr after bar In study 2, we used PET with [18F]fallypride to measure brain-wide testing showed no immobility. After 10 days, if both groups were DA release during the expression and inhibition of conditioned fear. tested following saline injection or following HAL injection, only the Twelve healthy volunteers underwent a baseline PET scan, after paired group was immobile. Results show that immobility is which an electric shock was paired with a visual cue. During a 2nd conditioned by the pairing of HAL with specific environmental stimuli PET scan, participants were exposed to the shock-paired cue. and does not simply result from a history of HAL injections. We Finally, a 3rd PET scan was performed following a reversal learning observed the same effect with spiroperidol (0.025–0.25 mg/kg). D1- procedure to study DA release during the inhibition of conditioned like receptors are implicated in IIL by the observation that groups fear. A priori region-of-interest analyses identified a main effect of day given SCH 23390 (SCH, 0.05–0.25 mg/kg) during testing fail to show (p = 0.04) in bilateral hippocampus reflecting increased DA release immobility on the saline test. Groups given SCH 15, but not 0 or 60, during the inhibition of conditioned fear, compared to the expression min after bar testing with HAL each day show weaker increases in of conditioned fear (p = 0.028) and compared to baseline (p = 0.045). immobility over days suggesting that post-testing D1-like receptor Brain-wide voxel-wise analyses also identified increased DA release blockade disrupted learning. In subsequent studies pre-exposure to in the left hippocampus during the inhibition of conditioned fear, the bar-test environment for 5 min during each of 6 days prior to the compared to baseline (p < 0.001, uncorrected), and in the posterior initiation of testing resulted in greater increases in immobility in the cingulate gyrus, compared to the expression of conditioned fear paired group. This suggests that habituation enhances the effects of (p < 0.001, uncorrected). pairing a test environment with low-dose HAL; SCH (0.05 mg/kg) 30 Together, these findings suggest that pharmacological and psycho- min after each pre-ex eliminates the enhancement. The results logical events can modulate DA responses to stress and stress- implicate D1-like receptors in habituation learning. Taken together, related cues. These forms of dopaminergic plasticity may be involved the results suggest that stimuli paired with decreased DA neurotrans- in the development and maintenance of stressor-related disorders, mission, in this case the bar test apparatus and associated environ- such as post-traumatic stress disorder (PTSD), psychoses, and mental stimuli, gradually lose their ability to elicit approach and other addictions. responses. The ability of SCH to block conditioned increases in immobility and the additive effects of habituation implicate D1-like Dopamine and neurodegeneration receptors in IIL. Stimuli associated with negative prediction error signals in DA neurons may similarly be subject to IIL. Funded by NSERC. A9.1 Reference Axonal arborization and energetic metabolism of nigral 1. Schmidt WJ, Tzschentke TM, Kretschmer BD: State-dependent dopamine neurons: a window into selective vulnerability in blockade of haloperidol-induced sensitization of catalepsy by Parkinson’s disease MK-801. Eur J Neurosci, 1999; 11(9):3365–3368. Louis-Éric TRUDEAU*, Consiglia PACELLI and Nicolas GIGUÈRE doi:10.1046/j.1460-9568.1999.00794.x Departments of Pharmacology and Neurosciences, Université de Montréal, QC, Canada *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A9.1 http://www.intrinsicactivity.org/2016/4/S2/A9.1

Although the mechanisms underlying the loss of neurons in A10.2 Parkinson’s disease are not well understood, impaired mitochondrial Hypothalamic – VTA circuits for motivated behavioral states function and pathological protein aggregation are suspected as Garret STUBER* playing a major role. Why dopamine neurons and a select small Department of Psychiatry and Neuroscience Center, University of subset of brain nuclei are particularly vulnerable to such ubiquitous North Carolina, Chapel Hill, NC, United States of America cellular dysfunctions is presently one of the key unanswered *E-mail: [email protected] questions in Parkinson’s disease research. This talk will present Intrinsic Activity, 2016; 4(Suppl. 2): A10.2 recent data testing the intriguing hypothesis that the heightened http://www.intrinsicactivity.org/2016/4/S2/A10.2 vulnerability of these neurons is a consequence of the particular morphological characteristics of these cells, which are long range In order to survive and effectively navigate an ever-changing and projections neurons with a highly elaborate axonal arborization and unpredictable environment, organisms must readily adapt their elevated bioenergetic requirements. We find that vulnerable nigral behavior to seek out needed resources, while simultaneously dopamine neurons differ from less vulnerable dopamine neurons avoiding life-threatening situations. These opposing processes are such as those of the ventral tegmental area by having a higher basal controlled by neural circuitry that is readily engaged by both rate of mitochondrial oxidative phosphorylation, a higher density of environmental and physiological factors to promote behavioral axonal mitochondria, an elevated level of basal oxidative stress and output. The work of my lab studies the precise neural circuits that a considerably more complex axonal arborization. We found that control both reward- and aversion-related behavioral responses. By reducing axonal arborization by acting on axon guidance pathways utilizing optogenetic and other circuit-mapping tools, we aim to reduces in parallel the basal rate of mitochondrial oxidative delineate the precise functional synaptic connections between phosphorylation and the vulnerability of nigral dopamine neurons. molecularly distinct neuronal populations that are critical for the Our data argue that the heightened vulnerability of nigral dopamine generation of these critical behavioral states. In this presentation I will neurons in Parkinson’s disease is directly due to their particular discuss ongoing unpublished work from my investigating the function bioenergetic and morphological characteristics. and encoding properties of hypothalamic cell groups that act to regulate the activity of midbrain dopamine neurons. A holistic under- Dopamine and basal ganglia standing of the interconnected neural circuit elements that mediate diverse motivational behaviors will likely provide important insight into a variety of complex neurological and neuropsychiatric illnesses such A10.1 as drug and alcohol addiction, anxiety, and depression. Illuminating axonal gatekeepers of dopamine transmission Stephanie J. CRAGG*, Sarah THRELFELL, Polina KOSILLO and A10.3 Yan-Fen ZHANG Differential cholinergic control of striosomal and matrix Department of Physiology, Anatomy and Genetics, University of projection neurons in mice with amphetamine-induced Oxford, United Kingdom repetitive behavior *E-mail: [email protected] Jill R. CRITTENDEN*, Carolyn J. LACEY, Eddie Feng-Ju WENG, Intrinsic Activity, 2016; 4(Suppl. 2): A10.1 Yingxi LIN and Ann M. GRAYBIEL http://www.intrinsicactivity.org/2016/4/S2/A10.1 Massachusetts Institute of Technology, Cambridge, MA, Dopamine transmission in the mammalian striatum is critically United States of America involved in the selection of actions by the basal ganglia. Dysregu- *E-mail: [email protected] lation of dopamine underlies a range of psychomotor disorders, Intrinsic Activity, 2016; 4(Suppl. 2): A10.3 including addiction and Parkinson’s disease. Dopamine neurons form http://www.intrinsicactivity.org/2016/4/S2/A10.3 extensive axonal fields, and it is becoming clear that a variety of local The neuropil of striatal projection neurons (SPNs) typically observes mechanisms within striatum and on dopamine axons are of funda- striosome / matrix borders, raising the question of whether there are mental importance in determining dopamine transmission. Axoaxonic specialized mechanisms for cross-compartmental interactions. inputs from striatal cholinergic interneurons to nicotinic receptors on Cholinergic interneurons are candidates for mediating local inter- dopamine axons may be particularly important. Striatal cholinergic compartmental communication because of their frequent location at interneurons function as a synchronized network showing phasic striosomal borders. New evidence in rodents shows that cholinergic changes in neuron activity that are time-locked to those in dopamine interneurons extend neuropil into both compartments, whereas neurons, suggesting key interactions. The advent of optogenetic cholinergic afferents from brainstem nuclei preferentially target the techniques has enabled us to address directly the effects of striatal matrix. To directly test whether both striosomal and matrix SPNs are inputs, including cholinergic inputs to dopamine axons, in determining regulated by striatal cholinergic interneurons, we recorded activity in dopamine transmission. We show for example that cholinergic striatal slice preparations from double-transgenic mice in which the interneurons and their inputs can directly trigger axonal dopamine compartments could be identified by green fluorescent protein release, bypassing activity in dopamine neurons. This axoaxonic and the cholinergic interneurons could be activated by channel- control can even override activity in dopamine axons, and can also rhodopsin. provide a clamp that suppresses the frequency response of dopamine Data from animal models suggest that cholinergic interneurons of the signals. This axonal triggering and gating of dopamine transmission dorsal striatum help re-set motor systems to allow for behavioral could place axoaxonic gatekeeping mechanisms as central to flexibility in the face of relevant environmental cues. Stereotypy, a dopamine function. severely repetitive behavior that is difficult to disrupt, exemplifies an extreme loss of behavioral flexibility. Psychomotor stimulants such as amphetamine induce stereotypies that are correlated with differential immediate-early gene activation in striosomes relative to matrix (e. g. [1]). This compartmental imbalance in gene induction is blocked by ablation of cholinergic inputs to the striatum (along with somatostatin inputs), but whether cholinergic interneurons modulate SPN spiking

during stereotypy is unknown. We optogenetically activated cholin- References ergic interneurons and recorded striosomal and matrix SPN 1. Kawaguchi Y, Wilson CJ, Emson PC: Intracellular recording of responses in brain slices from double-transgenic mice that were identified neostriatal patch and matrix spiny cells in a slice engaged in amphetamine-induced stereotypy just prior to tissue preparation preserving cortical inputs. J Neurophysiol, 1989; collection. Our findings suggest that whereas both striosomal and 62(5):1052–1068. matrix SPNs normally respond to cholinergic stimulation with a pause 2. Tajima K, Fukuda T: Region-specific diversity of striosomes in the in current-induced repetitive firing, the pause is differentially blocked mouse striatum revealed by the differential immunoreactivities for in matrix, but not striosomal, SPNs of mice with amphetamine- mu-opioid receptor, substance P, and enkephalin. Neuroscience, induced stereotypy. 2013; 241:215–228. doi:10.1016/j.neuroscience.2013.03.012 Supported by: The Simons Foundation, CHDI Foundation, James and Pat Poitras Research Fund, NICHD R01-HD28341. Dopamine and Parkinson’s disease Reference 1. Graybiel AM, Canales JJ, Capper-Loup C: Levodopa-induced dyskinesias and dopamine-dependent stereotypies: a new A11.1 hypothesis. Trends Neurosci, 2000; 23(10 Suppl):S71–S77. An LRRK2 postsynaptic role in the striatum projection neurons doi:10.1016/S1471-1931(00)00018-5 in response to dopamine signaling Loukia PARISIADOU1,*, Carmelo SGOBIO2, Jia YU2, Chengsong XIE2, Guoxiang LIU1, Makariou-Pikis CHRISSY1, David LOVINGER3 and A10.4 Huaibin CAI2 Not all striosomes are equal — it depends on where they are 1 Takaichi FUKUDA1,*, Yuta MIYAMOTO1 and Kae TAJIMA2 Radiation Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL, United States of America; 2Transgenics 1Department of Anatomy and Neurobiology, Kumamoto University, Section, Laboratory of Neurogenetics, NIA, Bethesda, MD, United Kumamoto, Japan; 2Department of Anatomy and Neurobiology, States of America; 3Laboratory of Integrative Neuroscience, NIAAA, Kyushu University, Fukuoka, Japan Bethesda, MD, United States of America *E-mail: [email protected] *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A10.4 Intrinsic Activity, 2016; 4(Suppl. 2): A11.1 http://www.intrinsicactivity.org/2016/4/S2/A10.4 http://www.intrinsicactivity.org/2016/4/S2/A11.1 The striatum has intricate but fascinating internal structure called Mutations in LRRK2 represent a strong genetic risk for both striosomes / matrix compartments. Their existence has been known hereditary and sporadic forms of Parkinson’s disease (PD). However, for long time since the discovery by Dr. Graybiel in 1978, and great there are still several fundamental aspects of LRRK2 function that efforts have been devoted to realize the significance of such compart- remain unresolved at this time. LRRK2 is significantly enriched in mentalization. Numerous molecular markers have been found to spiny projection neurons (SPN) in the dorsal striatum. This cellular visualize striosomes, and both compartments are present in all expression pattern argues that LRRK2 mutations contribute to striatal species studied, including human. However, it still remains obscure pathophysiology in PD. The function of LRRK2 in the striatum has how striosomes / matrix organization is involved in the execution of remained relatively under-investigated; however, we recently showed striatal functions. This will be partly because of the heterogeneity that LRRK2 directs PKA signaling in SPNs. Lrrk2 loss and the inherent in this dichotomous organization; the distributions of various pathogenic LRRK2R1441C mutation that impairs the binding of PKA with molecular markers are not uniform but depend on the three- LRRK2 lead to increased levels of PKA in the dendritic spines that in dimensional positions of the striosomes and matrix. For example, turn results to aberrant synaptic PKA signaling. The components of some striosomes in dorsolateral position are rich in substance P (SP)- PKA enzyme in neurons are confined to sub-cellular compartments, containing axon terminals but poor in enkephalin (Enk)-containing ensuring for a spatial and temporal regulation of PKA signaling that axon terminals, whereas other striosomes especially in the ventral leads to specificity in fundamental striatal functions. Therefore, our position are rich in both substances. Importantly, SP and Enk are data suggest that LRRK2 is strategically located in the dendritic shaft contained in dopamine D1 receptor-expressing striatonigral neurons to organize signaling events in a spatiotemporal way and we propose and D2 receptor-expressing striatopallidal neurons, respectively. that increased synaptic PKA activity in Lrrk2−/− and LRRK2R1441C Because these neuropeptide-positive terminals are thought to be in SPNs stems from altered subcellular compartmentalization of derived from axon collaterals of medium spiny neurons located inside PKA. the same striosome [1], the apparent intricacy in diverse SP- and As PKA is a critical effector of dopamine receptors, we provide Enk-immunoreactivities in a particular striosome may be associated evidence that Lrrk2 loss and the LRRK2R1441C mutation impacts with the site-specific heterogeneity in the composition of striatonigral dopaminergic signaling in SPNs. Specifically, Lrrk2−/− SPNs or SPNs and striatopallidal neurons in that striosome. To address these harboring LRRK2R1441C mutation show an abnormal elevation in PKA issues, we revealed a region-specific diversity of striosomes in mice activity in response to dopamine receptor Drd1 activation. In addition, focusing differential immunoreactivities for μ-opioid receptor, SP, and Lrrk2−/− and LRRK2R1441C SPNs show increased sensitivity to dopa- Enk [2]. To further extend the analysis, we are investigating three- mine depletion as well as altered behavioral responses suggesting a dimensional distributions of diverse striosomes using a computer- novel role of LRRK2 in dopaminergic signaling that in turn may direct assisted reconstruction from the complete serial sections covering the physiology of SPNs as well as the striatal related motor functions. the entire striatum. The reconstructed image shows that diverse Overall, our data reveal a novel LRRK2-based pathogenic mecha- striosomes are distributed to form multiple territories inside the nism of striatal dysfunction in PD. striatum, which in turn may become a clue to parcel the striatum into unknown, functionally different subdivisions in rodents. The relationship between the composition of two neuronal populations and immunohistochemical diverse properties in individual striosomes are also analyzed.

A11.2 in PD can be observed by MRI and this method for imaging neuronal Differential sensitivity of SN and VTA neurons to stress in vitro loss is becoming a new tool to confirm PD diagnosis. Se Joon CHOI1, Ori J. LIEBERMAN1, Ellen KANTER1, D. James NM is contained in special autophagolysosomes together with lipid SURMEIER2, David SULZER1 and Eugene V. MOSHAROV1,* bodies and proteins. The synthesis of NM is controlled by cytosolic concentration of catecholamines which depends on vesicular mono- 1Department of Neurology, Columbia University, New York, NY, amine transporter 2 (VMAT2) expression. In cytosol, catecholamine United States of America; 2Department of Physiology, Northwestern adducts with beta-sheet proteins are formed, then these are oxidized University Feinberg School of Medicine, Chicago, IL, United States to produce protein-melanin that is accumulated in autophagolyso- of America somes, where it is cleaved by proteases and reacts with dolichols to *E-mail: [email protected] give NM. Synthesis of NM is a protective process because the Intrinsic Activity, 2016; 4(Suppl. 2): A11.2 melanic component is generated through the removal of reactive http://www.intrinsicactivity.org/2016/4/S2/A11.2 quinones that would otherwise cause neurotoxicity. Neurons with Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro- high NM content have high cytosolic catecholamine content, high pyridine (MPTP), a neurotoxin that was first identified to induce vulnerability and low levels of VMAT2; the vice versa is also found. parkinsonism in humans, selectively kills dopaminergic neurons of NM can be protective also through its ability to chelate toxic metals substantia nigra (SN), while leaving the neighboring ventral and from stable non toxic complexes. Metals accumulated by NM tegmentum area (VTA) neurons relatively intact, closely mimicking include highly toxic ones like Pb and Hg, in addition to Fe, Zn, Al, Cr the difference in vulnerability of these neuronal populations in and Mo. Parkinson’s disease. It is, however, controversial whether higher However, NM can also play a toxic role in PD. Extracellular NM sensitivity of SN neurons to MPTP results from extra-neuronal toxin released by degenerating neurons of SN can activate microglia with metabolism and transport, or from the intrinsic properties of these production of H2O2, NO and pro-inflammatory factors then inducing cells that predispose them to neurodegeneration. Here, we compared further neurodegeneration, with release of NM, microglia activation metabolic changes induced by MPP+ in cultured mouse SN and VTA and so on. This generates a vicious cycle of neuroinflammation / neurons. In this system, which excludes extra-neuronal metabolism neurodegeneration which contributes to progression of PD. The and transport of MPTP and MPP+, we found significantly higher major histocompatibility class I complex (MHC-I) is highly concen- concentrations of cytosolic Ca2+, NO and DA in toxin-treated SN trated in NM-containing organelles of the SN and LC neurons which neurons, which resulted in higher mitochondrial oxidation and neuro- degenerate in PD. MHC-I can bind antigens derived from foreign toxicity. Contribution of complex I inhibition and DA production to proteins, presenting them on the neuronal membrane. Then CD8+ MPP+-induced toxicity was different between the two neuronal cytotoxic T cells, observed in proximity of MHC-I-presenting neurons populations with a larger role played by disrupted DA homeostasis in of SN and LC in PD subjects, can target these neurons inducing SN neurons and energy depletion in VTA neurons. DAT activity was neuronal death. higher in SN compared to VTA neurons, although this was not sufficient to fully explain the difference in toxicity. Importantly, A11.4 2+ + pharmacological blockade of L-type Ca channels normalized MPP - Dissecting the role of functional selectivity of dopamine induced alterations in Ca2+, NO and DA in SN neurons, also D3 receptor agonists in L-DOPA-induced dyskinesia and decreasing mitochondria oxidation and neurotoxicity. Overall, our Parkinson’s disease + data suggest that higher sensitivity of SN neurons to MPP reflects Wei XU1, Courtney MARSHALL1, Courtney WILLIAMS2, general susceptibility of these cells to some types of insults, a large Jay SCHNEIDER2 and Sandhya KORTAGERE1,* portion of which is dictated by the presence of L-type Ca2+ channels 1Drexel University College of Medicine, Philadelphia, PA, United and downstream upregulation of NO and DA production. States of America; 2Thomas Jefferson University College of Medicine, Philadelphia, PA, United States of America A11.3 *E-mail: [email protected] Protective and toxic role of neuromelanin in brain aging and Intrinsic Activity, 2016; 4(Suppl. 2): A11.4 Parkinson’s disease http://www.intrinsicactivity.org/2016/4/S2/A11.4 Luigi ZECCA1,*, Ioannis U. ISAIAS2,3, Luigi CASELLA4, David SULZER5 1 and Fabio A. ZUCCA The dopamine D3 receptor (D3R) has been suggested to play a critical role in the etiology of both Parkinson’s disease (PD) and levodopa 1Institute of Biomedical Technologies, CNR, Segrate (Milano), Italy; (L-DOPA)-induced dyskinesia (LID). Several dopaminergic agents 2Neurologische Klinik und Poliklinik, University of Würzburg, including L-DOPA have been used as therapeutic agents for Germany; 3Centro Parkinson-ICP, Milano, Italy; 4Department of Parkinson’s disease with limited success for therapy. We have Chemistry, University of Pavia, Italy; 5Departments of Psychiatry, recently designed a class of atypical D R agonists (SK 609 and Neurology and Pharmacology, Columbia University Medical Center, 3 SK 608) with functional selectivity to G protein-dependent, but New York, NY, United States of America β-arrestin-independent signaling features. In addition, these atypical *E-mail: [email protected] agonists do not induce desensitization of D Rs but induce dose- and Intrinsic Activity, 2016; 4(Suppl. 2): A11.3 3 time-dependent internalization of D Rs over-expressed in CHO cells. http://www.intrinsicactivity.org/2016/4/S2/A11.3 3 These results are in complete contrast with the signaling properties

Neuromelanins (NMs) are a family of compounds occurring in all of other known D3R agonists such as dopamine and PD 128907, regions of human brain. In particular, NMs accumulate in catechol- which induce desensitization but not internalization of D3Rs. D3Rs are amine neurons of substantia nigra (SN) and locus coeruleus (LC), only known to undergo pharmacological sequestration in response to which preferentially degenerate in Parkinson’s disease (PD). Then these known D3R agonists. Our compounds improved motor impair- the presence of NM in these neurons has been associated to their ments associated with PD-like symptoms in a 6-OHDA induced vulnerability and a number of studies have shown that NM can play hemiparkinson rat model of PD. SK 609 and SK 608 demonstrated either a protective or toxic role in PD depending on cellular context. synergistic effects with L-DOPA in improving the motor symptoms in In aging, the concentrations of NM attain high values like 3–4 mg/g the MPTP-induced non-human primate model of PD. In rodents, wet tissue in SN and LC; the decrease of NM concentration occurring chronic treatment of SK 609 or SK 608 did not induce abnormal

involuntary movements (AIMs) but significantly reduced AIMs A12.2 induced by L-DOPA when used adjuvantly with L-DOPA. Our results Investigating the heterogeneous effects of neuromodulators on suggest that the internalization of D3Rs induced by SK 608 striatal dopamine release: what is the role of the illusive contributes to the re-sensitization of D3R signaling and receptor striosome and matrix sub-territories? trafficking which may explain its novel therapeutic efficacy observed Katherine R. BRIMBLECOMBE* and Stephanie J. CRAGG in alleviating the symptoms of PD in rodent and non-human primate Department of Physiology, Anatomy and Genetics, University of models and LID in a rodent PD model. Oxford, United Kingdom

*E-mail: [email protected] Dopamine and reward Intrinsic Activity, 2016; 4(Suppl. 2): A12.2 http://www.intrinsicactivity.org/2016/4/S2/A12.2

A12.1 The striatum is organised not only topographically, but also Phospho-proteomic analysis of the dopamine pathway enables biochemically, into striosomes and matrix compartments. Striosomes discovery of a novel reward signal in vivo form patchy, labyrinth-like structures that are enriched in μ-opioid 1, 2 2 Taku NAGAI *, Keisuke KURODA and Kozo KAIBUCHI receptors (MORs) and Substance P (SP) relative to the surrounding 1Department of Neuropsychopharmacology, Nagoya University, matrix, which is enriched in calbindin-D28K and acetylcholinesterase. Nagoya, Japan; 2Department of Cell Pharmacology, Nagoya Evidence for a third ring-like “annulus” or “peristriosomal” region University, Nagoya, Japan surrounding striosomes, has been proposed from high met-enkepha- *E-mail: [email protected] lin expression, and at least in primates, by overlap of NK1 receptors Intrinsic Activity, 2016; 4(Suppl. 2): A12.1 with their endogenous agonist SP. Despite being first described http://www.intrinsicactivity.org/2016/4/S2/A12.1 almost 30 years ago, the functional implications of this organisation are poorly understood. Dopamine transmission occurs throughout Dopamine type 1 receptor (D1R) signaling in the striatum presumably striosomes and matrix, and is reported to be modulated by SP. regulates neuronal excitability and reward-related behaviors through However, reported effects are conflicting, ranging from facilitation to PKA. However, whether and how D1R and PKA regulate neuronal inhibition. We addressed whether dopamine transmission is modu- excitability and behavior remains largely unknown. To identify PKA lated differently in striosome / matrix compartments by SP. substrates that regulate the excitability changes that are associated We paired detection of electrically evoked dopamine release at with rewarding experiences and to more broadly examine PKA carbon-fibre microelectrodes using fast-scan cyclic voltammetry in substrates, we developed a phospho-proteomic analysis method that mouse striatal slices with post-hoc immunolabelling for MOR uses affinity beads coated with 14-3-3 proteins to enrich phosphory- immunoreactivity to define striosomes. SP modulated dopamine lated proteins. Using this approach, we comprehensively identified release via NK1 receptors; however, the effect of SP on extracellular PKA substrates downstream of D1Rs in the striatum of mice, and dopamine ranged from a 70 % enhancement to 50 % reduction. The found more than one hundred candidate substrates of PKA, including direction of modulation was determined by location within the Rap1 GEF (Rasgrp2). PKA directly phosphorylated Rasgrp2, and striosomal–matrix axis: Dopamine release was boosted in striosome PKA-mediated Rasgrp2 phosphorylation enhanced its guanine centres, diminished in striosomal–matrix borders and unaffected in nucleotide exchange activity on Rap1. Treatment with a single dose the matrix. These diametric effects of SP on dopamine release in of cocaine significantly and dose-dependently increased the level of striosome and boundary regions result in an apparent centre– Rasgrp2 phosphorylation and activated Rap1 in the nucleus accum- surround contrast of striosomal dopamine signals. These data reveal bens (NAc) of mice. Cocaine-induced phosphorylation of Rasgrp2 that dopamine transmission can be differentially modulated within the was detected in accumbal D1R-expressing medium spiny neurons striosomal–matrix axis, and furthermore, indicate a functionally (D1R-MSNs). The AAV-mediated expression of constitutively active distinct zone at the striosome–matrix interface which may have key PKA or Rap1 in accumbal D R-MSNs enhanced neuronal firing rates 1 impact on striatal integration. and behavioral responses to cocaine exposure through MAPK.

Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to A12.3 decrease these phenotypes. The deficit in cocaine-induced behav- Modulation of nucleus accumbens dopamine release during ioral response in Rap1 conditional knockout mice was restored by co- cue-motivated behavior transfection with constitutively active MAP2K1 (MEK1). These Kate M. WASSUM1,*, Anne L. COLLINS1, Sean B. OSTLUND2, findings demonstrate a novel DA–PKA–Rap1–MAPK intracellular Tara J. AITKEN1 and Venuz Y. GREENFIELD1 signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors [1]. We believe that our 1Department of Psychology, University of California, Los Angeles, phospho-proteomic screening is a powerful and useful tool to CA, United States of America; 2Department of Anesthesiology increase molecular-level understanding of multifarious brain func- and Perioperative Care, University of California, Irvine, CA, tions by elucidating the function of the dopamine. United States of America Reference *E-mail: [email protected] 1. Nagai T, Nakamuta S, Kuroda K, Nakauchi S, Nishioka T, Takano T, Intrinsic Activity, 2016; 4(Suppl. 2): A12.3 Zhang X, Tsuboi D, Funahashi Y, Nakano T, Yoshimoto J, Kobayashi http://www.intrinsicactivity.org/2016/4/S2/A12.3 K, Uchigashima M, Watanabe M, Miura M, Nishi A, Kobayashi K, Environmental reward-predictive stimuli provide a major source of Yamada K, Amano M, Kaibuchi K: Phosphoproteomics of the motivation for adaptive reward-seeking behaviors, which can become dopamine pathway enables discovery of Rap1 activation as a amplified in the addicted state, allowing cues to become potent reward signal in vivo. Neuron, 2016; 89(3):550–565. ‘triggers’ for maladaptive behavior (e. g. drug seeking). We have doi:10.1016/j.neuron.2015.12.019 recently shown that phasic dopamine signaling in the nucleus accumbens core (NAc) tracks this cue-induced incentive motivation process and that cue-evoked NAc dopamine responses are sensitive to need state, one critical variable that determines the current adaptive utility of cue-motivated behavior. Excitingly, signaling at

striatal cholinergic receptors has been demonstrated in vitro to A12.5 terminally modulate the release of dopamine. But whether this occurs Sex differences in and sex hormone effects on the in awake, behaving animals receiving natural sensory input is un- glutamatergic mechanisms that regulate dopamine levels known, precluding a full understanding of the functional significance of the prefrontal cortex in adult rats of this modulation for motivated behavior. We tested the hypothesis Mary KRITZER1,*, Mallory LOCKLEAR1, Erika BETANCOURT2, that NAc acetylcholine receptor activation mediates the motivational Jonathan WACHTEL2, Dana LENGEL1 and William F. COLLINS1 impact of reward-predictive cues over reward-seeking actions and 1Department of Neurobiology and Behavior, Stony Brook University modulates corresponding cue-evoked dopamine signaling. Blockade School of Medicine, Stony Brook, NY, United States of America; of muscarinic acetylcholine receptors was found to attenuate both 2Stony Brook University School of Medicine, Stony Brook, NY, cue-induced invigoration of reward seeking and cue-evoked dopa- United States of America mine signaling. Disruption of nicotinic acetylcholine receptor signaling *E-mail: [email protected] had the opposite effect, augmenting both the motivating influence of Intrinsic Activity, 2016; 4(Suppl. 2): A12.5 the cue and cue-evoked dopamine signaling. These data suggest that http://www.intrinsicactivity.org/2016/4/S2/A12.5 NAc cholinergic receptors gate cue-motivated behavior via modulation of cue-evoked dopamine signaling and, perhaps, suggest one Our lab examines sex differences and hormone effects on prefrontal mechanism whereby cue-evoked dopamine signaling is regulated to cortex (PFC) and its top-down modulation of midbrain dopamine (DA) moderate the motivating influence cues according to the current systems in rats. The PFC is responsible for executive operations adaptive value of reward seeking. including working memory and behavioral flexibility. Impairments in these functions number among the most disabling symptoms of A12.4 Parkinson’s disease, schizophrenia and other disorders. Given that Dopamine regulation of synaptic plasticity underlying reward normal operations of the PFC depend on intracortical DA levels being and aversive behavior maintained within precise limits, it is not surprising that imbalances in John A. DANI*, Manivannan SUBRAMANIYAN, Theodoros TSETSENIS midbrain DA systems contribute to the pathophysiologies underlying and Kechun YANG the PFC and non-PFC deficits seen in disorders where executive functions are at risk. However, treatment of these cognitive deficits is Department of Neuroscience, University of Pennsylvania, difficult and complex. For example, in schizophrenia, the DA antago- Philadelphia, PA, United States of America nists that quell hallucinations and other positive symptoms tend to *E-mail: [email protected] worsen cognitive deficits. Unfortunately, this is not an uncommon Intrinsic Activity, 2016; 4(Suppl. 2): A12.4 scenario. Our work could lead to new ways to resolve this problem by http://www.intrinsicactivity.org/2016/4/S2/A12.4 defining the sex-specific mechanisms that influence the functional Dopamine release during reward-driven behaviors influences synap- regulation of mesoprefrontal DA systems. Sex differences in and sex tic plasticity. However, dopamine innervation and release in the hormone effects on DA-dependent PFC function in healthy human hippocampus and its role during aversive behaviors are controversial. and animal subjects and on PFC dysfunction in patient populations Here, we present that dopamine influences in vivo synaptic plasticity and preclinical models of hyper- and hypo-dopaminergic disease in hippocampal circuits, and that this plasticity underlies contextual have been well described. However, there are significant gaps in learning during a behavioral task. Immunohistochemistry and molec- knowledge about the basic neurobiology of DA-dependent PFC ular techniques indicate that there is sparse dopaminergic innervation function particularly in females, and about the mechanisms of of the hippocampus from the midbrain. The long-term synaptic poten- hormone action especially in the male PFC. In addressing these tiation (LTP) induced by the addictive drug, nicotine, or underlying the areas, we discovered functionally relevant, androgen-driven sex learning of the aversive task, inhibitor avoidance, was assessed with differences among the intracortical glutamate and GABAergic a D1-like dopamine receptor agonist or antagonist in ex vivo hippo- systems that regulate PFC projections to the ventral tegmental campal slices and in vivo from freely-moving mice. Inhibition of D1- area — where mesoprefrontal and mesolimbic DA cells of origin like dopamine receptors impaired drug-induced synaptic plasticity reside. This presentation will describe behavioral, biochemical and and impaired memory of the inhibitory avoidance (IA) task. The electrophysiological data that support NMDA receptor-mediated overall results suggest that dopaminergic signaling serves as a glutamate signaling within the PFC as a major axis of hormone action functional label of salient events by enabling and scaling synaptic that is relevant for DA modulation of both PFC function and PFC plasticity that underlies drug-induced associative memory [1]. The dysfunction in rat models of schizophrenia and Parkinson’s disease. results further suggest that dopamine-receptor signaling during an Recent efforts to identify brain-specific androgen actions in hopes of aversive contextual task regulates memory retention and regulates identifying DA-correcting therapies that benefit brain and behavior the associated synaptic mechanisms of the hippocampus that and avoid cardiovascular and other health risks associated with underlie the learning [2]. hormone treatment will also be presented. Funded mainly by NIH NINDS and NIDA. References A12.6 1. Tang J, Dani JA: Dopamine enables in vivo synaptic plasticity The role of estrogen and progesterone in reinforcement associated with the addictive drug nicotine. Neuron, 2009; learning and impulsive choice: evidence from behavioral 63(5):673–682. doi:10.1016/j.neuron.2009.07.025 and neuroimaging research 2. Broussard JI, Yang K, Levine AT, Tsetsenis T, Jenson D, Cao F, Luise REIMERS1, Melanie RATNAYAKE1, Christian BÜCHEL2 and Garcia I, Arenkiel BR, Zhou FM, De Biasi M, Dani JA: Dopamine Esther K. DIEKHOF1,* regulates aversive contextual learning and associated in vivo 1Human Biology, Biocenter Grindel, University of Hamburg, synaptic plasticity in the hippocampus. Cell Rep, 2016; 14(8): Germany; 2Department of Systems Neuroscience, University 1930–1909. doi:10.1016/j.celrep.2016.01.070 Medical Center Hamburg-Eppendorf, Hamburg, Germany *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A12.6 http://www.intrinsicactivity.org/2016/4/S2/A12.6

Dopamine (DA) plays a major role in reinforcement learning. While cigarette while being imaged. Brief DA responses (lasting only an increase in DA promotes reward sensitivity during learning (Go minutes) were extracted from the dynamic PET data. Voxel-wise learning), a decrease facilitates the avoidance of negative outcomes analyses produced spatio-temporal patterns of DA release, visualized (NoGo learning). Variations in DA level are also reflected by the ability as “DA movies”. Parametric images of magnitude and timing of DA to adapt response time (RT) to changing reward value. A higher DA release were compared between groups. level favors the capacity to speed up for reward maximization, Results: Men were significantly more likely than women to release whereas lower DA promotes the ability to postpone reward-related DA in some fraction of the right ventral striatum in response to responses. In rodents, the hormone estradiol (E2) enhances smoking. The male activation pattern in right ventral striatum in dopaminergic transmission, while progesterone (PROG) opposes this response to cigarettes was characterized by a faster and greater effect and attenuates DA tone. This led us to hypothesize that these peak response to cigarettes than the female pattern. Additionally, hormones may also antagonistically modulate Go vs. NoGo learning women responded faster than men in a discrete sub-region of the and related processes. dorsal putamen. Here, we assessed to what extent cycle-related changes in E2 and Conclusions: This is the first demonstration of sex differences in the PROG level affect reinforcement learning and impulsive choice in DA response to tobacco smoking in the living human brain. We healthy women. In the first study, we found that women became more demonstrate that the magnitude and the timing of the DA response sensitive to positive as opposed to negative feedback in the state of differs between men and women. Our finding, that men activate more heightened E2 (follicular phase, FP), and exhibited a greater need to ventrally than women, is consistent with the established notion that compensate for their reduced capacity to wait for a reward. The men smoke for the reinforcing drug effect of cigarettes. Women follicular Go bias was accompanied by reduced activation in the smoke for other reasons, such as mood regulation and cue reactivity, rostral cingulate zone (RCZ) for negative feedback. In contrast, when which may be more habit-driven, and dorsally localized. The DAergic PROG peaked (luteal phase) women became more sensitive to signature of smoking may represent an important multi-dimensional negative outcomes and activation of the RCZ increased (luteal NoGo biomarker of smoking dependence and a tool for the development of bias). In sum, these data suggest that cycle-dependent variations in new gender-sensitive medications for smoking cessation. E2 and PROG antagonistically modulate reinforcement learning and mesolimbic activation in humans. A12.8 In the second study, we assessed the isolated effect of endogenous Sex difference in midbrain dopamine D2-type receptors and E2 on impulsive choice by comparison of the early and late FP (e-FP in association with nicotine dependence and l-FP). Surprisingly, women acted more impulsively in the e-FP, Kyoji OKITA1,*, Nicole PETERSEN1, Chelsea L. ROBERTSON1, when E2 level was still rather low. Early follicular E2 level further Andy C. DEAN1, Mark A. MANDELKERN2 and Edythe D. LONDON1 correlated with an enhanced capacity to speed up for reward maximi- 1Department of Psychiatry and Biobehavioral Sciences, UCLA, zation, while the ability to wait was compromised. Interestingly, this Los Angeles, CA, United States of America; 2Greater Los Angeles finding was restricted to women with low trait impulsiveness and thus Veterans Affairs Hospital and Health Care System, Los Angeles, supposedly lower baseline DA level. In contrast, E2 level and CA, United States of America optimized RT failed to correlate in impulsive women or in the l-FP, *E-mail: [email protected] despite a generally higher E2 level. These data support the idea that Intrinsic Activity, 2016; 4(Suppl. 2): A12.8 E2 may act as an endogenous DA agonist, at least in women with http://www.intrinsicactivity.org/2016/4/S2/A12.8 presumably low baseline DA. Yet, choices became less impulsive in the state of heightened E2 (l-FP), suggesting a non-linear relationship Women differ from men in smoking-related behaviors and neuro- between E2 and impulsive choice. chemical findings, and have more difficulty in quitting smoking. Unlike female smokers, however, male smokers have lower striatal A12.7 dopamine D2-type receptor availability (binding potential, BPND) than Sex differences revealed in the dopaminergic signature of nonsmokers, and exhibit greater smoking-induced striatal dopamine tobacco smoking release. Because D2-type dopamine autoreceptors in the midbrain Kelly P. COSGROVE1,*, Shuo WANG2, Erin MCGOVERN1 and influence striatal dopamine release, a biochemical function that has Evan D. MORRIS2 been linked to addictions, we tested for sex differences in midbrain dopamine D -type receptor BP , and in how midbrain BP is related 1Department of Psychiatry, Yale University, New Haven, CT, 2 ND ND to nicotine dependence and striatal dopamine D -type receptor BP . United States of America; 2Departments of Diagnostic Radiology 2 ND Positron emission tomography was used with [18F]fallypride to and Biomedical Engineering,Yale PET Center, New Haven, CT, measure BP in the dorsal and ventral striatum and in a midbrain United States of America ND region encompassing the substantia nigra and ventral tegmental area *E-mail: [email protected] in 24 daily smokers (10 women, 14 men) and 26 nonsmokers (14 Intrinsic Activity, 2016; 4(Suppl. 2): A12.7 women, 12 men). A significant sex-by-group interaction reflected http://www.intrinsicactivity.org/2016/4/S2/A12.7 greater midbrain D2-type BPND in female but not male smokers than

Background: Women have more difficulty quitting smoking than in corresponding nonsmokers (F1,44 = 5.189, p = 0.03). Midbrain BPND men. The neurobiological basis for this difference is unknown. was positively correlated with dorsal and ventral striatal BPND across Dopamine (DA) is a critical neurochemical that is implicated in groups (smokers, nonsmokers) and with nicotine dependence in nicotine reinforcement and smoking cessation treatment success, female but not in male smokers. An exploratory analysis indicated an with evidence supporting stronger associations for men compared to interaction of sex with smoking status on dopamine D2-type receptor women. A recent methodological innovation allows us to probe the BPND in the amygdala, insula, and temporal cortex, with higher values DA response to smoking in highly localized striatal regions. This is in female smokers and lower values in male smokers than in the first investigation to examine sex differences in anatomical and nonsmokers. These findings extend observations on D2-type temporal signatures of smoking-induced DA release. dopamine receptors in smokers, and suggest a sex difference in how

Methods: Sixteen healthy smokers (8 male, 8 female) abstinent midbrain D2-type autoreceptors influence nicotine dependence. overnight, participated in one [11C]raclopride positron emission tomography (PET) scan. Subjects smoked their preferred brand of

Dopamine and addiction A13.2 On the existence of multiple A2A, D2 and sigma1 allosteric

receptor–receptor interactions in sigma1–D2 and A2A–D2–sigma1 A13.1 heteroreceptor complexes: role in brain plasticity and cocaine The role of striatal adenosine A – dopamine D interactions in 2A 2 actions cocaine addiction Dasiel O. BORROTO-ESCUELA1,*, Manuel NARVAEZ2, Julia OFLIJAN1, Karolina WYDRA1,*, Agata SUDER1, Kjell FUXE2 and Małgorzata FILIP1 Karolina WYDRA3, Luigi F. AGNATI1, Małgorzata FILIP3 and Kjell FUXE1 1Department of Pharmacology, Institute of Pharmacology Polish 1Department of Neuroscience, Karolinska Institutet, Stockholm, 2 Academy of Sciences, Kraków, Poland; Department of Sweden; 2Department of Physiology, School of Medicine, Neuroscience, Karolinska Institutet, Stockholm, Sweden University of Málaga, Spain; 3Institute of Pharmacology, *E-mail: [email protected] Polish Academy of Sciences, Kraków, Poland Intrinsic Activity, 2016; 4(Suppl. 2): A13.1 *E-mail: [email protected] http://www.intrinsicactivity.org/2016/4/S2/A13.1 Intrinsic Activity, 2016; 4(Suppl. 2): A13.2 Cocaine is one of the most addictive substances in humans and its http://www.intrinsicactivity.org/2016/4/S2/A13.2 dependence is characterized by high risk of relapse following periods The field of dopamine D2 receptors and cocaine addiction changed of abstinence. Cocaine is accepted as to its rewarding properties markedly with the discovery of many types of D2 heteroreceptor resulting from the activation of the mesolimbic dopamine (DA) system complexes. We report the existence of D2R–D2R, A2AR–A2AR and including DA neuron projections from ventral tegmental area to the sigma1R–sigma1R homoreceptor and A2AR–D2R, D2R–sigma1R and nucleus accumbens (NAc) and prefrontal cortex (PFc) [1]. Recent A2AR–D2R–sigma1R heteroreceptor complexes in subcortical limbic studies indicate that adenosine (ADO) may influence DA neurotrans- areas as well as the dorsal striatum, with different distribution mission through A2A receptors antagonistically interacting with D2 patterns using the in situ proximity ligation assay. These heteromers receptors [2]. In the present study we examined the effects of were demonstrated also through BRET in HEK 293 cells. Antagonistic selective A2A receptor ligands (the agonist CGS 21680 and the A2AR–D2R-like receptor–receptor interactions in heteroreceptor com- antagonists KW 6002 or SCH 58261) as well as of the D2-like receptor plexes in the rat ventral striatum play a role in cocaine addictive ligands (the agonist quinpirole and antagonist raclopride) in cocaine behaviors and are differentially affected by cocaine self-administra- self-administration and reinstatement procedures in rats. For tion versus those in the dorsal striatum. More specifically, the comparison, effects of the A2A receptor ligands on rewarding effects activation of the A2AR protomer of this ventral striatal heteroreceptor of food and food seeking were also analyzed. The obtained results complex inhibits the development and maintenance of cocaine demonstrate the lack of tonic A2A receptor activation in rewarding addictive behavior through inhibition of the D2R protomer recognition effects of artificial and natural rewards because none of the tested and Gi/o mediated signaling. The anti-cocaine actions of A2A agonists A2A antagonists affected their behaviors in rats. On the other hand, may involve the restoration of the balance of signaling in the A2A and the selective A2A receptor agonist CGS 21680 reduced both D2 homoreceptor complexes and of their integrated signaling in the rewarding effects of intravenous cocaine and food. A2A receptors are striatal A2A–D2 heteroreceptor complexes and increased inhibitory also implicated in relapses to cocaine behavior since their blockade allosteric modulation. Sigma1–D2: Saturation binding assay demon- induced cocaine and food seeking, while their stimulation inhibited strated that in membrane preparations of HEK 293 D2R–sigma1R not only cocaine and food seeking but also reinstatement induced by cells, cocaine (1 nM) significantly increased the D2R Bmax values conditioned cue. A potent reduction toward the cocaine-, quinpirole-, (998 ± 40 fmol/mg protein) over D2R-alone cells (664 ± 37 fmol/mg cue- or A receptor antagonist-induced reinstatement of cocaine 2A protein). CREB reporter luc-gene assay indicated that the presence seeking was seen after raclopride administration. The results indicate of sigma1R significantly reduced the potency of the D2R-like agonist that A2A activation and D2-like receptor blockade counteract cocaine quinpirole to inhibit the forskolin-induced increase of the CREB signal and food relapse. As shown by using microinjection technique, (antagonistic allosteric receptor–receptor interaction). In contrast, the CGS 21680-mediated inhibitory actions towards cocaine reward and presence of a low concentration of cocaine (100 nM) was found to seeking effects depend on stimulation of A2A receptors localized in markedly increase the quinpirole potency to inhibit the forskolin the NAc. In conclusion, our results indicate the significance of agonist induced increase of the CREB signal in the D2R–sigma1R cells induced A2A receptor stimulation in counteracting the rewarding (synergistic allosteric receptor–receptor interaction). These dual actions of artificial and / or natural rewards. The study points to conformational changes induced by cocaine in the D2R–sigma1R A receptors as a new drug target in cocaine abuse treatment 2A heteroreceptor complexes may be associated to enhanced allosteric strategies. interactions and a redistribution of both protomers from the intra- Supported by the Polish National Science Centre in Kraków (grant cellular compartment to the plasma membrane. Overall, a potential no. 2011/03/N/NZ7/06294). functional role of the balance between the homoreceptor and Reference heteroreceptor complexes is presented to regulate the integration of 1. Koob GF, Le Moal M: Drug addiction, dysregulation of reward, and multiple signals, specifically in relation to cocaine use disorder. allostasis. Neuropsychopharmacology, 2001; 24(2):97–129. doi:10.1016/S0893-133X(00)00195-0 A13.3 2. Filip M, Zaniewska M, Frankowska M, Wydra K, Fuxe K: The A role for the dopamine D4 receptor in mediating expectations importance of the adenosine A2A receptor – dopamine D2 receptor of reward on a rodent slot machine task interaction in drug addiction. Curr Med Chem, 2012; 19(3):317–355. Paul COCKER* and Catharine WINSTANLEY doi:10.2174/092986712803414231 Department of Psychology, University of British Columbia, Vancouver, BC, Canada *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A13.3 http://www.intrinsicactivity.org/2016/4/S2/A13.3

Gambling is an enjoyable and innocuous past-time for many, but for model. Study 2: In a pilot study, another 16 problem gamblers (PrG) some it can become a maladaptive compulsion akin to drug or alcohol and another 24 HC performed the same LA task with changing addiction. Despite increasing recognition that the phenomenological pictures in the background (without MRI but peripheral-physiological process underlying both substance and behavioural addictions may measurements). Pictures were gambling-related, emotional or be similar, treatment options for problem gambling are still limited, neutral. and of questionable efficacy. Results: Study 1: PG patients showed reduced LA compared to HC Animal models offer an opportunity to not only study the underlying subjects (p < 0.01). PG showed reduced loss-related functional neurobiology of disorders such as gambling, but may also facilitate connectivity between left DLPFC and left striatum (peak at MNI: −26, the development of novel pharmacotherapies. To that end we have 11, −9; pFWE < 0.05). The functional connectivity was related to a developed a rodent slot machine task (rSMT) that suggests rats share clinical scale of gambling persistence (peak at MNI: −8, 7, 12; key behavioural features with human gamblers. The dopamine D2- r = −0.71, pFWE = 0.038). Study 2: Reduction in LA in PrG was similar like receptor family is critically involved in modulating animals’ as in study 1. Compared to HC, PrG showed increased gamble performance on the rSMT. Specifically, the D4 receptor appears to acceptance during gambling pictures compared to neutral pictures contribute to animals’ attributions of salience to reward-related (HC: 52 %, PrG: 65 %; p = 0.03). Level of modulation of LA via stimuli. D4 receptors are principally located within prefrontal cortical individual electro-dermal activity during stimulus watching preceding regions and consequently represent an intriguing target for the gamble correlated with PG severity (r = −0.60, p = 0.03). modulating higher order cognitive processes. In addition to systemic Discussion and Outlook: We replicated lowered LA in PG and PrG pharmacology, we have demonstrated that disruption of neural using two independent samples [5]. PG subjects’ lowered functional regions that are relatively rich in D4 receptors, such as the anterior connectivity from left DLPFC to left putamen may lead to an alteration cingulate cortex and insular cortex, impact animals’ ability to in the basal ganglia action selection process when deciding between accurately respond to reward-related stimuli on the rSMT; further accepting and rejecting a gamble [6]. We have found evidence that emphasising a role for these receptors in gambling-related decision- PDT effects may play a role in PG. The PDT, similar to the PIT, may making. be mediated by phasic Pavlovian value inputs of the amygdala to the Additionally, we have recently used the rSMT to provide insight into ventral striatum during the action selection process [7]. We are the iatrogenic gambling observed in patients with Parkinson’s disease currently testing this hypothesis in an fMRI study, using a modified (PD). This form of compulsive gambling tends to arise de novo in a LA task. small but significant sub-section of patients following adjunctive References therapy with D2-like agonists. Chronic administration of a D2-like 1. Corbit LH, Nie H, Janak PH: Habitual alcohol seeking: time course agonist galvanized performance on the rSMT, a finding that could be and the contribution of subregions of the dorsal striatum. Biol considered translationally analogous to the compulsive play exhibited Psychiatry, 2012; 72(5):389–395. doi:0.1016/j.biopsych.2012.02.024 by some PD patients. Preliminary results suggest that these 2. Leeman RF, Potenza MN: Similarities and differences between alterations in performance may be attributable to augmented pathological gambling and substance use disorders: a focus on activation of the D2-like receptor-mediated Akt–GSK3 intra-cellular impulsivity and compulsivity. Psychopharmacology, 2012; 219(2): signalling cascade. 469–490. doi:10.1007/s00213-011-2550-7 Ultimately, gambling is a heterogeneous disorder that is unlikely to 3. Garbusow M, Schad DJ, Sebold M, Friedel E, Bernhardt N, Koch SP, have a single underlying aetiology; however, these data indicate that Steinacher B, Kathmann N, Geurts DE, Sommer C, Müller DK, Nebe S, aberrant dopaminergic signalling within the D2-like receptor family, Paul S, Wittchen HU, Zimmermann US, Walter H, Smolka MN, Sterzer particularly the D4 receptor may underlie at least some of the cognitive P, Rapp MA, Huys QJ, Schlagenhauf F, Heinz A: Pavlovian-to- perturbations observed in problem gambling. instrumental transfer effects in the nucleus accumbens relate to relapse in alcohol dependence. Addict Biol, 2016; 21(3):719–731. A13.4 doi:10.1111/adb.12243 Cue-induced changes in decision-making in pathological 4. Tom SM, Fox CR, Trepel C, Poldrack RA: The neural basis of loss gamblers: alterations in loss aversion and their relation to aversion in decision-making under risk. Science, 2007; 315(5811): symptom severity 515–518. doi:10.1126/science.1134239 Alexander GENAUCK1,*, Saskia QUESTER2, Andreas HEINZ1 and 5. Lorains FK, Dowling NA, Enticott PG, Bradshaw JL, Trueblood JS, Nina Romanczuk-SEIFERTH1 Stout JC: Strategic and non-strategic problem gamblers differ on decision-making under risk and ambiguity. Addiction, 2014; 109(7): 1Department of Psychiatry and Psychotherapy, Charité 1128–1137. doi:10.1111/add.12494 Universitätsmedizin Berlin, Germany; 2Berlin School of Mind 6. Stewart TC, Bekolay T, Eliasmith C: Learning to select actions with and Brain, Berlin, Germany spiking neurons in the basal ganglia. Front Neurosci, 2012; 6:2. *E-mail: [email protected] doi:10.3389/fnins.2012.00002 Intrinsic Activity, 2016; 4(Suppl. 2): A13.4 7. Guitart-Masip M, Talmi D, Dolan R: Conditioned associations and http://www.intrinsicactivity.org/2016/4/S2/A13.4 economic decision biases. Neuroimage, 2010; 53(1):206–214. Background: Decision-making biases and Pavlovian-to-instrumental doi:10.1016/j.neuroimage.2010.06.021 transfer (PIT) have been shown related to addiction [1, 2]. Yet, in pathological gambling (PG), influences of cue reactivity on decision- A13.5 making (Pavlovian-to-decision-making transfer; PDT) have not been Cell-type-specific dysregulation of GABAergic plasticity investigated with regards to cue-induced changes in loss aversion in the ventral pallidum after extinction from cocaine (LA). We hence tried to answer the following questions: Is LA reduced self-administration in PG and what are the neural substrates of such a reduction? Is there Daniela NEUHOFER1,*, Jasper HEINSBROEK1, Yonatan KUPCHIK2 a clinically relevant PDT effect similar to the PIT effect seen in alcohol and Peter W. KALIVAS1 dependency [3]? 1Department of Neuroscience, Medical University of South Carolina, Experiments: Study 1: Nineteen PG subjects and 17 matched Charleston, SC, United States of America; 2Department of Medical healthy controls (HC) performed an LA task in an fMRI setting [4]. LA Neurobiology, The Institute for Medical Research Israel-Canada, scores were estimated using a logistic regression mixed effects Faculty of Medicine, The Hebrew University of Jerusalem, Israel

*E-mail: [email protected] A13.7 Intrinsic Activity, 2016; 4(Suppl. 2): A13.5 Neurobiological correlates of learning and decision-making http://www.intrinsicactivity.org/2016/4/S2/A13.5 in alcohol dependence: the LeAD study Miriam SEBOLD1,*, Maria GARBUSOW1, Daniel J. SCHAD2, The advent of Cre mice in combination with opto- and chemogenetic Stephan NEBE3, Christian SOMMER3, Ulrich S. ZIMMERMANN3, strategies have allowed investigators to probe D1 and D2 MSNs of the Michael N. SMOLKA3, Michael A. RAPP2, Quentin J. M. HUYS4 nucleus accumbens for their distinct or overlapping roles in regulating and Andreas HEINZ1 motivated behaviors. A fairly consistent functional portrait has 1 emerged showing that the activation of D1 MSNs promotes the Department of Psychiatry and Psychotherapy, Charité execution of motivationally relevant behaviors such as sucrose or Universitätsmedizin Berlin, Germany; 2Cognitive Sciences, addictive drug seeking, while stimulation of D2 MSNs has the opposite Department of Sports and Health Sciences, University of Potsdam, effect. In general, these studies are interpreted as distinct roles for Germany; 3Department of Psychiatry and Psychotherapy, University the direct projections to the ventral mesencephalon and indirect Hospital Carl Gustav Carus, Technische Universität Dresden, 4 projections to the VP. However, the fact that both D1 and D2 MSNs Germany; Institute for Biomedical Engineering, Zürich and strongly innervate the VP and that inhibition of these inputs prevents University of Zürich, Switzerland reinstatement of cocaine seeking indicates that a more nuanced *E-mail: [email protected] understanding of this neurocircuitry is needed. Intrinsic Activity, 2016; 4(Suppl. 2): A13.7

We aimed to investigate how D1 and D2 inputs to the VP are modu- http://www.intrinsicactivity.org/2016/4/S2/A13.7 lated before and after extinction from cocaine self-administration and The mesolimbic dopaminergic system has long been associated with how modulation of these inputs could control cue-induced cocaine two functions in reward processing, one in reinforcement learning seeking. Using a cre-dependent optogenetic approach we find that (e. g. prediction error) and another in incentive motivation (e. g. cue- following extinction from cocaine self-administration synaptic plasti- elicited reward-seeking). Both functions have been implicated in city in NAcore inputs to the dlVP is blunted selectively in D and not 2 alcohol dependence with the former contributing to the persistence of D MSN afferents. This effect is mediated by a constitutive increase 1 chronic alcohol intake despite unfavorable life experiences and the in enkephaline tone on μ-opioid receptor on D afferents. Employing 2 latter playing a crucial role in craving and relapse, which are often a chemogenetic strategy using designer receptors exclusively triggered by alcohol-associated cues. activated by designer drugs (DREADDs) coupled to either G (G - i i The bicentric study “Learning in alcohol dependence (LeAD)” aims to DREADD) or G (G -DREADD) signaling we show that both D and s s 1 bridge a gap between these processes by investigating reinforcement D MSN regulate reinstated cocaine seeking via their innervation of 2 learning mechanisms and cue-induced automatic approach behavior the dlVP. such as Pavlovian-to-instrumental transfer (PIT) in 120 alcohol- Our results indicate that hat the modulation of GABAergic trans- dependent subjects and approximately 300 healthy control subjects. mission differs between D and D inputs to the VP and might be an 1 2 We here demonstrate that alcohol-dependent subjects show altera- important regulator of cue-induced cocaine seeking. tions in goal-directed, model-based reinforcement learning [1] and demonstrate that healthy young adults show increased model-free A13.6 neural signatures in the striatum when they start drinking alcohol at a Nicotinic modulation of dopaminergic activity and exploration younger age (Nebe et al., in preparation). Moreover, we show that in Philippe FAURE*, Steve DIDIENNE, Marie Louise DONGELMANS, alcohol-dependent patients compared to healthy controls non- Jérémie NAUDÉ and Samir TAKILLAH alcohol-associated cues exert pronounced control over behavior Neuroscience Paris Seine, CNRS Université Pierre et Marie Curie, (PIT; [2]). Crucially, these cue-associated responses were signifi- Paris, France cantly related with nucleus accumbens activity in subsequent *E-mail: [email protected] relapsers, but neither in subsequent abstainers nor healthy controls, Intrinsic Activity, 2016; 4(Suppl. 2): A13.6 and were predictive of critical clinical outcomes such as alcohol intake http://www.intrinsicactivity.org/2016/4/S2/A13.6 during a 3-months follow-up period in patients. These findings point to resilience of dopamine function as a predictor of good treatment Nicotine is the main addictive component that drives continued outcome. tobacco use. It exerts its reinforcing effects by acting on nAChRs, a This work was supported by the German Research Foundation family of pentameric ligand-gated ion channels with different types of (Deutsche Forschungsgemeinschaft, DFG, FOR 1617; grants HE subunits expressed throughout the mammalian brain. The neuro- 2597/13-1, HE 2597/14-1, HE 2597/15-1, RA 1047/2-1, SM 80/7-1, circuitry underlying nicotine addiction is broad, complex, and ZI 1119/3-1, WI 709/10-1, SCHA 1971/1-2, HE 2597/13-2, HE depends on the stage of the disease process. Nicotine, like all drugs 2597/14-2, HE 2597/15-2, RA 1047/2-2, SM 80/7-2, ZI 1119/3-2, WI of abuse, activates mesolimbic dopamine neurons in the ventral 709/10-2). tegmental area (VTA). The midbrain dopamine system serves many References functions through the modulation of DA cells activity at short time 1. Sebold M, Deserno L, Nebe S, Schad DJ, Garbusow M, Hägele C, scales such as fast responses related to reward, but also at larger Keller J, Jünger E, Kathmann N, Smolka MN, Rapp MA, Schlagenhauf time scales, such as motor activity or the likelihood of switching F, Heinz A, Huys QJ: Model-based and model-free decisions in among alternatives. In this talk we will discuss the role of β2- alcohol dependence. Neuropsychobiology, 2014; 70(2):122–131. containing nAChRs in the modulation of DA system at various time doi:10.1159/000362840 scale and the consequence of nicotine exposure. Using behavior and 2. Garbusow M, Schad DJ, Sebold M, Friedel E, Bernhardt N, Koch SP, in vivo electrophysiology in freeely moving animals we will show that Steinacher B, Kathmann N, Geurts DE, Sommer C, Müller DK, Nebe S, these nicotinic modulation were involved in exploration and decision- Paul S, Wittchen HU, Zimmermann US, Walter H, Smolka MN, Sterzer making under uncertainty but not in reinforcement learning and P, Rapp MA, Huys QJ, Schlagenhauf F, Heinz A: Pavlovian-to- motivation for certain rewards. Furthermore, nicotine, by acting on instrumental transfer effects in the nucleus accumbens relate to this pathway, will increase the value-sensitivity and thus the exploit- relapse in alcohol dependence. Addict Biol, 2016; 21(3):719–731. ative behavior of the animal. doi:10.1111/adb.12243

A13.8 A13.10

Role of dopamine D2 receptors in cocaine-induced plasticity Lack of dopamine-mediated effects differentiate modafinil from and relapse methylphenidate actions on cocaine-reinforced behavior in rats Ja-Hyun BAIK* Maddalena MEREU1,*, Takato HIRANITA2, Lauren E. CHUN2, 2 2 2 School of Life Sciences and Biotechnology, Korea University, Seoul, Jessica LOPEZ , Mark COGGIANO , Juliana C. QUARTERMAN , 2 2 2 Republic of Korea Amy H. NEWMAN , Jonathan L. KATZ and Gianluigi TANDA *E-mail: [email protected] 1Università degli Studi di Padova, Italy; 2NIDA-IRP, NIH/DHHS, Intrinsic Activity, 2016; 4(Suppl. 2): A13.8 Baltimore, MD, USA http://www.intrinsicactivity.org/2016/4/S2/A13.8 *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A13.10 Dopamine (DA) regulates motivational behavior including feeding http://www.intrinsicactivity.org/2016/4/S2/A13.10 behaviors through DA receptors which belong to the G protein- coupled receptor family. We have recently found that dopamine D2 Modafinil (MOD) and methylphenidate are drugs approved for the −/− receptor null (D2R ) mice manifest enhanced anxiety-like and treatment of narcolepsy and attention-deficit disorders, respectively. depressive behaviors after chronic stress compared with wild-type Similar to cocaine, both drugs inhibit dopamine (DA) reuptake by (WT) mice. We also found that repeated stress exposure attenuated blocking the DA transporter, and have been extensively tested in cocaine-induced addictive behavior in association with changes in clinical studies to treat cocaine dependence. MOD and methyl- −/− synaptic plasticity in the nucleus accumbens (NAc) in D2R mice, phenidate have been described as smart drugs, especially used by suggesting that D2R plays a key role in the interaction between stress students in order to increase their cognitive performance. Importantly, and addiction-induced plasticity during withdrawal period. We have this young population is also at risk for abuse of illicit drugs like used an optogenetic approach to examine the role of NAc medium cocaine and thus it is important to understand how these drugs spiny neurons (MSNs) expressing D2Rs in cocaine-induced behav- interact with cocaine and other illicit drugs. We have investigated the ioral sensitization. Adeno-associated viral vectors encoding channel- effects of MOD, methylphenidate and cocaine, alone and in rhodopsin-2 were delivered into the NAc of D2R-Cre transgenic mice. combination, in self-administration and nucleus accumbens shell DA

Photostimulation of NAc D2R-MSNs in vivo affected neither the microdialysis studies using Sprague Dawley rats. MOD (0.1–10 initiation nor the expression of cocaine-induced behavioral sensiti- mg/kg, i.v.) failed to maintain self-administration behavior in rats, zation. However, photostimulation during the drug withdrawal period while methylphenidate self-administration was maintained at the attenuated expression of cocaine-induced behavioral sensitization. same doses used for cocaine self-administration (0.1–1.0 mg/kg, i.v.).

