Positive Allosteric Modulators of the Alpha7 Nicotinic

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Positive Allosteric Modulators of the Alpha7 Nicotinic Acetylcholine Receptor Potentiate Glutamate in Prefrontal Cortex: In Vivo Evidence for a Novel Class of Schizophrenia Treatments

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

David Michael Bortz

Graduate Program in Psychology

The Ohio State University

2015

Dissertation Committee:

Dr. John Bruno, Advisor

Dr. Rene Anand

Dr. Kathryn Lenz

Dr. Gary Wenk

Dr. Howard Gu

Copyrighted by

David Michael Bortz

2015

Abstract

The cognitive deficits of schizophrenia are the core symptom class of the disease, but they remain largely untreated by current pharmacotherapeutic strategies. Initiatives by the NIMH, such as MATRICS, have highlighted the alpha7 nicotinic acetylcholine (alpha7) receptor as a leading target for the development of novel cognition-enhancing treatments due to its unique ability to modulate many key executive function-related neurotransmitter systems, its theorized role in the portrayal of the cognitive symptoms of schizophrenia, and its agonist’s ability to reverse such deficits in preclinical models of schizophrenia.

However, clinical trials conducted with several alpha7 agonists have produced mixed results. Some have theorized that these heterogeneous results in clinical trials may be caused by the indiscriminate, temporally- disengaged activation produced by direct agonists. Positive allosteric modulators (PAMs) potentiate afferent signaling without carrying any intrinsic activity, thus enhancing receptor function without mistimed, false signals. This mechanism may be more conducive to improving executive functioning, but there is a paucity of data to support this claim. Early studies in vitro indicate that alpha7 PAMs do indeed potentiate excitatory post-synaptic potentials without directly activating receptors, but no studies have tested this in vivo. The purpose of this project was to address this need by determining if two novel alpha7 PAMs would be able to

ii potentiate glutamate release in PFC (as measured by the glutamate-sensitive microelectrode array; MEA) as a function of and dependent upon afferent stimulation. This experiment required an assay where glutamate release in the

PFC was driven by afferent activation (choline) at the site where the PAMs are active (the alpha7 receptor). The mesolimbic stimulation assay, which involves stimulating the shell of the nucleus accumbens (NAcSh) with NMDA and had previously been shown to result in the dose-dependent release of glutamate in

PFC (Bortz et al., 2014), was shown to also produce dose dependent increases in choline in PFC (chapter 3). Additionally, the dose-dependent glutamate increases are driven locally in PFC via cholinergic activation of the alpha7 receptor (chapter 4). This data confirmed that the mesolimbic stimulation assay could be used to characterize the potentiating effects of two alpha7 PAMs. The results presented in this dissertation demonstrated that both type I (AVL3288) and II (PNU120596) PAMs were able to potentiate glutamate release in the PFC.

Additionally, the degree of potentiation of both PAMs interacted with the level of

NMDA-NAcSh stimulation (i.e. the amount of choline in the PFC, see chapter 3) and the type of PAM, and neither PAM produced any change in PFC glutamate levels in the absence of an afferent signal that increased basal choline levels.

Collectively, these findings demonstrate the usefulness of the MEA and mesolimbic stimulation assay to study novel alpha7 agonists and PAMs; as well as confirm, for the first time, that alpha7 PAMs operate in vivo in a similar fashion as in vitro. In conclusion, this data supports the notion that alpha7 PAMs may be

iii more effective treatments for the cognitive deficits of schizophrenia than agonists and should be further investigated in preclinical and clinical trials.

Acknowledgments

I would like to express my utmost gratitude to Dr. Bruno for all of the knowledge and support he has given me over the last 5 years. You have set me up for success and I could not have asked for a better advisor.

I would like to thank my other colleagues who have taught me techniques and provided additional guidance through this process:

Julie Brooks

Katie Alexander

Asa Konradsson-Gueken

CR Gash

Michelle Pershing

Sarah Vunck

I would also like to thank all the undergraduates who helped me in performing these experiments throughout the years, especially:

Brian Upton

Seva Khambadkone

Jackson Schumacher

Vita

May 2005 ...... Steubenville Catholic Central High School

June 2009 ...... B.S. Psychology, Ohio State University

May 2012 ...... M.A. Psychology, Ohio State University

March, 2014 to present ...... Graduate Candidate, Psychology, Ohio

State University

Publications

Bortz, D.M.; Mikkelsen, J.D.; Bruno, J.P.(2013) Localized infusions of the partial alpha7 nicotinic receptor agonist SSR 180711 evoke rapid and transient increases in prefrontal glutamate release. Neuroscience 255, 55-67.

Bortz, D.M.; Jorgensen, C.V.; Mikkelsen, J.D.; Bruno, J.P. (2014) Transient inactivation of the ventral hippocampus in neonatal rats impairs the mesolimbic regulation of prefronatal glutamate release in adulthood. Neuropharmacology 84, 19-30.

Pershing, M.; Bortz, D.M.; Pocivavsek, A.; Fredericks, P.J.; Jørgensen, C.V.; Vunck, S.A.; Leuner, B.; Schwarcz, R.; Bruno, J.P. (2015) Elevated levels of kynurenic acid during gestation produce neurochemical, morphological, and cognitive deficits in adulthood: implications for schizophrenia. Neuropharmacology 90, 33-41.

Fields of Study

Major Field: Psychology vi

Table of Contents

Abstract ...... ii

Acknowledgements ...... v

Vita...... vi

List of Tables ...... viii

List of Figures ...... ix

Chapter 1: Introduction ...... 1

Chapter 2: General Methods ...... 20

Chapter 3: Mesolimbic Regulation of Prefrontal Choline Release ...... 25

Chapter 4: Mesolimbic Regulation of Glutamate Release in PFC is Mediated by Local

Alpha7 Nicotinic Acetylcholine Receptor Activation ...... 47

Chapter 5: Positive Allosteric Modulators of the Alpha7 Receptor Potentiate

Mesolimbically-Stimulated PFC Glutamate Release Dependent Upon Afferent

Activity ...... 75

Chapter 6: General Discussion ...... 113

References ...... 136

Appendix A Tables and Figures ...... 160 vii

List of Tables

Table 1. Means and standard errors for chapter 4 ………………………………161

Table 2. Means and standard errors for chapter 5 ………………………………162

viii

List of Figures

Figure 1. Schematic representation of the distributed neural system believed to control executive functioning ……………………………………….………...……163

Figure 2. Diagram describing MEA signal transduction scheme and organization

….....………………………………………………………………………………...…164

Figure 3. Representative in vitro calibration of the choline MEA …………....…165

Figure 4. Representative photomicrograph of the regions within the PFC and the

NAcSh where the choline MEA and infusion cannula were situated ……….…166

Figure 5. Representative tracings describing the dose-dependent release of

Choline following NMDA stimulation of the NAcSh …………………………..…167

Figure 6. Group data describing the dose-dependent release of Choline following

NMDA stimulation of the NAcSh ……………………………………………...... …169

Figure 7. Representative in vitro calibration of the glutamate MEA ……………171

Figure 8. Expanded view of MEA to identify cannula termination zone ….……172

Figure 9. Representative photomicrograph of the regions within the PFC and the

NAcSh where the choline MEA and two infusion cannulae were situated.…….173

Figure 10. Representative tracings describing the dose-dependent inhibition of glutamate by MLA following NMDA stimulation of the NAcSh ……………….…174

Figure 11. Group data describing the dose-dependent inhibition of glutamate by

MLA following NMDA stimulation of the NAcSh …………………………….……176

Figure 12. Representative tracings describing the inhibition of glutamate by MLA, but not DHE, following NMDA stimulation of the NAcSh ………………………177

Figure 13. Group data describing the inhibition of glutamate by MLA, but not

DHE, following NMDA stimulation of the NAcSh..……………………………....179

Figure 14. Representative photomicrograph of the regions within the PFC and the NAcSh where the glutamate MEA and infusion cannula were situated ..…180

Figure 15. Representative tracings comparing the difference in degree of potentiation by AVL3288 (1mg/kg) following NMDA stimulation of the NAcSh..181

Figure 16. Representative tracings comparing the difference in degree of potentiation by AVL3288 (3mg/kg) following NMDA stimulation of the NAcSh..183

Figure 17. Group data comparing the difference in degree of potentiation by

AVL3288 following NMDA stimulation of the NAcSh ……...... …..185

Figure 18. Representative tracings comparing the difference in degree of potentiation by PNU120596 (3mg/kg) following NMDA stimulation of the

NAcSh…………………………………………………………...……………………187

Figure 19. Representative tracings comparing the difference in degree of potentiation by PNU120596 (9mg/kg) following NMDA stimulation of the NAcSh

……………………………….…………………………………………...…..…..……189

Figure 20. Group data comparing the difference in degree of potentiation by

PNU120596 following NMDA stimulation of the NAcSh ………………………..191

Figure 21. Flouro-Jade stain of an NMDA infusion cannula track ………...... 193

CHAPTER 1

INTRODUCTION

1.1 Schizophrenia

Schizophrenia is a devastating psychological disorder that is a very serious world health problem for a number of reasons. First, it occurs in approximately 1% of the world’s population, which was about 24 million worldwide as of 2002 (Lewis & Lieberman, 2000). Second, it decreases life expectancy by 10-25 years, due mainly to substance abuse comorbidities and a

10% lifetime incidence of suicide (Lewis & Lieberman, 2000). Third, it accounts for billions of dollars- worth of healthcare costs and lost productivity, annually.

Finally, the core symptoms of the disorder remain largely untreated (Elvevag &

Goldberg, 2000), which is a major burden on both patients and their caretakers.

As such, it has commanded much attention and research in the mental health industry, including an initiative put forth by the NIMH called MATRICS

(measurement and treatment research for the improvement of cognition in schizophrenia; Green et al., 2004).

Schizophrenia, which was first characterized in the late 1800s by Emil

Kraepelin, occurs in both men and women at approximately the same rate, but some reports indicate a slightly higher incidence in men (Picchioni & Murray,

2007). The first psychotic episode typically occurs when the patient is between

18 and 30 years old, with average age of onset being earlier in that range for men and later for women. The cause of schizophrenia is largely unknown, but it is believed to have a strong genetic component. In fact, a recent meta-analytic study showed a heritability of 81% for schizophrenia, with concordance rates being as high as 45% for identical twins and 14% for dizygotic twins (Sullivan et al., 2003). Environmental risk factors also play a major role in the development of schizophrenia. For example, growing up in an urban environment, experiencing childhood abuse, being socially isolated, maternal infection, experiencing negative life events, using cannabis during adolescents, as well as several other things have all been shown to increase the risk for developing schizophrenia (see

Fatemi & Folsom, 2009 for review). However, while many genetic and environmental risk factors have been identified, few, individually, increase risk by more than a couple of percent. Therefore, it is generally believed that an interaction between several genetic vulnerabilities and several environmental factors leads to the development of schizophrenia, rather than any single one.

1.2 Schizophrenia symptoms and subtypes

The notion that schizophrenia can be developed in a number of different ways via a combination of a number of different factors is supported by the

2 heterogeneous expression of the disorder. For example, the symptoms of schizophrenia are classified into three clusters: positive, negative, or cognitive deficits, with the degree and severity of each varying from person to person

(American Psychiatric Association., 2000). Positive symptoms include auditory and visual hallucinations, delusions, and disorganized speech and behavior

(American Psychiatric Association., 2000). They are the most obvious, external symptoms and are termed “positive” because they represent the addition of characteristics not normally seen in unaffected people. Negative symptoms include flat affect, catatonic behavior, poverty of speech, loss of pleasure in previously pleasurable things, loss of motivation, and decreased desire for social interaction (American Psychiatric Association., 2000). These get their name because they reflect a loss or subtraction of something seen in unaffected people. Cognitive symptoms refer to deficits in executive functions, such as attention, working memory, short and long-term memory, verbal fluency, episodic memory, and cognitive flexibility (Keefe, 2007; Kerns et al., 2008). These symptoms represent the core symptom cluster of the disease (Elvevag &

Goldberg, 2000) because they can be detected during the prodromal period, long before frank psychosis; they can be detected to a less severe degree in first- degree relatives of patients; and their severity is most predictive of the long-term functional outcome of the patient (Gold, 2004).

Another example of the heterogeneous expression of the disease is that schizophrenia is categorized into subtypes based on which symptoms patients show and to what degree. A patient with the paranoid type displays a high

3 degree of auditory and visual hallucinations and delusions, but less disorganization and flat affect. Additionally, the hallucinations and delusions tend to be persecutory or fear- inducing in nature (American Psychiatric Association.,

2000). A patient with the disorganized type does not show paranoia, but shows a high degree of disorganized thought and behavior, typically along with inappropriate affect (mistimed emotionality, such as laughing during a sad moment; American Psychiatric Association., 2000). The catatonic subtype is characterized by long periods of immobility or purposeless movement (American

Psychiatric Association., 2000). Finally, the undifferentiated subtype displays typical positive and negative symptoms without meeting the classification of the other three subtypes, and the residual subtype displays atypically low levels of positive and negative symptoms (American Psychiatric Association., 2000).

1.3 Schizophrenia treatment

The search for effective treatments for this dreaded disease requires an understanding of the underlying neuropathology that produce the symptoms. This understanding has evolved over the last 100 years and so too have the pharmacological treatments. The first theory of schizophrenia was that the disease was caused by excessive dopamine release in the striatum. This theory was brought on by several observations. First, drugs that increase dopamine release, such as amphetamine, produce schizophrenia- like psychoses in normal individuals and worsen symptoms in patients (Lieberman et al., 1987), and drugs that deplete dopamine levels, such as reserpine, reduce psychotic symptoms

(Arnold & Freeman, 1956). Second, drugs that block the dopamine D2 receptor alleviated the positive symptoms of schizophrenia, and the degree of their effectiveness was directly related to their affinity for the D2 receptor (Creese et al., 1976). Third, many PET studies measuring the reuptake of radiolabeled L-

DOPA in the striatum, the bio precursor for dopamine, demonstrated a large increase in L-DOPA re-uptake in schizophrenics (reviewed in Howes et al.,

2015). Fourth, tyrosine hydroxylase, the rate-limiting enzyme for the synthesis of dopamine, was shown to be elevated in the substantia nigra of schizophrenics

(Howes et al., 2013). Both of the previous two studies indicate a much greater capacity for dopamine synthesis in the schizophrenic brain. Fifth and related to the previous two points, six different studies all found evidence of significantly increased striatal dopamine release using radiolabeled raclopride displacement

(reviewed in Howes et al., 2015). The striatal dopamine release was approximately two times greater in patients compared to unaffected people, and the degree of raclopride displacement correlated with the severity of psychotic symptoms (reviewed in Howes et al., 2015).

Based on this data, D2 antagonist antipsychotics have been by far the most prescribed medicine for schizophrenia, and they are quite effective at alleviating positive symptoms in most patients. There are, however, two major problems with this strategy. First, about one third of patients do not respond to traditional D2 antagonist treatments, regardless of the treatment’s ability to decrease striatal dopamine levels, indicating a significant number of patient’s symptoms involve more than a hyperdopaminergic state in the striatum (Mortimer

5 et al., 2010). Second, although studies in both humans (Howes et al., 2009) and rodents (Simpson et al., 2010) indicate that elevated levels of dopamine synthesis and release in the striatum are correlated with worsening negative and cognitive symptoms, D2 antagonist antipsychotics vary in their effectiveness and ability to treat either symptom cluster (Desamericq et al., 2014). In fact, some studies even indicate that traditional antipsychotics may worsen negative and cognitive symptoms (Kim et al., 2013). This is a serious problem because, as mentioned above, it is the cognitive deficits that occur first and are most predictive of patient outcome. Therefore, the search for drug therapies that do overcome these two major problems are critically important and require knowledge of the underlying neuropathology of schizophrenia that goes beyond dopamine dysregulation in the striatum. Current understanding now points to multiple disruptions within a distributed neural system as all playing roles in schizophrenia symptomatology.

1.4 The prefrontal cortex as the core of the distributed neural system

The distributed system mentioned above refers to multiple cortical and sub-cortical brain regions networked via connecting neurotransmitter pathways

(figure 1). This network includes brain regions such as: the ventral hippocampus

(VH; spatial and context information), the ventral tegmental area (VTA; reward processing), the nucleus accumbens (NAc; limbic-motor integrator), the basal forebrain (BF; attentional resources), the medial dorsal thalamus (MDthal; sensory information), the basolateral amygdala (BLA; negative emotionality), and

6 the prefrontal cortex (PFC; cognitive control). It is this system that many believe mediates the executive functions that are deficient in schizophrenia; however, the

PFC is thought to be the central region of this system.

The PFC shares reciprocal connections with the VTA (Sesack et al., 1989;

Hoover & Vertes, 2007), BF (Zaborszky et al., 1997; Luiten et al., 1987), BLA

(Hoover & Vertes, 2007; Gabbott et al., 2005), and MDthal (Groenewegen,

1988); receives a direct efferent projection from the VH (Jay et al., 1992); and sends a direct afferent projection to the NAc (Gabbott et al., 2005). Thus, it is uniquely able to control information flow within the entire system. For example, it was recently demonstrated that the PFC maintains a gating influence over information flow from the VH to the NAc (Belujon & Grace, 2008). This study showed that if the PFC was inactivated (tetrodotoxin), VH-mediated increases in spike probability within the NAc were significantly reduced (Belujon & Grace,

2008). In another study, PFC stimulation evoked EPSPs in 95% of NAc neurons tested as well as triggered spike discharge in 47% of those neurons. On the other hand, VH and BLA stimulation resulted in spike discharge in only 20% and

29%, respectively, of NAc neurons tested (O'Donnell & Grace, 1995). Similarly, stimulation of the PFC led to increases in burst firing in the VTA (Gariano &

Groves, 1988). Changes in burst firing rates in the VTA are known to affect phasic dopamine release in the NAc, which, in turn, gates NAc-Limbic throughput

(Goto & Grace, 2005). Finally, the glutamatergic projection from the PFC to the

BF is known to contribute to activation of the BF corticopetal projections to the cortex (Fadel et al., 2001). Thus, due to its unique position of control, the PFC

7 clearly plays a major role in the proper functioning of this distributed system, making it a new focal point for study in schizophrenia research.

1.5 Glutamate and schizophrenia

Within the PFC, several neurotransmitter systems are highlighted in schizophrenia research. The first is glutamate. Glutamate is a small amino acid and mediates the vast majority of fast synaptic transmission in the central nervous system (Lambert & Kinsley, 2005). Glutamate synapses are formed onto dendrites and other axons, as well as onto astrocytes, microglia, and oligodendrocytes, where release of glutamate increases excitatory post-synaptic currents (Lambert & Kinsley, 2005). Glutamate receptors in the brain can be ionotropic, where they mediate fast opening of ion channels, or metabotropic where they activate intracellular signaling cascades via g-proteins. The most commonly studied ionotropic glutamate receptor is the n-methyl-d-aspartate or

NMDA receptor. This receptor is unique from other glutamate receptors because it allows the influx of calcium when activated (Lambert & Kinsley, 2005). The

NMDA receptor is critical for many higher order cognitive functions, including memory formation and learning, via its role in long-term potentiation (See Gnegy,

2000 for review).

The earliest indication that glutamate played a role in schizophrenia came from the observation that NMDA receptor antagonists produce symptoms in normal individuals and rodents that mirror the positive, negative, and cognitive symptoms of schizophrenic patients (Javitt, 2007; Morgan & Curran, 2006;

Egerton et al., 2008), and worsen symptoms in patients (Lahti et al., 1995). In fact, both the absolute concentrations and the rank-order potency with which a range of compounds induce psychotomimetic effects in humans and animal models conforms to their rank order of potency at the NMDA receptor, and this was not mediated by any effect on dopamine (Javitt & Zukin, 1991; Seeman,

2010). Therefore, NMDA receptor blockade, in itself, was sufficient to produce the schizophrenia-like symptoms. Further investigation into the possibility of an

NMDA receptor dysfunction in schizophrenia revealed polymorphisms in the genes that code both the NR1 and NR2B subunit of the NMDA receptor (Qin et al., 2005; Martucci et al., 2006), as well as alterations in NMDA receptor subunit proteins (Kristiansen et al., 2007) and decreases in kainate receptor binding in the dorsolateral PFC (Konradi & Heckers, 2003). These findings were critical because animal studies have demonstrated that glutamate transmission, especially in the PFC, is necessary for the performance of many higher order cognitive functions, such as cognitive flexibility (Stefani & Moghaddam, 2005b), working memory (Aultman & Moghaddam, 2001), and the top-down control of sustained attention (Parikh et al., 2008). Indeed, schizophrenic patients performing cognitive tasks during fMRI scans show decreased functional activation of the PFC, labeled a hypoglutamatergia, which correlates with a decrement in performance (See Ragland et al., 2007 for review). Taken together, evidence points towards the importance of normalizing glutamatergic transmission in the PFC in treating the cognitive deficits of schizophrenia.

