Exploring Diversity in the Midbrain Dopamine System with Emphasis on the Ventral Tegmental Area

Home , ALDH1A1, Mesostriatal system, Midbrain

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1985

BIANCA VLCEK

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-1057-2 UPPSALA urn:nbn:se:uu:diva-423730 2020 Dissertation presented at Uppsala University to be publicly examined in Ekmanssalen, Evolutionsbiologiskt centrum, Norbyvägen 16, Uppsala, Wednesday, 16 December 2020 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Associate Professor Eva Hedlund.

Abstract Vlcek, B. 2020. Exploring Diversity in the Midbrain Dopamine System with Emphasis on the Ventral Tegmental Area. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1985. 72 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1057-2.

Midbrain dopamine neurons of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) are important for motor, cognitive and limbic functions through substantial projections to forebrain structures. Dysfunction of the midbrain dopamine system is associated with several disorders, including Parkinson´s disease (PD) and substance use disorder. PD is characterized by the loss of dopaminergic neurons leading to severe motor dysfunction and by the brain distribution of aggregated α-Synuclein protein. Mutations in the α-Synuclein gene (SNCA) have been associated with PD. The overall aim of this thesis was to increase the understanding of anatomical and histological features of dopamine neurons by identifying and characterizing gene expression differences between dopamine neurons, primarily those located within VTA but also the SNc. This knowledge should help towards increased understanding of the roles exerted by different dopamine neurons in behavior during healthy conditions and in disease. For this purpose, unbiased microarray analysis was first performed to identify genes that are differentially expressed in the VTA and SNc. Identified genes, e.g. Trpv1, NeuroD6, Calbindin and Grp, were analyzed on mRNA level primarily in mouse brain sections to determine their distribution patterns. Several were found to localize only in restricted neurons within the VTA, thus defining subpopulations of dopamine neurons in the mouse VTA. Further analysis revealed several interesting findings. For example, in clinical samples of PD biopsies, most dopamine neurons were degenerated, but those remaining were positive for Grp. VTA dopamine neurons positive for NeuroD6 or Calbindin2 were investigated using optogenetics. Activation of the NeuroD6-neurons, but not Calbindin2-neurons, was unexpectedly revealed as sufficient to drive place preference. To address the integrity of midbrain dopamine neurons in mouse models of PD, fluorescent in situ hybridization analyses was performed in two transgenic mouse lines expressing the human SNCA gene and in one dopamine degeneration lesion model, the 6- OHDA method. Despite near-ubiquitous presence of SNCA mRNA, all markers for midbrain dopamine cells remained intact in both transgenic models. Dopamine neurons thus showed an unanticipated robustness towards α-Synuclein pathology not described before. In contrast, most dopamine neurons were lost in the lesion model. In summary, by identifying and characterizing dopamine neurons using established and new markers, histological analyses revealed the presence of distinct gene expression patterns within the VTA that allowed anatomical and functional assessments of discrete subpopulations of midbrain neurons. The thesis contributes new knowledge of the midbrain dopamine system.

Bianca Vlcek, Department of Organismal Biology, Comparative Physiology, Norbyv 18 A, Uppsala University, SE-75236 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-513-1057-2 urn:nbn:se:uu:diva-423730 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-423730)

“Most people say that it is the intellect which makes a great scientist. They are wrong, it is character”

Albert Einstein

Till min familj Para a minha família To my family

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Viereckel, T., Dumas, S., Smith-Anttila, C.J.A., Vlcek, B., Bimpisidis Z., Lagerström, M. C., Konradsson-Geuken, Å., Wal- lén-Mackenzie, Å. (2016): Midbrain gene screening identifies a new mesoaccumbal glutamatergic pathway and a marker for dopamine cells neuroprotected in Parkinson’s disease. Scientific Reports, 6: 35203.

II Bimpisidis, Z., König, N., Stagkourakis, S., Zell, V., Vlcek, B., Dumas, S., Giros, B., Broberger, C., Hnasko, S., T., Wallén-Mac- kenzie, Å. (2019): The NeuroD6 subtype of VTA neurons contributes to psychostimulant sensitization and behavioral reinforcement. eNeuro, 6 (3).

III Vlcek, B., Dumas, S., Ekmark-Lewén, S., Ingelsson, M., Wallén- Mackenzie, Å. (2020): Preserved gene expression patterns in midbrain dopamine neurons of two different transgenic mouse lines expressing the human α-synuclein (SNCA) gene Manuscript.

IV Vlcek, B., Guillaumin, A., Wallén-Mackenzie, Å. (2020): Ven- tral tegmental area neuronal subpopulations are strongly affected in a 6-OHDA mouse model. Manuscript.

Reprints were made with permission from the respective publisher.

Additional Paper

My work has also contributed to the following paper that is beyond the focus of this thesis:

Wallén-Mackenzie, Å., Dumas, S., Papathanou, M., Martis Thiele, M.M., Vlcek, B., König, N., Björklund, ÅK. (2020): Spatio-molecular domains identified in the mouse subthalamic nucleus and neighboring glutamatergic and GABAergic brain structures. Communications Biology - Nature, 3 (338).

Contents

Introduction ...... 11 The Midbrain Dopamine System ...... 12 Dopamine Neurons ...... 12 Anatomical Organization ...... 13 The Substantia Nigra (SN) ...... 15 The Ventral Tegmental Area (VTA) ...... 16 Functions of the Midbrain Dopamine System ...... 18 The Basal Ganglia ...... 18 Motor Loop ...... 19 Limbic Loop ...... 20 Diversity within Circuitries of the Midbrain Dopamine System ...... 21 Dysfunction in the Midbrain Dopamine System ...... 22 Substance Use Disorder (Drug addiction) ...... 22 Parkinson’s Disease (PD) ...... 23 Animal models of PD ...... 27 Toxin-induced models ...... 27 6-OHDA mouse model ...... 28 Genetic models ...... 29 Diversity within the VTA ...... 31 Dopamine and Glutamate Co-phenotype ...... 31 GABA and GABA/Dopamine Co-phenotype ...... 32 Gene Expression Diversity ...... 33 Overall Aim ...... 37 Materials and Methods ...... 38 Mice ...... 38 Stereotaxic 6-OHDA injection ...... 39 Post-operative care ...... 40 Tissue preparation for histology ...... 40 In Situ Hybridizzzation ...... 40 Synthesis of PCR Products and Riboprobes ...... 41 Fluorescent In Situ Hybridization (FISH) ...... 43 Immunohistochemistry ...... 45 Study I ...... 47 Study II ...... 50

Study III ...... 53 Study IV ...... 56 Concluding Remarks ...... 58 Acknowledgements ...... 59 References ...... 62

Abbreviations

αSyn alpha-Synuclein protein 6-OHDA 6-hydroxydopamine AADC Aromatic L-aminoacid decarboxylase Ab Antibody Aldh1a1 Aldehyde dehydrogenase 1 family member a1 AP Anterior-posterior Calb1 Calbindin-D28K Calb2 Calretinin, Calbindin2 ChR2 Channel rhodopsin 2 CLi Caudal linear nuclei of the raphe COMT Catechol-O methyl transferase Cre Cre recombinase enzyme D1 D1-like dopamine receptor D2 D2-like dopamine receptor DAT Dopamine transporter DBS Deep brain stimulation DIG Digoxigenin DV Dorsal-ventral eYFP Enhanced yellow fluorescent protein FA Formaldehyde FISH Fluorescent in situ hybridization Fst Follistatin GABA Gamma-aminobutyric acid GFP Green fluorescent protein Girk2 G-protein coupled inwardly rectifying K channel 2 GP Globus pallidus GPi Globus pallidus pars externa GPi Globus pallidus pars interna Grp Gastrin releasing peptide H2O2 Sodium hydroxide HRP Horseradish peroxidase IF Interfascicular nuclei L-DOPA L-3, 4-dihydroxy-phenylalanine Lmx1a LIM homeobox transcription factor 1 alpha Lmx1b LIM homeobox transcription factor 1 beta M Medial group of dopamine neurons MABT Maleic acid buffer with tween-20 MAO Monoamine oxidase MFB Median forebrain bundle ML Medial-lateral

MPP+ 1-methyl-4-phenylpyridinuim MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydrodropyridine mRNA Messenger RNA MSN Medium spiny neurons Mv Medioventral group of dopamine neurons NAc Nucleus accumbens NAcC Nucleus accumbens core NaCl Sodium chloride NAcSh Nucleus accumbens shell NeuroD6 Neuronal differentiation 6 Ntf3 Neurotrophic factor 3 OT Olfactory tubercle Otx2 Orthodenticle homeobox 2 P3 Postnatal day 3 PBP Parabrachial pigmented nuclei PBS Phosphate-buffered saline PCR Polymerase chain reaction PIF Parainterfascicular nuclei Pitx3 Paired like homeodomain 3 PN Paranigral nuclei rAAV Recombinant associated adenovirus RLi Rostral linear nuclei RRF Retrorubral field Six3 Sine-oculis-related homeobox-3 SN Substantia nigra SNc Substantia nigra pars compacta SNCA Alpha-Synuclein human gene SNpd Substantia nigra pars dorsalis SNpl Substantia nigra pars lateralis SNr Substantia nigra pars reticulata Sox6 SRY-box transcription factor 6 SSC Saline-sodium citrate STN Subthalamic nucleus szPBP Subzone of the PBP Tacr3 Tachykinin-receptor-3 TH Tyrosine hydroxylase TSA Tyramide signal amplification Trpv1 Transient receptor potential cation channel subfamily V member 1 Vglut2 Vesicular glutamate transporter 2 Viaat Vesicular inhibitory amino acid transporter VMAT Vesicular monoamine transporter VMAT 2 Vesicular monoamine transporter VP Ventral pallidum VTA Ventral tegmental area VTAR Ventral tegmental area rostral part

Introduction

Dopamine is popularly known as the “pleasure” molecule. This is highly due to the association of dopamine with the rewarding effects of natural rewards, such as chocolate (my favorite) and addictive drugs. Apart from its role in the reward system, dopamine is essential for the modulation of movement and integration of emotional states that drive decision-making. The structure providing dopamine in the reward system is the ventral tegmental area (VTA), while the structure providing dopamine in the motor system is the substantia nigra pars compacta (SNc). Together with a third dopaminergic structure called retrorubral field, the VTA and the SNc form the midbrain dopamine system. Dysfunction of the midbrain dopamine system can have severe consequences and lead to several different neurological diseases, such as Parkin- son’s disease (PD) and neuropsychiatric disorders such as schizophrenia, substance use disorder and attention deficit hyperactive disorder.

In neuroscience, while the circuitry driving motor function is quite well established, the circuitry driving affective functions, which in turn influence motor behavior, are still under intense investigation. Dopamine signaling from the VTA is known to influence structures involved in emotions and memory formation, driving the association of certain actions with emotional outcome. The VTA is a highly heterogeneous structure and its dopamine neurons are, for some biological reason, less vulnerable to degeneration in PD than dopamine neurons of the SNc. However, as the disease progresses, also the VTA dopamine neurons degenerate. Microarray and single-cell screenings, attempting to find gene expression differences between the VTA and SNc have resulted in lists of genes pinpointing differential expression separating the VTA and SNc. Histological and functional investigation of subpopulations of neurons represented by different molecular profiles will aid in furthering the understanding of normal neurobiological processes and how to approach novel therapy targets in the range of disorders that implicate the midbrain dopamine system.

In this thesis, I present how my studies have contributed to the histological identification and characterization of genes differentially expressed between the SNc and VTA, both under normal circumstances and in three different mouse models commonly used for the study of PD.

11 The Midbrain Dopamine System

Dopamine Neurons

Arvid Carlsson first described dopamine as a neurotransmitter in 1958. Dopa- mine is a monoamine synthesized from a naturally occurring amino-acid in the body, L-tyrosine (Carlsson et al., 1958). The first step in the synthesis starts with the conversion of L-tyrosine into L-3, 4-dihydroxy-phenylalanine (L-DOPA) by tyrosine hydroxylase (TH), the rate-limiting enzyme, followed by L-DOPA conversion into dopamine by aromatic L-aminoacid decarboxylase (AADC) (Figure 1).

Today, dopamine neurons are defined by their expression of the Th gene, along with the ability to release dopamine. Dopamine neurons use the vesicular monoamine transporter (VMAT), in the brain mainly the subtype 2 (VMAT2), for packaging dopamine into vesicles for release (Figure 1) (Björ- klund and Dunnett, 2007). VMAT2 is also used by noradrenaline, serotonin and histamine neurons of the brain for the same purpose (Fei and Krantz, 2009). Dopamine mediates its effects by binding to dopamine receptors, D1- like (D1 and D5) and D2-like (D2, D3 and D4), which are metabotropic G- coupled receptors (Figure 1). After signaling, dopamine is either returned to the presynaptic neuron through the dopamine transporter (DAT) (Figure 1) or metabolized. Once returned to the presynaptic neuron, dopamine is recycled into vesicles by VMAT2. Glial cells break down dopamine into inactive metabolites by monoamine oxidase and catechol-O methyl transferase. Dopa- mine molecules that accumulate in the cytosol of dopamine neurons due to leakage from the storage vesicles can also be degraded by monoamine oxidase (Meiser et al., 2013).