These results show that D2R-MSNs of NAc play a key role in However, combinations of low doses of MOD (10–32 mg/kg, i.p.) or withdrawal-induced plasticity and may contribute to relapse after methylphenidate (1–10 mg/kg, i.p.) potentiated cocaine self- cessation of drug abuse. We are currently investigating the effect of administration, shifting the dose–effect curves to the left. This effect photoinhibition of D2R-MSNs by delivering archaerhodopsin into the of methylphenidate was paralleled by a larger cocaine-induced

NAc of D2R-Cre transgenic mice, but also are trying to identify the stimulation of DA, while MOD did not enhance the stimulation of DA synaptic connectivity between D2R-MSNs and interacting neural by cocaine. Lack of a DA action in the effects of MOD triggered circuit to identify neural substrates of D2R-dependent signaling in ongoing experiments to test whether electronic coupling or orexin- cocaine / withdrawal-induced plasticity. mediated effects might underlie MOD-induced potentiation of cocaine This work was supported by a Korea University Grant, by the Brain reinforcing effects. In summary, MOD showed a unique stimulant Research Program (grant no. 2013M3C7A1056101), and by Mid- profile compared to cocaine and methylphenidate. Considering its Career Researcher Program (grant no. 2014R1A2A2A01003337) relatively low potential for abuse and positive results from recent through the National Research Foundation of Korea (NRF) funded by clinical trials, MOD might be effective as a potential agonist the Ministry of Science, ICT & Future Planning, Republic of Korea. substitution therapy for the treatment of cocaine-use disorders in certain patient populations. A13.9 Addiction and corticotropin-releasing factor: from the A13.11 amygdala to the prefrontal cortex and beyond Effects on neurochemistry and behavior of modafinil and its Olivier GEORGE* analogs in rodents: new clues for the next generation of The Scripps Research Institute, La Jolla, CA, United States medications for the treatment of psychostimulant use of America disorders *E-mail: [email protected] Gianluigi TANDA*, Claudio ZANETTINI, Zheng-Xiong XI, Leandro Intrinsic Activity, 2016; 4(Suppl. 2): A13.9 VENDRUSCOLO, Chelsea HO, Apre DIXON-GLEAVES, Juliana C. http://www.intrinsicactivity.org/2016/4/S2/A13.9 QUARTERMAN, Mark COGGIANO, Jacqueline KEIGHRON, Jianjing CAO, Rachel D. SLACK and Amy H. NEWMAN This talk will present recent results demonstrating the critical role of Medication Development Program, National Institute on CRF neurons in alcohol and nicotine addiction, with a particular Drug Abuse (NIDA) / National Institutes of Health (NIH), emphasis on the CRF neurons in the prefrontal cortex, central Baltimore, MD, United States of America nucleus of the amygdala and ventral tegmental area. Pharmacolo- *E-mail: [email protected] gical, optogenetic and pharmacogenetic evidence will be presented Intrinsic Activity, 2016; 4(Suppl. 2): A13.11 and the role of newly identified CRF neurons in the ventral tegmental http://www.intrinsicactivity.org/2016/4/S2/A13.11 area will be discussed. Clinical trials have shown promising yet inconsistent results with oral modafinil (MOD) as a treatment for psychostimulant use disorders. MOD blocks the dopamine (DA) transporter (DAT) like abused psychostimulants, such as cocaine. However, the resulting stimulation of DA levels and its efficacy as a reinforcer are among the lowest in its class. In order to dissect the pharmacologic activity that

contributes to its mechanism of action, we have recently synthesized (±)-Modafinil (200 mg) was tested in a randomized, double-blind, several structural analogs of MOD, including its (R)-enantiomer ((R)- placebo-controlled cross-over study in abstinent, MA-dependent and MOD). Here we show behavioral studies in rats aimed to test these healthy control subjects. Modafinil and placebo were each analogs and their potential therapeutic effects when administered in administered as a single oral dose 2 h before testing. Across combination with cocaine or methamphetamine under different subjects, modafinil improved sustained attention, with no significant schedules of self-administration behavior. We also have performed improvement in tests of other cognitive functions. In MA-dependent microdialysis and fast-scan cyclic voltammetry (FSCV) tests in the subjects, modafinil enhanced inhibitory control and processing speed nucleus accumbens shell (NAcS), a brain area related to reward, in more in participants who reported higher baseline use of MA than Sprague Dawley rats and Swiss Webster mice, in order to better those with lower use. Modafinil did not affect learning in controls understand the dopaminergic effects of these drugs alone and in subjects on a reversal learning task, but boosted performance in MA combination with cocaine. We show that pretreatment with several users to the level of control subjects. There also was a greater structural analogs of MOD attenuates cocaine and / or methamphet- modafinil-induced enhancement of brain activation in executive amine self-administration behavior in rats. Also, like the atypical DAT control regions in MA users than in controls. The findings suggest that blockers, when administered alone these MOD analogs show modafinil improves learning in stimulant users, and that modafinil, reduced facilitation of medial-forebrain-bundle-induced maximal now available for testing in the active (R)-isomeric form, may be a stimulation of DA in the NAcS, in FSCV experiments in mice. Further, suitable pharmacological adjunct for enhancing efficiency of the MOD analogs reduce the maximal stimulation of DA elicited by cognitive-based therapies for stimulant-use disorders. cocaine when administered in combination with it. In microdialysis tests, we have confirmed the limited stimulation of DA levels induced A13.13 by MOD and (R)-MOD and extended this finding to the MOD-analogs. Dopamine, effort, and behavioral activation: animal models of So far, the MOD analogs that show atypical DAT-blocker activity in effort-related motivational symptoms in psychopathology FSCV tests and small, if any, increases in extracellular DA in our John D. SALAMONE1,*, Samantha E. YOHN1, Noemi SAN MIGUEL2, microdialysis studies are also those providing better results as Laura LÓPEZ CRUZ2 and Mercè CORREA2 potential blockers of cocaine or methamphetamine reinforcing 1Department of Psychological Sciences, University of Connecticut, effects. In conclusion, our results confirm the potential therapeutic Storrs, CT, United States of America; 2Department of Psycho- effects of (R)-MOD for the treatment of cocaine and methamphet- biology, Universitat Jaume I, Castelló de la Plana, Spain amine dependence. We also extend these findings to support *E-mail: [email protected] potential therapeutic effects of selected analogs of MOD, which may Intrinsic Activity, 2016; 4(Suppl. 2): A13.13 provide important clues in the search for a pharmacotherapeutic http://www.intrinsicactivity.org/2016/4/S2/A13.13 treatment of psychostimulant use disorders. These experiments have been funded by the Medication Develop- Deficits in behavioral activation, exertion of effort, and other psycho- ment Program, NIDA-IRP, NIH / DHHS. motor / motivational dysfunctions are frequently seen in people with depression and other disorders. People with major depression show A13.12 alterations in effort-related decision-making, and a bias towards

D2-type dopamine receptors and executive functioning in selection of low effort activities. Animal tests of effort-related stimulant users: modafinil as a potential therapeutic agent decision-making are being developed as models of motivational Edythe D. LONDON1,*, Dara GHAHREMANI1, Andy C. DEAN1, Milky dysfunctions. In view of basic research implicating nucleus KOHNO1, Angelica M. MORALES1 and Mark A. MANDELKERN2 accumbens dopamine (DA) in effort-based decision-making, and clinical studies showing that inhibition of catecholamine uptake may 1Department of Psychiatry and Biobehavioral Sciences, University be a useful strategy for treatment of motivational symptoms, the of California, Los Angeles, CA, United States of America; 2Greater present research assessed the ability of various monoamine uptake Los Angeles Veterans Affairs Health Care System, Los Angeles, inhibitors to reverse the effort-related effects of the VMAT-2 inhibitor CA, United States of America tetrabenazine (TBZ), and to increase work output in rats responding *E-mail: [email protected] on a test of effort-related decision-making (i. e. a progressive ratio Intrinsic Activity, 2016; 4(Suppl. 2): A13.12 (PROG) / chow feeding choice task). Effort-related effects of TBZ http://www.intrinsicactivity.org/2016/4/S2/A13.12 were attenuated by the catecholamine uptake inhibitor bupropion,

Striatal dopaminergic deficits are common features of stimulant-use and this effect of bupropion was reversed by either D1 or D2 family disorders involving methamphetamine (MA) or cocaine use. antagonism. The effort-related effects of TBZ also were attenuated Individuals who are MA-dependent are impulsive and on average by the selective DA uptake blocker GBR 12909, as well as modafinil perform below control levels on tests of executive functioning. and methylphenidate. In contrast, the selective 5-HT uptake inhibitors Positron emission tomographic assessment of dopamine (DA) fluoxetine and (S)-citalopram, as well as the selective norepinephrine receptors and functional magnetic resonance imaging (fMRI) have uptake inhibitor desipramine, failed to reverse the effects of TBZ. shown that in healthy controls, striatal D1- and D2-type DA receptor Acute administration of bupropion and GBR 12909 shifted choice availability is positively correlated with inhibitory control task perfor- behavior in rats tested on a concurrent PROG / chow feeding choice mance, and that D2-type receptor availability is correlated with task, increasing markers of PROG lever pressing but decreasing activation linked to inhibition on the task. Striatal D2-type receptor chow intake. In contrast, fluoxetine, desipramine, and atomoxetine availability also is related to modulation of prefrontal cortical and failed to increase lever pressing output, and actually decreased it at striatal function during risky decision-making, but this modulation is higher doses. In the behaviorally effective dose range, GBR 12909 disrupted in MA users, who exhibit a striatal D2-type receptor deficit. elevated extracellular DA levels in nucleus accumbens core

Medications that target D2 receptors directly, however, have been measured by microdialysis. An additional study examined the effects unsuccessful as treatments for stimulant abuse, possibly because of of repeated daily injections (up to 7 days) of each drug. GBR 12909 downregulation and reduced responsivity of D2 receptors. As and bupropion were the only drugs that increased PROG output with stimulant-use disorders comprise a host of problems, a combination repeated treatment. These results indicate that blockade of DA of pharmacological and behavioral therapies may be needed, and a transport reverses the effects of TBZ and increases selection of the medication that improves cognition may be particularly useful. high effort activity (PROG lever pressing), while inhibition of SERT or

NET do not. These findings are consistent with the hypothesis that We performed a combination of behavioural testing and molecular drugs that enhance DA transmission may be effective at treating analysis in rodents. The reactivation of a cued-fear memory activated effort-related psychiatric symptoms in humans. the MAPK pathway and extracellular signal-regulated kinase (ERK) in the basolateral amygdala, and not in other brain regions as A13.14 investigated by western blotting for the phosphorylated form of ERK. Cross-talk of corticotropin-releasing hormone receptor We analysed the regulation of this pathway, post memory reactivation, downstream of glutamate and dopamine receptors. subtype 1 with dopamine D1 receptor: functional relevance in alcohol dependence The results will further our understanding of the molecular mecha- Laura BROCCOLI1, Kay JÜNGLING2, Dasiel O. BORROTO-ESCUELA3, nisms downstream of dopamine receptor signalling for retrieval and Rainer SPANAGEL1, Wolfgang H. SOMMER1, Hans C. PAPE2, destabilisation of memories. Kjell FUXE3, Jan M. DEUSSING4 and Anita C. HANSSON1,* Reference 1. Yokoyama M, Suzuki E, Sato T, Maruta S, Watanabe S, Miyaoka H: 1Department of Psychopharmacology, Central Institute of Mental Amygdalic levels of dopamine and serotonin rise upon exposure Health, Mannheim, Germany; 2Institute of Physiology I to conditioned fear stress without elevation of glutamate. (Neurophysiology), Westfälische Wilhelms-Universität Münster, Neurosci Lett, 2005; 379(1):37–41. doi:10.1016/j.neulet.2004.12.047 Germany; 3Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; 4Molecular Neurogenetics, Max Planck Institute of Psychiatry, Munich, Germany A14.2 *E-mail: [email protected] Updating memories: the importance of dopaminergic prediction Intrinsic Activity, 2016; 4(Suppl. 2): A13.14 error signaling in memory reconsolidation 1, 2 2 http://www.intrinsicactivity.org/2016/4/S2/A13.14 Amy REICHELT *, Marc EXTON-MCGUINNESS and Jonathan LEE 1School of Psychology, University of New South Wales, Sydney, Upregulated corticotropin-releasing hormone (CRH) and CRH NSW, Australia; 2School of Psychology, University of Birmingham, receptor subtype 1 (CRF ) activity within the amygdala play a crucial 1 Edgbaston, Birmingham, United Kingdom role in the development of a chronic negative affective state, which is *E-mail: [email protected] recognized as a major driving force for perpetuating the vicious cycle Intrinsic Activity, 2016; 4(Suppl. 2): A14.2 of alcohol dependence. Although the role of the neurotransmitter http://www.intrinsicactivity.org/2016/4/S2/A14.2 dopamine (DA) is mainly seen in the acquisition of alcohol reinforcement and maintenance of voluntary alcohol consumption, increasing Memories are not static imprints of past experience, but rather are evidences also indicate that amygdala DA receptors are highly dynamic entities which enable us to predict outcomes of future involved in the modulation of emotional responses. Within the situations and inform appropriate behaviours following experiences. amygdala nuclei, GABAergic cluster of interneurons, known as By updating existing memories with new information, their relevance intercalating cell masses (ITC). These neuronal populations are can be maintained, and this process is known as reconsolidation. highly enriched in DA D1 and CRH CRF1 receptors, and exert This talk describes recent experimental advances in the reconsolida- important gating functions for intra-amygdala signal flow. We have tion of both appetitive and aversive memories, and explores the established a functional interaction between the CRF1 and D1 neuronal mechanisms that underpin the conditions under which receptor involved in the regulation of affective behavior, which reconsolidation will occur. It is proposed that a prediction error signal, represent a novel mechanism of amygdala function. Specifically, originating from dopaminergic midbrain neurons in the ventral

CRF1-driven increased stress vulnerability and stress-induced tegmental area, is critical for destabilisation and the subsequent alcohol seeking in dependent animals is due to the simultaneous reconsolidation of a memory. activation of D1 receptor. Based on these findings we suggest that CRF1–D1 receptor cross-talk in the amygdala neurons is involved in A14.3 the hyper-responsiveness to behavioral stress that importantly adds Reconciling striatal dopamine’s roles in reward-based learning to maintaining the dependent state. and cognitive control Roshan COOLS* Dopamine and cognition Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands A14.1 *E-mail: [email protected] Dopamine signalling in the basolateral amygdala and the Intrinsic Activity, 2016; 4(Suppl. 2): A14.3 reactivation of a reconsolidating fear memory http://www.intrinsicactivity.org/2016/4/S2/A14.3 Emma CAHILL*, Barry J. EVERITT and Amy L. MILTON Striatal dopamine has been implicated in a wide range of cognitive Department of Psychology, University of Cambridge, functions, ranging from reward / punishment learning to cognitive United Kingdom control. Can these various roles be reconciled? I will begin by *E-mail: [email protected] presenting evidence from neurochemical PET / MRI studies strongly Intrinsic Activity, 2016; 4(Suppl. 2): A14.1 implicating striatal dopamine in reward / punishment learning. Next, http://www.intrinsicactivity.org/2016/4/S2/A14.1 I will reframe the problem of cognitive control as a problem involving reward / punishment-based choice. Finally, I will assess the hypo- Fear is a strong emotional experience. A memory of learned fear can thesis that dopamine alters cognitive control, not only by modulating be quantified using a Pavlovian fear-conditioning task. Once reac- the ability to implement cognitive control, but also by biasing reward / tivated the fear memory can be expressed or destabilised and punishment-based learning and choice about whether to exert restabilised (in a nonexclusive manner). Fear reminders engage the cognitive control. The work shifts the question of whether people are dopamine system in the basolateral amygdala [1], but the contribution capable of exerting cognitive control to whether they choose to do so of dopamine signalling to the retrieval and destabilisation of fear based on its costs and benefits. Moreover, it provides a novel memory is not fully understood. We explored the idea that dopamine integration of current ideas about striatal dopamine, reward / punish- is an essential modulator of fear memory through its ability to regulate ment learning and cognitive control. synaptic mechanisms.

A14.4 Previously, we found a dichotomy in the anatomical representation of A role for striatal presynaptic dopamine in model-based hierarchically coupled precision-weighted prediction errors (PEs) decision-making used to update learning during a probabilistic sensory associative Lorenz DESERNO* and Florian SCHLAGENHAUF learning task; precision-weighted PEs about the outcome, ε2, were represented in the SN / VTA, whereas higher-level precision-weighted Max Planck Institute for Human Cognitive and Brain Sciences, PEs about the probability of the outcome, ε , were represented in the Leipzig, Germany 3 cholinergic septum. Here, we used the same Bayesian model, the *E-mail: [email protected] hierarchical Gaussian filter, to model participants’ behavior in two Intrinsic Activity, 2016; 4(Suppl. 2): A14.4 pharmacological fMRI studies with a double-blind between-subject http://www.intrinsicactivity.org/2016/4/S2/A14.4 design. Healthy participants received either a single dose of a Human decision-making shows hallmarks of habitual and goal- dopaminergic (DA) or cholinergic (ACh) substance or placebo. We directed behavior. Computational neuroscience describes these investigated whether precision-weighted PEs in toto and / or the systems as ‘model-free’ and ‘model-based’. Model-free reward precision weights (ψ2, ψ3) were modulated by these substances. prediction errors were traditionally associated with phasic dopamine Restricting the analysis to a mask containing the dopaminergic (DA) signals while it is conceivable that model-based control is midbrain, the pontine cholinergic nuclei and the basal forebrain, we related to ‘prefrontal’ (PFC) functioning. A pharmacological challenge only found a trend for a cholinergic effect on the higher-level with L-DOPA enhanced model-based choices during sequential precision-weighted PE, ε3 (peak-level p = 0.06, FWE-corrected) in the decision-making and some studies indicate that higher striatal nucleus basalis: in this region, galantamine (an acetylcholinesterase presynaptic DA facilitates cognitive ‘PFC’ functions. Here, we inhibitor) decreased ε3-related activity compared to placebo. By demonstrate a relationship between presynaptic striatal DA and contrast, we found significant effects for the precision weights: behavioral and neural signatures of model-free and model-based compared to galantamine, levodopa (a dopamine precursor) de- decision-making. creased activity related to the precision of outcome PEs (ψ2) in both Using a two-step sequential decision task during fMRI, we separated the substantia nigra and left basal forebrain. Furthermore, activity in model-free and model-based control over choices in 29 participants. the substantia nigra related to the precision of the probability estimate

All participants also underwent FDOPA PET. Computational models (ψ3) was enhanced by amisulpride (a D2 / D3 antagonist) compared to differing in for first-step choice values were implemented and trial-by- placebo. This pattern of results is only partially compatible with the trial trajectories were used for analysis of fMRI data. Presynaptic hypothesis that different precision-weighted PEs may map onto striatal dopamine levels were quantified as DA synthesis capacity distinct neuromodulatory systems. Instead, the findings suggest that from FDOPA PET scans. PE and precision components of precision-weighted PE signals may First, right ventral striatal (VS) presynaptic dopamine levels were originate from different neuromodulatory nuclei and may be combined positively associated with the degree of model-based decisions. locally. This possibility could be examined by means of further Second, we demonstrate that VS presynaptic dopamine levels computational modelling of our data. Overall, the present combination correlate negatively with ventral striatal coding of model-free of computational modeling and pharmacological fMRI represents an predictions errors. Third, we show a positive correlation between VS important first step to explore how local activity related to different presynaptic dopamine levels and model-based signatures in right computational quantities may depend on neuromodulatory (dopamin- lateral PFC. ergic and cholinergic) transmitters. This, in turn, is a crucial problem In the present study, we demonstrate that VS presynaptic reflects for understanding maladaptive learning and decision-making in behavioral and neural signatures of model-free and model-based psychiatric disorders. choice behavior, thus, pointing towards a role of ventral striatal presynaptic DA as acting at the interface of behavioral control A14.6 systems. We replicate some previous findings. Our correlational The dopaminergic midbrain encodes shifts in beliefs findings are supported by causal study design using pharmacological Philipp SCHWARTENBECK1,*, Thomas H. B. FITZGERALD2 and challenges with L-DOPA and the same task in healthy volunteers and Ray DOLAN2 in patients with Parkinson’s disease as well as PFC brain stimulation 1Centre for Cognitive Neuroscience, University of Salzburg, Austria; studies. In summary, our data suggests an integrative view of 2Max Planck University College London Centre for Computational behavioral control systems with ventral striatal presynaptic DA Psychiatry and Ageing Research, London, United Kingdom playing a pivotal role in higher-order control over decision-making. *E-mail: [email protected] Putatively underlying mechanisms (e. g. uncertainty-based compe- Intrinsic Activity, 2016; 4(Suppl. 2): A14.6 tition via hierarchical Bayesian learning) are a target of ongoing http://www.intrinsicactivity.org/2016/4/S2/A14.6 studies. Dopamine is implicated in a diverse range of cognitive functions A14.5 including cognitive flexibility, task switching, signaling novel or Hierarchical prediction error responses modulated by unexpected stimuli as well as advance information. There is also a dopaminergic and cholinergic substances longstanding line of thought that links dopamine with belief formation Sandra IGLESIAS1,*, Christoph MATHYS1,2,3 and and, crucially, aberrant belief formation in psychosis. Integrating Klaas Enno STEPHAN1,3,4 these strands of evidence would suggest dopamine plays a central role in belief updating and more specifically in encoding of meaningful 1Translational Neuromodeling Unit, University of Zurich and ETH information content in observations. The precise nature of this Zurich, Switzerland; 2Max Planck UCL Centre for Computational relationship has remained unclear. To directly address this question Psychiatry and Ageing Research, London, United Kingdom; we developed a paradigm that allowed us to decompose two distinct 3Wellcome Trust Centre for Neuroimaging, Institute of Neurology, types of information content, information-theoretic surprise that University College London, United Kingdom; 4Max Planck Institute reflects the unexpectedness of an observation, and epistemic value for Metabolism Research, Cologne, Germany that induces shifts in beliefs or, more formally, Bayesian surprise. *E-mail: [email protected] Using functional magnetic resonance imaging in humans we show Intrinsic Activity, 2016; 4(Suppl. 2): A14.5 that dopamine-rich midbrain regions encode shifts in beliefs whereas http://www.intrinsicactivity.org/2016/4/S2/A14.5

surprise is encoded in prefrontal regions, including the pre-supple- of this pathway is, therefore, key in regulating experience-dependent mentary motor area and dorsal cingulate cortex. By linking putative behavioral changes. In particular, the individual response to early life dopaminergic activity to belief updating these data provide a link to adverse social experience depends upon an organism’s innate false belief formation that characterises hyperdopaminergic states biological make-up. How certain temperaments can be deregulated associated with idiopathic and drug induced psychosis. following early life adverse social experience, which independently lead to enduring changes in behavior in adulthood via perturbations A14.7 of dopamine transmission during adolescence, will be discussed. In Cell-type-specific modulation of behaviorally relevant and particular, synaptic changes of the mesocorticolimbic dopamine system following early life adverse experience will be presented to distracting stimuli by dopamine D1-like receptors in primate prefrontal cortex enable us to develop new pharmacological tools for the prevention Simon N. JACOB1,*, Wolfgang M. STALTER2 and Andreas NIEDER2 and treatment of aberrant affective functioning. 1Institute of Neuroscience, Technical University of Munich, Germany; 2Department of Animal Physiology, University of A15.2 Tübingen, Germany Gene regulation in dopamine circuitry and stress-related *E-mail: [email protected] behaviors Intrinsic Activity, 2016; 4(Suppl. 2): A14.7 François TRONCHE* http://www.intrinsicactivity.org/2016/4/S2/A14.7 CNRS INSERM, Université Pierre et Marie Curie, Institut de biologie Paris-Seine, Paris, France The prefrontal cortex (PFC) is a crucial brain structure for maintaining *E-mail: [email protected] behaviorally relevant (target) information in working memory. Pre- Intrinsic Activity, 2016; 4(Suppl. 2): A15.2 frontal cognitive functions are strongly regulated by dopamine, but it http://www.intrinsicactivity.org/2016/4/S2/A15.2 is unknown whether dopamine receptors are involved in protecting target memories from distracting stimuli. In monkeys trained to ignore Traumatic experiences and social stress may contribute to the onset interfering stimuli in a delayed-match-to-sample task, we addressed of psychiatric disorders. These situations trigger stress responses the local circuit dynamics of target and distractor representations in including the orchestrated release of hormones, including gluco- PFC and investigated their regulation by dopamine using a combina- corticoids (GCs) and prolactin (PRL). This mechanism is beneficial tion of single-unit recordings and micro-iontophoretic application of when normally working, but disproportionate or excessively long-lived the D1-like receptor (D1likeR) antagonist SCH 23390 to the local neural stress responses can precipitate the development of pathological network. Blocking D1likeR with SCH 23390 enhanced the represent- anxiety, depression, inability to socially perform or addiction. GCs tation of both target and distractor stimuli in putative interneurons, i. e. activate directly the glucocorticoid receptor (GR) and PRL binding to both stimuli were encoded similarly in these cells without particular its receptor activates STAT5 two widespread transcription factors. To weighting. In contrast, only target stimuli were modulated in putative address their respective roles in stress-related behaviors, we pyramidal neurons, resulting in a disproportionate increase in task- generated mice with either GR or STAT5 genes ablation targeted to relevant information after SCH 23390 application. Thus, stimulus specific brain cell populations, focusing on the dopamine circuitry. context and function, not physical identity, govern the extent of We reported that GR gene ablation in dopamine neurons had neither dopaminergic regulation in this cell class. These results show that effects on studied behaviors nor on VTA dopamine neurons activity. dopaminergic neuromodulation of prefrontal circuits is a highly These results were surprising given the reported effects of stress and adaptable and context-dependent process that biases mental GCs on dopamine neurons. However, GR gene ablation in post- presentations towards behaviorally relevant input and could play a synaptic dopaminoceptive neurons markedly decreased the activity central role for cognitive functioning in health and disease. of pre-synaptic dopamine neurons of the VTA, demonstrating that GC action on dopamine neurons firing is indirect. This mutation deeply Dopamine and affective disorders affected behavioral responses to cocaine and abolished enduring social aversion induced by repeated social defeats, shifting all defeated animals toward resiliency. Induced social aversion requires A15.1 enduring activation of dopamine neurons firing, abolished by GR Synaptic changes within the mesocortical circuit following gene inactivation. Induced anxiety and fear memories remained early life adverse social experience unaffected. Acute inhibition of the activity of DA-releasing neurons 1, 2 Miriam MELIS * and Marco BORTOLATO fully restored social interaction in socially defeated wild-type mice. 1Department of Biomedical Sciences, University of Cagliari, Italy; Our data suggest a GR-dependent neuronal dichotomy for the 2Department of Pharmacology and Toxicology, University of Utah, regulation of emotional and social behaviors, and clearly implicate Salt Lake City, UT, United States of America GR as a link between stress resiliency and dopaminergic tone. *E-mail: [email protected] Upon stress, the release of PRL is concomitant to that of GCs. Intrinsic Activity, 2016; 4(Suppl. 2): A15.1 Comparison of mice carrying brain-targeted GR and STAT5 gene http://www.intrinsicactivity.org/2016/4/S2/A15.1 mutations suggest that these two hormones have opposing effects on a large panel of stress-related behaviors but act on distinct structures Psychiatric disorders mirror affective imbalances within the brain. in the mesocorticolimbic dopamine pathway. Affects are essential for well-being, and once primary physiological needs and safety are satisfied, interpersonal interaction and acceptance represent a need that has to be met, and drives motivation. In fact, these latter not only act as natural reinforces, but are also important for social and cognitive development. Derangements from these, hence, lead to discrete affective disorders including anxiety, depression, schizophrenia and addiction. The mesocorticolimbic dopamine system, being a key component of the brain reward circuitry and processing value-related signals, is considered an important circuit for affective functioning. Modulation

A15.3 The dopamine D2 receptors (D2R), which are members of the large Concomitant VTA – PFC neural encoding of anxiety-related G protein-coupled receptor (GPCR) family, are the main target of modulation of goal-directed behavior clinically effective antipsychotics. Although GPCR pharmacology Junchol PARK1,*, Byron YU2, Alberto DEL ARCO1 and Bita affords selectivity of action, current antipsychotics do not adequately MOGHADDAM1 correct the postulated cortical hypodopaminergia and striatal hyperdopaminergia in schizophrenia. The recent observation that GPCRs 1Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, can signal not only through G proteins but also through the ability of United States of America; 2Carnegie Mellon University, Pittsburgh, the β-arrestin components of the desensitization machinery to PA, United States of America scaffold distinct intracellular signaling complexes provides an *E-mail: [email protected] opportunity to develop more effective therapies with fewer side Intrinsic Activity, 2016; 4(Suppl. 2): A15.3 effects. Accordingly, a β-arrestin-2 (βarr2)-biased D R ligand, http://www.intrinsicactivity.org/2016/4/S2/A15.3 2 UNC 9994A, based on the scaffold of aripiprazole was developed [1].

In real-life situations, motivated behavior leading to reward may UNC 9994A is a partial agonist at βarr2–D2R interaction but virtually involve a risk of aversive outcomes. Appropriate behavior adjust- devoid of antagonism at the D2R–G protein signaling. Using neuron- ments in these circumstances may be critical to survival and specific (βarr2)-knockout mice we show that the antipsychotic-like reproduction. We have designed a task for rodents to model this effects of the UNC 9994 tool compound in the amphetamine or situation and have recorded from two brain regions, the ventral phencyclidine (PCP) mouse models of psychotic-like behaviors, are tegmental area (VTA) and the medial prefrontal cortex (mPFC), which driven through antagonism of D2R–βarr2 interactions in the striatum comprise a critical circuitry that may encode potential dangers in but agonism of D2R / βarr2 in the prefrontal cortex. Interestingly, reward seeking. Especially, the VTA dopaminergic input to the mPFC similar to quinpirole, local injection of UNC 9994A in the prefrontal is spotlighted as selective activation of this input induces anxiety-like cortex inhibits PCP-induced responses and that effect is blocked by behavior. The task involved a motivated behavior (instrumental action inhibition of the GPCR kinase 2 (GRK-2). This paradoxical cortical leading to reward) that intermittently led to an aversive foot shock with D2R / βarr2 agonism can be attributed to elevated cortical expression varying levels of risk in different task blocks. Single unit activity and of βarr2 and GRK-2 compared to striatum. Direct electrophysiological LFPs were recorded from the two regions while animals engaged in corroboration of the agonist vs. antagonist role of UNC 9994A in the the task. We found significant anxiety-like behavioral alterations as a prefrontal cortex vs. striatum provides further credence to the in vivo function of the risk of punishment, indicated by a marked increase in behavioral pharmacological profile of this antipsychotic-like tool the mean action latency. Substantial proportions of single neurons in compound. Therefore, βarr2-biased D2R ligands like UNC 9994A that both regions encoded the risk, preceding and during the action. exert region-selective actions based on the neuronal complement of Distinct temporal patterns and tuning properties were observed from βarr2 / GRK-2, could possibly provide a path to develop better VTA dopaminergic, non-dopaminergic and PFC neuronal encoding of therapies, which correct both cortical and striatal dopamine the risk. A prominent increase in trial-to-trial behavioral variability was dysfunctions. observed as rats became anxious of the risk. This motivated a trial- Reference by-trial analysis of behavioral and neural data. Trial-by-trial neural 1. Allen JA, Yost JM, Setola V, Chen X, Sassano MF, Chen M, Peterson trajectories were extracted from simultaneously recorded VTA or S, Yadav PN, Huang XP, Feng B, Jensen NH, Che X, Bai X, Frye SV, mPFC populations using the Gaussian process factor analysis. In all Wetsel WC, Caron MG, Javitch JA, Roth BL, Jin J: Discovery of

VTA and mPFC populations, strong correlations between neural and β-arrestin-biased dopamine D2 ligands for probing signal behavioral data were detected, indicating that the trial-to-trial transduction pathways essential for antipsychotic efficacy. variation in behavior was well represented in the neural population Proc Natl Acad Sci U S A, 2011; 108(45):18488–18493. activity. We then examined how the risk altered the interareal neural doi:10.1073/pnas.1104807108 communication through coherent LFP oscillations. During risk-free actions, coherent oscillations (10–15 Hz) were observed in the VTA– A16.2 mPFC circuit at the time of the action. This oscillation was driven by Elevated striatal dopamine function in immigrants and their VTA, and temporally organized the spike activity in both regions. children: a risk mechanism for psychosis During risky actions, however, the power, coherence and directional- Romina MIZRAHI*, Alice EGERTON, Oliver HOWES, Sylvain HOULE, ity of the oscillations were markedly diminished, suggesting reduced Kwame MCKENZIE, Lucia VALMAGGIA, Michael BAGBY, Sagnik communication between the two regions. Together these findings BHATTACHARYYA, Andreas MEYER-LINDENBERG and Philip MCGUIRE reveal individual- and population-level neural representations for Centre for Addiction and Mental Health and University of Toronto, anxiety-related behavioral modulation in the VTA–mPFC circuit. Ontario, Canada

*E-mail: [email protected] Dopamine and schizophrenia Intrinsic Activity, 2016; 4(Suppl. 2): A16.2 http://www.intrinsicactivity.org/2016/4/S2/A16.2

A16.1 Importance: Migration is a major risk factor for schizophrenia but the Neuron-selective deletion of β-arrestin-2 uncovers unique neurochemical processes involved are unknown. One candidate mechanism of dopamine D2 receptor-biased ligands mechanism is through the elevations in striatal dopamine synthesis 1,2, 1 Marc G. CARON * and Nikhil M. URS and release that occur in schizophrenia and during the clinical high- 1Department of Cell Biology, Duke University Medical Center, risk (CHR) state that precedes psychosis. Durham, NC, United States of America; 2Department of Objective: To determine whether striatal dopamine function is Neurobiology, Duke University Medical Center, Durham, NC, elevated in immigrants compared to non-immigrants and the relation- United States of America ship with psychosis. *E-mail: [email protected] Design: Two complementary case-control studies of dopamine Intrinsic Activity, 2016; 4(Suppl. 2): Aold20.2 function in vivo (stress-induced dopamine release and dopamine http://www.intrinsicactivity.org/2016/4/S2/Aold20.2 synthesis capacity) in immigrants compared to non-immigrants. Two

complementary studies carried out in Academic research centres in Introduction: Dopamine synthesis capacity is elevated in patients Canada and the UK. with schizophrenia. However, it is not known how this alters during Participants: The Canada dopamine release study included 25 the development of psychosis, or in people at risk of psychosis due immigrant and 31 non-migrant Canadians. These groups included 23 to exposure to cannabis, or how this relates to treatment and changes CHR subjects, 9 antipsychotic naïve patients with schizophrenia and during the course of illness. 24 healthy volunteers. The UK dopamine synthesis study included 32 Methods: [18F]DOPA PET imaging was used to index dopamine immigrants and 44 non-immigrant British. These groups included 50 synthesis capacity in people with prodromal symptoms of schizo- CHR subjects and 26 healthy volunteers. phrenia who were re-scanned as they developed psychosis, in a Main outcome measures: The main effects of immigration status cohort of people who experienced sub-clinical psychotic-like symp- and clinical group on stress-induced dopamine release and on toms when they were exposed to cannabis, and in another cohort of dopamine synthesis capacity in the striatum. patients with first episode psychosis who were re-scanned during the Results: Both striatal dopamine release and dopamine synthesis course of their illness as well as matched controls. capacity were significantly elevated in immigrants compared to non- Results: Dopamine synthesis capacity was elevated in people immigrants, independent of clinical status. prior to the onset of psychosis (effect size = 0.8, p < 0.001), and Conclusions and relevance: These data provide the first evidence increased longitudinally with the development of psychosis (p < 0.05). that the effect of migration on the risk of developing psychosis may Dopamine synthesis capacity was elevated in first episode patients be mediated by an elevation in brain dopamine function. who responded to treatment (effect size = 1.3, p < 0.05) and did not change during the early course of illness. Furthermore, dopamine A16.3 synthesis capacity was related to treatment response (r = 0.6, Targeting neurosteroidogenesis as therapeutic strategy for p < 0.05). People who experienced psychotic-like symptoms when dopamine-related neuropsychiatric disorders exposed to cannabis showed reduced dopamine synthesis capacity. Roberto FRAU1,*, Alessandra PARDU1, Silvia FANNI1, Pierluigi SABA1, Discussion: These data suggest that the dopamine synthesis Paola DEVOTO1 and Marco BORTOLATO2 capacity is linked to the development of psychosis and response to treatment in the first episode. Cannabis use is associated with 1Departments of Neuroscience and Clinical Pharmacology, reduced dopamine synthesis. University of Cagliari, Italy; 2Department of Pharmacology and

Toxicology, School of Pharmacy, University of Kansas, Lawrence, KS, United States of America Dopamine and genetic disorders *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A16.3 A17.1 http://www.intrinsicactivity.org/2016/4/S2/A16.3 Impact of dopamine transporter-mediated, non-vesicular DA Neurosteroids are a subclass of steroids synthesized de novo in the release on ADHD traits as studied in the DAT Val559 mouse brain, which modulate the actions of several neurotransmitters, model including dopamine. The involvement of neurosteroids in brain Randy D. BLAKELY* function has recently attracted much interest with respect to their Department of Pharmacology, Vanderbilt University, Nashville, TN, therapeutic potential in psychiatric treatment. In particular, we have United States of America investigated the role of 5α-reductase (5AR), the key enzyme in *E-mail: [email protected] neurosteroidogenesis, in animal models of neuropsychiatric dis- Intrinsic Activity, 2016; 4(Suppl. 2): A17.1 orders linked to dopaminergic dysfunctions. Our preclinical and http://www.intrinsicactivity.org/2016/4/S2/A17.1 clinical findings suggest that 5AR inhibitors, such as finasteride, may Genetic, pharmacological and imaging studies support a role for elicit antidopaminergic effects in a number of disorders associated dopamine (DA) signaling dysfunction in establishing risk for attention- with dopaminergic hyperactivity, including Tourette syndrome and deficit hyperactivity disorder (ADHD). In this regard, the presynaptic schizophrenia. Unlike benchmark antidopaminergic therapies, how- DA transporter (DAT, SLC6A3) has been of particular interest, given ever, the effects of 5AR inhibitors are not accompanied by extra- that the most commonly prescribed ADHD medications, methyl- pyramidal symptoms, and appear to be contributed by the negative phenidate and amphetamine derivatives, target DAT, either by modulation of D1 and D3 receptor signaling in the nucleus accumbens. blocking DAT-mediated DA clearance, or the induction of transporter Notably, we identified that inhibitors of 17α-hydroxylase / 17,20-lyase reversal or non-vesicular DA release. In an effort to establish a causal (CYP450-C17), the rate-limiting enzyme in androgen synthesis, elicit link between DAT dysfunction and ADHD risk, we pursued a search behavioral effects akin to those produced by finasteride. Taken for rare, but functionally penetrant, DAT coding variants in ADHD together, these data suggest that 5α-reduced androgens may play a subjects [1, 2]. One of these variants, DAT Val559, a variant previous- critical role in the modulation of dopaminergic signaling in the nucleus ly reported in a female subject with bipolar disorder [3] and recently accumbens, and offer a potential theoretical platform to account for in two unrelated males with autism spectrum disorder [4], when the male predominance of Tourette syndrome and schizophrenia. expressed in HEK 293 cells, established an anomalous DA efflux (ADE) indicative of spontaneous DAT reversal. Furthermore, the ADE A16.4 of DAT Val559-transfected cells could be blocked either by methyl- Dopamine synthesis in psychosis: relationship to cannabis and phenidate or amphetamine, suggesting that the actions of these treatment response therapeutic psychostimulants in some ADHD subjects could arise Oliver HOWES*, Michael BLOOMFIELD, Ilaria BONOLDI, Sameer from ADE. To test the hypothesis that DAT Val559 could induce ADE JAUHAR, Philip MCGUIRE and Maria ROGDAKI in vivo and provoke ADHD-associated behavioral traits, we generat- MRC Clinical Science Centre, Imperial College and King’s College, ed knock-in mice expressing the Val559 variant from the endogenous London, United Kingdom Slc6a3 locus [5, 6], with studies supporting the institution of tonically *E-mail: [email protected] elevated extracellular DA, constitutive activation of DA autorecep- Intrinsic Activity, 2016; 4(Suppl. 2): Aold32.1 tors, and altered spontaneous and amphetamine / methylphenidate- http://www.intrinsicactivity.org/2016/4/S2/Aold32.1 induced locomotor behavior. In more recent studies with these mice, to be discussed in the presentation, we have obtained evidence

for disturbances in cognitive and reward behaviors, as well as implication of brain lipid composition in the etiology of psychiatric anomalous behavioral responses to cocaine. These studies support endophenotypes has been overlooked. the in vivo functional status of the DAT Val559 variant and the Using operant conditioning tasks in mice, we show that develop- DAT Val559 knock-in mouse as a construct-valid model of perturba- mental n-3 PUFA deficiency leads to avolition and anhedonia in tions in DA homeostasis underlying ADHD risk, changes that may be adulthood with no change in locomotion. Moreover, n-3 PUFA- shared with other DA-linked syndromes. deficient animals display blunted response to amphetamine in moti- References vational tasks. These behavioral deficits are accompanied by altered

1. Mazei-Robison MS, Bowton E, Holy M, Schmudermaier M, Freissmuth dopamine D2 receptor-dependent transmission, selectively in the

M, Sitte HH, Galli A, Blakely RD: Anomalous dopamine release ventral striatum. More precisely, D2 receptor-dependent signaling in associated with a human dopamine transporter coding variant. the ventral striatum is blunted in n-3 PUFA-deficient animals, despite J Neurosci, 2008; 28(28):7040–7046. an increase in receptor expression, suggesting that the upregulation

doi:10.1523/JNEUROSCI.0473-08.200808.2008 of D2 receptor is a compensatory mechanism to impaired signaling.

2. Sakrikar D, Mazei-Robison MS, Mergy MA, Richtand NW, Han Q, Accordingly, downregulation of D2 receptor by shRNA expression in Hamilton PJ, Bowton E, Galli A, Veenstra-VanderWeele J, Gill M, adulthood does not rescue — and even worsens — behavioral deficits. Blakely RD: Attention deficit/hyperactivity disorder-derived coding Interestingly, both the behavioral and neurobiological alterations variation in the dopamine transporter disrupts microdomain induced by n-3 PUFA deficiency recapitulate some endophenotypes targeting and trafficking regulation. J Neurosci, 2012; 32(16): of schizophrenia. Indeed, avolition and anhedonia are two main

5385–5397. doi:10.1523/JNEUROSCI.6033-11.2012 negative symptoms of schizophrenia, and an increase in D2 receptor 3. Grünhage F, Schulze TG, Müller DJ, Lanczik M, Franzek E, Albus M, expression in the striatum — which seem to participate to those Borrmann-Hassenbach M, Knapp M, Cichon S, Maier W, Rietschel M, symptoms — has been consistently described in schizophrenic Propping P, Nöthen M: Systematic screening for DNA sequence patients. Therefore, it is tempting to propose that the decreased n-3 variation in the coding region of the human dopamine transporter PUFA in schizophrenia could directly participate to some symptoms. gene (DAT1). Mol Psychiatry, 2000; 5(3):275–282. The genetic and / or environmental factors responsible for such 4. Bowton E, Saunders C, Reddy IA, Campbell NG, Hamilton PJ, Henry alteration in lipid metabolism should therefore be more profoundly LK, Coon H, Sakrikar D, Veenstra-VanderWeele JM, Blakely RD, explored. Sutcliffe J, Matthies HJ: SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and Poster presentations trafficking. Transl Psychiatry, 2014; 4:e464. doi:10.1038/tp.2014.90 5. Mergy MA, Gowrishankar R, Davis GL, Jessen TN, Wright J, Stanwood GD, Hahn MK, Blakely RD: Genetic targeting of the amphetamine A18.1 and methylphenidate-sensitive dopamine transporter: on the path Heterodimerization between dopamine D2 receptor and M1 to an animal model of attention-deficit hyperactivity disorder. muscarinic acetylcholine receptor 1, 1 Neurochem Int, 2014; 73:56–70. doi:10.1016/j.neuint.2013.11.009 René Albert Johan CRANS *, Elise WOUTERS , Lakshmi 1 1 2 6. Mergy MA, Gowrishankar R, Gresch PJ, Gantz SC, Williams J, Davis VASUDEVAN , Karen HEYNINCK , Jeanelle PORTELLI , Sofie 2 2 2 2 GL, Wheeler CA, Stanwood GD, Hahn MK, Blakely RD: The rare DAT DAELEMANS , Robrecht RAEDT , Alfred MEURS , Paul BOON , 3 3 4 coding variant Val559 perturbs DA neuron function, changes Debby VAN DAM , Peter Paul DE DEYN , Francisco CIRUELA 1 behavior, and alters in vivo responses to psychostimulants. Proc and Kathleen VAN CRAENENBROECK Natl Acad Sci U S A, 2014; 111(44):E4779–E4788. 1Department of Biochemistry and Microbiology, University of doi:10.1073/pnas.1417294111 Ghent, Belgium; 2Laboratory for Clinical and Experimental Neurophysiology, Neurobiology and Neuropsychology, A17.2 Department of Neurology, Ghent University, Ghent University 3 Impact of brain lipid composition on reward processing and Hospital, Ghent, Belgium; Laboratory of Neurochemistry and mesolimbic dopamine transmission: a role in schizophrenia Behavior, Institute Born-Bunge, University of Antwerp, Belgium; 4 endophenotypes? Unitat de Farmacologia, Departament de Patologia i Terapèutica Fabien DUCROCQ1,*, Roman WALLE1, Suzanne VAN DER VELDT1, Experimental, Facultat de Medicina, IDIBELL, Universitat de Clementine BOSCH-BOUJU1, Agnès AUBERT1, Rolando MELONI2, Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain Sophie LAYÉ1, Véronique DE SMEDT-PEYRUSSE1 and Pierre *E-mail: [email protected] TRIFILIEFF1 Intrinsic Activity, 2016; 4(Suppl. 2): A18.1 http://www.intrinsicactivity.org/2016/4/S2/A18.1 1INRA UMR 1286, University of Bordeaux, France; 2Institut du Cerveau et de la Moelle épinière (ICM), UPMC / INSERM About 30–50 % of all drugs target G protein-coupled receptors U 1127 / CNRS UMR 7225, Department of Biotechnology and (GPCRs), which is the largest receptor family and is involved in many Biotherapy, Paris, France (patho-)physiological processes. Since the last two decades, various *E-mail: [email protected] studies have led to the acceptance that GPCRs do not primarily Intrinsic Activity, 2016; 4(Suppl. 2): A17.2 reside in the plasma membrane as a monomer, but are capable to http://www.intrinsicactivity.org/2016/4/S2/A17.2 form homodimers, heterodimers or multimers. Interestingly, various interacting protomers with the dopamine D receptor (D R) have been Various though very distinct psychiatric disorders, such as 2 2 described and a change in G-protein coupling with the D R has been schizophrenia, obsessive–compulsive disorder or attention-deficit 2 observed upon heterodimer formation. However, most studies cannot hyperactivity disorder are associated with a dysfunction of the reward unambiguously conclude a direct interaction between GPCRs in system linked to an alteration of dopamine transmission. Further- native tissue. more, these pathologies are also accompanied by changes in brain The cholinergic system plays an important role in neurological lipid composition and in particular by a decrease in the content of processes, a network involved in arousal, cognitive processes (e. g. docosahexaenoic acid (DHA), the main n-3 polyunsaturated fatty acid learning and memory) and attention. In pharmacological animal (PUFA) in the brain. However, despite that n-3 PUFA supplementa- models of epilepsy and Alzheimer’s disease (AD) the cholinergic tion seems to improve or prevent some psychiatric symptoms, the

GPCRs are targeted with pilocarpine (agonist) and (methyl-)scopol- for the Unknown, Lisbon, Portugal; 8Wellcome Trust Centre for amine (antagonist), respectively. The administration of a high dose of Human Genetics, University of Oxford, United Kingdom; 9Institut du pilocarpine causes status epilepticus (SE), which progresses into Fer à Moulin, Institut National de la Santé et de la Recherche spontaneous seizures, resembling temporal lobe epilepsy (TLE) in Médicale, Unité Mixte de Recherche S839, Université Pierre et humans. The administration of scopolamine induces memory impair- Marie Curie, Paris, France; 10Department of Language and ment, which relates to the intellectual dysfunction in AD patients. Till Genetics, Max Planck Institute for Psycholinguistics, Nijmegen, 11 now, literature only mentions the formation of M1 muscarinic The Netherlands; Departments of Developmental and acetylcholine receptor (mAChR) homodimers and heterodimers with Comparative Psychology and Primatology, Max Planck Institute for

M2 and M3 mAChRs. A novel interaction with the D2R could modulate Evolutionary Anthropology, Leipzig, Germany the function of this GPCR; both rececptors are co-distributed in *E-mail: [email protected] several brain regions and both are located at post-synaptic Intrinsic Activity, 2016; 4(Suppl. 2): A18.3 membranes. The M1 mAChR is coupled to the Gαq/11 and D2R to the http://www.intrinsicactivity.org/2016/4/S2/A18.3

Gαi/o protein. The cholinergic system is able to modulate the ligand What enables the human nervous system to acquire language and affinity of the D2R, which could be due to GPCR heterodimerization. speech and which genetic candidates might have contributed to this On the other hand this suggests that the dopaminergic system could capacity? Two human-specific amino acid substitutions in the tune the cholinergic system, thereby ascribing a more prominent role transcription factor FOXP2 are outstanding candidates, given that for dopamine in diseases which are less related to dopamine. they might have been positively selected during human evolution and In our studies, we have identified heterodimerization between D2R given that FOXP2 is currently the only gene firmly linked to speech and M1 mAChR by co-immunoprecipitation (coIP), bioluminescence and language development. When these two substitutions are resonance energy transfer (BRET) and complementation (NanoBiT) introduced into endogenous Foxp2 of mice (Foxp2hum), cortico-basal assays in transfected HEK 293 cells. The next steps will be the ganglia circuits are specifically affected. Here we show that validation of the heterodimer in native tissue. The D2R–M1 mAChR humanized Foxp2 alters dopamine levels, learning and cortico- interaction will be tested by coIP, proximity ligation assay (PLA) and striatal synaptic plasticity. Foxp2hum/hum mice learn stimulus–response time-resolved fluorescent resonance energy transfer (TR-FRET). associations faster than their wild-type littermates in situations in which declarative (i. e. place-based) and procedural (i. e. response- A18.2 based) forms of learning could compete. Striatal districts known to be Local striatal dopamine governs psychomotor behavior in mice differently related to these two modes of learning are affected Wenlin LIAO* differently in the Foxp2hum/hum mice, as judged by measures of Institute of Neuroscience, National Cheng-Chi University, Taipei, dopamine levels, gene expression patterns, and synaptic plasticity, Taiwan including an NMDA receptor-dependent form of long-term depress- *E-mail: [email protected] sion. These findings suggest that dopamine-processing in cortico- Intrinsic Activity, 2016; 4(Suppl. 2): A18.2 basal ganglia circuits might be altered in mice with the humanized http://www.intrinsicactivity.org/2016/4/S2/A18.2 form of Foxp2 and raise the possibility that the humanized Foxp2 phenotype reflects a different tuning of corticostriatal systems Psychomotor dysfunction caused by aberrant dopamine content in involved in declarative and procedural learning, a capacity potentially the striatum has been associated with numerous neurological and contributing to adapting the human brain for speech and language psychiatric disorders, such as Parkinson’s disease, attention-deficit acquisition. hyperactivity disorder (ADHD) and Rett syndrome (RTT). Using genetically manipulated mice modeling RTT and ADHD separately, we found that the relative content of dopamine in the rostral versus A18.4 caudal striatum is critical for psychomotor control. The underlying Splice-variant and isoform-dependent isradipine inhibition of 2+ neuronal and circuit mechanisms are currently under investigation. recombinant L-type Ca currents evoked by substantia nigra dopamine neuron-like activity patterns Nadine J. ORTNER1,*, Gabriella BOCK1, Antonios DOUGALIS2, A18.3 Maria KHARITONOVA1, Johanna DUDA2, Petronel TULUC1, Humanized Foxp2, a gene involved in language acquisition, Thomas POMBERGER1, Nadia STEFANOVA3, Florian PITTERL4, alters dopamine levels, cortico-striatal synaptic plasticity and Thomas CIOSSEK5, Herbert OBERACHER4, Henning J. DRAHEIM5, accelerates transitions from declarative to procedural learning Birgit LISS2 and Jörg STRIESSNIG1 Christiane SCHREIWEIS1,*, Ulrich BORNSCHEIN2, Eric BURGUIÈRE1, 1 Cemil KERIMOGLU3, Sven SCHREITER4, Michael DANNEMANN2, Department of Pharmacology and Toxicology, University of 2 Shubhi GOYAL5, Ellis REA6, Catherine A. FRENCH7, Rathi PULIYADI8, Innsbruck, Austria; Institute of Applied Physiology, University of 3 Matthias GROSZER9, Simon E. FISHER10, Roger MUNDRY11, Christine Ulm, Germany; Department of Neurology, Medical University of 4 WINTER6, Wulf HEVERS2, Svante PÄÄBO2, Wolfgang ENARD3 and Innsbruck, Austria; Institute of Legal Medicine and Core Facility Ann M. GRAYBIEL5 Metabolomics, Medical University of Innsbruck, Austria; 5CNS Research, Boehringer Ingelheim Pharma GmbH & Co KG, 1Brain and Spine Institute, Paris, France; 2Department of Biberach, Germany Evolutionary Genetics, Max Planck Institute for Evolutionary *E-mail: [email protected] Anthropology, Leipzig, Germany; 3Laboratory of Anthropology and Intrinsic Activity, 2016; 4(Suppl. 2): A18.4 Human Genetics, Department of Biology II, Ludwig-Maximilians http://www.intrinsicactivity.org/2016/4/S2/A18.4 University Munich, Martinsried, Germany; 4DFG Research Center for Regenerative Therapies, Technical University Dresden, Parkinson’s disease (PD) is a neurodegenerative disorder charac- Germany; 5McGovern Institute for Brain Research and Department terized by the selective loss of substantia nigra dopamine (SN DA) of Brain and Cognitive Sciences, Massachusetts Institute of neurons. Inhibition of brain L-type Ca2+ channels (LTCCs), especially