1.6 Acetylcholine and schizophrenia

A second system that has been highlighted in schizophrenia research is the cholinergic system. Acetylcholine (ACh) is a neurotransmitter well known for its action as the primary excitatory transmitter at the neuromuscular junction. In the brain, however, ACh performs a modulatory role where it changes neuronal excitability, alters presynaptic release of neurotransmitters, and coordinates the firing of groups of neurons (Rice & Cragg, 2004; Wonnacott, 1997; Zhang &

Sulzer, 2004; dos Santos Coura & Granon, 2012). The actions of ACh released in the brain are mediated via both ionotropic (nicotinic) and metabotropic

(muscarinic) receptors that are located both pre- and postsynaptically. The nicotinic classes of receptors mediate fast synaptic transmission via a cation channel pore, and are so named because of their binding of the neuro-active substance nicotine (Changeux et al., 1998). They are formed as homomeric or heteromeric assemblies by five subunits (α2-7, β2-4), and their function, kinetics, and location change based on which subunits are present (see Picciotto et al.,

2012 for review).

A role for nicotinic receptors in schizophrenia was also inferred some time ago based on patient’s unique cigarette smoking habits. Not only do about 90% percent of schizophrenic patients smoke, but they smoke more cigarettes per day, inhale more deeply, and smoke their cigarettes to the butt more often than non-schizophrenic smokers (Olincy et al., 1997). This behavior is not seen in other mentally-ill patients or in other people taking the same medications, suggesting this self-medicating practice is unique to schizophrenia and not

10 caused by anti-psychotics (Olincy et al, 1997). A role for nicotinic receptors in schizophrenia was further supported by genome-wide association studies linking polymorphisms in CHRNA7, the gene that codes for the alpha7 nicotinic acetylcholine receptor (alpha7), to an elevated risk for schizophrenia (Stefansson et al., 2008). Post-mortem studies done on schizophrenics also support a role of cholinergic dysfunction in schizophrenia. For example, alpha7 receptor protein expression (Freedman et al., 1995; Court et al., 1999; Guan et al., 1999) and binding (Marutle et al., 2001) are decreased in the schizophrenic reticular nucleus of the thalamus, hippocampus, cingulate cortex, and frontal lobe.

Interestingly, the degree of these reductions correlates to the degree of overall cognitive dysfunction that was seen in the patients (Martin-Ruiz et al., 2003).

Additional studies in animals and humans confirm the importance of these receptor reductions, as cholinergic activity in the PFC is critical for cognitive abilities that are disrupted in schizophrenia, such as selective attention (St Peters et al., 2011) and working memory (Levin & Simon, 1998; Levin et al., 1998).

Likewise, manipulations that reduce cholinergic tone or functioning produce cognitive deficits in rodents that are similar to those in schizophrenia (Alexander et al., 2012), while nicotinic agonists improve cognitive deficits in schizophrenia animal models (Alexander et al., 2012; Brooks et al., 2012; See Freedman, 2014 for further review). Therefore, manipulation of the cholinergic system is another major focal point in schizophrenia research, and nicotinic agonists make up a large proportion of the drugs being tested currently in clinical trials (See

Freedman, 2014 for review).

1.7 Glutamate and acetylcholine interactions in executive functioning

As detailed above, both glutamate and ACh in PFC are necessary for the normal expression of the executive functions that are disrupted in schizophrenia.

Importantly, however, studies have indicated that interactions between the two systems are necessary for portrayal of the abovementioned cognitive functions.

First, Parikh et al. (2008) demonstrated that ionotropic glutamate receptor antagonists, administered directly into the PFC, blocked the choline transients that had previously been shown to be necessary and sufficient for cue detection during an attention task. Through their studies, they purported that glutamatergic neurons from the MDthal synapsed onto the terminals of cholinergic neurons from the BF in the PFC and that this interaction mediated attention through cue- detection (Parikh et al., 2008; Parikh et al., 2010). A second example examining glutamate-ACh interactions in learning and memory comes from a study performed by Li et al. (2013). They initially demonstrated that choline-induced upregulation of LTP, the hypothesized neural mechanism of learning and memory, was NMDA receptor-dependent, and that alpha7 receptors formed coupled complexes with the NMDA receptors, which were responsible for this effect (Li et al., 2013). Next, they demonstrated that disruption of this NMDA- alpha7 coupling produced deficits in the novel object recognition task, a rodent correlate of memory (Li et al., 2013). Third, Alexander et al. (2012) demonstrated that a systemic injection of the alpha7 receptor antagonist kynurenic acid produced a 30% reduction in glutamate levels in PFC. Next, they showed that

12 injections of kynurenic acid also produced deficits in cognitive flexibility with a task known to require NMDA receptor activity in the PFC (Stefani & Moghaddam,

2005a). Finally, they showed that perfusion of the alpha7 positive allosteric modulator (PAM) galantamine, directly into the PFC, reversed the deficits in cognitive flexibility (Alexander et al., 2012). Glutamate-ACh interactions have been shown to be critical for executive functions in many other studies as well

(Sarter, 1994; Verma & Moghaddam, 1996; Stefani et al., 2003; Chan et al.,

2007; Timofeeva & Levin, 2011). Taken together, interactions between ACh and glutamate, particularly in the PFC, are critical for the cognitive functions that are deficient in schizophrenia. Therefore, the search for cognitive-enhancers must focus on receptor targets that link these two systems. A great deal of evidence, as well as the data that will be reported in this document, points toward the alpha7 receptor as one promising drug target that links these two systems. In fact, even the MATRICS, or measurement and treatment research to improve cognition in schizophrenia, initiative put forth by the NIMH has designated the alpha7 receptor as a key target for the treatment of the cognitive deficits of schizophrenia (Buchanan et al., 2007).

1.8 The alpha7 nicotinic acetylcholine receptor as a treatment target

The alpha7 receptor is ubiquitous in the brain, and belongs to the family of nicotinic (ionotropic) ACh receptors. It is formed by five alpha7 subunits and has five potential ligand-binding sites, one between each pair of alpha7 subunits

(Palma et al., 1996). This makes the alpha7 receptor unique from heteromeric

13 nicotinic receptors, as those are made up of both alpha and beta subunits and have only two ligand binding sites (Blount & Merlie, 1989). Sub-maximal binding activates both homomeric alpha7 receptors as well as heteromeric receptors; however, only heteromeric receptors have an increased probability of opening with maximal binding (Williams et al., 2011a). Alpha7 receptors, on the other hand, rapidly desensitize under conditions of saturation. This rapid desensitized state is unique to alpha7 receptors compared to the desensitized state of heteromeric receptors because it is rapidly induced with agonist presentation and readily reversible once the agonist is removed (Papke et al., 2000).

Alpha7 receptors have been highlighted by the NIMH and others as potential cognitive-enhancers to treat schizophrenia for several reasons. The first is because they have the ability to influence many neurotransmitter systems throughout the brain, namely glutamate. Alpha7 receptor stimulation in the PFC has been shown to induce glutamate release (Konradsson-Geuken et al., 2009;

Bortz et al., 2013) via stimulation of receptors that are present both on glutamatergic terminals and cell bodies (Dickinson et al., 2008; Livingstone et al.,

2009), as well as dopamine release through dopamine-glutamate interactions

(Livingstone et al., 2009). Alpha7 receptors also influence glutamate release in the cerebellum (Reno et al., 2004), amygdala (Barazangi & Role, 2001), and hippocampus (Barik & Wonnacott, 2006). Additionally, alpha7 receptors have been shown to enhance the release of ACh, GABA, serotonin, and possibly norepinephrine, typically through presynaptic enhancement of release (reviewed in dos Santos Coura & Granon, 2012). Therefore, each and every

14 neurotransmitter associated with schizophrenia has the potential to be modulated by the alpha7 receptor.

The second reason is alpha7 receptor’s high relative permeability to calcium and ability to initiate calcium-induced calcium release (CICR), which is unique from other nicotinic receptors (Mansvelder et al., 2006; Dickinson et al.,

2008). Alpha7 receptors are typically found on nerve terminals that also contain ryanodine receptors; therefore, calcium that enters the cell via an activated alpha7 receptor is able to induce more calcium release from intracellular stores

(Dickinson et al., 2008; Bancila et al., 2009). This CICR was shown to phosphorylate synapsin-1, which has a well-established role in vesicle tethering and release, via ERK 1/2 activation (Dickinson et al, 2008). Therefore, alpha7 receptor activation has the ability to increase vesicle fusion and neurotransmitter release or generate other ERK 1/2-related long-term changes (Gotti et al., 2007).

It is this unique characteristic that has perpetuated the view that alpha7 receptors are critical for synaptic plasticity and the cellular events underlying executive function, such as LTP (Mansvelder et al., 2006). In fact, as mentioned above, they can form complexes with NMDA receptors where they play a direct role in learning and memory (Li et al., 2013).

Finally, both direct and indirect evidence points to alpha7 receptor dysfunction as playing a key role in the portrayal of cognitive deficits. First, genome-wide association studies have linked polymorphisms in the CHRNA7 gene, which codes for alpha7 protein, with an increased risk for schizophrenia

(Stefansson et al., 2008). Second, a link has been demonstrated between

15 deficient P50 auditory gating, a schizophrenia endophenotype, and a locus on chromosome 15q14 less than 120 kilo-base pairs from the alpha7 gene

(Freedman et al., 1997). Third, alpha7 receptor protein expression (Freedman et al., 1995; Court et al., 1999; Guan et al., 1999) and binding (Marutle et al., 2001) are decreased in the reticular nucleus of the thalamus, hippocampus, cingulate cortex, and frontal lobe. Importantly, the degree of the reduction seen in the dentate gyrus and CA3 region of the hippocampus correlates to the degree of overall cognitive dysfunction that was seen in the patients (Martin-Ruiz et al.,

2003). Fourth, kynurenic acid, an endogenous alpha7 antagonist, has been shown to be elevated in plasma and cerebrospinal fluid of schizophrenic patients

(Erhardt et al., 2001; Schwarcz et al., 2001). Finally, animal models of schizophrenia that utilize decrease or loss of alpha7 function, such as alpha7 knock outs and perinatal elevations of brain kynurenic acid, display many of the cognitive deficits seen in the disease (Young et al., 2007; Alexander et al., 2012).

Taken together, the alpha7 receptor is seen as one of the primary targets for cognition- enhancing drug development.

1.9 Positive allosteric modulators of the alpha7 receptor

The majority of drugs that are being designed for the alpha7 receptor are direct agonists, but indirect agonists called positive allosteric modulators (PAMs) have begun to be developed as well. Constructing a selective alpha7 receptor agonists can be done in several ways, but it begins with a tetramethyl-ammonium cationic center (Horenstein et al., 2008). In order to increase the selectivity of the

16 agonist for the alpha7 receptor, usually at the cost of potency, one or more hydrophophic elements are added to the cationic center (Horenstein et al., 2008).

The hydrophobic elements are quite large in some drugs, but studies on alpha7 receptor selectivity have shown that they can be as simple as a single methyl group (Horenstein et al., 2008). The pharmacophore of alpha7 PAMs are similar to agonists in that they contain hydrogen bond acceptors and donors as well as large hydrophobic elements to ensure selectivity (Capelli et al., 2010). A major difference, however, is that PAMs are neutral under physiological conditions and do not contain the cationic ammonium center that is required to bind to the orthosteric site (Horenstein et al., 2008). Interestingly, the one PAM that is not neutral, galantamine, binds at a location very near the orthosteric site that is unique from other PAMs (Hansen & Taylor, 2007).

The investigation of alpha7 PAMs is in its infancy, but there are important reasons for their use instead of direct agonists, which will be discussed in greater detail in chapter 5. Alpha7 PAMs have been shown, in vitro, to increase the amount of time the alpha7 channel pore remains open when the endogenous ligand is bound to the orthosteric site, thus potentiating the effects of the endogenous ligand (Gronlien et al., 2007; Ng et al., 2007; Williams et al., 2011a).

Importantly, the degree of the PAM-mediated potentiation was shown to interact with levels of the PAM and levels of the endogenous ligand (Dinklo et al., 2011;

Williams et al., 2011a). In contrast, alpha7 PAMs produce no change in current in the absence of the endogenous ligand, indicating they carry no intrinsic activity

(Gronlien et al., 2007; Ng et al., 2007; Williams et al., 2011a). Therefore, PAMs

17 have the potential to be a unique way to increase activity at the alpha7 receptor without stimulating it directly. However, no experiments have been done to determine if PAMs will potentiate neurotransmitter release in vivo in the same manner as they do excitatory currents in vitro. Therefore, the examination of the effects of novel alpha7 PAMs on endogenous neurotransmitter release remains a pressing need for their further development.

1.10 Experimental purpose

Based on the aforementioned importance of the interaction between glutamate and ACh transmission in the PFC to cognitive functions known to be deficient in schizophrenia, and the proposed role of the alpha7 receptor as a possible treatment target, this project seeks to characterize the ability of two novel alpha7 PAMs to potentiate glutamate release in vivo in the PFC as a function of varying levels of local choline. This will be accomplished with a series of experiments. First, an assay that uses mesolimbic stimulation to evoke endogenous glutamate release in the PFC will be further characterized to determine if it also evokes endogenous choline in the PFC, and if that choline release is dose-dependent (reasons for choline measurement instead of ACh discussed in chapter 3). Second, it will be determined whether alpha7 and/or alpha4beta2 nicotinic ACh receptors are necessary for the production of the mesolimbically stimulated glutamate release in the PFC, thus linking the local choline release to the glutamate release. Finally, the ability of two alpha7 PAMs

18 to potentiate the mesolimbically stimulated glutamate release will be determined as a function of differing choline levels in the PFC.

Taken together, this project will provide three things. First, it will further characterize and expand the use of a novel, rapid electrochemical method that is very valuable in examining phasic, in vivo neurotransmitter release. Second, it will provide a useful assay that evokes alpha7-mediated, endogenous glutamate release in the PFC, which can be used to examine the neurochemical effects of an array of novel alpha7 ligands in future studies. Third, it will, for the first time, demonstrate that alpha7 PAMs potentiate in vivo neurotransmitter release similarly to in vitro, i.e. only in the presence of afferent signaling and to varying degrees dependent upon that signaling.

CHAPTER 2

GENERAL METHODS

2.1 Subjects

Male Wistar rats, weighing 280-420 g, were used as subjects in these experiments. Animals were maintained in a temperature and humidity controlled room on a 12:12-hour light:dark cycle (lights on at 06:00 a.m.) and were group housed (pre-surgery) in pairs in plastic cages lined with corn cob bedding (Harlan

Teklad, Madison, WI). After microelectrode array implantation, and for the duration of experimentation, animals were singly housed. Animals had access to food and water ad libitum. The Ohio State University Institutional Animal Care and Use Committee, in accordance with the NIH Guide for the Care and Use of

Laboratory Animals, approved all procedures involving animals. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to consider alternatives to in vivo techniques.

2.2 Drugs

Solutions used for intracranial infusion were prepared in artificial cerebrospinal fluid (aCSF; NMDA) or saline (MLA; DHβE) and pHed to 7.1-7.4. NMDA

(n-Methyl-D-Aspartate) and MLA (methyllycaconitine citrate salt) were purchased from Sigma Aldrich Corp. (St. Louis, MO). DHβE (dihydro-β-erythroidine hydrobromide) was purchased from Tocris Bioscience (Minneapolis, MN). Both

PAMs, delivered intraperitoneally, (PNU120596; AVL3288, also known as compound 6 and XY4083) were acquired from Neurosearch (Copenhagen,

Denmark). These were dissolved in 5% DMSO (by volume; dimethyl sulfoxide) and Solutol (8% by weight), which were purchased from Sigma Aldrich Corp (St.

Louis, MO). All solutions used to prepare and calibrate the glutamate-sensitive microelectrode array (MEA) were prepared using distilled, de-ionized water.

These include the following: m-Phenylenediamine dihydrochloride (m-PD), purchased by Acros Organics (New Jersey, USA), L-ascorbic acid (AA), 3- hydroxytyramine (DA), L-glutamate monosodium salt, glutaraldehyde [25% (w/w) in water], bovine serum albumin (BSA), H2O2, choline chloride, and choline oxidase from Alcaligenes sp., obtained from Sigma Aldrich Corp. (St. Louis, MO), and L-glutamate oxidase, purchased from Seikaghaku America, Inc (East

Falmouth, MA).

2.3 Detection of glutamate-generated signals

Microelectrode arrays (MEA) were composed of a paddle that interfaces with a preamplifier and produces electrical readout that is translated by the

FAST-16 MKII electrochemical recording system (Quanteon, LLC, Nicholasville,

KY), and a ceramic tip bearing four 15 x 333 µm platinum recording sites (see

Rutherford et al., 2007 for further details on assemblage). For glutamate experiments, each pair of recording sites was designated to be either glutamate- sensitive (Gluox) or not (sentinel). The Gluox channels were coated with the glutamate oxidase (2%, 1 unit/1 uL, 100 nL), (BSA, 1%), and glutaraldehyde

(0.125%), whereas the sentinel channels were coated with just the BSA and glutaraldahyde. Sentinel channels are sensitive to every electro-active substance in the microenvironment, except glutamate, while glutamate channels are also sensitive to glutamate. This coating design allowed us to isolate electrical signal driven solely by glutamate by subtracting the sentinel channel activity from the

Gluox channel activity. For choline experiments, recording site designations and design remain the same, except the enzyme used is choline oxidase. This allows us to isolate the signal driven by choline oxidation, similar to that described above with glutamate.

Enzyme- coated MEAs were allowed to dry for 48 hours at room temperature prior to in vitro calibration. The enzyme detection scheme responsible for the generation of current due to the selective oxidation of glutamate or choline has been described in greater detail in previous publications

(Burmeister & Gerhardt, 2001; Rutherford et al., 2007). Briefly however, glutamate is oxidized by the glutamate oxidase, generating α-ketoglutarate and

H2O2. Choline is oxidized by choline oxidase, generating betaine and H2O2.

Because the MEA is maintained at a constant potential of +0.7 V vs. Ag/AgCl

22 reference electrode, in both cases the H2O2 reporting molecule is reduced, yielding two electrons. The resulting current is then amplified and recorded by the

FAST-16 MKII recording system (Quanteon, LLC, Nicholasville, KY). On the sentinel channels, extracellular glutamate or choline reaches the platinum surface, but in the absence of the converting enzymes, no oxidation current is generated. Any current detected is due to endogenous electro-active molecules other than glutamate or choline.

MEAs were electro-plated with m-PD to block the oxidation of some endogenous electro-active molecules that are present in higher concentrations.

The m-PD barrier excludes larger molecules from reaching the platinum surface, such as dopamine and ascorbic acid. Therefore, some of the molecules that would confound the sole detection of glutamate are blocked from reaching the platinum sites and the rest are subtracted using the sentinel channels.

2.4 Histology

At the conclusion of each experiment, animals were given an overdose of sodium pentobarbital and trans-cardially perfused. Brains were removed and stored in formalin (10%) for at least 2 days, and then transferred to a sucrose solution (30%) for at least 2 days. Brains were sectioned using a cryostat; coronal and sagittal sections (70 µm) were mounted on gelatin-coated slides, stained using Cresyl Violet, and examined under a light microscope for verification of microelectrode and cannula placement.

2.5 Data analysis

Measurements derived from the FAST-16 data file included: (i) basal glutamate/choline levels, (ii) maximum peak amplitude (µM) of the glutamate/choline signal, (iii) the latency (seconds) to peak onset, (iv) time to clear 50 (T50; seconds) and 80 (T80; seconds) percent of the total peak, and (v) total peak duration measured from peak onset to total clearance (seconds). The glutamate/choline signal, initially measured in pA, was transformed to a concentration equivalent (µM) on the basis of each sensor’s individual calibration slopes generated immediately before surgery. Slopes of sentinel channels were replaced with slopes of Gluox/Cholox channels to ensure equal comparison. The signal derived exclusively by the oxidation of glutamate/choline was isolated using a self-referencing procedure as we have described above and elsewhere

(Burmeister & Gerhardt, 2001; Rutherford et al., 2007; Konradsson-Geuken et al., 2009). Comparisons were performed on all dependent measures derived from the FAST-16 data file mentioned above (basal, maximum amplitude, time to peak onset, T50, T80, and duration) using the IBM SPSS statistics program

(version 22, IBM Corporations, Armonk, NY).

Chapter 3

MESOLIMBIC REGULATION OF PREFRONTAL CHOLINE RELEASE

3.1 Introduction

The alpha7 receptor, as discussed in chapter one, has been established as one of the leading targets for cognition- enhancing drug development.

Specifically, positive allosteric modulators (PAMs) of the alpha7 receptor are beginning to be examined, but their study is in its infancy. There is a wealth of in vitro data that confirm PAMs’ ability to potentiate the alpha7 receptor without intrinsic activity, but no in vivo studies have tested this. Thus, an appropriate assay is needed to confirm that PAMs will potentiate neurotransmission in vivo in the same manner. For this purpose, we selected an assay that involves stimulating the shell of the nucleus accumbens (NAcSh) with the ionotropic glutamate agonist, NMDA. Our reasons for selecting this assay, as well as a description of its value, are detailed in the introduction below.