Figure 1. Illustration showing dopamine synthesis and where in the neuron TH, VMAT2 and DAT act. DAT, dopamine transporter; D1, dopamine 1 receptor; D2, dopamine 2 receptor; AADC, aromatic L-aminoacid decarboxylase; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2. Illustration by B. Vlcek

In 1964, Annica Dahlström and Kjell Fuxe published a histological study where they identified dopaminergic neurons in the substantia nigra (SN) pars compacta (SNc) of the rat brain using a method developed by Bengt Falck and Nils-Åke Hillarp, the Falck-Hillarp method. This method allows visualization of monoamine neurons based on freeze-drying tissue and exposing it to formaldehyde vapor, converting dopamine into iso-quinoline molecules that then emit a specific yellow-green fluorescence color in the cell body of these neurons which allows their detection. Dahlström and Fuxe were also able to identify terminals from the SNc in the caudate nucleus, part of the dorsal striatum, suggesting for the first time a nigrostriatal pathway, which was a few years later described by Urban Ungerstedt (Dahlström and Fuxe, 1964; Falck, 1962; Ungerstedt, 1971).

Anatomical Organization The midbrain dopamine system consists of three structures containing dopamine neurons, the VTA (group A10), SNc (group A9) and the retrorubral field (group A8) (Björklund and Dunnett, 2007; Dahlström and Fuxe, 1964) (Figure 2).

Figure 2. Illustration of the three different dopamine groups in the midbrain dopamine system of the mouse brain. Bregma: -3.64 mm. VTA, ventral tegmental area; SNc, substantia nigra pars compacta; RRF, retrorubral field. Illustration by B. Vlcek based on The Mouse Brain In Stereotaxic Coordinates by Franklin and Pax- inos (Paxinos, George).

In 1971, Ungerstedt published his study where, through lesion methods in addition to histological analysis using the Falck-Hillarp method, he was able to identify different groups of monoaminergic neurons in the rat brain. Unger- stedt was also able to identify the projections of these neurons, describing for the first time the major pathways that arise from the dopaminergic neurons of the midbrain. He described that the axons arising from the dopamine neurons of the SNc travel ventro-medially along the median forebrain bundle (MFB), enter the capsula interna, pass through the globus pallidus (GP) and innervate the dorsal striatum (caudate/putamen in humans), forming the nigrostriatal pathway. The axons coming from the VTA (then known as area ventralis teg- menti, cell group dorsal to the nucleus interpeduncularis) travel instead in an antero-lateral direction. Also along the MFB, innervating the nucleus accumbens (NAc) and the olfactory tubercle (tuberculum olfactorium) of the ventral striatum, forming the mesolimbic pathway (Ungerstedt, 1971) (Figure 3). Do- pamine receptors in the brain were described for the first time in the following year, supporting Ungerstedt’s findings (Kebabian et al., 1972).

Figure 3. Illustration of the nigrostriatal and mesolimbic pathways in the mouse brain described by Ungerstedt in 1971. VTA, ventral tegmental area; SNc, substantia nigra pars compacta; RRF, retrorubral field; NAc, nucleus accumbens; OT, olfactory tubercle. Illustration by B. Vlcek

The Substantia Nigra (SN) The SN is a ventrolateral area in the midbrain composed of two different structures, the SN reticulata (SNr), which is a gamma-aminobutyric acid (GABA) releasing structure and the SNc, which is mainly dopaminergic (Alexander et al., 1991). The nigrostriatal pathway arising from the SNc structure plays an important role in the modulation of movement through its influence on the basal ganglia circuitry of the motor system. Degeneration of this pathway due to SNc dopamine neuron death is the main pathological hallmark of PD (Mhyre et al., 2012; Nelson and Kreitzer, 2014).

In humans, the SNc can easily be recognized due to its characteristic pigmen- tation of neuromelanin, which is a byproduct of dopamine auto-oxidation that is lacking in mice. The SNc is commonly anatomically divided into a ventral and a dorsal tier. This division is based on the difference that the dopamine neurons within these two subdivisions present in PD, where the dopamine neurons in the ventral tier are more vulnerable while the dopamine neurons in the dorsal tier are less vulnerable (Figure 4A, B) (González-Hernández et al., 2010; Halliday et al., 2005). About a decade ago, the ventral tier was further categorized into nigral matrix and nigrosomes, corresponding to the SN in mice, while the dorsal tier was described as being part of the VTA rather than the SN. A more dorsolateral group of neuromelanin positive dopamine neurons is described as SN pars lateralis (SNpl), which corresponds to the tip of the SNc in mice (Figure 4C, D) (Damier et al., 1999a, 1999b; Reyes et al., 2012).

Figure 4. The SNc in human and in mice. A Illustration of the human SNc divided into ventral and dorsal tier. B Illustration of the mouse SNc divided into ventral and dorsal tier. C Illustration of the human SNc divided into matrix, nigrosomes and SNpl. D Illustration of the mouse SNc corresponding to the human SNc in F. SNc, substantia nigra pars compacta; NM, neuromelanin; SNr, substantia nigra pars reticulata; D, dorsal; V, ventral; SNpl, substantia nigra pars lateralis. Illustrations by B. Vlcek based on published atlas (Paxinos, George) and (Damier et al., 1999a)

The Ventral Tegmental Area (VTA) The VTA is a ventromedial dopaminergic area of the midbrain located between left and right SN (Björklund and Dunnett, 2007). The pathway between the VTA and NAc plays an important role in incentive salience (motivation for reward stimuli), reinforcement and learning (Björklund and Dunnett, 2007; Hyman and Malenka, 2001). The pathway formed mainly by the projections from the VTA to the prefrontal cortex is crucial for regulating motivation, emotions and cognitive control. Together these pathways form the mesocorticolimbic pathway of the so called reward system (Figure 5) (Björklund and Dunnett, 2007; Sesack and Grace, 2010).

Figure 5. Illustration of the mesocorticolimbic pathway in the mouse brain. VTA, ventral tegmental area; SNc, substantia nigra pars compacta; RRF, retrorubral field; NAc, nucleus accumbens; OT, olfactory tubercle. Illustration by B. Vlcek.

Cytoarchitectural studies have enabled the division of the VTA into different subregions in the rodent brain. The following subregions have been described: parabrachial pigmented nuclei (PBP), ventral tegmental area rostral part (VTAR), paranigral nuclei (PN), interfascicular nuclei (IF), parainterfascicular nuclei (PIF), the rostral linear nuclei (RLi) and caudal linear nuclei of the raphe (CLi) (Fu et al., 2012; Paxinos, George; Swanson, 1982) (Figure 6A).

In humans, the VTA comprises the SN pars dorsalis (SNpd), which corresponds to the PBP, and the Medioventral (Mv) area, corresponding to the PN and PIF areas of the VTA (Damier et al., 1999a; Halliday et al., 2005; Hirsch et al., 1988). The Medial (M) area corresponds to the RLi and CLi, which in humans this area is considered part of the raphe nucleus (Figure 6B) (Allen Brain Atlas; Reyes et al., 2012). In the SNpd and Mv about half of the dopamine neurons are melanized (Halliday et al., 2005; Hirsch et al., 1988).

Figure 6. Illustration of the VTA in mouse and human. A Mouse VTA and its subdivisions. B Human VTA and its subdivisions. PBP, parabrachial nucleus; PIF, parainterfascicular nuclei; PN, paranigral nuclei; IF, interfascicular nuclei; M, medial; Mv, ventromedial; SNpd, substantia nigra pars dorsalis. Illustration by B. Vlcek based on published atlas (Paxinos, George) and (Damier et al., 1999a)

Functions of the Midbrain Dopamine System As mentioned above, the nigrostriatal pathway plays an essential role in the modulation of voluntary movement through its influence on the basal ganglia circuitry. The mesocorticolimbic pathway plays an essential role in the reward system, which in turn is part of the limbic system (Nelson and Kreitzer, 2014; Sesack and Grace, 2010). Both the motor and the limbic system are driven by structures of the basal ganglia along with other structures. These other structures include the amygdala and hippocampus, important for encoding emotional context and declarative and spatial memory. Also the thalamus, prefrontal, entorhinal and motor cortex as well as the pedunculopontine tegmentum are important for the motor and limbic systems. These systems, together with the cognitive system, drive goal-directed behavior (Nelson and Kreitzer, 2014; Sesack and Grace, 2010; White, 2011).

The Basal Ganglia The basal ganglia form a group of subcortical structures involved in motor, limbic and cognitive functions. Initiation of movement is the most well characterized function. In addition, motor planning, motor learning, goal-directed behavior as well as cognitive drive triggered by memory-encoded cues or ex- ternal cues, are also functions regulated by the basal ganglia (Graybiel et al., 1994; Nelson and Kreitzer, 2014).

The main input structure of the basal ganglia is the striatum, mainly composed of GABAergic neurons, where 90% are medium spiny neurons (MSN’s). The

18 main output structures are the SNr and the structures of the pallidum, the GP pars interna (GPi), also known as entopeduncular nucleus in rodents, the GP pars externa (GPe), known as GP in rodents) and the ventral pallidum (VP), all primarily GABAergic structures. Additionally, some, but not, all studies include the glutamatergic subthalamic nucleus (STN) as part of the basal ganglia. Others discuss the STN as a structure associated with the basal ganglia. Dopaminergic projections from the ventral midbrain to the dorsal and ventral striatum are essential for the functions executed by the basal ganglia (Graybiel et al., 1994; Nelson and Kreitzer, 2014; Sesack and Grace, 2010).

Motor Loop According to the current model of the motor loop of the basal ganglia, the striatum receives glutamatergic inputs from the cortex and dopaminergic inputs from the SNc, which either acts on MSN’s via D1 receptors, activating the direct pathway leading to initiation of movement, or via D2 receptors, activating the indirect pathway leading to suppression of movement. Both pathways work in a “push and pull” manner in order to produce movement in the just right amount. When dopamine acts on D1 receptors, it upregulates the signals coming from the cortex, which results in the striatum inhibiting the GPi and SNr, consequently leading to disinhibition of the thalamus. This in turn results in thalamic excitation of the frontal cortex for initiation of movement (Figure 7). In contrast, when dopamine acts on D2 receptors, it down- regulates the effects of cortical excitation on MSN’s, leading to inhibition of the GPe, which in turn stops inhibiting the STN. As result, the STN sends glutamatergic inputs to the GPi, which inhibits the thalamus for suppression of movement (Figure 7). A hyperdirect pathway originating in the cortex di- rectly to the STN is a more recently established pathway that plays a significant role in rapidly inhibiting unwanted movement (Figure 7) (Nelson and Kreitzer, 2014; Simonyan, 2019).

Figure 7. Schematic figure of the motor loop of the basal ganglia. Arrows represent direction of pathways. Dopamine structure and projections are in blue, glutamatergic structures and projections are in green, and GABAergic structures and projections are in red. SNc, substantia nigra pars compacta; GPe, globus pallidus pars externa; GPi, globus pallidus interna; STN, subthalamic nucleus; SNr, substantia nigra pars reticulata; D1, dopamine 1 type receptor; D2, dopamine 2 type receptor. Figure by B. Vlcek based on (Nelson and Kreitzer, 2014).

Limbic Loop Similar to the motor loop, the limbic loop is composed of striatal, pallidal and midbrain structures. Its main input structure is the NAc, whereas the main output structures are the VP and SNr. The major dopamine input is derived from the VTA. Additional structures known to influence this loop are the amygdala, hippocampus and prefrontal cortex, among others (Alexander et al., 1991; Sesack and Grace, 2010).

The limbic loop works in a similar way as the direct pathway of the motor loop. In the limbic loop, glutamatergic cortical projections signal onto MSN’s of the NAc, which in turn inhibits the VP and SNr. This leads to disinhibition of the mediodorsal nucleus of the thalamus. Disinhibited, the thalamus sends glutamatergic input to the limbic forebrain cortical areas. Dopaminergic projections from the VTA to the NAc serve to reinforce the effects from the cortical input. The VTA also sends dopaminergic projections to the prefrontal cortex, which also receives glutamatergic input from the amygdala. The signals coming from the VTA and amygdala to the cortex serve to reinforce the signals arriving from the thalamus (Figure 8) (Alexander et al., 1991; Sesack and Grace, 2010).

Figure 8. Schematic figure of the limbic loop of the basal ganglia. Arrows represent direction of projections. Dopamine structure and projections are in blue, glutamatergic structures and projections are in green and GABAergic structures and projections are in red. VTA, ventral tegmental area; NAc, nucleus accumbens; VP, ventral pallidum; SNr, substantia nigra pars reticulata. Figure by B. Vlcek based on (Alexander et al., 1991)

Diversity within Circuitries of the Midbrain Dopamine System The loops of the basal ganglia described above represent a small part of the circuitries involving the midbrain dopamine system. For instance, studies have shown that both VTA and SNc also have other target structures in addition to the striatum, and that both are involved in limbic, motor and cognitive functions (Björklund and Dunnett, 2007; Sesack and Grace, 2010). The NAc shell (NAcSh), which primarily receives projections from VTA dopamine neurons, is a ventral extension of the dorsal striatum and receives input also from the SNc. Additionally, the NAcSh is also considered as an extension of the amyg- daloid complex, because it sends projections to the hypothalamus and brainstem having functions in visceral motor control and affective behavior. Fur- thermore, the NAcSh sends projections back to the VTA that regulate dopaminergic inputs from the VTA to the NAc core (NAcC) (Sesack and Grace, 2010).