Technology, Cambridge, MA, United States of America; CaV1.3, by the antihypertensive drug isradipine (ISR) is currently 6Department of Psychiatry and Psychotherapy, Faculty of Medicine tested as a neuroprotective approach in a phase 3 clinical trial Carl Gustav Carus, Technical University Dresden, Germany; (NCT02168842). 7Champalimaud Neuroscience Programme, Champalimaud Centre

We show that neither global CaV1.3 knockout nor in vivo pretreatment remains unclear. The aim of this study was to determine the relation- with therapeutic plasma concentrations of ISR can rescue SN DA ships between NS and impulsivity on one hand, and D2/3R density and neurons in a 6-OHDA PD mouse model. In knockouts, upregulation DA release in striatum on the other hand. of other voltage-gated Ca2+ channels [1] may explain lack of We used the Roman low- and high-avoidance rat (RLA vs. RHA) neuroprotection. Instead, in wild-type mice it may be due to weaker lines, which show divergent phenotypes with regards to impulsivity ISR inhibition of LTCCs in SN DA neurons than of its original drug and NS. RHAs (n = 15) and RLAs (n = 15) were tested for impulsivity target, CaV1.2 channels in arterial smooth muscle (aSM). using the 5-choice serial reaction time task (5-CSRTT) and for NS

We confirmed expression of CaV1.2 and CaV1.3 variants in micro- using the novelty-induced place preference (NIPP) test. Rats were 123 dissected mouse SN DA neurons and generated stable HEK 293 cell scanned with SPECT using [ I]IBZM to measure D2/3R density and lines expressing human long (hCaV1.3L) or short (hCaV1.3S) CaV1.3 amphetamine (AMPH)-induced DA release in striatum. Binding splice variants or CaV1.2 (+β3, α2δ1). Using whole-cell patch-clamp potential (BPND) and gamma (γ) values were quantified as indexes of recordings LTCC currents and ISR sensitivity during SN DA- and D2/3R density and AMPH-induced DA release, respectively, using a aSM-like activity were investigated. Sustained SN DA-like pace- single scan and the linearized simplified reference region model. making (2.5 Hz) resulted in a reduced steady-state availability Compared to RLAs, RHAs were more impulsive as shown by a

(15–24 % remaining ICa) and only CaV1.3 isoforms also conducted ICa greater number of premature responses (p < 0.01) and more novelty during the interspike interval. Simulated burst activity led to increased seeker as shown by the time spent in a novel compartment (p < 0.05). integrated ICa during a 3-spike burst (hCaV1.3S+L: ~ 1.9-fold, hCaV1.2: In addition, RHAs showed lower BPND (p < 0.001) and released more 2.9-fold) and during the first spike following the post-burst pause striatal DA in response to AMPH (p < 0.01) than RLAs. Importantly,

(hCaV1.3S+L: 1.5-fold). CaV1.3 ICa during pacemaking was inhibited in individual values of premature responding were negatively correlated a splicing-dependent manner (IC50 hCaV1.3L: 7.7 [6.3–9.6] nM; (r = −0.65, p < 0.01) with BPND, but positively correlated with γ hCaV1.3S: 17.0 [13.8–20.9] nM; mean [95 % CI]) and was less (r = 0.64, p < 0.01), while pilot data showed that NS scores correlated sensitive than hCaV1.2 (3.1 [2.3–4.3] nM). During aSM-like activity with γ (r = 0.41, p < 0.03) but not BPND (p < 0.05). Interestingly, indivi- hCaV1.2 ICa was inhibited at 2-fold lower concentrations (IC50 = 1.6 dual BPND were negatively correlated with γ (r = −0.59, p < 0.01). [1.3–1.9] nM). Our study confirms and extends previous data of an association 2+ We show that during and after SN DA-like bursts LTCC Ca load was between D2/3R deficits in striatum and impulsivity by showing that increased significantly. The lack of neuroprotective effects of ISR can presynaptic DA release could be important in underlying this be explained by its weaker state-dependent inhibition of LTCCs in SN personality trait. Furthermore, although NS has been associated to

DA neurons as compared to aSM CaV1.2. CaV1.3 channels, which are impulsivity, both personality traits may not share the exact same considered the main target for ISR-mediated neuroprotection, require neurochemical substrates. Those findings provide further information

5–10-fold higher ISR concentrations for inhibition than aSM CaV1.2. on the mechanism by which altered transmission at D2/3R may Our data therefore predict that supratherapeutic doses of ISR, likely contribute to impulsive behaviours. to cause cardiovascular side effects, may be required for CaV1.3- mediated neuroprotection in PD. A18.6 Support: FWF and DFG (F4402, P27809, W1101-B12, LI 1754/1). Behavioral deficits and related in vivo electrophysiological Reference recordings in a mouse model of schizophrenia with genetic 1. Poetschke C, Dragicevic E, Duda J, Benkert J, Dougalis A, DeZio R, reduction of dysbindin-1 and D2L 2+ Snutch TP, Striessnig J, Liss B: Compensatory T-type Ca channel Simone GUADAGNA1,*, Huiping HUANG1, Diego SCHEGGIA1, activity alters D2-autoreceptor responses of Substantia nigra Emiliana BORRELLI2, Theresa BALLARD3 and Francesco PAPALEO1 dopamine neurons from Cav1.3 L-type Ca2+ channel KO mice. 1Department of Neuroscience and Brain Technology, Italian Sci Rep, 2015; 5:13688. doi:10.1038/srep13688 Institute of Techonlogy, Genova, Italy; 2Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, United A18.5 States of America; 3Neuroscience, Ophthalmology and Rare Sriatal dopamine D2/3 receptors and amphetamine-evoked Diseases, Roche Pharma Research and Early Development, dopamine release in impulsivity and novelty-seeking traits: Roche Innovation Center Basel, Switzerland 123 a SPECT imaging study using [ I]IBZM *E-mail: [email protected] 1, 2 1 Diego P. PANDOLFO *, Benjamin B. TOURNIER , Andrea DIMIZIANI , Intrinsic Activity, 2016; 4(Suppl. 2): A18.6 2 2 1 Marouane BEN AMMAR , Maria DEMIRI , Sarah GARDI , Stergios http://www.intrinsicactivity.org/2016/4/S2/A18.6 TSARTSALIS2, Philippe MILLET2 and Nathalie GINOVART1 The pathophysiology of schizophrenia and its treatment have been 1Department of Psychiatry, University of Geneva, Switzerland; consistently linked to dysregulation of dopaminergic neural trans- 2Department of Psychiatry, University Hospital of Geneva, mission and in particular of dopamine D receptor pathways. The D Switzerland 2 2 gene codes for a short form (D ) and a long form (D ) of the D *E-mail: [email protected] 2S 2L 2 receptor. Interestingly, recent results indicate that functional genetic Intrinsic Activity, 2016; 4(Suppl. 2): A18.5 variants in the D gene might modulate schizophrenia-related pheno- http://www.intrinsicactivity.org/2016/4/S2/A18.5 2 types by modifying the D2S / D2L ratio. Levels of dystrobrevin-binding Impulsivity and novelty-seeking (NS) have been studied for their protein 1 gene expression and its protein dysbindin 1 (dys1) are potential role in the vulnerability to drug abuse. Impulsivity has been significantly reduced in patients with schizophrenia. This reduction of linked to a decreased density of dopamine (DA) D2/3 receptors (D2/3R) dys1 impacts cognitive abilities and leads to up-regulation of D2 in striatum whereas studies trying to link D2/3R to NS, which has an receptors on the neural surface. Based on this biological evidence, impulsive component, have provided mixed data. Clinical imaging we generated a novel mouse model with reduced expression of both studies have shown that D2/3R binding and presynaptic DA release in D2L and dys1. We then predicted that synergistic effects of a reduced striatum are lowered in addicted subjects. However, understanding dys1 expression and an increased D2S / D2L ratio in the same subject whether those deficits are a cause or a consequence of addiction might alter dopamine / D2 signaling, thus triggering cognitive- and schizophrenia-relevant symptoms.

Executive function deficits are core enduring symptoms in schizo- in right VS (k = 275, T = 4.4), left insula (k = 215, T = 4.1), and vmPFC phrenia; thus, we tested our dys1 / D2L genetically modified mice in a (k = 195, T = 4.2). Furthermore, all correlations remained significant modified version of the 5-choice serial reaction time task. This test when nonresponders were included and after controlling for DAMPH revealed increased impulsivity in dys1 / D2L 2-het mice. Dys1, D2L and dose, plasma amphetamine levels, age, and sex. dys1 / D2L 2-het mice showed also deficits in visuospatial attention. This work indicates that D2 receptor signaling in vmPFC is associated Furthermore, testing in our novel automated task able to measure with individual differences in subjective responses to DAMPH: attentional set-shifting in mice revealed that mice with reduction of placebo vmPFC BPND was positively correlated with subsequent dys1 have a selective impairment on extradimensional shift (EDS) DAMPH High and vmPFC %ΔBPND tracked with Want More ratings. abilities, while mice with a reduction of D2L show deficits in reversal The observation that differences in DA release in the insula correlated learning. Dys1 / D2L 2-het mice exhibited more severe deficits and with drug wanting converges with data suggesting the importance of were not able to reliably perform the test. To investigate possible the insula in drug craving. Together these data highlight the impor- underlying deficits in neuronal activity, we implanted microelectrode tance of variability in DA signaling in specific paralimbic cortical arrays for extracellular recording of multi-unit activity in the mPFC of regions in the subjective response to DAMPH, which may confer risk our mutant mice and recorded neuronal activity while they performed for abusing psychostimulants. the attentional set-shifting task. Dys1 het mice showed a reduced number of activated neurons in the first trials of the EDS stage, while A18.8 learning the rule of this stage, compared to wild-type mice. These Photostimulation of novel dopaminergic circuit elicits results suggest that the behavioral deficits observed in the dys1 het locomotor activity in mice mice in the EDS may be possibly due to altered firing activity in the Kathrin KOBLINGER*, Céline JEAN-XAVIER, Jillian EJDRYGIEWICZ, mPFC. Tamás FÜZESI, Sandeep SHARMA, Jaideep S. BAINS and Taken together, these results begin to unravel the role of a genetic Patrick J. WHELAN interaction between dys1 and D in cognitive and schizophrenia- 2L Hotchkiss Brain Institute, University of Calgary, Alberta, Canada related deficits. *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A18.8 A18.7 http://www.intrinsicactivity.org/2016/4/S2/A18.8 Variability in paralimbic dopamine signaling correlates with subjective responses to D-amphetamine Dopaminergic neurons in the posterior hypothalamus have been Christopher SMITH1,*, Linh DANG1, Ronald COWAN1,2, Robert suggested to play a role in pain, restless legs syndrome and spinal KESSLER3 and David ZALD1,2 locomotor network modulation. However, the contribution of these neurons to movement control is unknown. Our goal was to look at the 1Department of Psychology, Vanderbilt University and Vanderbilt functional connectome of the A11 dopaminergic cells to the lumbar University School of Medicine, Nashville, TN, United States of spinal cord (SC) and other brain locomotor regions. Also, we wanted America; 2Department of Psychiatry, Vanderbilt University and to explore specific activation of A11 neurons and their role in loco- Vanderbilt University School of Medicine, Nashville, TN, United motor behaviour. States of America; 3Department of Radiology, University of Alabama Our data suggests projections from the A11 dopaminergic neurons to School of Medicine, Birmingham, AL, United States of America the medial reticular formation (MRF), an important locomotor region *E-mail: [email protected] within the brain. Using a TH-IRES-Cre mouse we transfected A11 Intrinsic Activity, 2016; 4(Suppl. 2): A18.7 dopamine (DA) neurons with channelrhodopsin 2 (ChR2) and investi- http://www.intrinsicactivity.org/2016/4/S2/A18.7 gated the effects of photostimulation on these neurons. We found that D-Amphetamine (DAMPH)-induced dopamine (DA) release in the stimulation (20 Hz, 10 ms) of A11 neurons with blue light (470 nm) ventral striatum (VS) has been positively associated with either initiated and modulated locomotion in awake behaving adult mice. DAMPH-induced euphoria or drug wanting, but these studies were We also observed a modulation of the Hoffman reflex (H-reflex) with limited in measuring DA D2/3 receptor availability (binding potential, light stimulation reflecting a modulation of reflex function within the 11 BPND) only in the striatum with [ C]raclopride. The radiotracer lumbar SC. Taken together, this DAergic nucleus may be part of the 18 [ F]fallypride allows estimation of D2/3 BPND in extrastriatal regions diencephalic locomotor region which is suggested to play an and can index DAMPH-induced DA release, measured as %ΔBPND important role in goal-directed behaviours. from baseline / placebo. Using fallypride PET, we searched for associations between A18.9 subjective effects of DAMPH and DA signaling across the brain. Involvement of cJun N-terminal kinase (JNK) signaling in Participants (n = 46, 23 male, age: 22 ± 2.9) completed PET scans dopamine transmission after receiving either placebo or oral DAMPH (0.43 mg/kg). A Giada SPIGOLON1, Anna CAVACCINI2, Massimo TRUSEL2, simplified reference tissue (cerebellum) model was used to estimate Raffaella TONINI2 and Gilberto FISONE1,* BP across the brain. %ΔBP [%ΔBP = (placeboBP − DAMPHBP ) / ND ND ND ND ND 1Department of Neuroscience, Karolinska Institutet, Stockholm, (placeboBP ) × 100 %] maps were generated with positive values ND Sweden; 2Italian Institute of Technology, Genova, Italy indicating increased release. Peak positive subjective responses to *E-mail: [email protected] DAMPH vs. placebo were measured via the Drug Effects Question- Intrinsic Activity, 2016; 4(Suppl. 2): A18.9 naire (DEQ) Feel, Like, High, and Want More ratings. Initial analyses http://www.intrinsicactivity.org/2016/4/S2/A18.9 focused on 35 DEQ responders (22 male, age: 21.9 ± 2.7) as participants lacking subjective responses (nonresponders) could Parkinson’s disease (PD) is treated with pharmacological agents that hinder correlational analyses. DEQ ratings were regressed against promote dopamine (DA) transmission in the basal ganglia. Here, we both placebo BPND and %ΔBPND maps using SPM 8. Cluster-level used a mouse model of PD to study the effects produced by activation significance was set at p < 0.05 family-wise error corrected. of DA D1 receptors (D1Rs) on the c-Jun N-terminal kinase (JNK)

DEQ High positively correlated with placebo BPND in a large cluster signalling pathway. We found that in the DA-depleted striatum, (k, number of voxels) in ventromedial prefrontal cortex (vmPFC; administration of the antiparkinson drug L-DOPA, results in a large k = 388, T = 4.5). DEQ Want More positively correlated with %ΔBPND increase in JNK phosphorylation. This effect is blocked by a D1R

antagonist and is localized in the striatal projection neurons (SPNs) neurons. We observed a large number of pairs (76 of 197) that exhibit of the direct pathway. The activation of JNK in direct SPNs is millisecond-timescale synchrony, which is, according to our modeling accompanied by concomitant phosphorylation of the early response results, more than necessary to achieve an increase in DA neuron transcription factor, cJun. This, in turn, leads to accumulation of burstiness. Our results provide a mechanistic understanding of the phospho-cJun at the promoter of fosB, another transcription factor diverse mechanisms whereby GABA neurons regulate DA neuron implicated in the effects of dopaminergic drugs. Pharmacological burst firing. inhibition of JNK reduces the synaptic and behavioral responses to This work was supported by NIAAA (grant R01A022821), ANR-13- activation of D1Rs in the PD disease state. Thus, our findings identify NEUC-0003-01, IdEx ANR-11-0001-02 PSL and LabEx ANR-10- the JNK signaling pathway as a novel player in dopamine trans- LABX-0087 (France), Russian Foundation for Basic Research (grant mission and provide information for the development of antiparkinson 14-02-00916-a). therapies. A18.11 A18.10 Swapping the selectivity of ring-substituted methcathinones Contribution of synchronized GABAergic neurons to for human dopamine and serotonin transporter dopaminergic neuron firing and bursting Marco NIELLO1,*, Eva HELLSBERG2, Daniel SZÖLLŐSI1, Marion Ekaterina MOROZOVA1,2,*, Maxym MYROSHNYCHENKO3, HOLY1, Oliver KUDLACEK1, Nicolas V. COZZI3, Michael BAUMANN4, Denis ZAKHAROV4, Matteo DI VOLO5, Boris GUTKIN5, Richard A. GLENNON5, Thomas STOCKNER1, Gerhard F. ECKER2 Christopher LAPISH6 and Alexey KUZNETSOV2 and Harald H. SITTE1 1Department of Physics, Indiana University, Bloomington, IN, 1Department of Pharmacology, Medical University of Vienna, United States of America; 2Department of Mathematical Sciences, Austria; 2Department of Pharmaceutical Chemistry, University of Indiana University – Purdue University, Indianapolis, IN, United Vienna, Austria; 3Department of Cell and Regenerative Biology, States of America; 3Program in Neuroscience, Indiana University, School of Medicine and Public Health, University of Wisconsin, Bloomington, IN, United States of America; 4Institute of Applied Madison, WI, United States of America; 4Designer Drug Research Physics, Russian Academy of Sciences, Nizhny Novgorod, Russia; Unit, National Institute on Drug Abuse (NIDA), Baltimore, MD, 5Group of Neural Theory, INSERM U960 LNC, IEC, École Normale United States of America; 5Department of Medicinal Chemistry, Superieure PSL University, Paris, France; 6Addiction Neuroscience Virginia Commonwealth University, Richmond, VA, United States Program, Indiana University – Purdue University, Indianapolis, IN, of America United States of America *E-mail: [email protected] *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A18.11 Intrinsic Activity, 2016; 4(Suppl. 2): A18.10 http://www.intrinsicactivity.org/2016/4/S2/A18.11 http://www.intrinsicactivity.org/2016/4/S2/A18.10 Dopamine and serotonin transporters (DAT and SERT, respectively) In the ventral tegmental area (VTA), interactions between dopamine are members of the solute carrier 6 (SLC6) transporter family. Both (DA) and γ-aminobutyric acid (GABA) neurons are critical for are associated with a number of human disorders and therefore are regulating DA neuron activity, and thus DA efflux throughout the important targets for clinically relevant therapeutics. In addition, DAT brain. We explored how time-patterned activity of the inhibitory and SERT are directly involved in the action of and for psycho- network modulates the dynamics of DA neuron firing. For this stimulant drugs, including cathinones and their methylated deriva- purposes, we developed a local circuit model of the VTA, which is tives. Methcathinone (MCAT) and congeners are amphetamine-type based on feed-forward inhibition and recreates canonical features of drugs, which are characterized by high abuse potential. Evaluation of the VTA neurons. In particular, the DA neuron fires at relatively low a large set of analogs showed that adding a bulky chemical group to firing rates spontaneously and when driven by tonic AMPA receptor the 4-position of the phenyl ring can change the selectivity between activation and increases the frequency to the levels observed in DAT and SERT in a synaptosomal release assay [1]. In particular, the bursts following application of NMDA or pauses in GABA receptor serine in position 149 (S149) of the human DAT and the alanine in stimulation (by disinhibition). Our simulations challenge the classical position 169 (A169) in the human SERT were proposed to act as key view that GABA neurons exclusively reduce DA neuron firing and amino acids in the selectivity of these compounds [1, 2]. However, no bursting. We found that a synchronous GABAergic input enables an functional studies are present in the literature to corroborate this increase in DA neuron firing and bursting. Distinct from previous hypothesis. Sequence alignments indicate that S149 in hDAT is mechanisms, the increases were not based on a lowered firing rate homologous to A169 in hSERT. Therefore, we decided to generate of GABA neurons or weaker hyperpolarization by the GABA receptor mutations to exchange both amino acids and test whether this change synaptic current. A mechanism of GABA-mediated increases in firing swapped transporter selectivity among the following 4-MCATs: is based on a dynamic reduction of a long-lasting hyperpolarization MCAT, 4-OCH3MCAT, 4-BrMCAT, 4-CF3MCAT. A robust selectivity due to the SK-type Ca2+-dependent K+ current. Our model suggests profile is maintained between the rat and the human transporters: for that GABA-mediated hyperpolarization replaces the hyperpolarize- all the compounds tested, the selectivity conforms with previous data tion produced by the SK current; however, the pulsatile pattern of this obtained from a synaptosomal release assay [1]. The only exception inhibitory input allows the DA neuron to fire during the pauses. was seen with 4-OCH3MCAT suggesting that possible species Furthermore, we found that DA neuron bursting and transient DA differences can be present. Surprisingly, no difference was found release can be produced in the absence of bursty glutamatergic input, between the wild-type and mutant transporters for all the 4-MCATs if the neuron receives transiently synchronized GABA input. This tested, both in hDAT and hSERT suggesting that the S149 in hDAT suggests that rewarding stimuli need not be encoded in increases in and A169 in hSERT are, on their own, not sufficient to determine the afferent excitatory drive directly to DA neurons. The presented selectivity of 4-MCAT analogs and that other residues, or more likely data add to a number of studies that explored how synchronous a combination of residues, are involved. Our conclusion is supported activity of GABA interneurons alters the computational properties of by pharmacoinformatics which suggests alternative mutations that neural networks. The biological relevance of our modeling prediction can be combined with the mutations tested so far. Our study may was validated by calculating shuffle-corrected cross-correlograms provide important information for the synthesis of new therapeutic between pairs of simultaneously recorded putative VTA GABA and possibly more selective compounds.

References may be related more to behavioral effects of altered DA activity in 1. Bonano JS, Banks ML, Kolanos R, Sakloth F, Barnier ML, Glennon RA, associative regions mediating action-outcome contingencies than to Cozzi NV, Partilla JS, Baumann MH, Negus SS: Quantitative drug sensitivity per se. structure–activity relationship analysis of the pharmacology of Supported by PHS grant DA022433. para-substituted methcathinone analogues. Br J Pharmacol, 2015; References 172(10):2433–2444. doi:10.1111/bph.13030 1. Brewer RD, Swahn MH: Binge drinking and violence. JAMA, 2005; 2. Sakloth F, Kolanos R, Mosier PD, Bonano JS, Banks ML, Partilla JS, 294(5):616–618. doi:10.1001/jama.294.5.616 Baumann MH, Negus SS, Glennon RA: Steric parameters, molecular 2. Centers for Disease Control and Prevention (CDC): Vital signs: binge modeling and hydropathic interaction analysis of the drinking prevalence, frequency, and intensity among adults – pharmacology of para-substituted methcathinone analogues. United States, 2010. MMWR Morb Mortal Wkly Rep, 2012; 61(1): Br J Pharmacol, 2015; 172(9):2210–2218. doi:10.1111/bph.13043 14–19. Available from: http://www.cdc.gov/mmwr/preview/mmwrhtml/ mm6101a4.htm A18.12 3. Ichise M, Liow JS, Lu JQ, Takano A, Model K, Toyama H, Suhara T, Preliminary examination of the role of striatal dopamine Suzuki K, Innis RB, Carson RE: Linearized reference tissue 11 function in risk-taking behavior in binge drinkers parametric imaging methods: Application to [ C]DASB positron Lynn OSWALD1,*, Gary WAND2, Hiroto KUWABARA3, Dean WONG3 emission tomography studies of the serotonin transporter in and James BRAŠIĆ3 human brain. J Cereb Blood Flow Metab, 2003; 23(9):1096–1112. doi:10.1097/01.WCB.0000085441.37552.CA 1Department of Family and Community Health, University of 4. Stopper CM, Floresco SB: Contributions of the nucleus accumbens Maryland School of Nursing, Baltimore, MD, United States of and its subregions to different aspects of risk-based decision America; 2Department of Medicine, Johns Hopkins University making. Cogn Affect Behav Neurosci, 2011; 11(1):97–112. School of Medicine, Baltimore, MD, United States of America; doi:10.3758/s13415-010-0015-9 3Department of Radiology and Radiological Science, Johns 5. Palm S, Momeni S, Lundberg S, Nylander I, Roman E: Risk- Hopkins University School of Medicine, Baltimore, MD, United assessment and risk-taking behavior predict potassium- and States of America amphetamine-induced dopamine response in the dorsal striatum *E-mail: [email protected] of rats. Front Behav Neurosci, 2014; 8:236. Intrinsic Activity, 2016; 4(Suppl. 2): A18.12 doi:10.3389/fnbeh.2014.00236 http://www.intrinsicactivity.org/2016/4/S2/A18.12 6. Xiao L, Bechara A, Gong Q, Huang X, Li X, Xue G, Wong S, Lu ZL, Purpose: Binge drinking accounts for almost ¾ of US economic costs Palmer P, Wei Y, Jia Y, Johnson CA: Abnormal affective decision related to excessive alcohol consumption and is associated with making revealed in adolescent binge drinkers using a functional profound public health consequences [1, 2]. Nevertheless, surprising- magnetic resonance imaging study. Psychol Addict Behav, 2013; ly little is known about underlying biobehavioral mechanisms. In this 27(2):443–454. doi:10.1037/a0027892 study, we evaluated the role of striatal dopamine (DA) function in this 7. Worbe Y, Irvine M, Lange I, Kundu P, Howell NA, Harrison NA, behavior. Bullmore ET, Robbins TW, Voon V: Neuronal correlates of risk- Methods: Forty-four healthy M / F drinkers, ages 18–29 years, seeking attitudes to anticipated losses in binge drinkers. completed the Iowa gambling task (IGT) and underwent two 90-min Biol Psychiatry, 2014; 76(9):717–724. PET studies with [11C]raclopride (RAC). Twenty-one (M = 16) were doi:10.1016/j.biopsych.2013.11.028 classified as non-bingers and 23 (M = 13) were classified as bingers based on a history of at least one binge episode in the past year. The A18.13 first scan was preceded by injection of 10 ml 0.9 % NaCl; the second Neuronal correlates of goal-directed and habitual reward by 0.3 mg/kg amphetamine (AMPH). D2 receptor nondisplaceable seeking in dorsal striatum binding potential (BPND) was obtained by multilinear reference tissue Youna VANDAELE*, Jocelyn M. RICHARD and Patricia H. JANAK method (MRTM) [3]. ΔBP was estimated as % change from the ND Department of Psychological and Brain Sciences, Johns Hopkins placebo to the AMPH scan. Six volumes of interest (VOIs) were University, Baltimore, MD, United States of America defined: left (LvS) and right (RvS) ventral striatum, anterior (aPU) and *E-mail: [email protected] posterior (pPU) putamen, and anterior (aCN) and posterior (pCN) Intrinsic Activity, 2016; 4(Suppl. 2): A18.13 caudate nucleus. http://www.intrinsicactivity.org/2016/4/S2/A18.13 Results: The two groups did not differ by gender, age of first drink or first intoxication, sensation-seeking traits, or IGT performance. How- The balance between goal-directed and habitual response strategies ever, bingers consumed a greater quantity of alcohol than non- is necessary for efficient and flexible decision-making. An inability to bingers, 2.7 (3.2) vs. 0.46 (0.57) drinks / week (p = 0.003). ΔBPND was “break the habit” and re-engage in a goal-directed strategy when lower in bingers than in non-bingers in all VOIs, but differences were circumstances change may render the behavior impervious to not significant. The two groups also did not differ in baseline BPND or negative consequences, and could be involved in several psycho- subjective drug effects. Nevertheless, findings of regression analyses pathologies including alcoholism and substance use disorder. While adjusted for gender and drinks / week revealed significant interactions the dorsomedial striatum (DMS) has been implicated in goal-directed (p < 0.003) between IGT scores and binge drinking status in pre- behavior and the dorsolateral striatum (DLS) in the expression of dicting regional ΔBPND. Findings of subsequent stratified analyses habitual responding, the neural mechanisms and circuits controlling showed that greater risk-taking was associated with greater DA the shift between these two response strategies remain unclear. To responses in the aPU (p = 0.001) and aCN (p = 0.005) in bingers, address this question, we developed a new instrumental procedure whereas no relationship was found between IGT scores and ΔBPND allowing simultaneous in vivo single unit recording in the DMS and in non-bingers. DLS while rat’s lever pressing is either habitual or goal-directed. We Conclusion: Although preliminary, the findings are consistent with compared the neural activity in DMS and DLS during the acquisition evidence of DA involvement in risk-taking in rodents [4, 5] and of of the task, after overtraining, and during the satiety-induced altered corticolimbic brain activation during decision-making tasks in devaluation test, in order to disentangle the relative contribution of binge drinkers [6, 7]. The findings suggest that risks for binge drinking these structure in the transition between goal-directed and habitual

response strategies. Preliminary results suggest that the dynamic of A18.15 activity changes in DMS and DLS during learning correlate with shifts Ventral tegmental area dopamine neuron activity in a closed between goal-directed and habitual response strategies. economy and choice under chronic nicotine Marie Louise DONGELMANS*, Steve DIDIENNE, Samir TAKILLAH, A18.14 Nicolas TORQUET, Jérémie NAUDÉ and Philippe FAURE Rewarding behavior and decision-making processes in healthy Université Pierre et Marie Curie, Paris, France and lesioned Lewis and Fischer 344 rats *E-mail: [email protected] 1 2 1 2 Elena ESPA , Augusta PISANU , Silvia CORONGIU , Cristina CADONI Intrinsic Activity, 2016; 4(Suppl. 2): A18.15 1, and Sandro FENU * http://www.intrinsicactivity.org/2016/4/S2/A18.15 1Department of Biomedical Sciences, University of Cagliari, Italy; Mice were single-housed in operant boxes for the duration of the 2Institute of Neuroscience, National Research Council, Cagliari, Italy experiment while DA neuron activity was continuously recorded with *E-mail: [email protected] a polytrode implanted in the ventral tegmental area (VTA). Food Intrinsic Activity, 2016; 4(Suppl. 2): A18.14 pellets were delivered after lever presses under a fixed ratio 1 http://www.intrinsicactivity.org/2016/4/S2/A18.14 schedule. Mice were primarily nocturnally active, which was also Pramipexole (PPX) is a dopamine agonist widely used to alleviate reflected in a significant increase in bursting activity during the dark motor dysfunction in Parkinson’s disease (PD). Dopamine agonists vs. light period. The active lever in the operant box would either shift therapy can lead to behavioral disorders in those patients who have every 24 hours or every 4 days. In the first setting mice had to update reward-related personality trait or impulsive trait. These disorders their lever choice every 24 hours, which resulted in increased include impulse control disorders (ICDs) defined as a failure to resist exploration of both levers and a slow evolving choice discrimination an impulse, drive or temptation to perform an act that is harmful to overnight, while in the second setting mice learned to discriminate the self or others, and dopamine dysregulation syndrome (DDS), both levers and focused on the active lever. We demonstrate that an which involves drug craving, seeking and overuse which are increase in VTA DA activation between the lever press and reward observed in drug addiction. delivery (0.5 s) appeared when a lever was associated with rewards. In order to assess the existence of a correlation between impulsive- However, 24 hours was too short to update reward expectation and compulsive personality, Parkinson’s disease and the onset of ICDs therefore both levers evoked a response. When mice were chronical- or DDS, we studied in the addiction-prone Lewis (LEW) and the ly treated with nicotine (drinking water or minipump 10 mg/kg/day) addiction-resistant Fischer 344 (F344) inbred rat strains the reward- they displayed more exploitative behaviour by means of a significant ing effects of PPX in a conditioned place preference (CPP) paradigm increase in active lever presses and reduction in inactive lever after bilateral 6-hydroxydopamine lesions of striatal dorso-lateral presses. Next, we will apply electrophysiology to study the involve- DAergic terminals. Furthermore, we assessed the decision-making ment of VTA DA activity in this phenomenon. performance on rat Iowa gambling task (rIGT) in the same rat strains. The rIGT has been designed to model decision-making processes A18.16 through a conflict between immediate gratification and long-term Loss of PICK1 in mice causes hyperdopaminergia associated outcomes. with enhanced dopamine release and attenuated behavioral High amount of rewards (3 sugar pellets) was combined with the response to cocaine incurrence of a high amount of punishment (3 quinine pellets) in the Mattias RICKHAG1,*, Gunnar SØRENSEN1, Kathrine Louise JENSEN1, long-term disadvantageous choice (9 punishments out of 10 choices), Ditte DENCKER2, William Anthony OWENS3, Annika THOMSEN1, as opposed to the low amount of reward (1 sugar pellet) combined to Troels RAHBEK-CLEMMENSEN1, Mette RATHJE1, Chunyu JIN4, low amount of punishment (1 quinine pellet) in the long-term advanta- Birgitte HOLST4, Anders FINK-JENSEN2, Kenneth MADSEN1, geous one (2 punishment out of 10 choices) [1]. Lynette DAWS3 and Ulrik GETHER1 PPX (1 mg/kg, s.c.) was able to induce significant CPP both in healthy 1Molecular Neuropharmacology and Genetics Laboratory, University and lesioned LEW and F344 rats, without any significant difference of Copenhagen, Denmark; 2Laboratory of Neuropsychiatry, between strains. However, in the rIGT LEW rats, known to be more Psychiatric Center Copenhagen and Department of Neuroscience impulsive then F344 rats, either healthy or lesioned, showed a and Pharmacology, Faculty of Health and Medical Sciences, significant lower number of advantageous choices compared with University of Copenhagen, Denmark; 3University of Texas Health F344 rats. and Science Center at San Antonio, TX, United States of America; These results suggest that neither different behavioral phenotype nor 4Molecular Pharmacology Laboratory, Department of Neuroscience striatal DA terminals lesion affect PPX rewarding properties at least and Pharmacology, Faculty of Health and Medical Sciences, at this dose of PPX utilized. However, only the impulsive and reward- University of Copenhagen, Denmark sensitive trait may be a critical factor affecting the decision-making *E-mail: [email protected] process. Apparently DDS and ICDs may not share a common Intrinsic Activity, 2016; 4(Suppl. 2): A18.16 mechanism even if further studies will be needed to clarify this issue. http://www.intrinsicactivity.org/2016/4/S2/A18.16 Reference 1. van den Bos R, Lasthuis W, den Heijer E, van der Harst J, Spruijt B: Tuning dopaminergic signaling is critical for several physiological Toward a rodent model of the Iowa gambling task. Behav Res functions of the brain including motor behavior, reward and cognitive Methods, 2006; 38(3):470–478. doi:10.3758/BF03192801 function. The presynaptic plasma membrane dopamine transporter (DAT) sequesters dopamine from the extracellular space, and thereby sustains physiological dopamine levels. DAT contains at its C-terminus a PSD-95 / Discs-large / ZO-1 (PDZ)-domain binding sequence, shown to interact with the scaffolding protein, protein interacting with C-kinase 1 (PICK1). PICK1 regulates subcellular trafficking of its binding partners but the explicit role of PICK1 for DAT function and dopamine homeostasis has remained elusive. Here, we present a detailed investigation of the dopaminergic system in mice

lacking PICK1. Compared to wild-type (WT) mice, we found that DAT uptake is dependent on basal PKC activity. These findings surface expression was unaltered in striatal terminals from PICK1 demonstrate that His547 plays a crucial role in enhancing basal DA knock-out (KO) mice together with DAT localization to membrane transport and the Tat–DAT interaction. This study may provide novel microdomains. However, PICK1 KO mice were characterized by insights into identifying targets on DAT for therapeutic interventions, hyperlocomotion and attenuated locomotor response to cocaine improving DAT-mediated DA transmission of HAND in concurrent although unaltered postsynaptic dopamine receptor activation. cocaine abusers. Intriguingly, PICK1 KO mice showed increased levels of tyrosine Supported by NIH grant R01DA035714. hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis in striatum and elevated striatal dopamine content. Chronoampero- A18.18 metric recordings in striatum revealed elevated dopamine release in In vivo systems pharmacology of IRL752, a novel compound PICK1 KO mice supporting a hyperdopaminergic phenotype. PICK1 for treatment of BPSD and cognitive impairment in dementia, KO mice showed impaired behavioral sensitization to a cocaine- enhancing prefrontal synaptic activity by modulation of challenge and reduced chronic self-administration of cocaine sug- dopamine transmission gesting long-term maladaptive plasticity changes. Lentiviral knock- Susanna WATERS1,*, Clas SONESSON1, Peder SVENSSON1, down of PICK1 in midbrain dopaminergic cultures resulted in elevated Johan KULLINGSJO1, Joakim TEDROFF2 and Nicholas WATERS1 TH expression and thus imply that PICK1 acts as negative regulator 1Integrative Research Laboratories, Gothenburg, Sweden; 2Institute of dopamine synthesis. We infer that loss of PICK1 alters subcellular of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden distribution of TH in striatum leading to elevated striatal dopamine *E-mail: [email protected] content and development of a hyperdopaminergic phenotype. Intrinsic Activity, 2016; 4(Suppl. 2): A18.18 http://www.intrinsicactivity.org/2016/4/S2/A18.18 A18.17 Mutation of histidine 547 of human dopamine transporter In Alzheimer’s disease (AD) and other forms of dementia, impaired increases dopamine reuptake and attenuates HIV-1 cortical monoamine transmission is a core feature associated with Tat-induced inhibition of dopamine transporter progressive cognitive decline and behavioural and psychiatric Jun ZHU1,*, Pamela QUIZON1, Wei-Lun SUN1, Yaxia YUAN2, symptoms (BPSD). Available medications can improve cognition by Narasimha MIDDE1 and Chang-Guo ZHAN2 enhancing cortical synaptic activity, by increasing acetylcholine (ACh esterase inhibitors), or by low-affinity NMDA receptor antagonism 1Drug Discovery and Biomedical Sciences, University of South presumably acting extrasynaptically (memantine). Also, antipsychotic Carolina, Columbia, SC, United States of America; 2College of drugs are used, largely off-label, to control BPSD. The efficacy on Pharmacy, University of South Carolina, Columbia, SC, United cognitive functions is limited, and antipsychotics are associated with States of America severe side effects. Furthermore, there are indications that anti- *E-mail: [email protected] dopaminergic compounds may be detrimental with respect to Intrinsic Activity, 2016; 4(Suppl. 2): A18.17 phenotype and rate of decline in neurodegenerative disorders http://www.intrinsicactivity.org/2016/4/S2/A18.17 including AD. Estimated thirty-four million people worldwide are living with HIV. IRL752 was discovered using a phenotypic screening approach, More than 30–50 % of HIV-1-positive individuals suffer from neuro- based on broad data arrays generated in vivo (gene expression, logical complications collectively referred to as HIV-1-associated monoamine biomarkers, behaviour). The in vivo profile was bench- neurocognitive disorders (HAND). Abnormal dopaminergic trans- marked against an in-house database covering most CNS compound mission has been implicated as a risk determinant of HAND. Human classes. IRL752 displays a novel profile, with region-selective dopamine (DA) transporter (hDAT) activity is strikingly reduced in enhancement of cortical dopamine (DA) and norepinephrine (NE), HIV-1-infected cocaine-using patients, correlating with the severity of and net effects in vivo addressing pathological dysregulations in HIV-1-associated cognitive deficits. HIV-1 transactivator of transcrip- multiple transmitter systems affected in dementia. IRL752 induces tion (Tat) protein and cocaine synergistically increase synaptic DA Arc mRNA in the frontal cortex, limbic and striatal areas. The effect levels by directly inhibiting DAT activity, ultimately leading to on cortical Arc is interpreted as enhanced synaptic activity, secondary dopaminergic neuron damage. Therefore, an intervention for HIV to increased DA or NE levels leading to downstream activation of DA infection-induced dysfunction of DA systems has the potential to D1 and / or α receptors. The increase of subcortical Arc is presumably improve neurocognitive function in patients with the early stage of related to activation of cortico-striatal pathways. The Arc expression HAND. Through integrated computational modeling prediction and profile by IRL752 resembles that of memantine. IRL752 also induces experimental validation, we have identified key residues in DAT with a modest increase in cortical ACh. Behaviourally, IRL752 does not which Tat interacts, which are critical for Tat-induced inhibition of affect motor activity patterns in normal rats, and does not enhance DAT and transporter conformational transitions. This study investi- locomotor activity under the influence of psychostimulants. It gated the functional influences of mutations of histidine 547 (H547A) reverses tetrabenazine-induced hypomotility, indicating efficacy in and its associated residues tyrosine 548 and tyrosine 551 of human conditions with impaired dopamine transmission, and hence potential DAT in basal DA transport and Tat-induced inhibition of DA transport. to improve motor function in dementia where mild parkinsonian Compared to wild-type human DAT, H547A-hDAT displayed a 197 % features are frequently observed. IRL752 reverses deficits in novel increase in the Vmax with no change in the Km. The increased Vmax in object recognition in a subchronic PCP model, suggesting potential H547A was not accompanied by change in DAT surface expression. to enhance short-term memory. Furthermore, IRL752 reverses

Results from other substitutions of His547 show that the Vmax was not MK-801-induced hyperactivity, suggesting antipsychotic properties. altered in H547R but decreased by 99 % in H547P and 60 % in Main receptor level targets involved in the effects of IRL752 are

H547D, respectively. Importantly, Tat-induced inhibition of DA 5-HT7, α2 and σ1 receptors. IRL752 is currently in phase I. transport observed in wild-type hDAT was attenuated in H547A, H547R and H547D. PMA, a PKC activator, produced a 40 % inhibition of Vmax in wild-type DAT and 61 % in H547A, whereas BIM, a PKC inhibitor, increased Vmax in wild-type and H547A by 198 % and 142 %, respectively. This indicates that H547A-induced increase in DA

A18.19 The development of molecular detection that allows rapid responses Neuromelanin-sensitive MRI as a proxy measure of dopamine with high sensitivity and selectivity remains challenging. Here, we function in neuropsychiatric illness demonstrate the strategy of novel bio-nanotechnology to successfully Clifford CASSIDY1,*, Luigi ZECCA2, Fabio A. ZUCCA2, Jodi WEINSTEIN1, fabricate a high-performance dopamine (DA) biosensor using DA Emanuele FERRARI2, Seth BAKER1, Caridad BENAVIDES1, Un KANG3, receptor-containing uniform-particle-shaped nanovesicles-immobi- David SULZER3, Anissa ABI-DARGHAM1 and Guillermo HORGA1 lized carboxylated poly(3,4-ethylenedioxythiophene) (CPEDOT) NTs (DRNCNs). DA molecules are commonly associated with serious 1Department of Psychiatry, Columbia University, New York, NY, diseases, such as Parkinson’s and Alzheimer’s diseases. For the first United States of America; 2Institute of Biomedical Technologies – time, nanovesicles containing a human DA D receptor (hD R) were CNR, Segrate (Milano) Italy; 3Department of Neurology, Columbia 1 1 successfully constructed from HEK 293 cells stably expressing hD R. University, New York, NY, United States of America 1 The nanovesicles containing hD R as gate-potential modulator on the *E-mail: [email protected] 1 conducting polymer (CP) nanomaterial transistors provided high- Intrinsic Activity, 2016; 4(Suppl. 2): A18.19 performance responses to DA molecule owing to their uniform, http://www.intrinsicactivity.org/2016/4/S2/A18.19 monodispersive morphologies and outstanding discrimination ability. Neuromelanin-sensitive magnetic resonance imaging (NM-MRI) Specifically, the DRNCNs were integrated into a liquid-ion-gated purports to detect the content of neuromelanin, a product of field-effect transistor (FET) system via immobilization and attachment dopamine metabolism found in the neurons of substantia nigra (SN). processes, leading to high sensitivity and excellent selectivity toward Neuromelanin formation rescues neurons from toxicity caused by DA in liquid state. Unprecedentedly, the minimum detectable level excess dopamine. NM-MRI has proven to be very effective at (MDL) from the field-induced DA responses was as low as 10 pM in detecting neurodegeneration of dopamine neurons in Parkinson’s real-time, which is 10 times more sensitive than that of previously disease but its utility as a marker of dopamine function in non- reported CP-based DA biosensors. Moreover, the FET-type DRNCN neurodegenerative conditions is unclear, making its application to biosensor had a rapid response time (< 1 s) and showed excellent other neuropsychiatric illness dubious. selectivity in human serum. We performed NM-MRI scans on 20 healthy controls, 7 patients with Parkinson’s disease, 20 patients with schizophrenia, 10 individuals at A18.21 risk of schizophrenia and 3 post-mortem sections of human midbrain. Dynamic updating of choice strategy by dopamine reward Dopamine release capacity in the striatum was estimated for 17 of prediction errors these subjects based on amphetamine-induced displacement of the Emily HUESKE1,*, Hae Yoon JUNG1, Gishnu DAS1, Corey PURYEAR2, 11 radiotracer [ C]raclopride, with positron emission tomography (PET). Timothy O’CONNOR1, Vinod RAO3, Naoshige UCHIDA4 and Susumu To validate NM-MRI as a neuromelanin-sensitive measure, tissue TONEGAWA1 concentrations of neuromelanin were estimated in post-mortem 1Massachusetts Institute of Technology, Cambridge, MA, United samples using spectrophotometry. States of America; 2Forum Pharmaceuticals, Waltham, MA, USA; NM-MRI signal variation in different regions of a post-mortem 3Department of Psychiatry, Massachusetts General Hospital, midbrain sample was highly correlated to neuromelanin concentration Boston, MA, USA; 4Department of Molecular and Cellular Biology, in the same regions (Pearson r = 0.81, p = 0.0004). Consistent with Harvard University, Cambridge, MA, United States of America prior reports, we observed a profound reduction in the NM-MRI signal *E-mail: [email protected] (contrast-to-noise ratio in the whole SN) in patients with Parkinson’s Intrinsic Activity, 2016; 4(Suppl. 2): A18.21 disease compared to controls (t16 = 4.5, p = 0.0003). Voxelwise http://www.intrinsicactivity.org/2016/4/S2/A18.21 analysis within the SN found a cluster in which NM-MRI signal intensity was positively correlated to striatal dopamine release It has long been observed that recent reward history can bias capacity (p < 0.05, uncorrected). Similar voxelwise analyses examin- decisions even in perceptual decisions when a sensory stimulus is ing clinical groups identified an SN cluster where patients with the sole arbiter of current choice. In a perceptual decision-making schizophrenia had higher NM-MRI signal than matched controls task, we observe a trial-by-trial influence of prior rewards, and this (p < 0.05, uncorrected) and a partially overlapping cluster was influence of prior outcome is further modulated by a novel decision observed to have higher signal in individuals at risk of schizophrenia variable, an influence of prior decision difficulty. In other words, when compared to controls (p < 0.05, uncorrected). the animal is rewarded for difficult decisions, its subsequent decisions These preliminary data indicate that NM-MRI indeed appears to be are more biased than following easy rewards. Given the reward- sensitive to neuromelanin content, even in the absence of neuro- dependent and graded nature of the phenomenon, we hypothesized degeneration. The method shows promise as an imaging tool in that phasic dopamine neuron activity is updating the decision neuropsychiatric illness since it may be able to capture interindividual boundary differentially according to prior decision difficulty. Record- variability in dopamine system function and psychopathology; ing from dopamine neurons during revealed that reward responses however, further work is needed to confirm this. are modulated according to trial difficulty in accordance with reward prediction error-like signaling. In order to determine if dopamine plays A18.20 a causal role in this phenomenon, we selectively perturbed phasic Human dopamine receptor nanovesicles for gate-potential dopamine activity by knocking out NMDA receptors in dopamine modulators in high-performance field-effect transistor neurons, a model that spares baseline firing rates and thus motor biosensors function; we found a lack of influence of prior difficulty in mice with Oh Seok KWON* reduced phasic dopamine signaling demonstrating a causal role for dopamine in this phenomenon. BioNano Research Center, Korea Research Institutes for Bioscience and Biotechnology, Daejeon, Republic of Korea *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A18.20 http://www.intrinsicactivity.org/2016/4/S2/A18.20

A18.22 A18.23 (S)-ketamine increases prefrontal cerebral blood flow but not The serotonin innervation of the striatum is significantly glutamate levels in anterior cingulate cortex in healthy increased in parkinsonian monkeys volunteers Dave GAGNON, Therese DI PAOLO and Martin PARENT* 1, 1 Kirsten B. BOJESEN *, Kasper A. ANDERSEN , Sophie N. Department of Psychiatry and Neuroscience, Université Laval, 1 1 2 RASMUSSEN , Lone BAANDRUP , Line M. MADSEN , Birte Québec, QC, Canada 1 3 1 Y. GLENTHØJ , Egill ROSTRUP and Brian V. BROBERG *E-mail: [email protected] 1Center for Neuropsychiatric Schizophrenia Research (CNSR), Intrinsic Activity, 2016; 4(Suppl. 2): A18.23 University of Copenhagen, Denmark; 2Department of Anaesthesia, http://www.intrinsicactivity.org/2016/4/S2/A18.23 University Hospital Copenhagen, Glostrup, Denmark; 3Functional Chronic use of L-DOPA to treat Parkinson’s disease often produces Imaging Unit, Department of Clinical Physiology and Nuclear adverse side effects, such as L-DOPA-induced dyskinesia. Un- Medicine, Copenhagen University Hospital, Glostrup, Denmark regulated release of dopamine by serotonin (5-HT) axons following *E-mail: [email protected] L-DOPA administration is a major presynaptic determinant of dys- Intrinsic Activity, 2016; 4(Suppl. 2): A18.22 kinesia. Our study was designed to characterize the reorganization of http://www.intrinsicactivity.org/2016/4/S2/A18.22 5-HT striatal afferent projections following dopaminergic denervation Background: Glutamatergic disturbances might be more pro- in a primate model of Parkinson’s disease. Eight cynomolgus nounced than dopaminergic dysfunction in a subgroup of patients monkeys were used. Four received MPTP and four served as with schizophrenia. Increased levels of glutamate can cause loss of controls. The state of striatal 5-HT and dopamine innervation was brain tissue seen in some patients, either due to glutamate-mediated evaluated by unbiased stereological approach using immunohisto- excitotoxicity or alterations in resting cerebral blood flow (rCBF). chemistry. Our investigation revealed a 34 % increase in the number However, the relationship between levels of glutamate and rCBF is of 5-HT axon varicosities in the dorsolateral putamen of MPTP largely unknown in humans. monkeys compared to controls. This increase was particularly Ketamine is a non-competitive N-methyl-D-aspartate receptor anta- obvious in the sensorimotor territory of the striatum, where the gonist that transiently induces schizophrenia-like symptoms in male dopamine denervation was the most severe. Electron microscopic healthy volunteers (HV) as well as increased cortical glutamate levels examination of the dorsolateral putamen showed that the SERT+ and rCBF alterations in some studies. Here, we used (S)-ketamine axon varicosities established about twice as many synaptic contacts infusion in HV to test the hypothesis that increased levels of in MPTP monkeys than in controls. No significant difference in the glutamate in anterior cingulate cortex (ACC) precede rCBF altera- density of TpH+ cell bodies between MPTP and control monkeys was tions. observed. Our findings demonstrate the highly plastic nature of 5-HT Methods: Twenty-five healthy male volunteers (age 25.4 ± 3.3) striatal afferent projections, a feature that becomes obvious in the received constant infusion of 0.375 mg/kg (S)-ketamine over 40 min absence of dopamine. Although the number of dorsal raphe neurons during a magnetic resonance scan at 3 Tesla. Five sequential remains constant in parkinsonian monkeys, their ascending axonal acquisitions of [1H] magnetic resonance spectroscopy in ACC and projections undergo marked proliferative and synaptic adaptive whole brain pseudo-continuous arterial spin labelling were obtained changes that might play a significant role in the expression of before, during, and after (S)-ketamine infusion to measure the time L-DOPA-induced dyskinesia. course of glutamate levels and rCBF. Psychopathology was assessed with Positive and Negative Syndrome Scale (PANSS). A18.24 Results: (S)-ketamine significantly induced a transient increase in all Investigating the associations of flow proneness with the DRD2 PANSS sub scores and rCBF in prefrontal cortex and ACC, but C957T polymorphism and attentional control unexpectedly not in glutamate and glx (glutamate + glutamine) levels Máté GYURKOVICS1,*, Eszter KÓTYUK1, Enikő Rózsa KATONAI1, in ACC. Erzsébet Zsófia HORVÁTH2, Andrea VERECZKEI2, David A. BALOTA3 Discussion: Although the levels of glutamatergic metabolites were and Anna SZÉKELY1 unaffected by (S)-ketamine, we speculate if the increase in rCBF 1Institute of Psychology, Eötvös Loránd University, Hungary; reflects increased glutamatergic activity. Glutamate plays a key role 2Department of Medical Chemistry, Molecular Biology and in the regulation of rCBF and the majority of neurons in cortex are Pathobiochemistry, Semmelweis University, Budapest, Hungary; glutamatergic. Levels of glutamatergic metabolites prior to ketamine 3Department of Psychology, Washington University, St. Louis, MO, infusion were significantly higher compared to findings in another United States of America group of 14 male HV scanned on the same scanner as part of another *E-mail: [email protected] study. Thus, increased baseline levels of glutamatergic metabolites Intrinsic Activity, 2016; 4(Suppl. 2): A18.24 might have caused the ketamine-induced increase to be below the http://www.intrinsicactivity.org/2016/4/S2/A18.24 detection-limit of 10 % for within-subject differences on the scanner used in this study. Flow is a mental state that occurs when one devotes their fullest Further studies are needed to clarify the relationship between attention to a challenging task. It is characterized by deep, yet effort- glutamatergic metabolites and rCBF alterations and to elucidate if less concentration, and often a distortion of sense of time [1]. ketamine challenge mimics the pathophysiology of a ‘non-dopamin- Proneness to experience such a state in everyday life has been ergic’ subgroup of patients with primarily glutamatergic disturbances. shown to be moderately heritable [2]; however, to date there have been no empirical attempts to determine which genes affect this phenotype. Our study is the first such attempt. As flow is a highly rewarding experience, genetic variations of the dopamine neurotransmitter system are potentially relevant. A recent PET-scan study linked

flow proneness to dopamine D2 receptor availability in the striatum [3], and according to Hirvonen et al. [4, 5] the DRD2 C957T (rs6277)

polymorphism influences striatal dopamine D2 receptor availability.