3.1.1 Mesolimbic stimulation assay

The first reason to use the mesolimbic stimulation assay is that it evokes the endogenous release of glutamate and ACh in the PFC (Zmarowski et al.,

2005; Bortz et al., 2014). As detailed in the introduction, both ACh and glutamate release in the PFC are critical for executive functions such as memory (Levin &

Simon, 1998; Aultman & Moghaddam, 2001; Levin & Rezvani, 2002), attention

(Parikh et al., 2008; St Peters et al., 2011), and cognitive flexibility (Stefani &

Moghaddam, 2005a; Alexander et al., 2012), validating the selection of these neurotransmitter systems for study using a potential cognitive-enhancer.

Additionally, the glutamate release evoked by this mesolimbic stimulation is hypothesized to be related to and possibly secondary to the PFC ACh release.

Data supporting this hypothesis will be detailed in chapter 4. Thus, this assay allows the possibility to manipulate endogenous PFC ACh levels and determine what effect that has on PFC glutamate in both the presence and absence of an alpha7 PAM. This would mirror, in vivo, the studies that have been done in vitro, which is the goal of this project.

The second reason is this assay evokes PFC neurotransmitter release by mimicking excitatory input into the NAcSh (NMDA stimulation), and, thus, does so by tapping into a cortical-subcortical distributed neural system (figure 1) that is involved in executive functioning (described in chapter 1). The NAc is central to this distributed system because it receives efferent projections from several cortical and limbic regions within the system, such as the hippocampus, amygdala, VTA, and PFC (Voorn et al., 1996; Groenewegen et al., 1999). As

26 such, the NAc mediates goal-directed behavior through integrating contextual information from the hippocampus, affective information from the amygdala, top- down executive information from the PFC, and incentive-reward information from the VTA, and then converting this to a motor plan via its afferent projection to the ventral pallidum (Zahm et al., 1996). In support of this notion, the NAc has been implicated in goal-directed locomotor activity necessary for feeding, attack, survival, and sexual behavior; oral motor responses associated with feeding, drinking, and vocalizing; as well as reward, spatial learning, and sensorimotor gating (Mogenson et al., 1980; Annett et al., 1989; Damsma et al., 1992; Wan &

Swerdlow, 1996). Additionally, the NAc directly contacts the basal forebrain cholinergic system (Mogenson et al., 1983; Zaborszky & Cullinan, 1992), which has been shown to be critical in the top-down control of attention (St Peters et al.,

2011) and the processing of incoming thalamo-cortical sensory information

(Groenewegen & Berendse, 1994). Taken together, tapping into this very important distributed neural system, by stimulating the NAcSh, increases the likelihood that the resultant endogenous neurotransmitter release is cognitively relevant.

Third, additional studies further support the notion that the assay is cognitively relevant. As mentioned above, NAcSh stimulation with NMDA is hypothesized to mimic excitatory input. One of the excitatory inputs to the NAcSh is from the PFC, which is a mechanism by which the PFC exerts top-down control over this sub-cortical region. Therefore, NMDA stimulation of the NAcSh is hypothesized to be a model of top-down regulation. This hypothesis is

27 supported by a series of studies. First, it was shown that stimulation of the

NAcSh with NMDA results in increases in ACh release within the PFC

(Zmarowski et al., 2005). Second, ACh levels in PFC increased in animals performing a sustained attention task, which was associated with the increased attentional effort required to perform the task (Kozak et al., 2006; St Peters et al.,

2011). Third, the presentation of a distractor during the attention task caused rodent performance to decline dramatically. Finally, they showed that the same stimulation of the NAcSh that increased PFC ACh levels was able to reverse the rodent’s failing performance during the presentation of the distractor (St Peters et al., 2011). Thus, these studies supported the notion that NAcSh stimulation mimics the mechanism by which the PFC, via excitatory projections to the NAc, recruits top-down control processes, displayed as increases in PFC ACh release, to filter distracting information.

Fourth, disruptions within the distributed system (figure 1) that we tap into with this assay are demonstrated in schizophrenia, and are, likely, the cause of the cognitive deficits. Both human and animal studies provide evidence to support this statement. For example, increased dopamine release in the striatum is a hallmark of schizophrenia. Interestingly, animal studies have shown that increases in dopamine activity in the NAc results in the preferential shunting of ventral hippocampus throughput to NAc at the expense of PFC throughput (Goto

& Grace, 2005). This could contribute to the top-down cognitive control deficit often seen in patients. Additionally, dysregulated glutamatergic transmission has been shown in the PFC of patients, which often is portrayed in fMRI scans as a

28 hypofrontality (see Ragland et al., 2007 for review). This, likely, stems from

NMDA receptor hypofunction (see general introduction), and is tightly correlated with deficient performance during cognitive tasks (see Ragland et al., 2007 for review). Finally, there are several indices of irregularities in the anterior hippocampus (akin to the VH in rodents) of patients, such as cellular disorganization and decreased GABAergic interneuron markers (Zhang &

Reynolds, 2002). This region plays a major role in organizing the PFC during development (Kruger et al., 2012), and is responsible for driving theta and gamma oscillations in the PFC (see Sun et al., 2011 for review). Additionally, animal models of schizophrenia that manipulate the VH during development have shown many similar PFC-mediated cognitive deficits as those seen in patients

(Marquis et al., 2008; Brooks et al., 2012). Therefore, there are many indications that disruptions within this distributed system (figure 1) are responsible for the cognitive deficits of schizophrenia, making the use of an assay that taps into this distributed system more interesting from a therapeutic standpoint.

3.1.2 Experimental rationale and hypothesis

Based on the fact that this assay evokes the endogenous release of the neurotransmitters that are of interest to this project via activation of a cognitively- relevant, multi-regional neural network that may contribute to the cognitive deficits of schizophrenia, it was determined to be the right assay to examine the in vivo effects of alpha7 PAMs. Although previous studies have indicated that this assay results in ACh release within the PFC, these studies used microdialysis to

29 both administer the NMDA into the NAcSh (reverse dialysis) and measure the release of ACh in the PFC (Zmarowski et al., 2005). Additionally, it has not been determined that this stimulation results in the dose-dependent release of choline in the PFC. In order to test the hypothesis outlined in chapter one concerning the alpha7 PAMs, it is necessary to determine that varying concentrations of NMDA into the NAcSh, delivered via bolus infusion, result in dose-dependent increases in PFC choline as measured by the choline-sensitive microelectrode. Therefore, this experiment will address these needs.

It is hypothesized that if bolus infusions of NMDA into the NAcSh result in dose-dependent increases in choline release within the PFC, then choline levels in the PFC, as measured by the choline-sensitive microelectrode, will change after NMDA stimulation of the NAcSh based on the dose of NMDA delivered.

3.2 Methods

3.2.1 In vitro calibration of MEAs

Microelectrode arrays (MEAs) were calibrated in vitro using the FAST-16

MKII electrochemical recording system just prior to implantation. Constant potential amperometry was conducted using an applied potential of +0.7 V vs an

Ag/AgCl reference electrode just as is done in the brain. Calibrations were performed in a stirred solution of PBS (0.05 M, 40 ml, pH 7.4, 37 C). After a stable baseline was established, ascorbic acid (AA; 500 µL, 20 mM), glutamate

(40 µL, 3x 20 mM), dopamine (DA; 40 µL, 20 mM), and hydrogen peroxide (H2O2,

40 µL, 8.8 mM), were sequentially added to the calibration beaker for final beaker concentrations of 250 µM, 60 µM, 20 µM, and 8.8 µM, respectively.

Amperometric signals were acquired at a rate of 1.0 Hz. The slope (sensitivity of the MEA, nA/µM choline), limit of detection (L.O.D, µM choline), selectivity (ratio of choline sensitivity versus ascorbic acid sensitivity), and linearity of choline responsiveness (R2) were calculated. In order to be used for subsequent in vivo recordings, the MEAs had to conform to the following calibration criteria: (i) similar background current (i.e., no greater than a 20 pA difference) between the

Cholox and sentinel channels, (ii) linear response to increasing concentrations of choline (R2 > 0.998), (iii) a minimum slope of -0.003 nA/µM choline, (iv) a minimum L.O.D of < 0.5 μM, (v) a high selectivity for choline over either AA or DA

(i.e. > 50:1), and (vi) similar sensitivity to the reporting molecule H202 on all four channels (> 80% similarity for each channel pair).

Figure 3 depicts a representative in vitro calibration. The tracings represent change in current in response to the addition of the chemicals listed above (indicated by arrows). The top channel represents the choline- sensitive channel (Cholox), and the bottom represents the background channel (sentinel).

The addition of AA produces little to no change in current on either channel.

Successive additions of choline produce large, definitive increases in current on only the Cholox channel, and these increases are linear as the beaker concentration of choline gets progressively higher. The addition of DA produces no change in current indicating the strength of the m-PD layer. Finally, the addition of H202 produced equal increases in current across both channels,

31 indicating both channels are similarly sensitive to the reporting molecule. An important result to note is that the addition of every chemical except choline produced an equal response on both channels. This point is of paramount importance as it is the basis of the self-reference technique by which the choline signal is isolated in vivo.

3.2.2 Implantation of MEAs and infusion cannulae

Animals were anesthetized using isofluorane (2%, 0.6 L/min) and implanted with an MEA unilaterally in the PFC (in mm from bregma: AP +2.7, ML

+ 0.65, DV -3.4; hemispheres counterbalanced). Stainless steel guide cannulae

(Plastics One, Roanoke, VA), used for intra-NAcSh infusions of NMDA, were implanted ipsilaterally in the NacSh (at 10 degree angle, in mm from bregma: AP

+ 0.4, ML + 0.70, DV – 7.4). The actual ventral termination of the guide cannula is -6.4 mm, accounting for 1 mm of the infusion cannula extension beyond the tip of the guide. All infusion cannulae used for a particular animal were measured to extend equally from the bottom of the guide cannula to ensure consistency of delivery location. A dummy cannula was inserted into the guide cannula and extended 0.7 mm beyond the tip of the guide. The Ag/AgCl reference electrode was implanted in the contralateral side at a site distant from the recording area.

All coordinates were determined using a stereotaxic atlas (Paxinos & Watson,

1998).

3.2.3 In vivo recordings

After MEA and cannula implantation, rats were tested in 3 consecutive recording sessions. During recording sessions rats were allowed to move freely in a wooden recording box (dimensions: H 57.2 cm; W 341.9 cm; L 317.0 cm).

The first day after MEA implantation surgery consisted of placing the rats in the wooden testing box, without connecting them to the preamplifier, to allow them to habituate to their environment prior to testing. On the second day after implantation, recordings of cortical choline began. At the onset of each testing day, animals were placed in the recording box and connected to the preamplifier.

Animals then remained undisturbed for 1 - 3 hours to allow for signal stabilization to occur before any drugs were delivered. Once the baseline period had concluded, the dummy cannula was removed and 1.0 µL of vehicle (aCSF) was delivered into the NAcSh to serve as an infusion control and to clean away any blood or tissue from the bottom of the guide cannula. Then, following a 10-minute delay, one of three doses of NMDA (0.05, 0.15, 0.30 µg in 0.5 µL, pH 7.1-7.4) was delivered into the NAcSh. NMDA was delivered over about 2 using an infusion cannula (extended 1mm beyond the guide) attached to a Hamilton

PB600-1 manual dispenser (Hamilton Company, Reno, NV). Doses of NMDA not given on the first day were delivered on the two subsequent days using the same procedure. Dose order was counterbalanced amongst all animals.

3.2.4 Data analysis

Comparisons were performed on all dependent measures derived from the FAST-16 data file mentioned in chapter 2 (basal choline levels, maximum choline peak amplitude, time to choline peak onset, T50, T80, and choline peak duration) using analysis of variance (ANOVA) by the IBM SPSS statistics program (version 22, IBM Corporations, Armonk, NY). An initial omnibus ANOVA was performed for each dependent measure using dose of NMDA (0.05, 0.15,

0.30/ .5L) as a within-subjects factor. If significance was determined, post-hoc comparisons were performed to determine specific locations of significance. The

Huyen-Feldt correction was used to minimize the occurrence of type II errors.

Significance was defined as p < 0.05.

3.3 Results

3.3.1 MEA and cannula placement

All subjects included in this analysis had confirmed MEA placements within the prelimbic/infralimbic region of the medial PFC and cannula placements within the anterior portion of the NAcSh. Figure 4 depicts representative coronal photomicrograph sections of a PFC sensor placement and a NAcSh cannula placement along with a descriptive cartoon for comparison. As shown, the representative placements fall within their respective desired regions, and arrows indicate the ventral termination of both items. The representative

34 photomicrographs also illustrate the minimal tissue damage produced by both the

MEA and infusion cannula compared to other in vivo methods.

3.3.2 NMDA infusions into the NAcSh dose-dependently increase choline levels in the PFC

In order to determine whether or not NMDA infused into the NAcSh would dose-dependently increase choline levels in the PFC, NMDA infusions were administered into the NAcSh at three counterbalanced doses on three consecutive days (0.05, 0.15, 0.30 µg in 0.5 µL). Figure 5 displays representative tracings of the MEA’s electrochemical signal generated by each of the three doses, as well as an example vehicle (aCSF) infusion, which was delivered each day prior to NMDA. Artificial CSF vehicle infusions were not included in the remaining figures as they did not result in any detectable change from baseline.

In this figure, the black tracings represents the signal generated by the choline- sensitive channels (Cholox), and the green tracings represent the signal generated from the sentinel channels. The sentinel channels are sensitive to every electro-active molecule (at +0.7 V) that the Cholox sites are except for choline. The red tracings represent the resulting signal generated from subtracting the respective sentinel channel from its Cholox channel. Thus, these tracings indicate the self-referenced (Self Ref) signal or the signal generated exclusively by the oxidation of choline. This procedure to isolate the choline- driven signal is critical because not everything detected by the MEA is choline.

This is illustrated well by the figure as some elements in the Cholox tracing are

35 clearly also seen in the sentinel tracing, while others are not. Then, examination of the Self Ref tracing reveals that only the unique elements of the Cholox waveforms remain, indicating the self-referencing procedure was successful.

The Self Ref tracing for each dose of NMDA (aCSF, 0.05, 0.15, 0.30 µg in

0.5 µL) reveal the stability of each baseline (aCSF: 0.71 µM, 0.05: 0.70 µM, 0.15:

0.40 µM, 0.30: 0.99 µM) both before and after the drug effect. Baselines are displayed uniformly for ease of comparison. The low dose of NMDA (0.05 µg) resulted in a robust, multi-phasic increase in PFC choline that occurred 71 seconds post injection and persisted for 46 seconds. The amplitudes of each peak (0.63 and 0.40 µM) were summed and expressed as total amplitude (1.03

µM). All multi-phasic waveforms were handled in this manner. The middle dose of NMDA (0.15 µg) also resulted in a clear increase in PFC choline, but it had only a single phase, occurred significantly faster (40 seconds), and was significantly higher in amplitude (1.85 µM) than the peak produced by the low dose of NMDA. The total duration of the peak was also longer (116 seconds) as clearance of the final 20% of the peak was significantly protracted (T80, time to clear 80% of the peak; 23 seconds). The high dose of NMDA (0.30 µg) resulted in a multi-phasic peak, similar to the low dose of NMDA, but occurred significantly faster (33 seconds), was significantly higher in amplitude (1.73 µM), and had a significantly longer duration (2,175 second), similar to the middle dose.

Again, the lengthy peak duration with this dose was caused by a protracted time to clear the final 20% of the peak as the time to clear the first 80% of the peak

(T80) was 95 seconds.

3.3.3 Group data

Overall, the group data (N=6) were consistent with the individual data depicted in figure 5. Basal choline levels did not differ significantly before or after any NMDA infusion (0.05 µg: 2.45 + 1.14 µM, 0.15 µg: 2.97 + 1.34 µM, 0.30 µg:

2.35 + 1.10 µM; p > .9), indicating that basal choline levels did not affect the stimulated choline increases. However, as indicated above, the latency to peak onset (mean + SEM) did differ between NMDA doses (0.05 µg: 83.57 + 18.76 sec, 0.15 µg: 41.29 + 4.24 sec, 0.30 µg: 36.29 + 2.32 sec; F2,20 = 5.395, P =

.015; figure 6), with the low dose of NMDA having significantly longer peak onset latencies than either the middle dose (F1,13 = 4.834, P = .048) or the high dose

(F1,13 = 6.258, P = .028). The peak onset latency did not differ between the middle and high doses of NMDA (p > .3). Similarly, peak amplitude differed significantly between groups (0.05 µg: 0.87 + 0.15 µM, 0.15 µg: 1.61 + 0.17 µM,

0.30 µg: 1.73 + 0.31 µM; F2,20 = 4.448, P = .027; figure 6), with the low dose of

NMDA producing significantly smaller amplitudes than either the middle dose

(F1,13 = 10.781, P = .007) or the high dose (F1,13 = 6.318, P = .027). Again, peak amplitude did not differ between the middle and high doses of NMDA (p > .7).

Finally, high doses of NMDA produced peaks with significantly longer durations

(0.05 µg: 151.29 + 74.07 sec, 0.15 µg: 151.00 + 58.30 sec, 0.30 µg: 787.71 +

267.24 sec F2,20 = 5.046, P = .018; figure 6) than either middle (F1,13 = 5.419, P =

.038) or low (F1,13 = 5.267, P = .041) doses of NMDA. These differences, however, were produced solely by the protracted clearance of the final 20% of

37 the high dose peaks as T50 (time to clear 50% of the peak; 0.05 µg: 16.29 + 4.56 sec, 0.15 µg: 10.86 + 2.22 sec, 0.30 µg: 12.29 + 2.62 sec) times were not significant (p > .4), and T80 times (time to clear 80% of the peak; 0.05 µg: 33.71

+ 5.21 sec, 0.15 µg: 62.71 + 33.61 sec, 0.30 µg: 145.29 + 63.16 sec) only approached significance (p > .1). Vehicle infusions produced no detectable change from baseline in any animal; therefore, no statistics were performed on any of the dependent measures mentioned above.

3.4 Discussion

The results from this chapter indicate, for the first time, that bolus infusions of NMDA into the NAcSh evoke choline release in the PFC, as measured by the choline-sensitive MEA. Furthermore, the choline release in PFC is dose- dependent among several of the dependent measures. First, both the high and middle doses of NMDA resulted in peak amplitudes that were larger and occurred quicker than the low dose of NMDA. Second, the choline peaks produced by the high dose of NMDA lasted much longer than the low or middle doses of NMDA. In contrast, basal choline values and the time to clear 50 and

80% of the choline peaks were not affected by NMDA dose. Infusions of aCSF vehicle did not produce any detectable change from baseline. This data confirms that NMDA infused into the NAcSh is sufficient to dose-dependently increase choline levels in the PFC that are measurable with the choline-sensitive MEA.

These results are similar to what has been previously shown with glutamate

38 release in the PFC, as well as confirms previous work measuring ACh with microdialysis after mesolimbic stimulation.

The following section will focus on a two important methodological questions related to this data, a discussion of possible explanations and significance of the unique characteristics of the PFC choline release, and finally some concluding statements about how this data sets the stage for use of this assay with alpha7 PAMs.

3.4.1 Methodological justifications

The first methodological question is why measure choline instead of ACh?

ACh is difficult to detect in vivo because it is hydrolyzed very rapidly once released into the synapse, and because many in vivo techniques do not measure

ACh directly. For example, microdialysis collects ACh samples, but the ACh is converted to choline prior to the reporting molecule hydrogen peroxide by AChE on a postcolumn reaction coil. Similarly, the MEA detects choline, not ACh directly. This can be endogenous choline or ACh after it is hydrolyzed by endogenous AChE or AChE coated on the platinum surface. Then the choline is converted to the reporting molecule hydrogen peroxide by choline oxidase.

Importantly to this experiment, choline is actually a neuro- active metabolite that is highly selective for the alpha7 receptor. In fact, due to the speed with which

ACh is hydrolyzed by AChE and the fact that alpha7 receptors can exist extra- synaptically (See Higley & Picciotto, 2014 for review), it is likely that choline signaling represents a meaningful mode of neural communication. Therefore,

39 since the purpose of this dissertation was to characterize the interaction between the potentiating effects of two alpha7 PAMs and levels of the endogenous ligand, and choline is much more selective for the alpha7 receptor than ACh, then determination of endogenous choline levels is actually the more interesting result. For these reasons, endogenous choline levels were measured by the

MEA.

The second methodological question is why measure in the prelimbic/infralimbic (PreL/InfL) cortices instead of other regions? The prefrontal cortex of rodents has many sub-regions, and each shows differential distributions of afferent and efferent contacts and can be associated with different behaviors.