There is increasing evidence that the dorsal striatum plays a role in goal-directed behavior, and that it is essential in habit formation. For instance, natural rewards, such as food and caffeine, as well as drugs of abuse, are known to

21 activate the NAc initially, leading to reinforcement. However, long term exposure is associated with habit formation and enhanced activity of the dorsal striatum. Further, the frontal cortex seems to play a role in coordinating this transition between reinforcement and habit, which might be related to the inputs that it sends back to the VTA, which have been suggested to regulate dopamine signaling in the mesocorticolimbic pathway, in a feedback-like manner (Sesack and Grace, 2010).

Dopamine neurons in the VTA are known to mediate different behaviors depending on the target structures (Chuhma et al., 2014; Trudeau et al., 2014). In mice, these neurons have been shown to send projections to other structures besides the NAc and olfactory tubercle, such as the cortex, hippocampus, amygdala, VP, periaqueductal grey, bed nucleus of the stria terminalis and locus coeruleus (Aransay et al., 2015; Morales and Margolis, 2017). Further- more, during the past decade, it has become increasingly clear that the VTA is not only composed of dopamine neurons, but also glutamatergic, GABAer- gic as well as co-releasing neurons, neurons that play a range of different roles in VTA circuitries (Bimpisidis and Wallén-Mackenzie, 2019; Morales and Margolis, 2017; Pupe and Wallén-Mackenzie, 2015). Understanding how the different neurons of the VTA influence its many different functions is crucial for understanding the role that these neurons might play in the various disorders that more or less strongly implicate the midbrain dopamine system.

Dysfunction in the Midbrain Dopamine System Dysfunction in the midbrain dopamine system affects motor, limbic and cognitive systems. It can lead to a series of different neurological diseases and neuropsychiatric disorders, such as PD, substance use disorder, schizophrenia and attention deficit hyperactive disorder, among others (Berke and Hyman, 2000; Goldstein and Deutch, 1992; Parkinson, 2002). These diseases and disorders give strongly disabling symptoms and any treatment available affects the whole dopamine system in an indiscriminate manner, often leading to un- desirable side effects. Understanding how the VTA is affected in these severe conditions might be crucial for identifying new therapeutic targets.

Substance Use Disorder (Drug addiction) Substance use disorder or drug addiction is classified as a neuropsychiatric disorder where the individual has developed a compulsive use of substances despite the negative consequences associated with it (Hyman and Malenka, 2001; Zou et al., 2017). Drug addiction is a problem increasing worldwide, having a great negative impact on general health but also on economy. Current

22 treatments are not very effective (Hyman and Malenka, 2001; Zou et al., 2017).

Drugs of abuse, such as amphetamine, cocaine and opioids are known for ex- erting their actions on both VTA and striatal neurons, mainly in the NAcSh but also in the dorsal striatum. These substances enhance dopamine levels in the mesocorticolimbic pathway (Di Chiara and Imperato, 1988; Hyman and Malenka, 2001). Repetitive exposure to drugs of abuse results in long-lasting alterations in synaptic plasticity within the dopamine and glutamatergic circuitries involved. These alterations lead to reinforcement of drug-seeking behavior, resulting in compulsive use (Hyman and Malenka, 2001). Certain substances, such as the ones listed above, can lead to tolerance, defined as decreased effect produced by a constant dose. Tolerance makes the user inclined to increase the dose in order to get a desirable effect (Hyman and Malenka, 2001). Substances such as cocaine and amphetamine can also lead to sensitization, which means that the brain systems become more sensitive to the effects of a dose, which leads to increased responses (Hyman and Malenka, 2001).

Drug addiction therapies often consist of rehabilitation, where patients have to stop using the substance for a long period, defined as abstinence. Due to molecular changes in the mesocorticolimbic system caused by drug abuse, abstinence often leads to withdrawal symptoms (Hyman and Malenka, 2001; Zou et al., 2017). Withdrawal symptoms develop as a result of dependence and can be either cognitive, emotional or physical. Importantly, dependence and withdrawal symptoms are not features unique to addictive drugs, but can also result from the use of medical substances aimed for treatment, such as adrenaline-agonist-based drugs used in the treatment of asthma and heart diseases (Berke and Hyman, 2000).

Substance abuse is known to have profound effects in the prefrontal cortex, amygdala and memory processing. These circuitry effects have been proposed to drive cue-induced relapse, which is when the user associates a cue with use of the drug and becomes emotionally conditioned. Relapse can occur even after several years of abstinence (Hyman and Malenka, 2001). All these factors together with the variety of effects that different drugs of abuse exert, makes the treatment process highly challenging and difficult. Understanding the circuitries underlying drug addiction is a key component in the development of more accurate therapies.

Parkinson’s Disease (PD) PD is the second most common neurodegenerative disease after Alzheimer’s disease and is the most common neurodegenerative motor disease affecting

23 millions of people worldwide (Dorsey et al., 2018; Mhyre et al., 2012). PD is defined by the degeneration of dopamine neurons in the SNc, leading to severe motor dysfunction due to loss of dopamine release in the basal ganglia and additional target areas (Lees et al., 2009; Williams-Gray and Worth, 2016). This loss of dopaminergic signaling leads to akinesia, bradykinesia and/or hypokinesia, tremor and rigidity, which are the cardinal symptoms of PD along with loss of posture and gait (Lees et al., 2009; Mhyre et al., 2012). In addition to motor dysfunction, many PD patients also suffer from non-motor symptoms resulting from dysfunction in the limbic, cognitive and autonomic systems, which severely affects the patient’s quality of life. Two secondary hallmarks of PD are Lewy body pathology and neuroinflammation, which are believed to contribute to degeneration along with mitochondrial dysfunction and oxidative stress (Mhyre et al., 2012; Rocha et al., 2018). There is still no cure for PD. Treatments available today are mainly based on dopamine replacement therapy which helps slowing down disease progression. However, the main focus of dopamine replacement is to alleviate motor symptoms (Lees et al., 2009; Williams-Gray and Worth, 2016).

Types of PD There are two main types of PD, sporadic/idiopathic and familial/hereditary. Sporadic/idiopathic PD is the most common type often affecting the elderly, where patients are diagnosed around 60-80 years of age. Disease progression is relatively slow and most patients pass away from other causes. However, by the time of diagnosis, which is based on the presence of the cardinal symptoms, most of the SNc dopamine neurons are already lost (60-80%). The main cause of sporadic PD is yet unknown, but genetic and environmental risk factors have been identified (Lees et al., 2009; Williams-Gray and Worth, 2016). Familial PD is less common but usually more aggressive. In most cases, patients are diagnosed around 25-30 years of age. Familial PD shows a fast progression, often leading to death (Williams-Gray and Worth, 2016). The familial type of PD is caused by gene mutations, which can be autosomal dominant. Examples of genes that have been found associated with PD includes alpha- Synuclein (SNCA), for example mutations A53T and A30P, and PARKIN and PINK1 (Klein and Schlossmacher, 2007; Krüger et al., 1998; Williams- Gray and Worth, 2016).

Motor symptoms Bradykinesia is defined as a slowness of movement, for instance reduced speed when executing movements such as opening and closing hands, or fin- ger tapping or walking. The term also includes impaired fine motor control resulting in difficulties to, for example, write and sew. Hypokinesia is defined as reduced frequency of movements and decreased amplitude presented as decreased arm swing during walking, decreased facial expression and shortened gait (Lees et al., 2009; Mhyre et al., 2012; Williams-Gray and Worth, 2016).

24 In the majority of the PD patients, tremor is the first symptom to emerge, but the presence of bradykinesia is required for diagnosis of this disease (Lees et al., 2009; Mhyre et al., 2012). Motor symptoms are usually unilateral in the early stages of the disease, but with progression, they tend to extend to the contralateral side and patients eventually develop postural instability (Mhyre et al., 2012).

Non-motor symptoms The majority of PD patients present one or more non-motor symptoms that can vary in category, but also in intensity and have often a profoundly negative effect on the quality of life (Mhyre et al., 2012). Non-motor symptoms can be divided into autonomic, sensory, sleep-related, cognitive and neuropsychiatric symptoms. Some of these symptoms may appear before motor symptoms, develop at the same time as motor symptoms or appear in later stages of the disease. Many of the non-motor symptoms result from the degeneration of neurons in the midbrain dopamine system, but also in other systems such as serotonin, noradrenalin and cholinergic systems, or as side-effects of dopamine replacement therapy, for example dyskinesia (Fields, 2017; Mhyre et al., 2012).

Autonomic, sensory and sleep related symptoms usually appear in the form of constipation, loss of olfactory function, hypotension and REM sleep behavior disorder (Mhyre et al., 2012; Williams-Gray and Worth, 2016). Sleep dysfunction is highly correlated with dysfunction in the striatal-thalamo-cortical pathway of the basal ganglia. Autonomic and sensory related symptoms have shown correlation with the presence of alpha-Synuclein pathology in the peripheral nervous system, but also with degeneration of cholinergic neurons of the brainstem. The most common cognitive symptom is dementia, which can be mild or aggressive and is often accompanied by the presence of depression. Hallucinations, mostly in the visual form, are common in patients diagnosed with PD with dementia (Fields, 2017; Mhyre et al., 2012).

Common neuropsychiatric symptoms in PD include depression and anxiety, but also apathy, psychosis, mania and impulse control disorders. Both depression and anxiety have been suggested to result from loss of dopamine and noradrenalin innervations of the cortical areas associated with the limbic system, which might be associated with the presence of alpha-Synuclein pathology observed in limbic areas (Braak et al., 1994, 1994; Fields, 2017; Mhyre et al., 2012; Remy et al., 2005). These symptoms might also be partially due to degeneration of the dopamine neurons of the VTA, resulting in decreased dopamine signaling in the mesocorticolimbic system (Alberico et al., 2015; Lees et al., 2009). Dysfunction in the mesocorticolimbic system is evident in PD patients suffering from addiction to natural rewards, such as gambling, excessive shopping and hypersexuality, usually observed in patients receiving

25 dopamine replacement therapy, the most effective treatment of motor impairments at early stages of PD (Williams-Gray and Worth, 2016). alpha-Synuclein and Lewy Body Pathology alpha-Synuclein (αSyn) is a neuron-specific protein, involved in normal neuronal function including synaptic vesicle transportation and release, lipid binding and dopamine homeostasis (Lotharius et al., 2002; Murphy et al., 2000; Perez et al., 2002; Perrin et al., 2000). Null mutation of the Snca gene in mice has been shown to lead to dysfunction in the nigrostriatal dopamine system and giving rise to cognitive dysfunctions (Abeliovich et al., 2000; Kokhan et al., 2012).

In PD, αSyn tends to form fibrils and oligomers, which are toxic for the neurons, including dopamine neurons of the SNc (Mhyre et al., 2012). These secondary structures easily bind to each other along with other proteins and cellular products, forming aggregates known as pale bodies, paradoxically shown to be neuroprotective (Breydo et al., 2012; Takahashi et al., 1994). As the aggregates develop further and become Lewy bodies, they interfere with the cell’s metabolism contributing to cell death (Breydo et al., 2012). In addition, αSyn accumulation has been implied as a trigger of neuroinflammation in PD. The activation of microglia as well as astrocytes may occur as a result of this accumulation (Dexter and Jenner, 2013; Rocha et al., 2018). The different factors leading to αSyn misfolding include the nature of the protein structure, environmental and genetic factors (Lee et al., 2007; Spillantini et al., 1997; Uversky et al., 2001). These factors vary between sporadic and familial PD, but also between patients within the same type of PD (Lee et al., 2007; Uver- sky et al., 2001).

Lewy body pathology has been observed in numerous types of neurons, such as dopaminergic, noradrenergic, serotonergic, histaminergic, glutamatergic and cholinergic neurons (Braak et al., 2003; Williams-Gray and Worth, 2016). Lewy body pathology has also been implicated in limbic dysfunction in PD, as aggregates have been identified in limbic regions in postmortem tissue from patients (Braak et al., 1994, 2003; Williams-Gray and Worth, 2016). Apart from cytoplasmic, Lewy body pathology can also develop in neurites, commonly known as Lewy neurites. Importantly, Lewy body pathology is not an exclusive feature of PD and there are PD patients that do not develop these aggregates. Therefore, Lewy body pathology is considered as a secondary hallmark.