Thus, we investigated whether individual differences in flow frequen- Experimental and Clinical Pharmacology and Toxicology, University cy during everyday mandatory and leisure activities are linked to the of Lübeck, Germany; 4Department of Molecular Neuropharma- DRD2 C957T polymorphism. cology, Institute of Pharmacology, Polish Academy of Sciences, Using the Flow Proneness Questionnaire [6] in a sample of 236 Kraków, Poland; 5Department of Neurology, Beth Israel Deaconess healthy Hungarian adults, we found that CC homozygotes reported Medical Center and Harvard Medical School, Boston, MA, United higher flow frequency during mandatory activities (i. e. studying and States of America working) as compared to T-allele carriers (β = −0.19, p < 0.005). The *E-mail: [email protected] T allele has been shown to be associated with higher self-reported Intrinsic Activity, 2016; 4(Suppl. 2): A18.25 impulsivity and lower response inhibition efficiency in a stop-signal http://www.intrinsicactivity.org/2016/4/S2/A18.25 paradigm [7]. Preliminary results from our ongoing studies also Systemic inflammation causes malaise and general feelings of suggest a negative correlation between flow proneness and self- discomfort. This fundamental aspect of the sickness response reported attentional impulsivity. Associations between flow prone- reduces the quality of life for people suffering from chronic ness and attentional control are also investigated using behavioral inflammatory diseases and is a nuisance during mild infections like performance on a Go / No-Go task as the indicator of the latter the common cold or the flu. To investigate how inflammation is construct. perceived as unpleasant and causes negative affect, we used a The theoretical implications of our results (e. g. an attention control behavioral test based on Pavlovian conditioning (conditioned place model of flow, a potential gene × environment interaction) are dis- aversion) where mice avoid an environment which they have learned cussed, and the necessity to replicate our findings using a different to associate with inflammation-induced discomfort. By using a combi- measure of flow proneness is also addressed. nation of cell-type-specific gene deletions, pharmacology and chemo- The present work was supported by the Hungarian Scientific genetics, we examined the role of specific signaling molecules and Research Fund (OTKA K100845) and the postgraduate scholarship neural circuits. We demonstrate that systemic inflammation induced awarded to M. Gy. by the Hungarian-American Fulbright Commission. by lipopolysaccharide (derived from E. coli) or interleukin-1β triggers References aversion through MyD88-dependent activation in brain endothelial 1. Nakamura J, Csikszentmihalyi M: Catalytic creativity. The case of cells followed by cerebral prostaglandin E synthesis mediated by Linus Pauling. Am Psychol, 2001; 56(4):337–441. 2 cyclooxygenase 1. Furthermore, we found that inflammation-induced doi:10.1037/0003-066X.56.4.337 prostaglandin E targets EP receptors on striatal dopamine D 2. Mosing MA, Pedersen NL, Cesarini D, Johannesson M, Magnusson 2 1 1 receptor-expressing neurons and that this signaling sequence PK, Nakamura J, Madison G, Ullén F: Genetic and environmental induces aversion through GABA-mediated inhibition of dopaminergic influences on the relationship between flow proneness, locus of cells. DREADD G -mediated activation of dopaminergic cells in the control and behavioral inhibition. PLoS One, 2012; 7(11):e47958. q midbrain prior to the LPS challenge abolished the inflammation- doi:10.1371/journal.pone.0047958 induced aversion, suggesting that a decrease in dopaminergic firing 3. de Manzano Ö, Cervenka S, Jucaite A, Hellenäs O, Farde L, Ullén F: is the direct cause of LPS-triggered aversion. Collectively, these Individual differences in the proneness to have flow experiences findings demonstrate that prostaglandin E -mediated modulation of are linked to dopamine D2-receptor availability in the dorsal 2 the dopaminergic motivational circuitry is a key mechanism behind striatum. Neuroimage, 2013; 67:1–6. the negative affect induced by inflammation. doi:10.1016/j.neuroimage.2012.10.072 4. Hirvonen M, Laakso A, Någren K, Rinne JO, Pohjalainen T, Hietala J: C957T polymorphism of the dopamine D2 receptor (DRD2) gene A18.26 affects striatal DRD2 availability in vivo. Mol Psychiatry, 2004; The disease-causing mutation G108Q destabilizes the 9(12):1060–1061. doi:10.1038/sj.mp.4001561 Drosophila dopamine transporter in molecular dynamic 5. Hirvonen M, Laakso A, Någren K, Rinne JO, Pohjalainen T, Hietala J: simulations 1, 1 2 Corrigendum: C957T polymorphism of the dopamine D2 receptor Daniel SZÖLLŐSI *, Ameya KASTURE , Ali EL-KASABY , 1 3 (DRD2) gene affects striatal DRD2 availability in vivo. Mol Hafiz Muhammad Mazhar ASJAD , Alexandra GRIMM , 3 1 1 Psychiatry, 2005; 10(9):889. doi:10.1038/sj.mp.4001707 Thomas HUMMEL , Michael FREISSMUTH , Sonja SUCIC 1 6. Ullén F, de Manzano Ö, Almeida R, Magnusson PKE, Pedersen NL, and Thomas STOCKNER Nakamura J, Csíkszentmíhalyi M, Madison G: Proneness for 1Institute of Pharmacology, Medical University of Vienna, Austria; psychological flow in everyday life: associations with personality 2Department of Pharmacology, Faculty of Veterinary Medicine, and intelligence. Personality and Individual Differences, 2012; Mansoura University, Mansoura, Egypt; 3Department of 52(2):167–172. doi:10.1016/j.paid.2011.10.003 Neurobiology, University of Vienna, Austria 7. Colzato LS, van den Wildenberg WP, Van der Does AJ, Hommel B: *E-mail: [email protected] Genetic markers of striatal dopamine predict individual Intrinsic Activity, 2016; 4(Suppl. 2): A18.26 differences in dysfunctional, but not functional impulsivity. http://www.intrinsicactivity.org/2016/4/S2/A18.26 Neuroscience, 2010; 170(3):782–788. The dopamine transporter (DAT) is a member of the solute carrier 6 doi:10.1016/j.neuroscience.2010.07.050 (SLC6) protein family. Its physiological role is to remove dopamine from the synaptic cleft, thereby terminating neurotransmission. A18.25 Several psychiatric disorders ranging from schizophrenia to attention- Inflammation-induced aversion is elicited by prostaglandin- deficit hyperactivity disorder (ADHD) originate at least partially from dependent modulation of dopaminergic neurotransmission DAT malfunction. Several point mutations distributed at the proteins– 1, 1 1 Michael FRITZ *, Anna M. KLAWONN , Anna NILSSON , Anand Kumar membrane interface have been identified in patients suffering from 1 1 1 SINGH , Joanna ZAJDEL , Daniel BJÖRK WILHELMS , Michael infantile dystonia / Parkinson’s disease. One of these mutation is the LAZARUS2, Markus SCHWANINGER3, Jan RODRIGUEZ PARKITNA4, G140Q mutation. The homologous mutation (G108Q) in Drosophila 5 1 1 Clifford B. SAPER , Anders BLOMQVIST and David ENGBLOM melanogaster causes a sleepless phenotype and shows a lack of 1Department of Clinical and Experimental Medicine (IKE), Linköping DAT surface expression. To analyze how the mutation abolishes University, Linköping, Sweden; 2International Institute for Integrative protein function (e. g. folding mutant, trafficking mutant) we started Sleep Medicine, University of Tsukuba, Japan; 3Institute of molecular dynamics simulations with the available fruit fly crystal

structure (PDB ID: 4XP1) of wild-type and the G108Q mutant. Reference A homology model of dDAT (WT and G108Q) based on the PDB ID: 1. Hoerbelt P, Lindsley TA, Fleck MW: Dopamine directly modulates

4XP1 was built using MODELLER. The best selected models were GABAA receptors. J Neurosci, 2015; 35(8):3525–3536. inserted into a pre-equilibrated POPC membrane. We also ex- doi:10.1523/JNEUROSCI.4390-14.2015 changed the residue type 108 between glycine and glutamine in all six systems after membrane insertion (resulting in a total of 12 A18.28 simulations) to rule out the effects of subtle differences among the Rescuing dysfunctional dopamine transporter trafficking: starting systems. Every system was independently equilibrated and a role for the non-receptor tyrosine kinase, Ack1 MD simulations were carried for 100 ns. We found that the C-terminal Sijia WU1, Karl BELLVE2, Kevin E. FOGARTY2 and Haley MELIKIAN1,* part of TM12 was pushed away from TM3 in the simulations that 1Department of Psychiatry / BNRI, University of Massachusetts carried the G108Q mutation. The six simulations harbouring the wild- Medical School, Worcester, MA, United States of America; type glycine residue showed lower RMSD throughout the simulations 2Biomedical Imaging Group, Program in Molecular Medicine, and also relative to each other, indicating a stable conformation of University of Massachusetts Medical School, Worcester, MA, TM12 in contrast to the G108Q mutant showing higher RMSD values. United States of America These movements indicate that TM12 fails to adopt a stable con- *E-mail: [email protected] formation in G108Q due to the presence of glutamine. Analysis of the Intrinsic Activity, 2016; 4(Suppl. 2): A18.28 distribution of water indicated a higher water density in the vicinity of http://www.intrinsicactivity.org/2016/4/S2/A18.28 residue 108 in the G108Q mutants which also contributes to the destabilization effect. The observed changes in the dynamics of the The dopamine (DA) transporter (DAT) facilitates high-affinity protein as well as the modified micro environment due to the mutation presynaptic DA reuptake that temporally and spatially shapes DA suggest that G108Q cannot adopt a stable conformation indicating a neurotransmission. DAT is the primary target for addictive and folding deficiency. therapeutic psychostimulants and the rewarding properties of these This work was supported by project program grant SFB35-10, grant drugs is strictly dependent upon their binding to DAT. Recent genetic no. P27518-B27 and funded by the Austrian Science Fund FWF, as studies have identified a variety of DAT coding variants in patients well as by the doctoral program ‘Cell Communication in Health and presenting with attention-deficit hyperactivity disorder (ADHD), Disease’ and the Medical University of Vienna. infantile parkinsonism and autism spectrum disorder. While many of these coding variants result in anomalous dopamine efflux via DAT, A18.27 some identified DAT mutants exhibit membrane trafficking dys-

Dopamine effects on GABAA receptor subtypes: searching function that perturbs DAT plasma membrane stability. Thus, under- for the binding sites standing the mechanisms that facilitate DAT surface expression and Margot ERNST*, Philip SCHMIEDHOFER, Marco TREVEN and stability may shed light onto how these mutations impact normal DA Konstantina BAMPALI neurotransmission and may offer possible opportunities for therapeutic intervention. Wild-type DAT surface expression is acutely Department of Molecular Neurosciences, Medical University of modulated by dynamic membrane trafficking at the presynaptic Vienna, Austria terminal and a PKC-sensitive negative endocytic mechanism, or *E-mail: [email protected] “endocytic brake”, controls DAT plasma membrane stability. Interest- Intrinsic Activity, 2016; 4(Suppl. 2): A18.27 ingly, the ADHD DAT coding variant R615C lacks influence from the http://www.intrinsicactivity.org/2016/4/S2/A18.27 endocytic brake and exhibits profound plasma membrane instability.

Dopamine was found recently to act directly on GABAA receptors in However, the molecular basis for the DAT endocytic brake is not cultured striatal neurons [1]. In recombinant GABAA receptors of known, nor is it known whether this braking mechanism is unique to different subunit composition, dopamine exerts different effects on DAT or common to monoamine transporters. Here, we report that the GABA-elicited currents: In some subtypes, dopamine enhances cdc42-activated, non-receptor tyrosine kinase, Ack1, is an endocytic GABA currents, while in others it reduces them. A small population of brake that stabilizes DAT at the plasma membrane and is released in

GABAA receptor subtypes can even be gated by dopamine [1]. Thus, response to PKC activation. Pharmacologic and shRNA-mediated dopamine can modulate and produce chloride currents in GABAA Ack1 silencing enhanced basal DAT internalization and blocked PKC- receptors. It may affect receptors either at postsynaptic or at extra- stimulated DAT internalization, but had no effects on SERT endo- synaptic sites, or both. The receptor binding sites by which these cytosis. Both cdc42 activation and PKC stimulation converge to effects are elicited are unknown. The physiological role of this control Ack1 activity and DAT membrane stability, and Ack1 inactiva- completely novel mechanism of dopamine signaling by direct action tion is required for stimulated DAT internalization in response to PKC on ligand gated chloride channels in mammalian brains is also activation. Moreover, constitutive Ack1 activation is sufficient to unknown. rescue the gain-of-function endocytic phenotype exhibited DAT In the present study, dopamine is tested on an extended panel of R615C. These findings reveal a unique endocytic control switch that

GABAA receptor subtypes to achieve a more complete understanding is highly specific for DAT. Moreover, the ability to rescue the DAT of subtype selectivity of the effect and to obtain first hints towards the R615C coding variant suggests that manipulating DAT trafficking binding sites that dopamine uses to either allosterically modulate or mechanisms may be a potential therapeutic approach to correct DAT to activate them. Structural models of invertebrate dopamine-gated coding variants that exhibit trafficking dysregulation. chloride channels are compared with those of dopamine-sensitive

GABAA receptors. Site-directed mutagenesis is aimed at testing binding-site hypotheses derived from the structural studies. Under- standing the molecular mechanism behind the dopamine effect on

GABAA receptors will lead to the identification of novel tools, such as antagonists of these sites, which will help in delineating the physiological role of this crosstalk between transmitter systems.

A18.29 Dopamine (DA) transmission plays multiple roles in movement AKTing / acting up in the dopamine hypothesis of psychosis initiation and execution, reward signaling, cognition, and motivation. and probability-based decision-making: new insights and links It is well-established that DA is released from terminals of dopamin- from Akt1 mutant mice and schizophrenic patients ergic neurons (DANs) in dorsal striatum, nucleus accumbens, Wen-Sung LAI1,*, Ching CHEN1, Da-Zhong LUO1, Hung-Hsiang LIU1, prefrontal cortex, and many other brain regions. However, whether, Chia-Tzu LI1, Yao-Chu CHEN1, Chih-Ming LIU2 and Ming-Hsien how, when and to what physiological relevance DA is released via HSIEH2 vesicular exocytosis from the somata of DANs in the substantia nigra (SN) remains elusive. Here, we reveal depolarization-evoked Ca2+- 1Department of Psychology, National Taiwan University, Taipei, dependent somatic DA release from single vesicles in SN slices using Taiwan; 2Department of Psychiatry, National Taiwan University combined recordings of path clamp and amperometry of electro- Hospital, Taipei, Taiwan chemical micro-carbon-fiber electrodes for the first time. On average, *E-mail: [email protected] 7.8 ×104 DA molecules were released per vesicle. The latency of the Intrinsic Activity, 2016; 4(Suppl. 2): A18.29 evoked DA release was 340 ms, or 44 times slower in the somata of http://www.intrinsicactivity.org/2016/4/S2/A18.29 DANs than axonal terminal release in the striatum. The D2 receptor Abnormalities in the dopamine (DA) system have long been imply- was expressed in SNc neural somata, and served as auto-receptor cated in the explanation of schizophrenia / psychosis, and the DA D2 to inhibit cell activity. Thus, the longer latency of somatic DA release receptor continues to be the key target for many antipsychotics. AKT permits strong modulation of action potentials patterns through its (protein kinase B) is a key signaling intermediate downstream of DA autoreceptors in DA somata versus terminals.

D2 receptors, and its signaling cascade is important for the expression Supported by grants from NSFC, MOST and DOE 2011 program. of DA-associated behaviors. Accumulating evidence from human genetic studies suggests AKT1 is one of susceptibility genes for A18.31 schizophrenia and stimulation of PI3-kinase / AKT signaling pathway Optical control of midbrain dopamine neurons using a light- might be related to delusional ideation. “How does genetic deficit lead inhibited nicotinic receptor to DA dysfunction and psychotic phenomena?” is a million-dollar Romain DURAND-DE CUTTOLI1,*, Fabio MARTI1, Damien LEMOINE1, question. It was proposed that dysregulation of DA systems could Dirk TRAUNER2, Richard KRAMER3, Uwe MASKOS4, Philippe FAURE1 alter the appraisal of stimuli through a process of aberrant salience and Alexandre MOUROT1 and eventually lead to psychosis. Accordingly, the assessment of 1Neuroscience Paris Seine, Université Pierre et Marie Curie, Paris, reward prediction error in decision-making could provide a potential France; 2Department of Chemistry, Ludwig Maximilians University behavioral index for dopaminergic activity in the brain that allows for Munich, Germany; 3Department of Molecular and Cell Biology, the evaluation of psychosis. Taking advantage of probability-based University of California, Berkeley, CA, United States of America; decision-making and model-fitting, we tackled this issue in Akt1 4Department of Neurosciences, Institut Pasteur, Paris, France mutant mice and schizophrenic patients. In the mouse studies, we *E-mail: [email protected] examined the role of Akt1 in the regulation of DA sensitivity, Intrinsic Activity, 2016; 4(Suppl. 2): A18.31 motivational salience, and decision-making. We found that (1) Akt1 http://www.intrinsicactivity.org/2016/4/S2/A18.31 mutant mice revealed a sex- and region-specific effect in the regulation of DA-dependent behaviors and methamphetamine sensitivity; Dopamine (DA) neurons of the ventral tegmental area (VTA) show (2) mutant mice normally attributed their motivational salience to the two different firing patterns: a regular pattern associated with a tonic stimulus but they updated their reward values more rapidly and have (constant and low) release of DA in the target structures and a “burst” more exploratory decisions than controls in the probabilistic reward- pattern associated with a phasic release of DA and involved in ing task; (3) age-specific effects of Akt1 on the regulation of striatal reinforcement learning and reward prediction. Previous studies in our DA D2 receptor activity using micro-PET scan; and (4) alterations of laboratory have identified the β2-containing nicotinic acetylcholine neural oscillations activity and event-related potentials in the striatum receptor (β2*nAChR) as a major player of the bursting activity: of Akt1 mutant mice during decision-making. In a similar vein, we nicotine injection switches the activity of the DA neuron from tonic to developed a probabilistic rewarding task for schizophrenic patients phasic, while deletion of the β2 subunit results in DA neurons that and found that patients with low and high psychosis show higher only fire tonically and don’t respond to nicotine. Assessing the precise learning rates and more exploratory decisions as we reported in Akt1 role of the β2*nAChR in the formation of bursts and in behavioral mutant mice. We also found that the degree of exploration increases decision-making processes requires methods for the acute, with the severity of the psychotic symptoms. Together, our studies reversible control of this receptor subtype at time scales compatible revealed epistatic effects of Akt1 variations on the regulation of DA- with synaptic transmission. To this aim, we are developing new associated functions and provided a potential link from a genetic technologies for the optical control of specific nAChR subtypes deficiency, to neurobiological abnormalities, to higher cognitive in vivo. Notably, we have engineered a β2-containing, light-inhibited functions. nAChR (β2*LinAChR). This designed receptor presents at its surface a free cysteine amino acid for the anchoring of a chemical photo- A18.30 switch. The photoswitch maleimide azobenzene homocholine Long latency of patch clamp-evoked quantal DA release from (MAHoCh) is made of three components: a maleimide group for bio- the somata of dopaminergic neurons in rat substantial nigra conjugation to the cysteine mutant, an azobenzene photoswitch that slices can be photoisomerized between its cis and trans states using violet Zhuan ZHOU*, Li WANG, Ruiying JIAO, Feipeng ZHU, Quanfeng and green light, respectively, and a homocholine competitive antago- ZHANG, Bin LIU, Lianghong ZHENG and Changhe WANG nist. After covalent attachment of MAHoCh, β2*LinAChRs respond normally to nicotine and acetylcholine, but can be rapidly antagonized Peking University, Peking, China using violet light (390 nm). Illumination with green light (520 nm) with- *E-mail: [email protected] draw the homocholine group from its binding pocket, and restores the Intrinsic Activity, 2016; 4(Suppl. 2): A18.30 function of β2*nAChRs. We are using a lentiviral vector strategy to http://www.intrinsicactivity.org/2016/4/S2/A18.30 express β2*LinAChRs selectively in the VTA. Patch clamp recordings of VTA DA neurons show that LinAChRs can be used to optically

inhibit nicotinic inputs to the VTA. Furthermore, using in vivo extra- A18.33 cellular recordings, we show that β2*LinAChR can be used to rapidly The modulation of left frontal activity and dopamine activity by and reversibly modulate not only the spontaneous bursting activity of induced positive mood: the role of appetitive motivation DA neurons, but also the bursts induced by intravenous injection of Jessica WEAVING* and Ian THARP nicotine. Our findings establish a causal role for β2*nAChRs in Department of Psychology, University of Greenwich, regulating the excitability of VTA DA neurons. United Kingdom *E-mail: [email protected] A18.32 Intrinsic Activity, 2016; 4(Suppl. 2): A18.33 Parkin knockout rats develop Parkinsonian-like motor http://www.intrinsicactivity.org/2016/4/S2/A18.33 impairments upon exposure to psychostimulant methamphetamine Greater left frontal asymmetry (LFA) has been proposed as a neural Anna MOSZCZYNSKA*, Jickssa M. GEMECHU and marker of positive affect. However, findings are inconsistent. An Bryan A. KILLINGER alternative account of LFA, as an index of approach motivation, suggests the association between LFA and positive affect might be Department of Pharmaceutical Sciences, Wayne State University, dependent on an appetitive (reward-related) component of positive Detroit, MI, United States of America affect. To date, this has not been directly tested. Furthermore, LFA *E-mail: [email protected] has been linked to activity in the mesocorticolimbic dopamine system Intrinsic Activity, 2016; 4(Suppl. 2): A18.32 that is known to play a key role in appetitive motivation. Therefore, http://www.intrinsicactivity.org/2016/4/S2/A18.32 the present study examined the moderating role of appetitive motiva- Young adult chronic users of the psychostimulant methamphetamine tion in relation to the influence of positive mood on LFA and dopamin- (METH) are at risk to develop Parkinson’s disease (PD) later in life. ergic activity. Loss-of-function mutations in Park2, a gene encoding the E3 ligase Participants (n = 42) underwent a neutral mood induction and either parkin, have been found in patients with familial PD. Despite intense an appetitive or pleasant (non-appetitive) positive mood induction investigation, the exact role of parkin in the development of PD is still using mental imagery and music. Positive affect (activated and unclear as parkin knockout (PKO) rats and mice do not display deactivated) was reported before and after mood inductions. Electro- dopaminergic (DAergic) deficits and progressive nigrostriatal encephalogram (EEG) and spontaneous eye blink rate (EBR) — dopamine (DA) neuron degeneration. High doses of METH damage a putative psychophysiological measure of dopaminergic activity — striatal DAergic terminals and decrease parkin levels in striatal were recorded during the 8-minute mood induction sessions. synaptosomes while overexpression of parkin protects these Results showed an increase in deactivated affect only for the terminals from METH neurotoxicity. It is not known whether a parkin pleasant condition, and an increase in activated affect only for the deficit mediates susceptibility of METH users to develop PD. We have appetitive condition, indicating that the mood induction method was hypothesized that a METH-mediated deficit in parkin potentiates effective. LFA and spontaneous EBR were both found to be greater susceptibility of DA neurons to other parkin deficit-inducing for the appetitive compared to neutral induction, whereas no such environmental insults eventually leading to development of PD. Our difference was observed between the pleasant compared to neutral 1st objective was to characterize DAergic system in the striatum of induction. This suggests that, in the context of induced positive mood young adult (2 months old) Long Evans PKO rats. Our 2nd objective states, modulation of LFA and related dopamine activity is contingent was to determine whether PKO rats are hypersensitive to the upon appetitive motivation. neurotoxic effects of METH. Drug-naïve PKO rats displayed lower activity of monoamine oxidase B and higher levels of its preferred A18.34 substrate, phenylethylamine (PEA), than their WT counterparts. Neurobiological correlates of alcohol-related Pavlovian-to- These changes were accompanied by deficits in trace amine- instrumental transfer and relapse behavior in alcohol associated receptor 1 and postsynaptic DA D2 receptor (D2Long), as dependence: the LeAD study well as by increased levels of DA transporter (DAT), in the striatum. Maria GARBUSOW1,*, Daniel J. SCHAD2, Miriam SEBOLD1, The in D2Long and DAT levels negatively correlated with PEA levels in Stephan NEBE3, Christian SOMMER3, Ulrich S. ZIMMERMANN3, PKO rats, suggesting downregulation of both proteins by PEA. During Michael N. SMOLKA3, Florian SCHLAGENHAUF1,4, Quentin J. M. the exposure to binge METH, we observed Parkinsonian-like motor HUYS5, Michael A. RAPP2 and Andreas HEINZ1 impairments in PKO, but not in WT, rats. The PKO rats regained 1Department of Psychiatry and Psychotherapy, Charité ability to move; however, they continued to display tremors, Universitätsmedizin Berlin; 2Cognitive Sciences, Department of movement incoordination and rigidity, and difficulty with movement Sports and Health Sciences, University of Potsdam, Germany; initiation for at least 3 days after cessation of METH administration. 3Department of Psychiatry and Psychotherapy, University Hospital These impairments were suggestive of substantial deficit in striatal Carl Gustav Carus, Technische Universität Dresden, Germany; DA. In agreement, METH-treated PKO rats displayed larger deficit in 4Max Planck Institute for Human Cognitive and Brain Sciences, striatal DA than WT rats (−77 % vs. −41 %). In summary, we have Leipzig, Germany; 5Institute for Biomedical Engineering, ETH Zürich demonstrated that the loss of the Park2 gene increases PEA neuro- and University of Zürich, Switzerland transmission in young drug-naïve rats, leading to severe DA deficit *E-mail: [email protected] and motor impairments upon exposure to high doses of METH. Our Intrinsic Activity, 2016; 4(Suppl. 2): A18.34 findings suggest that METH-induced parkin deficit may be a pre- http://www.intrinsicactivity.org/2016/4/S2/A18.34 disposing factor for development of PD in human METH users. Pavlovian-to-instrumental transfer (PIT) is an important phenomenon that may help understanding relapse behavior. Exposure to alcohol- related environments may induce craving and play a causal role in initiating relapses. Drug-associated cues are thought to acquire motivational properties via Pavlovian conditioning and to then control and generate drug-seeking behavior. At a neurobiological level, animal and human studies showed that PIT recruits the mesolimbic

dopaminergic reward system including the nucleus accumbens. The Parkinson’s disease. J Neurosci, 2009; 29(30):9651–9659. research group “Learning in alcohol dependence (LeAD)” (funded by doi:10.1523/JNEUROSCI.0833-09.2009 the German Research Council) investigated these learning mecha- 3. Airavaara M, Harvey BK, Voutilainen MH, Shen H, Chou J, Lindholm P, nisms using a PIT paradigm with alcohol-related stimuli during Lindahl M, Tuominen RK, Saarma M, Hoffer B, Wang Y: CDNF functional magnetic resonance imaging. Here, subjects were asked protects the nigrostriatal dopamine system and promotes to conduct an instrumental response (approach or non-approach recovery after MPTP treatment in mice. Cell Transplant, 2012; towards initially neutral stimuli that were rewarded by money) while 21(6):1213–1223. doi:10.3727/096368911X600948 alcoholic drink pictures were displayed in the background. Here, we report first data of 31 recently detoxified patients, followed up over a A18.36 period of six months and 24 control subjects. We found that alcohol- Effects of ADHD medications on brain metabolism: a microPET related background stimuli inhibited approach behavior in alcohol- imaging study in rats dependent patients but not in healthy controls. On a neural level, this Pierre CASTELNAU1, Emilie DESFOSSES2, Clovis TAUBER3, Sylvie effect was associated with a stronger nucleus accumbens activation CHALON2 and Laurent GALINEAU4,* in the group of patients suffering from alcohol dependence. Crucially, 1Neuropediatric Department, University of Tours, INSERM U930, both effects were present in low severe dependent patients and Imaging and Brain, Tours, France; 2INSERM U930, Imaging and subsequent abstainers but absent in high severe dependent patients Brain, Tours, France; 3Biophysics Department, University of Tours, and subsequent relapsers. The results point to potential resilience INSERM U930, Imaging and Brain, Tours, France; 4Neurosciences factors for relapse in alcohol dependence and will be discussed with Department, University of Tours, France respect to possible therapeutical implications. *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A18.36 A18.35 http://www.intrinsicactivity.org/2016/4/S2/A18.36 CDNF knockout mice show age-dependent changes in the function of dopaminergic terminals Introduction: Attention-deficit hyperactivity disorder (ADHD) is a Anne PANHELAINEN1,*, Erik PALM1, Vootele VÕIKAR2, Maria LINDAHL1 developmental disorder including inattention, hyperactivity and / or and Mart SAARMA1 impulsivity. Psychostimulants such as methylphenidate (MPH) are the gold standard treatments for this disorder and mainly target the 1Institute of Biotechnology, University of Helsinki, Finland; dopamine neurotransmission. In this study, we used positon emission 2Neuroscience Center / Laboratory Animal Center, University tomography (PET) to better understand the impact on brain of Helsinki, Finland metabolism of an injection of MPH (i) in naive Wistar rats at 28 days *E-mail: [email protected] of postnatal life (P28) and (ii) in the same rats at 70 days of postnatal Intrinsic Activity, 2016; 4(Suppl. 2): A18.35 life (P70) after a daily treatment that started at P28. The effects of http://www.intrinsicactivity.org/2016/4/S2/A18.35 MPH were compared to those of another psychostimulant, i. e. lisdex- In Parkinson’s disease, the nigrastriatal dopamine (DA) pathway amphetamine (LDX), which targets the dopamine and noradrenergic degenerates due to mechanisms not well known and there is no neurostransmissions, and to those of a non-psychostimulant ADHD treatment that is proven to slow or stop the disease progression; only medication, i. e. guanfacine (GFC), which acts on the noradrenergic symptoms can be alleviated. Recently, a novel protein called the system. cerebral dopamine neurotrophic factor (CDNF) was found to support Methods: Wistar rats were treated with MPH (2 mg/kg, i.p.), LDX the survival and function of midbrain DA neurons in animal models of (0.3 mg/kg, i.p.), GFC (0.3 mg/kg, i.p.) or saline from P28 to P70. The Parkinson’s disease [1–3]. impact of such treatments on brain metabolism were evaluated using Mice lacking the Cdnf gene (CDNF KO) show increased locomotor [18F]2-fluoro-2-desoxy-D-glucose ([18F]DG) on freely moving Wistar activation in response to amphetamine (unpublished data by Dr. rats performed after the first (P28) and last (P70) injection of each Lindahl, group of Dr. Saarma). This suggests an alteration in their compound. DAergic transmission. Here we have studied the function of dorso- Results: An acute MPH injection at P28 increased [18F]DG uptake in lateral striatal DAergic terminals in acute slices of adult and aged the orbitofrontal cortex, thalamus and dorsal hippocampus, and CDNF KO mice and their wild-type litter mate controls, with fast-scan decreased [18F]DG uptake in the dorsal striatum, amygdala, and cyclic voltammetry. We studied the release of DA in response to ventral hippocampus. In contrast, LDX and GFC increased [18F]DG single electrical stimulations as well as to paired stimulations and uptake in the ventral striatum, globus pallidus and amygdala, and bursts of stimuli, to evaluate the basal release, recovery of release decreased [18F]DG uptake in thalamus and dorsal hippocampus. At after previous exocytosis and the initial release probabilities at P70, MPH, LDX and GFC injections increased [18F]DG uptake in the DAergic terminals. In order to reveal the mechanisms behind the ventral striatum. MPH and GFC also increased [18F]DG uptake in the sensitized locomotor response of CDNF KO mice to amphetamine, orbitofrontal cortex and thalamus. Conversely, LDX induced we perfused the slices with amphetamine while recording its effects decreases in [18F]DG uptake in the cingular, orbitofrontal and parietal on stimulated and non-stimulated DA release. This study allowed us cortices and also in the thalamus. to compare the function of DA terminals in adult vs. aged mice, and Discussion: We showed distinct effects of acute MPH vs. LDX and furthermore to evaluate the role of CDNF in DA terminal function and GFC injections at P28, while modifications in brain metabolism during aging. observed at P70 were more similar. Overall, these data suggest a References crucial role for the ventral striatum in the effects of LDX and GFC 1. Lindholm P, Voutilainen MH, Laurén J, Peränen J, Leppänen VM, injections at P28, and MPH, LDX and GFC injections at P70. The Andressoo JO, Lindahl M, Janhunen S, Kalkkinen N, Timmusk T, similar effects of GFC and MPH treatments at P70 despite their Tuominen RK, Saarma M: Novel neurotrophic factor CDNF protects distinct actions on the dopamine neurotransmissions are highly and rescues midbrain dopamine neurons in vivo. Nature, 2007; interesting. These results may help to better understand how 448(7149):73–77. doi:10.1038/nature05957 dopamine and non-dopamine medications improve ADHD patients 2. Voutilainen MH, Bäck S, Pörsti E, Toppinen L, Lindgren L, Lindholm P, care and follow-up. Peränen J, Saarma M, Tuominen RK: Mesencephalic astrocyte- derived neurotrophic factor is neurorestorative in rat model of

A18.37 were used in rats. The expression of zif-268 mRNA (qRT-PCR) and Transient dopaminergic signaling in health and in Parkinson’s vGLUT1, vGLUT2, GAD-65 and GAD-67 proteins (western blot) in the disease: simultaneous cAMP / PKA imaging in D1 and D2 MSNs striatum, thalamus and cerebellum were determined. Liliana CASTRO*, Cédric YAPO, Lorna CLEMENT, Anu NAIR, Administration of TBZ (2 mg/kg, i.p.) and PIM (1 mg/kg, i.p.) led to a Aurélie KY, Jérôme TANGE, Bryon SILVA, Dahdjim BETOLNGAR, significant induction of TJMs, caused catalepsy, increased expres- Elia MOTA, Danièle PAUPARDIN-TRISCH and Pierre VINCENT sion of zif-268 mRNA and GAD-67 and vGLUT2 proteins in the striatum. SCH 58261 (5 mg/kg, i.p.) reduced TJMs and catalepsy in Institut de Biologie Paris Seine IBPS, Université Pierre et Marie TBZ-treated rats. Harmaline (15 mg/kg, i.p.) induced generalized Curie, Paris, France tremor, measured in the Force Plate Actimeters. ENBA (0.5, 0.1, 0.05 *E-mail: [email protected] mg/kg, i.p.) in a dose-dependent manner inhibited harmaline-induced Intrinsic Activity, 2016; 4(Suppl. 2): A18.37 tremor and at the same time lowered locomotor activity. ENBA in the http://www.intrinsicactivity.org/2016/4/S2/A18.37 lowest dose (0.01 mg/kg, i.p.) also inhibited rats locomotion but had We used FRET biosensors to image intracellular cAMP concentration no effect on tremor. Furthermore, harmaline raised the expression of or PKA-dependent phosphorylation in striatal brain slice preparations zif-268 mRNA and GAD-67 protein in the cerebellum, lowered the from 8–12-day-old mice: a flash of UV light was used to release active expression of GAD-65 protein in the thalamus and did not influenced dopamine from NPEC-dopamine while monitoring the biosensor vGLUT1 or vGLUT2 proteins in examined structures. signal simultaneously in spiny neurons of both types. In direct The present results suggest different role of adenosine A1 and A2A pathway neurons, transient DA produced a transient increase in receptors in ET vs. PD tremors and involvement of GABA and intracellular cAMP with an EC50 of 0.9 µM. In indirect pathway glutamatergic transmission in both types of tremors. neurons, transient DA produced a transient decrease in intracellular The study was supported by the grant of the National Science Center cAMP with an IC50 of 0.4 µM from a cAMP level that was beforehand (no. 2013/11/B/NZ4/04565). B. K. is a holder of scholarship from the increased by applying an agonist of the adenosine A2A receptors or a KNOW (Ministry of Science and Higher Education, Poland). low concentration of forskolin. Downstream of cAMP, at the level of PKA-dependent phosphorylation, positive D1 responses showed a A18.39 lower EC50, whereas the activation of D2 receptors appeared of little Lentiviral-mediated local siRNA-induced inactivation of Gpr88 efficacy in decreasing PKA-dependent phosphorylation level. The in the medium spiny neurons modulates the behavioral and integration of the cAMP signal into a PKA / phosphatase equilibrium molecular effects of striatal dopamine depletion induced by thus makes D1 neurons highly responsive to brief dopamine stimula- 6-OHDA lesions in the rat tions whereas transient DA has little effect on D2 neurons. Manuela INGALLINESI1, Jonathan PEGON1, Nicole FAUCON BIGUET1, In Parkinson’s disease, DA release in the striatum progressively Anh DO THI1, Benjamin GALET1, Philippe RAVASSARD1, Jacques diminishes. Using biosensor imaging, we showed that D1 neurons MALLET1, Mark J. MILLAN2, Clotilde MANNOURY LA COUR2 and from 6-hydroxydopamine-lesioned mice responded strongly to Rolando MELONI1,* transient DA, contrary to neurons from healthy mice. 1Department of Biotechnology and Biotherapy, Institut du Cerveau et de la Moelle épinière (ICM), Paris, France; 2Institut de A18.38 Recherches Servier, Croissy-sur-Seine, France Differential involvement of adenosine receptors in rodent *E-mail: [email protected] models of parkinsonism and essential tremors Intrinsic Activity, 2016; 4(Suppl. 2): A18.39 1, 2 1 Barbara KOSMOWSKA *, Agnieszka ZELEK-MOLIK , Tomasz LENDA , http://www.intrinsicactivity.org/2016/4/S2/A18.39 Urszula GŁOWACKA1, Krystyna OSSOWSKA1 and Jadwiga WARDAS1 Gpr88, a G protein-coupled orphan receptor, is highly and almost 1Department of Neuropsychopharmacology, Institute of exclusively represented in the GABAergic medium spiny neurons Pharmacology, Polish Academy of Sciences, Kraków, Poland; (MSN) of the striatum. The expression of Gpr88 in this structure is 2Department of Brain Biochemistry, Institute of Pharmacology, regulated by dopamine (DA) and glutamate as well as psychotropic Polish Academy of Sciences, Kraków, Poland drugs. Interestingly, changes in Gpr88 mRNA reported in rats after *E-mail: [email protected] 6-OHDA lesions are also reversed by L-DOPA. In addition, Gpr88 Intrinsic Activity, 2016; 4(Suppl. 2): A18.38 knock-out mice exhibit a schizophrenic-like phenotype with deficits http://www.intrinsicactivity.org/2016/4/S2/A18.38 that are normalized by antipsychotics. These findings support a role Harmaline is a well-known tremorgenic compound which has been of GPR88 in the pathophysiology of basal-ganglia-related diseases suggested to model essential tremor (ET) in animals. The main cause and in the response to drugs that modulate dopamine and glutamate of harmaline tremor is activation of olivo-cerebellar climbing fibers neurotransmission. and excessive glutamate release. Furthermore, mechanisms We have recently shown that lentiviral-mediated knock-down (KD) of underlying this behaviour involve GABAergic and glutamatergic Gpr88 expression by a specific shRNA in ventral striatum (nucleus connections to the ventral motor thalamic nuclei. accumbens) reduces amphetamine-induced motor hyperactivity and Tremulous jaw movements (TJMs), defined as rapid vertical ameliorates cognitive deficits in a rat model of schizophrenia. Further- deflections of the lower jaw that are not directed at any particular more, the Gpr88 KD modifies the expression of the dopamine and stimulus, are used as a rodent model of parkinsonian (PD) resting cAMP-regulated phosphoprotein of 32 kDa (Darpp-32), suggesting tremor. TJMs can be induced by striatal dopamine depletion cross-talk between dopamine signaling and Gpr88 [1]. (6-OHDA, reserpine) or typical antipsychotics and are dependent In order to further evaluate the interaction between Gpr88 and upon the striatum. dopamine signaling, Gpr88 expression was inactivated in the dorsal The aim of our study was to examine the involvement of adenosine striatum after unilateral injection of 6-OHDA in the medial forebrain receptors in models of ET (harmaline) and PD (TJMs) tremors. We bundle in rats. As expected, the loss of DA neurons induced by used two highly selective adenosine ligands: (±)-5′-chloro-5′-deoxy- 6-OHDA and the concomitant depletion of DA in the striatum resulted

ENBA (A1 agonist) and SCH58261 (A2A antagonist). For modeling PD in a strong amphetamine-induced turning behavior ipsilateral to the tremor acute tetrabenazine (TBZ), a selective and reversible VMAT-2 lesioned side and increased, as assessed by in situ hybridization, the inhibitor, and subchronic pimozide (PIM), a typical antipsychotic drug expression of glutamic acid decarboxylase 67 (GAD67), a key marker

of the activity of the MSN. Moreover, as assessed by western blot 2Beroendekliniken, Sahlgrenska University Hospital, Gothenburg, analysis, striatal DA depletion decreased the expression of Darpp32 Sweden while increased protein levels of the GluA1 subunit of the AMPA *E-mail: [email protected] receptor. Inactivation of Gpr88 in the dorsal striatum ipsilateral to Intrinsic Activity, 2016; 4(Suppl. 2): A18.41 the 6-OHDA lesion significantly reduced amphetamine-induced http://www.intrinsicactivity.org/2016/4/S2/A18.41 turning behavior and restored the normal expression of GAD67. The rewarding effects of alcohol are thought to be related to elevated Furthermore, the Gpr88 KD restored toward normal levels the levels of dopamine in the nucleus accumbens (NAc), an important expression of both Darpp32 and GluA1 in lesioned animals. In part of the mesolimbic dopamine system. The exact mechanism by conclusion, these observations show that extinguishing expression of which ethanol induces this dopamine elevation has been difficult to Gpr88 moderates the impact of DA loss on MSN activity and suggest determine. We have previously demonstrated that ethanol increases that Gpr88 could be a relevant target for modulating dopaminergic dopamine via a neuronal circuitry involving glycine receptors in the neurotransmission. NAc and nicotinic acetylcholine receptors in the ventral tegmental Reference area. We have also shown that in order for ethanol to increase 1. Ingallinesi M, Le Bouil L, Biguet NF, Thi AD, Mannoury la Cour C, dopamine in the NAc an initial enhancement of extracellular levels of Millan MJ, Ravassard P, Mallet J, Meloni R: Local inactivation of the endogenous amino acid taurine is required. Taurine can be Gpr88 in the nucleus accumbens attenuates behavioral deficits released into the extracellular space via volume-regulated anion elicited by the neonatal administration of phencyclidine in rats. channels (VRACs) which is why we hypothesized that blocking Mol Psychiatry, 2015; 20(8):951–958. doi:10.1038/mp.2014.92 VRACs will prevent ethanol from elevating NAc dopamine. Thus by means of in vivo microdialysis in awake, freely moving male Wistar A18.40 rats we measured extracellular levels of dopamine and taurine in the Dopamine-receptive neurons in the medial amygdala regulate NAc following administration of the VRAC inhibitor DCPIB (100 µM) innate fear and / or ethanol (300 mM). Local perfusion with DCPIB partially Samara MILLER*, Marta SODEN and Larry ZWEIFEL blocked the ethanol-induced dopamine and taurine release. Thus we Department of Pharmacology, University of Washington, Seattle, suggest that the observed release of extracellular taurine after WA, United States of America ethanol is mainly mediated via VRACs. These data also support *E-mail: [email protected] previous findings that the elevations of dopamine and taurine in the Intrinsic Activity, 2016; 4(Suppl. 2): A18.40 NAc are closely linked with regards to ethanol. http://www.intrinsicactivity.org/2016/4/S2/A18.40

Innate fear is a critical mechanism animals rely on to avoid danger A18.42 and survive. Dopamine and dopamine-receptive neurons have been Dependence of accumbens shell dialysate dopamine on the 2+ broadly implicated in conditioned fear, but little is known about activity of apamin-sensitive slow-conducting Ca -activated + whether dopamine-receptive neurons influence innate fear behavior. K channels Cristina MILIANO*, Valentina VALENTINI, Giovanna PIRAS and We find that dopamine D1 receptor (D1R)-expressing neurons regulate innate threat processing through distinct amygdalar-hypo- Gaetano DI CHIARA thalamic circuits. Specifically, D1R neurons localized within the Department of Biomedical Sciences, University of Cagliari, Italy posteroventral medial amygdala (MEAPV) project to the dorsomedial *E-mail: [email protected]

VMH (VMHDM) and the bed nucleus of the stria terminalis (BNST), Intrinsic Activity, 2016; 4(Suppl. 2): A18.42 and are selectively activated by both predator and conspecific threat http://www.intrinsicactivity.org/2016/4/S2/A18.42 stimuli. We find that MEAPV D1R neurons are heterogeneous and Introduction: Midbrain dopaminergic neurons have two different release both GABA and glutamate at downstream target regions. firing patterns: single-spike firing, associated with tonic dopamine Optogenetic activation of MEAPV D1R neurons attenuates defensive (DA) release and burst firing, associated with phasic DA release. behaviors in response to threat stimuli; reduced freezing and hiding Burst firing in DA neurons is controlled by apamin-sensitive Ca2+- in response to predator odor and visual threat, and increased activated K+ (SK) channels. Electrophysiological studies showed that territorial aggression. In contrast to direct cell body stimulation in the the blockade of SK channels by apamin increases DA burst firing MEAPV, optogenetic stimulation of MEAPV D1R terminals in the VMHDM while activation reduces it. reciprocally influences defensive behaviors to threat stimuli, Aim: In order to demonstrate that microdialysis technique is able to increased freezing and hiding in response to predator odor and visual detect phasic dopamine release, we evaluated the effect of intra- threat, and reduced territorial aggression. These data suggest a ventral tegmental area (VTA) administration of apamin on dialysate hierarchical circuit that serves to fine-tune the processing of nuanced DA in the nucleus accumbens (NAc) shell and core. In addition we threat cues presented in complex environmental contexts. The studied the effect of systemic administration of the allosteric activator collective activation of MEAPV D1R neurons reduces innate threat of SK channels, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl- responses, whereas activation of a specific circuit connection pyrimidin-4-yl]-amine (CyPPA) on the increase of dialysate DA in the (MEAPV → VMHDM) enhances fear. Future experiments designed to NAc shell and core induced by systemic Raclopride, a pure D2 antag- identify the circuit connections of the MEAPV that reduce innate fear onist known to activate DA burst firing and to increase dialysate DA will help to resolve the nature of this hierarchy. in the NAc shell and core. Materials and methods: By in vivo microdialysis studies we evaluate A18.41 the effects of apamin (1.7 pmol / 1 µl; 3.3 pmol/µl) locally injected into The VRAC inhibitor DCPIB partially blocks the dopamine- and the VTA and the effects of CyPPA (3.3 mg/kg, i.v.) 5 min before the taurine-elevating properties of ethanol in the rat nucleus administration of raclopride (75 µg/kg, i.v.). accumbens Results: Intra-VTA apamin, at doses of 1.7 and 3.3 pmol, dose- 1, 1 1,2 Lisa ULENIUS *, Louise ADERMARK , Bo SÖDERPALM and dependently increased dialysate DA in the NAc shell but not in the 1 Mia ERICSON NAc core. Doses of 3.3 pmol apamin increased NAc shell dialysate 1Department of Psychiatry and Neurochemistry, Institute of DA by 75 % over basal after 40 min, reaching a plateau of 100–125 % Neuroscience and Physiology, University of Gothenburg, Sweden; over basal at 80 min post-drug. CyPPA, given i.v. at a dose of

3.3 mg/kg did not affect basal dialysate DA but reduced to half the Here we investigated the role of the melanocortin 4 receptor (MC4R) maximal increase of NAc shell dialysate DA induced by raclopride in aversion. Normal mice showed a significant conditioned place (75 µg/kg, i.v.). CyPPA did not affect the increase of dialysate DA aversion to the inflammatory stimulus lipopolysaccharide, the toxin induced by raclopride in the NAc core. lithium chloride and the κ-opioid receptor agonist U 50.488. In Conclusions: Collectively these observations are consistent with the contrast, these stimuli induced a robust place preference in mice idea that NAc shell dialysate DA can provide a correlate of phasic lacking MC4Rs. Similarly, normal mice displayed a strong place stimulation of in vivo DA transmission as a result of activation of DA aversion to formalin injections in the hind paw whereas mice lacking neuron burst firing. Our observations contradict the common belief MC4Rs showed no pain aversion. All behaviors were reverted back to that brain microdialysis only reflects the tonic modality of DA trans- aversion by rescuing the MC4Rs selectively on dopamine D1 receptor mission in vivo. expressing neurons in adult mice. We found that MC4Rs in the striatum are responsible for the rescue of LPS aversion. This was A18.43 elucidated with AAV5-Cre rescue of MC4Rs in this area of KO mice. Loss of NMDA receptor-dependent activity of dopaminergic Further, administration of a dopamine D1 receptor antagonist together neurons leads to development of depressive-like symptoms with the aversive stimuli restored LPS, U 50.488 and formalin-paw- in mice pain aversions in the mice lacking MC4Rs. Chemogenetic inhibition Jan RODRIGUEZ PARKITNA1,*, Kamila JASTRZĘBSKA1, Przemysław of midbrain dopaminergic neurons caused conditioned place aversion CIEŚLAK1, Magdalena WALCZAK2, Łukasz SZUMIEC1, David ENGBLOM1 in both wild-type and MC4R KO mice. Collectively, these findings and Tomasz BŁASIAK2 indicate that MC4Rs on dopamine D1-expressing neurons of the striatum are key regulators of aversive signaling, and that the deletion 1Laboratory of Transgenic Models, Institute of Pharmacology, of these receptors allows aversive stimuli to activate dopaminergic Polish Academy of Sciences, Kraków, Poland; 2Department of reward circuits and cause a “flip” in motivational value of the aversive Neurophysiology and Chronobiology, Institute of Zoology, stimuli. This is consistent with the idea that reward and aversion are Jagiellonian University, Kraków, Poland intimately coupled and processed in the mesolimbic dopamine *E-mail: [email protected] system. Intrinsic Activity, 2016; 4(Suppl. 2): A18.43 http://www.intrinsicactivity.org/2016/4/S2/A18.43 A18.45

The role of abnormal activity of dopaminergic neurons in the A role for β-arrestin-2 in the coordination of D1 dopamine development of affective disorders remains controversial. Recent and μ-opioid receptors responses in the rat nucleus studies in animal models show that altered activity of the midbrain accumbens shell dopamine system, changes in burst firing in particular, are Simona SCHEGGI1, Alberto FERRARI1, Teresa PELLICCIA1, responsible for many of the depressive-like behaviors. We found that Paola DEVOTO2, Maria Graziella DE MONTIS1 and inactivation of NMDA receptors on dopamine neurons in adult Carla GAMBARANA1,* animals abolished burst firing of midbrain dopamine neurons which 1Department of Molecular and Developmental Medicine, resulted in increased immobility during the forced swim test and University of Siena, Italy; 2Department of Biomedical Sciences, decreased frequency of social interactions. The mutation had no University of Cagliari, Italy effect on saccharin preference, even though it significantly reduced *E-mail: [email protected] saccharin intake. Mutant mice were slower to learn instrumental food Intrinsic Activity, 2016; 4(Suppl. 2): A18.45 self-administration but were not different in effort discounting of an http://www.intrinsicactivity.org/2016/4/S2/A18.45 operant task. Accordingly, the mutation had no significant effect on effort-discounting in a T-maze task; mutant mice chose to cross a Background: β-Arrestins (β-arrs) are multifunctional adaptors that barrier baring access to a larger reward with similar frequency as mediate desensitization, internalization and some signaling functions controls. Finally, mutant mice had normal sensitivity to negative of G protein-coupled receptors. In mice acute morphine administra- reinforcement in conditioned place aversion or a probabilistic reversal tion increases motility in a dopamine D1 receptor (D1R)-dependent task. Thus, the loss of NMDA receptor-dependent activity of manner, via a β-arr-2 signaling complex that functionally connects dopamine neurons led to increase in depressive-like behaviors such μ-opioid receptors (MORs) to D1Rs, which are co-expressed in as behavioral despair and reduced social activity, but without medium spiny neurons of the NAc. In rats acute morphine administra- significantly affecting effort-discounting, causing anhedonia or tion increases extraneuronal dopamine levels in the NAc shell altered sensitivity to negative reinforcement. (NAcS), yet motility is decreased. Sucrose consumption increases extraneuronal dopamine levels in the NAcS that, in turn, stimulate A18.44 D1Rs. Moreover, MOR stimulation mediates palatability in different Melanocortin 4 receptors in aversion: flipping motivational brain areas, including the NAcS. Thus, MORs mediate sucrose and value morphine hedonic value, and sucrose consumption and acute Anna M. KLAWONN*, Michael FRITZ, Anna NILSSON, morphine administration simultaneously activate MORs and D1Rs in Kiseko SHIONOYA, Joanna ZAJDEL, Anders BLOMQVIST the NAcS. and David ENGBLOM Aims: In order to further assess the physiological role of β-arr-2 in functionally connecting MORs and D Rs in the NAcS, we studied Institute for Clinical and Experimental Medicine, Linköping 1 NAcS D R signaling following sucrose consumption in non-food- University, Linköping, Sweden 1 deprived (NFD) and food-deprived (FD) control rats and rats with *E-mail: [email protected] reduced NAcS β-arr 2 expression. Moreover, we studied motility and Intrinsic Activity, 2016; 4(Suppl. 2): A18.44 NAcS D1R signaling after acute morphine administration in the same http://www.intrinsicactivity.org/2016/4/S2/A18.44 experimental conditions. Symptoms such as negative affect, malaise, discomfort and Results: In NFD rats, morphine administration induced sedation and depressive mood decreases life quality of many patients. Activation did not modify D1R signaling, in terms of phosphorylation pattern of of aversive neurocircuitry is critical for these symptoms, yet the dopamine and cAMP-regulated phosphoprotein, Mr32 (DARPP-32). molecular mechanism controlling aversive signaling remain elusive. Reduced β-arr-2 expression did not modify these responses. In FD

rats, morphine increased motor activity, extraneuronal dopamine and Intrinsic Activity, 2016; 4(Suppl. 2): A18.47 phospho-Thr34 DARPP-32 levels in the NAcS. These responses http://www.intrinsicactivity.org/2016/4/S2/A18.47 were blunted in rats with reduced β-arr-2 expression. In NFD rats, Nicotine is the tobacco neuroactive compound mediating its sucrose consumption induced a transient increase in phospho-Thr34 rewarding / reinforcing properties by acting on the dopaminergic DARPP-32 levels. This effect was long lasting when β-arr-2 system. The lateral habenula (LHb) is a structure known to modulate expression was reduced, i. e. MOR-dependent inhibition on adenylyl the DA system activity both directly and indirectly, suggesting that it cyclase activity was only observed when the two receptors were might represent a possible target for the action of nicotine in the CNS. connected through β-arr-2. In FD rats, sucrose induced a transient We used an in vivo electrophysiological recording approach to increase in phospho-Thr34 DARPP-32 levels, an effect blunted in rats investigate the nicotine-induced response of single LHb / DA neurons. with reduced β-arr-2 expression. Systemic administration of nicotine increased the LHb neuronal Conclusions: Responses to sucrose consumption are mediated by activity in vivo in rats. Following nicotine chronic treatment, this NAcS MORs and D1Rs functionally connected through a β-arr-2 response was drastically decreased. To further elucidate the LHb role complex in NFD and FD rats, whereas in response to acute morphine in central nicotine effects, we recorded the activity of VTA putative- the two receptors act independently in NFD rats and interconnected DA neurons following LHb electrolytic lesion in both drug-naïve and via β-arr-2 in FD rats. nicotine chronically treated animals. Systemic administration of nicotine induced a significant increase in the neuronal activity of A18.46 putative-DA neurons located in the paranigral nucleus (PN) of the Brain dopamine systems modulate cost–benefit decisions VTA. This effect was completely abolished by LHb electrolytic lesion. in rats Conversely, neurons located in the parabrachial pigmented nucleus Wolfgang HAUBER* (PBP) of the VTA responded significantly to nicotine administration Department of Animal Physiology, Institute of Biomaterials and only after LHb lesion. Following chronic nicotine treatment, putative- Biomolecular Systems, University of Stuttgart, Germany DA VTA neurons recorded from LHb sham-lesioned rats showed a *E-mail: [email protected] nicotine-induced response pattern similar to LHb-lesioned drug-naïve Intrinsic Activity, 2016; 4(Suppl. 2): A18.46 animals, while nicotine failed to increase the neuronal activity of both http://www.intrinsicactivity.org/2016/4/S2/A18.46 PN and PBP putative-DA neurons in LHb-lesioned rats. Our evidence strongly suggests that nicotine modulates LHb activity and plays an Rodent studies suggest that subregions of the medial frontal cortex, important role in mediating the effects of nicotine on the midbrain DA the basolateral amygdala, and the nucleus accumbens are key system thus participating in the mechanisms of addiction and components of an interconnected neural system that supports aversion to this drug. different forms of cost–benefit decisions such as effort-based or risk- based decisions in T-maze or instrumental tasks. For instance, in rats not only inactivation of the anterior cingulate cortex (ACC), A18.48 basolateral amygdala (BLA) or nucleus accumbens (NAc) but also Optogenetic stimulation of the VTA – nucleus accumbens disconnection between the ACC and the BLA or between the ACC projection reduces impulsive responding on the 5-choice and the NAc impaired effort-based decision-making indicating that an serial reaction time task information transfer between these structures is essential to guide Rebecca BARLOW, Wiebke NISSEN, Anne-Kathrin ZELL, Anna decisions requiring an assessment of costs and benefits. Further- MENZEL, Johannes FREUDENREICH, Janet NICHOLSON, Roberto more, there is compelling evidence that brain dopamine (DA) ARBAN, Anton PEKCEC and Moritz VON HEIMENDAHL* systems, in particular in the NAc, regulate cost–benefit decisions. For Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany instance, relative to sham controls, rats with NAc DA depletion had a *E-mail: [email protected] low preference for effortful but large-reward action. Likewise, relative Intrinsic Activity, 2016; 4(Suppl. 2): A18.48 to vehicle controls, rats with a NAc core DA D1/2 receptor blockade http://www.intrinsicactivity.org/2016/4/S2/A18.48 displayed a lower preference for risky but large-reward action. In turn, Impulsivity is a multi-faceted behavioural construct which can broadly DA stimulant drugs such as amphetamine or methylphenidate be defined as a tendency to act prematurely and without foresight. It increased the preference for effortful or risky large-reward action. is symptomatic of several neuropsychiatric disorders including Recent data further demonstrate that cost–benefit decisions are schizophrenia, ADHD and substance abuse disorders, and can be sensitive to stress. For instance, the pharmacological stressor observed behaviourally as impaired response inhibition (motor yohimbine increased the preference of rats for the risky large-reward impulsivity) or the preferential choice of risky or immediate rewards action, in part by stimulating DA transmission. Furthermore, cost– (choice impulsivity). Dysfunction of the midbrain dopaminergic benefit decisions are influenced by motivational states, e. g. a system has been implicated in several forms of impulsive behavior, motivational downshift by satiety manipulations reduced the and there has been much investigation into the associated preference of rats for effortful large-reward action. Of note, in rats abnormalities in fronto-striatal networks seen in highly impulsive with NAc DA depletion effort-based decision-making was still subjects. However, there has been limited work investigating the role sensitive to motivational shifts. Thus, NAc DA seems not to mediate of the dopaminergic VTA–NAc projection in modulating motor effects of motivational shifts on decision-making policies. impulsivity. We hypothesise that a hyperdopaminergic state within the nucleus accumbens may drive impulsive behavior, and may arise as A18.47 a consequence of increased dopaminergic input from the VTA. To Role of the lateral habenula in nicotine addiction: interaction functionally investigate this hypothesis, a cohort of male Lister- with the dopaminergic system hooded rats (n = 32) were trained, and assessed for motor impulsivity 1, 1 2 Massimo PIERUCCI *, Roberto COLANGELI , Caitlin DAVIES , on the 5-choice serial reaction time task (5-CSRTT). To overcome the 1 1 Francis DELICATA and Giuseppe DI GIOVANNI confounding factor of continual light stimulation on the 5-CSRTT, 1Department of Physiology and Biochemistry, University of Malta, which involves the detection of a brief light stimulus, a stabilised step Msida, Malta; 2Neuroscience Research Division School of function opsin (SSFO) was used. Rats received bilateral injections of Biosciences, Cardiff University, United Kingdom a virus expressing the SSFO (AAV5-hSyn-SSFO-eYFP) or control *E-mail: [email protected] virus into the VTA and a bilateral implantation of a dual-fibre stub in

the NAc shell. Contrary to our expectation, we observed that opto- Nicotinic receptors (nAChRs) containing an α5 subunit are found in genetic stimulation of the VTA-NAc shell projection resulted in a the prefrontal cortex (PFC) and ventral striatum (vSTR), key nodes in decrease in premature responding on the 5-CSRTT. These results a neural circuit whose activity is modulated by dopamine (DA). A loss- indicate that increased input from the VTA may result in an of-function mutation in the gene coding for the α5 subunit is amelioration of impulsive behaviour. associated with an increase risk for addiction to nicotine, alcohol and opiates [1–3]. This suggests that the α5 subunit is well-positioned to A18.49 modulate activity in cortico-striatal circuits that when dysfunctional Modelling dopamine transporter deficiency syndrome with leads to cognitive and reward-processing deficits that are hallmarks patient-derived induced pluripotent stem cells of several neurologic and psychiatric disorders. To investigate the Serena BARRAL1,*, Sonja HEASMAN1, Gabriele LIGNANI2, function of these subunits in cortico-striatal circuits, we utilized a short Carmen DE LA FUENTE BARRIGÓN1, Joanne NG1,3, Esther MEYER1, hairpin viral strategy to selectively knock-down (KD) α5 expression in Sally A. COWLEY4, Richard WADE-MARTINS5, Simon HEALES1 the mesolimbic DA system (VTA) or the PFC of rats. We measured and Manju A. KURIAN1,3 nAChR modulation of DA release in the nucleus accumbens shell region of the vSTR using fast scan cyclic voltammetry and consis- 1ICH Developmental Neurosciences Prog, UCL Institute of Child tently observed lower baseline levels (54 % reduction) of electrically Health, London, United Kingdom; 2Institute of Neurology, University evoked DA release in both in vivo anesthetized as well as ex vivo College, London, United Kingdom; 3Great Ormond Street Hospital slice preparations. In addition, the kinetics of DA release was slowed NHS Trust, London, United Kingdom; 4Sir William Dunn School of in VTA-α5 KD with both a slower rise time to peak as well as an Pathology, University of Oxford, United Kingdom; 5Department of extended decay time. In ex vivo experiments VTA-α5 KD also Physiology, Anatomy and Genetics, University of Oxford, United increased the frequency dependency of DA release. Confirming the Kingdom significant functional impact of α5 subunits on DA signaling we found *E-mail: [email protected] reduced DA transporter function in α5 KO mice compared to wild Intrinsic Activity, 2016; 4(Suppl. 2): A18.49 types. In the neocortex, PFC-α5 KD reduces the response of http://www.intrinsicactivity.org/2016/4/S2/A18.49 pyramidal neurons to agonist application and increases the rate of Dopamine transporter deficiency syndrome (DTDS) is an early onset desensitization to repeated agonist application. At a behavioral level, neurological disorder presenting with infantile parkinsonism-dystonia, PFC-α5 KD impairs cognitive control during performance of an due to loss-of-function mutations in SLC6A3, encoding the dopamine attention task. Specifically, PFC-α5 KD rats have a sustained transporter (DAT). In order to further investigate the cellular mecha- decrement (> 2 weeks) in task performance on the days following the nisms underpinning DTDS, we generated induced pluripotent stem session where a distractor is introduced but had no effect on standard cells (iPSCs) from two patients with different homozygous SLC6A3 task performance prior or during the distractor. This suggests that the missense mutations (p.L368Q and p.P395L) as well as from an age- loss of the α5 subunit affects recovery of cognitive functions following matched healthy control. During reprogramming, all patient lines a stressor. In sum, the α5 subunit is a critical player in nAChR control retained the specific SLC6A3 mutation and displayed robust pluri- of cortical-striatal circuitry. Targeting the α5 subunit presents a novel potency. We differentiated patient and control iPSCs into midbrain means to modulate processes that mediate not just the liability to dopaminergic (mDA) neurons. Both control and patient lines differen- addiction but also play a role in the cognitive, reward processing and tiated into midbrain precursors at high efficiency and with no signifi- motivational deficits observed in a range of neuropsychiatric and cant differences. iPSC-derived mDA precursors further matured into neurological disorders. dopaminergic neurons, confirmed by expression of tyrosine hydrox- References ylase (TH) and the substantia-nigra-specific potassium channel 1. Bierut LJ, Stitzel JA, Wang JC, Hinrichs AL, Grucza RA, Xuei X, GIRK2. A mature neuronal phenotype was confirmed on electro- Saccone NL, Saccone SF, Bertelsen S, Fox L, Horton WJ, Breslau N, physiological analysis. As seen in cerebrospinal fluid analysis of Budde J, Cloninger CR, Dick DM, Foroud T, Hatsukami D, Hesselbrock affected patients, high-performance liquid chromatography confirmed V, Johnson EO, Kramer J, Kuperman S, Madden PA, Mayo K, an increased homovanillic acid / 5-hydroxyindoleacetic acid ratio in Nurnberger J Jr, Pomerleau O, Porjesz B, Reyes O, Schuckit M, Swan patient-derived mDA neurons. In addition, patient-derived mDA G, Tischfield JA, Edenberg HJ, Rice JP, Goate AM: Variants in neurons seemed to show decreased morphological complexity when nicotinic receptors and risk for nicotine dependence. Am J compared to control lines. Furthermore, iPSCs-derived neurons from Psychiatry, 2008; 165(9):1163–1171. one patient showed an overall decreased neuronal population when doi:10.1176/appi.ajp.2008.07111711 compared to control lines, possibly reflecting a neurodegenerative 2. Erlich PM, Hoffman SN, Rukstalis M, Han JJ, Chu X, Linda Kao WH, process. In conclusion, we show the effective generation of iPSC Gerhard GS, Stewart WF, Boscarino JA: Nicotinic acetylcholine from two DTDS patients and the ability of these cells to further receptor genes on chromosome 15q25.1 are associated with differentiate into dopaminergic neurons that can be used to both nicotine and opioid dependence severity. Hum Genet, 2010; study cellular characteristics of the disease, as well as to develop 128(5):491–499. doi:10.1007/s00439-010-0876-6 novel therapeutic strategies. 3. Wang JC, Grucza R, Cruchaga C, Hinrichs AL, Bertelsen S, Budde JP, Fox L, Goldstein E, Reyes O, Saccone N, Saccone S, Xuei X, Bucholz A18.50 K, Kuperman S, Nurnberger J Jr, Rice JP, Schuckit M, Tischfield J, Influence of the α5 subunit of nicotinic receptors on the Hesselbrock V, Porjesz B, Edenberg HJ, Bierut LJ, Goate AM: function of cortico-striatal circuitry: effects on dopamine Genetic variation in the CHRNA5 gene affects mRNA levels and release and attention is associated with risk for alcohol dependence. Mol Psychiatry, Patrick TIERNEY*, William HOWE, Damon YOUNG, Jonathan 2009; 14(5):501–510. doi:10.1038/mp.2008.42 GARST-OROZCO, Amie ROSSI, Edward GUILMETTE and Rouba KOZAK Pfizer, United States of America *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A18.50 http://www.intrinsicactivity.org/2016/4/S2/A18.50