We decided to measure choline release specifically within the medial PFC because it is associated with behaviors such as attentional processing, working memory, and goal-directed behavior (Kolb & Gibb, 1990; Rakic et al., 1994;

Repovs & Baddeley, 2006), which are among the cognitive deficits of schizophrenia. The medial PFC is, however, broken down to two more subregions, the ventromedial PFC: infralimbic and prelimbic cortices (InfL/PreL), and the dorsomedial PFC: agranular cortex and anterior cingulate. Measurement in the InfL/PreL cortices was chosen for a number of reasons. First, the InfL/PreL cortices have a high degree of afferent and efferent connectivity with other regions within the distributed neural system (figure 1), such as the BLA, MDthal, hippocampus, NAc, and the VTA (Gabbott et al., 2005; Hoover & Vertes, 2007).

Importantly, they also receive dense cholinergic innervation from BF (Hoover &

Vertes, 2007). The high degree of connectivity within this distributed neural system is important because, as previously stated, it is likely responsible for executive functioning and is disrupted in schizophrenia. Second, basal ACh increases in this region were associated with an increase in “readiness” to perform a cognitive task (St. Peters et al, 2011), and choline transients measured in this region predicted the detection of visual cues during a sustained attention task (Parikh et al., 2007). Thus, there is evidence to confirm that this region of the PFC is involved in the executive functions that are disrupted in schizophrenia.

Third, the glutamate release after mesolimbic stimulation was also measured in the InfL/PreL cortices. This is important because it has been hypothesized that the PFC glutamate release is secondary to the measured release of choline via its activation of local alpha7 receptors (see chapter 4). Fourth, the previous microdialysis studies that initially indicated mesolimbic stimulation would increase

ACh levels demonstrated that this occurred in the medial PFC (InfL/PreL;

Zmarowski et al., 2005) and not in other regions of the cortex (Zmarowski et al.,

2007). Therefore, based on these reasons, choline release was measured in

InfL/PreL cortices.

3.4.2 Discussion of results

A number of questions may arise when examining the data presented in this chapter. First, where does the choline signal detected by the MEA originate?

Previous studies have indicated that a lesion of the BF eliminates increases in

PFC ACh after NMDA stimulation of the NAcSh (St Peters et al., 2011). Even

41 though this ACh was measured with microdialysis, the choline measured in the present study is likely to also be from the BF because both stimulation (NAcSh) and measurement (InfL/PreL cortices) occurred in the same place as the microdialysis study. Additionally, the BF projections to the PFC (Luiten et al.,

1987) are the same that are contacted by direct projections from the NAcSh

(Zaborszky & Cullinan, 1992), thus completing the most likely pathway by which

NAcSh stimulation results in PFC choline release.

Second, why are the choline peaks so small in comparison to the glutamate peaks previously reported (Bortz et al., 2014) and those seen in subsequent chapters? This is an important question because the choline release is hypothesized to be related to, and possibly supersede, the glutamate release in PFC via its activation of local alpha7 receptors. The possible answers for this question are two-fold. First, it is possible that the actual endogenous cholinergic signal was greater than the choline signal that was measured. ACh gets rapidly hydrolyzed in the synapse after it is released, making the choline available for detection. However, choline is subject to high-affinity re-uptake by choline transporters as well as diffusion away from the synapse. Thus, the choline molecules that make contact with the surface of the electrode may be less than what was released. This is a common problem with in vivo neurotransmitter detection, but does not negate the hypothesis that the choline is driving the glutamate release locally. Another possible explanation is that the cholinergic signaling is occurring via volume transmission. It is well established that volume

42 transmission is a mode by which cholinergic signaling occurs in the brain, and if this were so, the amount of choline detected by the MEA would likely be small.

Again, this does not negate the hypothesis that the choline is driving glutamate release locally because alpha7 receptors are able to be activated when only one of their five possible binding sites are bound by agonist (Andersen et al., 2013).

Furthermore, activation by a single binding site results in maximal activation by the receptor (Andersen et al., 2013). Therefore, a small choline signal, possibly due to delivery by volume transmission, has the potential to generate full activation of alpha7 receptors locally. This idea is further supported by a study performed by Papke & Porter Papke (2002) where they reported that maximum amount of alpha7 channel opening was obtained with low agonist concentrations.

Third, why do the choline peaks have such protracted durations after the high dose of NMDA, and what does that might mean in vivo? Even though the peak amplitudes did not differ between the middle and high dose of NMDA, the peak durations did. This may indicate that more choline was, in fact, released with the high dose. If this were the case, the protracted peak clearance could simply have been caused by saturation of the choline transporters in the area around the MEA. This is supported by the fact that the difference in peak duration was caused by the time to clear the final 20% of the choline peak.

Alpha7 receptors can be maximally activated via binding of a single site, and binding of additional sites does not produce additive or synergistic affects

(Papke & Porter Papke, 2002). In fact, strong activation of alpha7 receptors

43 results in receptor desensitization that is either readily reversible upon removal of the agonist or more stable and not as readily reversible, depending on the degree and length of the activation (Williams et al., 2011a). Therefore, it is likely that the protracted duration of the choline peak after the high dose of NMDA indicates that this level of cholinergic stimulation would not be cognitively beneficial. This is supported by the study performed by St. Peters et al. (2011) that was referenced in the introduction. In the study, the cognitive improvement produced by the NMDA infusions into the NAcSh had an inverted U distribution, with the highest doses of NMDA no longer producing cognitive benefit in the sustained attention task (St Peters et al., 2011).

Finally, if the sentinel channels are not sensitive to choline, what are the sentinel channels detecting? The MEAs are electroplated with an exclusion barrier called m-Phenylenediamine dihydrochloride or m-PD prior to implantation.

This barrier prevents larger molecules, such as dopamine and ascorbic acid, from reaching the surface of the MEA and being converted to the reporting molecule hydrogen peroxide. Therefore, the sentinel channel must be detecting a molecule small enough to penetrate this barrier, and the most likely candidate is nitric oxide. NOS-I is the neuronal isoform of nitric oxide synthase, which generates nitric oxide. NOS-I forms complexes with NMDA receptors on post- synaptic densities in the PFC, and is activated when calcium enters the NMDA receptor (see Weber et al., 2014 for review). Therefore, if choline release is stimulating glutamate locally via activation of alpha7 receptors on glutamatergic

44 terminals (see chapter 4), then nitric oxide is likely being released when that glutamate stimulates local NMDA receptors. Interestingly, the choline-sensitive channels often register choline before the sentinel channels begin to rise, giving further credence to this possibility. Another possibility is that the m-PD exclusion barrier weakens in vivo compared to when it is tested in vitro prior to implantation. If this is the case, the sentinel channel could be detecting other molecules that would reduce to hydrogen peroxide under a 0.7-volt electrical potential, including dopamine. Dopamine is also mediated by alpha7 receptor activation in the PFC (Livingstone et al., 2009), and would likely be present near the platinum recording sites of the MEA. Therefore, the most likely answer to this question is that the sentinel is detecting nitric oxide and dopamine, as well as a handful of other molecules present near the MEA. It is important to point out, however, that even though the answer to this question still remains unclear, all four channels are detecting anything that is being detected by the sentinel.

Therefore, those things are being subtracted during the self-referencing procedure, thus isolating the signal driven by choline.

3.4.3 Conclusions

The purpose of this experiment was to determine whether or not NMDA infused into the NAcSh would dose-dependently increase choline release in the

PFC. The reason for this is to set the stage for the final experiment of this dissertation, which is to determine if PAMs will potentiate neurotransmitter release (glutamate) in vivo in a similar fashion as they have done in vitro (see

General Introduction for further detail). Answering this question requires an assay where release of the reporting neurotransmitter (glutamate) is driven by endogenous afferent activity (choline) at the alpha7 receptor. The first step in determining if the mesolimbic stimulation assay meets that requirement is to demonstrate that choline levels change in the PFC according to the amount of stimulation of the NAcSh. Having accomplished this, it is now known that there is a means to control endogenous choline levels in the PFC. Thus, it is possible to determine whether or not those choline levels will interact with the ability of a

PAM to potentiate glutamate release in the subsequent experiments. What has not yet been accomplished is to confirm that the dose-dependent choline release in PFC, described above, drives the PFC glutamate release, shown previously, via activation of the alpha7 receptor. As stated, this is required to answer the question laid out in the General Introduction, and the next chapter in this dissertation addresses this need.

Chapter 4

MESOLIMBIC REGULATION OF GLUTAMATE RELEASE IN PFC IS

MEDIATED BY LOCAL ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR

ACTIVATION

4.1 Introduction

The previous chapter demonstrated that NMDA stimulation of the NAcSh resulted in dose-dependent increases in choline in the PFC. However, the purpose of this dissertation is to measure alpha7 PAMs’ effects on glutamate release in the PFC. It is, therefore, necessary to demonstrate that the mesolimbically-stimulated glutamate release in PFC is related to the choline release from chapter 3 via the alpha7 receptor as a proof of principle. The following introduction section will discuss the reasons for measuring glutamate release in PFC, the evidence to suggest that it is mediated locally by nicotinic receptors, and, finally, anything that is currently known to distinguish between alpha7 and alpha4beta2 receptors in the PFC.

4.1.1 The importance of PFC glutamate transmission

This dissertation will use glutamate release in the PFC as its principle outcome measure for two main reasons. The first is the critical nature of PFC glutamate to many executive functions, such as cognitive flexibility (Stefani &

Moghaddam, 2005a), working memory (Aultman & Moghaddam, 2001), and sustained attention (Parikh et al., 2008). One mechanism by which it mediates these cognitive functions is through its role in oscillatory activity. Populations of neurons firing at synchronized rates generate a rhythmic pattern of activity that can be measured at a couple of different frequencies. It has been suggested that normal cognitive functioning depends on this synchronized firing, particularly at the gamma frequency (30-80 hz, see Sun et al., 2011 for review). GABAergic interneurons mediate this synchronous firing via patterns of selective inhibition

(Fries et al., 2007); however, excitatory glutamatergic neurons synapsing onto these local GABAergic interneurons control their pattern of inhibition (reviewed in

Rotaru et al., 2012). Glutatmatergic inputs into the PFC also synapse onto pyramidal cells, which generates excitation of local circuits. An example of these two patterns of activity working jointly to generate executive function is seen with the cognitive operation spatial working memory. To accurately hold a location in working memory requires the maintenance of firing of the direction-specific neurons in the dorsolateral PFC via feed forward recurrent excitation (Wang,

1999), while inhibiting neurons of the incorrect direction via excitation of

GABAergic interneurons (Yang et al., 2013). Thus, a combination of GABA- mediated inhibition and glutamate-mediated recurrent excitation, which are both

48 generated by glutamate transmission in PFC, are needed for this cognitive operation.

The second reason to focus on glutamate release in PFC is because it is disrupted in schizophrenia. This was initially characterized as a “hypofrontality” because patients displayed reduced activity in the PFC during tasks, such as working memory and cognitive flexibility (see Ragland et al., 2007 for review), but it is now believed to be more of a dysregulated glutamate transmission. This dysregulation is perpetrated by connectivity deficits between glutamatergic inputs to the PFC, local GABAergic interneurons, and their respective pyramidal cell targets (Reviewed in Rotaru et al., 2012); and as described in the paragraph above, proper connectivity and cooperative functioning of all of these circuits are needed for executive functioning. Evidence of dysregulated glutamatergic transmission in the PFC is seen through a myriad of changes in multiple PFC regions. They include NR1 NMDA subunit decreases, decreased kainate receptor binding, robust changes in NMDA and AMPA-associated intracellular proteins, such as PSD95 and SNAP102, reduced neuropil (fewer axon terminals), reduced dendritic spine presence and density, and decreased mean somal volume of pyramidal cells (Glantz & Lewis, 1997; 2000; Konradi &

Heckers, 2003). Interestingly, indicators of reduced GABA activity in the PFC have been shown as well, including reductions in the GABA synthesizing enzyme

GAD67 and a calcium binding protein specific to GABAergic interneurons called parvalbumin (Hashimoto et al., 2003; Gonzalez-Burgos et al., 2010). Importantly,

GAD67 and parvalbumin have been shown to be activity dependent, indicating

49 there is a deficit in the excitation of these GABAergic interneurons (reviewed in

Lewis, 2012). Therefore, there is abundant evidence of dysregulated glutamate transmission in the PFC, and, together with the importance of PFC glutamate transmission to normal executive function, these make up the reasons for measuring glutamate transmission in the PFC in these experiments.

4.1.2 Glutamate and nicotinic acetylcholine receptors

Glutamate release within the PFC is mediated, at least in part, by nicotinic

ACh receptors. This class of receptors belongs to the ionotropic receptor family and mediates fast synaptic transmission via the influx of positively charged ions, such as sodium or calcium. Both alpha7 and alpha4beta2 receptor subtypes are found in the PFC, and both have been shown to increase glutamate release in the rodent PFC both in in vitro (Dickinson et al., 2008; Livingstone et al., 2009) and in in vivo electrophysiological (Gioanni et al., 1999; Lambe et al., 2003) and neurochemical studies (Konradsson-Geuken et al., 2009; Bortz et al., 2013). The studies by Lambe et al. (2003) and Gioanni et al. (1999) indicated that nicotinic- mediated glutamate release was from beta2 containing receptor subtypes on the glutamatergic terminals of projection neurons from the mediodorsal thalamus

(MDthal). In fact, a lesion of this region of the thalamus decreased the nicotine- mediated glutamate increases by 80% (Lambe et al., 2003). However, other studies that examined layer-specific excitatory inputs to the PFC demonstrated that both alpha7 and alpha4beta2 receptors mediated excitatory transmission, but in a layer and cell-specific manner (Poorthuis et al., 2013). The notion that

50 both receptor subtypes are involved in glutamate transmission in the PFC is supported by the ability of alpha7-selective agonists to initiate glutamate release in an MLA/αBGT -sensitive manner both in vitro (Dickinson et al., 2008;

Livingstone et al., 2009) and in vivo (Bortz et al., 2013).

4.1.3 Alpha7 versus alpha4beta2 nicotinic acetylcholine receptors

Although it is clear that both alpha7 and alpha4beta2 receptor subtypes are involved in glutamate transmission in the PFC, studies have shown that they can be distinguished based on cell type, location on the neuron, cortical layer, where the neuron originated, and the mechanism by which they influence neurotransmission. First, alpha4beta2 receptors are found on both glutamatergic and GABAergic cells in the PFC (Poorthuis et al., 2013). However, GABAergic interneurons can be divided into fast-spiking and nonfast-spiking, and alpha4beta2 receptors tend to be seen to a much greater degree on the nonfast- spiking interneurons (Poorthuis et al., 2013). Alpha4beta2 receptors are also frequently seen presynaptically on glutamatergic terminals, but less so postsynaptically (Gioanni et al., 1999; Lambe et al., 2003; Poorthuis et al., 2013).

Alpha7 receptors are also present on both glutamatergic and GABAergic cell types, but they tend to be primarily on the fast-spiking interneurons, though they are seen on both fast and nonfast-spiking interneurons, and postsynaptically on pyramidal cell bodies (Poorthuis et al., 2013). There is some debate about whether or not alpha7 receptors are located presynaptically, with some studies indicating only presynaptic alpha4beta2 receptors (Lambe et al., 2003; Poorthuis

51 et al., 2013) and others indicating both (Gioanni et al., 1999; Dickinson et al.,

2008).

Second, location and presence of each receptor subtype changes based on the cortical layer examined. For example, glutamatergic projection neurons in layer V and VI are modulated by presynaptic beta2-containing receptors (Lambe et al., 2003; Poorthuis et al., 2012), whereas neuronal inputs into other layers are not (Poorthuis et al., 2012). Similarly, there are fewer postsynaptic alpha7 receptors on layer II/III pyramidal cells than in layers V and

VI, and alpha7-mediation of fast-spiking interneurons goes from high in layers

II/III to low in layer VI (Poorthuis et al., 2012).

Third, presynaptic mediation by each subtype changes depending on the region the efferent originates. The PFC receives inputs from all over the brain, and each subregion of the PFC differs (Hoover & Vertes, 2007). Some of the major glutamatergic inputs that innervate many regions of the PFC are from the

MDthal, basolateral amygdala (BLA), and ventral hippocampus (VH; Hoover &

Vertes, 2007). The projections from the MDthal are mediated, in large part, by presynaptic beta2 containing receptors (Lambe et al., 2003; Gioanni et al., 1999), but some alpha7 mediation is possible as well (Dickinson et al., 2008; Gioanni et al., 1999). Presynaptic receptor enhancement has been less well studied for the other glutamatergic projections, but likely involves some contribution from both subtypes.

Fourth, both alpha7 and alpha4beta2 receptors affect transmitter release and produce long-term changes by increasing intracellular calcium levels;

52 however, they do so via decidedly different mechanisms. Alpha4beta2-mediated calcium influx is dependent upon voltage-gated calcium channels that are opened by the local depolarization created when sodium flows into the open receptor channel (Dickinson et al., 2008). Alpha7 receptors, on the other hand, are permeable to calcium themselves and co-localize with intracellular ryanodine receptors, which bind calcium (Dajas-Bailador & Wonnacott, 2004; Dickinson et al., 2008). Thus, calcium influx through alpha7 receptors activates the ryanodine receptors, and further calcium is released from intracellular stores (calcium- induced calcium release, CICR). CICR has been shown to increase transmitter release via ERK 1/2’s phosphorylation of synapsin-1 (Dickinson et al., 2008), a protein that tethers reserve vesicles to the cytoskeleton for release (Matsubara et al., 1996). ERK 1/2 phosphorylation has also been linked to plasticity-dependent learning, providing the mechanism by which alpha7 receptors can aid in this process (Kushner et al., 2005). The effects of alpha4beta2 receptors were not mediated by ERK or synapsin-1 (Dickinson et al., 2008). In conclusion, both alpha7 and alpha4beta2 receptors mediate glutamate transmission in the PFC, but often to differing degrees, on different cellular locations, and via different mechanisms.

4.1.4 Experimental rationale and hypothesis

Based on their joint, but differing roles in mediating glutamate transmission in the PFC, the purpose of this experiment is to determine to what

53 degree alpha7 and alpha4beta2 receptors each mediate the PFC glutamate release seen after stimulation of the NAcSh with NMDA.

It is hypothesized that 1if alpha7 receptors are necessary for the mesolimbically-stimulated PFC glutamate release, then the alpha7-specific antagonist, MLA, delivered directly into the PFC just before NMDA stimulation of the NAcSh will attenuate or completely block the resultant glutamate release in the PFC; and 2if alpha4beta2 receptors are not necessary for the mesolimbically- stimulated PFC glutamate release, then the alpha4beta2-specific antagonist,

DHβE, delivered directly into the PFC just before NMDA stimulation of the NAcSh will not block the resultant glutamate release in the PFC.

4.2 Methods

4.2.1 In vitro calibration of MEAs

Microelectrode arrays (MEAs) were calibrated in vitro using the FAST-16

MKII electrochemical recording system just prior to implantation. Constant potential amperometry was conducted using an applied potential of +0.7 V versus an Ag/AgCl reference electrode just as is done in the brain. Calibrations were performed in a stirred solution of PBS (0.05 M, 40 ml, pH 7.4, 37 C). After a stable baseline was established, ascorbic acid (AA; 500 µL, 20 mM), glutamate

(40 µL, 3x 20 mM), dopamine (DA; 40 µL, 20 mM), and hydrogen peroxide (H2O2,

40 µL, 8.8 mM), were sequentially added to the calibration beaker for final beaker concentrations of 250 µM, 60 µM, 20 µM, and 8.8 µM, respectively.

Amperometric signals were acquired at a rate of 1.0 Hz. The slope (sensitivity to glutamate, nA/µM glutamate), limit of detection (L.O.D, µM glutamate), selectivity

(ratio of glutamate sensitivity over ascorbic acid sensitivity), and linearity of glutamate sensitivity (R2) were calculated. In order to be used for subsequent in vivo recordings, the MEAs had to conform to the following calibration criteria: (i) similar background current (i.e., no greater than a 20 pA difference) between the glutamate-sensitive and sentinel channels, (ii) linear response to increasing concentrations of glutamate (R2 > 0.998), (iii) a minimum slope of -0.003 nA/µM glutamate, (iv) a minimum L.O.D of < 0.5 M, (v) a high selectivity for glutamate over either AA or DA (i.e., > 50:1), and (vi) similar sensitivity to the reporting molecule H202 on all four channels (>80% similarity for each channel pair).

Figure 7 depicts a representative in vitro calibration. The tracings represent change in current in response to the addition of the chemicals listed above (indicated by arrows). The top channel is the glutamate- sensitive channel

(Gluox), and the bottom channel is the background channel (sentinel). The addition of AA produces little to no change in current on either channel.

Successive additions of glutamate produce large, definitive increases in current on only the Gluox channel, and these increases are linear as the beaker concentration of glutamate gets progressively higher. The addition of DA produces no change in current indicating the strength of the m-PD layer. Finally, the addition of H202 produced equal increases in current across both channels, indicating all channels are similarly sensitive to the reporting molecule. An important result to note is that the addition of every chemical, except glutamate,

55 produced an equal response on both channels. This point is of paramount importance as it is the basis of the self-reference technique by which the glutamate signal is isolated in vivo.