Treatment of PD The treatment of PD during early stages is mainly based on dopamine replacement therapy in order to ameliorate motor symptoms. The most used types of medications include L-DOPA, dopamine agonists and MAO inhibitors, but

26 COMT inhibitors and peripheral AADC inhibitors may also be used (Mhyre et al., 2012). Arvid Carlsson described L-DOPA as a potential treatment for PD back in the 1950s, and it is still today the main medication used (Abbott, 2010; Mhyre et al., 2012; Williams-Gray and Worth, 2016b). L-DOPA is highly effective in the early years of treatment when it is used by the remaining dopamine neurons of the SNc to synthesize dopamine. However, as the disease progresses, degeneration advances and the treatment becomes less effective. In addition, prolonged use of L-DOPA induces dyskinesia (involuntary movements) as a side-effect of the so called “on-off” effect of the dosage (Obeso et al., 2000). Therefore, dopamine agonists rather than L-DOPA are usually the first line of treatment offered, especially in the case of PD with early onset (Mhyre et al., 2012). In later stages of the disease, when pharmacological treatment does not give a desirable effect anymore, patients may be treated with electrical stimulation of basal ganglia structures, for example the STN. This treatment is known as deep brain stimulation (DBS) (Hamani et al., 2017). However, both pharmacological and DBS treatments can lead to unde- sirable side-effects. In addition to hyperkinesia and addictions, other side-effects of dopamine replacement include nausea and vomiting, hallucinations, hypotension, excessive daytime sleepiness, depression and confusion (Wil- liams-Gray and Worth, 2016b). Some of the side-effects of DBS of the STN include impulsivity, cognitive dysfunction, depression and mood changes (Buhmann et al., 2017; Hamani et al., 2017). Therefore, major efforts are aimed at identifying novel therapies for PD, including the identification of neuroprotective factors that could be useful in the early stages in order to slow down disease progression.

Animal models of PD Because PD is a multifactorial disease, different kinds of animal models are needed for its study. For example, dopamine neuron degeneration and αSyn pathology are often studied using different animal models. Animal models are also used for testing of promising novel therapies.

Toxin-induced models Two commonly used methods to induce degeneration, or lesion, of dopamine cells and/or their projections are the toxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydrodropyridine (MPTP) models (Bové et al., 2005). These methods are applied to non-human primates (MPTP) and rodents (both), mostly rats and mice. While 6-OHDA has to be injected intra- cranially, MPTP can be administered systemically as it has the ability to pass through the brain-blood-barrier. Once through the blood-brain-barrier, MAO converts MPTP into 1-methyl-4-phenylpyridinuim (MPP+), mainly in glial cells. MPP+ is then able to enter dopamine neurons through DAT and exerts

27 its toxic effects leading to oxidative stress and finally resulting in cell death (Blum et al., 2001; Bové et al., 2005). MPTP was discovered in the early 1980’s when a population of young substance abusers in California developed a synthetic drug aiming to mimic an opioid. Accidently, they developed a deri- vate of fentanyl containing MPTP, which resulted in Parkinsonian symptoms (Blum et al., 2001). However, neither 6-OHDA nor MPTP models mimic Lewy body and aSyn pathology (Blum et al., 2001; Bové et al., 2005). 6- OHDA is further described below.

6-OHDA mouse model 6-OHDA was first described by Senoh in 1959 as a DOPA metabolite in the rat, later shown by Porter in 1963 to cause noradrenaline depletion in the heart of mice when administered systemically (Porter et al., 1963). In 1968, Tranzer and Thoenen reported the molecule to cause degeneration selectively in adrenergic nerve terminals of the sympathetic system (Tranzer and Thoenen, 1973). In the same year, Ungerstedt showed the effects of the toxin in the brain for the first time by injecting it in the SNc and striatum of rats. This caused depletion of dopamine leading to “marked motor asymmetry”, presented by the animals as turning to the ipsilateral side to the injection (Ungerstedt, 1968). The actions of 6-OHDA were described by Jonsson in 1980 as “chemical denervation of a given neuron type”, a concept that was novel to neurobiology (Jonsson, 1980). The 6-OHDA lesion method was one of the methods used by Ungerstedt to study and describe the midbrain dopamine system (Ungerstedt, 1971).

6-OHDA is a dopamine analogue, a molecule of similar structure to that of dopamine. Mechanisms of action described for this toxin include: 1) Uptake by dopamine neurons through the plasma membrane transporter due to high affinity; 2) Accumulation in the cytosol leading to auto-oxidation, which can also occur for extracellular 6-OHDA, leading to the formation of reactive ox- ygen species (ROS); 3) Degradation by monoamine oxidase forming ROS; 4) Inhibition of the mitochondrial respiratory chain reaction leading to decreased production of adenosine triphosphate. Altogether, this leads to oxidative stress, decreased energy availability, and finally to cell death (Blum et al., 2001; Hernandez-Baltazar et al., 2017).

The 6-OHDA lesion strategy has been used as method to induce dopamine depletion to facilitate PD research since Ungerstedt’s findings and it is still today a useful tool in this research area. The model does not present broad brain pathology, no αSyn pathology and it is quite aggressive. However, the method has several features in addition to nigrostriatal pathway damage that are relevant for PD (Blum et al., 2001). For example, early findings showed the presence of endogenous 6-OHDA in both rat and human brains as well as

28 in the urine from PD patients (Andrew et al., 1993; Blum et al., 2001; Curtius et al., 1974).

In this thesis, the 6-OHDA lesion strategy was implemented in mice (Study IV).

Genetic models In order to understand how gene mutations might cause PD and how Lewy body and αSyn pathology might contribute to disease progression, a wide range of genetic animal models have been created using transgenic methodol- ogy. These models are primarily based on the introduction of a gene or gene mutation known to cause PD in familial cases, but also genes that are associated with a higher risk for developing PD (Fernagut and Chesselet, 2004; Lee et al., 2007, 2012). An example is the A30P mutation in the SNCA gene, in which a point mutation on the position 88 of the coding sequence of exon 3 exchanges guanine to cytosine, which causes a shift from alanine to proline in the αSyn protein (Krüger et al., 1998). The A30P mutation has been detected in a few families, where all members develop an aggressive and early-onset PD and die at a young age (50-60 years) (Krüger et al., 1998). It is unknown how the mutation affects αSyn functionality, but it has been suggested that it may alter synaptic vesicle binding and enhance the propensity of αSyn to ag- gregate (Breydo et al., 2012).

In mice, the effects of the A30P mutated SNCA gene have shown differences depending on the promoter used. For instance, introduction of A30P-SNCA using the rat Th promoter induces progressive dopamine neuron loss, motor phenotype, but no aggregates in dopamine neurons (Lee et al., 2012; Richfield et al., 2002). Introduction of A30P-SNCA under the mouse prion promoter does not lead to dopamine neuron loss, but causes degeneration of other neurons and αSyn aggregates (Dawson et al., 2010).

Other mutations have also been induced in transgenic animal models such as the A53T-SNCA, which when introduced under the rat TH promotor has resulted in similar effects as the A30P-SNCA under the same promotor (Lee et al., 2012; Richfield et al., 2002). Expressed under control of the brain-specific Thy-1 promoter, A53T-SNCA expression leads to degeneration of motor neurons in the spinal cord causing motor dysfunction (van der Putten et al., 2000). Leucine-rich repeat kinase 2 mutation, which causes autosomal dominant forms of familial PD, but is also considered as a risk gene for developing sporadic PD, was shown to decrease amphetamine-induced hyperlocomotion but no dopamine neuron degeneration in mice (Lee et al., 2012). Further, PAR- KIN, PINK1 and other PD-associated genes have also been used to model PD

29 using transgenic approaches in mice (Dawson et al., 2010; Fernagut and Ches- selet, 2004; Lee et al., 2012).

In this thesis, two genetic mouse models were used (Study III): The Thy1- h[A30P]αSyn and Thy1-h[wt]αSyn mouse lines, further described below.

Thy1-h[A30P]αSyn The Thy1-h[A30P]αSyn (also referred to as A30P mice) mouse line was generated and characterized by Phillip Kahle and colleagues. The human A30P- SNCA sequence was amplified by PCR and subcloned into the XhoI (deoxy- ribonuclease restriction enzyme) site of pTSC221k (plasmid-vector Thy-1 promoter construct). The DNA fragment containing only the transgene sequence was isolated from the rest of the plasmid and injected into fertilized mouse eggs (Kahle et al., 2000). The line characterized with highest expression of the transgene, line 31, was further intercrossed generating a stable col- ony of homozygous mice. The eggs were implanted into surrogate mothers whose offspring was genotyped with primers specific for Thy-1 sequences inserted on both sides of the SNCA insert. The mice positive for Thy1- h[A30P]SNCA were then backcrossed into C57BL/6 mice (> 10 generations) (Neumann et al., 2002a).

Behavioral characterization of the model showed that these mice develop motor impairment already at 2 months of age, when they make more errors per step in the challenging beam test, which shows progression with ageing (EkmarkLewén et al., 2018). In other tests, such as the automated home cage, the catwalk and rotarod tests, A30P mice also show a decrease in voluntary motor activity, a need to take more steps to cross, but also impaired motor coordination in comparison to wild type controls (Casadei et al., 2014; Freichel et al., 2007; Rotermund et al., 2017).

Pathological forms of αSyn, such as Proteinase-K resistant, hyper-phosphorylated, fibrils and aggregates of αSyn has been observed in the neocortex, hippocampus, brainstem, midbrain and spinal cord with variations between these forms (Freichel et al., 2007; Neumann et al., 2002b; Schell et al., 2009). The first studies published on this model have shown that the dopamine neurons of the SN seem highly intact (Freichel et al., 2007; Kahle et al., 2000; Neu- mann et al., 2002b). However, a more recent study showed decreased detection of TH by immunohistochemistry (EkmarkLewén et al., 2018), suggesting dopamine cell loss or down-regulation of the Th promoter in A30P mice.

Thy1-h[wt]αSyn mice The Thy1-h[wt]αSyn mouse line (also referred to as L61 mice) was generated and characterized by Eliezer Masliah, Edward Rockeinstein and colleagues. The wild-type human SNCA mRNA was used to synthesize a cDNA fragment (53-475 bp) by RT-PCR, that was then ligated in a pCRII plasmid vector. The

30 cDNA fragments were inserted into a mouse Thy1 expression cassette between exons 2 and 4. After purification, the fragments were microinjected into one- cell embryos of a mixed background (C57BL/6 X DBA/2F1) subsequently implanted into surrogate mothers. PCR was used to genotype the offspring, where the highest expressing line, line 61, was selected for breeding. These mice were maintained on a mixed background (Chesselet et al., 2012; Rock- enstein et al., 2002).

Behavioral characterization of this model showed that L61 mice develop motor impairment already at a young age (3 months), displayed as more errors per step in the challenging beam test. Additionally, at the same age, L61 mice further show motor impairments in the pole and cylinder tests where L61 mice take a longer time to descend the pole and decreased limb movements and rearing (Fleming et al., 2004). In older ages, L61 mice show motor dysfunction when tested in the inverted grid and when given amphetamine, in which the mice took shorter steps to cross the grid and did not respond with hyperlocomotion, respectively (Fernagut et al., 2007; Fleming et al., 2004, 2006). The same pathological forms of αSyn identified in the A30P has been also identified in the L61 mice (Chesselet et al., 2012). Dopamine neuron loss has yet not been shown in the L61 mice, but decreased levels of dopamine, dopamine metabolites and TH content in the striatum has been shown (Chesselet et al., 2012).

Diversity within the VTA In addition to neurons releasing dopamine, the VTA also contains neurons capable of both producing and releasing GABA and/or glutamate as well as neurons co-releasing more than one of these neurotransmitters (Bimpisidis and Wallén-Mackenzie, 2019; Kawano et al., 2006; Pupe and Wallén-Mac- kenzie, 2015; Tritsch et al., 2012; Yamaguchi et al., 2007). This heterogeneity was originally identified in the VTA but subsequent studies have pinpointed some degree of heterogeneity also in the SNc (Poulin et al., 2018; Root et al., 2016; Yamaguchi et al., 2013). The capacity of co-releasing dopamine and glutamate has also been observed in humans and non-human primates (Root et al., 2016). Yet another level of heterogeneity is present within the dopamine neurons. There are differences in gene expression between the VTA and SNc and between the different subdivisions of each of these two structures (Chung et al., 2005; Greene et al., 2005; Poulin et al., 2014).

Dopamine and Glutamate Co-phenotype Neurons in the midbrain with a dual dopamine and glutamate phenotype were found to co-localize Th and the Vesicular glutamate transporter 2 (Vglut2) mRNAs in the mouse brain already at 12.5 days old embryos (Birgner et al.,

31 2010; Nordenankar et al., 2015). In earlier stages, Vglut2 mRNA can be detected by in situ hybridization in most non-differentiated dopamine neurons prior to detection of Th mRNA (Dumas and Wallén-Mackenzie, 2019). In cell culture of SNc dopamine neurons, glutamate was shown to control dopamine neuron growth rate and axonal branching (Schmitz et al., 2009). Together these studies suggest that the VGLUT2 protein has an important role in midbrain dopamine neuron development (Dumas and Wallén-Mackenzie, 2019; Schmitz et al., 2009). In adult rodents, Vglut2 mRNA was found in both VTA and SNc. Most Vglut2-positive neurons are in the VTA, mainly in the medial aspect with few neurons scattered more laterally (Kawano et al., 2006; Yama- guchi et al., 2007). Midbrain neurons positive for Vglut2 mRNA have also been identified in non-human primates as well as in humans (Root et al., 2016).