A18.51 defined VTA dopamine, GABA, or glutamate neurons. This approach HCN channels constrain synaptic excitability in substantia enabled specific rabies virus replication and transynaptic transport in nigra dopaminergic neurons in vitro and promote their each of the VTA neuronal populations and the characterization of cell- survival in vivo type-specific afferent inputs. Surprisingly, we find that these three Alessio MASI*, Carmen CARBONE, Francesco RESTA, VTA populations received qualitatively similar inputs, with dominant Gustavo PROVENSI and Guido MANNAIONI and comparable projections from the lateral hypothalamus, raphe, and ventral pallidum. However, notable differences were observed, Neurofarba, University of Florence, Italy with striatal regions and globus pallidus providing a greater share of *E-mail: [email protected] input to VTA dopamine neurons, cortical input preferentially on to Intrinsic Activity, 2016; 4(Suppl. 2): A18.51 glutamate neurons, and GABA neurons receiving proportionally more http://www.intrinsicactivity.org/2016/4/S2/A18.51 input from the lateral habenula and laterodorsal tegmental nucleus. Aims: The molecular mechanisms underlying the differential vulnera- By comparing inputs to each of the transmitter-defined VTA cell bility between substantia nigra pars compacta (SNc) and ventral types, this study sheds important light on the systems-level organiza- tegmental area (VTA) dopaminergic (DAergic) neurons in Parkinson’s tion of diverse inputs to VTA. disease (PD) are still unclear. We recently demonstrated that MPP+, a neurotoxin able to cause selective nigrostriatal degeneration in A18.53 rodents and primates, alters the electrophysiological properties of The clearance of dopamine from the striatum following SNc DAergic neurons in vitro by inhibiting the hyperpolarization- methamphetamine stimulation is strain-dependent activated current (Ih). The goal of this work is to identify physiological Jacqueline S. WOMERSLEY1,2,*, Lauriston A. KELLAWAY1, determinants of differential vulnerability within DAergic neurons. Dan J. STEIN3, Greg A. GERHARDT4,5 and Vivienne A. RUSSEL1 Methods: Whole-cell recordings were performed in acute midbrain 1Department of Human Biology, University of Cape Town, South slices from juvenile WH rats or TH-GFP mice. Simultaneous determi- Africa; 2Department of Psychiatry, University of Stellenbosch, South nation of changes in cytosolic calcium concentration was achieved by Africa; 3Department of Psychiatry, University of Cape Town, South loading the recorded neuron with Fluo-4 or Oregon Green. Inactiva- Africa; 4Center for Microelectrode Technology, University of tion of Ih in vivo was obtained by stereotaxic intranigral injection of Kentucky Chandler Medical Center, Lexington, KY, United States of ZD 7288 or ivabradine in adult WH rats or TH-GFP mice. America; 5Department of Anatomy and Neurobiology, University of Results: In midbrain DAergic neurons from TH-GFP mice, pharma- Kentucky Chandler Medical Center, Lexington, KY, United States of cological suppression of Ih increases the amplitude and duration of America evoked excitatory post-synaptic potentials (EPSPs) leading to *E-mail: [email protected], [email protected] temporal summation of multiple EPSPs. The extent of this response Intrinsic Activity, 2016; 4(Suppl. 2): A18.53 depends on postsynaptic Ih magnitude and is significantly greater in http://www.intrinsicactivity.org/2016/4/S2/A18.53 SNc compared to VTA DAergic neurons. In vivo, local administration of specific Ih blockers causes a DAergic degeneration pattern reminis- Introduction: The psychostimulant methamphetamine increases cent of MPTP-intoxication. synaptic dopamine concentration to produce euphoria and height- Conclusion: These results indicate that Ih regulates synaptic excita- ened energy. Although the abuse potential of this drug is high, only a bility differentially within midbrain DAergic neurons and suggest that subset of users progress to addiction, suggesting that gene and Ih loss of function, possibly resulting from metabolic stress in early environmental factors influence the response to methamphetamine. phases of PD, may act in concert with SNc-specific connectivity to This study used in vivo chronoamperometry and in vitro superfusion promote selective vulnerability. in male adolescent rats to examine the influence of developmental stress and genetic predisposition on methamphetamine-stimulated A18.52 dopamine release and reuptake using a model of attention-deficit Afferent inputs to transmitter-defined cell types in the ventral hyperactivity disorder, the spontaneously hypertensive rat (SHR), tegmental area and Wistar Kyoto (WKY) and Sprague Dawley (SD) comparator Lauren FAGET1,*, Fumitaka OSAKADA2,3, Jinyi DUAN1, Reed strains. RESSLER1, Alexander B. JOHNSON1, James A. PROUDFOOT4, Materials and methods: SHR, WKY and SD litters were exposed to Ji Hoon YOO1, Edward M. CALLAWAY5 and Thomas S. HNASKO1 either standard rearing (nMS) or the maternal separation (MS) model of developmental stress, in which litters are separated from the dam 1University of California, San Diego, CA, United States of America; for 3 hr/day from postnatal day 2 to 14. In vivo chronoamperometry 2Laboratory of Cellular Pharmacology, Graduate School of Pharma- was employed in nMS and MS SHR, WKY and SD rats to determine ceutical Sciences, Nagoya University, Nagoya, Japan; 3Laboratory real-time changes in extracellular striatal dopamine in response to of Neural Information Processing, Institute for Advanced Research, methamphetamine, thereby providing a measure of dopamine trans- Nagoya University, Nagoya, Japan; 4Clinical and Translational porter function and synaptic vesicle dopamine release. Strain Research Institute, University of California, San Diego, La Jolla, CA, differences in striatal [3H]dopamine release in response to meth- United States of America; 5Systems Neurobiology Laboratories, amphetamine were further analysed using in vitro superfusion. Salk Institute for Biological Studies, La Jolla, CA, United States Results: Methamphetamine produced positive deflections from of America baseline dopamine concentration in both chronoamperometric and *E-mail: [email protected] superfusion experiments (p < 0.0001). Analysis of dopamine peak Intrinsic Activity, 2016; 4(Suppl. 2): A18.52 amplitude revealed no significant differences between groups. How- http://www.intrinsicactivity.org/2016/4/S2/A18.52 ever, the chronoamperometry experiments revealed that the k−1 rate The ventral tegmental area (VTA) plays a central role in the neural constant, a measure of the rate at which dopamine is taken up by the circuit control of behavioral reinforcement. Though considered a dopamine transporter, was higher in SHR than WKY (p = 0.0139). dopaminergic nucleus, the VTA contains substantial heterogeneity in No effect of developmental stress on methamphetamine-induced neurotransmitter type, containing also GABA and glutamate neurons. dopamine release was found. Here, we used a combinatorial viral approach using glycoprotein- Conclusions: Methamphetamine administration reliably induced deleted rabies virus (Rb-ΔG) to transsynaptically label afferents to dopamine release in both chronoamperometric and superfusion

experiments producing peaks in dopamine concentration that were permeable L-type Ca2+ channel (LTCC) blockers of the dihydro- equivalent across experimental groups. However, SHR striatum pyridine-type reduce the risk for PD by about 30 %. However, while cleared dopamine at a faster rate than WKY. This suggests that the LTCC blockers are already in clinical trials as neuroprotective PD efficiency of the dopamine transporter may differ between strains. therapy, the age-dependent functional roles of distinct types of VGCCs in SN DA neurons remain unclear. A18.54 We addressed this by combining electrophysiological (patch clamp Dopamine transporter availability cognitive function in different and multi-electrode array approaches) and cell-specific molecular subgroups of alcohol dependence analysis of postnatal juvenile and adult mice with pharmacological Chih-Yun HUANG, Mei-Chen SHIH and San-Yuan HUANG* and genetic tools. Our findings suggest that (CaV1.3) LTCCs are not crucial for SN DA pacemaker-activity at either postnatal age, but their Department of Psychiatry, Tri-Service General Hospital, Taipei, activity rather stabilizes pacemaker-precision. Moreover, they can Taiwan control SN DA activity by sensitizing inhibitory dopamine D -auto- *E-mail: [email protected] 2 receptor (D -AR) responses [1]. In addition, we identified that voltage- Intrinsic Activity, 2016; 4(Suppl. 2): A18.54 2 gated T-type Ca2+ channels (TTCCs) can also modulate SN DA http://www.intrinsicactivity.org/2016/4/S2/A18.54 neuron activity, and they can compensate for CaV1.3 LTCC D2-AR The dopamine transporter (DAT) plays a crucial role in the modulation in SN DA neurons. Accordingly, SN DA neurons from pathogenesis of alcohol dependence (AD) and major depression juvenile CaV1.3 KO mice displayed significantly larger amplitudes of (MD), and males have more risk factors for the development of AD. TTCC-currents. The increase in current was accompanied by a

However, imaging studies on brain DAT availability in males with AD corresponding 2-fold increase in CaV3.1 TTCC mRNA. Moreover, comorbid with MD (AD / MD) are limited, and the association of DAT with unbiased stereology, we detected an about 30 % lower number availability with cognitive function and depressive scores in patients of SN DA neurons already in juvenile CaV1.3 KO mice. In summary, with AD / MD has not been analyzed. Hence, this study examined the the here identified homeostatic interplay of CaV1.3 and CaV3.1 relationship between brain DAT availability, cognitive function and VGCCs, and their complex modulation of SN DA activity pattern depressive symptoms in different subgroups of males with AD. provides new insights into flexible age- and Ca2+-dependent activity Single-photon emission tomography imaging with [99mTc]TRODAT-1 control of SN DA neurons, and has implications for PD pathophysio- as a ligand was used to measure striatal DAT availability in 49 logy and its pharmacological therapy. patients with AD (28 pure AD and 21 AD / MD) and 24 age- and sex- Reference matched healthy volunteers. The Wisconsin Card Sorting Test 1. Dragicevic E, Poetschke C, Duda J, Schlaudraff F, Lammel S, (WCST) and 17-item Hamilton Depression Rating Scale were used to Schiemann J, Fauler M, Hetzel A, Watanabe M, Lujan R, Malenka RC, assess neurocognitive function and depressive scores, respectively. Striessnig J, Liss B: Cav1.3 channels control D2-autoreceptor Patients with AD showed a significant reduction of DAT availability in responses via NCS-1 in substantia nigra dopamine neurons. three brain regions (p < 0.001), and this reduction was more pro- Brain, 2014; 137(8):2287–2302. doi:10.1093/brain/awu131 nounced in the pure AD patients compared to healthy controls. The patients with AD showed significantly poorer performance on the A18.56 WCST, but only in the control group was DAT availability significantly The fast-off hypothesis revisited: a functional kinetic study of negatively correlated with total errors and perseverative errors antipsychotic antagonism of the dopamine D2 receptor (p < 0.001). Kristoffer SAHLHOLM*, Hugo ZEBERG, Johanna NILSSON, Sven Ove ÖGREN, Kjell FUXE and Peter ÅRHEM A18.55 Karolinska Institutet, Stockholm, Sweden Flexible and age-dependent modulation of pacemaker activity *E-mail: [email protected] of substantia nigra dopaminergic neurons by distinct types of Intrinsic Activity, 2016; 4(Suppl. 2): A18.56 voltage-gated calcium channels http://www.intrinsicactivity.org/2016/4/S2/A18.56 Christina PÖTSCHKE1,*, Alina GLEBOVA1, Julia BENKERT1, Antonios DOUGALIS1, Johanna DUDA1, Terrance P. SNUTCH2, Newer, “atypical” antipsychotics carry a lower risk of motor side Jörg STRIESSNIG3 and Birgit LISS1 effects than older, “typical” compounds. It has been proposed that a ~ 100-fold faster dissociation from the dopamine D receptor (D R) 1Institute of Applied Physiology, University of Ulm, Germany; 2 2 distinguishes atypical from typical antipsychotics. Furthermore, 2Michael Smith Laboratories, University of British Columbia, differing antipsychotic D R affinities have been suggested to reflect Vancouver, BC, Canada; 3Department of Pharmacology and 2 differences in dissociation rate constants (k ), while association rate Toxicology, Center for Molecular Biosciences, University of off constants (k ) were assumed to be similar. However, it was recently Innsbruck, Austria on demonstrated that lipophilic accumulation of ligand in the cell interior *E-mail: [email protected] and / or membrane can cause underestimation of k , and as high- Intrinsic Activity, 2016; 4(Suppl. 2): A18.55 off affinity D2R antagonists are frequently lipophilic, this may have been http://www.intrinsicactivity.org/2016/4/S2/A18.55 a confounding factor in previous studies. In the present work, a Dopamine neurons within the substantia nigra (SN DA) are functional electrophysiology assay was used to measure the recovery particularly important, as their selective and progressive degenera- of dopamine-mediated D2R responsivity from antipsychotic antago- tion causes the major motor-related symptoms of Parkinson’s nism, using elevated concentrations of dopamine to prevent the disease (PD). While metabolic stress, mitochondrial dysfunction and potential bias of re-binding of lipophilic ligands. The variability of 2+ impaired Ca homeostasis have been identified as PD trigger antipsychotic kon was also reexamined, capitalizing on the temporal 2+ factors, aging is the most prominent risk factor. Voltage-gated Ca resolution of the assay. kon was estimated from the experimental channels (VGCCs), especially those of the CaV1.3 L-type, generate recordings using a simple mathematical model assumed to describe an activity-related oscillatory Ca2+ burden selectively in SN DA the binding process. The time course of recovery from haloperidol neurons that contributes to their high vulnerability to degeneration. In (typical antipsychotic) was only 6.4–2.5-fold slower than that of the humans, epidemiological studies indicate that blood–brain barrier- atypical antipsychotics amisulpride, clozapine, and quetiapine, while

antipsychotic kons were found to vary more widely than previously

suggested. Finally, affinities calculated using our kon and koff Intrinsic Activity, 2016; 4(Suppl. 2): A18.58 estimates correlated well with functional potency and with affinities http://www.intrinsicactivity.org/2016/4/S2/A18.58 reported from radioligand binding studies. In light of these findings, it Epidermal growth factor (EGF) is one of ligands for the ErbB appears unlikely that typical and atypical antipsychotics are primarily receptors expressed by midbrain dopamine neurons. Excessive distinguished by their D2R binding kinetics. signals of this cytokine EGF in the perinatal stage perturb the postnatal development of dopamine neurons, resulting in the post- A18.57 pubertal behavioral endophenotypes relevant to schizophrenia (such The impact of clozapine on the dopamine D2 receptor binding as reduced PPI and social deficits). To explore the contribution of in ketamine induced attentional set shift task model their dopaminergic abnormality to social behaviors as well as to Marta DUBIEL*, Agata FARON-GÓRECKA, Paulina PABIAN, Maciej stress vulnerability, we investigated the firing activity of dopamine KUŚMIDER, Magdalena KOLASA, Joanna SOLICH, Dariusz ŻURAWEK neurons in the ventral tegmental area (VTA). Single unit recording in and Marta DZIEDZICKA-WASYLEWSKA an anesthetic condition revealed that the mean firing rate of VTA Department of Pharmacology, Institute of Pharmacology, Polish dopaminergic neurons was increased in EGF-pretreated rats (EGF Academy of Sciences, Kraków, Poland rats). Subchronic treatment with the antipsychotic drug risperidone *E-mail: [email protected] ameliorated both social deficits and the firing abnormality. Social Intrinsic Activity, 2016; 4(Suppl. 2): A18.57 defeat stress mimicked the effects of EGF pretreatment; it elevated http://www.intrinsicactivity.org/2016/4/S2/A18.57 the mean firing rate and conversely reduced social interaction scores in control rats. In contrast, the stress responses were less evident in Deficits in cognitive abilities are widely recognized as a core feature those of EGF rats. In order to further explore the above patho- of schizophrenia. One of the cognitive deficits model in rodent is the physiological phenomena, we performed single-unit recordings and attentional set-shifting task (ASST). In this test, the mouse has to in vivo microdialysis in free-moving conditions. We confirmed that the learn to pay attention and respond to the relevant cue (i. e. digging firing activity of EGF rats was higher in the basal state than that of medium) and ignore an irrelevant cue (i. e. odor), and pairing a food controls but was less elevated than control during social interactions. reward with the medium. In the crucial phase (extra dimension shift, In agreement, microdyalysis revealed that dopamine efflux in the EDS) leading dimension (i. e. digging medium) is changed and then medial prefrontal cortex was less responsive to the social interaction the odor becomes a new leading dimension. Ketamine (KET) evokes stimuli. These results suggest that the dynamic range of dopamine- cognitive impairments, observed in the ASST as selective deficits of ergic responses to stressors is one of the crucial determinants for mice EDS performance. Therefore, modulation of the behavioral their social interaction ability and its reduction contributes to the effects induced by KET in ASST provides good animal model to study social negativity of the schizophrenia model. the mechanism of action of antipsychotic agents. First aim of our study was to investigate if clozapine (CLO), an atypical antipsychotic, could reverse ketamine-induced impairments A18.59 + and improve cognitive function. KET in a dose of 20 mg/kg was Kinetics of Na release from the functional Na2 site of the administered repeatedly i.p. for 7 consecutive days, then exchanged dopamine transporter regulated by the N-terminus 1 for CLO in two doses (0.3 and 1 mg/kg, i.p.) for the next 7 days. ASST George KHELASHVILI*, Asghar RAZAVI and Harel WEINSTEIN was performed following 14 days of drug administration. Since the Department of Physiology and Biophysics, Weill Cornell Medical dopamine D2 receptor (D2R) is one of main targets of antipsychotic College, New York, NY, United States of America pharmacotherapy for the treatment of schizophrenia, the second goal *E-mail: [email protected] was to examine the effect of KET and CLO to the D2R binding. In this Intrinsic Activity, 2016; 4(Suppl. 2): A18.59 part of study we used the same paradigm of drug treatment as in the http://www.intrinsicactivity.org/2016/4/S2/A18.59 behavioral study. For the autoradiography study on the mouse brain The dopamine transporter (DAT) belongs to the neurotransmitter / section the [3H]domperidone was used as radioligand. sodium symporter (NSS) family of proteins that are responsible for The biochemical results indicate that KET administered repeatedly reuptake of neurotransmitters from the synaptic cleft, thus helping affects the level of D2 receptors in the lateral striatum. Decrease of terminate a neuronal signal and enabling subsequent neurotrans- D2R after administration of CLO at 0.3 mg was observed in the cortex mitter release from the presynaptic nerve. The release of a sodium and lateral striatum. On the other hand, CLO at 1 mg induced the ion from one of the functional sites identified crystallographically, the increase of D2R in lateral striatum. Repeated administration of KET Na2 site, has been identified as important mechanistic step in the and CLO (0.3 or 1 mg) induced increase of D2R in lateral striatum. In transport cycle, which prepares NSS for substrate translocation by other brain regions an increase of D2R was observed following KET stabilizing an inward-open conformation. Here, we used extensive and CLO (1 mg) administration. The biochemical results seem to molecular dynamics simulations combined with Markov state models confirm the results observed in the ASST test, where CLO (1 mg) to explore the mechanism of Na+ release from the Na2 site of the after ketamine administration potentiated cognitive impairments human dopamine transporter (hDAT). Our results show that the observed in the EDS phase. It seems that the observed effects may initiation of the release process is triggered by hydration of the Na2 be related to the phenomenon of dopamine supersensitivity, which site, concomitant with conformational transition from an outward- can lead to cognitive difficulties. facing to inward-facing state. Using the Markov model approach we Supported by grant NCN OPUS 8 UMO-2014/15/B/NZ7/01019. quantify the kinetics of the release process and identify most probable Na+ release pathways, revealing the importance of various modes of A18.58 interaction of the N-terminus of hDAT in controlling these pathways. Abnormal firing activity of midbrain dopaminergic neurons of a Furthermore, an intermediate state is discovered in the release schizophrenia animal model established by perinatal EGF pathway of Na+ and its kinetics are determined. treatment: implication for their social interaction ability Hidekazu SOTOYAMA*, Hisaaki NAMBA and Hiroyuki NAWA Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan *E-mail: [email protected]

A18.60 Leg Med (Tokyo), 2003; 5(Suppl 1):S221–S224. The dopamine hypothesis is linked with the neural stem cell doi:10.1016/S1344-6223(02)00117-7 dysfunction hypothesis by the D-cell hypothesis (trace amine 6. Kitahama K, Ikemoto K, Jouvet A, Araneda S, Nagatsu I, Raynaud B, hypothesis) in the pathogenesis of schizophrenia Nishimura A, Nishi K, Niwa S: Aromatic L-amino acid Keiko IKEMOTO* decarboxylase-immunoreactive structures in human midbrain, pons, and medulla. J Chem Neuroanat, 2009; 38(2):130–140. Department of Psychiatry, Iwaki Kyoritsu General Hospital, Iwaki, doi:10.1016/j.jchemneu.2009.06.010 Fukushima, Japan 7. Zucchi R, Chiellini G, Scanlan TS, Grandy DK: Trace amine- *E-mail: [email protected] associated receptors and their ligands. Br J Pharmacol, 2006; Intrinsic Activity, 2016; 4(Suppl. 2): A18.60 149(8):967–978. doi:10.1038/sj.bjp.0706948 http://www.intrinsicactivity.org/2016/4/S2/A18.60 8. Wolinsky TD, Swanson CJ, Smith KE, Zhong H, Borowsky B, Seeman Although the molecular basis of mesolimbic dopamine (DA) hyper- P, Branchek T, Gerald CP: The trace amine 1 receptor knockout activity has not yet been clarified, the DA hypothesis [1] has still been mouse: an animal model with relevance to schizophrenia. a major pathophysiological hypothesis of schizophrenia. In the Genes Brain Behav, 2007; 6(7):628–639. congress, the author shows a theory which links mesolimbic DA doi:10.1111/j.1601-183X.2006.00292.x hyperactivity with the neural stem cell (NSC) dysfunction hypothesis 9. Bradaia A, Trube G, Stalder H, Norcross RD, Ozmen L, Wettstein JG, [2] of schizophrenia. A patent cooperation treaty (PCT) patent- Pinard A, Buchy D, Gassmann M, Hoener MC, Bettler B: The selective required histochemical method was used to specify subgroups of so- antagonist EPPTB reveals TAAR1-mediated regulatory called D-neurons (trace amine [TA] neurons) in the human brain [3, 4], mechanisms in dopaminergic neurons of the mesolimbic system. and to show D-neuron decrease in the nucleus accumbens (NAcc) Proc Natl Acad Sci U S A, 2009; 106(47):20081–20086. (D16, subgroup of D-neurons) of postmortem brains with schizo- doi:10.1073/pnas.0906522106 phrenia [5]. It was shown that the human D-neuron system is far more 10. Sanai N, Tramontin AD, Quiñones-Hinojosa A, Barbaro NM, Gupta N, developped in the forebrain in comparison with that of other species, Kunwar S, Lawton MT, McDermott MW, Parsa AT, Manuel-García including non-human primates [6]. The TAAR1 (TA-associated Verdugo J, Berger MS, Alvarez-Buylla A: Unique astrocyte ribbon in receptor type 1), the exclusive receptor of trace amines in humans, is adult human brain contains neural stem cells but lacks chain shown to have a large number of ligands, including tyramine, migration. Nature, 2004; 427(6976):740–744. β-phenylethylamine and methamphetamine which influence on doi:10.1038/nature02301 human mental states [7]. The “D-cell hypothesis (TA hypothesis)” is 11. Ikemoto K: [Localization and functions of the D-neuron: that the striatal D-neuron decrease in schizophrenia and consequent significance in pathogenesis of schizophrenia]. TAAR1 stimulation decrease onto terminals of midbrain ventral Nihon Shinkei Seishin Yakurigaku Zasshi, 2013; 33(4):141–147. tegmental area (VTA) DA neurons induces mesolimbic DA hyper- 12. Kippin TE, Kapur S, van der Kooy D: Dopamine specifically inhibits activity [8, 9] of schizophrenia. Dysfunction of subventricular NSC, forebrain neural stem cell proliferation, suggesting a novel effect located in NAcc [10], is the cause of D-neuron decrease in NAcc [11]. of antipsychotic drugs. J Neurosci, 2005; 25(24):5815–5823. DA hyperactivity, which inhibits NSC proliferation [12], causes doi:10.1523/JNEUROSCI.1120-05.2005 disease progression of schizophrenia. The highlight is the rational 13. Revel FG, Moreau JL, Pouzet B, Mory R, Bradaia A, Buchy D, Metzler that the “D-cell hypothesis of schizophrenia” is a pivotal theory to link V, Chaboz S, Groebke Zbinden K, Galley G, Norcross RD, Tuerck D, NSC dysfunction hypothesis to the DA hypothesis. From a therapeu- Bruns A, Morairty SR, Kilduff TS, Wallace TL, Risterucci C, Wettstein tic direction, (1) TAAR1 agonists or TAAR1 partial agonists [13], JG, Hoener MC: A new perspective for schizophrenia: TAAR1

(2) DA D2 antagonists, and (3) neurotropic substances (e. g. brain- agonists reveal antipsychotic- and antidepressant-like activity, derived neurotrophic factor (BDNF), lithium, anticonvulsants and improve cognition and control body weight. Mol Psychiatry, 2013; antidepressants) have potential to normalize mesolimbic DA hyper- 18(5):543–556. doi:10.1038/mp.2012.57 activity. To further develop novel therapeutic strategies, TAAR1 signals, TAAR1 ligands, and NSC and D-neuron pathophysiology of A18.61 neuropsychiatric illnesses remain to be explored. Accumbal D-neuron Role of dopamine D2 receptors in human reinforcement decrease is a key to mesolimbic DA hyperactivity of schizophrenia. learning References Christoph EISENEGGER1,*, Michael NAEF2, Anke LINSSEN3, 1. Hökfelt T, Ljungdahl A, Fuxe K, Johansson O: Dopamine nerve Luke CLARK4, Ulrich MÜLLER5 and Trevor W. ROBBINS6 terminals in the rat limbic cortex: aspects of the dopamine 1Department of Basic Psychological Research and Research hypothesis of schizophrenia. Science, 1974; 184(4133):177–179. Methods, University of Vienna, Austria; 2Department of Economics, doi:10.1016/0014-2999(74)90101-0 Royal Holloway, University of London, Egham, United Kingdom; 2. Reif A, Fritzen S, Finger M, Strobel A, Lauer M, Schmitt A, Lesch KP: 3Department of Neuropsychology and Psychopharmacology, Neural stem cell proliferation is decreased in schizophrenia, but University of Maastricht, The Netherlands; 4Centre for Gambling not in depression. Mol Psychiatry, 2006; 11(5):514–522. Research at UBC, Department of Psychology, University of British doi:10.1038/sj.mp.4001791 Columbia, Vancouver, BC, Canada; 5Cambridgeshire and 3. Ikemoto K, Kitahama K, Jouvet A, Arai R, Nishimura A, Nishi K, Peterborough NHS Foundation Trust, Adult ADHD Service, Nagatsu I: Demonstration of L-dopa decarboxylating neurons Cambridge, United Kingdom; 6Behavioural and Clinical specific to human striatum. Neurosci Lett, 1997; 232(2):111–114. Neuroscience Institute and Department of Psychology, University of doi:10.1016/S0304-3940(97)00587-9 Cambridge, United Kingdom 4. Ikemoto K: Involvement of so-called D-neuron (trace amine neuron) *E-mail: [email protected] in pathogenesis of schizophrenia: D-cell hypothesis. In: Farooqui Intrinsic Activity, 2016; 4(Suppl. 2): A18.61 T, Farooqui AA (eds): Trace Amines and Neurological Disorders: http://www.intrinsicactivity.org/2016/4/S2/A18.61 Potential Mechanisms and Risk Factors. 1st edn., Academic Press, 2016 (in press). Influential neurocomputational models emphasize dopamine (DA) as 5. Ikemoto K, Nishimura A, Oda T, Nagatsu I, Nishi K: Number of striatal an electrophysiological and neurochemical correlate of reinforcement D-neurons is reduced in autopsy brains of schizophrenics. learning. However, evidence of a specific causal role of DA receptors

in learning has been less forthcoming, especially in humans. Here, A18.63 we combine, in a between-subjects design, administration of a high Dominant negative variant of the dopamine transporter dose of the selective DA D2/3 receptor antagonist sulpiride with associated with early-onset parkinsonism and psychiatric genetic analysis of the DA D2 receptor in a behavioral study of disease reinforcement learning in a sample of 78 healthy male volunteers. In Freja HANSEN1,*, Natascha ARENDS1, Sasha TOLSTOY1, contrast to predictions of prevailing models emphasizing DA’s pivotal Mattias RICKHAG1, Troels RAHBEK-CLEMMENSEN1, Kevin ERREGER2, role in learning via prediction errors, we found that sulpiride did not Lars FRIBERG3, Aurelio GALLI4, Lisbeth MØLLER5, Lena HJERMIND6 disrupt learning, but rather induced profound impairments in choice and Ulrik GETHER1 performance. The disruption was selective for stimuli indicating 1Department of Neuroscience and Pharmacology, University of reward, whereas loss avoidance performance was unaffected. Copenhagen, Denmark; 2Department of Physiology and Biophysics, Effects were driven by volunteers with higher serum levels of the Weill Medical College of Cornell University, New York, NY, United drug, and in those with genetically determined lower density of striatal States of America; 3Department of Neurology, Bispebjerg Hospital, DA D receptors. This is the clearest demonstration to date for a 2 Copenhagen University Hospital, Denmark; 4Department of causal modulatory role of the DA D receptor in choice performance 2 Molecular Physiology and Biophysics, Vanderbilt University, that might be distinct from learning. Our findings challenge current Nashville, TN, United States of America; 5Center for Applied reward prediction error models of reinforcement learning, and Human Genetics, Kennedy Center, Copenhagen University suggest that classical animal models emphasizing a role of post- Hospital, Glostrup, Denmark; 6Department of Neurology, synaptic DA D receptors in motivational aspects of reinforcement 2 Copenhagen University Hospital and Department of Cellular learning may apply to humans as well. and Molecular Medicine, Section of Neurogenetics, University of Copenhagen, Denmark A18.62 *E-mail: [email protected] Dissociable effects of dopamine and serotonin on goal-directed Intrinsic Activity, 2016; 4(Suppl. 2): A18.63 action initiation and inhibition http://www.intrinsicactivity.org/2016/4/S2/A18.63 Oliver HÄRMSON*, Laura Lucy GRIMA, Sean James FALLON, Masud HUSAIN and Mark WALTON The dopamine transporter (DAT) exerts a critical function in dopamine homeostasis by mediating reuptake of dopamine for Department of Experimental Psychology, University of Oxford, subsequent storage and release. An increasing number of missense United Kingdom mutations in the dopamine transporter (DAT) have been identified in *E-mail: [email protected] patients suffering from psychiatric disorders. Moreover, homozygote Intrinsic Activity, 2016; 4(Suppl. 2): A18.62 and compound heterozygote loss-of-function mutations in DAT have http://www.intrinsicactivity.org/2016/4/S2/A18.62 been associated with infantile / childhood onset parkinsonism-dys- Dopamine (DA) and serotonin (5-HT) neurotransmission have tonia. We recently described the first patient, carrying DAT missense commonly been associated with, respectively, reward-related initia- mutations, who suffered from both early-onset parkinsonism and tion and inhibition of responses. However, the roles of DA and 5-HT ADHD. Here, we further expand the clinical spectrum of DAT- transmission have seldom been studied in a context where animals associated disease by presenting an additional patient that presented have to switch between goal-directed inhibition or initiation of actions with the unique combination of early-onset parkinsonism and on a trial-by-trial basis. concurrent psychiatric disorder. This patient is heterozygote for a We therefore investigated the effects of manipulation of DA and 5-HT missense mutation in the C-terminal PDZ-binding domain of DAT. A neurotransmission on a symmetrically reinforced Go / No-Go task. To characterization of the mutant in heterologous cells revealed reduced first look at the role of DA receptor signalling in goal-directed action dopamine uptake capacity (60 % of WT DAT), attenuated amphet- initiation and inhibition, we examined the individual and combined amine-induced efflux, and slightly reduced expression of DAT- effects of blocking (D1/D2-like receptor antagonist flupenthixol, K619N. Strikingly, the mutant exerts a dominant negative effect on 0.5 mg/kg) or enhancing dopamine (amphetamine, 1.5 mg/kg). WT DAT upon viral expression in mice, seen as a significant reduction Amphetamine administration had a detrimental effect on No-Go trial in synaptosomal dopamine uptake. Furthermore, two SPECT scans performance. In contrast, flupenthixol administration reduced accura- of the patient performed seven years apart, uncovered reduced DAT cy on Go trials. The attenuation of No-Go responding is causally binding along with a mild progressive worsening during the seven- linked to DA receptor signalling, as No-Go responding was rescued year period. The identification of yet another patient with DAT- when flupenthixol was administered prior to amphetamine. associated parkinsonism and neuropsychiatric disorder further To contrast the role of 5-HT neurotransmission in goal-directed action supports that abnormal DAT function may constitute a risk factor for inhibition and initiation, we also tested the effect of a 5-HT2C receptor both psychiatric disorders and parkinsonism. selective ligand SB 242084 (0, 0.1, 0.5 mg/kg, i.p.), which is known to influence dopamine neuron activity and dopamine release. A18.64 SB 242084 robustly reduced the animals’ ability to withhold move- Synaptic vesicle glycoprotein 2C modulates vesicular ment for reward, but only in the case of small reward trials. In dopamine release and contributes to behaviors associated contrast, SB 242084 significantly increased accuracy on Go trials. with reward This improvement in performance was due to a significant decrease Kristen STOUT*, Amy DUNN, Minzheng WANG, and Gary MILLER in Go trial response latency without a detrimental effect on choice Department of Molecular and Systems Pharmacology, Emory precision, breaking the speed-accuracy trade-off. These results point University, Atlanta, GA, United States of America to a role for the 5-HT receptor in promoting goal-directed action 2C *E-mail: [email protected] initiation, particularly for less rewarding options. Considered in the Intrinsic Activity, 2016; 4(Suppl. 2): A18.64 light of previous work showing attenuation of dopamine release when http://www.intrinsicactivity.org/2016/4/S2/A18.64 withholding an action, the findings suggest that serotonin may also play an important role in inhibition for reward via modulation of the The synaptic vesicle glycoprotein 2C (SV2C) localizes to synaptic dopaminergic system. vesicles within the basal ganglia. The importance of this protein and its contribution to reward behavior, which is dictated by basal

ganglia structures, are unknown. To investigate these questions, we A18.66 generated mice with genetically ablated SV2C (SV2C-KO). SV2C-KO A tamoxifen analog is an asymmetric dopamine transporter animals have reduced dopamine tone compared to wild-type mice, modulator and reduces amphetamine in vitro and in vivo shown by: decreased stimulated dopamine release in the ventral effects striatum and ventral pallidum by fast-scan cyclic voltammetry, Colleen CARPENTER1,*, Alexander ZESTOS2, Roderick SORENSON3, diminished radioactive vesicular uptake storage capacity, reduced Robert KENNEDY2, Hollis SHOWALTER3 and Margaret GNEGY1 total dopamine level by high performance liquid chromatography, and 1Department of Pharmacology, University of Michigan, Ann Arbor, lessened methamphetamine-stimulated dopamine overflow by micro- MI, United States of America; 2Department of Chemistry, University dialysis. Behavioral metrics support the observed neurochemical of Michigan, Ann Arbor, MI, United States of America; 3Vahlteich deficits; SV2C-KO animals have decreased methamphetamine- Medicinal Chemistry Core, Ann Arbor, MI, United States of America stimulated locomotor activity, conditioned place preference, and *E-mail: [email protected] locomotor sensitization compared with their wild-type counterparts. Intrinsic Activity, 2016; 4(Suppl. 2): A18.66 The mechanism by which SV2C augments vesicular capacity was http://www.intrinsicactivity.org/2016/4/S2/A18.66 also investigated. SV2C is a heavily glycosylated protein. Glyco- proteins within the vesicle lumen are hypothesized to interact with The reinforcing properties of amphetamine (AMPH) stem from its dopamine, removing it from the concentration gradient, and allowing reversal of the dopamine transporter (DAT) which greatly increases for enhanced vesicular packaging. To investigate the contribution of extracellular dopamine (DA) in the brain. Inhibition of protein kinase C glycosylation to SV2C function, we compared radioactive uptake from (PKC) reduces these AMPH effects. The CNS-permeant estrogen cells expressing recombinant site-directed SV2C glycomutants receptor (ER) modulator, tamoxifen, inhibits PKC and reduces AMPH versus normal SV2C. Additionally, SV2C may be involved in vesicle effects. Therefore, revisiting the tamoxifen scaffold may fill the unmet trafficking, particularly in regulation of recruitment of vesicles from need for a therapeutic against AMPH abuse. reserve pools. We addressed this mechanism using stimulus trains We aim to utilize reported structure–activity relationship studies to to deplete vesicular stores in brain slices from WT and SV2C-KO create CNS-permeant tamoxifen analogs with increased selectivity animals using fast-scan cyclic voltammetry. The above data indicate for PKC inhibition and reduced ER affinity. Here, we evaluate that SV2C is an important player in dopamine-mediated reward reduction in AMPH-stimulated neurochemical and behavioral effects behavior. in rats by our lead compound, CCG-215103. We also investigated more thoroughly the effects of CCG-215103 on DAT. A18.65 CCG-215103 inhibits PKC activity (IC50, 0.2 μM) but does not bind Characterization of [3H]LS-3-134, a novel arylamide to ERα at ≤ 3 μM. Further, CCG-215103 did not inhibit other kinase modulators of DAT, namely Ca2+ / calmodulin-dependent protein phenylpiperazine D3 dopamine receptor-selective radioligand Robert R. LUEDTKE1, Claudia RANGEL-BARAJAS1, Maninder MALIK1, kinase II and protein kinase B. We find that CCG-215103 is an Michelle TAYLOR1 and Robert H. MACH2,* asymmetric DAT modulator because it more potently blocks dopamine efflux than uptake. Treatment of rat striatal synaptosomes 1University of North Texas Health Science Center, Fort Worth, TX, with 0.3–3 μM CCG-215103 robustly reduces DA efflux by 10 μM United States of America; 2Department of Radiology, University of AMPH. However, only 3 µM CCG-215103 blocks [3H]DA uptake. Pennsylvania, Philadelphia, PA, United States of America CCG-215103 does not displace [3H]WIN 35428 binding showing that *E-mail: [email protected] it does not bind near the active site of DAT. Injection of 1 μM Intrinsic Activity, 2016; 4(Suppl. 2): A18.65 CCG-215103 into the nucleus accumbens reduces by ~ 40% both DA http://www.intrinsicactivity.org/2016/4/S2/A18.65 release and locomotion induced by 2 mg/kg i.p. AMPH measured with

We reported that LS-3-134 exhibits (1) high affinity binding (Ki value microdialysis in freely-moving rats. Yet, CCG-215103 does not

0.2 nM) at human D3 dopamine receptors, (2) > 150-fold D3 vs. D2 increase basal DA or locomotion in vivo, suggesting it will not have dopamine receptor subtype binding selectivity, (3) > 1400-fold selec- an abuse potential. tivity for D3 vs. D4 and (4) low-affinity binding (Ki values > 5,000 nM) The novel compound CCG-215103 reduces both AMPH in vitro and at σ1 and σ2 receptors. Based upon a forskolin-dependent activation in vivo effects with a bias towards efflux. CCG-215103 action could of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial be due to allosteric modulation of DAT and/or its interaction with a agonist at both D2 and D3 dopamine receptor subtypes (29 % and mediator of DAT functioning, such as PKC. 35 % of full agonist activity, respectively). Funded by NIH grant 1R01 DA11697, HHMI International Student [3H]-labeled LS-3-134 was prepared and evaluated to further Research Fellowship, UM EDGE award. characterize its use as a D3 dopamine receptor-selective radioligand. This radioligand rapidly reaches equilibrium (10–15 min at 37 °C) and A18.67 it binds with high affinity to both human (Kd = 0.06 ± 0.01 nM) and rat Dopamine gene therapy for Parkinson’s disease: preclinical ® (Kd = 0.2 ± 0.02 nM) D3 receptors expressed in HEK 293 cells. safety and efficacy of OXB-102, the enhanced ProSavin vector Scatchard transformation of data from direct binding studies exhibited Romina ARON-BADIN1,*, Katie BINLEY2, Nadja VAN CAMP1, 2 linear correlation coefficients with r values > 0.95. Competitive radio- Caroline JAN1, Jeanne GOURLAY1, Hannah J. STEWART2, ligand binding studies using rat caudate and nucleus accumbens Scott RALPH2, Yatish LAD2, Koichi HOSOMI3, Stéphane PALFI3, 3 tissue indicate that [ H]LS-3-134 selectively binds a homogeneous Philippe HANTRAYE1 and Kyriacos A. MITROPHANOUS2 population of binding sites with a dopamine D receptor pharmaco- 3 1Molecular Imaging Research Center (MIRCen), CEA, Fontenay- logical profile. Based upon these studies we propose that [3H]LS-3- aux-Roses, France; 2Oxford BioMedica (UK) Ltd, Oxford, United 134 represents a novel D dopamine receptor-selective radioligand 3 Kingdom; 3Department of Neurosurgery, Henri Mondor Hospital, that can be used to study the expression and regulation of the D 3 APHP / UPEC, Créteil, France dopamine receptor subtype. It also provides supportive evidence *E-mail: [email protected] for using [18F]LS-3-134 as a radiotracer for imaging D dopamine 3 Intrinsic Activity, 2016; 4(Suppl. 2): A18.67 receptors independently of the D receptor subtype in vivo with PET. 2 http://www.intrinsicactivity.org/2016/4/S2/A18.67

ProSavin® is an EIAV lentiviral-based vector expressing the 3 key exhibited behavioral slowing when challenged by a motivational enzymes in dopamine biosynthesis: tyrosine hydroxylase, dopamine context that required vigilance, i. e. after priming a past prevention decarboxylase (AADC), and GTP cyclohydrolase. The therapeutic failure. By contrast, Met / Met individuals, typically characterized by action of vector-mediated dopamine production in non-dopaminergic relative cognitive rigidity, exhibited behavioral slowing in response to cells of the sensorimotor putamen has been demonstrated in rodents, a context that required eagerness and openness to opportunities for non-human primates (NHPs) and, more recently, in Parkinson’s positive feedback, i. e. after priming a past promotion failure. The disease (PD) patients. Oxford BioMedica has now generated OXB- findings suggest facilitation effects between COMT Val / Val genotype 102, an improved version of ProSavin®, expressing the same and promotion goal pursuit, and between COMT Met / Met genotype enzymes but able to generate 5–10 times more dopamine per and prevention goal pursuit. Individual differences in cognitive genetically modified cell. flexibility appear to interact with self-regulatory context in a way that In order to assess the behavioural efficacy and safety of OXB-102 can be adaptive or maladaptive depending on match with task compared to ProSavin®, two MRI-guided bilateral stereotaxic demands. injections of null vector (n = 4), ProSavin® (n = 4), and OXB-102 at a high (n = 4) or low dose (n = 4) were performed into the putamen of A18.69 stably parkinsonian MPTP NHPs. The follow-up consisted of Orexin A in the ventral tegmental area enhances saccharin- longitudinally clinical ratings and video-based analysis of motor induced conditioned flavor preference in a dose-dependent 18 activity before and after vector administration. [ F]FMT positron manner emission tomography (PET) scans were performed before and after Cristina MEDIAVILLA1,* and Severiano RISCO2 vector delivery to specifically assess striatal AADC metabolic activity 1Department of Psychobiology, and Mind, Brain, and Behavior changes in vivo. MRI scans were acquired after surgery to assess Research Center (CIMCYC), University of Granada, Spain; potential adverse reactions. 2Department of Pharmacology, and Centro de Investigación Behavioural results at 6 months post-administration showed similar Biomédica (CIBM), University of Granada, Spain efficacy with all therapeutic vectors compared to the null vector- *E-mail: [email protected] treated controls that remained parkinsonian. The quantification of Intrinsic Activity, 2016; 4(Suppl. 2): A18.69 activity showed that OXB-102 delivered at a low dose (at which http://www.intrinsicactivity.org/2016/4/S2/A18.69 ProSavin® was previously not efficacious) is efficacious at increasing locomotor activity. [18F]FMT PET showed that OXB-102 administra- It has been suggested that the orexinergic system intervenes in tion generated higher AADC activity than ProSavin®, which was reward processes through excitatory projections to the ventral further confirmed in post-mortem analysis by a qualitatively greater tegmental area (VTA) and that orexin may contribute to obesity and AADC immunoreactivity in OXB-102-treated NHPs. addiction by modifying mesolimbic dopaminergic system activity. A 6-month toxicology and biodistribution study in naïve NHPs further Orexin neurons of the lateral hypothalamus (LH) are also known to confirms that the OXB-102 vector is safe and well tolerated following play a role in reward-related learning through these anatomical bilateral stereotactic administration into the putamen, without connections with the VTA. Thus, LH and VTA orexin neurons may be evidence of spreading beyond the site of administration. The GMP critical regions in reward-based learning and memory. In fact, the manufacture of OXB-102 is complete and a clinical trial in PD patients orexinergic system is involved in conditioned place preference (CPP) will initiate in 2016. and conditioned flavor preference (CFP). With this background, we investigated whether orexin in the VTA facilitates the acquisition of a A18.68 saccharin-induced CFP. This type of learning develops a preference Individual differences (COMT Val158Met genotype) in cognitive for a neutral flavor that is associated with a previously preferred taste. flexibility interact with self-regulatory context to predict Male Wistar rats were unilaterally implanted by a cannulae into the divergent task performance VTA. The animals were deprived of water and trained to drink tap Elena DAVIS1, Ian THARP2,*, Ahmad HARIRI3 and Timothy STRAUMAN3 water for 15 min a day. Water consumption, food intake and body weights were measured daily and a period of re-hydration took place 1Department of Psychiatry and Behavioral Sciences, Stanford in the afternoon. After the training sessions, animals underwent four University, Stanford, CA, United States of America; 2Department of acquisition sessions (eight days) to develop flavor preference. Two Psychology, University of Greenwich, United Kingdom; 3Department doses (53 and 107 ng in 0.3 µl) of orexin-A or saline (control group) of Psychology and Neuroscience, Duke University, Durham, NC, were unilaterally administered into the ATV 10 min prior to a 15-min United States of America flavor intake period. The preference was tested on day 9 and 10 by *E-mail: [email protected] two bottle-test sessions containing flavors used for CS+ and CS− in Intrinsic Activity, 2016; 4(Suppl. 2): A18.68 a counterbalanced left-right position. Our results show that orexin-A http://www.intrinsicactivity.org/2016/4/S2/A18.68 in the ATV increases CFP in a dose-dependent manner and may The present study investigated how a specific, challenging self- contribute to the positive reinforcing signals required to develop a regulatory context can impact cognitive flexibility based on genotype taste preference. Further studies are required to determine the in the COMT Val158Met functional polymorphism. The study used a relationships between dopaminergic and orexinergic systems in taste cognitive priming technique to create a motivationally salient self- learning. regulatory context of either promotion goal pursuit (related to ideals) Supported by MEYC grant PSI2012-37987. or prevention goal pursuit (related to oughts) to assess the impact of individual differences in cognitive flexibility on task performance. We A18.70 used the COMT genotype to index individual differences in cognitive Effects of alpha-synuclein fibrils on dopamine flexibility that are associated with Val- or Met-like dopaminergic neurodegenration in young and one-year-old mice: signaling profiles. We predicted a trade-off in the adaptiveness of neuroprotective study with CDNF relative cognitive flexibility vs. stability depending on the self-regula- Katrina ALBERT1,*, Merja H. VOUTILAINEN1, Jenni SIEKKINEN1, tory context of either promotion or prevention, and our results showed Kelvin C. LUK2, Virginia M.-Y. LEE2, Mart SAARMA1 and that genotype group indeed modulated task performance. Val / Val Mikko AIRAVAARA1 individuals, typically characterized by relative cognitive flexibility,

1Institute of Biotechnology, University of Helsinki, Finland; striatal projection target specificity, and large sampling region 2Department of Pathology and Laboratory Medicine, Institute necessary to adequately assess functional dynamics across popula- on Aging and Center for Neurodegenerative Disease Research, tions of dopamine axons projecting to specific striatal subregions. To University of Pennsylvania School of Medicine, Philadelphia, PA, overcome these limitations, we have utilized a new 2-photon calcium United States of America imaging approach to measure dynamics across the dopaminergic *E-mail: [email protected] projection population to dorsal striatum with single axon resolution in Intrinsic Activity, 2016; 4(Suppl. 2): A18.70 behaving mice. This method has revealed phasic, acceleration- http://www.intrinsicactivity.org/2016/4/S2/A18.70 locked dopaminergic signals that indicate a role for dopamine in modulating rapid changes in motor output, independently of reward Parkinson’s disease is a progressive neurodegenerative disorder that contingencies. Moreover, we have identified significant functional and affects over 7 million people worldwide. Alpha-synuclein (α-syn) is anatomical heterogeneity in the axonal projection population with mutated in certain patients with familial forms of Parkinson’s disease respect to acceleration and unpredicted reward signaling. Based on (PD), and it is a major component of insoluble protein aggregates, these findings, we propose a new model for dopamine signaling in called Lewy bodies, present in all patients. Therefore, α-syn is the striatum that explains the dual roles of dopamine in regulating strongly considered to be one of the major causes of dopamine movement and reinforcement learning. neuron degeneration in PD. Injection of preformed fibrils consisting of purified α-syn (PFFs) into rodents is a promising model of the disease due to the presence of Lewy bodies and the slow onset of A18.72 motor symptoms. Long-term impact of sucrose overconsumption at adolescence Recently discovered cerebral dopamine neurotrophic factor (CDNF) on the mesolimbic dopamine system 1, 1 1 has been shown to be neurorestorative in neurotoxin models of PD. Fabien NANEIX *, Florence DARLOT , Coralie HERROUIN , 2 2 1 Importantly, it is able to rescue neurons from endoplasmic reticulum- Adeline CATHALA , Umberto SPAMPINATO , Étienne COUTUREAU 1 induced cell death, a process associated with aggregation of α-syn. and Martine CADOR The objective was first to create an α-syn-based model of dopamine 1Institut de neurosciences cognitives et intégratives d'Aquitaine neurodegeneration in our laboratory using PFFs and then to test the (INCIA), UMR 5287 CNRS, Université de Bordeaux, France; ability of CDNF to decrease α-syn-induced neurodegeneration. 2NeuroCentre Magendie, Physiopathologie de la plasticité Mouse PFFs were injected to the striatum of 3-month- or 1-year-old neuronale, INSERM U862, Université de Bordeaux, France mice and their behaviour was measured at 1, 3 and 6 months. At *E-mail: [email protected] 1 month, CDNF was also injected to the striatum. For behaviour, Intrinsic Activity, 2016; 4(Suppl. 2): A18.72 cylinder test, rotarod, wire hanger and beam walking tests were http://www.intrinsicactivity.org/2016/4/S2/A18.72 performed. Brains were immunostained for tyrosine hydroxylase (TH) Adolescence represents a critical developmental period character- and α-syn. ized by major neurobiological changes which allow the transition into From 1 to 3 months there was a tendency for PFF-injected mice to adulthood. However this brain immaturity also constitutes a vul- stay on the rotarod for less time, indicating motor deficits. For the nerability window to external insults. Several evidences demonstrate cylinder test performed at 3 months with mice injected with CDNF or that adolescents are more sensitive to rewarding effects of palatable vehicle, CDNF-injected mice showed significant restoration in food like sweet diet. However, the protracted impact of the ipsilateral vs. contralateral paw use (i. e. mice were using both paws (over)consumption of these rewards remains unclear. Interestingly, equally). The direct outcome measures for quantifying dopamine loss the dopamine (DA) system, which plays a central role in reward- are striatal dopamine concentration, TH optical density and number related processes, presents a delayed maturation during adoles- of TH+ cells from substantia nigra, and will be presented in the poster. cence. Based on this, we hypothesize that sucrose consumption during adolescence could alter reward processes and dopamine A18.71 system. Calcium imaging of dopaminergic projections to dorsal Adolescent rats were given free and continuous access to water and striatum with single-axon resolution in behaving animals a 5 % sucrose solution from post-natal day 30 to 46. At adulthood Mark HOWE* and Daniel DOMBECK (> 70 days), we investigated the impact of adolescence diet on Department of Neurobiology, Northwestern University, Evanston, IL, behavioral mechanisms associated with sweet reward processing United States of America and on the functioning of DA system. We first demonstrated that *E-mail: [email protected] sucrose-exposed rats consumed less sweet rewards than control Intrinsic Activity, 2016; 4(Suppl. 2): A18.71 water-exposed rats in a free-access consumption test. This decrease http://www.intrinsicactivity.org/2016/4/S2/A18.71 was associated with a lower c-Fos immunostaining in both the nucleus accumbens and the ventral tegmental area. Moreover, we Dopamine signaling in the striatum has long been recognized as observed using microdialysis that sucrose-exposed rats present a critical for regulating movement on immediate timescales, and for lower release of DA in the nucleus accumbens during sucrose driving learning-related plasticity over extended timescales. Current consumption. theories of dopamine function, derived primarily from midbrain In a second experiment, we found that sucrose-exposed rats present electrophysiological recordings in the midbrain SNc and VTA, a decreased motivation to obtain sweet rewards in a progressive ratio propose that dopamine neurons homogenously transmit phasic task. Interestingly, systemic injection of the DA D1 agonist SKF-38393 (hundreds of ms) signals to unpredicted rewards or reward-predictive induced dose-dependent motivational changes in control rats but not cues. These signals are believed to enhance representations of in sucrose-exposed rats, which is consistent with the decrease in the environmental stimuli or actions that lead to reward, driving future expression of D1 receptor in the nucleus accumbens that we recently goal-directed learning and behavior. Regulation of ongoing move- reported. Taken together, these results demonstrate that adolescent ment, on the other hand, is believed to be enabled by the ongoing sugar diet induces long-term deficits in consummatory and motiva- tonic or slowly varying (several seconds) firing within these same tional responses to sweet reward related to a hypo-functioning of the neurons. Recent electrophysiological and voltammetric recordings in DA system. different striatal subregions have called these theories into question, but lack the combination of fine (micron scale) spatial resolution,

Finally, we are currently trying to restore a normal consummatory and compacta (SNc), a separate dopaminergic region with distinct motivational behavior in sucrose-exposed rats using pharmaco- efferent and afferent projections, toward guiding motivated behavior. genetic reversible stimulation (activatory DREADD hM3Dq) of the However, it is not known whether dopamine neurons in the VTA and mesolimbic pathway. SNc process reward-related events in the same manner. Here we aimed to directly compare the response pattern of VTA and SNc A18.73 neurons within the same animal during reward-mediated instrumental behavior. Adult rats were implanted bilaterally with fixed micro- The interactive role of dopamine D1 and adenosine A1 receptors in the nucleus accumbens on methamphetamine relapse electrode arrays to conduct single-unit recordings in the VTA and SNc Ryan BACHTELL* and Madeline WINKLER through task acquisition and maintenance. During this task, rats learned to execute a naturalistic nose poke after presentation of a Department of Psychology and Neuroscience, University of cue to receive a single sugar pellet. Electrophysiological data from Colorado, Boulder, CO, United States of America these animals demonstrate that neurons within the VTA and SNc with *E-mail: [email protected] dopamine-like characteristics exhibit similar phasic responses to Intrinsic Activity, 2016; 4(Suppl. 2): A18.73 cues predicting the availability of reward and reward delivery. This http://www.intrinsicactivity.org/2016/4/S2/A18.73 parallel population activity persists across all sessions of the task. Methamphetamine abuse is a global health problem, however the Additionally, baseline firing rates for both regions are comparable. neuropharmacological mechanisms of its use and abuse are not well These data suggest that reward responsiveness of VTA putative understood. Previous work has shown that dopamine and adenosine dopamine neurons generalizes to the SNc. This finding implies that receptors are important for methamphetamine’s rewarding and dopamine neurons supply similar information about reward-related reinforcing effects. The studies presented here examine the stimuli to both the mesolimbic and nigrostriatal pathways. Ongoing interactive role of dopamine and adenosine receptors in the nucleus work is extending these studies to adolescent animals to further accumbens (NAc) on the reinstatement of methamphetamine seeking understand how reward-related learning develops in dopaminergic following extinction. Rats were trained to lever press for methamphet- regions. amine in daily 2-hr self-administration sessions on a fixed-ratio-1 schedule for 10 consecutive days. Lever pressing was extinguished A18.75 in at least 6 daily extinction sessions prior to reinstatement testing. TAAR1 modulates cortical glutamate NMDA receptor function We first assessed the effects of adenosine A1 or A2A receptor Stefano ESPINOZA1,*, Gabriele LIGNANI1, Lucia CAFFINO2, Silvia stimulation on reinstatement induced by a methamphetamine prime MAGGI1, Ilya SUKHANOV1, Lucian MEDRIHAN1, Anja HARMEIER3, (1.0 mg/kg, i.p.). Systemic administration of the adenosine A1 agonist, Valter TUCCI1, Marius C. HOENER3, Fabio BENFENATI1, Fabio CPA, but not the adenosine A2A agonist, CGS 21680, dose- FUMAGALLI2 and Raul GAINETDINOV1 dependently inhibited methamphetamine-induced reinstatement. We 1Neuroscience and Brain Technologies, Istituto Italiano di then assessed whether stimulation of dopamine D or D receptors in 1 2 Tecnologia, Genova, Italy; 2Dipartimento di Scienze Farmacologiche the NAc is sufficient to induce methamphetamine seeking. Micro- e Biomolecolari, Università degli Studi di Milano, Italy; infusion of a dopamine D agonist (SKF 81297), but not a dopamine 1 3Neuroscience Research, Pharmaceuticals Division, F. Hoffmann- D agonist (quinpirole), exhibited a dose-dependent increase in 2 La Roche Ltd., Basel, Switzerland methamphetamine seeking. We then identified whether SKF 81297- *E-mail: [email protected] induced reinstatement would be altered by co-administration with an Intrinsic Activity, 2016; 4(Suppl. 2): A18.75 adenosine A agonist (CPA) in the NAc. SKF 81297-induced re- 1 http://www.intrinsicactivity.org/2016/4/S2/A18.75 instatement was blunted when CPA was co-administered in the NAc.