4.2.2 Implantation of MEAs and infusion cannulae

Animals were anesthetized using isofluorane (2%, 0.8 L/min) and implanted with an MEA unilaterally in the PFC (in mm from bregma: AP +2.7, ML

+ 0.65, DV -3.9; hemispheres counterbalanced). Stainless steel guide cannulae

(Plastics One, Roanoke, VA), used for intra-cortical infusions of DHβE and MLA, were secured anterior to the MEAs (20 degrees; wax), such that the tip of the infusion cannula was positioned 40-70 µm from the upper portion of the more ventral pair of recording sites (see figure 8), and implanted with the MEA. A second guide cannula (Plastics One, Roanoke, VA), used for intra-NAcSh infusions of NMDA, was implanted ipsilaterally (at a 24 degree angle, in mm from bregma: AP – 1.105, ML + 0.70, DV – 7.88). The actual ventral termination of the guide cannula is -6.88 mm, accounting for a 1 mm extension of the infusion cannula beyond the tip of the guide. All infusion cannulae used for a particular animal were measured to extend equally from the bottom of the guide cannula to ensure consistency of delivery location. A dummy cannula was inserted into both guide cannulae and extended 0.7 mm beyond the tip of the guides. The Ag/AgCl reference electrode was implanted in the contralateral side at a site distant from the recording area. All coordinates were determined using a stereotaxic atlas

(Paxinos & Watson, 1998).

4.2.3 In vivo recordings and intra- cortical infusions

After MEA and cannulae implantation, rats were tested in 3 consecutive recording sessions. During recording sessions, rats were allowed to move freely in a wooden recording box (dimensions: H 57.2 cm; W 341.9 cm; L 317.0 cm).

The first day after MEA implantation surgery consisted of placing the rats in the wooden testing box, without connecting them to the preamplifier, to allow them to habituate to their environment prior to testing. On the second day after implantation recordings of cortical glutamate began. At the onset of each testing day, animals were placed in the recording box and connected to the preamplifier.

Animals then remained undisturbed for 1 - 3 hours, to allow for signal stabilization to occur, before any drugs were delivered. Once the baseline period had concluded, the dummy cannula was removed and 1.0 µL of vehicle (aCSF) was delivered into the NAcSh to serve as an infusion control and to clean away any blood or tissue from the bottom of the NAcSh guide cannula. Then, following a 10-minute delay, either MLA (saline vehicle, 3.38, or 6.75 μg, in 0.5 μL, pH 7.1-

7.4) or DHβE (saline vehicle, 3.38, or 6.75 μg, in 0.5 μL, pH 7.1-7.4) was infused directly into the PFC (MLA or DHβE drug between subjects, MLA or DHβE dose within subjects). Two minutes after the intra-PFC infusion, NMDA (0.15 μg, in 0.5

μL) was infused into the NAcSh. The two- minute delay post intra-PFC infusion was to allow for drug diffusion and signal re-stabilization. All drugs were delivered over about 2 seconds using an infusion cannula attached to a Hamilton PB600-1 manual dispenser (Hamilton Company, Reno, NV). On the subsequent two test

57 days, the doses of MLA or DHβE not given on the first day were delivered using the same procedure. MLA and DHβE dose order was counterbalanced among animals, and no animal received both MLA and DHβE.

4.2.4 Data analysis

Comparisons were performed on all dependent measures derived from the FAST-16 data file mentioned above (basal glutamate, maximum glutamate amplitude, time to glutamate peak onset, T50, T80, and glutamate peak duration) using analysis of variance (ANOVA) by the IBM SPSS statistics program (version

22, IBM Corporations, Armonk, NY). An initial, omnibus ANOVA was performed for each dependent measure using dose (saline vehicle, 3.38, or 6.75 μg, in 0.5

L) as a within-subjects factor for each drug separately (MLA, DHβE). If significance was determined, post-hoc comparisons were performed to determine specific locations of significance. The Huyen-Feldt correction was used to minimize the occurrence of type II errors. Significance was defined as p <

0.05.

4.3 Results

4.3.1 MEA and cannulae placements

All subjects included in this analysis had confirmed MEA and cannula placements within the prelimbic/infralimbic region of the medial PFC and cannula placements within the anterior portion of the NAcSh. Figure 9 depicts a

58 representative sagittal photomicrograph section of a PFC sensor and cannula placement and a NAcSh cannula placement along with a descriptive cartoon for comparison. The black arrows on the cartoon mirror the placements of the two cannulae and MEA shown in the photomicrograph for ease of comparison.

Colored arrows on the photomicrograph mark the ventral termination of all items that were implanted (see figure legend for identification of each color). As shown, the representative placements fall within their respective desired regions. The representative photomicrograph also illustrates the minimal tissue damage produced by both the MEA and infusion cannulae.

4.3.2 NMDA- induced PFC glutamate increase attenuated by MLA

In order to determine whether or not the mesolimbically-stimulated glutamate release in PFC is mediated locally by alpha7 receptors, NMDA (0.15

µg in 0.5 µL) was administered into the NAcSh of rats two minutes after one of three, counterbalanced, doses of MLA was delivered into the PFC (saline vehicle, 3.38, 6.75 µg in 0.5 µL). Figure 10 displays representative tracings of the

MEA’s electrochemical signal generated by the combination of NMDA and each of the three doses of MLA, as well as an example local glutamate control infusion. Glutamate control infusions were delivered on days where no signal was detected after administration of NMDA and MLA to ensure proper functioning of the MEA. In this figure, the red tracing (top) represents the signal generated by the glutamate-sensitive channel (Gluox) following a saline/ NMDA combination. The blue tracings represent the signal generated from the sentinel

59 channel. The sentinel channels are sensitive to every electro active molecule (at

+0.7 V) that the Gluox channels are except for glutamate. The top green tracing represents the resultant signal after the sentinel channel is subtracted from the

Gluox channel. Thus, all of the green tracings indicate the self-referenced (Self

Ref) signal or the signal generated exclusively by the oxidation of glutamate.

The self-referenced tracings for each NMDA/MLA combination (0.15 µg

NMDA with vehicle (saline), 3.38, 6.75 µg MLA in 0.5 µL) reveal the stability of each baseline both before and after the drug effect. Baselines are displayed uniformly for ease of comparison (for actual mean values, see table 1). NMDA

(0.15 µg) delivered to the NAcSh two minutes after saline was delivered into the

PFC resulted in a sharply-peaked increase in PFC glutamate (3.90 µM) that occurred 65 seconds post injection and persisted for 47 seconds. However, when

NMDA (0.15 µg) was delivered in to the NAcSh two minutes after the low dose of

MLA (3.38 µg) was delivered into the PFC (same animal, 24 hours later), the resultant glutamate release was attenuated (1.89 µM). Peak duration (21 seconds) was also attenuated, while the time to peak onset was not affected (55 seconds). When NMDA (0.15 µg) was delivered into the NAcSh two minutes after the high dose of MLA (6.75 µg), the resultant glutamate release was completely blocked. Subsequent infusion of glutamate (500 µM; immediately following the

MLA/NMDA infusions) directly into the PFC resulted in a glutamate peak similar in amplitude, shape, and duration as the one seen after saline/NMDA, which confirmed that the MLA-induced blockade was not due to a loss in MEA sensitivity. Importantly, MLA dose order was counterbalanced across animals as

60 an additional precaution to ensure MLA’s effects were not due to a loss in sensor sensitivity. For example, this representative animal received the high dose of

MLA on the first session and the saline dose on the third.

4.3.3 Group data

Overall, the group data (N=6) were consistent with the individual data depicted in figure 10. Basal glutamate levels did not differ significantly before or after any NMDA infusion (0.15 µg NMDA/ saline: 1.78 + 1.46 µM, 0.15 µg NMDA/

3.38 µg MLA: 1.27 + 0.92 µM, 0.15 µg NMDA/ 6.75 µg MLA: 1.26 + 1.01 µM; p >

.9). Thus, basal glutamate levels did not affect the stimulated glutamate increases, nor were they affected by MLA infusions. However, as indicated above, peak amplitude (figure 11) differed significantly between groups (0.15 µg

NMDA/ saline: 3.54 + 1.35 µM, 0.15 µg NMDA/ 3.38 µg MLA: 0.67 + 0.29 µM,

0.15 µg NMDA/ 6.75 µg MLA: 0.19 + 0.06 µM; F2,17 = 5.186, P = .019), with the high dose of MLA significantly attenuating glutamate amplitudes (F1,11 = 6.194, P

= .032) and the low dose of MLA producing a trend towards a significant attenuation that did not quite reach statistical significance (F1,11 = 4.345, P =

.064). Time to peak onset, T50 and T80 clearance, and glutamate peak duration were only able to be calculated when glutamate peaks reached the criteria of 3x noise. Instances where MLA infusions produced a complete blockade of the glutamate peak were not included as the abovementioned values were impossible to calculate. For identifiable glutamate peaks, MLA infusions did not affect time to peak onset (F2,14 = .053, p > .9), T50 (F2,14 = .060, p > .6), or T80

(F2,14 = .477, p > .6) clearances (see table 1 for MEAN and SEM values). There was a trend towards a significant reduction in peak duration (F2,14 = 1.987, P =

.180), but due to a reduced number of data points, statistical significance could not be reached. Infusions of aCSF into the NAcSh produced no detectable change from baseline in any animal tested, therefore, a visual representation was not included and no statistics were performed on any of the dependent measures mentioned above.

4.3.4 NMDA- induced PFC glutamate increase unaffected by DHE

In order to determine whether or not the mesolimbically-stimulated glutamate release in the PFC is mediated locally by alpha4beta2 receptors,

NMDA (0.15 µg in 0.5 µL) was administered into the NAcSh of rats two minutes after one of three, counterbalanced, doses of DHE was delivered into the PFC

(saline vehicle, 3.38, 6.75 µg in 0.5 µL). Figure 12 displays the self- referenced signal of two representative animals comparing the difference in effects between the high dose of DHE (top, red; 6.75 µg) and the high dose of MLA (bottom, green; 6.75 µg). The self-referenced tracings for each animal reveal the stability of each baseline both before and after the drug effect. Baselines are displayed uniformly for ease of comparison (For actual mean values, see table 1).

NMDA (0.15 µg) delivered to the NAcSh two minutes after saline was delivered into the PFC resulted in a sharply- peaked, multiphasic increase in PFC glutamate (top, red tracing; 4.49 µM) that occurred 53 seconds post injection and persisted for 39 seconds. This peak was quite similar in total amplitude (3.90 62

µM), peak onset latency (65 seconds), and duration (47 seconds) to that produced in the second animal (bottom, green tracing), which is described above. However, when NMDA (0.15 µg) was delivered in to the NAcSh two minutes after the high dose of DHE (6.75 µg) was delivered into the PFC, the resultant glutamate release was only slightly attenuated (3.81 µM). This is in marked contrast to the complete abolishment of the NMDA-stimulated glutamate peak that is seen following a local infusion of MLA (6.75 µg). Peak onset (43 seconds) and duration (40 seconds) were not affected by the presence of DHE.

4.3.5 Group data

Overall, the group data (N=6) were consistent with the individual data depicted in figure 12. Basal glutamate levels did not differ significantly before or after any NMDA infusion (0.15 µg NMDA/ saline: 0.59 + 0.19 µM, 0.15 µg NMDA/

3.38 µg DHE: 0.55 + 0.26 µM, 0.15 µg NMDA/ 6.75 µg DHE: 0.81 + 0.23 µM; p

> .7). Thus, basal glutamate levels did not affect the stimulated glutamate increases, nor were they affected by DHE infusions. Additionally, and in complete contrast to MLA infusions, DHE infusions into the PFC did not significantly attenuate glutamate peaks produced by NMDA stimulation of the

NAcSh (see figure 13). Mean glutamate peak amplitudes did trend downward slightly with increasing doses of DHE (0.15 µg NMDA/ saline: 4.05 + 1.23 µM,

0.15 µg NMDA/ 3.38 µg DHE: 3.01 + 0.76 µM, 0.15 µg NMDA/ 6.75 µg DHE:

2.74 + 0.58 µM) but this downward trend did not approach significance (F2,17 =

.595, P = .564). Similar to basal and stimulated glutamate levels, time to peak 63 onset (F2,17 = 1.205, p > .3), T50 (F2,17 = .823, p > .4) and T80 (F2,17 = .466, p >

.6) clearance, and glutamate peak duration (F2,17 = .102, p > .9) did not differ between any dose of DHE (see table 1 for MEAN and SEM values). Infusions of aCSF into the NAcSh produced no detectable change from baseline in any animal tested, therefore, a visual representation was not included and no statistics were performed on any of the dependent measures mentioned above.

4.4 Discussion

The results from this chapter indicate, for the first time, that the glutamate released after mesolimbic stimulation is mediated locally in the PFC via alpha7 receptors. This was determined because the selective alpha7 antagonist MLA, infused directly into the PFC just prior to NMDA stimulation of the NAcSh, produced a dose-dependent blockade of the stimulated glutamate release in

PFC. The high dose of MLA produced a nearly complete blockade of the glutamate release (~95%), which was not due to a loss in sensitivity of the MEA.

Furthermore, these results indicate that alpha4beta2 receptors play only a limited role in the mesolimbically-stimulated PFC glutamate release. This was determined because the selective alpha4beta2 antagonist DHE, infused directly into the PFC just prior to NMDA stimulation of the NAcSh, produced only a slight attenuation of the mesolimbically-stimulated PFC glutamate release that was not dependent on the dose of DHE. In contrast, local infusions of DHE and MLA did not result in any change in basal glutamate levels, time to glutamate peak onset, or glutamate peak clearance (T50, T80, peak duration; determined only for 64 glutamate peaks that met the criteria of 3x noise). Thus, this data confirms that alpha7, and not alpha4beta2, receptors are primarily responsible for the local mediation of the PFC glutamate released after NAcSh stimulation with NMDA.

The following discussion section will focus on an important methodological question related to this data, a discussion of possible explanations and significance of the results reported in this chapter, and finally some concluding statements about how this data supports the use of the mesolimbic stimulation assay for the final set of experiments characterizing the two alpha7 PAMs.

4.4.1 Methodological justifications

A very important methodological question that may arise when examining this data is in reference to the selection of MLA and DHE doses. Many of the studies that have compared nicotinic receptor regulation of PFC glutamate release have not been in vivo (Lambe et al., 2003; Dickinson et al., 2008).

Therefore, dose selection was based on a number of other studies, as well as several preliminary observations. A number of previous studies determined behaviorally- relevant doses of MLA and DHE when delivered intra-cranially

(Bettany & Levin, 2001; Addy et al., 2003; Nott & Levin, 2006). These studies determined that the low dose of DHE used in this study (3.38 g), infused directly into the VH (Levin et al., 2002) or BLA (Addy et al., 2003), was able to significantly impair spatial working memory as determined by the 16-arm radial maze task. The high doses of DHE and MLA used in this study (6.75 g) also impaired working memory when infused into the hippocampus (dorsal and 65 ventral; Nott & Levin, 2006; Levin et al., 2002) and BLA (Addy et al., 2003).

Lower doses of MLA did not produce significant deficits in working memory.

However, doses of MLA that were higher than 6.75 g, which did produce memory impairments, also show decreased specificity for the alpha7 receptor and can cause seizures (Felix & Levin, 1997).

Some preliminary observations also guided dose selection. Preliminary studies for this experiment examined a commonly- used, behaviorally- impairing dose of each drug that was the most similar in concentration (3.38 g DHE:

18.97 mM; 6.75 g MLA: 15.43 mM). Since a near maximal blockade of the mesolimbically-stimulated PFC glutamate release was demonstrated with the

6.75 g dose of MLA in preliminary studies, and higher doses can produce nonspecific effects and seizures, a lower rather than higher dose of MLA was used as the second dose. Contrarily, since 3.38 g DHE did not block glutamate in preliminary studies, and higher doses produce more severe memory- impairing effects, a higher dose was selected as the second dose. Lastly, and importantly, these doses were also in accordance with the few studies that examined nicotinic receptor’s effects on PFC glutamate release in vivo (Gioanni et al., 1999).

Therefore, to conclude, these doses were the best that could be used for the present study.

4.4.2 Discussion of results

Three questions that may arise when examining the data in this chapter are addressed in this section. First, is it surprising that DHE still produced a 66 slight attenuation of the mesolimbically- stimulated glutamate release in PFC considering MLA’s strong attenuation? This was not a surprising result because, when examining the group data, it is clear that even the high dose of MLA did not completely block the glutamate release, on average. Additionally, if the assumption is being made that the endogenous signaling that is activating the nicotinic receptors is ACh/ choline (Zmarowski et al., 2005; chapter 3 of this dissertation), then it is unlikely that only alpha7 receptors would be activated.

Choline is selective for the alpha7 receptor, but a study by Papke & Porter Papke

(2002) showed that ACh has relatively equal affinity for both alpha7 and alpha4beta2 receptors, which confirms the likely activation of both receptor subtypes. Therefore, it would be expected for the glutamate release to mediated, at least in part, by both receptor subtypes. This notion is supported by a number of studies that demonstrate the involvement of both subtypes in glutamate release that is stimulated both in vivo (Konradsson-Geuken et al., 2009; Bortz et al., 2013; Gionnani et al., 1998) and in vitro (Gionnani et al., 1998; Dickinson et al., 2008; Lambe et al., 2003). However, the largest piece of confirmation comes from a study performed by Valentini et al. (in preparation). This study utilized the same mesolimbic stimulation assay that is described in this dissertation, except glutamate was measured in the PFC with microdialysis. They show that local perfusion of the alpha7 antagonist BGT produces a nearly complete blockade of the mesolimbically-stimulated PFC glutamate release, while local perfusion of

DHE produced a slight attenuation (Valentini et al., In preparation). Thus, their data mirrors the data described in this chapter exactly, simply using a different

67 alpha7 antagonist and a different method to measure PFC glutamate release. To conclude, it was expected that local infusion of DHE would still produce some small degree of attenuation even if the glutamate release was mediated, in large part, via the alpha7 receptor.

Second, where does the glutamate signal measured in this experiment originate? As described previously, the most likely mechanism by which NAcSh stimulation leads to choline release in the PFC is via BF cholinergic projections to the PFC. A subset of BF cholinergic neurons are contacted directly by the medium spiny projections from the NAcSh (Zaborszky & Cullinan, 1992) and terminate in the medial PFC (Luiten et al., 1987), and ablation of these BF cholinergic neurons eliminates the ACh release in PFC after mesolimbic stimulation (St Peters et al., 2011). Thus, it is easy to see how NAcSh stimulation can lead to ACh/ choline increases in the PFC (as demonstrated in Zmarowski et al., 2005; and chapter 3). Data from this chapter, for the first time, completes the abovementioned circuit by demonstrating that ACh/choline drives the glutamate release locally via its activation of nicotinic (mainly alpha7) receptors in the PFC.

What is not known is where the glutamate neurons that are activated by alpha7 receptors in the PFC originate from. Though the answer to this question has yet to be experimentally determined, there is a major clue from the data in this chapter that may begin to narrow down the search. This clue is that the glutamate release evoked after mesolimbic stimulation is not, in large part, driven by alpha4beta2 receptors.

There are many smaller glutamatergic inputs from other regions within the

PFC that terminate where the MEAs are implanted (medial PFC), but the largest projections originate from the hippocampal formation (HF), basolateral amygdala

(BLA), and medial dorsal thalamus (MDthal; Hoover & Vertes, 2007). Previous studies have indicated the MDthal’s glutamatergic projections to PFC do contain nicotinic receptors on their terminals, but they are beta2-containing receptor subtypes (Lambe et al., 2003). For example, ibotenic acid lesions of the MDthal prevented local alpha4beta2 agonist-stimulation of PFC glutamate release

(Parikh et al., 2010). Also, inputs into PFC layer V have been shown to be mainly from the MDthal, and a study by Poorthuis et al. (2012) showed that layer V inputs to the PFC are modulated by presynaptic beta2-containing receptor subtypes only. Therefore, since alpha4beta2 receptors did not mediate the glutamate released after mesolimbic stimulation, it is unlikely that this glutamate came from neurons originating in the MDthal. This hypothesis is further supported by a pilot study performed by this lab that showed that ibotenic acid lesioning of the MDthal did not block the glutamate released in PFC after mesolimbic stimulation (Bortz et al., unpublished observations).

The only caveat that may preclude this assumption from being true is the potential presence of alpha7beta2 receptors. Recent studies have demonstrated that alpha7 subunits do have the ability to co-assemble with beta2 subunits forming a heteromeric alpha7 receptor, but it is not known if this occurs endogenously, to what degree, or where in the brain (Murray et al., 2012).

However, the studies mentioned above identified alpha4beta2 receptors primarily

69 via the beta2 subunit, thus allowing the possibility that the receptors identified in the studies above could have been alpha7beta2 instead of alpha4beta2. If this were the case, and functional alpha7beta2 receptors do exist in the PFC, then the possibility still exists that the glutamate could originate from the MDthal.

Though the pilot study mentioned above likely negates this possibility, a complete experiment is still needed to confirm the initial observation that MDthal lesions do not block mesolimbically-stimulated PFC glutamate release.