Vglut2-positive neurons of the VTA medial aspect have been shown to send projections to the NAcSh (Hnasko et al., 2012). Ablation of Vglut2 gene expression in Dat-expressing dopamine neurons in mice leads to an altered risk- taking behavior and altered locomotor response to addictive drugs, such as amphetamine and cocaine, in adult mice (Alsiö et al., 2011; Birgner et al., 2010). Further, this targeted knockout of Vglut2 was also shown to increase self-administration of both high sugar-food and cocaine. These results suggested that the glutamate/dopamine co-phenotype is relevant for addiction susceptibility (Alsiö et al., 2011). When Vglut2 gene expression is knocked out in mature dopamine neurons, using an inducible DAT-Cre mouse line, decreased glutamate neurotransmission in the D1 expressing MSN’s of the NAc is observed with enhanced baseline in the AMPA/NMDA ratio. These findings suggested alterations in synaptic plasticity caused by the targeted loss of Vglut2. (Papathanou et al., 2018).

GABA and GABA/Dopamine Co-phenotype Neurons positive for Glutamate decarboxylase (GABA synthesizing enzyme) or the Vesicular inhibitory amino acid transporter (Viaat), were shown to be scattered throughout the VTA. Most of these presumptive GABA neurons seem to have a GABA/dopamine co-phenotype (Margolis et al., 2012; Mo- rales and Margolis, 2017). Some of these neurons send projections to cholinergic interneurons of the striatum, and have been suggested to play a role in associative learning (Brown et al., 2012; Morales and Margolis, 2017). A small GABA population found positive for the TH protein has projections to the lateral habenula, promoting reward via GABA activity (Morales and Mar- golis, 2017; Stamatakis et al., 2013). Viaat mRNA in ventral midbrain neurons can be detected at embryonal day 11.5 (Dumas and Wallén-Mackenzie, 2019).

32 Gene Expression Diversity Dopamine neurons of the VTA are more resistant to PD than dopamine of the SNc. About 45% of VTA dopamine neurons are lost while the SNc loses around 60-80% of its dopamine neurons during disease progression (Alberico et al., 2015; Damier et al., 1999b; McRitchie et al., 1997). In efforts to identify factors that protect dopamine neurons in the VTA, microarray and tran- scriptomics analyses have been performed with the idea to identify gene that encode neuroprotective factors. A number of genes have been identified that show significant differences in their expression levels between the SNc and VTA (Chung et al., 2005; Greene et al., 2005; Grimm et al., 2004; Poulin et al., 2014). Most genes expressed at higher levels in the SNc than VTA dopamine neurons encodes for proteins important for cell metabolism, vesicle-me- diated transport and apoptosis. Genes higher expressed in dopamine neurons of the VTA than the SNc are more related to synaptic plasticity, learning, neu- ropeptide and hormone activity, cell survival and protection (Brichta and Greengard, 2014; Chung et al., 2005; Greene et al., 2005; Grimm et al., 2004).

To summarize some main findings from these gene expression analyses, mRNAs that were found elevated in dopamine neurons of the SNc included: G-protein coupled inwardly rectifying K channel 2 (Girk2), gamma-Synuc- lein, alpha-Synuclein (Snca), Aldehyde dehydrogenase 1 family member a1 (Aldh1a1), SRY-box transcription factor 6 (Sox6), Paired like homeodomain 3 (Pitx3), and Dat.

A different profile was found for mRNAs found elevated in the VTA, which included: Gastrin releasing peptide (Grp), Neuronal differentiation 6 (Neu- roD6), Calbindin-D28K (Calb1), Calretinin (Calbindin2, Calb2), Neurotrop- hic factor 3 (Ntf3), Orthodenticle homeobox 2 (Otx2), LIM homeobox transcription factor 1 alpha (Lmx1a), Engrailed homeobox 1 and Vglut2 (Chung et al., 2005; Greene et al., 2005; Grimm et al., 2004; Poulin et al., 2014).

While higher expressed in one area or the other, most genes are to some degree expressed in both, for example Girk2 and Aldh1a1 (Greene et al., 2005; Grimm et al., 2004).

Dopamine neuron markers in both VTA and SNc Th, Vmat2 and Dat mRNA are highly distributed in both structures, where TH, VMAT2 and DAT proteins have crucial roles in dopamine metabolism and, are therefore used as general markers of dopamine neurons (Ciliax et al., 1999; Meiser et al., 2013; Morales and Margolis, 2017). Dat mRNA has a more lateral distribution pattern in the VTA compared to Th and Vmat2, where the most medial aspect lacks Dat while Th and Vmat2 mRNAs can still be detected (Bimpisidis and Wallén-Mackenzie, 2019; Morales and Margolis,

33 2017). As previously mentioned, higher distribution of Dat has been observed in the SNc compared to the VTA in both mice and humans, which has led to its association with neurons that are more vulnerable in PD (Brichta and Greengard, 2014; Chotibut et al., 2012; Ciliax et al., 1999). Additionally, because the DAT protein is used by 6-OHDA and MPTP to enter dopamine neurons, neurons positive for DAT are more vulnerable than dopamine neurons negative for DAT in toxin models of PD (Blum et al., 2001).

In contrast, the VMAT2 protein has been implicated as a “neuroprotective” factor by studies showing VMAT2 to be dysfunctional in PD, while a familial mutation in the Vmat2 gene was shown to cause infantile PD (Guillot and Miller, 2009; Lohr and Miller, 2014). Further, studies have shown that a gain of function in the Vmat2 gene seems to be protective against the development of the disease in humans at the same time that mice with elevated levels of the protein show more resistance to MPTP (Guillot and Miller, 2009; Lohr and Miller, 2014). The orphan nuclear receptor 1 (Nurr1, also known as Nr4a2) and the LIM homeobox transcription factor 1 beta (Lmx1b) are both necessary for the differentiation of the midbrain dopamine neurons. Nurr1 is required for maturation of the precursors and expression of Th, but also for maintenance of adult dopamine neurons (Kadkhodaei et al., 2009; Wallén and Perl- mann, 2003; Wallén et al., 1999; Zetterström et al., 1997). Lmx1b is involved in the specification of the progenitors but not necessary for Th expression (Arenas et al., 2015). Studies have shown that NURR1 protein deficiency in dopamine neurons of PD patients is associated with accumulation of dysfunctional αSyn, while ablation of Lmx1b and Lmx1a genes in mice leads to impaired mitochondrial respiration, DNA damage, oxidative stress as well as accumulation of αSyn and, finally dopamine cell death (Chu et al., 2006; Dou- cet-Beaupré et al., 2016).

The Snca gene codes for the αSyn protein that, as mentioned earlier, is a neuron-specific protein involved in normal neuronal function including synaptic vesicle transportation and release, lipid binding and dopamine homeostasis (Lotharius et al., 2002; Murphy et al., 2000; Perez et al., 2002; Perrin et al., 2000). In PD, αSyn tends to form fibrils and oligomers that are toxic for the neurons. Different mutations as well as duplication and triplication of the human SNCA gene has been associated with aggressive early onset of hereditary PD forms (Chartier-Harlin et al., 2004; Krüger et al., 1998b; Polymeropoulos et al., 1997; Singleton et al., 2003). Additionally, certain haplotypes of the human SNCA gene have been associated with sporadic cases of PD (Farrer et al., 2001; Pals et al., 2004). A recent study has shown that Snca mRNA can be detected co-localizing with Th in the midbrain of mice already at 11.5 days old embryos (Dumas and Wallén-Mackenzie, 2019; Poulin et al., 2014).

34 Dopamine neuron markers of the SNc The PITX3 protein is a transcription factor involved in the development of dopamine neurons (Nunes et al., 2003). Pitx3 mRNA has been found elevated in the SNc, but, it can also be detected in the VTA (Poulin et al., 2014). How- ever, Pitx3 gene expression has been reported as crucial for the development of SNc dopamine neurons but not VTA dopamine neurons. This is based on studies where mice with a null mutation of Pitx3 fail to develop SNc dopamine neurons while VTA neurons are highly intact (Li et al., 2009; Nunes et al., 2003; Wallén and Perlmann, 2003). In 6-OHDA lesion mouse model as well as in PD patients, Pitx3 mRNA distribution was found decreased, related to the decrease in dopamine neurons, while polymorphisms of the Pitx3 gene has been associated with sporadic and early onset of PD (Li et al., 2009). SOX6 is also a transcription factor elevated in the SNc with few cells within the VTA in mice, while in humans, it can be detected in dopamine neurons containing neuromelanin, which are more vulnerable in PD (Anderegg et al., 2015).

The GIRK2 protein has for many years been considered as a marker of vulnerable SNc dopamine neurons in PD. This is based on that most studies show Girk2 mRNA or GIRK2 protein mainly in the ventral tier of the SNc and, in overall elevated in the SNc in comparison with the VTA (Chung et al., 2005; Karschin et al., 1996; Schein et al., 1998). Additionally, mutation of the Girk2 gene leads to developmental loss of most SNc dopamine neurons and Girk2 mRNA-positive neurons have also shown higher vulnerability in MPTP mice (Chung et al., 2005; Graybiel et al., 1990; Slesinger et al., 1996). However, in more recent studies, Girk2 mRNA and its protein were shown to be distributed in both SNc and VTA. In the SNc, Girk2 was found was equally distributed throughout the structure, while in the VTA, it was more prominent in the PBP and PN in both mice and humans (Lammel et al., 2008; Reyes et al., 2012).

Aldh1a1 mRNA detection is specific for dopamine neurons of the ventral midbrain, mainly in the SNc but also VTA in both rodents and humans, where the distribution patterns are similar (Anderson et al., 2011; Galter et al., 2003; Wallén et al., 1999). Most of the Aldh1a1-positive neurons are located in the ventral aspect of both the SNc and VTA, with some neurons scattered in the PBP (Anderson et al., 2011; Galter et al., 2003; Liu et al., 2014). Aldh1a1 was recently implicated in SNc dopamine neuron vulnerability based on analyses in MPTP mice, contrasting findings from a study on a transgenic mouse model of Snca overexpression (Liu et al., 2014; Poulin et al., 2014). Further, a role in neuroprotection rather than vulnerability has been suggested based analyses in tissue from PD patients that ALDH1A1-positive SNc dopamine neurons are relatively speared in comparison with ALDH1A1-negative neurons (Galter et al., 2003; Liu et al., 2014). In addition, the ALDH1A1 protein is involved in the degradation of excessive L-DOPA in dopamine neurons, a process known to decrease cytotoxicity. Accumulation of L-DOPA was shown to promote

35 polymerization of αSyn as well as in compromising the function of certain proteins necessary for the survival of dopamine neurons (Cai et al., 2014). Decreased levels of ALDH1A1 have been found in the brain of some PD patients, suggesting that lack of this protein could lead to higher vulnerability (Cai et al., 2014; Galter et al., 2003; Liu et al., 2014).

Dopamine neuron markers of the VTA Otx2 mRNA is restricted to the VTA in both adult mice and humans. The OTX2 protein is a transcription factor required for proliferation, fate and identity of all midbrain dopamine neurons during development (Anderegg et al., 2015; Di Salvio et al., 2010a). The OTX2 protein has been implied as neuroprotective in the MPTP mouse model, however, in the adult human brain OTX2 protein seems to be lacking (Di Salvio et al., 2010b; Reyes et al., 2013). Calb1, Calb2, Grp and NeuroD6 mRNA have been suggested as markers of neuroprotected neurons due to their higher expression levels in dopamine neurons of the VTA (Catoni et al., 2019; Chung et al., 2005; Damier et al., 1999b; Kramer et al., 2018; Mouatt-Prigent et al., 1994). Both Calb1 and Calb2-positive neurons have shown higher survival rate in MPTP mice, while Calb1 was further shown relatively speared in PD. Calb1 and Girk2 are often used to discriminate between VTA and SNc neurons (Chung et al., 2005; Dopeso- Reyes et al., 2014; Reyes et al., 2012). Recent studies have shown that GRP- positive neurons were more resistant to MPTP toxin in cell culture of cells expressing high levels of Snca, while NEUROD6-positive neurons were resistant to 6-OHDA in mice (Chung et al., 2005; Kramer et al., 2018). Both these factors are of interest to this thesis and will be discussed further in the context of Study I and II.