Lastly, we evaluated the effects of an adenosine A1 antagonist Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled (DPCPX) administered into the NAc alone or in combination with receptor expressed in the mammalian brain and known to influence SKF 81297. DPCPX had no effect when administered alone, but subcortical monoaminergic transmission. Monoamines, such as significantly enhanced SKF 81297-induced methamphetamine seek- dopamine, play also an important role within the prefrontal cortex ing. Collectively, these data suggest that dopamine D1 and adenosine (PFC) circuitry, which is critically involved in high order cognitive

A1 receptors in the NAc play an interactive role in methamphetamine- processes. TAAR1-selective ligands have shown potential anti- seeking behavior. psychotic, antidepressant and pro-cognitive effects in experimental animal models; however, it remains unclear if TAAR1 can affect PFC- A18.74 related processes and functions. In this study, we document distinct Dopamine neurons in ventral tegmental area and substantia patterns of expression of TAAR1 in the mouse PFC, as well as altered nigra pars compacta exhibit similar responses to reward- subunit composition and deficient functionality of the glutamate related cues and events N-methyl-D-aspartate (NMDA) receptors in the pyramidal neurons of Meredyth A. WEGENER* and Bita MOGHADDAM layer V of PFC in mice lacking TAAR1. The dysregulated cortical glutamate transmission in TAAR1-KO mice was associated with Department of Neuroscience, Center for Neuroscience at the aberrant behaviors in several tests, indicating a perseverative and University of Pittsburgh, PA, United States of America impulsive phenotype of mutants. Conversely, pharmacological active- *E-mail: [email protected] tion of TAAR1 with selective agonists reduced premature impulsive Intrinsic Activity, 2016; 4(Suppl. 2): A18.74 responses observed in the fixed-interval conditioning schedule in http://www.intrinsicactivity.org/2016/4/S2/A18.74 normal mice. Our study indicates that TAAR1 plays an important role Research investigating motivation and psychiatric disorders has in the modulation of NMDA receptor-mediated glutamate trans- identified dopamine neurons as a crucial part of the mechanism mission in the PFC and related functions. Furthermore, these data behind reward-mediated behavior. Much of this work has revolved suggest that development of TAAR1-based drugs could provide a around the rodent limbic system by closely examining the activity of novel therapeutic approach for the treatment of disorders related to dopamine neurons in the ventral tegmental area (VTA) and their aberrant cortical functions. influence on the nucleus accumbens. Recently, research has recognized the potential contribution of the substantia nigra pars

A18.76 pathway downstream of dopamine D2 receptors. Moreover, several Optogenetic activation of ventral tegmental area dopamine genetic risk factors for mental illnesses encode proteins which either cells enhances extinction of fear conditioning comprise or converge on this pathway. However, Gsk3 has up to 200 Jen WENZEL*, Anthony COLE, Willard GOVE, Vivian CHIOMA, substrates, which makes it a non-specific drug target for management Erik OLESON and Joseph CHEER of specific psychiatric symptoms. Moreover, it is not clear which targets of Gsk3 regulate particular behaviors. Thus, identification of Department of Anatomy and Neurobiology, University of Maryland specific targets of Gsk3 could greatly advance our understanding of School of Medicine, Baltimore, MD, United States of America mental disorders and open new avenues for specific drug develop- *E-mail: [email protected] ment. Intrinsic Activity, 2016; 4(Suppl. 2): A18.76 Aims: Identify and characterize novel Gsk3 targets that regulate http://www.intrinsicactivity.org/2016/4/S2/A18.76 specific behaviors. A wide body of research implicates the mesolimbic dopamine (DA) Results: We identified a new target of Gsk3, fragile X mental system in the control of appetitive conditioning. Interestingly, retardation syndrome-related protein 1 (FXR1P). By using AAV viral emerging evidence suggests that midbrain DA systems are integral delivery we study regulation of anxiety by FXR1P. Here we also to aversive conditioning as well. Utilizing a Pavlovian fear condition- use AAV mediated CRISPR / Cas9 technology to knockout genes ing paradigm and fast-scan cyclic voltammetry, our laboratory has (Gsk3, Fxr1) in vivo and investigate regulation of anxiety by the shown that DA release in the nucleus accumbens core (NAcc) Gsk3–FXR1P pathway. Moreover, by combining CRISPR / Cas9 negatively correlates with the incidence of freezing behavior to an technology in vivo and in vitro with electrophysiology we study single aversive conditioned stimulus (CS). Specifically, we observed a neuron activity and neuronal network activity regulations by the suppression of NAcc DA transients at the presentation of a CS Gsk3–FXR1P pathway. previously paired with footshock. Further, with repeated exposure to Conclusions: FXR1P is regulating anxiety downstream of Gsk3 by the CS alone, freezing behavior extinguished, and coincident with regulating activity of neuronal networks implicated in anxiety-related behavioral extinction, NAcc DA release returned to baseline levels. behaviors. Dopamine is implicated in regulation of mood and Gsk3 is

These data suggest that phasic NAcc DA release signals the presen- a downstream of D2 receptor signaling. Having said this, is regulation tation of an aversive CS, and therefore may control an animal’s of Gsk3–FXR1P pathway a new mechanism of how dopamine may reaction to an aversive stimulus. Here, we examined whether phasic regulate anxiety? activation of DA cells plays a causal role in the reaction to an aversive +/− −/− CS. TH::Cre rats and TH::Cre controls received bilateral trans- A18.78 duction with a Cre-inducible channelrhodopsin-2 virus into the ventral Cocaine-evoked bi-directional plasticity at nucleus accumbens tegmental area (VTA) as well as chronic bilateral optical fibers aimed to ventral pallidum synapses: mechanisms and behavioral at the VTA. On day one of our fear conditioning protocol, animals implications were conditioned to associate the delivery of footshock (2 s, 0.8 mA) Meaghan C. CREED* and Christian LÜSCHER with the last 2 s of a 20 s audio cue, over three presentations. On day Department of Basic Neuroscience, University of Geneva, two, in a novel environment, rats were exposed to the audio cue only Switzerland at 3 min intervals for a total of 18 cue presentations. Throughout each *E-mail: [email protected] audio cue rats received optical stimulation to the VTA (20 Hz, Intrinsic Activity, 2016; 4(Suppl. 2): A18.78 10 pulses, 5 ms duration, at 3 s intervals). All behavioral sessions http://www.intrinsicactivity.org/2016/4/S2/A18.78 were recorded, coded to ensure blind analysis, and hand scored for freezing behavior during each cue presentation. In congruence with The nucleus accumbens (NAc) and ventral pallidum (VP) are critically our previous work, optical stimulation of VTA DA cells in TH::Cre+/−, involved in hedonic and motivational processing of both natural but not control, rats resulted in a significant decrease in freezing rewards and of guiding motivationally-relevant behaviour. The NAc is behavior during each subsequent CS presentation, effectively primarily composed of two classes of projection neurons, which enhancing the extinction of freezing behavior and illustrating a causal express either the dopamine D1 or D2 receptor (D1- or D2-MSNs), role for phasic mesolimbic DA activation in the processing of an which undergo distinct forms of plasticity after cocaine exposure. We aversive CS. This enhanced extinction of fear conditioning could not first report that cocaine treatment increases inhibitory tone to the be accounted for by locomotor enhancing effects of DAergic stimula- ventral medial VP (vmVP), and that the vmVP receives projections tion, as a control experiment revealed that this stimulation protocol from D1- and D2-MSNs in equal proportion. Using mutated channel- does not increase locomotor activity. rhodopsin (chETA; capable of following stimulation frequencies of up

to 100 Hz) expressed selectively in D1- or D2-MSNs of the NAc shell A18.77 to dissect the contribution of each of these projections to cocaine- Regulation of anxiety by the Gsk3 – FRX1P pathway: a role for evoked plasticity in the vmVP. We found that D1- and D2-MSNs dopamine? express distinct forms of activity-dependent pre-synaptic plasticity at Jivan KHLGHATYAN*, Simon CHAMBERLAND, Katalin TÓTH and their VP terminals. Specifically, high-frequency stimulation applied Jean-Martin BEAULIEU selectively at of D1-MSN inputs to the vmVP induces long-term potentiation (LTP) of these inputs whereas and the same stimulation Centre de recherché Institut universitaire en santé mentale de protocol applied at D -MSNs induced a long-term depression (LTD) Québec, Université Laval, Montréal, QC, Canada 2 at the stimulated inputs. Critically, both forms of plasticity were *E-mail: [email protected] occluded by cocaine treatment, suggesting that cocaine increases Intrinsic Activity, 2016; 4(Suppl. 2): A18.77 output of D -MSNs and decreases output of D -MSNs. We next used http://www.intrinsicactivity.org/2016/4/S2/A18.77 1 2 in vivo optogenetic stimulation to normalize transmission at either D1

Background: Neuropsychiatric disorders such as bipolar disorders, or D2 inputs to the vmVP following cocaine exposure. Our results depression and schizophrenia represent a major public health implicate D1-MSNs in sensitization to cocaine whereas D2-MSNs problem, and a heavy burden for patients and their relatives. Drugs regulate impaired motivation for natural rewards (i. e. sucrose) (such as mood stabilizers) used for management of these diseases induced by cocaine exposure. Taken together, our results suggest may exert part of their therapeutic action by regulating Akt / Gsk3 that cocaine shifts the balance of D1 and D2 input to the vmVP through

distinct dopamine-dependent plasticity mechanisms, and these The current axiom in psychopharmacology states that repeated synaptic modifications differentially contribute to behavioral symp- treatment with antidepressants lasting at least several weeks is toms of addiction. required for clinical response. This axiom follows the so-called steady state paradigm: a psychoactive drug needs to saturate its target A18.79 receptors to exert its activity in the brain. Some authors, among them How does the N-terminus of the human serotonin transporter members of our laboratory, have reported phenomena called time- regulate the conformational cycle of the transporter? dependent sensitization (TDS). It is a unique property of the central Carina KERN*, Fatma Aslı ERDEM, Ali EL-KASABY, Walter SANDTNER, nervous system to respond to a single stressful event after a Michael FREISSMUTH and Sonja SUCIC prolonged response-free time. In case of antidepressant drugs (ADS), it turned out to be true for some biochemical changes reported Department of Pharmacology, Medical University of Vienna, Austria so far only after repeated treatment with ADS. Such data lead to the *E-mail: [email protected] concllusion that at least some biochemical changes observed after Intrinsic Activity, 2016; 4(Suppl. 2): A18.79 chronic treatment with antidepressants may occur not due to http://www.intrinsicactivity.org/2016/4/S2/A18.79 repeated treatment, but because of time passing since the first drug The serotonin transporter (SERT) mediates serotonin re-uptake to exposure. terminate neurotransmission. Substrate and co-transported ions bind Calcyon is a cellular protein initially reported as one of the dopamine the outward-facing state of SERT, which then alters conformation to receptor-interacting proteins (DRIPs). Later it turned out that it inter- reach the inward-facing state, where the cycle is complete by acts with distinct elements of the endosomal and synaptic scaffolding returning to the outward-facing state. SERT can operate in the machinery. Electrophysiological and cell-biological studies indicate exchange mode: substrate and ions are bound by the inward-facing that its integration with the vesicle-trafficking system is required for state, which then returns to the outward-facing state without complet- synaptic plasticity, as well as for transcytosis and targeting of axonal ing the cycle. This is triggered by amphetamines (AMPH): SERT cargos. Therefore it was interesting to investigate whether changes seesaws through half-cycles, where AMPH is transported inward to (in the TDS paradigm) are related only to dopamine receptors or to drive serotonin efflux. The switch between forward and reverse other elements of signaling pathway as well. modes is accounted for by the alternate access model. Regulatory We examined the expression of calcyon after chronic and acute input (e. g. N-terminal phosphorylation) and oligomeric state modify imipramine (IMI) treatment, as well as during 3 weeks of that the extent of AMPH-induced release, supporting the N-terminal lever treatment’s discontinuation. Wistar male rats were treated with IMI hypothesis: efflux relies on oligomers, where the N-terminus of one (10 mg/kg, i.p.) or saline (2 ml/kg, i.p.) for 20 days. On the 21st day AMPH-bound SERT moiety triggers the counter-transport of some animals, treated previously with saline, received an acute dose substrate by the adjacent one. The two models are not mutually of IMI (10 mg/kg). Then, the 3 weeks discontinuation phase began, exclusive. Here, we explored how the hypothetical lever action of the during which, at certain time points (3 h, 72 h, 7 days, 21 days), a N-terminus affects the transition of SERT through the conformational number of animals were sacrificed and the tissue was collected. In cycle. Limited tryptic digestion showed that the N-terminal conforma- these brains the expression of calcyon mRNA (in situ hybridization) tion differed in the empty inward- and outward-facing and the AMPH- was measured. Chronic IMI treatment increased the expression of bound state: in the absence of Na+ (i. e. inward-facing state), the calcyon in certain basal ganglia. Similar effects were observed at N-terminus was rapidly cleaved, but the presence of AMPH protected some time points during discontinuation. The results will be presented it against trypsin. This showed that the conformational state of the with detailed time and anatomical resolution. hydrophobic core was relayed to the N-terminus, predicting a Research funded by NCN grant no. UMO-2012/07/B/NZ4/01811. segment in the N-terminus that binds the hydrophobic core of a SERT moiety. Thus, we created truncation mutants of the N-terminus. A18.81 Surface expression, bound inhibitors and transported substrate in the Characterization of striatally-mediated motor and cognitive forward mode with affinities and turnover numbers were similar to function in the DJ-1 knockout rat model of Parkinson’s disease WT. In contrast, efflux was greatly reduced in cells expressing SERT Danielle GIANGRASSO*, Teri M. FURLONG and Kristen A. KEEFE missing 32 residues (ΔN32) or more. The individual steps of the Department of Pharmacology and Toxicology, University of Utah, transport cycle were assessed by measuring currents associated with Salt Lake City, UT, United States of America substrate binding (i. e. capacitive gating currents) and transport in the *E-mail: [email protected] forward mode (i. e. steady-state currents) using whole-cell patch Intrinsic Activity, 2016; 4(Suppl. 2): A18.81 clamp. The recovery rate of the currents was significantly lower for http://www.intrinsicactivity.org/2016/4/S2/A18.81 ΔN32 than for WT, consistent with ΔN32 operating less likely in reverse mode. These findings are consistent with the lever hypothe- Parkinson’s disease (PD) arises from various genetic and environ- sis and allow for a unified model, where the role of the N-terminus is mental factors. Genetic animal models of PD grant a high degree of to impose a switch in the kinetic cycle. translational validity to the investigation of inherited PD pathology. The recently developed DJ-1 knockout (KO) rat models the loss-of- A18.80 function mutation of DJ-1 in one form of autosomal recessive PD. Changes in rat brain expression of calcyon, after chronic or Studies of DJ-1 KO rats show that dopamine (DA) cell death in the acute imipramine treatment: evidence of time-dependent substantia nigra pars compacta begins at 4 months of age, becoming sensitization significant at 8 months. As some motor deficits emerge before the Maciej KUŚMIDER*, Agata FARON-GÓRECKA, Joanna SOLICH, Paulina significant loss of DA, this mutation is suggested to yield a period of PABIAN, Dariusz ŻURAWEK, Magdalena KOLASA and DA neuron dysfunction preceding cell death. This dysfunction period Marta DZIEDZICKA-WASYLEWSKA may also give rise to early cognitive impairments, a common feature of PD. Given the novelty of this model, we assessed the performance Department of Pharmacology, Institute of Pharmacology, Polish of DJ-1 KO rats and controls on a battery of striatally mediated motor Academy of Sciences, Kraków, Poland and cognitive tasks. On both the tapered balance beam and open *E-mail: [email protected] field tests, there was no difference between DJ-1 KOs and controls Intrinsic Activity, 2016; 4(Suppl. 2): A18.80 at 4 months. At 6 months, however, DJ-1 KOs slipped significantly http://www.intrinsicactivity.org/2016/4/S2/A18.80

more on the tapered balance beam and had a significantly lower A18.83 rearing frequency in the open field than controls. We also assessed Cerebellar modulation of substantia nigra instrumental learning, which is impaired in human PD patients and Samantha KEE* and Kamran KHODAKHAH other rodent models of striatal DA loss. Typically, instrumental Dominick P. Purpura Department of Neuroscience, Albert Einstein learning is initially driven by action–outcome associations and then College of Medicine, Bronx, NY, United States of America transfers to stimulus–response associations with repeated training. *E-mail: [email protected] Our preliminary data, however, suggest that both DJ-1 KO and control Intrinsic Activity, 2016; 4(Suppl. 2): A18.83 rats display action–outcome behavior at the 4 and 6 month tests. As http://www.intrinsicactivity.org/2016/4/S2/A18.83 controls did not transfer to stimulus–response behavior, the lack of transition may be due to the extended time frame of our instrumental The basal ganglia and cerebellum are two nodes in a complex system learning paradigm. Additionally, although DJ-1 KOs are behaviorally of interconnected brain regions that coordinate body movement. The similar to controls, there may be underlying neurochemical and long-held view that these structures interact primarily at the level of signaling abnormalities in the DJ-1 KO rat that have led them to rely the cerebral cortex through closed-loop circuits has been challenged on different neural structures as a means of compensation, as occurs by a growing body of evidence suggesting the presence of a number in other models of partial striatal DA loss. Lastly, DA transporter of cortex-independent pathways that mediate direct communication densities in striatum did not differ significantly from controls at between these two structures. Understanding how the cerebellum 4 months. Assessments of monoamine transporter density in striatum communicates with different nuclei in the basal ganglia may provide and prefrontal cortex of 4, 6, and 8-month DJ-1 KO and control tissue insight into motor disorders caused by dysfunction of these brain are currently underway. Taken together, this work further character- regions, including Parkinson’s disease and dystonia. The substantia izes the state of striatal DA integrity as well as striatally mediated nigra pars compacta (SNc) is a nucleus in the basal ganglia that gives motor and cognitive function in the DJ-1 KO rat model. rise to the nigrostriatal tract, which provides dopaminergic input to the striatum and degenerates in Parkinson’s disease, leading to motor A18.82 deficits. Multiple studies using lesions of the cerebellar nuclei and Investigation of human TAAR1 (trace amine-associated viral tracing have suggested that the cerebellar nuclei send receptor 1) brain expression monosynaptic projections to dopaminergic neurons of the SNc. The Boris FERGER1,*, Ulrike GROSS2, Eric SIMON3 and strength of this connection and its relevance to motor activity is Birgit STIERSTORFER3 unknown. It has also been shown that trains of electrical stimulation to the cerebellar nuclei can alter dopamine levels in both the 1CNS Diseases Research Germany, Boehringer Ingelheim Pharma substantia nigra and the striatum. Whether this is mediated directly, GmbH & Co KG, Biberach, Germany; 2Medicinial Chemistry through the monosynaptic cerebellar projection to the SNc, or Germany, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, indirectly, via other brain regions, remains to be established. Here, Germany; 3Target Discovery Research Germany, Boehringer we test the hypothesis that the cerebellum can drive the activity of Ingelheim Pharma GmbH & Co KG, Biberach, Germany neurons in the substantia nigra through a direct, monosynaptic *E-mail: [email protected] connection. To test this hypothesis, channelrhodopsin (ChR2), a Intrinsic Activity, 2016; 4(Suppl. 2): A18.82 light-sensitive cation channel, was expressed in cerebellar nuclei http://www.intrinsicactivity.org/2016/4/S2/A18.82 neurons. Antibody staining for GFP, which was expressed in ChR2- TAAR1 (trace amine-associated receptor 1) is an emerging target in positive cells, confirmed that cerebellar fibers expressing ChR2 were CNS diseases research, and data in genetic and non-genetic animal present in substantia nigra. Using an optrode, cerebellar fibers in models are supportive to believe that TAAR1 agonists may play a role substantia nigra were activated by light, and the responses of in psychiatric disorders. However, knowledge of human TAAR1 brain neurons in substantia nigra were recorded. We found in vivo that expression in the public domain is limited and analysis of the short (1–2 ms) light pulses on average increased the firing of neurons intronless TAAR1 is challenging. Here, we like to share and discuss in substantia nigra by 0.883 extra spikes with a median latency of results from a recent human TAAR1 expression study which was 2.5 ms. Taken together, these data provide support for a mono- carried out using fresh frozen samples and formalin-fixed paraffin- synaptic, cortex-independent pathway capable of relaying informa- embedded post mortem brain tissue from 5 donors (age at autopsy tion directly from the cerebellum to the substantia nigra. Future 61–69 years). Two brain areas, prefrontal cortex (PFC) and midbrain studies aim to characterize this pathway in more detail and scrutinize including ventral tegmental area / substantia nigra, were selected for its role in motor control. next-generation sequencing (NGS), PCR, in situ hybridization and immunohistochemistry. All samples passed the quality control check A18.84 and were suitable for analysis. No relevant TAAR1 signal was Temporal and spatial changes in striatal neurotransmission detected by NGS RNA-Seq and PCR analysis although the positive and dopamine receptor expression initiated by nicotine in control using TARR1-overexpressing cells worked well. In situ hybrid- Wistar rats dization (RNAscope technology) showed a low TAAR1 expression Louise ADERMARK*, Julia MORUD, Amir LOTFI, Bo SÖDERPALM signal in the human pancreatic islets (positive control tissue) but no and Mia ERICSON relevant TAAR1 expression in the brain areas selected. Immunohisto- Department of Psychiatry and Neurochemistry, Institute of chemistry staining with commercially available polyclonal TAAR1 Neuroscience and Physiology, University of Gothenburg, Sweden antibodies resulted in specific staining in human TAAR1 over- *E-mail: [email protected] expressing cells and in pancreatic islets but not in the human PFC Intrinsic Activity, 2016; 4(Suppl. 2): A18.84 and midbrain samples. In conclusion, we were unable to detect http://www.intrinsicactivity.org/2016/4/S2/A18.84 human TAAR1 brain expression in two brain areas, which is in contrast to our prediction and should encourage the discussion on Drug addiction has been conceptualized as maladaptive recruitment human TAAR1 target engagement in CNS diseases. of integrative circuits coursing through the striatum, facilitating drug- seeking and drug-taking behavior. The aim of this study was to define temporal neuroadaptations in striatal subregions of rat initiated by 3 weeks of intermittent nicotine exposure followed by 3 months of

abstinence. By means of in vivo microdialysis in nicotine-naïve Wistar in dopaminergic tone. Moreover, individual differences in terms of rats we found that nicotine administration increased extracellular chronotype and trait impulsiveness may further modulate this effect. dopamine levels in a subregion-specific manner. In addition, following 3 weeks of intermittent nicotine treatment (0.36 mg/kg, s.c.), striatal A18.86 output, as measured by electrophysiological field potential record- Habit learning is negatively regulated by HDAC3 in lateral and ings, was selectively depressed in the dorsomedial striatum. The medial dorsal striatum decrease in striatal output sustained even after one month of Melissa MALVAEZ1,*, Venuz Y. GREENFIELD1, Dina P. MATHEOS2, abstinence, and was concomitant with an increase in spine density, Marcelo A. WOOD2, Pamela J. KENNEDY1 and Kate M. WASSUM1 decreased transmitter release and elevated levels of dopamine D 1 1Department of Psychology, University of California, Los Angeles, and D receptor mRNA. Following 3 months of abstinence, spine 2 CA, United States of America; 2Department of Neurobiology and density was normalized, while transmitter release was enhanced and Behavior, University of California, Irvine, CA, United States of dopamine D receptor mRNA expression suppressed. After 3 months 1 America of abstinence, striatal output was normalized in the dorsomedial *E-mail: [email protected] striatum, but significantly depressed in the nucleus accumbens and Intrinsic Activity, 2016; 4(Suppl. 2): A18.86 dorsolateral striatum. The data presented here suggest that, even http://www.intrinsicactivity.org/2016/4/S2/A18.86 though not self-administered, nicotine may produce progressive neuronal alterations in brain regions associated with goal-directed Optimal behavior and decision-making results from a balance of and habitual performance, which might contribute to the development control between two systems, one cognitive and one habitual. These of compulsive drug seeking and the increased vulnerability to relapse, systems rely on the dorsomedial (DMS) and dorsolateral (DLS) which are hallmarks of drug addiction. striatum, respectively. But almost nothing is known about the molecular mechanisms that regulate encoding of the distinct A18.85 associative memories at the core of each behavioral control system. Are owls more patient? The ability to wait for a reward is Here we examined the role of one epigenetic mechanism, histone modulated by daytime and chronotype acetylation regulated by histone deacetylases (HDACs), in this Luise REIMERS*, Malika MÜLLER and Esther K. DIEKHOF process. Rats were trained to lever press for a food reward and were administered the non-specific histone deacetylase (HDAC) inhibitor Human Biology, Biocenter Grindel and Zoological Museum, sodium butyrate (NaB) immediately following each training session. University of Hamburg, Germany Following training, devaluation of the food outcome was used to *E-mail: [email protected] probe cognitive versus habitual control of instrumental behavior. Intrinsic Activity, 2016; 4(Suppl. 2): A18.85 Although both groups learned the task, those rats treated with NaB http://www.intrinsicactivity.org/2016/4/S2/A18.85 showed insensitivity to outcome devaluation earlier in training than Dopamine (DA) plays a major role in reinforcement learning with vehicle-treated rats, suggesting that HDAC inhibition potentiated increases leading to enhanced sensitivity to positive outcomes (Go habit formation. Insensitivity to degradation in the action-outcome learning) and decreases facilitating learning to avoid negative options contingency was also observed and the potentiation of habit occurred (NoGo learning). This is also reflected in response-time adaptions: regardless of training schedule (i. e. random interval vs. ratio). high levels of DA enhance speeding up to get reward, whereas low Systemic HDAC inhibition increased histone H4 lysine 8 acetylation levels favor slowing down. Since both time perception and DA levels specifically in the dorsal striatum and altered transcription of key show circadian shifts, we aimed to examine possible effects of dopamine-related plasticity throughout the dorsal striatum. Decreas- daytime on preferences for Go and NoGo learning in terms of ing HDAC3 function, either by pharmacological inhibition or expres- response time adaption. sion of a mutated HDAC3, in either the DMS or DLS recapitulated Thirty-nine subjects completed a response time adjustment paradigm this, while HDAC3 overexpression in either region prevented habit (i. e. the ‘clock task’) during which they had to explore the optimal formation. These data identify chromatin modification by histone response time (RT) to stop a running clock to win a reward. Different acetylation as an important mechanism in the development of clock faces were presented: in the FAST clock fast responses yielded stimulus–response associative memories. Moreover, the results high reward (i. e. Go learning), whereas in the SLOW clock subjects suggest that HDAC inhibition can potentiate habitual over cognitive had to wait longer in order to win maximum points (i. e. NoGo instrumental learning processes, a finding with important implications learning). Subjects were tested twice at different times of day: once for the therapeutic application of HDAC inhibitors. at 11 a.m, when DA levels are low, and a second time in the evening at 21 p.m. when DA levels increase again. A18.87 We found an interaction between clock type and daytime when Pharmacochaperoning of the dopamine transporter considering the optimized RTs towards the end of the task, which Ali EL-KASABY*, Ameya KASTURE, Hafiz Muhammad Mazhar ASJAD, represent the best learning outcome. Subjects learned better to Michael FREISSMUTH and Sonja SUCIC speed up in the FAST clock condition in the evening than at 11 a.m. Institute of Pharmacology, Medical University of Vienna, Austria suggesting that the presumed increase of DA towards the night *E-mail: [email protected] facilitates Go learning. Furthermore, at 11 a.m. subjects adapted Intrinsic Activity, 2016; 4(Suppl. 2): A18.87 better to the SLOW than to the FAST clock which is also in line with http://www.intrinsicactivity.org/2016/4/S2/A18.87 the proposed positive influence of low DA levels on NoGo learning. In addition, we also investigated the impact of individual differences The dopamine transporter (DAT) is a member of the solute carrier 6 in chronotype (i. e. circadian preference) and trait impulsiveness (i. e. gene (SLC6) family. DAT is localised at the presynaptic specialisa- indicative of dopaminergic tone). Impulsiveness was negatively tion, where it rapidly retrieves the previously released dopamine from correlated with initial RT adjustment in the SLOW clock at the early the synaptic cleft, and operates in relay with the vesicular monoamine test time. As to the chronotype, a tendency towards eveningness was transporter to replenish vesicular stores. Mutations in the coding associated with better adaption at 11 a.m. to the SLOW clock. sequence of the DAT gene are known to cause protein misfolding. In summary, our data suggest that daytime influences the preference The aim of our study was to characterise and rescue misfolded / for Go and NoGo learning in RT adaption, possibly due to variations dysfunctional DAT mutants by pharmacochaperoning. Mutations in

Drosophila melanogaster DAT (dDAT) lead to a sleepless phenotype methyl-4-isoxazolepropionic acid receptor (AMPAR)- and N-methyl- in flies. We identified a single-point mutation in dDAT, i. e. G108Q, D-aspartate receptor (NMDAR)-evoked EPSC and found that signals which arises due to defective protein folding, based on the following from AMPAR were higher in DdcKI mice (p = 0.03), indicating an lines of evidence: (i) dG108Q is retained in the endoplasmic reticulum increment of pre-and post-synaptic excitatory signals. (ER), as illustrated by confocal laser scanning microscopy images Conclusions: Our data suggest that compensatory regulation of and co-localisation with the ER-resident chaperone calnexin. (ii) The afferent excitatory signals plays a role in the control of dopaminergic mutant displayed no specific dopamine uptake in either HEK 293 or neuron firing possibly through a feedback mechanism in AADC Schneider (S2) cells. (iii) The glycine residue equivalent to G108 in deficiency mice. dDAT is absolutely conserved among all DAT species, from C. elegans to H. sapiens, but also among the related monoamine A18.89 transporters. (iv) G108 resides juxtamembrane to the second A causal role for phasic dopaminergic activity in perceptual membrane-spanning helix, i. e. at the very end of the first cytoplasmic decisions loop, which is known to play a key role in the folding of SLC6 Armin LAK*, Sylvia SCHRÖDER, Christopher BURGESS, Elina transporters. (v) The mutant associated with significantly higher JACOBS, Kenneth HARRIS and Matteo CARANDINI levels of HSP70-1A and calnexin than the wild-type dDAT. This Institute of Ophthalmology – Visual Neuroscience, University interaction was markedly reduced upon treatment with noribogaine or College London, United Kingdom pifithrin-μ, and a combined action of the two compounds resulted *E-mail: [email protected] in an additive effect, i. e. enhanced cell surface expression. The Intrinsic Activity, 2016; 4(Suppl. 2): A18.89 pharmacochaperoning action of noribogaine can be accounted for by http://www.intrinsicactivity.org/2016/4/S2/A18.89 its ability to stabilise the inward-facing conformation of DAT, whereas pifithrin-μ acts by inhibiting HSP70. Moreover, both noribogaine and Phasic dopamine (DA) activity is necessary and sufficient for value pifithrin-μ restored sleep in vivo, in dDAT-G108Q-expressing flies. In estimation in free-choice tasks. However, it is not known whether DA addition, we examined a series of human DAT (hDAT) mutations activity can govern perceptual decisions, which require integration of causing infantile dystonia / parkinsonism. Intracellular retention of the value signals with sensory evidence. Moreover, it is not clear whether hDAT variants in the ER compartment leads to dramatic reductions DA cells projecting to frontal cortex play the same role in perceptual in their functional activity. We observed that a number of these choices as other DA cells. clinically relevant DAT mutations can also be functionally rescued by We reasoned that if phasic DA activity is necessary and sufficient for pharmacochaperoning. Collectively, our data provide a proof-of- signalling the value of perceptual decisions, then increasing or principle that mutations in DAT are amenable to rescue by pharmaco- decreasing it for a given choice should bias an animal’s decision chaperoning in vitro and in vivo. towards or against that choice. We expressed channelrhodopsin-2 (ChR2) or archaeorodopsin-3 (Arch3) in midbrain DA neurons of A18.88 DAT-Cre mice, and implanted optical fibers over ventral tegmental Compensatory regulation of afferent modulation on area and medial prefrontal cortex (mPFC). We trained these mice to dopaminergic neurons in mouse model of aromatic perform a perceptual decision task rewarded with water. In each trial, L-amino acid decarboxylase (AADC) deficiency a grating appeared on the left or right side of the monitor, and head- Ni-Chung LEE1,*, Shih-Yin HO2, Yin-Hsiu CHIEN1, Horn-Huei LIOU2 fixed mice indicated its position by turning a steering wheel. By and Wuh-Liang HWU1 varying stimulus contrast across trials we obtained high-quality psychometric curves. We paired some correct choices with phasic 1Department of Medical Genetics, National Taiwan University optogenetic manipulation of midbrain DA cells or their axons in Hospital, Taipei, Taiwan; 2Department of Neurology, National mPFC. Taiwan University Hospital, Taipei, Taiwan We found that phasic DA activity is both sufficient and necessary to *E-mail: [email protected] determine the value of perceptual choices. In alternating blocks of Intrinsic Activity, 2016; 4(Suppl. 2): A18.88 trials we rewarded correct choices toward one side not only with http://www.intrinsicactivity.org/2016/4/S2/A18.88 water but also with phasic activation of DA cells. The psychometric Background: Aromatic L-amino acid decarboxylase deficiency curves shifted towards the side paired with DA stimulation. A similar (AADC deficiency) is a rare neurotransmitter disorder caused by a effect could be seen on a trial-by-trial basis: when we stimulated DA deficiency of the AADC enzyme. Because of the deficiency in cells in 50 % of correct trials, we observed a choice bias towards the dopamine, serotonin and catecholamine, patients usually present side paired with DA stimulation on the past trial. Phasic DA activity with hypotonia, developmental delay, oculogyric crisis and autonomic was necessary to encode the value of choice options: when we briefly dysfunction. We have created a knock-in mouse model of AADC suppressed DA cells in mice expressing Arch3, their choices shifted (DdcKI mice) and show that gene therapy at the neonatal stage can away from the side paired with the DA suppression. rescue the phenotype. Finally, we explored the effects of activating DA axons in mPFC. Mice Methods: Three-weeks-old AADC deficiency (n = 12) and control expressing ChR2 in DA cells developed a bias toward the side paired mice (n = 17) were used for this study. Horizontal brain slices of with DA activation not only when the laser targeted DA cell bodies substantia nigra (SNc) were sectioned to perform patch clamp but also — albeit to a lesser extent — when it targeted their mPFC electrophysiology studies on dopaminergic neurons. Spontaneous axons. firing patterns and excitatory postsynaptic currents (EPSC) were Together, our results show that DA phasic activity is necessary and recorded. sufficient for signaling the value of perceptual choices. Moreover, DA Results: The in vitro whole-cell recording showed that current– cells projecting to mPFC play a similar role as DA cells projecting voltage relationships are indistinguishable between DdcKI mice and elsewhere in the brain. controls, but the spontaneous firing rates of dopaminergic neurons of DdcKI mice were slower and irregular (p = 0.08). The action potential and afterhyperpolarization (AHP) of DdcKI mice were wilder than in control mice. In addition, the sEPSC frequency was higher in DdcKI mice (p = 0.04). We further measured the α-amino-3-hydroxy-5-

A18.90 reach the cell surface, whereas the closely related serotonin trans-

Reward alterations, dopamine D2 receptor blockade and porter SERT does not. metabolic side effects in first-episode schizophrenia patients HEK 293 (a cell line of fibroblast origin) or CAD (a Cath.a-cell-derived Mette O. NIELSEN1,*, Egill ROSTRUP2, Sanne WULFF1, line of neuronal origin) cells were transiently co-transfected with Birte GLENTHØJ1 and Bjørn EBDRUP1 plasmids encoding the wild-type dopamine transporter (DAT) and serotonin transporter (SERT) along with different components of the 1Center for Neuropsychiatric Schizophrenia Research, Mental exocyst using jetPRIME (Polyplus). Radioligand uptake, confocal Health Center Glostrup, Denmark; 2Functional Imaging Unit, Mental laser scanning microscopy and immunoprecipitation experiments Health Center Glostrup, Denmark were performed 48 h after transfection to study the effect of exocyst *E-mail: [email protected] components on trafficking of DAT and SERT. Intrinsic Activity, 2016; 4(Suppl. 2): A18.90 Overexpression of the C-terminal fragment of EXO70 reduced http://www.intrinsicactivity.org/2016/4/S2/A18.90 dopamine uptake in HEK 293 and CAD cells, which heterologously Background: Dopamine receptor antagonists are the cornerstone of expressed DAT. Confocal microscopy verified that this was due to the pharmacological treatment of psychoses. Numerous side-effects retention of DAT in the endoplasmic reticulum (ER). ER retention was have been associated with D2 receptor blockade, but the precise confirmed by immunoblotting for core-gycosylated DAT. This effect mechanisms underlying some of these are still poorly understood. We was phenocopied by overexpression of EXO70, SEC6 and SEC8. In have previously shown a relation between reward disturbances in contrast, surface expression of SERT was not affected by over- schizophrenia patients and weight gain in relation to treatment with a expression of any component of the exocyst, regardless of whether selective D2 receptor antagonist. In a subsample of the same patients the experiments were performed in HEK&thinnsp;293 cells or in CAD we here tested potential associations between reward disturbances, cells. weight gain and the paraclinical metabolic measures, such as Trafficking of DAT, but not of SERT, in the secretory pathway requires cholesterols and glucose. assembly of the exocyst at an early stage. This may explain why the Methods: As a part of a multimodal study, 39 patients went through PDZ motif of DAT must be present for DAT to reach the cell surface. a functional magnetic resonance imaging (fMRI) while playing a This work was supported by FWF project SFB 35-10 (to M. F.). monetary reward task, before and after they received individual doses of amisulpride for six weeks (mean dose 272 mg, range 50–800 mg). A18.92 In a subsample of 27 patients, blood samples including glucose, LINE-1 retrotransposition in midbrain dopaminergic neurons blood lipids and se-prolactin were obtained. Gabriela-Oana BODEA* and Geoffrey J. FAULKNER Results: After six weeks patients had a significant weight gain of 2.3 Mater Research Institute – University of Queensland, kg (SD 2.8; range −4 to +8, p < 0.001). All metabolic measurements Woolloongabba, Queensland, Australia numerically increased, and significant increases were observed in *E-mail: [email protected] total cholesterol (mean 0.36, SD 0.69, p = 0.01) and se-prolactin Intrinsic Activity, 2016; 4(Suppl. 2): A18.92 (mean 1.78, SD 0.81, p < 0.001). In this subsample, we confirmed http://www.intrinsicactivity.org/2016/4/S2/A18.92 previous observations of a negative correlation between baseline reward activity during anticipation of salient events and weight gain; Long interspersed element-1 (LINE-1 or L1) retrotransposon is the and also between normalization of the reward activity and weight only active and autonomous mobile element in the human genome, gain. We found no relations between reward activity or weight change and accounts for about 17 % of the human genome. L1 can ‘jump’ and any of the metabolic measures or prolactin. from one place in the genome to another by first transcribing itself Conclusions: As expected, antipsychotic treatment on average into RNA and then reverse transcribing this template RNA to DNA, caused weight gain and cholesterol changes in the patients. The generating a new L1 copy elsewhere on the genome, with the weight gain was predicted by reward alterations at baseline and was potential to impact gene structure and function through insertional correlated with changes in the reward system during treatment. mutagenesis. L1 mobilisation occurs during normal neuronal differen- However, weight gain and reward alterations were not directly tiation, and potentially in mature post-mitotic neurons. L1 activity has associated with any changes in metabolic measures or prolactin. been recently linked to several neurological diseases, such as Rett syndrome and schizophrenia. When normal epigenetic and post- A18.91 transcriptional silencing mechanisms are dysfunctional, L1 elements Role of exocyst in trafficking of the dopamine transporter may become hyperactive, contributing to disease. Furthermore, there Hafiz Muhammad Mazhar ASJAD*, Ali EL-KASABY, Ameya KASTURE, is evidence that the environment can also stimulate L1 activity, and Oliver KUDLACEK, Sonja SUCIC and Michael FREISSMUTH as a stress-responsive element, L1 may provide the bridge from environmental stress to genetic change in neurons. Midbrain Institute of Pharmacology, Medical University of Vienna, Austria dopaminergic neurons (MbDN) are involved in motor control, reward *E-mail: [email protected] associated behavior, cognition and modulation of emotion. Dys- Intrinsic Activity, 2016; 4(Suppl. 2): A18.91 functions in MbDN can lead to various diseases such as Parkinson’s http://www.intrinsicactivity.org/2016/4/S2/A18.91 disease (PD) and schizophrenia. The susceptibility of MbDN to Uptake through the dopamine transporter (DAT) represents the disease is believed to be induced by environmental and genetic primary mechanism used to terminate dopaminergic transmission in triggers, however the associated mechanisms are not well under- the brain. Intracellular membrane trafficking of the dopamine trans- stood. Our preliminary data suggest that L1-encoded ORF1p RNA- porter at the presynaptic membrane is critical for synaptic function. binding protein, necessary for L1 mobility, is abundantly expressed in However, little is known about the specialized trafficking events substantia nigra (SN) MbDN and co-localizes with TH in aged mice. occurring at the presynaptic membrane. Here, we tested the These data suggest that L1 is expressed in SN MbDN and is hypothesis that DAT requires the exocyst for reaching the cell potentially hyperactive during ageing. We are currently investigating surface. The exocyst is a multiprotein complex required by many whether L1 is normally expressed during MbDN development and membrane proteins for delivery to and insertion into the plasma whether L1 levels change with time, using an L1-EGFP reporter membrane. DAT requires an intact C-terminal PDZ-binding motif to mouse. To examine the potential link between L1 hyperactivation in MbDN and neurotoxic factors that can lead to disease, we will assess

changes in L1 activity in an MPTP mouse model of PD. Additionally, 3University of North Texas Health Science Center, Fort Worth, we are investigating whether ablation of methyl-CpG-binding protein 2 TX, United States of America (MeCP2), a known repressor of L1 mobilisation, correlates with high *E-mail: [email protected] levels of L1 in SN as it has been previously reported that SN is Intrinsic Activity, 2016; 4(Suppl. 2): A18.94 dysfunctional in MeCP2 null mice. This study will provide important http://www.intrinsicactivity.org/2016/4/S2/A18.94 insights into L1 mobilization in MbDN, and potentially contribute to a Preclinical studies in rats suggest that dopamine D receptor antago- better understanding of MbDN vulnerability to disease. 3 nists show promise as treatments for cocaine dependence. We have synthesized drugs with varying degrees of selectivity and intrinsic A18.93 activity at dopamine D3 vs. D2 receptors and tested them for their Dopamine synthesis during neuroinflammation: a focus on effects on cocaine self-administration in rats. The drugs tested are tetrahydrobiopterin listed here followed by their selectivity ratio in D3 vs. D2 receptor- 1, 1 2 Hortense FANET *, Sophie LAYÉ , Frédéric CALON and expressing cell lines and their intrinsic activity in forskolin-dependent 1 Sylvie VANCASSEL adenylyl cyclase stimulation assay, respectively: WC-44: 23, 96 %; 1INRA, NutriNeuro – University of Bordeaux, France; 2Centre WC-10: 42, 20 %; OS-3-106: 115, 59 %; LS-3-134: 168, 35 %; WW-III- Hospitalier de l’Université Laval (CHUL) Research Center, Faculty 55: 860, 68 %. Male rats were trained and tested either under a of Pharmacy, Université Laval, Québec, QC, Canada multiple VI 60 sec schedule of cocaine (0.375 mg/kg, i.v.) and *E-mail: [email protected] sucrose (45 mg) reinforcement or under a progressive ratio schedule Intrinsic Activity, 2016; 4(Suppl. 2): A18.93 of cocaine reinforcement (0.375 mg/kg, i.v.). Our results show that http://www.intrinsicactivity.org/2016/4/S2/A18.93 the compounds with selectivity ratios ≤ 115 decreased reinforcement rates and increase latency to the first response for both sucrose and A growing body of evidence suggests that neuroinflammation, cocaine, although response latency for cocaine was affected more especially through cytokines, acts on the dopaminergic system and than that for sucrose in some cases. In contrast, the compounds with related behaviours. In particular, pro-inflammatory cytokines have selectivity ratios > 115 had no effect on cocaine or sucrose reinforce- been shown to induce dopamine synthesis impairment. The pathway ment rates under the low effort multiple schedule of reinforcement, that mediates protein synthesis is pretty well-known, involving three but decreased break points on the high effort, progressive ratio major enzymes: phenylalanine hydroxylase (PAH), tyrosine hydroxyl- schedule of cocaine reinforcement. Collectively, these results lase (TH) and DOPA-decarboxylase. suggest that relatively less selective D preferring compounds Even though it has been less studied, tetrahydrobiopterin (BH ) is 3 4 attenuate cocaine and sucrose reinforcement, perhaps through an also an essential component of this pathway. Indeed, BH is a 4 action at D receptors or via an interaction between D and D mandatory co-factor for PAH and TH activity and therefore essential 2 2 3 receptors. In contrast, compounds that are relatively more selective for dopamine synthesis. Inhibition of BH production in the brain leads 4 for D receptors do not alter the reinforcing effects of cocaine; to dramatic decrease in dopamine levels. Interestingly, inflammation 3 however, their ability to reduce break points under a high effort can influence BH availability by acting on its synthesis and degra- 4 progressive ratio schedule suggests that these compounds may dation. Despite its importance, the effects of BH on dopamine 4 selectively decrease motivation to seek cocaine. These results synthesis and the potential effect of neuroinflammation on BH have 4 support the idea that selective D receptor partial agonists and been poorly characterized. 3 antagonists may be useful therapeutically for reducing motivation for Here, we used lipopolysaccharide (LPS) to model acute inflammation cocaine. in mice and evaluated the effects on BH and dopamine levels by 4 Supported by DA023957. HPLC and electrochemical detection. We also tested whether administration of BH4 could reverse inflammation-induced alterations of dopamine levels. The related protein levels in mesolimbic dopamine- A18.95 ergic system were evaluated by western blots. Dopamine release in Circuit architecture of ventral tegmental area dopamine nucleus accumbens in response to palatable food and amphetamine neurons revealed by systematic input–output mapping Kevin T. BEIER1,2,*, Elizabeth E. STEINBERG1, Katherine E. injection using in vivo microdialysis after LPS and BH4 injections was 2 2 2 also evaluated. DELOACH , Stanley XIE , Kazunari MIYAMICHI , Lindsay 2 2 3 We show that LPS injection induces a strong neuroinflammation in SCHWARZ , Xiaojing J. GAO , Eric J. KREMER , Robert C. 1 2 the dorsal striatum, nucleus accumbens and ventral tegmental area. MALENKA and Liqun LUO 1 Only enzymes involved in BH4 synthesis are affected by neuro- Nancy Pritzker Laboratory, Department of Psychiatry and inflammation, contrary to TH and PAH which are not affected by LPS Behavioral Sciences, Stanford University School of Medicine, 2 injection. Preliminary data show that BH4 administration induces a Stanford, CA, United States of America; Howard Hughes Medical slight increase in basal dopamine levels and an important increase Institute and Department of Biology, Stanford University, Stanford, after amphetamine injection compared to control mice. Moreover LPS CA, United States of America; 3Institut de Génétique Moléculaire de seems to decrease dopamine release after amphetamine injection. Montpellier, CNRS, UMR 5535, Université de Montpellier, France *E-mail: [email protected] A18.94 Intrinsic Activity, 2016; 4(Suppl. 2): A18.95 Comparisons of dopamine antagonists and partial agonists: http://www.intrinsicactivity.org/2016/4/S2/A18.95 selectivity for dopamine D3 receptors produces selective Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) effects on motivation for cocaine in rats integrate complex inputs to encode multiple signals that influence 1, 1 1 Janet L. NEISEWANDER *, Timothy CHEUNG , Brian NOLAN , motivated behaviors via diverse projections. Here, we combine axon- 1 1 1 John Paul BONADONNA , Gregory POWELL , Andrew CARLSON , initiated viral transduction with rabies-mediated transsynaptic tracing 2 2 3 Kuiying XU , Robert H. MACH and Robert R. LUEDTKE and Cre-based cell-type-specific targeting to systematically map 1School of Life Sciences, Arizona State University, Tempe, AZ, input–output relationships of VTA DA neurons. Using a novel United States of America; 2Department of Radiology, University technique termed TRIO, we found that VTA DA neurons projecting to of Pennsylvania, Philadelphia, PA, United States of America; different forebrain regions exhibit specific biases in their input

selection, and display unique projections to forebrain nuclei, innervat- A18.97 ing largely non-overlapping striatal and extra-striatal targets. Using Change of the excitability type of dopamine neurons in electrophysiology and behavioral pharmacology, we validated new response to synaptic stimuli circuits identified with TRIO, including a novel top-down reinforcing Denis ZAKHAROV1,*, Ekaterina MOROZOVA2,3, Boris GUTKIN4 circuit from anterior cortical regions to lateral nucleus accumbens via and Alexey KUZNETSOV3 VTA DA neurons. We are now investigating the function of these 1Department of Nonlinear Dynamics, Institute of Applied Physics, differential inputs to each unique VTA DA neuron subpopulation. This Russian Academy of Sciences, Nizhny Novgorod, Russian study highlights the utility of our viral-genetic tracing strategies to Federation; 2Department of Physics, Indiana University, elucidate the complex neural substrates that underlie motivated Bloomington, IN, United States of America; 3Department of behaviors. Mathematical Sciences, Indiana University – Purdue University, Indianapolis, IN, United States of America; 4Group of Neural A18.96 Theory, INSERM U960 LNC, IEC, École Normale Superieure Sex differences in dorsolateral prefrontal cortex dopamine PSL University, Paris, France release and the relationship to tobacco smoking treatment *E-mail: [email protected] outcomes Intrinsic Activity, 2016; 4(Suppl. 2): A18.97 1, 2 3 Yasmin ZAKINIAEIZ *, Ansel HILLMER , Laura GOETZ , Evan D. http://www.intrinsicactivity.org/2016/4/S2/A18.97 MORRIS4, David MATUSKEY2,4, Nabeel NABULSI5, Yiyun Henry HUANG5, Sherry MCKEE4 and Kelly P. COSGROVE2 The type of neuronal excitability determines the neuron’s response to stimuli, its synchronization and resonance properties (the first and the 1Interdepartmental Neuroscience Program, Yale University, New second types of excitability correspond to integrator and resonator Haven, CT, United States of America; 2Department of Radiology behavior, respectively) and, ultimately, the computations it performs and Biomedical Imaging,Yale University, New Haven, CT, United in the brain. We investigated the dynamical mechanisms underlying States of America; 3Department of Molecular Biophysics and the excitability type of dopamine (DA) neurons, using a conductance- Biochemistry Yale University, New Haven, CT, United States of based biophysical model, and its regulation by intrinsic and synaptic America; 4Department of Psychiatry, Yale University, New Haven, currents. By calibrating the model to reproduce low frequency tonic CT, United States of America; 5Yale PET Center, Yale University, firing, NMDA excitation is balanced by GABA-mediated inhibition and New Haven, CT, United States of America leads to integrator behavior (type I) characterized by a continuous *E-mail: [email protected] decrease in firing frequency in response to hyperpolarizing currents. Intrinsic Activity, 2016; 4(Suppl. 2): A18.96 Furthermore, we analyzed how excitability type of the DA neuron http://www.intrinsicactivity.org/2016/4/S2/A18.96 model is influenced by changes in the intrinsic current composition.

Sex differences exist in the behavioral and molecular mechanisms In particular, enhancing an anomalous rectifier Ih current and blocking underlying tobacco smoking. For example, men tend to smoke for the of a subthreshold sodium current makes the neuron to be a resonator reinforcing effects of nicotine whereas women tend to smoke for (type II). Key characteristics of synaptic conductances that may be stress and mood regulation. While the mesolimbic dopamine (DA) observed in vivo also change the type of excitability: a depolarized system drives the reinforcing effects of tobacco smoking, the meso- GABAR reversal potential or co-activation of AMPARs leads to an cortical dopamine system — including dorsolateral prefrontal cortex abrupt frequency drop to zero, which is typical for resonators. (dlPFC) — is critical for inhibitory control and working memory Coactivation of NMDARs together with AMPARs and GABARs shifts function, which are both compromised by stress. Guanfacine, an the boundary separated integrator and resonator behaviors of the

α2-adrenergic agonist, enhances inhibitory control and reduces pre- neuron toward more hyperpolarized GABAR reversal potentials. To frontal cortical DA release. The goals of this study were to investigate better understand how altering each of the aforementioned currents sex differences in amphetamine-induced cortical DA release in leads to changes in the excitability profile of DA neurons, we provide tobacco smokers and to examine whether the magnitude of DA a thorough dynamical analysis. Collectively, these results imply that release predicted treatment outcomes. In this study, 25 tobacco integrator properties (type I excitability) in dopamine neurons might smokers (12 females) participated in two same-day [11C]FLB-457 be important for low firing rates and fine-tuning basal dopamine PET scans before and 3 hours after amphetamine administration levels, while switching behavior to resonator mode (type II (0.4–0.5 mg/kg, p.o.). After their PET scans, subjects participated in excitability) during NMDAR and AMPAR activation may facilitate a a 3-week guanfacine (3 mg, p.o., daily) trial. Toward the end of the transient increase in dopamine concentration, as resonators are more guanfacine trial, in order to model the ability to resist smoking, amenable to synchronization. subjects underwent a smoking-lapse paradigm following a psycho- This work was partially supported by the Russian Foundation for logical stressor. We measured time lapsed before the first cigarette. Basic Research (grant 14-02-00916-a), NIAAA (grant R01A022821), We compared percent change in binding potential (%ΔBP), an ANR-13-NEUC-0003-01, IdEx ANR-11-0001-02 PSL and LabEx indirect measure of dopamine release, between males and females ANR-10-LABX-0087 (France). in dlPFC. Preliminary analyses suggested that female smokers have a smaller amphetamine-induced DA release in dlPFC (%ΔBP = 2.60 ± 3.19 %) than male smokers (%ΔBP = 18.26 ± 5.91 %; p = 0.03). In female smokers, smaller amphetamine-induced DA changes were associated with shorter time to delay smoking following a stressor, p = 0.026, suggesting that the more blunted this response, the greater the inability to delay smoking. This relationship was not found in male smokers. This finding is consistent with previous literature showing that blunted DA responses predict poorer treatment outcomes.