With the probable exclusion of the MDthal, the glutamate likely originates from either the BLA or HF. Interestingly, the HF inputs contact the medial PFC at a higher density than the BLA where the platinum recording sites of the MEA terminate (approximately layer II/III; Hoover & Vertes, 2007). Some studies have indicated that glutamatergic inputs to layer II/III do not contain alpha7 receptors

(Poorthuis et al., 2013); but these studies utilized nicotine as their stimulus, which has a low affinity for alpha7 receptors. Additionally, local application of alpha7- selective agonists delivered to the region directly adjacent to the MEA’s platinum recording sites has been shown to induce glutamate release (Bortz et al., 2013).

Another possibility is that the glutamate does not come from local presynaptic excitation, but from disinhibition of pyramidal cells. Alpha7 receptors are also present on GABAergic interneurons in layer II/III of the medial PFC (Poorthuis et al., 2013), which can inhibit each other via lateral synaptic contacts. Thus, the consequence of alpha7 receptor activation in the PFC by ACh/choline could be disinhibition of pyramidal cells via recurrent collateral inhibition of GABAergic interneurons by other interneurons. Interestingly, there are no indications of

70 alpha4beta2 receptor expression on presynaptic glutamatergic terminals that contact the medial PFC in layer II/III, and only very limited expression of this receptor subtype postsynaptically on GABAergic interneurons in these layers

(Poorthuis et al., 2013). Though the answer to this question remains to be experimentally determined, the selective mediation of the stimulated PFC glutamate release in this experiment by alpha7 receptors, and not alpha4beta2 receptors, indicates the glutamate may be from a source other than the MDthal.

Third, what is the functional significance of the glutamate released after mesolimbic stimulation being mediated by alpha7 instead of alpha4beta2 receptors? As was previously mentioned in this dissertation, the mesolimbic stimulation assay has been hypothesized to be a model of top-down regulation because it was able to improve rodents’ failing performance when a distractor was introduced during a sustained attention task (St Peters et al., 2011). Thus, this data supported the notion that the neurochemical consequence of mesolimbic stimulation, i.e. the release of ACh/choline and glutamate in PFC, was indeed cognitively- relevant in terms of sustained attention. However, this cognitive benefit is likely due to alpha4beta2 receptor enhancement of MDthal inputs into the PFC (Parikh et al., 2008; Parikh et al., 2010). As was established above, the glutamate measured in this experiment is not likely to be from the

MDthal and is not mediated by the alpha4beta2 receptor. Therefore, the glutamate increases measured in this experiment may have relevance to other cognitive operations.

Per the discussion in the previous section, one of the potential sources of the glutamate measured in this experiment is the HF. Interestingly, the glutamatergic projections from the ventral hippocampus (VH) to the PFC have been shown to drive gamma rhythmicity in the PFC via excitatory synapses onto

GABAergic interneurons (Takita et al., 2007; Korotkova et al., 2010; Brockmann et al., 2011). This is important because gamma rhythmicity has been shown to be necessary for key cognitive functions, such as perception, attention, memory, and synaptic plasticity (see Phillips & Uhlhaas, 2015 for review). Therefore, because the glutamate measured in this experiment is alpha7 mediated and not alpha4beta2-mediated, it could be involved in or the result of recruitment of VH entrainment of gamma in the PFC via a top-down regulatory mechanism. This hypothesis is supported by the fact that alpha7 receptor activation in the hippocampus has already been shown to play a major role in the maintenance of gamma rhythmicity (Song et al., 2005), and that alpha7 receptors are present on the GABAergic interneurons that regulate gamma rhythmicity in the PFC

(Timofeeva & Levin, 2011; Poorthuis et al., 2013).

In terms of schizophrenia, evidence from other studies suggests that gamma oscillatory activity is disrupted in this disease. This is evidenced by reductions in PFC markers of receptors and neurons that are critical for the generation of gamma, such as GAD67, parvalbumin, and alpha7 receptors; as well as EEG recordings in patients that show reduced low and high frequency activity during working memory and cognitive control tasks (see Phillips &

Uhlhaas, 2015 for review). Thus, an assay that could tap into the neural

72 mechanisms involved with the production of gamma in the PFC would be very valuable in terms of testing the efficacy of alpha7 agonists and PAMs in animal models of schizophrenia. Further testing is required to determine if the mesolimbic assay actually fits this criteria, but the determination that the evoked glutamate release is alpha7 mediated and not alpha4beta2 mediated raises the possibility that this could be the case.

4.4.3 Conclusions

The purpose of this experiment was to determine whether or not the glutamate produced by NMDA-stimulation of the NAcSh would require the activation of alpha7 and/or alpha4beta2 receptors. The reason for this was twofold. First, this dissertation sought to find an assay where endogenous glutamate release in the PFC was mediated by the alpha7 receptor. This is beneficial because glutamate release in the PFC is critical for executive functions that are deficient in schizophrenia and because alpha7 ligands make up a leading class of drugs that are being investigated to treat those executive function deficits. Therefore, based on the data reported in this chapter, this assay could be used to examine novel alpha7 ligands in the future. Second, this experiment sets the stage for the final, and primary, experiment of this dissertation, which is to determine if PAMs will potentiate neurotransmitter release (glutamate) in vivo in a similar fashion as they have done in vitro (see

General Introduction for further detail). Answering this question requires an assay where release of the reporting neurotransmitter (glutamate) is driven by

73 endogenous afferent activity (choline) at the alpha7 receptor. After determining that mesolimbic stimulation is able to dose-dependently change choline levels in the PFC (chapter 3), it was necessary to demonstrate that cholinergic signaling mediated our previously measured glutamate release via the alpha7 receptor.

Having accomplished this, the next chapter in this dissertation seeks to address the final question as laid out in the general introduction.

CHAPTER 5

POSITIVE ALLOSTERIC MODULATORS OF THE ALPHA7 RECEPTOR

POTENTIATE MESOLIMBICALLY-STIMULATED PFC GLUTAMATE

RELEASE DEPENDENT UPON AFFERENT ACTIVITY

5.1 Introduction

Positive allosteric modulators (PAMs) of the alpha7 receptor are a novel class of drugs that are just beginning to gain traction in clinical and preclinical studies. The reason for this is because PAMs have the ability to potentiate the alpha7 receptor without activating it, which may be more cognitively beneficial than activating the receptor directly as conventional agonists do. However, all knowledge about the effects of PAMs on neurotransmission has come from in vitro studies, which greatly limits their translatability. Therefore, examining PAMs in vivo is a great need. This project seeks to characterize the ability of two novel alpha7 PAMs to potentiate glutamate release in vivo in the PFC as a function of varying local choline levels in order to meet that need. Accomplishing this required an assay where glutamate release in the PFC was driven by afferent activation (choline) at the site where the PAMs bind (the alpha7 receptor). The

75 mesolimbic stimulation assay has been shown to meet these two requirements, per the 3rd and 4th chapters of this dissertation; therefore, it was used for the experiments described in this chapter. The following introduction section will step more clearly and directly through the current state of alpha7 agonists, any evidence to suggest why PAMs may be more cognitively beneficial than agonists, and finally the limited data that currently exists on alpha7 PAMs.

5.1.1 Review of alpha7 agonists

As described in chapter 1, the alpha7 receptor has been highlighted as a leading target for cognitive-enhancer drug development for a number of reasons

(see General Introduction for description of reasons). A multitude of positive results from studies performed in intact and schizophrenia model animals have supported this position. For example, the partial alpha7 agonist, GTS-21, improved auditory gating in DBA/2 mice (a genetic model of impaired sensory gating; Simosky et al., 2001), and improved working memory in non-human primates (Briggs et al., 1997). Another partial agonist, SSR180711, improved intact rodent performance on the novel object recognition task, Morris water maze, and radial arm maze (Pichat et al., 2007; Hashimoto et al., 2008), which are tests of short-term and spatial working memory. SSR-180711 also reversed cognitive flexibility (attentional set-shifting) deficits produced by neonatal disruption of the ventral hippocampus (Brooks et al., 2012), a well-validated animal model of schizophrenia. The alpha7 agonist ABT-107 improved working memory performance when given to monkeys, and, similarly, was able to

76 improve social recognition, a memory task, in rats (Bitner et al., 2010). Repeated dosing of Tropisetron improved novel object recognition in a sub-chronic PCP schizophrenia mouse model, as well as apomorphine-induced pre-pulse inhibition (PPI) deficits (Hashimoto et al., 2006; Kohnomi et al., 2010). Another potent, full alpha7 agonist, TC-5619, reversed PPI deficits and restored social approach in a mouse model of schizophrenia (Swerdlow et al., 1994).

Additionally, TC-5619 was able to reverse apomorphine-induced deficits in PPI and improve novel object recognition in rats (Hauser et al., 2009). Finally, the alpha7 agonist CP-810123 reversed disruptions in sensory gating and novel object recognition in two different rat models of schizophrenia (acute amphetamine and scopolamine injections, respectively; O'Donnell et al., 2010).

Similar results have been seen with several other alpha7 agonists as well; therefore, the preclinical evidence certainly supports the alpha7 receptor as a promising target for drug development.

Several alpha7 agonists and partial agonists have gone to clinical trials as well, but the results have been heterogeneous. An initial study with the alpha7 agonist, Tropisetron, demonstrated that it could reverse sensory gating deficits, as well as improve immediate and delayed memory in schizophrenic patients

(Zhang et al., 2012). A second study, which had patients taking Tropisetron along with the antipsychotic Risperidone, revealed that this alpha7agonist generally improved negative symptoms (Noroozian et al., 2013). Another alpha7 agonist,

TC-5619, significantly improved performance on a working memory task called the Groton Maze Learning Task, as well as negative symptoms (Lieberman et al.,

2013). The most well studied alpha7 ligand to date is the partial agonist GTS-21 or DMXB-A. GTS-21 enhanced attention, working memory, and episodic memory in healthy human volunteers (Kitagawa et al., 2003), and general neurocognitive functioning and sensory gating in schizophrenic patients (Olincy et al., 2006).

These results are very promising, but they are marred by contradictory data as well. For example, a larger phase II trial with GTS-21 failed to reproduce any of the cognitive-enhancing effects seen in the smaller, first trial (Freedman et al., 2008), and results seen with Tropisetron and TC-5619 were varied depending on whether the patient was a smoker or nonsmoker (Zhang et al., 2012;

Lieberman et al., 2013). Additionally, several agonists have been discarded due to troublesome side effects. For example, an agonist by memory pharmaceuticals was discontinued because it caused patients to become irritable and combative (see Freedman, 2014 for review), and two agonists from Pfizer,

PHA-543613 and PHA-568487, were discontinued from clinical trials because of an occasional incidence of asymptomatic ventricular tachycardia in healthy volunteers (Wishka et al., 2006). In conclusion, though there are some very promising results, the inconsistencies with direct agonists remain troubling, and it is clear that the reason for the negative results requires investigation.

5.1.2 Possible explanations

There are a number of possibilities for why alpha7 agonists have shown mixed results in clinical trials. One possibility is the manner by which the clinical trials are carried out. First, clinical trials are not carried out uniformly in the use of

78 smokers, nonsmokers, or both. For example, the GTS-21 clinical trials were performed with non-smokers (Olincy et al., 2006; Freedman et al., 2008), whereas trials with TC-5619 and Tropisetron enrolled both smokers and nonsmokers (Zhang et al., 2012; Lieberman et al., 2013). Importantly, both TC-

5619 and Tropisetron showed differing and opposite effects in smokers versus nonsmokers, such that TC-5619 was more effective in smokers and Tropisetron’s effects were not seen in smokers (Zhang et al., 2012; Lieberman et al., 2013).

Therefore, if GTS-21 is later discovered to be more effective in smokers, similar to TC-5619, then its negative phase II trial data could simply have been caused by their use of only nonsmokers. Thus, it is critical to enroll both smokers and nonsmokers in clinical trials and to differentiate effects between the two.

A second issue with the way clinical trial are carried out is that alpha7 agonists are often given to patients who have been taken off their antipsychotic treatments for only a brief period of time. A recent study demonstrated that the ability of a GABA alpha5 positive allosteric modulator to reverse behavioral abnormalities in a well-established model of schizophrenia was no longer seen if the animals were initially given an antipsychotic and then taken off of it (Gill et al.,

2014). Thus, clinical trials must include patients who are either treatment-naïve or who have been taken off of their antipsychotic medications for a longer period of time.

Finally, the doses that are being used in clinical trials may be too high.

There is a wealth of data from preclinical studies in both rodents and primates that ultra-low doses of alpha7 agonists may be more effective than the doses

79 being used (Castner et al., 2011; Werkheiser et al., 2011; Yang et al., 2013). For example, Castner et al. (2011) demonstrated that very low doses of the alpha7 partial agonist, AZD0328 (0.0016 and 0.00048 mg/kg), improved spatial working memory performance in rhesus monkeys, while higher doses (0.48 mg/kg) actually impeded performance. Interestingly, the high dose in this test (0.48 mg/kg) was at the low end of the effective dose range seen in rodents as determined by the same experimenters (Castner et al., 2011). This study provides evidence that there could be a critical difference in effective doses between species (0.48 in rodents vs. 0.00048 in primates) that could be quite large. Therefore, though it is expensive, care and time must be taken to examine a range of doses when performing clinical trials.

5.1.3 Positive allosteric modulators

Although the abovementioned suggestions do present reasonable areas where clinical trials could be improved, another possible explanation for the heterogeneous clinical trial data could be the use of direct agonists. Direct agonists stimulate the receptor regardless of the activity of the afferent neuron, which may result in two things. First, this creates the possibility for false signals because the receptor is stimulated without afferent activation. Second, the primary effect of agonist administration is desensitization of the receptor. Thus, when an afferent signal does comes through, the receptor may be desensitized and offline due to the agonist being present. Together these two effects of direct agonists cause a temporal disengagement in critical neurocircuitry that may

80 counteract whatever benefits the agonist may provide. Positive allosteric modulators (PAMs), on the other hand, are theorized to not have any intrinsic activity, but only potentiate transmission under conditions of afferent activity.

Therefore, PAMs can synergize and enhance endogenous signals rather than competing with them. This is supported by several studies performed in vitro where alpha7 PAM administration increased agonist- induced excitatory currents

(typically ACh or nicotine), but did not produce any current changes in the absence of an agonist (Gronlien et al., 2007; Ng et al., 2007; Williams et al.,

2011a). Therefore, PAMs have the potential to eliminate the potential for false signals and preserve the temporal integrity of neurotransmission (Faghih et al.,

2008).

5.1.4 Positive allosteric modulators and endogenous neurotransmission

The difference between the mechanism of action of direct agonists and

PAMs may be critical when considering executive functioning because of the dynamic and temporally- precise nature of the neurotransmission that produces it. For example, there are two different, identifiable modes of neurotransmitter signaling, tonic and phasic. Both types play different, but individually important, roles in executive functioning. For example, animals performing an attention task show an initial tonic elevation in cortical ACh release that determines the general readiness of the rat to perform as well as the effort being put forth (Kozak et al.,

2006). They also show transient choline spikes that occur simultaneously with the detection of cues. In fact, the presence or absence of the choline transients

81 actually predict whether or not the animal will correctly identify the presence or absence of the cue, respectively (Parikh et al., 2007). Therefore, both the tonic and phasic components of the ACh release, as well as the appropriate timing of those components, are required for successful performance in the task. Direct agonists favor more steady state changes in neurotransmission, which might dampen the signal-to-noise ratio and be less effective at mimicking the timing- specific aspects of phasic signaling (see Sarter et al., 2007 for review). In contrast, a PAM, theoretically, would simply potentiate the signal that was already present, thus eliminating the pharmacologically- induced false signal and enhancing the signal-to-noise ratio. Therefore, a PAM may be more effective at maintaining the precisely-timed tonic-phasic balance needed for the enhancement of attention.

A second example is that neurotransmission in the brain changes depending on level of activation (i.e. task performing or not) of the person or animal. Numerous studies have shown that level of activation or arousal interacts with drug effects (Sarter & Bruno, 1994; Fadel et al., 2001). For example, a study in animals with a ventral hippocampal lesion demonstrated that D2 receptor antagonists only affected prefrontal ACh release when the animal was aroused by a stressor (Laplante et al., 2004). Therefore, a PAM’s ability to enhance neurotransmission specifically under conditions of afferent activity (task performance) could be important.

Third, inappropriate levels of receptor stimulation, whether the stimulation be too great in magnitude or mistimed, often lead to nonspecific effects that do

82 not benefit cognition. For example, a study performed in primates examined direction-specific delay cell activity in the dorsolateral PFC (dlPFC) during a spatial working memory task, and the ability of the primates to recall the location of a cue based on the direction-specific delay cell firing (Yang et al., 2013). What they found was that level of activation of a subset of delay cells that was specific for the location of the cue predicted whether or not the primate would correctly identify the cue location after a time delay (Yang et al., 2013). Next, they showed that high doses of the alpha7 agonist, PHA543613, produced large increases in delay cell firing that were not direction-specific and caused the primate’s performance to fall dramatically and, in some cases, stop altogether. Thus, inappropriate levels of receptor activation, as may be the case with the indiscriminate activation of direct agonists, can be damaging to cognition in some circumstances.

Fourth, the synchronization of neuronal activity has been hypothesized to be the mode by which sets of neurons, which may be distributed across brain regions, form functionally coherent ensembles to perform normal and executive brain functions (Singer, 1999). This synchronization is observable as unified increases and decreases in population activity at different frequencies, and is driven by specific sets of GABAergic interneurons. Importantly, alpha7 receptors have been shown to aid in mediating these neural oscillations through their activation of GABAergic interneurons (Arnaiz-Cot et al., 2008), mainly in the hippocampus and PFC. In fact, alpha7 activation in the hippocampus selectively enhances the activity of GABAergic interneurons (Arnaiz-Cot et al., 2008). This is

83 important because proper timing is the key to synchronizing neural oscillations, and disruption of that timing leads to the portrayal of deficits in executive functions in rodent models similar to those in schizophrenia (reviewed in Phillips

& Uhlhaas, 2015). There is currently no evidence in rodents that direct alpha7 agonists disrupt neuronal synchrony; however, alpha7 agonists also consistently improve cognitive performance in rodents. Therefore, based on PAMs’ ability to operate only under conditions of afferent activity, the possibility exists that a PAM will be better suited to maintain the precise timing needed to synchronize neural networks for multi-brain region communication.

5.1.5 Further justification for positive allosteric modulators

Beyond their closer conformity to the aspects of endogenous neurotransmission, there are two more reasons why PAMs may be more efficacious than direct agonists. The first is that PAM binding sites tend to be less evolutionarily conserved than orthosteric sites, giving them more structural diversity (Yang et al., 2012). For example, as opposed to the single, similar orthosteric binding site at each of the alpha7 subunits, there have been many different PAM binding sites discovered on this receptor. Several sites exist on the extracellular N-terminal domain and bind alpha7 PAMs such as galantamine

(Ludwig et al., 2010), calcium (Eisele et al., 1993), and 5-hydroxy-indole

(Gronlien et al., 2010). PNU120596, a PAM that has been well- characterized and is being used in this study, binds to a separate, intra-subunit transmembrane domain (Collins et al., 2011). This intra-subunit transmembrane domain also

84 binds other PAMs such as LY-2087101 (Young et al., 2008), Invermectin (Collins

& Millar, 2010), and NS-1738 (Collins et al., 2011), and the possibility exists that more than one binding site may exist here. This structural diversity of PAM binding sites may be very beneficial for drug development because it allows for more selectivity when synthesizing drugs.

The second reason is that some PAMs can block receptor desensitization

(Dinklo et al., 2011). PAMs are categorized as type I or type II based on this quality, such that type II PAMs are able to block and reverse receptor desensitization and type I PAMs are not (Gronlien et al., 2007). This is significant because alpha7 receptors desensitize very rapidly with agonist binding (Papke et al., 2000), which could lead to the development of tachyphylaxis and a loss of behavioral effects (Umana et al., 2013). Consequently, there may be a need to increase dosing and, thus, increase the likelihood for side effects. Due to their ability to reverse receptor desensitization, treatment with type II PAMs has been suggested as a mechanism to counteract the effects of tolerance.

5.1.6 Review of positive allosteric modulators

The use of PAMs instead of or in addition to agonists for the reasons mentioned above is being investigated for the treatment of many conditions

(reviewed in Nickols & Conn, 2014). The first and most obvious examples of this are benzodiazepines. These GABAA receptor PAMs, which are prescribed to treat anxiety, sleep, and seizure disorders, are used to avoid the side effects of direct agonists and are very common. PAMs of the mGLU4 receptor, a

85 metabotropic glutamate receptor subtype, have shown preclinical efficacy in reducing symptoms in Parkinson’s disease (Jones et al., 2012). This is very exciting research because these PAMs are reducing the effective dose of L-

DOPA, leading to a reduction in L-DOPA- induced dyskinesias. Alpha7 PAMs are being studied in cerebral ischemic stroke animal models as a way to enhance the cholinergic- mediated auto-therapy that is initiated internally to reduce damage done by injury (Kalappa et al., 2013; Sun et al., 2013). This alpha7-dependent auto therapy is spatially and temporally specific, i.e. the site and time of injury; thus, the indiscriminate activation of direct agonists are less effective and less safe (Kalappa et al., 2013; Sun et al., 2013). PAMs are also in development for the treatment of neuropathic pain, Huntington’s disease, Alzheimer’s, depression, and Fragile-X syndrome, all for the purpose of increasing temporal specificity of drug effects and reducing side effects (reviewed in Nickols & Conn, 2014).