The NTF3 protein is a neurotrophic factor with elevated mRNA levels in the VTA. There seems to be a decrease in the levels of NTF3 in the brain content of patients. Furthermore, the Brain derived neurotrophic factor, BDNF, has been shown to have certain neuroprotective properties and have been used in trials for treatment of PD, but without any significant improvement of the symptoms (Sampaio et al., 2017). The VGLUT2 protein has been implicated in dopamine cell development and survival (Dumas and Wallén-Mackenzie, 2019; Steinkellner et al., 2018). VGLUT2 enables packaging of glutamate into presynaptic vesicles, and is found throughout the brain in glutamatergic neurons. Some, but far from all, midbrain dopamine neurons are positive for VGLUT2 (Morales and Margolis, 2017; Trudeau et al., 2014). Viral injection of Vglut2-expressing DNA constructs in the midbrain of adult mice results in a progressive loss of dopamine neurons, mainly in the SNc, but also in the VTA, resulting in impaired motor behavior (Steinkellner et al., 2018). Condi- tional deletion of the Vglut2 gene in dopamine neurons increased vulnerability to the effects of 6-OHDA and MPTP (Steinkellner et al., 2018). Vglut2 mRNA can be detected in differentiating dopamine neurons prior to any dopamine

36 neuron markers in the mouse midbrain (Dumas and Wallén-Mackenzie, 2019). Vglut2 mRNA co-localizes extensively with Th mRNA at embryonic day 10-11, and is subsequently downregulated to be only found in a restricted number of cells in the adult ventral midbrain (Dumas and Wallén-Mackenzie, 2019).

Current information about genes that are higher expressed in the SNc versus VTA, and vice versa, contributes to the knowledge that there are molecular differences between the dopamine neurons that could contribute to differences in vulnerability in PD. In addition, recent findings clearly show that subtypes, or subpopulations, of midbrain dopamine neurons exist within both the SNc and VTA, defined by different profiles in gene expression patterns.

Overall Aim The overall aim of this doctoral thesis was first to identify diversity within the midbrain dopamine system with emphasis on the VTA, in order to establish subpopulations represented by distinct gene expression patterns. Based on such knowledge, the subsequent aim was to explore how midbrain dopamine neurons of the VTA are affected in Parkinson´s disease, and in three different mouse models of this disorder: Two different transgenic mouse lines overexpressing the human SNCA gene and the 6-OHDA model.

37 Materials and Methods

Mice All mice were housed in the local animal facility, kept at standard temperature and humidity conditions with a 12-hour dark/light cycle. All mice had access to food and water ad libitum. Experiments were performed in accordance with ethical permits obtained from the local Uppsala Animal Ethical Committee in accordance with the Swedish regulations and European Union legislation ap- proved all animal procedures.

In study I, DAT-Cre mice (Ekstrand et al., 2007) were bred with a red-fluorescent Cre-reporter mouse line (B6; 129S6-Gt(ROSA)26Sortm9(CAG- tdTOMATO)HZE/J mice (Jax Mice (Madisen et al., 2010)) to generate DAT-CretdTom mice for microarray experiments. C57BL/6 mice were used for histological mapping. Transient receptor potential cation channel subfamily V member 1 (Trpv1)-Cre mice (Lagerström et al., 2010) were bred with the Cre-reporter mice to generate Trpv1-CretdTom for histological mapping, electrophysiology and optogenetics experiments.

In study II, C57BL/6 mice were studied for further characterization of the NeuroD6 mRNA pattern within the ventral midbrain. DAT-Cre (Ekstrand et al., 2007), Vglut2-Cre (Borgius et al., 2010), Calb2-Cre (Jackson laboratory) and NeuroD6/Nex-Cre (Goebbels et al., 2006) transgenic mice were bred in- house. NeuroD6-Cre mice were crossed with Vmat2Lox/Lox mice (Narboux- Nême et al., 2011) in order to address the role of the NeuroD6 subpopulation in the context of the midbrain dopamine system. Vmat2Lox/Lox;Nex-Cre-tg were used as conditional knockout mice and Vmat2Lox/Lox;Nex-Cre-wt were used as control.

In study III, two different transgenic mouse lines overexpressing the human SNCA gene were used. Homozygous Thy1-h[A30P]αSyn transgenic mice expressing the human SNCA with the A30P mutation under the brain-specific Thy-1 promoter were studied along with age and sex matched wild type control mice of the same background (C57BL/6J). In total 30 animals, both males and females were analyzed between 16-18 months of age. Transgenic mice were generated as previously described (Kahle et al., 2000). Heterozygous Thy1-h[wt]αSyn transgenic mice expressing the full-length human wild type

38 SNCA were studied together with wild type littermates. In total, 20 male mice were analyzed at 5 and 9-11 months of age. Transgenic mice were generated as previously described and maintained on a C57BL6/DBA2 background (Rockenstein et al., 2002).

In study IV, C57BL/6NTac (Taconic) male mice were used for 6-OHDA injections. In total 15 animals were used.

Stereotaxic 6-OHDA injection Stereotaxic 6-OHDA injections were performed in mice anesthetized with isoflurane (pre-operative: 4 l/min, during operation: 0.5-2 l/min isoflurane-air mix v/v) and treated with the anti-inflammatory substances Carpofen (5 mg/ml; Norocarp) and topic Marcain (1.5 mg/kg; AstraZeneca) injected sub- cutaneously. The skull was exposed by incision in the skin of the head and a hole was drilled. Injections contained 1µl 6-OHDA (2.2 mg 6-OHDA-hydro- chloride dissolved in 0.9% NaCl, 0.02% ascorbic acid) or 1 µl sham solution (0.9% NaCl, 0.02% ascorbid acid). Injections were made at the coordinates anterior-posterior (AP): -1.20mm, medial-lateral (ML): -1.10mm and dorsal- ventral (DV): -4.75mm, targeting the MFB (Paxinos, George), using a Nano- Fil syringe (World Precision Instruments, Saracosta, FL, USA) at 100 nl/min. After the surgery, mice were given 1 ml saline. Carpofen was administered on the day after (20-24h post-surgery). Solution were prepared 24h before surgery.

Figure 9. Illustration of stereotaxic injection of 6-OHDA in the MFB. MFB, median forebrain bundle; AP, anterior-posterior; ML, medial-lateral; DV, dorsal-ventral. Bregma 1.20 mm. Illustration by B. Vlcek

39 Post-operative care Every day after 6-OHDA injection and previous to sacrifice, mice received 1ml of body-temperature saline. All animals were weighted and provided with solution (15%) and sucrose moistened pellets. Additional nutritionally forti- fied water and sunflower seeds were also provided.

Tissue preparation for histology Brains used for fluorescent in situ hybridization (FISH) were quickly dissected from animals sacrificed by cervical dislocation, snap-frozen in cold (-30 to - 35°C) 2-methylbutane (≥99%, Honeywell) and kept at -80°C until cryosec- tioning at 16µm thick coronal sections on glass slides. Brains used for immunohistochemistry were dissected from animals anesthetized with isoflurane (Baxter 4%), transcardially perfused with 25ml phosphate-buffered saline solution (1x PBS) followed by 25ml 4% formaldehyde (FA). After dissection brains were post-fixed in 4% FA for 4h, rinsed with 1x PBS and sectioned on a vibratome at 60µm thick coronal sections. Sections were kept at 4°C free- floating in 1x PBS with 0.01% sodium azide until further use.

In Situ Hybridizzzation In situ hybridization is a histological method that allows detection of mRNA by a probe in a tissue of choice, thus allowing the analysis of gene expression. In situ hybridization can be performed on tissue sections such as mouse brain sections, whole mount tissue such as zebra fish or mouse embryos, but also in cell cultures, allowing visualization of gene expression in specific cells groups (Matt Carte and Jenniffer Shieh). There are different types of probes that can be used for in situ hybridization, such as oligo, DNA and RNA probes (riboprobes), which can be labelled with either radioactive labels or non-radioactive labels such as digoxigenin (DIG) and fluorochromes. Riboprobes are known to have a higher sensitivity then the other two types and together with non-radioactive labels are preferred for achieving fast results with excellent cellular and subcellular resolution (Corthell, 2014). The main property of a probe is that it is single-stranded and complementary to the sequence of interest allowing it to hybridize with the target mRNA forming a labeled double- stranded structure in which selected nucleotides are labeled (chromogenic, fluorescent or radioactive) which allows their visualization using either bright field or fluorescent microscopy.

40 Synthesis of PCR Products and Riboprobes The sequence of the gene of interest encoding the mRNA to be detected is first identified so that the probe and primers can be designed. This can be done by using the gene database on the National Center for Biotechnology Information website. A plasmid containing the DNA sequence encoding the mRNA of interest is produced (Figure 10A). This DNA sequence is amplified by the polymerase chain reaction (PCR) using primers that contain promoter sequences for RNA polymerase, which enables in vitro transcription in the next step (Fig- ure 10B). After the amplification step, the PCR products are purified in order to isolate the target DNA fragments (Figure 10C). The amplified DNA is subsequently used as template for synthesis of the probes by in vitro transcription using T3 or T7 RNA polymerase. In the protocols used here, T7 RNA polymerase was used to generate the antisense probe (which binds to mRNA of interest), and T3 RNA polymerase was used to generate the sense probe (negative control) (Figure 10D). In the in vitro transcription process, selected nucleotides carry the molecule which allows detection of the probe. In the current studies, this was DIG and fluorescein that allowed the mRNA of interest to be visualized in the cells of brain sections (Figure 10E).

Figure 10. Different steps in the synthesis of PCR products and Probes. A Plasmid containing the DNA sequence coding for the mRNA of interest. B PCR reaction with DNA polymerase and primers containing the sequence for the T3 and T7 promoters for T3 and T7 RNA polymerase recognition. C PCR products synthesized by DNA polymerase. D Synthesis of antisense probe using T7 RNA polymerase. E Probes synthesized and labeled. Illustration by B. Vlcek

42 Protocol: Generation of probes Sylvie Dumas (Oramacell, Paris, France) provided DNA plasmids for each mRNA of interest. Synthesis of PCR products from plasmids was performed using a mix of T3 and T7 primers (10µM each) of sequence specified for each probe, and Redextract PCR mix (Sigma). Amplification was verified by gel electrophoresis and purification of products was done using a QIAquick PCR purification kit (Qiagen). In vitro transcription was performed in a mixture of 5x transcription buffer, 100mM DTT, 20U/µl Rnasin and 20U/µl T7 (or T3) RNA polymerase (Promega), 10x DIG- or Fluorescein-RNA labelling mix (Sigma). Riboprobes were purified using Illustra probequant G-50 micro col- umns (Sigma) and stored in hybridization buffer containing formamide at - 80°C. The primers used for the synthesis of the different probes used in this thesis are listed in Table 1 and Table 2 of the manuscripts for Study II and Study IV, respectively.

Fluorescent In Situ Hybridization (FISH) The FISH protocol used in this thesis was designed by Sylvie Dumas (Orama- cell). The protocol is based on the use fluorescein and DIG-labeled riboprobes (Figure 11A) together with immunohistochemical revelation, where an antibody against fluorescein or DIG, conjugated with a horseradish peroxidase is used followed by tyramide signal amplification (TSA) reagents, which will amplify the signal given by the labels added Figure 11B). Two different TSA reagents were used, one that is conjugated with biotin and one that is conjugated with Cy3. The one conjugated with biotin is further exposed to Neu- trAvidin Oregon green, a biotin-binding protein that is conjugated with Ore- gon green 488 that will emit a green fluorescence color when observed in the fluorescent microscope, while the one conjugated with Cy3 does not need further reaction and will emit a red fluorescent color. If two mRNA probes are detected in the same cell, this will give a yellow fluorescent color.

43 Figure 11. Illustration of the in situ hybridization process. A DIG or Fluorescein- labeled probes are introduced, probes hybridize with the mRNA and Anti-DIG or Anti-Fluorescein Ab’s conjugated with HRP are introduced. B Ab’s detect the epitopes (DIG or Fluorescein labels), TSA + Cy3 is introduced and reacts with HRP emitting red fluorescence. Or TSA + biotin is introduced and reacts with HRP, Neu- trAvidin Oregon-Green is introduced and binds to biotin emitting green fluorescence. Illustration by B. Vlcek. DIG, digoxigenin; Ab, antibody; HRP, horseradish peroxidase

44 FISH Protocol Cryosections were thawed at room temperature, fixed in 4% parafolmade- hyde, acetylated in 100mM triethanolamine acetic anhydride buffer (pH 8) and hybridized for 16-18h at 65°C with a hybridization buffer containing formamide and 75-100ng/100µl of each DIG- and fluorescein-labelled probes per sample. Sections were washed with 5x SSC, 0.2x SSC and MABT buffers. Immunological reactions were performed for each probe incubating sections with a FBS-based blocking reagent followed by incubation with a horse-radish peroxidase conjugated-antibody against fluorescein and one against DIG (Roche). Fluorescein signal was revealed using a TSA + biotin kit (Perkin Elmer), followed by incubation with neutravidin-oregon green. The enzyme was inhibited with 0.1M glycine (pH 2.1) and 3% H2O2. DIG signal was revealed using a TSA + Cy3 kit (Perkin Elmer) followed by incubation with DAPI. 1x PBS with 0.1% Tween-20 was used for washes when necessary. Slides were mounted using fluoromount, scanned with a Hamamatsu Nano- Zoomer 2.0-HT and analyzed using the Hamamatsu Ndp2.view software.

Immunohistochemistry Immunohistochemistry is a histological method that allows detection of proteins in tissue sections.