A18.98 Brain-derived neurotrophic factor (BDNF) has been shown to have a Association study of dopamine transporter gene polymorphism critical role not only on neurite growth during early stages of and adult obesity development, but also on physiological and pathological functions in Orçun AVŞAR1,*, Ayşegül KUŞKUCU2, Seda SANCAK3 and Ece GENÇ4 the adult brain. Several studies have explored the role of BDNF in addiction-related brain regions, like prefrontal cortex, ventral tegmen- 1Department of Biotechnology, Natural and Applied Sciences, tal area or shell and core (NAcore) of the nucleus accumbens. In Yeditepe University, Istanbul, Turkey; 2Department of Medical adults, the expression of BDNF in the NAcore is low, and the two Genetics, Yeditepe University, Istanbul, Turkey; 3Department of main sources of BDNF are glutamatergic projections from the PFC Endocrinology, Fatih Sultan Mehmet Education and Research and dopaminergic input from the VTA. Both D -and D -receptor- Hospital; 4Department of Medical Pharmacology, Yeditepe 1 2 expressing medium spiny neurons (MSNs) of the NAcore express the University, Istanbul, Turkey primary receptor for BDNF, TrkB. BDNF binding to TrkB induces *E-mail: [email protected] activation of several intracellular signaling cascades like MAPK, Intrinsic Activity, 2016; 4(Suppl. 2): A18.98 PI3K, phospholipase C-g. It has been proposed that BDNF affects on http://www.intrinsicactivity.org/2016/4/S2/A18.98 cocaine reward are mainly due to activation of TrkB on D2-expressing Obesity is increasingly spreading worldwide and a health problem MSNs, since specific TrkB gene deletion induces a decrease in which causes various diseases such as cancer, diabetes, and cardio- cocaine-induced place preference and profound neuronal firing vascular diseases. Several factors such as genes, physical activity, modifications [1]. food-seeking behavior are involved in the pathogenesis of obesity. Here, we seek to understand the rapid, acute effects of BDNF in the Furthermore, obesity is defined as a neurological disorder rather than NAcore on drug seeking, using the behavioral model of cocaine self- metabolic disease. The neurotransmitter dopamine is significant for administration in rats. To study the non-transcriptional effects of hedonism, mood, food intake, overeating, and addiction. An increas- BDNF in the NAcore, rats were trained to self-administer cocaine and ingly general agreement is that obesity is a form of food addiction extinguished. We then microinjected BDNF into NAcore 15 min which significantly similar to drug addiction. Additionally, it has been before cue-induced reinstatement. BDNF decreased reinstated lever supposed that disrupted dopamine neurotransmission may cause pressing. Concurrently to this acute effect, we also measured a long- weight gain and then obesity. lasting inhibitory effect that endured for days after BDNF administra- The dopamine transporter (DAT) is a transmembrane protein which tion that is likely due to transcriptional changes. Supporting a role for provides the reuptake of DA from the synaptic cleft and terminates endogenous BDNF-induced activation of TrkB we found that blocking the neurotransmission of dopamine. The DAT gene, the solute carrier TrkB activation by microinjecting TrkB-Fc 15 min before reinstatement family 6, member 3 gene (SLC6A3), is located on the short arm of the potentiated reinstated lever pressing. TrkB-Fc also prevented co- chromosome 5 in band 15.3. It is known that the rs27072 (2319G>A) administered BDNF from antagonizing reinstated cocaine seeking. polymorphism of the DAT1 gene is correlated with alcoholism and BDNF effects seem specific to cue-induced drug seeking, since no smoking. decrease in exploratory behavior or cocaine-induced locomotion was In this study, we have investigated the polymorphism of the DAT1 observed after BDNF microinjections in the NAcore. gene in 2 groups. Group 1 (n = 234), whose mean age was 31.4 ± 7.8 These results suggest that, in addition to the long-lasting transcript- years, consisted of overweight and obese individuals with a mean tional effects of BDNF shown in the literature, acute activation of the BMI of 33.8 ± 8.7 kg/m2. The mean age of the control group (n = 214) TrkB intracellular pathway just before reinstatement can prevent was 27.6 ± 5.8 years and the mean BMI was 21.7 ± 1.9 kg/m2. Both cocaine seeking. female and male volunteers were collected at the Department of Reference Endocrinology of Fatih Sultan Mehmet Education and Research 1. Lobo MK, Covington HE 3rd, Chaudhury D, Friedman AK, Sun H, Hospital in İstanbul, Turkey. DNA extraction was performed from the Damez-Werno D, Dietz DM, Zaman S, Koo JW, Kennedy PJ, Mouzon peripheral blood according to the DTAB-CTAB method. The PCR- E, Mogri M, Neve RL, Deisseroth K, Han MH, Nestler EJ: Cell type- RFLP method was used for the DAT1 SNP analysis. The PCR specific loss of BDNF signaling mimics optogenetic control of products were cleaved by the use of MspI RE, and then analyzed by cocaine reward. Science, 2010; 330(6002):385–390. 5 % agarose gel electrophoresis. doi:10.1126/science.1188472 The A allele was not cleaved, on the other hand, 107 and 93 bp fragments were produced for the G allele. The association between A18.100 obesity and distributions of allele frequencies were tested by Chi Chemogenetic inactivation of midbrain dopamine neurons and 2 Square (χ ) test. The statistical assesment of the data showed a their projections to the nucleus accumbens attenuates alcohol- significant association between DAT1 SNP and adult obesity seeking behaviour (p = 0,014). Milan D. VALYEAR*, Ivan TRUJILLO-PISANTY, Franca LACROIX, This study states that the AA genotype of the DAT1 gene might be a Peter SHIZGAL and Nadia CHAUDHRI risk factor for the devolepment of obesity in the Turkish population. Department of Psychology, Concordia University, Montreal, QC, Canada A18.99 *E-mail: [email protected] Role of intra-accumbens brain-derived neurotrophic factor on Intrinsic Activity, 2016; 4(Suppl. 2): A18.100 cue-induced reinstatement after cocaine self-administration http://www.intrinsicactivity.org/2016/4/S2/A18.100 Ana-Clara BOBADILLA*, Constanza GARCIA-KELLER, Jeff HYDE, Victoria CHAREUNSOUK and Peter W. KALIVAS Cues that predict alcohol influence the vigour with which alcohol is pursued. We examined the impact of an alcohol-context on alcohol- Department of Neuroscience, Medical University of South Carolina, seeking behaviour elicited by a discrete cue. Next, we investigated Charleston, SC, United States of America the role of dopamine receptors, midbrain dopamine neurons and *E-mail: [email protected] dopaminergic projections to the nucleus accumbens core in alcohol- Intrinsic Activity, 2016; 4(Suppl. 2): A18.99 seeking elicited by a discrete alcohol cue. Male, Long-Evans rats http://www.intrinsicactivity.org/2016/4/S2/A18.99 received 15 % ethanol (EtOH) in the home-cage, followed by Pavlovian conditioning sessions in which a 10 s conditioned stimulus

(CS+; 15 trials per session) was paired with EtOH (0.2 ml / CS+, 3 ml suspension and pole tests. None of them were altered following these per session). Conditioning occurred on alternating days in a distinct striatal proteins inhibitions, with the exception, interestingly, of the alcohol context where EtOH was dispensed into a fluid port during PKA / STEP61 pathway. Taken together, our data illustrate that each CS+ trial. On the intervening days rats were exposed to a mTOR and ERK proteins of the dorsal striatum are selectively different non-alcohol context where a distinct auditory stimulus was affecting the learning of a complex motor task but not movement played but EtOH was not delivered. In Exp 1, after equal exposure to executions. These findings reveal a novel and important action of both contexts, the CS+ was presented without EtOH in the non- striatal signaling pathways that could be a future target for selective alcohol and alcohol contexts. Alcohol-seeking elicited by the CS+ therapeutic interventions to support learning and recovery of was significantly elevated in the alcohol context and this effect movement resulting from injury or disease. persisted across several tests. Subsequently, systemic eticlopride (a D2/3 receptor antagonist), but not SCH 23390 (a D1-like receptor A18.102 antagonist) significantly attenuated alcohol-seeking elicited by the The role of glutamate receptor-dependent signaling in the CS+ in the non-alcohol context. Next, we used chemogenetics to dopamine system in reinforcement learning determine if midbrain dopamine neurons and their projections to the Przemysław E. CIEŚLAK* and Jan RODRIGUEZ PARKITNA nucleus accumbens core (NAcC) were needed for alcohol-seeking Institute of Pharmacology, Polish Academy of Sciences, Kraków, elicited by the CS+ in the non-alcohol context. Male, transgenic Poland TH::Cre rats received infusions of a cre-dependent viral vector *E-mail: [email protected] encoding the G -coupled (hM4Di) designer receptor into the ventral i Intrinsic Activity, 2016; 4(Suppl. 2): A18.102 tegmental area. This receptor induces neuronal silencing when http://www.intrinsicactivity.org/2016/4/S2/A18.102 bound by clozapine N-oxide (CNO). Behavioural training was as described above. At test, port-entries elicited by the CS+ in the non- Glutamate receptors regulate activity and plasticity of dopaminergic alcohol context were significantly attenuated by systemic CNO neurons. Impairments in glutamate receptor-dependent signaling (Exp 2; 0, 10 or 20 mg/kg, i.p.) or CNO microinfusions into the NAcC may lead to aberrant reinforcement learning and maladaptive (Exp 3; 0 or 3 mM, 0.3 μl per hemisphere). Thus, the level of alcohol- decision-making. To investigate the role of specific glutamate seeking elicited by a discrete alcohol cue depends crucially on the receptors in reward-based learning, we used two genetically modified context in which the discrete cue is encountered. When this mouse lines with inactivation of NMDA receptors in dopaminergic DATCreERT2 contextual influence is controlled for, alcohol-seeking elicited by a cells (the NR1 mice) and mGlu5 receptors in D1 receptor- discrete cue requires dopamine and dopaminergic projections to the expressing neurons (the mGluR5D1-KD mice). Animals were tested in nucleus accumbens core. a classical Pavlovian conditioning task in which they had to discriminate between reward-paired (CS+) and non-paired (CS-) stimuli, and A18.101 in an adaptive decision-making task (a ‘two-armed bandit’) in which Striatal signaling adaptation during motor skill learning they had to choose between two alternatives with different proba- Yan BERGERON, Geneviève BUREAU, Bruno OUIMET, bility of reward delivery. During the course of conditioning the DATCreERT2 Laure CHAGNIEL and Michel CYR* NR1 mice approached the place of reward delivery preferentially during the presentation of the reward-paired (CS+) stimulus. Department of Medical Biology, University of Trois-Rivières, However, mutant mice made significantly fewer approach responses Trois-Rivières, QC, Canada than their wild-type littermates. Moreover, when a CS+ was presented *E-mail: [email protected] contingent upon a novel instrumental response, the NR1DATCreERT2 Intrinsic Activity, 2016; 4(Suppl. 2): A18.101 mice made significantly fewer responses that resulted in CS+ http://www.intrinsicactivity.org/2016/4/S2/A18.101 presentation. Additionally, during the first few sessions of the ‘two- It is well accepted that the striatum structure is involved in motor skill armed bandit’ task, choices of richer alternative in the NR1DATCreERT2 learning. However, the underlying molecular mechanisms of this mice were at a chance level, suggesting that animals were unable to learning process are not clearly understood. In view of the known discriminate between the alternatives. Nevertheless, performance of importance of the striatal glutamatergic afferents, as well as the the NR1DATCreERT2 mice improved in subsequent sessions and reached dopaminergic nigrostriatal projection, our studies investigated the the same level of performance as in wild-type littermates. No deficit role of downstream targets of striatal dopamine and glutamate in discrimination between alternatives was observed in mGluR5D1-KD receptors activation in the learning processes associated with the mice. However, mGluR5D1-KD mice were less sensitive to reward rotarod task in mice. To this end, we used genetically altered mouse delivery as evidenced by decreased probability of ‘win-stay’ choices models or performed injections of genetic and pharmacological and had longer choice latency during all testing sessions. Taken inhibitors into mice dorsal striatum to impair functions of protein together, our results suggest that NMDA receptors in dopaminergic kinase A (PKA), 61 kDa striatal-enriched protein tyrosine phosphatase neurons are crucial for attribution of incentive salience to reward

(STEP61), mammalian target of rapamycin (mTOR), protein kinase B predicting stimuli and facilitation of learning, while mGlu5 receptors in gamma (Akt3), glycogen synthase kinase 3 (GSK3) and extracellular- D1 receptor-expressing neurons modulate reward sensitivity. signal-regulated kinases (ERK). The accelerating rotarod is a recognized test to study the rapid (acquisition) and slower (consolida- A18.103 tion) phases of motor skill learning. As previously demonstrated, wild- Relnrl-Orl/+ mice show behavioral abnormalities in emotion, type or vehicle-treated mice rapidly improved their performances on social interaction and motor learning: association with the rotarod task at the first day of training whereas at the second and dopaminergic dysfunction third day, their scores improved slowly and reached a plateau at the Akira SOBUE*, Yuki AOYAMA, Shan WEI, Taku NAGAI and fourth day. Impairing the PKA / STEP61 pathways was affecting both Kiyofumi YAMADA the fast and slower phases of rotarod learning. Inhibition of mTOR or Department of Neuropsychopharmacology and Hospital Pharmacy, ERK activity was disrupting the consolidation phase exclusively. On University of Nagoya, Japan the other hand, the Akt / GSK3 pathway was not significantly affecting *E-mail: [email protected] motor learning. To confirm that reduced performances were not due Intrinsic Activity, 2016; 4(Suppl. 2): A18.103 to impaired motor capacities, we performed the stepping, wire http://www.intrinsicactivity.org/2016/4/S2/A18.103

Reelin is an extracellular matrix protein, which plays a pivotal role in regulation of miRNA levels by neuronal stimulation. Finally, since the embryonic neuronal migration and is essential to the development of dopamine D1 receptor is centrally involved in mediating the effects of the laminar structure of the cerebral cortex. In the adult brain, reelin cocaine in the striatum and is essential for cocaine-induced is produced by GABAergic interneurons and concerned in synaptic behavioural sensitization, we analyzed the same subset of miRNAs density, dendritic spines, and axons in the hippocampus and cortex. specifically in D1-containing striatal projection neurons in the NAcc Reelin activates a number of neuronal signal transduction pathways using a combination of viral vectors and fluorescent-activated cell that subsequently modulate synaptic function and plasticity. It has sorting (FACS) strategy in mice chronically treated with cocaine. been reported that reelin mRNA and protein levels are significantly Better deciphering the role of gene expression regulators like reduced in several areas of the brains of patients with schizophrenia. miRNAs in specific neuronal populations in the striatum could help Moreover, we found the CNV of C-terminus of reelin in Japanese improve our understanding of the long-lasting changes that cocaine patients with schizophrenia. However, the pathophysiological role of induces in the brain’s reward circuitry and thus provide bases for the a truncated form of reelin in schizophrenia remains unclear. development of novel efficient therapeutic tools for drug addiction. Accordingly, we carried out comprehensive behavioral analyses in This work was supported by French Fondation pour la Recherche Relnrl-Orl/+ mice that produce a transcript with a 220 bp deletion. Médicale (FRM), LAbex Biopsy, INSERM and Centre National pour Relnrl-Orl/+ mice showed a reduction of natural aversion to illuminated la Recherche Scientifique. open areas in the open field test. The social approach behaviors were rl-Orl/+ rl-Orl/+ impaired in Reln mice. We also demonstrated Reln mice A18.105 have deficit in motor coordination and motor learning. Antipsychotics Low-anxiety rats are characterised by long-lasting greater (haloperidol 0.03 mg/kg, i.p., and clozapine 1.0 mg/kg, i.p.) amelio- susceptibility to amphetamine in the place preference test rl-Orl/+ rated impairment of rotarod performance in Reln mice. Małgorzata LEHNER1,*, Marek GRYZ1, Aleksandra WISŁOWSKA- Methamphetamine (METH)-induced hyperactivity was significantly STANEK2, Anna SKÓRZEWSKA1, Ewa TARACHA1 and Adam PŁAŹNIK1,2 reduced in Relnrl-Orl/+ mice compared with that in WT mice. To clarify 1Department of Neurochemistry, Institute of Psychiatry and whether reduction of METH-induced hyperactivity in Relnrl-Orl/+ mice is Neurology, Warsaw, Poland; 2Department of Experimental and associated with alteration of dopaminergic neurotransmission in the Clinical Pharmacology, Medical University of Warsaw, Poland nucleus accumbens (NAc), we examined METH-induced DA release *E-mail: [email protected] by in vivo microdialysis. METH-induced dopamine release in the NAc Intrinsic Activity, 2016; 4(Suppl. 2): A18.105 was markedly reduced in Relnrl-Orl/+ mice compared to WT mice. In http://www.intrinsicactivity.org/2016/4/S2/A18.105 addition to these functional changes in dopaminergic system, region- specific alterations of GABAergic markers were demonstrated in the This study utilised the conditioned place preference test (CPP) to brains of Relnrl-Orl/+ mice. These results suggest that Relnrl-Orl/+ may assess the behavioural effects of amphetamine. We used low-anxiety lead to abnormalities in emotion, social interaction and motor (LR) and high-anxiety (HR) rats that are known to have different fear- learning, which is accompanied by the functional changes in conditioned response strengths, different susceptibility to amphet- dopaminergic and the GABAergic systems. Relnrl-Orl/+ mouse could be amine in the TIPS procedure and different amphetamine-dependent a useful model for some of clinical symptoms and neuropatho- frequency-modulated (FM) 50-kHz ultrasonic vocalisation (USV) physiology in schizophrenia and for screens of antipsychotic efficacy. responses. We have found that during the CPP test and after repeated amphetamine administration (8 daily injections) followed by A18.104 a 14 day withdrawal period, the effects in a drug-paired compartment Cocaine differentially regulates plasticity-related microRNAs in and on USV were stronger in LR rats in comparison to HR rats. LR the ventral vs. dorsal striatum rats vocalised much more intensively and spent more time in the Benoit FORGET*, Arthur GODINO, Vincent KAPPES, Alexandra amphetamine-paired compartment. We also used immunocyto- GARCIA, Radhia KACHER and Jocelyne CABOCHE chemistry to examine changes in the expression of D1 and NMDA receptor 2B (GluN2B) subunit in the subcortical brain regions of HR Laboratory of Neuroscience, Université Pierre et Marie Curie; and LR rats in response to the re-exposition to amphetamine-paired INSERM UMRS 1130; CNRS-UMR 8246, Paris, France compartment in CPP test followed by a withdraw period. The LR rats *E-mail: [email protected] showed more D and GluN2B expression in the nucleus accumbens Intrinsic Activity, 2016; 4(Suppl. 2): A18.104 1 core, showed more GluN2B expression in the ventral tegmental area, http://www.intrinsicactivity.org/2016/4/S2/A18.104 and increased expression of D1 receptors in the central amygdala. Chronic cocaine exposure induces long-lasting alterations in the The data revealed that the differences between HR and LR rat are brain’s reward circuitry through modification of neuronal plasticity, a reflected not only in amphetamine sensitisation models, but also in phenomenon that may contribute to the development of addiction. the CPP model which measures the reinforcing potency of amphet- MicroRNAs (miRNAs) are emerging as central regulators of gene amine. These results indicate a long lasting greater susceptibility of expression in the brain and have an important role in modulating the LR rats dopaminergic system to amphetamine. Understanding the neuronal plasticity, but their involvement in processes related to neurobiological factors that contribute to emotional disorders and cocaine addiction is still not fully understood. sensitisation to psychostimulants is essential for developing future Here, we report that a subset of plasticity-related miRNAs are effective psycho- and pharmacotherapies. differentially upregulated between the nucleus accumbens (NAcc) and the dorsal striatum in response to either acute or chronic A18.106 (sensitization procedure) cocaine treatment in mice, without modifica- Anatomical, physiological and behavioral dissection of an tions in the expression of genes involved in their processing. We also amygdala – midbrain circuit investigated under the same conditions the expression of potential Elizabeth E. STEINBERG1,*, Madison D. TAYLOR1, Csaba FÖLDY1,#, target genes of specific miRNAs that were regulated by cocaine. In Kevin T. BEIER1,2, Liqun LUO2 and Robert C. MALENKA1 parallel we demonstrate using in vitro pharmacological and in vivo 1Nancy Pritzker Laboratory, Department of Psychiatry and genetic approaches the potential implication of the mitogen-activated Behavioral Sciences, Stanford University School of Medicine, protein kinase (MAPK) / extracellular signal-regulated kinase (ERK) Stanford, CA, United States of America; 2Howard Hughes Medical signaling cascade — a canonical cocaine-activated pathway — in the Institute and Department of Biology, Stanford University, Stanford,

CA, United States of America; #present address: Brain Research paradigm that induces Arc transcription and subcellular trafficking in Institute, University of Zurich, Switzerland a temporally constrained manner. Confocal imaging of Arc mRNA *E-mail: [email protected] expression in identified striatal efferent neuron subpopulations has Intrinsic Activity, 2016; 4(Suppl. 2): A18.106 been completed, and we currently are analyzing transcription of the http://www.intrinsicactivity.org/2016/4/S2/A18.106 Arc mRNA, as well as nuclear export of Arc mRNA into the perinuclear cytoplasm in identified striatonigral and striatopallidal The central amygdala (CeA) is known to orchestrate learned fear efferent neurons. Results from this project will determine whether responses via multiple subcortical projections. In addition to its well- L-DOPA treatment is sufficient to restore the normal Arc expression described role in fear, the CeA influences other cognitive processes phenotype in striatonigral efferent neurons of rats with METH-induced including reward learning. However, the nature of this influence, as neurotoxicity. Future studies will then determine whether such well as the underlying circuit and synaptic mechanisms, remain treatment restores cognitive functions mediated by striatum. largely unexplored. Midbrain dopamine neurons perform essential computations that are required for many aspects of reward learning. Accordingly, we hypothesized that the robust projection from the CeA A18.108 to the substantia nigra pars lateralis (SNL) — a heterogeneous region The selective protein kinase Cβ inhibitor, enzastaurin, containing dopamine, GABA and glutamate neurons — was well- decreases amphetamine stimulated locomotion and self- positioned to link amygdala signals with downstream effectors of administration in rats reward learning. Using multiple viral tracing strategies and single-cell Rachel ALTSHULER*, Margaret GNEGY and Emily JUTKIEWICZ gene expression analysis, we found that SNL-projecting CeA neurons Department of Pharmacology, University of Michigan, Ann Arbor, are anatomically and genetically distinct from those that project to MI, United States of America brain areas associated with fear and anxiety. Consistent with our *E-mail: [email protected] hypothesis, optogenetic activation of CeA–SNL projections was Intrinsic Activity, 2016; 4(Suppl. 2): A18.108 sufficient to support a specific subset of reward-related behaviors, http://www.intrinsicactivity.org/2016/4/S2/A18.108 establishing a selective functional role for CeA–SNL projections in Extracellular dopamine levels are regulated by the dopamine reward learning. Ongoing physiology experiments seek to identify the transporter (DAT), a transmembrane protein that takes up dopamine microcircuit architecture through which CeA activity influences from the synapse into the cell. Amphetamines are substrates of DAT synaptic transmission in the SNL and beyond. Collectively, these and reverse DAT function to release dopamine into the synapse. experiments provide valuable insight into the complex neural Amphetamines elicit their stimulating and reinforcing effects by interactions that endogenously regulate the brain’s reward circuitry. increasing extracellular dopamine levels in the brain. Protein kinase Cβ (PKCβ) is important for amphetamine’s effects on outward A18.107 transport without altering basic DAT function. Inhibition of PKCβ L-DOPA treatment for methamphetamine-induced basal ganglia reduces amphetamine-stimulated dopamine efflux through the dysfunction transporter in vitro and in vivo. The purpose of this study was to Anne S. GIBSON* and Kristen A. KEEFE examine if PKCβ inhibition would decrease key amphetamine- Department of Pharmacology and Toxicology, University of Utah, stimulated behaviors: locomotion and self-administration. The selec- Salt Lake City, UT, United States of America tive PKCβ inhibitor, enzastaurin (1–30 pmol), was administered to *E-mail: [email protected] male Sprague Dawley rats by intracerebroventricular injections Intrinsic Activity, 2016; 4(Suppl. 2): A18.107 18 hours before behavioral evaluation. Locomotor activity was http://www.intrinsicactivity.org/2016/4/S2/A18.107 measured in infrared beam break boxes following administration of single doses (0.32–3.2 mg/kg) of amphetamine. In self-administration Methamphetamine (METH) is a highly addictive psychostimulant. studies, rats earned infusions of amphetamine (0.032 mg/kg/infusion) Cognitive deficits are apparent in individuals with a history of METH or sucrose pellets under a fixed ratio 5 schedule of reinforcement for abuse, and targeting cognitive function may be an efficacious 60- or 20-minute sessions, respectively. Pretreatment with 10 pmol approach to managing METH abuse and addiction. However, to enzastaurin reduced locomotion following injections of 0.32 and develop a successful treatment for METH abuse, approaches to 1 mg/kg amphetamine but the effect was surmountable at 3.2 mg/kg mitigate the consequences of METH-induced toxicity must be better amphetamine, demonstrating a rightward shift in the amphetamine understood. METH exposure is known to be associated with damage dose–effect curve. Enzastaurin (1 pmol) was not sufficient to to central monoamine systems. Rodent models of such damage have decrease locomotor activity. Larger doses of enzastaurin (30 pmol) revealed a decrease in the amplitude of phasic dopamine signaling failed to further decrease amphetamine-stimulated locomotion. and significant striatal dysfunction. In particular, the regulation of Arc Enzastaurin decreased the number of amphetamine infusions earned (activity-regulated cytoskeleton-associated protein), particularly in by 60 % but did not alter the number of sucrose pellets earned, striatonigral efferent neurons, is disrupted. This disruption correlates suggesting that enzastaurin altered the reinforcing effects of amphet- with the degree of dopamine loss, which is further known to correlate amine but not natural rewards. The data demonstrate that low doses with the degree of disruption of phasic dopamine signaling. Arc is an of a specific PKCβ inhibitor attenuate amphetamine-mediated effector immediate early gene critically involved in experience- behaviors in a surmountable manner without non-selectively altering dependent plasticity; thus, impairments in Arc have significant behavior. This study demonstrates that inhibition of PKCβ serves to cognitive consequences. Together, these observations suggest that reduce amphetamine reinforcement and could have therapeutic utility disruption of striatal Arc expression may be a consequence of against amphetamine abuse. diminished phasic dopamine signaling in striatum and contribute to Funded by NIH grant RO1 DA11697 and T32-GM007767. cognitive dysfunction associated with METH abuse. We hypothesize that pharmacologic restoration of phasic dopamine signaling will reverse METH-induced deficits in striatal Arc expression and, ultimately, restore cognitive function. To test this hypothesis, rats were treated with a binge regimen of METH to induce neurotoxicity. Three weeks later, rats were treated with benserazide (40 mg/kg, i.p.) and L-DOPA (50 mg/kg, i.p.) and then exposed to a behavioral

A18.109 L-DOPA. We generated a mouse model to investigate the involve- Lateral habenula – rostromedial tegmental nucleus – ventral ment of distinct classes of dopamine receptors in affective and tegmental area circuit regulates voluntary ethanol consumption cognitive disorders associated with PD. Memory deficits, as well as by aversion-mediated learning depressive- and anxiety-like behaviors were examined in mice with a Shashank TANDON*, Chandni SETH, Teri M. FURLONG, Kristen A. partial bilateral loss of dopamine, induced by injection of the toxin KEEFE and Sharif TAHA 6-hydroxydopamine (6-OHDA). This procedure resulted in a deficit of novel object recognition, accompanied by disruption of long-term Department of Pharmacology and Toxicology, University of Utah, potentiation in the hippocampus. In addition, we observed increased Salt Lake City, UT, United States of America immobility time in the forced swim and tail suspension tests, two *E-mail: [email protected] behavioral paradigms of depression. The 6-OHDA lesion also exerted Intrinsic Activity, 2016; 4(Suppl. 2): A18.109 an anxiogenic effect, as shown by reduced time spent in the open http://www.intrinsicactivity.org/2016/4/S2/A18.109 arms of the elevated plus maze and increased thigmotaxis in the open

Drugs of abuse, including ethanol, have both rewarding and aversive field. Pharmacological treatment with L-DOPA or with the D1-type effects that play an important role in regulating drug intake. receptor agonist, SKF 81297, rescued the deficit in novel object Rewarding effects lead to further drug intake, whereas aversive recognition and hippocampal synaptic plasticity, but did not modify effects limit it. It is well known that dopamine (DA) neurons in the depressive- and anxiety-like behaviors. Conversely, the dopamine ventral tegmental area (VTA) play a crucial role in mediating ethanol D2/D3 receptor agonist pramipexole counteracted behaviors indica- reward. Studies indicate that increased DA is critical for ethanol tive of affective impairment, without rescuing the memory deficit. Pre- reward, whereas reduced DA may mediate aversion to ethanol. treatment with desipramine, which protects noradrenaline neurons However, the neural circuitry mediating the decrease in VTA DA firing from the toxic effect of 6-OHDA, did not modify depressive- and associated with ethanol-induced aversion is not well delineated. The anxiety-like behaviors. Notably, reboxetine, a selective noradrenaline lateral habenula (LHb) exerts a potent inhibitory influence on VTA DA reuptake inhibitor, reverted the depressive and anxiogenic effects neuron firing through a disynaptic pathway involving the rostromedial observed in the PD model. Taken together, these results indicate that tegmental nucleus (RMTg). The LHb sends excitatory projections to reboxetine acts by promoting dopamine transmission via blockade of the RMTg, a GABAergic structure, which then provides inhibitory the noradrenaline transporter, which is responsible for the bulk of input to VTA DA neurons. Both the LHb and RMTg are activated by dopamine clearance in extra-striatal regions, including the cerebral aversive stimuli. Given that the LHb–RMTg circuit negatively cortex and the hippocampus. These data contribute to the under- modulates VTA DA neurons, we hypothesized that the LHb–RMTg standing of the effects exerted by different classes of dopaminergic circuit may mediate aversion to ethanol and hence regulate ethanol drugs on non-motor symptoms of PD. intake. We addressed this fundamental question by investigating voluntary ethanol intake and ethanol-induced conditioned taste A18.111 aversion (CTA) in LHb- and RMTg-lesioned rats. We found that LHb Adenosine and dopamine receptor interaction on selection of and RMTg lesions significantly increase escalation of voluntary sedentary versus active sources of reinforcement: impact on ethanol consumption in an intermittent ethanol access paradigm and DARPP32 phosphorylation patterns also attenuate ethanol-induced CTA. Next, we determined the role of Mercè CORREA1,*, Laura LÓPEZ CRUZ1, Noemi SAN MIGUEL1, neuronal activity in the LHb in mediating aversion to ethanol. To Carla CARRATALA1, Régulo OLIVARES1, Lidón MONFERRER1 directly address this question, we used in vivo electrophysiology to and John D. SALAMONE2 record LHb activity during operant responding for saccharin in 1Department of Psychobiology, Universitat Jaume I, Castelló de la sessions before and after the development of ethanol-induced CTA. Plana, Spain; 2Department of Psychological Sciences, University of We found that baseline firing of the LHb neurons were elevated after Connecticut, Storrs, CT, United States of America the development of ethanol-induced CTA. Furthermore, cue-, lever- *E-mail: [email protected] press- and reward-evoked phasic activity of the LHb neurons was Intrinsic Activity, 2016; 4(Suppl. 2): A18.111 higher during CTA sessions as compared to pre-CTA sessions. http://www.intrinsicactivity.org/2016/4/S2/A18.111 Together, our findings suggest that LHb–RMTg excitatory activity is critical for expression of ethanol-induced CTA. Thus, the LHb–RMTg Vigor, persistence, and work output are fundamental features of circuit, through inhibition of VTA DA neurons, may provide an normal motivation. However, they are potentiated in drug addiction aversive teaching signal, which is crucial for negative regulation of as excessive seeking behavior. Dopamine (DA), in nucleus accum- ethanol intake. bens, regulates these activational aspects of motivation. In striatum

DA D2 receptors are co-localized with adenosine A2A receptors while A18.110 A1 are co-localized with D1 receptors interacting in an antagonistic

Differential involvement of dopamine D1- and D2-type receptors manner. In the present work, wild-type (WT) and A2A receptor in the cognitive and affective symptoms of Parkinson’s disease knockout (A2ARKO) mice were used to explore the impact of D2 Débora MASINI*, Alessandra BONITO-OLIVA, Giada SPIGOLON antagonism in the activational component of motivated behaviors. We and Gilberto FISONE also evaluated the effects of caffeine, a widely consumed psychostimulant that acts as a non-selective adenosine A /A receptor Department of Neuroscience, Karolinska Institutet, Stockholm, 1 2A antagonist, after DA depletion on preference between sedentary vs. Sweden active sources of reinforcement. Adenosine / DA receptor-activity- *E-mail: [email protected] related markers (pDARPP32-Thr75 and -Thr34) were assessed using Intrinsic Activity, 2016; 4(Suppl. 2): A18.110 immunoblotting, and c-Fos immunoreactivity measures were used to http://www.intrinsicactivity.org/2016/4/S2/A18.110 assess brain areas affected by this interaction. A2ARKO mice were Psychiatric disorders are increasingly recognized as a major resistant to the impact of haloperidol in shifting behavior from a high challenge in the treatment of Parkinson’s disease (PD). These non- density of reward–high effort option to a low density–low effort option motor symptoms, which often appear in the prodromal stage of the in a T-maze with a barrier. Moreover, in a T-maze task for the disease, affect a large number of patients and are only partly assessment of preference between physical activity (wheel running) responsive to conventional antiparkinsonian medications, such as in one arm and a dish with freely available sucrose pellets in the

other, WT animals that received haloperidol shift normal behavior A18.113 spending less time running, but increasing time consuming sucrose. Exploring the neural mechanism by which optogenetic A2ARKO mice did not shift to the less effort-requiring reinforcer after stimulation of ventral tegmental area dopamine neurons haloperidol administration. WT animals showed a higher increase in prevents extinction of cued approach behavior c-Fos expression after haloperidol administration than A2ARKO mice Cindy REYES* and Saleem NICOLA in cingulate cortex and accumbens core. DA depletion also produced Department of Neuroscience, Albert Einstein College of Medicine, a shift in the relative preference, as did haloperidol, reducing the Brony, NY, United States of America choice of the running wheel, but increasing consumption of sucrose, *E-mail: [email protected] and caffeine was able to reverse this shift in preferences. These Intrinsic Activity, 2016; 4(Suppl. 2): A18.113 behavioral effects were parallel to pDARPP32 changes. All these http://www.intrinsicactivity.org/2016/4/S2/A18.113 data suggest that adenosine antagonism modulates the role of DA in active seeking of reinforcers, and support the idea of a functional When an environmental stimulus predicts a reward at a specific adenosine / DA interaction at the receptor level that modulates location, animals learn to approach that location in response to the motivational processes. stimulus. When the stimulus no longer predicts reward, the approach behavior extinguishes. Recordings from dopamine (DA) neurons A18.112 during conditioned approach tasks have demonstrated that DA PKCβ inhibitors attenuate amphetamine- and cocaine- neurons in the ventral tegmental area (VTA) burst in response to the stimulated dopamine release delivery of an unexpected reward; conversely, when predicted Alexander ZESTOS*, Sarah MIKELMAN, Robert KENNEDY rewards are omitted, there is a characteristic pause in firing. This and Margaret GNEGY suggests that DA neurons encode reward prediction errors (RPEs), which serve to update the current state and alter the strength of cue– Department of Pharmacology, University of Michigan, Ann Arbor, reward associations depending on the valence of the RPE (positive MI, United States of America or negative). While RPEs presumably lead to changes in response *E-mail: [email protected] probability, the neural mechanisms downstream of the RPE signal Intrinsic Activity, 2016; 4(Suppl. 2): A18.112 that mediate this behavior remain unknown. http://www.intrinsicactivity.org/2016/4/S2/A18.112 Many nucleus accumbens (NAc) neurons exhibit cue-evoked excita- Amphetamine abuse afflicts over 13 million people, and there is tions that are required for approach behavior. We hypothesized that currently no universally accepted treatment for amphetamine addic- negative RPE signals carried by the pause in VTA DA neuronal tion. Amphetamine serves as a substrate for the dopamine trans- activity may result in decreased cue-evoked firing of NAc neurons in porter (DAT) and reverses the transporter to cause dopamine efflux subsequent trials, lowering the probability of cued approach. To test in addition to inhibiting the vesicular monoamine transporter (VMAT) this hypothesis, we used a conditioned approach task with a reward to promote dopamine exocytosis. Activation of protein kinase Cβ omission paradigm, and recorded from neurons in the NAc in Th::Cre enhances extracellular dopamine in the presence of amphetamine by rats that express channelrhodopsin in VTA DA neurons. During a 30 enhancing the reverse transport of dopamine and internalizing the D2 min baseline period, auditory cues predicted the availability of a autoreceptor. We previously demonstrated that PKCβ inhibitors block sucrose reward contingent on entry into a receptacle. Sucrose reward amphetamine-stimulated dopamine efflux in rat striatum in vitro. In was then omitted for the rest of the 90 min session, leading to a this study, we utilized in vivo microdialysis in live, behaving rats to decrease in responding. Using multi-electrode recording of NAc unit assess the effect of the PKCβ inhibitors enzastaurin and firing, we found a reduction in the magnitude of cue-evoked ruboxistaurin on amphetamine-stimulated increases in monoamines excitations of NAc neurons on trials subsequent to the reward and their metabolites. A 30 min perfusion of the nucleus accumbens omission. Additionally, we found that optical stimulation of VTA DA core with 1 µM enzastaurin or 1 µM ruboxistaurin reduced amphet- neurons at the time when reward was omitted was sufficient to amine-stimulated efflux of dopamine and its metabolite 3-methoxy- prevent extinction. By recording from NAc neurons during optical tyramine by approximately 50 %. The inhibitors also significantly stimulation trials, we will be able to determine if the reduction in cue- reduced extracellular levels of norepinephrine and its metabolite evoked excitations is prevented, thereby suggesting a mechanism by normetanephrine after amphetamine. The stimulation of locomotor which phasic dopamine release modulates cue-evoked NAc neuronal behavior by amphetamine, measured simultaneously with the excitations on subsequent trials. analytes, was comparably reduced by the PKCβ inhibitors. Ruboxistaurin also attenuated cocaine-stimulated extracellular A18.114 dopamine, a process that would not be dependent upon DAT Nicotinic and opioidergic modulation on inhibitory inputs to reversal. In order to see if this process was D2 autoreceptor- cholinergic interneurons in the striosomes and the matrix of mediated, we examined the effect of ruboxistaurin on cocaine the mouse striatum activation when D2 receptors were blocked with raclopride. The Ritsuko INOUE* and Masami MIURA inhibitory effect of ruboxistaurin was reduced in the presence of Department of Neurophysiology, Tokyo Metropolitan Institute of cocaine and raclopride, suggesting that ruboxistaurin action involved Gerontology, Tokyo, Japan D autoreceptors. Using a stable isotope label retrodialysis 2 *E-mail: [email protected] procedure, we determined that ruboxistaurin had no effect on basal Intrinsic Activity, 2016; 4(Suppl. 2): A18.114 levels of dopamine, norepinephrine, glutamate, or GABA. Our results http://www.intrinsicactivity.org/2016/4/S2/A18.114 support the utility of using PKCβ inhibitors to reduce the effects of amphetamine and cocaine. Striatal functions are strongly modulated by both cholinergic and Funding support comes from DA11697, T32DA07268, and NIHR37 opioidergic innervation; however, the modulations would be uneven EB003320. between the compartments, the striosomes (or patches) and the extrastriosomal matrix. The striosomes and the matrix are anatomically and neurochemically distinct compartments. Acetylcholinester- ase is expressed more abundantly in the matrix, and dopaminergic innervation is relatively dense in neonate striosomes. Furthermore,

μ-opioid receptors are expressed exclusively in the striosomal In contrast to previous findings, results indicate that dopamine in projection neurons. general is critical for acquisition and expression of both sign-tracking

We examined γ-aminobutyric acidergic (GABAergic) inhibitory inputs and goal-tracking behaviour. More specifically however, D1R antago- to cholinergic interneurons in both compartments, and found that nists impaired acquisition and expression of sign- and goal-tracking, nicotinic receptor (nAChR)-mediated GABAergic responses were while D2R antagonists impaired the expression of both behaviours but evoked more frequently in the matrix than in the striosomes. Most of only impacted the acquisition of sign-tracking, not goal-tracking. the matrix cholinergic neurons received polysynaptic inhibitory post- Results in general support the conclusion that sign-tracking may be synaptic currents, which had long latency and required the activation more sensitive to dopaminergic manipulations than goal-tracking. It of nicotinic receptors. A single action potential of cholinergic neurons is suggested that while D1 receptor activity may be critical for learning induced nAChR-mediated GABAergic inputs to the cholinergic a CS–US association, D2 receptor activity may play a more specific neurons themselves, suggesting mutual connections that shape the role in the motivational processes involved in making a CS a target temporal firing pattern of cholinergic neurons. The nAChR-mediated of approach. responses were attenuated by continuous application of acetylcholine References or the acetylcholinesterase inhibitor eserine, and were enhanced by 1. Tomie A, Grimes KL, Pohorecky LA: Behavioral characteristics and desformylflustrabromine, a positive allosteric modulator of the α4β2 neurobiological substrates shared by Pavlovian sign-tracking and subunit-containing nicotinic receptor. SCH-23390 and sulpiride, drug abuse. Brain Res Rev, 2008; 58(1):121–135. antagonists of dopamine receptors, had no effect on the nAChR- doi:10.1016/j.brainresrev.2007.12.003 mediated responses. We also evaluated the opioidergic effects on the 2. Huys QJ, Tobler PN, Hasler G, Flagel SB: The role of learning- GABAergic inhibitory inputs in both compartments. Although an related dopamine signals in addiction vulnerability. Prog Brain application of DAMGO, a μ-opioid receptor agonist, did not affect the Res, 2014; 211:31–77. doi:10.1016/B978-0-444-63425-2.00003-9 nAChR-mediated GABAergic inputs, a frequency of mIPSCs of 3. Flagel SB, Clark JJ, Robinson TE, Mayo L, Czuj A, Willuhn I, Akers CA, cholinergic interneurons was reduced by DAMGO only in the Clinton SM, Phillips PE, Akil H: A selective role for dopamine in striosomes, suggesting that continuous inhibitory inputs were stimulus–reward learning. Nature, 2011; 469(7328):53–57. provided by the striosomal projection neurons. doi:10.1038/nature09588 As acetylcholine and opioid are important modulators of striatal functions, the differences in nicotinic and opioidergic effects between A18.116 the striosomes and matrix might be involved in the physiology and Pharmacology of IRL790, a psychomotor stabilizer for the pathophysiology of the striatal compartments. treatment of L-DOPA-induced dyskinesias and psychosis in Parkinson’s disease A18.115 Nicholas WATERS1,*, Manolo CARTA2, Joakim TEDROFF3, 1 1 1 Dopamine D1 and D2 receptor involvement in Pavlovian Clas SONESSON , Johan KULLINGSJO , Peder SVENSSON conditioned approach and Susanna WATERS4 Stephanie ROUGHLEY* and Simon KILLCROSS 1Integrative Research Laboratories, Gothenburg, Sweden; School of Psychology, University of New South Wales, Sydney, 2Department of Biomedical Sciences, University of Cagliari, Italy; New South Wales, Australia 3Institute of Clinical Neuroscience, Karolinska Institutet, Stockholm, *E-mail: [email protected] Sweden; 4Sahlgrenska University Hospital, Gothenburg, Sweden Intrinsic Activity, 2016; 4(Suppl. 2): A18.115 *E-mail: [email protected] http://www.intrinsicactivity.org/2016/4/S2/A18.115 Intrinsic Activity, 2016; 4(Suppl. 2): A18.116 http://www.intrinsicactivity.org/2016/4/S2/A18.116 Pavlovian conditioned approach behaviour can be separated into 2 types: sign-tracking, in which an animal approaches and engages The key pathophysiology of PD involves loss of dopaminergic and with the conditioned stimulus (CS), and goal-tracking, in which noradrenergic neurons in the substantia nigra and locus coeruleus, behaviour is oriented towards the associated outcome (US). An respectively, which causes severe motor symptoms. Other sub- investigation into the neurochemical differences between these two cortical, cortical, and autonomic pathologies contribute to the non- processes is of interest because a propensity toward sign-tracking is motor symptoms in PD. It has been suggested that cortico-striatal suggested to reflect compulsive characteristics relating to addiction dysconnectivity and plasticity are key drivers for both core symptoms vulnerability, and may be a factor that contributes to the maintenance of Parkinson’s disease and adverse effects emerging with long-term of addictive behaviour [1, 2]. Recent research into the involvement of dopaminergic treatment. dopamine in sign- and goal-tracking has suggested that while it IRL790 is a novel DA D3 / D2 / σ1-modulating compound, aimed at appears to be important for both acquisition and expression of sign- reducing levodopa-induced dyskinesias and psychosis in PD through tracking responses, under a non-specific dopamine antagonist the its powerful psychomotor stabilizing properties. It is currently in expression of goal-tracking responses is impaired but acquisition phase I. IRL790 has been investigated in a series of in vivo pharma- remains intact [3]. The present experiments first aimed to replicate cological studies including models of disrupted dopaminergic or these findings, and subsequently investigate whether involvement glutamatergic transmission. Biological measures include monoamin- could be isolated to specific dopamine receptors. ergic biomarkers, gene expression and behaviour. Rats were trained for 12 days on one of two basic Pavlovian In the monoaminergic systems, indices of increased DA turnover conditioning paradigms with pellet reinforcers. The CS was either a appear in basal ganglia and cortical regions. IRL790 also increases 10-sec lever presentation or a 10-sec train of clicks. These different extracellular DA, NE and Ach levels measured by in vivo micro- stimuli are shown to result in the development of sign-tracking (lever dialysis. Furthermore, it induces frontal cortex and striatal Arc gene pressing) or goal-tracking responses (entry to food magazine) expression. The concomitant increase in striatal DA, DA metabolites respectively. Half the rats in each experiment received injections of and Arc indicates modulation of striatal DA transmission associated either the non-specific dopamine antagonist flupenthixol, the D1R with enhanced signaling onto medium spiny neurons from cortico- antagonist SCH 39166, or the D2R antagonist eticlopride prior to each striatal projection neurons, likely arising through inhibition of striatal of the first 7 sessions of training. DA D3 and D2 receptors.

In rat models of L-DOPA-induced adverse involuntary movements Intrinsic Activity, 2016; 4(Suppl. 2): A18.118 (AIMs), IRL790 significantly reduces abnormal motor behaviour, http://www.intrinsicactivity.org/2016/4/S2/A18.118 without affecting L-DOPA-induced therapeutic effects. Moreover, In both human and nonhuman primates, dopamine (DA) in the IRL790 has no effect on spontaneous locomotor activity over a wide prefrontal cortex (PFC) plays important roles in cognitive functions. dose range, but counteracts psychostimulant-induced hyperactivity. The relationship between cognitive performance and PFC DA levels Thus in addition to antidyskinetic effects, IRL790 appears to behave follows an “Inverted-U-shaped” function, where both too little and too as an antipsychotic. This indicates a novel compound profile with much DA impairs performance. However, there have been no studies potential to alleviate LIDs and psychosis while sparing normal motor to directly measure human PFC DA levels in relation to cognitive functions and L-DOPA efficacy. functions because of the relatively low basal PFC DA level and technical difficulties associated with measuring them. While the basal A18.117 PFC DA level in the monkey is also very low (about 1/10 of the CD24 expression does not affect dopamine neuron survival striatum level), it is possible to directly measure DA levels in the in a mouse model of Parkinson’s disease monkey PFC in relation to task performance using “in vivo micro- 1, 1 2 Simon R. W. STOTT *, Shaista HAYAT , Jonathan P. SLEEMAN dialysis”. We trained monkeys on the cognitively demanding working 1 and Roger A. BARKER memory (WM) task (delayed alternation task) as well as a cognitively 1Department of Clinical Neuroscience, University of Cambridge, non-demanding sensory-guided control task and conducted micro- United Kingdom; 2Medical Faculty Mannheim, University of dialysis experiments obtaining dialysate samples from several areas Heidelberg, Mannheim, Germany of the monkey PFC. We found a significant increase in DA release *E-mail: [email protected] during the WM task compared with that during the control task only Intrinsic Activity, 2016; 4(Suppl. 2): A18.117 at the dorsolateral, but not at the arcuate or orbitofrontal PFC area. http://www.intrinsicactivity.org/2016/4/S2/A18.117 As an extension of our monkey PET (positron emission tomography) study where we found default mode of brain activity (more activity Parkinson’s disease (PD) is a neurodegenerative condition that is during rest than during the cognitively demanding task period) in the characterised by the loss of specific populations of neurons in the medial PFC, we examined DA release in the medial PFC of the brain, including nigral dopaminergic cells. The mechanisms under- monkey during the WM task. We found a significantly smaller DA lying this selective cell death are unknown. We identified the glyco- release in the medial PFC during the WM task than during rest, protein CD24 as a potential marker of those neurons that are affected indicating the increased DA level during rest compared with that in PD and decided to investigate whether it had any role in PD. Using during the WM task. Human default mode of brain activity is immunohistochemistry and in situ hybridization on sections of adult considered to be related to internal thought processes. Considering mouse brain, we found that CD24 is robustly expressed by many of that DA in the dorsolateral PFC is associated with cognitive the specific subsets of cells affected by PD, such as the dorsal motor operations, and monkeys have enough social intelligence, DA in the nucleus of the vagus, locus coeruleus, substantia nigra, and amyg- medial PFC may be concerned with internal thought processes of the dala. In order to determine any functional role that CD24 may have in monkey although it may be of primitive level because of the lack of the disease course, we modelled PD in CD24 mutant mice using language. striatal delivery of 6-OHDA. The CD24 mutant mice had a normal midbrain dopamine system, suggesting that the absence of CD24 does not affect the development or maintenance of this population of A18.119 neurons. When 6-OHDA was delivered to the striatum of these mice, Development of a high-throughput assay to measure VMAT2- we found no difference in the loss of striatal fibre innervation or cell mediated vesicular dopamine transport loss in the midbrain. There was a significant reduction in the number Carlie HOFFMAN*, Kristen STOUT, Bethany WILSON, Lauren JONAS of dividing cells in the substantia nigra one week post surgery, but and Gary MILLER this had no long-term impact on the dopamine cell survival (at 70 days Department of Neuroscience, Emory University, Atlanta, GA, post surgery). Immunohistochemistry and in situ hybridization on United States of America sections of healthy adult human brain suggested that CD24 is *E-mail: [email protected] expressed by the dorsal motor nucleus of the vagus, locus coeruleus, Intrinsic Activity, 2016; 4(Suppl. 2): A18.119 and amygdala, but not the substantia nigra. Expression analysis http://www.intrinsicactivity.org/2016/4/S2/A18.119 suggested a reduction of CD24 in many regions of the PD brain, but Cytoplasmic dopamine is packaged into neuronal vesicles via the no difference in the substantia nigra. These results led us to conclude vesicular monoamine transporter 2 (VMAT2; SLC18A2). Perturba- that the expression of CD24 does not affect the survival of dopamine tions in VMAT2 function have been found to result in altered vesicular neurons in mice and may not necessarily play a direct role in PD. integrity within the nigrostriatal dopamine system, modified neuronal Despite this conclusion, we still believe that CD24 is worthy of further vulnerability to toxic insult, and increased risk for neurodevelopmen- investigation in both the dopamine system and PD. tal and neurodegenerative diseases such as Parkinson’s disease. Recent studies indicate that overexpression of VMAT2 using a BAC A18.118 construct increased VMAT2 expression, enhanced dopamine release, Dopamine release in the prefrontal cortex increases in the and conferred protection to toxicants. In an attempt to identify small dorsolateral (executive) area and decreases in the medial molecules that alter VMAT2 function, we previously developed an (default mode) area during the working memory task in the assay to spatially resolve VMAT2-mediated packaging of dopamine monkey: in vivo microdialysis studies utilizing high-content imaging with a fluorescent dye and mCherry- 1, 1 1 Masataka WATANABE *, Takashi KOJIMA , Yoshiko HONDA , tagged VMAT2, but this assay required significant image analysis to 2 1 Ken-ichiro TSUTSUI and Tohru KODAMA obtain suitable results. The recent development of the false fluores- 1Department of Physiological Psychology, Tokyo Metropolitan cent neurotransmitter, FFN206, by Sames and Sulzer [1] allowed us Institute of Medical Science, Tokyo, Japan; 2Division of Systems to adapt our assay to a fluorescent plate reader format. We have now Neuroscience, Tohoku University Graduate School of Life Sciences, optimized a cell-based high-throughput assay using FFN206 and Sendai, Japan HEK 293 cells stably transfected with VMAT2 in 96-well plates. This *E-mail: [email protected] assay has a dynamic range that allows detection of compounds that

inhibit or enhance vesicular uptake. We have demonstrated that the Reference assay yields appropriate values for dopaminergic drugs such as 1. Bassareo V, Cucca F, Frau R, Di Chiara G: Differential activation of tetrabenazine (TBZ) and environmental toxicants such as poly- accumbens shell and core dopamine by sucrose reinforcement chlorinated biphenyls (PCBs) when compared to values obtained with nose poking and with lever pressing. Behav Brain Res, 2015; from vesicular dopamine uptake in isolated synaptic vesicles. We are 294:215–213. doi:10.1016/j.bbr.2015.08.006 in the process of optimizing the assay for a 384-well plate format to permit library screening. This assay should provide a robust initial A18.121 screening of compounds that alter VMAT2 function that can then be Amphetamine in adolescence alters the expression of the verified using dopamine uptake in isolated vesicles and fast scan dopamine gene, Dcc, and its microRNA regulators cyclic voltammetry. Santiago CUESTA*, José María RESTREPO-LOZANO, Reference Dominique NOUEL and Cecilia FLORES 1. Hu G, Henke A, Karpowicz RJ Jr, Sonders MS, Farrimond F, Edwards Department of Psychiatry, McGill University, Montreal, QC, R, Sulzer D, Sames D: New fluorescent substrate enables Canada quantitative and high-throughput examination of vesicular *E-mail: [email protected] monoamine transporter 2 (VMAT2). ACS Chem Biol, 2013; 8(9): Intrinsic Activity, 2016; 4(Suppl. 2): A18.121 1947–1957. doi:10.1021/cb400259n http://www.intrinsicactivity.org/2016/4/S2/A18.121

A18.120 Initiation of drug use in adolescence is a strong predictor of the Differential c-fos activation in the nucleus accumbens shell incidence and severity of addiction throughout life. Adolescence is a and infralimbic cortex following acquisition of heroin self- period of dynamic refinement in the organization of prefrontal cortex administration using lever-pressing and nose-poking operant (PFC) circuitry, with dramatic changes occurring in the establishment responses of dopamine connectivity. We have shown that the guidance cue Annesha SIL1,*, Augusta PISANU2, Daniele LECCA1, Valentina receptor DCC, which is highly expressed by dopamine neurons, BASSAREO1, Roberto FRAU1, Andrea SCIFO1 and Gaetano DI CHIARA1 orchestrates the dopamine innervation to the medial PFC (mPFC) specifically in adolescence. Importantly, we showed that exposure 1Department of Biomedical Sciences, University of Cagliari, Italy; to amphetamine (AMPH) in adolescence, but not in adulthood, 2Institute of Neuroscience, National Research Council, Cagliari, Italy (a) downregulates DCC protein expression within ventral tegmental *E-mail: [email protected] area (VTA) dopamine neurons, (b) leads in turn to increased mPFC Intrinsic Activity, 2016; 4(Suppl. 2): A18.120 dopamine input, but to a drastic reduction in mesocortical dopamine http://www.intrinsicactivity.org/2016/4/S2/A18.120 axons presynaptic sites, and (c) causes exaggerated salience Nose-poking (NP) and lever-pressing (LP) represent two different attribution to a previously drug-paired environment. However, how operant responses which have been utilized in the self-administration AMPH in adolescence regulates DCC protein expression in dopamine (SA) paradigm to study behavioural and neurochemical effects neurons is currently unknown. Here, we sought to investigate whether induced by food or drug administration in an active contingent- AMPH in adolescence alters Dcc mRNA expression in the VTA and response manner. NP is part of an animal’s natural exploratory whether epigenetic mechanisms are involved. To this end, we treated repertoire which is used in SA as the required conditioned response adolescent C57BL/6 mice with saline or with the exact same to obtain a reward, whereas LP requires the animal to learn to the AMPH regimen (4 mg/kg, one injection per day, every other day, from activity of pressing the correct lever in order to receive a reward. PND 21 ± 1 to PND 31 ± 1) that downregulates DCC expression and Following the studies comparing differences in the mesolimbic disrupts PFC dopamine development [1, 2]. One week later, we dopaminergic responsiveness induced by sucrose reinforcement measured Dcc mRNA expression in the VTA using real time PCR. using LP and NP as operant responses [1], this study was designed Consistent with the effects of AMPH on DCC protein expression in to evaluate postsynaptic activation in limbic areas induced by heroin adolescence, we found a significant reduction in VTA Dcc mRNA SA under these two different protocols. expression in AMPH-treated mice in comparison to saline-treated Sprague-Dawley (SD) rats were trained for 10 days on an FR1 controls. Remarkably, this reduction was accompanied by increased schedule to acquire heroin self-administration (0.05 mg/kg) using NP expression of a microRNA which is expressed by dopamine neurons or LP modus operandi. After acquisition, some animals were sacri- and is known to repress Dcc expression in the rodent and human ficed within 1 hour of the end of the last session, while another brain. We are currently performing in situ hybridization to determine group was switched to an FR5 schedule for one week, and then coexpression of Dcc and the microRNA in VTA dopamine neurons. sacrificed similarly. The c-fos expression in nucleus accumbens and We are also assessing whether exposure to environmental infralimbic cortex of both groups was quantified using immunohisto- enrichment in adolescence alters Dcc and its epigenetic regulators, chemistry. potentially protecting and / or reversing enduring detrimental effects of Results revealed a robust, significant increase in the number of c-fos stimulant drugs of abuse. positive neurons in the nucleus accumbens shell and the infralimbic References cortex in the LP group compared to the NP group, only under the FR5 1. Yetnikoff L, Almey A, Arvanitogiannis A, Flores C: Abolition of the schedule. While the heroin intake did not differ between the LP and behavioral phenotype of adult netrin-1 receptor deficient mice by NP groups, the pattern of responding under the FR5 schedule, exposure to amphetamine during the juvenile period. showed that, compared to the NP group, the LP group had lower Psychopharmacology, 2011; 217(4):505–514. latency between responses, a possible indicator of a stronger doi:10.1007/s00213-011-2312-6 association between the conditioned response and the reward. Taken 2. Reynolds LM, Makowski CS, Yogendran SV, Kiessling S, Cermakian together, these results suggest that especially under tougher N, Flores C: Amphetamine in adolescence disrupts the schedules of responding such as FR5, the operant response utilised development of medial prefrontal cortex dopamine connectivity might have different effects on reinforcement and incentive learning in a DCC-dependent manner. Neuropsychopharmacology, 2015; and should be an important consideration while modelling different 40(5):1101–1112. doi:10.1038/npp.2014.287 aspects of self-administration behaviour.