In schizophrenia preclinical research, initial data with alpha7 PAMs is positive but very incomplete. The mostly widely- studied PAM in schizophrenia animal models is PNU120596. This type II PAM was able to reverse deficits in auditory gating in anesthetized rats after an acute injection of amphetamine

(Hurst et al., 2005), sensory gating (prepulse inhibition) deficits in MK-801- treated rats (Dunlop et al., 2009), cognitive flexibility (attentional set-shifting) deficits in a sub-chronic PCP animal model (McLean et al., 2012), and deficits in spatial working memory (12-arm radial maze) and short-term memory (novel object recognition) performance in scopolamine-treated rats (Pandya & Yakel,

2013). A type I PAM, SB-206553, reversed sensory gating (prepulse inhibition)

86 deficits in MK801-treated rats (Dunlop et al., 2009). NS-1738, which is the most studied type I PAM, reversed spatial learning deficits (Morris water maze) in scopolamine-treated rats, and improved short-term memory (social discrimination) in intact rats (Timmermann et al., 2007). In addition to pharmacological schizophrenia models, several PAMs have shown improvements in sensory gating in DBA/2 mice (Ng et al., 2007; Faghih et al.,

2008; Dinklo et al., 2011), as well as enhanced learning (social discrimination task) and memory (8-arm radial maze task) in intact rats (Ng et al., 2007;

Thomsen et al., 2011).

5.1.7 Experimental rationale and hypothesis

In conclusion, a wealth of data in animal models indicate that the alpha7 receptor is a good target for novel drug therapies, but clinical data suggest that direct agonists may not be the best strategy. In vitro studies with alpha7 PAMs indicate that they potentiate receptor activity as a function of the activity of the afferent neuron. This may be better at producing cognitive enhancement than the indiscriminate activation of direct agonists, considering the dynamic and temporally- precise nature of neurotransmission. However, in vivo characterization is limited with a paucity of in depth studies that examine alpha7

PAMs in well-validated developmental models of schizophrenia; as well as no studies that examine whether PAMs’ effects on in vivo neurotransmission will vary as a function of afferent activity, as indicated by the in vitro data.

Additionally, in vitro characterizations of PAMs have differed depending on

87 whether the tests were performed at room temperature or 37oC (Sitzia et al.,

2011); therefore, these studies may not be predictive of PAMs’ in vivo activity

(see Williams et al., 2011b for review). Therefore, in vivo characterization is necessary in order to support the notion that PAMs will be better cognitive- enhancers than direct agonists based on their ability to preserve the temporal integrity of neurotransmission.

The purpose of this experiment is to meet this need by examining whether

PAMs’ effects on glutamate release in vivo will mirror the mechanism seen in vitro. As such, this dissertation will seek to answer the following questions: 1. Are

PAMs able to potentiate mesolimbically-stimulated (see chapters 3 and 4) glutamate release in the PFC of awake, freely- moving rats similar to the EPSP currents seen in vitro? 2. Will the potentiation by the PAMs vary in magnitude as dose of PAM and level of choline in PFC varies, suggesting an interaction between the two? 3. Will the potentiation by the PAM only be apparent under conditions of choline level elevation, i.e. afferent activation, and not under basal choline conditions? 4. Will the potentiation profile differ between a type I

(increases channel open time, but does not affect desensitization kinetics) and a type II (increases channel open time and blocks receptor desensitization) PAM, such that the type II will produce a greater degree of potentiation due to its ability to block receptor desensitization?

It is hypothesized that 1.If the PAMs are able to potentiate mesolimbically- stimulated glutamate release in the PFC, then the amplitude of the glutamate peaks will be increased and/or the clearance times lengthened after PAM

88 injection compared to vehicle injection; 2 If the PAMs’ ability to potentiate glutamate release in PFC varies as a function of the activity at the orthosteric site, then the degree of PAM-mediated potentiation of glutamate release will interact with the dose of NMDA delivered into the NAcSh; 3 If the PAMs’ potentiation is dependent upon afferent activity, then the PAMs will only potentiate glutamate release in PFC after NMDA stimulation of the NAcSh and not under basal conditions; and 4 If type II PAMs produce a greater degree of potentiation than type I PAMs, then the mesolimbically-stimulated glutamate release after injection with PNU120596 will be larger in amplitude and/or have slower clearance times than the mesolimbically-stimulated glutamate release after injection with AVL3288.

5.2 Methods

5.2.1 In vitro calibration of MEAs

Microelectrode arrays (MEAs) were calibrated in vitro using the FAST-16

MKII electrochemical recording system just prior to implantation. Constant potential amperometry was conducted using an applied potential of +0.7 V vs an

(40 µL, 3x 20 mM), dopamine (DA; 40 µL, 20 mM), and hydrogen peroxide (H2O2,

40 µL, 8.8 mM), were sequentially added to the calibration beaker for final beaker

89 concentrations of 250 µM, 60 µM, 20 µM, and 8.8 µM, respectively.

Amperometric signals were acquired at a rate of 1.0 Hz. The slope (sensitivity to glutamate, nA/µM glutamate), limit of detection (L.O.D, µM glutamate), selectivity

(ratio of glutamate sensitivity over ascorbic acid sensitivity), and linearity of glutamate sensitivity (R2) were calculated. In order to be used for subsequent in vivo recordings, the MEAs had to conform to the following calibration criteria: (i) similar background current (i.e., no greater than a 20 pA difference) between the glutamate-sensitive and sentinel channels, (ii) linear response to increasing concentrations of glutamate (R2 > 0.998), (iii) a minimum slope of -0.003 nA/µM glutamate, (iv) a minimum L.O.D of < 0.5 uM, (v) a high selectivity for glutamate over either AA or DA (i.e., > 50:1), and (vi) similar sensitivity to the reporting molecule H202 on all four channels (>80% similarity for each channel pair).

(Gluox), and the bottom channel is the background channel (sentinel). The addition of AA produces little to no change in current on either channel.

Successive additions of glutamate produce large, definitive increases in current on only the Gluox channel and these increases are linear as the beaker concentration of glutamate gets progressively higher. The addition of DA produces no change in current indicating the strength of the m-PD layer. Finally, the addition of H202 produced equal increases in current across both channels, indicating all channels are similarly sensitive to the reporting molecule. An

90 important result to note is that the addition of every chemical, except glutamate, produced an equal response on both channels. This point is of paramount importance as it is the basis of the self-reference technique by which the current derived from glutamate is isolated in vivo.

5.2.2 Implantation of MEAs and infusion cannulae

Animals were anesthetized using isofluorane (2%, 0.8 L/min) and implanted with an MEA unilaterally in the PFC (in mm from bregma: AP +2.7, ML

+ 0.65, DV -3.9; hemispheres counterbalanced). Stainless steel guide cannulae

(Plastics One, Roanoke, VA), used for intra-NAcSh infusions of NMDA, were implanted ipsilaterally in the NAcSh (at 10 degree angle, in mm from bregma: AP

+ 0.4, ML + 0.70, DV – 7.40). The actual ventral termination of the guide cannula is -6.40 mm, accounting for 1 mm of the infusion cannula extension beyond the tip of the guide. All infusion cannulae used for a particular animal were measured to extend equally from the bottom of the guide cannula to ensure consistency of delivery location. A dummy cannula was inserted into the guide cannula and extended 0.7 mm beyond the tip of the guide. The Ag/AgCl reference electrode was implanted in the contralateral side at a site distant from the recording area.

All coordinates were determined from the atlas of Paxinos & Watson (1998).

5.2.3 In vivo recordings and intra-cortical infusions

After MEA and cannula implantation, rats were tested on 3 consecutive recording sessions. During recording sessions rats were allowed to move freely

91 in a wooden recording box (dimensions: H 57.2 cm; W 341.9 cm; L 317.0 cm).

The first day after MEA implantation surgery consisted of placing the rats in the wooden testing box, without connecting them to the preamplifier, to allow them to habituate to their environment prior to testing. On the second day after implantation, recordings of cortical glutamate began. At the onset of each testing day, animals were placed in the recording box and connected to the preamplifier.

Animals then remained undisturbed for 1 - 3 hours, to allow for signal stabilization to occur, before any drugs were delivered. Once the baseline period had concluded, the animals were given a systemic injection of either AVL3288

(type I PAM; 5% DMSO vehicle, 1, or 3 mg/kg, pH 7.1-7.4) or PNU120596 (type

II PAM; 5% DMSO vehicle, 3, or 9 mg/kg, pH 7.1-7.4). For this experiment, type of PAM was a between subjects variable, while dose of each PAM was within subjects. After 30 minutes, the dummy cannula was removed and 1.0 µL of vehicle (aCSF) was delivered into the NAcSh to serve as an infusion control and to clean away any blood or tissue from the bottom of the guide cannula. Then, following a 10-minute delay, NMDA (aCSF vehicle, 0.05, or 0.30 μg, in 0.5 μL, pH 7.1-7.4; between subjects) was infused into the NAcSh. NMDA was delivered over about 2 seconds using an infusion cannula attached to a Hamilton PB600-1 manual dispenser (Hamilton Company, Reno, NV). On the subsequent two test days, the doses of AVL3288 or PNU120596 not given on the first day were delivered using the same procedure. Individual animals received only one type of

PAM and one dose level of NMDA, and dose order of the PAMs was counterbalanced among animals.

5.2.4 Data analysis

22, IBM Corporations, Armonk, NY). Data for each type of PAM (type I,

AVL3288; type II, PNU120596) were analyzed separately. An initial, 2-way omnibus ANOVA, for each PAM individually, was performed for each dependent measure using PAM dose (AVL3288: 5% DMSO vehicle, 1, or 3 mg/kg;

PNU120596: 5% DMSO, 3, or 9 mg/kg) as a within-subjects factor and dose of

NMDA (aCSF vehicle, 0.05, or 0.30 μg, in 0.5 μL) as a between-subjects factor. If significance was determined, post-hoc comparisons were performed to determine specific locations of significance. The Huyen-Feldt correction was used to minimize the occurrence of type II errors. Significance was defined as p <

0.05.

5.3 Results

5.3.1 MEA and cannula placement

93 photomicrograph sections of a PFC sensor placement and a NAcSh cannula placement along with a descriptive cartoon for comparison. As shown, the representative placements fall within their respective desired regions, and arrows indicate the ventral termination of both items. The representative PFC photomicrograph also illustrates the minimal tissue damage produced by both the

MEA and infusion cannula compared to other in vivo methods.

5.3.2 AVL3288 differentially potentiates mesolimbically-stimulated PFC glutamate release as a function of NMDA dose

In order to determine whether or not an IP injection of AVL3288 would potentiate glutamate release in PFC dependent upon level of afferent stimulation

(NMDA stimulation of the NAcSh), NMDA infusions were administered at one of three dose levels (aCSF, 0.05, 0.30 µg in 0.5 µL; between subjects) 40 minutes after the injection of one of three, counterbalanced doses of AVL3288 (5%DMSO vehicle, 1, 3 mg/kg; within subjects). Figures 15 and 16 display the self- referenced signals of four representative animals comparing the difference in potentiating effects between the low dose of NMDA (0.05 g) and low dose of

AVL3288 (1mg/kg), the high dose of NMDA (0.30 g) and the low dose of

AVL3288 (1mg/kg), the low dose of NMDA (0.05 g) and the high dose of

AVL3288 (3mg/kg), and the high dose of NMDA (0.30 g) and the high dose of

AVL3288 (3 mg/kg). In all cases, the first glutamate effect of each tracing represents the amount of glutamate released after 5%DMSO vehicle injection and NMDA stimulation in the same animal as the second effect, separated by 24 94 hours. In order to capture the within subject, dose combination-dependent potentiation produced by AVL3288 in each individual animal, increases in amplitude produced by the PAM will be expressed as a percent change from

5%DMSO vehicle + NMDA. Thus, the percent change number represents the

“value added” by AVL3288 in reference to peak amplitude. Baselines are displayed uniformly for ease of comparison (For actual mean values, see table

2).

The low dose of NMDA (0.05 µg) delivered to the NAcSh, 40 minutes after an IP injection of vehicle (5% DMSO), resulted in a sharply-peaked, multi-phasic increase in PFC glutamate (2.83 µM) that occurred 35 seconds post injection and persisted for 32 seconds (figure 15; top). However, when the low dose of NMDA

(0.05 µg) was delivered in to the NAcSh forty minutes after an IP injection of the low dose of AVL3288 (1 mg/kg) the resultant glutamate release was potentiated

(3.46 µM; 22.3% increase). Peak onset (45 seconds) and duration (35 seconds) were not affected by the presence of AVL3288. When the high dose of NMDA

(0.30 µg) was delivered into the NAcSh forty minutes after an IP injection of vehicle (5% DMSO) in a different animal (figure 15; bottom), the resultant glutamate release was similar in shape to the low dose of NMDA, but the total glutamate amplitude was higher (5.15 µM). In this animal, an IP injection of

AVL3288 (1mg/kg) prior to the high dose of NMDA (0.30 µg) resulted in glutamate release that was significantly higher in amplitude (15.59 µM; 202.7% increase) than after an injection of vehicle. Peak onset (25 seconds) and duration

(57 seconds) after the vehicle injection was similar to after AVL3288 injection (28

95 and 51 seconds, respectively). Thus, IP injection of AVL3288 at the low dose

(1mg/kg) potentiated glutamate release after both the low and high doses of

NMDA, but to differing degrees.

In sharp contrast to the potentiation produced by AVL3288 reported above, the high dose of AVL3288 inhibited glutamate after both the low and high doses of NMDA. For example, figure 16 demonstrates that the high dose of

AVL3288 (3 mg/kg) injected prior to the low dose of NMDA (0.05 µg) actually attenuated the total glutamate amplitude (3.76 µM) as compared to the low dose of NMDA after a vehicle injection (5.23 µM; 28.1% decrease). Similarly, the high dose of AVL3288 (3 mg/kg) injected prior to the high dose of NMDA (0.30 µg) also attenuated the total glutamate amplitude (7.16 µM) as compared to the high dose of NMDA after a vehicle injection (9.44 µM; 24.2% decrease). In both cases, similar to the low dose, the high dose of AVL3288 did not affect peak onset or duration (see figure 15 legend for actual values).

5.3.3 Group data

Overall, the group data (aCSF: N=6, 0.05 µg: N=6, 0.30 µg: N=5) were consistent with the individual data depicted in figure 15 and 16. Two-way omnibus ANOVA’s were calculated for each dependent measure (baseline, latency to peak onset, percent change of glutamate amplitude, T50, T80, and peak duration) and if found to be significant, subsequent analysis were calculated to determine the loci of significance. A two-way (AVL3288 dose x NMDA dose)

ANOVA performed on basal glutamate levels confirmed that basal glutamate

96 levels (see Table 2 for Mean + SEM) did not differ significantly across AVL3288 dose or NMDA dose (p > .05), nor was there any AVL3288 dose x NMDA dose interaction (F4,18= .321, P=.860). This indicates that basal glutamate was not affected by the systemic injection of AVL3288. Similar to the representative tracings shown above, percent change of glutamate amplitude (See Table 2 for

Mean + SEM) had a significant main effect of AVL3288 dose (F2,28=9.128,

P=.001) and AVL3288 dose x NMDA dose interaction (F4,28= 5.008, P=.004; see figure 17). A subsequent one-way ANOVA comparing AVL3288 dose (5% DMSO vehicle vs. 1mg/kg vs. 3 mg kg) revealed that AVL3288 produced a dose- dependent potentiation of PFC glutamate when the high dose of NMDA (0.30 µg) was delivered into the NAcSh (F2,14= 11.873, P=.001); such that 1mg/kg

AVL3288 potentiated glutamate release significantly more than 5% DMSO vehicle (F1,9= 5.932, P=.041), 3mg/kg AVL3288 produced a significant inhibition of glutamate release compared to 5% DMSO vehicle (F1,9= 20.575, P=.002), and

1mg/kg AVL3288 potentiated glutamate release significantly more than 3mg/kg

AVL3288 (F1,9= 15.732, P=.004). AVL3288 did not produce a significant dose- dependent potentiation of PFC glutamate when the aCSF or the low dose of

NMDA (0.05 µg) was delivered into the NAcSh (p >.05). A second one-way

ANOVA comparing NMDA dose (aCSF vs. 0.05 µg vs. 0.30 µg) revealed that

AVL3288 produced a dose-dependent potentiation of PFC glutamate when the low dose (1 mg/kg) was injected (F2,16= 5.661, P=.016); such that 1mg/kg

AVL3288 potentiated glutamate release after the low dose of NMDA (0.05 µg) significantly more than after aCSF (F1,11= 17.471, P=.002) and 1mg/kg AVL3288

97 potentiated glutamate release after the high dose of NMDA (0.30 µg) significantly more than after aCSF (F1,10= 7.280, P=.024). AVL3288 (1mg/kg) potentiated glutamate release to a greater degree after the high dose of NMDA compared to the low dose of NMDA (0.05 µg: 24.12%, 0.30 µg: 84.70%), but this did not reach statistical significance (F1,10= 3.577, P=.091). AVL3288 did not potentiate PFC glutamate release at either the 5% DMSO vehicle or 3 mg/kg doses (p >.1). Two- way (AVL3288 dose x NMDA dose) ANOVAs performed on latency to peak onset, T50, T80, and peak duration confirmed that these dependent measures

(see Table 2 for Mean + SEM) did not differ significantly across AVL3288 dose,

NMDA dose, or AVL3288 dose x NMDA dose interaction (All p’s > .1).

5.3.4 PNU120596 differentially potentiates mesolimbically-stimulated PFC glutamate release as a function of NMDA dose

In order to determine whether or not an IP injection of PNU120596 would potentiate glutamate release in PFC dependent upon level of afferent stimulation

(NMDA stimulation of the NAcSh), NMDA infusions were administered at one of three doses (aCSF, 0.05, 0.30 µg in 0.5 µL; between subjects) 40 minutes after the injection of one of three counterbalanced doses of PNU120596 (5%DMSO vehicle, 3, 9 mg/kg; within subjects). Figure 18 and 19 display the self- referenced signals of four representative animals comparing the difference in potentiating effects between the low dose of NMDA (0.05 g) and low dose of

PNU120596 (3mg/kg), the high dose of NMDA (0.30 g) and the low dose of

PNU120596 (3mg/kg), the low dose of NMDA (0.05 g) and the high dose of 98

PNU120596 (9mg/kg), and the high dose of NMDA (0.30 g) and the high dose of PNU120596 (9 mg/kg). In all cases, the first glutamate effect of each tracing represents the amount of glutamate released after 5%DMSO vehicle injection and NMDA stimulation in the same animal as the second effect, separated by 24 hours. In order to capture the within subject, dose combination-dependent potentiation produced by PNU120596 in each individual animal, increases in amplitude produced by the PAM will be expressed as a percent change from

5%DMSO vehicle + NMDA. Thus, the percent change number represents the

“value added” by PNU120596 in reference to peak amplitude. Baselines are displayed uniformly for ease of comparison (For actual mean values, see table

2).

The low dose of NMDA (0.05 µg) delivered to the NAcSh, forty minutes after an IP injection of vehicle (5% DMSO), resulted in a typical increase in PFC glutamate (3.24 µM) that occurred 53 seconds post injection and persisted for 73 seconds (figure 18; top). However, when the low dose of NMDA (0.05 µg) was delivered in to the NAcSh forty minutes after an IP injection of the low dose of

PNU120596 (3 mg/kg) the resultant glutamate release was potentiated (4.32 µM;

33.3% increase). Peak onset (42 seconds) and duration (70 seconds) were not affected by the presence of PNU120596. When the high dose of NMDA (0.30 µg) was delivered into the NAcSh forty minutes after an IP injection of vehicle (5%

DMSO) in a different animal (figure 18; bottom), the resultant glutamate release was similar in shape to the low dose of NMDA, but the total glutamate amplitude was much higher (12.07 µM). Again in this animal, an IP injection of PNU120596

(3 mg/kg) prior to the high dose of NMDA (0.30 µg) resulted in glutamate release that was slightly higher in amplitude (16.22 µM; 34.4% increase) than after an injection of vehicle. Peak onset (77 seconds) and duration (22 seconds) after the vehicle injection was similar to after PNU120596 injection (72 and 28 seconds, respectively). Thus, IP injection of PNU120596 at the low dose (3mg/kg) produced a moderate potentiation of glutamate release in PFC after both the low and high doses of NMDA to similar degrees.