Two different protocols were used here, chromogenic immunohistochemistry and fluorescent immunohistochemistry. Sections were prepared on vibratome and maintained free-floating throughout the procedure. For colorimetric immunohistochemistry, sections were washed with 1% H2O2 in 1x PBS with 0.1% Triton-x for inhibition of the endogenous peroxidase. Sections were incubated for 90min with 5% goat-serum blocking solution followed by incubation with a primary antibody against TH (Rabbit anti-TH, ab152, millopore) at 4°C over night. Sections were incubated with a biotinylated anti-rabbit antibody (Vectastain ABC kit) for 90min followed by 90min incubation with ABC solution (Vectastain ABC kit), washed with 0.1M Tris-HCl buffer (pH 7.4) and exposed to DAB-nickel solution (DAB peroxidase substrate kit, Vec- tor Laboratories) for 4min. DAB was blocked with Tris-HCl and sections were mounted on glass slides and stained with cresyl violet. Slides were mounted with DPX mounting media (Sigma), scanned and analyzed with NanoZoomer 2.0-HT and Ndp2.view software (Hamamatsu). For fluorescent immunohistochemistry, sections incubated for 3h with 5% normal donkey-serum and 0.3% Triton-x blocking solution followed by incubation with a primary antibody against TH (Mouse anti-TH, MAB318, Millopore) and GFP (Chicken anti- GFP, Abcam, ab13970) at 4°C over night. Sections were incubated for 2h with secondary antibodies against mouse and chicken (Donkey anti-mouse Cy3, Millipore, AP192C and Donkey-chicken A488, JAX ImmunoResearch 703-

45 545-155), followed by 10min incubation with DAPI. Sections were mounted with DABCO mounting media (Sigma), analyzed and photographed using Leica’s CTR6000 microscope. Anatomical analysis was guided by The Mouse Brain In Stereotaxic Coordinates (Paxinos, George).

46 Study I

Aim The primary aim of this study was to identify gene expression patterns that differ between the SNc and VTA in mice, and subsequently to histologically analyze identified genes in mouse brain tissue to define precise location of corresponding mRNA within the SNc and VTA.

A follow-up aim was to address pathway and neurotransmitter release of the herein newly identified VTA subpopulation defined by expression of the Trpv1 gene. Another follow-up aim was to establish the anatomical position of some of the newly identified gene expression patterns in human midbrain sections derived from control and PD biopsies.

Results and discussion In order to establish specific markers representing the heterogeneity in the midbrain dopamine system, an unbiased microarray analysis comparing gene expression patterns of the VTA and SNc were performed. This was followed by histological analysis where patterns of expression for markers of interest were established. A restricted subpopulation of neurons identified represented by expression of the Transient receptor potential cation channel subfamily V member 1 (Trpv1) gene in the medial VTA was further investigated in order to establish target areas as well as neurotransmitter phenotype of this subpopulation. Selected genes identified in the screening were further analyzed in human brain tissue from controls and PD patients.

Microarray analysis was performed using whole cell RNA prepared from tissue of the VTA and SNc from postnatal day 3 (P3) DAT-CretdTom mice. Top candidate genes were selected for histological analysis. First, low-resolution in situ hybridization using radioactive oligo probes on brain sections from P3 C57BL/6J mice revealed genes restricted to either the SNc or the VTA. Among these Sine-oculis-related homeobox-3 (Six3) was restricted to the SNc while Grp, Trpv1, Calb1, Ntf3, Tachykinin-receptor-3 (Tacr3), NeuroD6 and Follistatin (Fst) were restricted to the VTA.

Analyses of the differentially expressed genes in adult C57BL/6J mice revealed interesting patterns. NeuroD6 and Grp mRNAs are restricted to the

47 ventromedial aspect of the VTA. Calb1 and Calb2 mRNAs have almost op- posite patterns of expression. Tacr3 and Ntf3 mRNAs were most prominent in the PBP/VTAR areas, but also in the SNc. Trpv1 mRNA was restricted to the medial VTA and appeared very weak in adult tissue, in accordance with previous studies (Cavanaugh et al., 2011). Girk2 mRNA was strong in the SNc and lateral VTA, according to a recent publication (Reyes et al., 2012). The Six3 riboprobe appeared non-selective and was removed from further analysis. All mRNAs co-localized with Th mRNA, while Ntf3 also showed co-localization with Viaat, suggesting it could be a potential marker for GABA or dopamine/GABA neurons of the VTA. Figure 12 illustrates some examples of the findings.

Figure 12. Illustration of the pattern of distribution of markers (mRNA) identified. A Calb2, B Girk2, C Grp, D NeuroD6, E Trpv1 and F Calb1. Illustration by B. Vlcek

To assess functionality of the newly identified Trpv1-population in the medial aspect of the VTA, Trpv1-CretdTom mice at the age of P3 were histologically analyzed. The results showed highly similar patterns between the Trpv1-Cre reporter and endogenous Trpv1 mRNA. The majority of Trpv1-Cre-positive neurons were found positive for Vglut2 mRNA and many where positive for

48 Th, including a medial-dorsal part of the PBP which we defined here as subzone of the PBP, the so called szPBP. Stereotaxic injection in the VTA with a viral-vector carrying a floxed construct encoding Channelrhodopsin (ChR2) conjugated with eYFP (rAAV-ChR2-eYFP) in Trpv1-Cre mice was performed. Analysis of projection patterns by eYFP-fluorescence showed that Trpv1-Cre neurons project to the lateral septum and medial NAcSh. Electro- physiological recordings revealed glutamatergic post-synaptic currents upon optogenetic stimulation.

To assess the levels of translation between mouse and humans in terms of the differentially expressed genes, TRPV1, GRP, NTF3, TH and VGLUT2 mRNA were analyzed in human brain biopsies encompassing the SN and VTA. NTF3 was not detected at all. TRPV1 and VGLUT2 were both weakly detected in the matrix, A8 and Mv regions, while TH was detected throughout the SN, A8, medial and Mv areas. GRP was found mainly in the SNpd, A8 and Mv, similar to our results in the mouse. In brain samples derived from PD patients, VGLUT2 and TRPV1 could barely be detected. TH was considerably decreased compared to controls, confirming dopamine neuron pathology in these patients. Interestingly, GRP distribution was strikingly preserved, where most GRP-neurons were non-melanized neurons of both VTA and SNc. Pre- vious studies have proposed neuroprotective properties of GRP. For example, in cultured cells over-expressing the Snca gene, and treated with MPTP, addition of GRP decreased cell vulnerability (Chung et al., 2005). GRP is a potential neuroprotective factor in PD. This factor might be important considering the higher resistance of VTA dopamine neurons to degeneration in PD.

In summary, this study identified a number of genes expressed at higher levels in the VTA than in the SNc in the newborn mouse. Careful histological analysis in brain sections allowed the precise localization of these newly found mRNAs in distinct neurons within the different VTA subareas. By identifying and defining subpopulations of dopamine neurons within the VTA, neurocircuitry and behavioral roles can be explored, as well as putative roles in disease. Here, this was exemplified by the use of optogenetics in Trpv1-Cre mice, which allowed the identification of a mesoaccumbal glutamatergic pathway defined by Trpv1. In terms of PD, histological analysis of human brain biopsies verified the loss of TH mRNA, and revealed the presence of GRP-positive dopamine neurons of non-melanized identity.

49 Study II

Aim The aim of this study, which was a direct follow-up of Study I, was to anatomically and functionally analyze the newly identified VTA subpopulations defined by NeuroD6 and Calbindin 2 (Calb2), respectively.

Results and discussion Different histological methods were applied for characterizing the pattern of expression, neurotransmitter phenotype and projection targets of neurons represented by NeuroD6 or Calb2 expression. In order to understand how the NeuroD6 subpopulation is involved in behavior, a new conditional knockout (cKO) mouse line was generated by crossing NeuroD6-Cre mice with Vmat2- Lox mice. The idea was to ablate NeuroD6 VTA neurons of their capacity to release dopamine. The cKO mice along with littermate control mice were assessed in different behavioral paradigms. To further understand how NeuroD6 VTA neurons contribute to behavior, optogenetics was implemented using NeuroD6-Cre mice. Also Calb2-Cre mice were also analyzed by optogenetics. DAT-Cre and Vglut2-Cre mice were included as controls, given their previous characterization. All mice were injected into the VTA with rAAV-ChR2- eYFP virus and fiberoptics was placed in the same area. All Cre mice were analyzed in different behavioral assays, and results compared.

Histological analyses in C57BL/6J mice confirmed the expression pattern identified in study I. NeuroD6 mRNA distribution was found mainly in the ventromedial VTA, where it co-localized with Th. Co-detection analysis further revealed some co-localization of NeuroD6 with Vglut2 mRNA, suggesting a small glutamate-dopamine co-releasing subpopulation within the Neu- roD6 subpopulation. Calb2 mRNA showed a broader distribution than Neu- roD6 within the VTA, but also modest distribution in the SNc. Calb2 showed 50% co-localization with Th, less co-localization with Viaat and very little co- localization with Vglut2. In addition, NeuroD6 mRNA showed partial co-localization with Calb2. These results show that the Calb2 subpopulation is not just broader anatomically, but also more heterogeneous than the NeuroD6 subpopulation, which is a restricted subpopulation of dopamine neurons.

50 Lack of Vmat2 mRNA in NeuroD6-Cre neurons in the newly produced cKO mouse line was confirmed by in situ hybridization. Results from immunohistochemistry using a TH antibody in brain sections suggested that gross anatomy of dopamine and noradrenaline systems are normal, set aside the loss of Vmat2 in NeuroD6-neurons.

Immunohistochemical analysis of eYFP and TH in the VTA of the different Cre-lines injected with rAAV-ChR2-eYFP enabled the analysis of projection patterns. As expected, DAT-Cre control mice had a strong labelling within the whole VTA, and projections positive for eYFP were dense in all striatal areas, in accordance with previous publications (Pascoli et al., 2015; Stuber et al., 2010). Patterns for the Vglut2-Cre projections also matched previous findings, with projections innervating the NAcC and OT (Hnasko et al., 2012; Qi et al., 2016; Yoo et al., 2016). Calb2-Cre and NeuroD6-Cre showed striking projection patterns originating from the VTA. Calb2-Cre neurons showed a similar labeling within the VTA as the DAT-Cre neurons, but with sparser density. Calb2-Cre-projections were few and restricted to the OT. In contrast, Neu- roD6-Cre neurons showed only restricted labeling in the VTA, in accordance with the restricted pattern of NeuroD6 mRNA. Projections from these sparse neurons were weakly detected in the NAcSh and OT.

In the behavioral context, ablation of dopamine signaling from the NeuroD6- Cre VTA neurons altered the animal’s response to amphetamine, cocaine and alcohol, while the response to sugar remained normal. This finding identified NeuroD6 dopamine neurons as critically important for the normal function of the brain reward system. While it is long known that VTA dopamine neurons indeed are crucial for the reward system, this finding identified the highly restricted NeuroD6 dopamine population, located within the medial aspect of the VTA, as an important player within larger VTA context.

Optogenetic stimulation of DAT-Cre and Vglut2-Cre VTA neurons, used as controls, confirmed previous results. DAT-Cre VTA neurons led to significant place preference, while the same stimulation of Vglut2-Cre mice caused place avoidance. Interestingly, optogenetic stimulation of NeuroD6-Cre VTA neurons, but not Calb2-Cre VTA neurons, was sufficient to drive significant place preference. The response was similar, albeit weaker, as when the whole dopamine system was activated using DAT-Cre mice. This was an important revelation as it shows that the NeuroD6 VTA subpopulation alone is sufficient to drive significant place preference behavior, an important aspect of goal-directed behavior in which dopamine neurons are known to play a critical role.

51 In summary, this study identified that a small NeuroD6-positive subpopulation within the medial aspect of the VTA possesses sufficient capacity to regulate distinct aspects of behaviors associated with the large and complex mesolimbic dopamine system.

52 Study III

Aim The aim of this study was to establish how the VTA and SNc are affected in two established transgenic mouse models of PD representing brain-specific expression of the human SNCA gene, encoding the protein αSyn.

Results and discussion The presence of αSyn pathological aggregates is a hallmark of PD. Several models aiming to mimic this pathology in mice show PD-like symptoms, including motor and non-motor phenotypes. However, not all of these models induce loss of dopamine neurons in the SNc, which is the main hallmark of clinical PD (Fernagut and Chesselet, 2004; Lee et al., 2012; Williams-Gray and Worth, 2016b). The correlation between αSyn pathology and the integrity of the midbrain dopamine system has remained poorly explored.

In this study, two transgenic mouse lines expressing the human SNCA gene under control of the brain-specific Thy-1 promoter were histologically analyzed by FISH and immunohistochemistry. The first mouse line is the Thy1- h[A30P]αSyn in which the A30P mutation has been introduced into the human SNCA gene. This model is also referred to as “A30P”. The second mouse line is the Thy1-h[wt]αSyn, also referred to as L61. The SNCA is here the wild type human gene. Both mouse lines have been extensively characterized in terms of biochemical and behavioral properties in several previous publications.