A18.122 the risk of relapse in addicts attempting to quit. In patients with The neuronal calcium sensor NCS-1 modulates age-dependent schizophrenia an ‘endogenous’ sensitization of the dopaminergic sensitization of dopamine D2 autoreceptor responses as well system has been proposed by several authors. Sensitization involves as survival of substantia nigra dopaminergic neurons changes in brain dopamine transmission. In healthy subjects, Julia BENKERT1,*, Alina GLEBOVA1, Johanna DUDA1, Christina dopaminergic sensitization can be induced by repeated amphetamine PÖTSCHKE1, Elena DRAGICEVIC1, Olaf PONGS2, Tobias FRANK3 administration. Studies in animals and humans have shown sex and Birgit LISS1 differences in dopamine release and behaviour after sensitization. We aimed at investigating behavioural effects of D-amphetamine 1Institute of Applied Physiology, University of Ulm, Germany; induced sensitization in stimulant-naïve healthy humans. 2Department of Physiology, University of the Saarland, Homburg, Objectives: Twenty (12 M / 8 F) healthy subjects underwent four oral Germany; 3Department of Neurology, University Medical Center administrations of D-amphetamine (0.4 mg/kg bodyweight) on four Göttingen, Germany separate days, with the first three administrations being at least one *E-mail: [email protected] day apart. The fourth D-amphetamine administration was performed Intrinsic Activity, 2016; 4(Suppl. 2): A18.122 two weeks later. Behavioral effects of D-amphetamine were assessed http://www.intrinsicactivity.org/2016/4/S2/A18.122 before D-amphetamine ingestion, and 60 min, 90–120 min, and The preferential and progressive degeneration of substantia nigra 210 min after D-amphetamine ingestion using the drug effects dopamine midbrain (SN DA) neurons causes the major motor-related questionnaire (DEQ) and the subjective states questionnaire (SSQ). symptoms of Parkinson’s disease (PD). While the molecular Changes in behavioral measures were calculated relative to baseline mechanisms of the particularly high vulnerability of SN DA neurons assessments. Sensitization effects were analyzed by comparing to degeneration in PD and during aging remain unresolved, a variety score change of the first and fourth amphetamine administration of disease triggering factors have been identified. Among them are using t-tests. e. g. genetic factors (PARK genes), metabolic stress, mitochondrial Results: At 90–120 min, there was a significant change in all items dysfunction, and activity-related Ca2+ load. However, age remains the of DEQ and SSQ pleasant stimulation after each amphetamine most prominent risk factor for PD. In addition, the electrical activity of administration for the whole sample (p < 0.05). Significant effects of SN DA neurons itself seems to affect their vulnerability to PD-triggers sensitization (dose 1 vs. dose 4 at 90–120 min) were found for the and to degeneration. This activity is intrinsically generated and whole sample for DEQ items “feel drug” and “want more“ (p < 0.05), controlled by a range of different ion channels and receptors, and it and for SSQ items “alert”, “focused”, “outgoing”, “energetic”, “lively” is crucial for presynaptic as well as somatodendritic dopamine (p < 0.005). Sensitization effects were stronger in males for SSQ release. Dopamine itself, in a negative feedback loop, modulates the items “outgoing” and “lively” (p < 0.05). activity of SN DA neurons by activating GIRK2 K+ channels via Conclusions: Our preliminary data show robust behavioural inhibitory dopamine autoreceptors of the D2 type (D2AR). dopamine sensitization effects in stimulant-naïve healthy humans. With cell-specific electrophysiological, pharmacological and molecu- Effects of sensitization in some of the behavioral domains were lar analysis of postnatal juvenile (PN13) and adult (PN90) mouse DA significantly stronger in males. Our findings may help explain the sex midbrain neurons, we identified that SN DA neurons — in contrast to differences in prevalence, symptom profile, severity, and course of neighboring and less vulnerable VTA DA neurons — display promi- addictive disorders and schizophrenia. nent somatodendritic D2AR responses that sensitize during postnatal 2+ maturation. This sensitization was Ca -dependent, and it required A18.124 the interaction of the neuronal calcium sensor NCS-1 with the D2AR. [18F]DOPA PET: quality of input functions obtained from Sensitized D2AR responses were absent in NCS-1 KO mice, and they arterial versus venous blood could provide a molecular mechanism for protecting SN DA neurons Anne SIGVARD1, Mette ØDEGAARD NIELSEN1, Albert GJEDDE2, from overexcitability and degeneration. Therefore, we analyzed Lars-Thorbjørn JENSEN3, Egill ROSTRUP4,* and Birte GLENTHØJ1 numbers of SN DA and VTA DA neurons in WT and NCS-1 KO mice 1Center for Neuropsychiatric Schizophrenia Research, CNSR, at PN13, PN90, PN360 and PN580, by combining immunohisto- Mental Health Centre Glostrup, Denmark; 2Faculty of Health and chemistry and unbiased stereology. We detected that the loss of Medical Sciences, University of Copenhagen, Denmark; NCS-1 indeed selectively affected the progressive age-dependent 3Department of Clinical Physiology and Nuclear Medicine, University loss of SN DA neurons. We currently investigate whether that is also of Copenhagen, Denmark; 4Functional Imaging Unit, Department of the case in the chronic MPTP PD mouse model. Clinical Physiology and Nuclear Medicine, Rigshospitalet – Glostrup, University of Copenhagen, Glostrup, Denmark A18.123 *E-mail: [email protected] Behavioural effects of amphetamine-sensitization in stimulant- Intrinsic Activity, 2016; 4(Suppl. 2): Aold36.52 naïve healthy humans http://www.intrinsicactivity.org/2016/4/S2/Aold36.52 Nicole PRASCHAK-RIEDER1,*, Ana POPOVIC1, Martin BAUER2, Lucie BARTOVA1, Ulrich SAUERZOPF1, Siegfried KASPER1 Introduction: Insufficient response to antipsychotic medication may and Matthäus WILLEIT1 be caused by differences in striatal dopamine synthesis capacity (DSC). The overall aim of this positron emission tomography (PET) 1Department of Psychiatry and Psychotherapy, Medical University study is to stratify antipsychotic-naïve first-episode psychotic patients of Vienna, Austria; 2Department of Clinical Pharmacology, Medical based on DSC and evaluate the prognostic value of stratification in University of Vienna, Austria terms of treatment response. To do this, we will recruit patients *E-mail: [email protected] suffering from psychotic symptoms who are not yet medicated at Intrinsic Activity, 2016; 4(Suppl. 2): A18.123 baseline. Working with a particularly vulnerable patient group raised http://www.intrinsicactivity.org/2016/4/S2/A18.123 the question of whether a less invasive procedure is applicable and Background: Sensitization is defined as a learning process in which input functions obtained from venous blood samples would be repeated administration of a stimulus results in the progressive comparable with arterial input functions. In the present preliminary amplification of a response. Drug sensitization can be seen as the work we compare the quality of input functions from arterial and opposite of drug tolerance. It may contribute to drug addiction and to venous blood respectively.

Background: DSC can be assessed using 3,4-dihydroxy-6- structure may affect the firing mode of DA cells. Excitatory and [18F]fluoro-L-phenylalanine ([18F]DOPA) PET. Different approaches inhibitory inputs are generated by the firing activity of a population of have been applied when interpreting [18F]DOPA PET data. In this glutamatergic and GABAergic neurons. The synchronization proper- study kinetic parameters are determined using the two-compartment ties of such populations affect the excitatory or inhibitory post- model. The input functions require measurement of peripheral radio- synaptic currents flowing in DA cells. Actually, the average neurons activity originating from intact [18F]DOPA and the primary metabolite firing rate drives the average of such currents, while their correlation 3-O-methyl-FDOPA (OMFD), respectively. Intact [18F]DOPA and drives the deviations of such currents from their mean. We investigate OMFD need to be distinguished in the blood. Arterial blood samples how these parameters drive the dynamics of DA neurons. To do so, collected through an arterial cannula are the standard in studies using we make use of a biophysically inspired conductance-based model, the two-compartment model. which is known to reproduce the main features of DA cells [3]. Methods: Eight healthy controls (HC) were scanned using Furthermore, we consider an experimentally validated model for [18F]DOPA. Blood samples were collected sequentially to measure synaptic DA release, given the firing pattern of DA neurons [1]. Our peripheral radioactivity over time and the fractions of [18F]DOPA and crucial finding is that the synchronization of both excitatory and OMFD. In addition to the arterial blood samples we simultaneously inhibitory synaptic drive is decisive for yielding a bursty regime. As a collected venous blood samples. Premedication was given one hour consequence, a non-trivial input structure appears to be a pivotal prior to the scan to minimize peripheral metabolism of [18F]DOPA. ingredient for producing phasic transients in DA release. Results: There is a remarkable difference in input functions from References arterial and venous blood. Venous samples fail to detect the initial 1. Wightman RM, Zimmerman JB: Control of dopamine extracellular peak visualized in the curves derived from arterial samples. This concentration in rat striatum by impulse flow and uptake. Brain finding is of potential significance for the interpretation of neuro- Res Brain Res Rev, 1990; 15(2):135–144. chemical PET data where input functions are based on venous blood doi:10.1016/0165-0173(90)90015-G measures. 2. Morikawa H, Morrisett RA: Ethanol action on dopaminergic neurons Conclusion: Arterial samples are the most reliable measurement of in the ventral tegmental area: interaction with intrinsic ion peripheral radioactivity originating from [18F]DOPA and OMFD. Based channels and neurotransmitter inputs. Int Rev Neurobiol, 2010; on the data future PET examinations in the study will be performed 91:235–288. doi:10.1016/S0074-7742(10)91008-8 with arterial samples. 3. Ha J, Kuznetsov A: Interaction of NMDA receptor and pacemaking mechanisms in the midbrain dopaminergic neuron. PLoS One, A18.125 2013; 8(7):e69984. doi:10.1371/journal.pone.0069984 Effects of inputs structure on dopamine realease in nucleus accumbens: a modelling approach A18.126 1, 1 2 Matteo DI VOLO *, Gregory DUMONT , Ekaterina MOROZOVA , Body mass index relationships with dopamine D3 receptor Maxym MYROSHNYCHENKO3, Christopher LAPISH4, Alexey availability as measured by [11C](+)PHNO KUZNETSOV5 and Boris GUTKIN1 Edward C. GAISER1,2, Ania M. JASTREBOFF3,4, Brian PITTMAN2, 1 2 1Group of Neural Theory, INSERM U960 LNC, IEC, École Normale Jean-Dominque GALLEZOT , Gustavo A. ANGARITA , Lauren 2 1,2 2,5 Superieure PSL University, Paris, France; 2Department of Physics, KANTROVITZ , Kelly P. COSGROVE , Marc N. POTENZA , Robert 2 1 1,2, Indiana University, Bloomington, IN, United States of America; T. MALISON , Richard E. CARSON and David MATUSKEY * 3Program in Neuroscience, Indiana University, Bloomington, IN, 1Department of Diagnostic Radiology, Yale University, New Haven, United States of America; 4Addiction Neuroscience Program, CT, United States of America; 2Department of Psychiatry, Yale Indiana University – Purdue University, Indianapolis, IN, United University, New Haven, CT, United States of America; 3Department States of America; 5Department of Mathematical Sciences, Indiana of Internal Medicine, Endocrinology, Yale University, New Haven, University – Purdue University, Indianapolis, IN, United States of CT, United States of America; 4Department of Pediatrics, Pediatric America Endocrinology, Yale University, New Haven, CT, United States of *E-mail: [email protected] America; 5Department of Neurobiology and Child Study Center, Intrinsic Activity, 2016; 4(Suppl. 2): A18.125 Yale University, New Haven, CT, United States of America http://www.intrinsicactivity.org/2016/4/S2/A18.125 *E-mail: [email protected] Intrinsic Activity, 2016; 4(Suppl. 2): A18.126 The release of dopamine (DA) by dopaminergic neurons of the ventral http://www.intrinsicactivity.org/2016/4/S2/A18.126 tegmental area (VTA) has strong implications for cognitive processing such as reward signaling and, eventually, plays a significant part in Background: Prior work with [11C]raclopride has implicated lower addiction. VTA DA neurons receive GABAergic inputs, notably from striatal dopamine 2/3 receptor (D2/3R) availability in obese (OB) interneurons present in the VTA, as well as glutamatergic inputs from compared to normal weight (NW) subjects. Further work utilizing the 11 different brain regions, e. g. indirectly from the prefrontal cortex. It is D3-preferring [ C](+)PHNO in non-OB subjects indicated BMI well-established that the input temporal structure changes with correlated positively with D2/3R availability in ventral striatum (VS). attentional modulation, anesthesia or drug exposure. In this study, we We sought to investigate D2/3R availability differences between OB focus on the impact of the input structure on the release of DA. The and NW subjects by acquiring [11C](+)PHNO PET. Additionally, where amount of DA released depends not only on the firing rate of D2/3R differences were observed, we added overweight (OW) dopaminergic neurons but also on their specific firing pattern [1]. subjects representative of the U. S. population based on BMI and

Indeed, DA neurons produce different types of activity such as examined the relationship between D2/3R availability and BMI in those pacemaking, irregular and bursting. The bursting mode is charac- regions. terized by high firing frequency episodes, which turn out to induce Methods: Fourteen NW subjects were compared to fourteen age- transients in dopamine concentration in the target structures. In the and gender-matched OB subjects. For the secondary analysis, nucleus accumbens (NAcc), such transients are of essence as they fourteen OW subjects were added and the association between are observed to be markers of rewarding events [2]. It is therefore of D2/3R availability and BMI was examined. Subjects underwent crucial interest to examine under which conditions DA neurons switch [11C](+)PHNO acquisition using a high-resolution research tomo- into a bursty mode. We therefore focus on how a change in the input

graph. Parametric images were computed using the simplified A18.128 reference tissue model with cerebellum as the reference region. Associations between dopamine D2 / D3 receptors, gray-matter Results: NW and OB groups differed in overall D2/3R availability volume, and temporal discounting in methamphetamine users (main effect of group, F1,26 = 4.77, p = 0.04). Post-hoc analyses Angelica M. MORALES1,*, Milky KOHNO1, Chelsea L. ROBERTSON1, revealed OB subjects exhibited increased binding potentials in the Andy C. DEAN1, Mark A. MANDELKERN2 and Edythe D. LONDON1 substantia nigra / ventral tegmental area (SN / VTA) (20 %; p = 0.03), 1Department of Psychiatry and Biobehavioral Sciences, University VS (11 %; p = 0.04), and pallidum (11 %; p = 0.02). BMI correlated of California, Los Angeles, CA, United States of America; 2Greater positively with D R availability in the pallidum (r = 0.30, p = 0.05) and 2/3 Los Angeles Veterans Affairs Health Care System, Los Angeles, SN / VTA (r = 0.34, p = 0.03) in the total sample. CA, United States of America Conclusion: These data suggest that obesity is associated with an *E-mail: [email protected] upregulation of D Rs in brain reward regions, thus potentially identi- 3 Intrinsic Activity, 2016; 4(Suppl. 2): A18.128 fying a novel pharmacologic target for the treatment of obesity. http://www.intrinsicactivity.org/2016/4/S2/A18.128

A18.127 Mesocorticolimbic dysfunction plays a critical role in addiction. How- Monoaminergic modulation of an amygdala fear extinction ever, despite evidence that midbrain dopamine receptors influence circuit amphetamine-induced dopamine release and that dopamine itself is James WOOD, Günther SPERK* and Ramon TASAN causally involved in neurotoxicity from methamphetamine (MA), associations between dopamine receptors and structural deficits in Department of Pharmacology, Medical University of Innsbruck, gray-matter volume (GMV) as related to MA use have been un- Austria explored. Relevant to the sustained use of MA despite obvious harm *E-mail: [email protected] are dopaminergic and structural abnormalities in the brain that may Intrinsic Activity, 2016; 4(Suppl. 2): A18.127 contribute to impairments in decision-making. http://www.intrinsicactivity.org/2016/4/S2/A18.127 Structural magnetic resonance and positron emission tomography The amygdala has a critical role in emotional learning and in with [18F]fallypride, respectively, were used to measure GMV and regulating physiological responses to fearful situations. Dysfunction dopamine D2 / D3 binding potential (BPND) in 31 MA users and 37 of neural projections to, and within the amygdala contributes to the control participants. Participants also completed the monetary choice pathophysiology of anxiety disorders and post-traumatic stress questionnaire. Although no difference in midbrain D2 / D3 BPND was disorder (PTSD). In particular, a deficit in fear extinction, the process detected between MA and control groups, midbrain D2 / D3 BPND was by which an association between a conditioned stimulus and an positively correlated with GMV in the striatum, prefrontal cortex, aversive stimulus is suppressed, is believed to underlie anxiety insula, hippocampus and temporal cortex in MA users, but not in disorders and PTSD. Synaptic plasticity within a microcircuit involving control participants (group-by-midbrain D2 / D3 BPND interaction, the basolateral amygdala (BLA), main intercalated nucleus (Im) and p < 0.05, corrected). Group-by-striatal D2 / D3 BPND interactions were medial subdivision of the central amygdala (CeM) is centrally involved detected on GMV of bilateral striatum, left pallidum, and temporal in fear extinction. This fear extinction circuit receives prominent cortex, reflecting positive correlations between striatal D2 / D3 BPND dopaminergic and noradrenergic innervations and these neuro- and GMV in the MA group, but no significant correlations in the modulatory systems have been suggested to play a central role in control group. In the MA group, striatal (β = 0.38, t24 = 3.00, p = 0.007) amygdala function; however, how dopamine and norepinephrine and midbrain D2 / D3 BPND (β = 0.28, t24 = 2.30, p = 0.03) were both 2 modulate synaptic transmission in this circuit is unknown. Using significant predictors of striatal GMV (adjusted R = 0.759, F6,24 = whole-cell patch clamp recordings we have found that both dopamine 16.75, p < 0.001). Lower midbrain D2 / D3 BPND (r = −0.619, p = 0.001) and the specific α2-adrenergic receptor agonist BHT-933 markedly and smaller caudate volume (r = −0.548, p = 0.005) were both and reversibly suppress feed-forward inhibition in the BLA–Im–CeM associated steeper discounting of delayed rewards in the MA group. microcircuit. Moreover, evoking dopamine and norepinephrine No associations between midbrain D2 / D3 BPND, caudate GMV, and release ex vivo by application of amphetamine resulted in a similar delay discounting behavior were detected in the control group suppression of feed-forward inhibition. Although the receptors (p’s > 0.16). involved in the dopamine-mediated suppression of feed-forward Lower D2 / D3 midbrain BPND may increase vulnerability to structural inhibition are not yet clear, we found that BHT-933 reduces the deficits in mesocorticolimbic circuitry in MA users, possibly due to frequency and amplitude of miniature inhibitory postsynaptic currents greater dopamine-induced toxicity. This study points to possible in CeM neurons indicating that α2-adrenergic receptor activation mechanisms that produce abnormalities in GMV in MA users and reduces inhibition of the CeM. Thus, we provide evidence that helps us understand the extent to which structural and chemical dopaminergic and noradrenergic signaling can modulate the function abnormalities co-occur. This information may be useful in under- of a central fear extinction circuit. standing behavioral abnormalities associated with drug use disorders, such as the preference for smaller sooner reward over larger later rewards.

Author Index (Numbers refer to abstract no.)

Abass, Doaa … A7.3 Benkert, Julia … A18.55, Calderon, Tina … A7.4 Correa, Mercè … A13.13, Abi-Dargham, Anissa … A1.1, A18.122 Callaway, Edward M. … A18.52 A18.111 A18.19 Bergeron, Yan … A18.101 Calon, Frédéric … A18.93 Cosgrove, Kelly P. … A12.7, Adermark, Louise … A18.41, Berman, Joan … A7.4 Cao, Jianjing … A13.11 A18.96, A18.126 A18.84 Berretta, Nicola … A6.1 Carandini, Matteo … A18.89 Costa, Kauê M. … A5.7 Agnati, Luigi F. … A13.2 Betancourt, Erika … A12.5 Carbone, Carmen … A18.51 Coutureau, Étienne … A18.72 Airavaara, Mikko … A18.70 Betolngar, Dahdjim … A18.37 Carlson, Andrew … A18.94 Cowan, Ronald … A18.7 Aitken, Tara J. … A12.3 Bhattacharyya, Sagnik … A8.2, Caron, Marc G. … A16.1 Cowley, Sally A. … A18.49 Albert, Katrina … A18.70 A16.2 Carpenter, Colleen … A18.66 Cozzi, Nicolas V. … A18.11 Allen, Paul … A8.2 Biever, Anne … A7.1 Carratala, Carla … A18.111 Cragg, Stephanie J. … A10.1, Altshuler, Rachel … A18.108 Binley, Katie … A18.67 Carson, Richard E. … A18.126 A12.2 Amara, Susan … A5.2 Björk Wilhelms, Daniel … A18.25 Carta, Manolo … A18.116 Crans, René Albert Johan … Anastos, Kathryn … A7.4 Blakeley, Randy D. … A17.1 Casella, Luigi … A11.3 A18.1 Andersen, Kasper A. … A18.22 Błasiak, Tomasz … A18.43 Cassidy, Clifford … A18.19 Creed, Meaghan C. … A18.78 Ang, Siew-Lan … A2.1, A2.2 Blomqvist, Anders … A18.25, Castelnau, Pierre … A18.36 Crittenden, Jill R. … A10.3 Angarita, Gustavo A. … A18.126 A18.44 Castro, Liliana … A18.37 Cuendet, Michel … A1.2 Aoyama, Yuki … A18.103 Bloomfield, Michael … A8.2, Cathala, Adeline … A18.72 Cuesta, Santiago … A18.121 Aragona, Brandon … A6.3 A16.4 Cavaccini, Anna … A18.9 Cyr, Michel … A18.101 Arban, Roberto … A18.48 Bobadilla, Ana-Clara … A18.99 Cepeda Prado, Efrain … A8.1 Daelemans, Sofie … A18.1 Arends, Natscha … A18.63 Bock, Gabriella … A18.4 Chaberland, Simon … A18.77 Dallto, Uillred … A3.1 Århem, Peter … A18.56 Bodea, Gabriela-Oana … A18.92 Chabrat, Audrey … A2.1, A2.2 Dang, Linh … A18.7 Aron-Badin, Romina … A18.67 Bojesen, Kirsten B. … A18.22 Chaddock, Christopher … A8.2 Dani, John A. … A12.4 Asjad, Hafiz Muhammad Mazhar Bonadonna, John Paul … A18.94 Chagniel, Laure … A18.101 Dannemann, Michael … A18.3 … A18.26, A18.87, A18.91 Bonito-Oliva, Alessandra … Chalon, Sylvie … A18.36 Darlot, Florence … A18.72 Aubert, Agnès … A17.2 A18.110 Charest, Julien … A2.2 Das, Gishnu … A18.21 Aversa, Daniela … A6.1 Bonoldi, Ilaria … A16.4 Chareunsouk, Victoria … A18.99 Davies, Caitlin … A18.47 Avşar, Orçun … A18.98 Booij, Linda … A8.5 Chaudhri, Nadia … A18.100 Davis, Elena … A18.68 Baandrup, Lone … A18.22 Boon, Paul … A18.1 Cheer, Joseph … A18.76 Daws, Lynette … A18.16 Bachtell, Ryan … A18.73 Bornschein, Ulrich … A18.3 Chen, Ching … A18.29 Day, Fern … A8.2 Bagby, Michael … A16.2 Borrelli, Emiliana … A18.6 Chen, Yao-Chu … A18.29 De Deyn, Peter Paul … A18.1 Baik, Ja-Hyun … A13.8 Borroto-Escuela, Dasiel O. … Cheung, Timothy … A18.94 de la Fuente Barrigón, Carmen … Bains, Jaideep S. … A18.8 A13.2, A13.14 Chien, Yin-Hsiu … A18.88 A18.49 Baker, Seth … A18.19 Bortolato, Marco … A15.1, A16.3 Chilcott, Jack … A8.2 De Montis, Maria Graziella … Ballard, Theresa … A18.6 Bosch-Boujou, Clementine … Chioma, Vivian … A18.76 A18.45 Balota, David A. … A18.24 A17.2 Choi, Se Joon … A11.2 De Smedt-Perusse, Véronique … Bampali, Konstantina … A18.27 Brašić, James … A18.12 Chrissy, Makariou-Pikis … A11.1 A17.2 Barker, Roger A. … A18.117 Brimblecombe, Katherine R. … Chuhma, Nao … A5.5 Dean, Andy C. … A12.8, A13.12, Barlow, Rebecca … A18.48 A12.2 Chun, Lauren E. … A13.10 A18.128 Barral, Serena … A18.49 Brisson, Guillaume … A2.2 Cieślak, Przemysław … A18.43, DeFrancesco-Lisowitz, Alicia … Bartova, Lucie … A18.123 Brix, Klaudia … A3.1 A18.102 A5.2 Bassareo, Valentina … A18.120 Broberg, Brian V. … A18.22 Ciossek, Thomas … A18.4 Del Arco, Alberto … A15.3 Batool, Zaina … A3.1 Broccoli, Laura … A13.14 Ciruela, Francisco … A18.1 Delicata, Francis … A18.47 Bauer, Martin … A18.123 Brown, Earl G. … A1.4 Clark, Luke … A18.61 DeLoach, Katherine E. … A18.95 Baumann, Michael … A18.11 Büchel, Christian … A12.6 Clark, Sam … A5.8 Demiri, Maria … A18.5 Beaulieu, Jean-Martin … A7.2, Budygin, Evgeny … A3.3 Clement, Lorna … A18.37 Dencker, Ditte … A18.16 A18.77 Bureau, Geneviève … A18.101 Cocker, Paul … A13.3 Deserno, Lorenz … A14.4 Beier, Kevin T. … A18.95, Burgess, Christopher … A18.89 Coggiano, Mark … A13.10, Desfosses, Emilie … A18.36 A18.106 Burgière, Eric … A18.3 A13.11 Deussing, Jan M. … A13.14 Bellve, Karl … A18.28 Burrell, Mark Henry … A5.3 Colangeli, Roberto … A18.47 Devoto, Paola … A16.3, A18.45 Ben Ammar, Marouane … A18.5 Caboche, Jocelyne … A18.104 Cole, Anthony … A18.76 Di Chiara, Gaetano … A18.42, Benavides, Caridad … A18.19 Cadoni, Cristina … A18.14 Collins, Anne L. … A12.3 A18.120 Benfenati, Fabio … A18.75 Cador, Martine … A18.72 Collins, William F. … A12.5 Di Giovanni … A18.47 Beninger, Richard J. … A8.4 Caffino, Lucia … A18.75 Collip, Dina … A8.3 Di Paolo, Therese … A18.23 Benkelfat, Chawki … A8.5 Cahill, Emma … A14.1 Cools, Roshan … A14.3 di Volo, Matteo … A18.10, Cai, Huaibin … A11.1 Corongiu, Silvia … A18.14 A18.125

Didienne, Steve … A13.6, Fenu, Sandro … A18.14 Ghahremani, Dara … A13.12 Hiranita, Takato … A13.10 A18.15 Ferger, Boris … A18.82 Giangrasso, Danielle … A18.81 Hjermind, Lena … A18.63 Diekhof, Esther K. … A12.6, Ferrari, Alberto … A18.45 Gibson, Anne S. … A18.107 Hnasko, Thomas S. … A18.52 A18.85 Ferrari, Emanuele … A18.19 Giguère, Nicolas … A9.1 Ho, Chelsea … A13.11 Dimiziani, Andrea … A18.5 Filip, Małgorzata … A13.1, A13.2 Gil, Carles … A7.3 Ho, Shih-Yin … A18.88 Dixon-Gleaves, Apre … A13.11 Fink-Jensen, Anders … A18.16 Ginovart, Nathalie … A18.5 Hoener, Marius C. … A3.3, Do Thi, Anh … A18.39 Fisher, Simon E. … A18.3 Giraldo, Jesús … A7.3 A18.75 Dolan, Ray … A14.6 Fisone, Gilberto … A18.9, Gjedde, Albert … A18.124 Hoffman, Carlie … A18.119 Dombeck, Daniel … A18.71 A18.110 Glebova, Alina … A18.55, Holst, Brigitte … A18.16 Dongelmans, Marie Louise … FitzGerald, Thomas H. B. … A18.122 Holy, Marion … A18.11 A13.6, A18.15 A14.6 Glennon, Richard A. … A18.11 Honda, Yoshiko … A18.118 Doucet-Beaupré, Hélène … A2.2 Flores, Cecilia … A2.3, A18.121 Glenthøj, Birte … A18.124 Horga, Guillermo … A18.19 Dougalis, Antonios … A18.4, Fogarty, Kevin E. … A18.28 Glenthøj, Birte … A18.22, A18.90 Horváth, Erzsébet Zsófia … A18.55 Földy, Csaba … A18.106 Głowacka, Urszula … A18.38 A18.24 Dragicevic, Elena … A18.122 Forget, Benoit … A18.104 Gnegy, Margaret … A18.66, Hosomi, Koichi … A18.67 Draheim, Henning J. … A18.4 Frank, Tobias … A18.122 A18.108, A18.112 Houle, Sylvain … A16.2 Dreyer, Jakob Kisbye … A6.3 Frau, Roberto … A16.3, A18.120 Godno, Arthur … A18.104 Howe, Mark … A18.71 Duan, Jinyi … A18.52 Freestone, Peter Stuart … A5.3 Goetz, Laura … A18.96 Howe, William … A18.50 Dubiel, Marta … A18.57 Freissmuth, Michael … A4.2, González-Sepúlveda, Marta … Howes, Oliver … A8.2, A16.2, Ducrocq, Fabien … A17.2 A18.26, A18.79, A18.87, A7.3 A16.4 Duda, Johanna … A18.4, A18.91 Gourlay, Jeanne … A18.67 Hsieh, Ming-Hsien … A18.29 A18.55, A18.122 French, Catherine A. … A18.3 Gove, Willard … A18.76 Huang, Chih-Yun … A18.54 Dumont, Gregory … A18.125 Freudenreich, Johannes … Goyal, Shubhi … A18.3 Huang, Huiping … A18.6 Dunn, Amy … A18.64 A18.48 Grandy, David K. … A3.1 Huang, San-Yuan … A18.54 Dunn, Matt … A5.8 Friberg, Lars … A18.63 Gravel, Booij … A8.5 Huang, Yiyun Henry … A18.96 Durand-de Cuttoli, Romain … Fritz, Michael … A18.25, A18.44 Graybiel, Ann M. … A10.3, A18.3 Hueske, Emily … A18.21 A18.31 Fukuda, Takaichi … A10.4 Greenfield, Venuz Y. … A12.3, Hummel, Thomas … A4.2, Dziedzicka-Wasylewska, Marta Fumagalli, Fabio … A18.75 A18.86 A18.26 … A18.57, A18.80 Furlong, Teri M. … A18.81, Grima, Laura Lucy … A18.62 Husain, Masud … A18.62 Ebdrup, Bjørn … A18.90 A18.109 Grimm, Alexandra … A18.26 Huys, Quentin J. M. … A13.7, Ecker, Gerhard F. … A18.11 Fuxe, Kjell …A13.1, A13.2, Gross, Ulrike … A18.82 A18.34 Edelmann, Elke … A8.1 A13.14, A18.56 Groszer, Matthias … A18.3 Hwu, Wuh-Liang … A18.88 Egerton, Alice … A8.2, A16.2 Füzesi, Tamás … A18.8 Gryz, Marek … A18.105 Hyde, Jeff … A18.99 Eisenegger, Christoph … A18.61 Gagnon, Dave … A18.23 Guadagna, Simone … A18.6 Iglesias, Sandra … A14.5 Ejdrygiewicz, Jillian … A18.8 Gainetdinov, Raul … A3.4, Guilmette, Edward … A18.50 Ikemoto, Keiko … A18.60 El-Kasaby, Ali … A4.2, A18.26, A18.75 Gutewort, Maya … A3.1 Ingallinesi, Manuela … A18.39 A18.79, A18.87, A18.91 Gaiser, Edward C. … A18.126 Gutkin, Boris … A18.10, A18.97, Inoue, Ritsuko … A18.114 Enard, Wolfgang … A18.3 Galet, Benjamin … A18.39 A18.125 Isaias, Ioannis U. … A11.3 Engblom, David … A18.25, Galineau, Laurent … A18.36 Gyurkovics, Máté … A18.24 Jacob, Simon N. … A14.7 A18.43, A18.44 Gallezot, Jean-Dominique … Hansen, Freja … A18.63 Jacobs, Elina … A18.89 Erdem, Fatma Aslı … A18.79 A18.126 Hansson, Anita C. … A13.14 Jan, Caroline … A18.67 Ericson, Mia … A18.41, A18.84 Galli, Aurelio … A18.63 Hantraye, Phlippe … A18.67 Janak, Patricia H. … A1.6, Ernst, Margot … A18.27 Gambarana, Carla … A18.45 Hariri, Ahmad … A18.68 A18.13 Erreger, Kevin … A18.63 Gao, Xiaojing J. … A18.95 Harmeier, Anja … A18.75 Jastreboff, Ania M. … A18.126 Espa, Elena … A18.14 Garbusow, Maria … A13.7, Härmson, Oliver … A18.62 Jastrzębska, Kamila … A18.43 Espinoza, Stefano … A3.4, A18.34 Harris, Kenneth … A18.89 Jauhar, Sameer … A16.4 A18.75 Garcia, Alexandra … A18.104 Hauber, Wolfgang … A18.46 Jean-Xavier, Céline … A18.8 Eugenin, Eliseo … A7.4 Garcia-Keller, Constanza … Hayat, Shaista … A18.117 Jensen, Kathrine Louise … Everitt, Barry J. … A14.1 A18.99 Heales, Simon … A18.49 A18.16 Exton-McGuiness, Marc … A14.2 Gardi, Sarah … A18.5 Heasman, Sonja … A18.49 Jensen, Lars-Thorbjørn … Faget, Lauren … A18.52 Garst-Orozco, Jonathan … Heinsbroek, Jasper … A13.5 A18.124 Fallon, Sean James … A18.62 A18.50 Heinz, Andreas … A13.4, A13.7, Jiao, Ruiying … A18.30 Fanet, Hortense … A18.93 Gaskill, Peter … A7.4 A18.34 Jin, Chunyu … A18.16 Fanni, Silvia … A16.3 Gemechu, Jickssa M. … A18.32 Hellsberg, Eva … A18.11 Johnson, Alexander B. … A18.52 Faron-Górecka, Agata … A18.57, Genauck, Alexander … A13.4 Henkie, Adam … A5.8 Jonas, Lauren … A18.119 A18.80 Genç, Ece … A18.98 Hernaus, Dennis … A8.3 Jung, Hae Yoon … A18.21 Faucon Biguet, Nicole … A18.39 George, Olivier … A13.9 Herrouin, Coralie … A18.72 Jüngling, Kay … A13.14 Faulkner, Geoffrey J. … A18.92 Gerhardt, Greg A. … A18.53 Hevers, Wulf … A18.3 Jutkiewicz, Emily … A18.108 Faure, Philippe … A13.6, A18.15, Gether, Ulrik … A4.3, A18.16, Heyninck, Karen … A18.1 Kacher, Radhia … A18.104 A18.31 A18.63 Hillmer, Ansel … A18.96 Kaibuchi, Kozo … A12.1

Kalivas, Peter W. … A13.5, Kuznetsov, Alexey … A18.10, Luo, Liqun … A18.95, A18.106 Miyamichi, Kazunari … A18.95 A18.99 A18.97, A18.125 Lüscher, Christian … A18.78 Miyamoto, Yuta … A10.4 Kang, Un … A18.19 Kwon, Oh Seok … A18.20 Ma, Guofen … A7.3 Mizrahi, Romina … A16.2 Kanter, Ellen … A11.2 Ky, Aurelie … A18.37 Mach, Robert H. … A18.65, Moghaddam, Bita … A2.4, A5.2, Kantrovitz, Lauren … A18.126 Lacey, Carolyn J. … A10.3 A18.94 A15.3, A18.74 Kappes, Vincent … A18.104 Lacroix, Franca … A18.100 Madsen, Kenneth … A18.16 Møller, Lisbeth … A18.63 Kasanova, Zuzana … A8.3 Lad, Yatish … A18.67 Madsen, Line M. … A18.22 Monferrer, Lidon … A18.111 Kasper, Siegfried … A18.123 Lai, Wen-Sun … A18.29 Maggi, Silvia … A18.75 Morales, Angelica M. … A13.12, Kasture, Ameya … A4.2, A18.26, Lak, Armin … A18.89 Malenka, Robert C. … A18.95, A18.128 A18.87, A18.91 Lapish, Christopher … A18.10, A18.106 Morgello, Susan … A7.4 Katonai, Enikő Rózsa … A18.24 A18.125 Malik, Maninder … A18.65 Morozova, Ekaterina … A18.10, Katz, Jonathan L. … A13.10 Lappin, Julia … A8.2 Malison, Robert E. … A18.126 A18.97, A18.125 Kee, Samantha … A18.83 Lataster, Johan … A8.3 Mallet, Jacques … A18.39 Morris, Evan D. … A12.7, A18.96 Keefe, Kristen A. … A18.81, Layé, Sophie … A17.2, A18.93 Malvaez, Mellissa … A18.86 Morud, Julia … A18.84 A18.107, A18.109 Lazarus, Michael … A18.25 Mandelkern, Mark A. … A12.8, Mosharov, Eugene … A11.2 Keighron, Jacqueline … A13.11 Lecca, Daniele … A18.120 A13.12, A18.128 Moszczynska, Anna … A18.32 Kellaway, Lauriston A. … A18.53 Lee, Jonathan … A14.2 Mannaioni, Guido … A18.51 Mota, Elia … A18.37 Kennedy, Pamela J. … A18.86 Lee, Ni-Chung … A18.88 Mannoury la Cour, Clothilde … Mourot, Alexandre … A18.31 Kennedy, Robert … A18.66, Lee, Virginia M.-Y. … A18.70 A18.39 Müller, Malika … A18.85 A18.112 Lehner, Małgorzata … A18.105 Marshall, Courtney … A11.4 Müller, Ulrich … A18.61 Kerimoglu, Cemil … A18.3 Lemoine, Damien … A18.31 Marti, Fabio … A18.31 Mundry, Roger … A18.3 Kern, Carina … A18.79 Lenda, Tomasz … A18.38 Martig, Adrian … A5.2 Murray, Robin … A8.2 Kessler, Robert … A18.7 Lengel, Dana … A12.5 Masi, Alessio … A18.51 Myin-Germeys, Inez … A8.3 Khan, Shabana … A2.1 Lessmann, Volkmar … A8.1 Masini, Débora … A18.110 Myroshnychenko, Maxym … Kharitonova, Maria … A18.4 Lévesque, Martin … A2.1, A2.2 Maskos, Uwe … A18.31 A18.10, A18.125 Khelashvili, George … A1.2, LeVine, Michael V. … A1.2 Matheos, Dina P. … A18.86 Nabulsi, Nabeel … A18.96 A18.59 Leyton, Marco … A8.5 Mathys, Christoph … A14.5 Naef, Michael … A18.61 Khlhatyan, Jivan … A18.77 Li, Chia-Tzu … A18.29 Matuskey, David … A18.96, Nagai, Taku … A12.1, A18.103 Khodakhah, Kamran … A18.83 Li, Jun-Xu … A3.2 A18.126 Nagano-Saito, Atsuko … A8.5 Khoshbouei, Habiheh … A4.4 Liao, Wenlin … A18.2 McGovern, Erin … A12.7 Nair, Anu … A18.37 Killcross, Simon … A18.115 Lieberman, Ori J. … A11.2 McGuire, Philip … A8.2, A16.2, Namba, Hisaaki … A18.58 Killinger, Bryan A. … A18.32 Lignani, Gabriele … A18.49, A16.4 Naneix, Fabien … A18.72 Klawonn, Anna M. … A18.25, A18.75 McKee, Sherry … A18.96 Narvaez, Manuel … A13.2 A18.44 Lin, Yingxi … A10.3 McKenzie, Kwame … A16.2 Naudé, Jérémie … A13.6, Koblinger, Kathrin … A18.8 Lindahl, Maria … A18.35 Mediavilla, Cristina … A18.69 A18.15 Kodama, Tohru … A18.118 Linssen, Anke … A18.61 Medrihan, Lucian … A18.75 Nawa, Hiroyuki … A18.58 Kohno, Milky … A13.12, A18.128 Liou, Horn-Huei … A18.88 Melikian, Haley … A18.28 Nebe, Stephan … A13.7, A18.34 Kojima, Takashi … A18.118 Lipski, Janusz … A5.3 Melis, Miriam … A15.1 Neisewander, Janet L. … A18.94 Kolasa, Magdalena … A18.57, Liss, Birgit … A6.2, A18.4, Meloni, Rolando … A17.2, Neuhofer, Daniela … A13.5 A18.80 A18.55, A18.122 A18.39 Newman, Amy H. … A13.10, Kortagere, Sandhya … A11.4 Lissemore, Jennifer I. … A8.5 Menzel, Anna … A18.48 A13.11 Kosillo, Polina … A10.1 Liu, Bin … A18.30 Mercuri, Nicola B. … A6.1 Ng, Joanne … A18.49 Kosmowska, Barbara … A18.38 Liu, Chih-Ming … A18.29 Mereu, Maddalena … A13.10 Nicholson, Janet … A18.48 Kótyuk, Eszter … A18.24 Liu, Guoxiang … A11.1 Metzakopian, Emmanouil … A2.2 Nicola, Saleem … A18.113 Kozak, Rouba … A18.50 Liu, Hung-Hsiang … A18.29 Meurs, Alfred … A18.1 Nieder, Andreas … A14.7 Kramer, Richard … A18.31 Locklear, Mallory … A12.5 Meyer, Esther … A18.49 Niello, Marco … A18.11 Kremer, Eric J. … A18.95 Lohani, Sweyta … A5.2 Meyer-Lindenberg, Andreas … Nielsen, Mette O. … A18.90 Kritzer, Mary … A12.5 London, Edythe D. … A12.8, A16.2 Nilsson, Anna … A18.25, A18.44 Kudlacek, Oliver … A18.11, A13.12, A18.128 Midde, Narasimha … A18.17 Nilsson, Johanna … A18.56 A18.91 Lopez, Jessica … A13.10 Mikelman, Sarah … A18.112 Nissen, Wiebke … A18.48 Kullingsjo, Johan … A18.18, Lopez, Lillie … A7.4 Mikhailova, Maria A. … A3.3 Nolan, Brian … A18.94 A18.116 López Cruz, Laura … A13.13, Miliano, Cristina … A18.42 Nouel, Dominique … A18.121 Kupchik, Yonatan … A13.5 A18.111 Millan, Mark J. … A18.39 O’Connor, Timothy … A18.21 Kurian, Manju A. … A4.1, A18.49 Lotfi, Amir … A18.84 Miller, Gary … A18.64, A18.119 Oberacher, Herbert … A18.4 Kuroda, Keisuke … A12.1 Lovic, Vedran … A6.3 Miller, Samara … A18.40 Ødegaard Nielsen, Mette … Kuşkucu, Ayşegül … A18.98 Lovinger, David … A11.1 Millet, Philippe … A18.5 A18.124 Kuśmider, Maciej … A18.57, Luedtke, Robert R. … A18.65, Milton, Amy J. … A14.1 Oflijan, Julia … A13.2 A18.80 A18.94 Mitrophanous, Kyriacos A. … Ögren, Sven Ove … A18.56 Kuwabara, Hiroto … A18.12 Luk, Kelvin C. … A18.70 A18.67 Okita, Kyoji … A12.8 Luo, Da-Zhong … A18.29 Miura, Masami … A18.114 Olesen, Erik … A18.76

Olivarez, Régulo … A18.111 Quarterman, Juliana C. … Schad, Daniel J. … A13.7, Stalter, Wolfgang M. … A14.7 Ortiz, Jordi … A7.3 A13.10, A13.11 A18.34 Stefanova, Nadia … A18.4 Ortner, Nadine J. … A18.4 Quester, Saskia … A13.4 Scheggi, Simona … A18.45 Stein, Dan J. … A18.53 Osakada, Fumitaka … A18.52 Quizon, Pamela … A18.17 Scheggia, Diego … A18.6 Steinberg, Elizabeth … A18.95, Ossowska, Krystyna … A18.38 Raedt, Robrecht … A18.1 Schlagenhauf, Florian … A14.4, A18.106 Ostlund, Sean B. … A12.3 Rahbeck-Clemmensen, Troels … A18.34 Stephan, Klaas Enno … A14.5 Oswald, Lynn … A18.12 A18.16, A18.63 Schlossmacher, Michael G. … Stewart, Hannah J. … A18.67 Ouimet, Bruno … A18.101 Ralph, Scott … A18.67 A1.4 Stiersdorfer, Birgit … A18.82 Owens, William Anthony … Rangel-Barajas, Claudia … Schmiedhofer, Philipp … A18.27 Stockner, Thomas … A4.2, A18.16 A18.65 Schmitz, Yvonne … A5.8 A18.11, A18.26 Pääbo, Svante … A18.3 Rao, Vinod … A18.21 Schneider, Jay … A11.4 Stott, Simon R. … A18.117 Pabian, Paulina … A18.57, Rapp, Michael A. … A13.7, Schreiter, Sven … A18.3 Stout, Kristen … A18.64, A18.80 A18.34 Schreiweis, Christiane … A18.3 A18.119 Pacelli, Consiglia … A9.1 Rasmussen, Sophie N. … A18.22 Schröder, Sylvia … A18.89 Strauman, Timothy … A18.68 Paladini, Carlos A. … A5.7 Rathje, Mette … A18.16 Schwaninger, Markus … A18.25 Striessnig, Jörg … A18.4, A18.55 Palfi, Stéphane … A18.67 Ratnayake, Melanie … A12.6 Schwartenbeck, Philipp … A14.6 Stuber, Garret … A10.2 Palm, Erik … A18.35 Ravassard, Philippe … A18.39 Schwarz, Lidsay … A18.95 Subramaniyan, Manivannan … Pandolfo, Diego P. … A18.5 Rayport, Stephen … A5.5 Scifo, Andrea … A18.120 A12.4 Panhelainen, Anne … A18.35 Razavi, Asghar … A18.59 Sebold, Miriam … A13.7, A18.34 Sucic, Sonja … A4.2, A18.26, Papaleo, Francesco … A18.6 Rea, Ellis … A18.3 Seth, Chandni … A18.109 A18.79, A18.87, A18.91 Pape, Hans C. … A13.14 Rehders, Maren … A3.1 Sgobio, Carmelo … A11.1 Suder, Agata … A13.1 Pardu, Alessandra … A16.3 Reichelt, Amy … A14.2 Sharma, Sandeep … A18.8 Sukhanov, Ilya … A18.75 Parent, Martin … A18.23 Reimers, Luise … A12.6, A18.85 Shih, Mei-Chen … A18.54 Sulzer, David … A5.8, A11.2, Parisiadou, Loukia … A11.1 Ressler, Reed … A18.52 Shionoya, Kiseko … A18.44 A11.3, A18.19 Park, David S. … A1.4 Resta, Francesco … A18.51 Shizgal, Peter … A18.100 Sun, Wei-Lun … A18.17 Park, Junchol … A15.3 Restrepo-Lozano, José María … Showalter, Hollis … A18.66 Surmeier, D. James … A11.2 Pasterkamp, R. Jeroen … A2.2 A18.121 Siekkinen, Jenni … A18.70 Svensson, Peder … A18.18, Paupardin-Trisch, Daniele … Reyes, Cindy … A18.113 Sigvard, Anne … A18.124 A18.116 A18.37 Richard, Jocelyn M. … A18.13 Sil, Annesha … A18.120 Székely, Anna … A18.24 Pegon, Jonathan … A18.39 Rickhag, Mattias … A18.16, Silva, Byron … A18.37 Szöllősi, Daniel … A4.2, A18.11, Pekcec, Anton … A18.48 A18.63 Simon, Eric … A18.82 A18.26 Pelliccia, Teresa … A18.45 Risco, Severiano … A18.69 Singh, Anand Kumar … A18.25 Szumiec, Łukasz … A18.43 Pereira, Daniela … A5.8 Robbins, Trevor W. … A1.7, Sitte, Harald H. … A18.11 Szumska, Joanna … A3.1 Petersen, Nicole … A12.8 A18.61 Skórzewska, Anna … A18.105 Taha, Sharif … A18.109 Pierucci, Massimo … A18.47 Robertson, Chelsea L. … A12.8, Slack, Rachel D. … A13.11 Tajima, Kae … A10.4 Piras, Giovanna … A18.42 A18.128 Sleeman, Jonathan P. … Takillah, Samir … A13.6, A18.15 Pisanu, Augusta … A18.14, Rodriguez Parkitna, Jan … A18.117 Tanda, Gianluigi … A13.10, A18.120 A18.25, A18.43, A18.102 Smith, Christopher … A18.7 A13.11 Pitterl, Florian … A18.4 Roeper, Jochen … A5.7 Smolka, Michael N. … A13.7, Tandon, Shashank … A18.109 Pittman, Brian … A18.126 Rogdaki, Maria … A16.4 A18.34 Tange, Jerome … A18.37 Płaźnik, Adam … A18.105 Romanczuk-Seiferth, Nina … Snutch, Terrance P. … A18.55 Taracha, Ewa … A18.105 Poewe, Werner … A1.5 A13.4 Sobue, Akira … A18.103 Tasan, Ramon … A18.127 Pomberger, Thomas … A18.4 Rossi, Amie … A18.50 Soden, Marta … A18.40 Tauber, Clovis … A18.36 Pongs, Olaf … A18.122 Rostrup, Egill … A18.22, A18.90, Söderpalm, Bo … A18.41, Taylor, Madison, R. … A18.106 Popovic, Ana … A18.123 A18.124 A18.84 Taylor, Michelle … A18.65 Portelli, Jeanelle … A18.1 Roughley, Stephanie … A18.115 Solich, Joanna … A18.57, Tedroff, Joakim … A18.18, Potenza, Marc N. … A18.126 Russel, Vivienne A. … A18.53 A18.80 A18.116 Pötschke, Christina … A18.55, Saarma, Mart … A18.35, A18.70 Sommer, Christian … A13.7, Tharp, Ian … A18.33, A18.68 A18.122 Saba, Pierluigi … A16.3 A18.34 Thomsen, Annika … A18.16 Powell, Gregory … A18.94 Sabria, Josefa … A7.3 Sommer, Wolfgang H. … A13.14 Threlfell, Sarah … A10.1 Praschak-Rieder, Nicole … Sahlholm, Kristoffer … A18.56 Sonesson, Clas … A18.18, Tierney, Patrick … A18.50 A18.123 Salamone, John D. … A13.13, A18.116 Tolstoy, Sasha … A18.63 Proudfoot, James A. … A18.52 A18.111 Sørensen, Gunnar … A18.16 Tomlinson, Julianna J. … A1.4 Provesi, Gustavo … A18.51 Sames, Dalibor … A5.8 Sorenson, Roderick … A18.66 Tonegawa, Susumu … A18.21 Pruessner, Jens … A8.3 San Miguel, Noemi … A13.13, Sotoyama, Hidekazu … A18.58 Tonini, Raffaella … A18.9 Puighermanal, Emma … A7.1 A18.111 Spampinato, Umberto … A18.72 Torquet, Nicolas … A18.15 Puliyadi, Rathi … A18.3 Sancak, Seda … A18.98 Spanagl, Rainer … A13.14 Tóth, Katalin … A18.77 Puryear, Corey … A18.21 Sandtner, Walter … A18.79 Sperk, Günther … A18.127 Tournier, Benjamin B. … A18.5 Qatato, Maria … A3.1 Saper, Clifford B. … A18.25 Spigolon, Giada … A18.9, Trauner, Dirk … A18.31 Sauerzopf, Ulrich … A18.123 A18.110 Treven, Marco … A18.27

Trifilieff, Pierre … A17.2 Verecskei, Andrea … A18.24 Whelan, Patrick J. … A18.8 Yu, Byron … A15.3 Tritsch, Nicolas … A5.6 Vincent, Pierre … A18.37 Wightman, R. Mark … A1.3 Yu, Jia … A11.1 Tronche, François … A15.2 Võikar, Vootele … A18.35 Willeit, Matthäus … A18.123 Yuan, Yaxia … A18.17 Truckenbrodt, Anna … A2.1 von Heimendahl, Moritz … Williams, Courtney … A11.4 Zajdel, Joanna … A18.25, Trudeau, Louis-Éric … A9.1 A18.48 Williams, Dionna … A7.4 A18.44 Trujillo-Pisanty, Ivan … A18.100 Voutilainen, Merja H. … A18.70 Wilson, Bethany … A18.119 Zakaniaez, Yasmin … A18.96 Trusel, Massimo … A18.9 Wachtel, Jonathan … A12.5 Winkler, Madeline … A18.73 Zakharov, Denis … A18.10, Tsartsalis, Stergios … A18.5 Wade-Martins, Richard … Winstanley, Catherine … A13.3 A18.97 Tsetsenis, Theodoros … A12.4 A18.49 Winter, Christine … A18.3 Zald, David … A18.7 Tsutsui, Ken-ichiro … A18.118 Walczak, Magdalena … A18.43 Winton-Brown, Toby … A8.2 Zanettini, Claudio … A13.11 Tucci, Valter … A18.75 Walle, Roman … A17.2 Wisłowska-Stanek, Aleksandra Zeberg, Hugo … A18.56 Tuluc, Petronel … A18.4 Walton, Mark … A18.62 … A18.105 Zecca, Luigi … A11.3, A18.19 Uchida, Naoshige … A18.21 Wand, Gary … A18.12 Womersley, Jacqueline S. … Zelek-Moli, Agnieszka … A18.38 Ulenius, Lisa … A18.41 Wang, Changhe … A18.30 A18.53 Zell, Anne-Kathrin … A18.48 Ullrich Gavilanes, E. Melissa … Wang, Huiling … A5.4 Wong, Dean … A18.12 Zestos, Alexander … A18.66, A3.1 Wang, Li … A18.30 Wood, James … A18.127 A18.112 Underhill, Suzanne … A5.1, A5.2 Wang, Minzheng … A18.64 Wood, Marcelo A. … A18.86 Zhan, Chang-Guo … A18.17 Urs, Nikhil M. … A16.1 Wang, Shuo … A12.7 Wouters, Elise … A18.1 Zhang, Quangfeng … A18.30 Valentini, Valentina … A18.42 Wardas, Jadwiga … A18.38 Wu, Sijia … A18.28 Zhang, Shiliang … A5.4 Valjent, Emmanuel … A7.1 Wassum, Kate M. … A12.3, Wulff, Sanne … A18.90 Zhang, Yan-Fen … A10.1 Valmaggia, Lucia … A8.2, A16.2 A18.86 Wydra, Karolina … A13.1, A13.2 Zheng, Lianghong … A18.30 Valyear, Milan D. … A18.100 Watanabe, Masataka … A18.118 Xi, Zheng-Xiong … A13.11 Zhou, Zhuan … A18.30 Van Camp, Nadja … A18.67 Waters, Nicholas … A18.18, Xie, Chengsong … A11.1 Zhu, Feipeng … A18.30 Van Craenenbroeck, Kathleen … A18.116 Xie, Stanley … A18.95 Zhu, Jun … A18.17 A18.1 Waters, Susanna … A18.18, Xu, Kuiying … A18.94 Zimmermann, Ulrich S. … A13.7, Van Dam, Debby … A18.1 A18.116 Xu, Wei … A11.4 A18.34 van der Veldt, Suzanne … A17.2 Weaving, Jessica … A18.33 Yamada, Kiyofumi … A18.103 Zucca, Fabio A. … A11.3, A18.19 Vancassel, Sylvie … A18.93 Wegener, Meredyth A. … A18.74 Yang, Kechun … A12.4 Żurawek, Dariusz … A18.57, Vandaele, Youna … A18.13 Wei, Shan … A18.103 Yapo, Cedric … A18.37 A18.80degaard Vander Weele, Caitlin … A6.3 Weinstein, Harel … A1.2, A18.59 Yee, Andrew Gregory … A5.3 Zweifel, Larry … A18.40 Vasudevan, Lakshmi … A18.1 Weinstein, Jodi … A18.19 Yohn, Samantha E. … A13.13 Veenstra, Mike … A7.4 Weng, Eddie Feng-Ju … A10.3 Yoo, Ji Hoon … A18.52 Vendruscolo, Leandro … A13.11 Wenzel, Jen … A18.76 Young, Damon … A18.50

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