For analysis of the SNc and VTA using FISH in these mouse lines, A30P mice were 16-18 months old while L61 mice were 5 months, as this line is considered to have a more aggressive phenotype (Fernagut and Chesselet, 2004). For immunohistochemistry experiments, A30P mice of the same age were used, while for the L61 line older mice (9-11 months) were used. A probe towards the rat Snca mRNA was used for analysis of distribution of mouse and human mRNA. This was possible due to high sequence homology between these species. Probes towards Th, Vmat2, Aldh1a1, Girk2, Calb1, Calb2 and Grp mRNA were used for addressing the dopamine neurons, while a probe towards Vglut2 was used for addressing the glutamate neurons of the VTA and SNc. Probes towards the Vesicular glutamate transporter 1 (Vglut1) and Viaat

53 mRNAs were used for addressing glutamate and GABA neurons of the cortex, respectively (Fei and Krantz, 2009). Chromogenic immunohistochemistry using antibodies against TH and GFAP was performed for addressing potential differences in the dopamine system and inflammatory processes, which are known to occur in PD (Rocha et al., 2018).

Results from FISH analyses showed readily detectable Snca mRNA throughout the cortex, hippocampus, SNc and VTA in the control mice. In the A30P and L61 mice, Snca/SNCA mRNA was strongly detected throughout the whole brain, much more prominent labeling in all the areas was observed. Also additional areas positive for Snca/SNCA mRNA in the transgenic mice were revealed. Minor differences between the two lines was observed. Results from analyses of the dopamine markers and Vglut2 in control mice from both lines were in accordance with the literature. Th and Vmat2 were strong throughout the SNc and VTA co-localizing extensively, Aldh1a1 mRNA was strong in the ventral SNc and in the ventral aspect of the VTA, while Girk2 mRNA was strong in the SNc but also in the lateral VTA. Calb1was strong in the VTA and Calb2 mRNA was present throughout the VTA and SNc, but weaker in the medial VTA. In addition to Vmat2, all markers co-localized extensively with Th, while Vglut2 mRNA was strong in the medial VTA, also according to the results from Study I (Anderson et al., 2011; Bimpisidis and Wallén-Mackenzie, 2019; Reyes et al., 2012). When analyzing the A30P and L61 mice, the same results were obtained, suggesting that the midbrain dopamine system is intact in these transgenic mouse lines. Results from the analyses using cortical markers showed intact cortical histology in both A30P and L61 mice.

TH immunohistrochemistry results confirmed FISH results for the A30P mice, possibly contradicting a previous study (Ekmark- Lewén et al., 2018), but supporting earlier publications (Kahle et al., 2000; Neumann et al., 2002a). However, individual differences between mice of the same genotype has been observed which makes conclusions difficult.

Interestingly, while normal histology of the dopamine system in A30P and L61 mice in most analyses, aged L61 mice showed a different result. De- creased TH immunoreactivity was detected in three out of six mice analyzed. This finding suggested that αSyn pathology in L61 mice may affect the dopamine system in a progressive manner. It would be of interest to address L61 mice at this age with FISH using the probes assessed in the younger mice in order to identify how the VTA and SNc are affected at this age. In addition, it would be of interest to address younger mice with TH immunohistochemistry to validate if differences are due to age.

54 GFAP immunohistochemistry showed increased immunoreactivity in the brainstem of A30P mice, thus, suggesting that αSyn pathology in this line may lead to neuroinflammation, a process known to preceded dopamine neuron death in PD (Rocha et al., 2018). GFAP immunohistochemistry in the L61 mice showed increased immunoreactivity in the brainstem of the mice that also showed decreased TH immunoreactivity, suggesting a correlation between possible dopamine neuron depletion and potential neuroinflammation in these mice.

In summary, this study shows that two different mouse lines expressing the human SNCA gene throughout most parts of the brain still maintains normal distribution of a range of markers for midbrain dopamine neurons. The distribution pattern and amount of SNc and VTA dopamine neurons appear highly normal. Also markers for glutamatergic neurons in the midbrain and cortex appeared normal. This study thereby identifies intact integrity of the midbrain dopamine system in the presence of αSyn pathology.

55 Study IV

Aim The aim of this study was to establish how the VTA is affected in the 6-OHDA mouse model of PD.

Results and discussion In this study, C57BL/6 mice were injected unilaterally in the MFB with the 6- OHDA toxin in order to induce dopamine neuron depletion, mimicking the degeneration that occurs in PD (Blum et al., 2001; Jonsson, 1980). These mice went through specific post-operative cares to increase the survival rate and brains were analyzed histologically using markers identified in study I, as well as other well-established markers of dopamine neurons of the VTA and SNc used in study III.

Mice showed ipsilateral rotation in the home cage confirming the 6-OHDA- induced lesion. The post-operative care provided resulted in a more than 90% survival rate of mice injected with 6-OHDA. Probes selected for this study were those detecting Th, Dat, Vmat2, Aldh1a1, Calb1, Grp, Snca and Vglut2 mRNA. Unilateral lesion of the MFB resulted in unilateral dopamine neuron depletion displayed by a decreased number of neurons detected with the Th probe on the lesioned side, confirming the success of the injections (Boix et al., 2015; Ungerstedt, 1968).

Results from FISH analyses in control mice were in accordance with the literature and with the results from study I and III. Th was strong throughout the SNc and VTA. Vmat2 and Snca were strong throughout the SNc and VTA co- localizing with Th extensively. Dat mRNA was strong in the SNc and lateral VTA co-localizing with Th to a lesser extent in the medial VTA. Aldh1a1 mRNA was strong in the ventral SNc and in the ventral aspect of the VTA, Calb1 was strong in the VTA and modest in the SNc, while Vglut2 mRNA was strong in the medial VTA.

In the 6-OHDA injected mice, Dat-positive neurons were highly depleted in both SNc and VTA, while Aldh1a1-positive neurons were highly depleted the SNc and PBP/VTAR, while neurons in the PN/PIF and IF were modestly depleted. Calb1-positive neurons were almost entirely depleted in the SNc, while

56 about half of them in the PBP/VTAR and PN/PIF areas were spared, and in the IF and RLi most of them were spared. Grp-positive neurons were depleted in the SNc, while in the VTA these neurons were almost entirely, but not com- pletely spared. Vglut2-positive neurons were spared in both SNc and the VTA subregions. These results suggest that the 6-OHDA toxin affects all the subregions of the VTA in addition to the SNc. Dopamine neurons located laterally were more affected by the lesion than those located more medially. Vglut2- positive neurons were spared, an expected finding based on their glutamatergic and not dopaminergic identity. DAT, present in dopamine neurons, is necessary for the toxin to exert its effects (Bimpisidis and Wallén-Mackenzie, 2019; Blum et al., 2001).

In summary, the 6-OHDA-lesion model, presented here in mice, show depletion of dopamine neurons in both the SNc and VTA. This toxin model thereby qualifies as a relevant mouse model for analysis of the contribution of midbrain pathology to behavioral phenotypes within the motor, associative and affective domains.

57 Concluding Remarks

The studies presented in this thesis show that the VTA can be divided into several subpopulations of neurons defined by gene expression profiles, for example Trpv1, NeuroD6 and Grp, among others. These subpopulations likely play different roles in the neurocircuitry and behavioral regulation, as exemplified with the Trpv1-subpopulation which releases glutamate into the ventral striatum (Study I) and the NeuroD6-population which directs place preference in response to optogenetic stimulation (Study II). Further, gene expression patterns helped increase the understanding of how the VTA and SNc structures are affected by pathology in PD where some GRP-positive neurons were found to remain (Study I) and also how well some different PD models in mice represent PD in terms of the VTA and SNc pathology (Study III, IV). While most dopamine neurons degenerate in PD as the disease progresses, no pathology of these neurons was detected in two different mouse lines expressing the human SNCA gene (Study III), while ample loss of both SNc and VTA dopamine neurons was found in the classical 6-OHDA toxin model of dopamine cell degeneration (Study IV).

Future studies would be of interest to place the results obtained in Studies III and IV in a broader context of additional mouse models that are commonly used to analyze mechanisms and pathology of PD. In addition, functional studies similar to Study I and II would be of interest in order to reveal any behavioral roles exerted by the newly identified VTA subpopulations in the large spectrum of dopamine-related behaviors and disorders.

58 Acknowledgements

During these years as a PhD student, I have learned so much that it is difficult to describe in words. I can say that I grew a lot, not only as a scientist but also as a person. I have met some cool people who I had the opportunity to share experiences with both in the scientific and social context. Therefore, I would like to take the opportunity to thank these people here.

First, I would like to thank my main supervisor Prof. Åsa Wallén-Mackenzie, who made it all possible. Thank you for the great opportunity of doing my PhD studies in your lab and for all the support that you have given me through these years. For encouraging me to do my best and showing me the amazing world that is the study of brain histology. Thank you for teaching me everything about in situ hybridization and histological data analyses, but also for letting me to try different methods, and for always helping me to improve my skills in the lab and in scientific communication. Thank you for the work en- vironment that you have provided, it was a pleasure working with you during these years. Thank you for believing in me, I could not have done this without you.

Thank you, Sylvie Dumas, for teaching me your FISH protocol, and for together with Åsa providing me with the tools necessary to succeed in my experiments. Thank you for all the great discussions, your support and for showing me the best side of Paris.

I also want to thank my dear colleague and friend Adriane Guillaumin, for being my PhD partner and for everything that you have thought me. Thank you for all the discussions and for always showing interest when I wanted to talk about research and interesting papers. You gave me confidence to believe in myself even when it was difficult. Thank you Gian Pietro Serra, my co- supervisor, for always encouraging me to ask for questions and all the support you have given me. I would also like to thank the previous members of the Mackenzie lab, especially Zisis Bimpisidis and Niclas König who I have worked close with. Thank you for all the discussions that we had about science and life in general, and for your support. Thank you Hanna Pettersson for teaching me how to plan my experiments and for all the support that you gave in the beginning of my PhD. Thank you Maria Papathanou, Thomas Vi-

59 ereckel, Nadine Schweizer and Åsa Konradsson-Geuken, who were members of the team when I joined and who made me feel welcome. Besides research, I also co-supervised some master and bachelor students in the lab, so I would like to thank Ross McLeod, Ellinor Betnér and Yu Zhang for the time that we worked together. Thank you also Tatjana Haitina, my co-supervisor, for always being so kind and friendly and for interesting discussions.

I also want to thank the people who are essential for the work that we do in the Mackenzie lab, Ulrika, Martina, Jenny and the rest of the special unit members. Thank you for taking care of our animals and for always being help- ful and kind.

Thank you, Prof. Martin Ingelsson for agreeing to our project on the alpha- synuclein transgenic mice and for all the fruitful discussions. Thank you Sara Ekmark-Lewén for your participation and together with the rest of In- gelsson’s team members for the interesting discussions.

A special thank you to the Zoological foundation that has provided me with grants so that I could go on conferences for presenting my research and meet- ing other scientists in the field. It was a great opportunity to learn and get more involved in research. Thank you also for the great lectures and dinners that you have provided and for inviting my partner and me to attend.

Thank you Prof. Irene Söderhäll as head of the department for welcoming me to be a part of the IOB departmental board and for always encouraging me to ask about how things work. Thank you for all the help that you have provided for the orderings and inventories and for always being kind and friendly. Thank you Prof. Monika Schmidt and Prof. Elena Jazin for always being kind and friendly, especially during the courses that you kindly invited me to teach. I also want to thank the current head of the department, Johan Ledin, who also did a great job as student coordinator. Thank you to the current student coordinator, Carina Schlebusch and to the administrative staff, Karin, Iva, Sandra and Yvonne, for making sure that the department works smoothly. Thank you Yvonne, for teaching me how to work with Raindance and for helping me sorting the bills of the lab. Thank you to everyone at In- tendenturen and at the IT department for your technical support. Thank you members of IBG for helping me with teaching matters, especially Banafsheh and Peter for all your help with preparing the labs.

A big thank you to my close friends from the Comfys squad Manolis and Gizem, for always being there for me. My PhD experience would not have been the same without the two of you. Thank you Cecile, Oskar, Laura, Martin, Hannah, Imke, Jake, François, Valeria, Philipp, Kingkamon,

60 Ratchanok, Chadanat, Luciana, João, Lore, Mahwash, Vincent, Moham- med, Sophie, Melanie, Dennis, Jordi, Daniel, Marie, Ehsan, Chrysa, Therese and Beata for all the time that we have spent together during these years and for all the interesting discussions.

I want to thank my family, especially my mom (Anna Vlcek), my brother (Martin Vlcek) and my uncle and godmother (Leif och Kinna), for all the support and interest that they have shared in my life as a PhD student. Thank you!

Thank you to my best friends, Lewar, Karro, Annie, Dilan, Elli, Shilan and Sabina for all your support, curiosity and for putting up with my nerd talk.

Finally, I would like to thank the best partner a young scientist could ask for, Sebastian. Thank you for your patience and kindness, for understanding and supporting my dreams. Thank you for being proud of what I have accom- plished and for always taking my side. These past 5 years would not have been as happy without you and our baby cats Tiger and Brooklyn by my side. Also, a big thank you to your family and friends who are the kindest and most sup- portive.

Thank you!

Tack!

Obrigada!

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