In contrast to the moderate potentiation produced by PNU120596 reported above, the high dose of PNU120596 (9 mg/kg) produced a large, significant potentiation of PFC glutamate release after the low dose of NMDA. The top tracing of figure 19 demonstrates that the high dose of PNU120596 (9 mg/kg) injected prior to the low dose of NMDA (0.05 µg) significantly increased the amount of glutamate released (4.27 µM) compared to the low dose of NMDA after a vehicle injection (1.72 µM; 148.3% increase). On the other hand, the high dose of PNU120596 (9 mg/kg) injected prior to the high dose of NMDA (0.30 µg) did not potentiate glutamate release (7.68 µM) compared to the high dose of

NMDA after a vehicle injection (8.43 µM; 8.9% decrease). In both cases, similar to the low dose, the high dose of PNU120596 did not affect peak onset or duration (see figure 19 legend for actual values).

5.3.5 Group data

Overall, the group data (aCSF: N=6, 0.05 µg: N=6, 0.30 µg: N=6) were consistent with the individual data depicted in figure 18 and 19. Two-way

100 omnibus ANOVA’s were calculated for each dependent measure (baseline, latency to peak onset, percent change of glutamate amplitude, T50, T80, and peak duration) and if found to be significant, subsequent analysis were calculated to determine the loci of significance. A two-way (PNU120596 dose x NMDA dose) ANOVA performed on basal glutamate levels confirmed that basal glutamate levels (see Table 2 for Mean + SEM) did not differ significantly across

PNU120596 dose or NMDA dose (p > .2), nor was there any PNU120596 dose x

NMDA dose interaction (F2,30= .332, P=.804). This indicates that basal glutamate was not affected by the systemic injection of PNU120596. Similar to the representative tracings shown above, percent change of glutamate amplitude

(See Table 2 for Mean + SEM) had a significant main effect of PNU120596 dose

(F2,30=11.43, p < .001) and a PNU120596 dose x NMDA dose interaction (F4,30=

7.020, p < .001; figure 20). A subsequent one-way ANOVA comparing

PNU120596 dose (vehicle 5% DMSO vs. 3 mg/kg vs. 9 mg kg) revealed that

PNU120596 produced a dose-dependent potentiation of PFC glutamate when the low dose of NMDA (0.05 µg) was delivered into the NAcSh (F2,17= 9.128,

P=.003); such that 9mg/kg PNU120596 potentiated glutamate release significantly more than 5% DMSO vehicle (F1,11= 17.404, P=.002) and 3 mg/kg

PNU120596 (F1,11= 6.958, P=.025). PNU120596 did not produce a significant dose-dependent potentiation of PFC glutamate when the aCSF or the high dose of NMDA (0.30 µg) was delivered into the NAcSh (p >.4). A second one-way

ANOVA comparing NMDA dose (aCSF vs. 0.05 µg vs. 0.30 µg) revealed that

PNU120596 produced a dose-dependent potentiation of PFC glutamate when

101 the high dose (9 mg/kg) was injected (F2,17= 9.827, P=.002); such that 9mg/kg

PNU120596 potentiated glutamate release after the low dose of NMDA (0.05 µg) significantly more than after aCSF (F1,11= 17.404, P=.002) or the high dose of

NMDA (0.30 µg; F1,11= 7.788, P=.019). PNU120596 did not potentiate PFC glutamate release at either the 5% DMSO vehicle or 3 mg/kg doses (p >.4). Two- way (PNU120596 dose x NMDA dose) ANOVAs performed on latency to peak onset, T50, T80, and peak duration confirmed that these dependent measures

(see Table 2 for Mean + SEM) did not differ significantly across PNU120596 dose, NMDA dose, or PNU120596 dose x NMDA dose interaction (All p’s > .05).

5.4 Discussion

The results of this chapter indicate that both alpha7 PAMs, PNU120596 and AVL3288, potentiate glutamate release in the PFC following a stimulus that evokes the release of choline (the endogenous ligand) in the PFC. Furthermore, the degree to which both PAMs potentiated glutamate release in the PFC was dependent upon and interacted with the dose of NMDA delivered, i.e. local choline levels. PNU120596 potentiated PFC glutamate release to the greatest degree (~200% on average) when the high dose of PNU120596 (9 mg/kg) was injected prior to the low dose of NMDA (0.05 g). Contrarily, AVL3288 potentiated glutamate release to the greatest degree (~80% on average) when the low dose of AVL3288 (1 mg/kg) was injected prior to the high dose of NMDA

(0.30 g). Other PNU/NMDA dose combinations still showed some small degree of potentiation, though it remained insignificant, while other AVL/NMDA dose

102 combinations ranged from a significant, but slightly smaller degree of potentiation

(~25% on average) after the low dose of AVL3288 and low dose of NMDA to a significant inhibition (~60% decrease on average) of PFC glutamate release after the high dose of AVL3288 and high dose of NMDA. Therefore, although both

PAMs potentiated PFC glutamate release as a function of the amount of choline in the PFC, the potentiation profiles of the type I (AVL3288) vs. the type II

(PNU120596) PAM were quite different. Importantly, neither PAM produced any potentiation of PFC glutamate release in the absence of a stimulus that evoked choline release in the PFC. No other dependent measures (basal glutamate levels, latency to glutamate peak onset, T50, T80, and glutamate peak duration) were affected by the injection of any PAM at any of the dose combinations.

The following discussion section will focus on an important methodological question related to this data, a discussion of possible explanations for the unique characteristics of each PAM potentiation profile, and finally some concluding statements about how this data relates to previous in vitro data.

5.4.1 Methodological justifications

A very important methodological question that may arise when examining this data is in reference to PAM dose selection. How were the PAM doses selected, are they comparable, and were they the right doses to use? The PAM doses were selected by examining the current behavioral literature. In vivo brain concentration assays have not been performed, to date, on these two PAMs after intraperitoneal (IP) injection; therefore, it was impossible to select doses based

103 on final brain concentrations. Thus, the most relevant option was to select doses based on which provided cognitive benefit in initial behavioral studies. AVL3288 has demonstrated cognitive benefit (improved spatial and short term memory) at two doses in rats, 0.3 and 1 mg/kg (Ng et al., 2007; Thomsen et al., 2011). This range is quite limited, as very few studies have been performed with this type I

PAM. More studies have been performed with PNU120596, as it is the most used

PAM at present. PNU120596 has been delivered subcutaneously (0.1 – 10 mg/kg; Hurst et al., 2005; McLean et al., 2012) and intravenously (0.1-1 mg/kg;

Hurst et al., 2005), in addition to IP in rats; however, these dose ranges were not considered as the amount of drug to reach the brain in these routes of administration would be expected to be quite different. Cognitive benefits

(improved sensory gating, spatial learning, short term memory, and episodic memory) with IP injection of PNU120596 were seen at a range of doses in rats from 1 to 30 mg/kg, with 3 and 10 mg/kg being the most common (Dunlop et al.,

2009; Callahan et al., 2013; Pandya & Yakel, 2013). Considering these behavioral studies, the doses selected for this dissertation were appropriate.

Obviously, however, the consequence of choosing doses based on behavioral outcomes is that it is not known if the doses are comparable. As suggested above, it is known that the doses for the respective PAMs are cognitively active, but it is impossible, at present, to directly compare the two until brain concentrations can be confirmed. For this reason, statistics were not performed comparing the two PAMs in this dissertation. Any mention of

104 comparison of “potentiation profile” is derived simply from researcher observation of the effect distribution.

Finally, considering the inverted U distribution of the results, it was determined that the doses selected were the best for the experiment described.

Of course a wider range of doses compared to a wider range of choline concentrations in PFC could have revealed additional results. However, this was beyond the scope of the study, and a complete revelation of the entire distribution of effects of these two PAMs was not the aim of this experiment.

5.4.2 Discussion of results

A number of questions may arise when examining the data presented in this chapter. First, what is the mechanism by which the PAMs are potentiating

PFC glutamate release? Based on the data from chapter three and four, it is proposed that ACh/choline, released after NAcSh stimulation, activates alpha7 receptors, locally, where the PAMs are already present (systemic injections occur

40 minutes prior to NMDA stimulation of the NAcSh). Then, an interaction between amount of choline and dose of PAM causes an increase in the amount of calcium (compared to after vehicle/NMDA) to enter the terminal through the receptor. This greater concentration of calcium within the cell causes more vesicles to fuse and release glutamate in the synapse via the phosphorylation of synapsin-1 (Dickinson et al., 2008), leading to a larger amount of glutamate detected by the MEA.

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The mechanism by which the PAM increases the amount of calcium that enters the terminal is hypothesized to include both an increase in the amount of time the channel pore remains open as well as possibly an increase in the affinity of ACh/choline for the alpha7 receptor (See Williams et al., 2011b for review).

Evidence that alpha7 PAMs increase the amount of time the channel pore remains open and, thus, allow more calcium to enter the cell has been demonstrated in nearly every in vitro study performed (See Williams et al., 2011b for review). However, evidence that PAMs increase agonist affinity has been a bit more controversial. One study demonstrated that the potency of agonist

PNU282987 was increased in the presence of higher concentrations of

PNU120596, in vitro (Gopalakrishnan et al., 2011). However, two other studies revealed that type II PAM JNJ1930942 did not increase the affinity of choline for the alpha7 receptor (Dinklo et al., 2011), and PNU120596 did not increase the affinity of agonist ABT107 for the alpha7 receptor (Malysz et al., 2010).

Type II PAMs are able to slow and reverse receptor desensitization in vitro, which provides an additional manner by which this PAM type can increase the amount of calcium that enters the cell (Gronlien et al., 2007). The mechanism of this is currently unknown, but may be because type II PAMs radically slow the dissociation of the alpha7 ligand from the receptor (Szabo et al., 2014).

Second, why don’t the PAMs have any effect on basal glutamate levels or glutamate levels after aCSF infusion into the NAcSh? In vitro studies indicate that alpha7 PAMs do not activate the receptor in the absence of the endogenous

106 ligand, and this is likely also the case in vivo. However, in contrary to in vitro, the endogenous ligand (choline) is present in vivo at basal levels. What makes this point irrelevant is that the endogenous basal levels of extracellular choline (<10

M, Uteshev et al., 2003; ~ 3 M in this study) in the rodent brain do not reach the affinity threshold to activate alpha7 receptors (~30 M, Papke & Porter

Papke, 2002). Additionally, it has been shown that PNU120596 has at least a hundred-fold lower affinity for the alpha7 receptor when it is in its resting state compared to after an agonist activates the receptor (Szabo et al., 2014). Thus,

PAMs’ lack of effect on glutamate levels in the absence of a stimulus that evokes choline increases beyond basal levels is expected if they indeed do not carry any intrinsic activity.

Third, why does PNU120596 cease to significantly potentiate PFC glutamate release when the high dose of NMDA is delivered? There are two possible explanations for this. The first is that the combination of the high dose of

NMDA and PNU120596 produces a prolonged stimulation (recall protracted choline peak durations after high dose of NMDA from chapter 3) of the alpha7 receptor that is too strong, causing the receptor to move to a more stable desensitized state. A Williams et al. (2011) study supports this by demonstrating that the combination of medium or low concentrations of agonist and PNU120596 caused the expected potentiation of agonist-induced excitatory currents, but high doses of either PNU102596 or agonist did not. They demonstrated that the lack of potentiation with high doses of agonist and/or PAM was caused by the alpha7

107 receptors moving to a further, more stable desensitized state that was not sensitive to the normal blockade/reversal effects of the type II PAM (Williams et al., 2011a).

Another possible explanation is that the extremely large increase in open time of the channel pore produced by the combination of PNU120596 and the high dose of NMDA leaves it susceptible to a voltage-dependent blockade by positively-charged molecules. This possibility has been demonstrated in a study performed by Kalappa & Uteshev (2013). They showed that the addition of

PNU120596 increased the open time of the channel pore from ~100 s to more than 1 second (Gusev & Uteshev, 2010), and that this massive increase in open time increased the likelihood of voltage-dependent inhibition of the receptor by positively charged molecules, such as bicuculline and choline, blocking the channel pore (Kalappa & Uteshev, 2013). Thus, these explanations may be why the potentiation of PNU120596 is no longer seen as the dose of NMDA, i.e. concentration of choline in the PFC, increases.

Fourth, why does the high dose of AVL3288 inhibit PFC glutamate release when it is injected prior to the high dose of NMDA? The most likely reason for this effect is that AVL3288 also shows some efficacy as a GABAA PAM. AVL3288 was originally characterized as a very weak GABAA PAM, but based on the similarity of this receptor with the alpha7, this drug was tested for efficacy at the alpha7 receptor (Ng et al., 2007). The Ng et al. (2007) study revealed that

AVL3288 (also known as compound 6), following a few modifications, had a

108 much higher affinity for the alpha7 receptor, and thus it was marketed as such.

However, some efficacy at the GABAA receptor remains, and, at high doses of

AVL3288, likely is still a factor. For this reason, the inhibition produced by the high dose of AVL3288 when combined with the high dose of NMDA likely reflects some GABA-mediated inhibition of glutamate. On the other hand, if the abovementioned reason was the only cause for the inhibition after the high dose of AVL3288 when combined with the high dose of NMDA, then there would likely also be inhibition of the glutamate peak when the high dose of AVL3288 was combined with the low dose of NMDA. Though there was no potentiation produced by this latter dose combination, there is no clear inhibition either.

Therefore, there is likely to be an additional cause for the degree of inhibition reported with the high/high dose combination.

It has recently been shown that a single binding site bound on the alpha7 receptor is sufficient for full receptor activation (Andersen et al., 2013).

Additionally, high concentrations of agonist can actually be inhibitory because alpha7 receptors desensitize rapidly under these conditions, and this desensitization is not reversed until the agonist is removed (Papke & Porter

Papke, 2002). This is why alpha7 agonists typically display inverted U dose response curves. Importantly, AVL3288 is a type I PAM, so, in contrast to

PNU120596, it does not block or reverse receptor desensitization (Gronlien et al.,

2007). Therefore, it is possible that the high dose of NMDA evokes a large enough concentration of choline release in the PFC that, when combined with the high dose of AVL3288, the resultant glutamate peak is inhibited both by receptor

109 desensitization and positive modulation of the GABAA receptor. This combination of factors would certainly explain how the high doses of AVL3288 and NMDA could result in such a profound inhibition of PFC glutamate release.

Fifth, why is there so much variability in some of the PAM/NMDA dose combinations? The answer to this question is not something that is knowable at present as no other experiments of this nature have been performed. However, the answer most likely lies in the fact that the PAMs’ effects truly are based on afferent signaling. There is natural variability from animal to animal when it comes to how much choline/glutamate is evoked in the PFC after NMDA stimulation of the NAcSh; therefore, of course the effects of the PAMs would co- vary with these differences. Importantly, some dose combinations are more variable than others, indicating that there may be a “most optimal” dose when considering a population of animals or patients. However, until comparative behavioral studies are performed with a sufficient range in dose selection, the actual functional meaning of the variability, or lack thereof depending on dose combination, remains largely unknowable.

Finally, why don’t either of the PAMs increase peak duration? Type I

PAMs do not alter normal peak decay kinetics when tested in vitro; therefore

AVL3288’s lack of effect on peak duration or clearance is not surprising.

Contrarily, type II PAMs do block receptor desensitization in vitro, so it is quite surprising that PNU120596 did not affect peak duration or clearance. However,

110 studies have reported that PNU120596 increases alpha7 receptor channel open time from ~100 s to ~1 second (Gusev & Uteshev, 2010), and average glutamate peak durations are ~30-50 seconds. Thus, the difference in time scales is quite pronounced when comparing the means of measuring in vivo neurotransmitter release described in this dissertation and measurement of current changes in vitro. When considering this time scale difference, the most likely place to actually see a diversion in potentiation profile between the two

PAM types, based on a blockade of receptor desensitization, is in the peak amplitudes, as the rise times of these peaks are typically 1-2 seconds.

Importantly, the highest degree of potentiation by PNU120596 (~200% on average) was much larger than that of AVL3288 (~80% on average), which supports this conclusion.

5.4.3 Conclusions

The purpose of this experiment was to determine if PAMs would potentiate neurotransmitter release (glutamate) in vivo, and if that potentiation would interact with the amount of the endogenous ligand (choline) present. Alpha7

PAMs, in vitro, increase the amount of time the channel pore remains open when the orthosteric site is bound by an agonist (Gronlien et al., 2007; Ng et al., 2007;

Williams et al., 2011a), and the degree of this increase interacts with levels of the endogenous ligand that are present (Dinklo et al., 2011; Williams et al., 2011a).

This experiment demonstrated, for the first time, that PAMs act in vivo similar to how they act in vitro in a number of different ways. First, it was confirmed that

111 both a type I and II PAM were able to potentiate endogenous glutamate release in the PFC, similar to how PAMs potentiate excitatory currents in vitro. Second, it was demonstrated that the degree of potentiation of both a type I and II PAM interacted with the amount of the endogenous ligand present (choline), similar to what has been shown in vitro. Third, the PAMs did not potentiate glutamate or produce any increase in glutamate in the absence of a stimulus that increased choline beyond basal levels. This mirrors PAMs’ lack of intrinsic activity in vitro.

Fourth, the potentiation profile differed between the type I and II PAMs, as is seen in vitro, but the manner by which they differed was not the same. In this experiment, the type II PAM potentiated glutamate amplitude to a greater degree than the type I PAM instead of lengthening peak duration compared to type I

PAMs as is seen in vitro. Thus, this experiment fulfilled its purpose of providing an in vivo characterization of a type I and II PAM, for the first time.

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CHAPTER 6

GENERAL DISCUSSION

6.1 Dissertation review

Schizophrenia is a devastating and costly psychological disorder whose core symptoms, deficits in executive functioning, remain largely untreated.

Current pharmacotherapeutic strategies have highlighted the alpha7 nicotinic acetylcholine (alpha7) receptor as a leading target for treatment of these symptoms, but early clinical trials with direct agonists have been heterogeneous.

Some believe this could be caused by non-specific increases in neuronal activity or receptor desensitization due to indiscriminate, mistimed activation produced by direct agonists. Positive allosteric modulators (PAMs) potentiate the activity of the afferent neuron without carrying any intrinsic activity, thus enhancing receptor function without mistimed false signals. This mechanism may make PAMs more effective cognitive treatments, but there is a paucity of data to support this claim.

Early studies in vitro indicated that alpha7 PAMs do indeed potentiate excitatory post-synaptic potentials without directly activating receptors, but no studies have tested this in vivo.

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The purpose of this dissertation was, for the first time, to determine if

PAMs would operate in the same fashion in vivo as they do in vitro. Specifically, this dissertation examined whether or not PAMs would potentiate glutamate release in PFC, and if that potentiation would interact with and be dependent upon the amount of endogenous choline present. This required an assay with a number of characteristics. First, the assay must result in the endogenous release of glutamate in the PFC. Second, this glutamate release must be contingent upon afferent activity mediated via the alpha7 receptor. Previous experiments performed by this lab revealed that NMDA stimulation of the NAcSh evoked endogenous glutamate release in PFC (Bortz et al., 2014). Next, the first experiment performed in this dissertation revealed that this same mesolimbic stimulation also evoked the dose-dependent release of choline (mean 0.05 =

0.87 + 0.15 M, 0.30 = 1.73 + 0.31 M, P = .027) in PFC. The second experiment determined that the glutamate was stimulated locally in PFC via cholinergic activation of the alpha7 receptor because the glutamate release was inhibited by local application of the alpha7 receptor antagonist MLA (saline = 3.54

+ 1.35 M; 6.75 g MLA = 0.19 + 0.06 M, P = .032), but not the alpha4beta2 receptor antagonist DHE (saline = 4.05 + 1.23 M; 6.75 g DHE = 2.74 + 0.58

M, P = .564).

Once this was established, it was possible to determine the ability of two novel alpha7 PAMs to potentiate glutamate release in the PFC as a function of varying levels of local choline. The experiments in chapter 5 revealed that, indeed, both the type I (AVL3288) and the type II (PNU120596) PAM potentiated 114 glutamate release in the PFC, but to varying degrees dependent upon on the amount of the endogenous ligand (choline) present. AVL3288 resulted in the largest degree of potentiation (84.70%) when the low dose (1 mg/kg) was injected IP prior to the high dose of NMDA into the NAcSh (0.30 g, mean 1.73

M choline increase above baseline), compared to 24.12% potentiation when the low dose of AVL was injected prior to the low dose of NMDA (0.05 g, mean 0.87

M choline increase above baseline). Contrarily, the high dose of AVL (3 mg/kg) resulted in a significant inhibition of glutamate release (64.24% decrease) when delivered before the high dose of NMDA. PNU120596 resulted in a large degree of potentiation (211.95%) when the high dose (9mg/kg) was injected prior to the low dose of NMDA, but lower, insignificant degrees of potentiation with every other dose combination. Importantly, neither PAM potentiated glutamate release in PFC in the absence of a stimulus that increased choline above basal levels.

Therefore, it was determined that these two PAMs do, indeed, potentiate in vivo glutamate release in PFC based on afferent activity and without directly stimulating the receptor, confirming that their in vitro mechanism is mirrored in vivo.

6.2 Discussion outline

The following discussion section will address a number of translational questions related to the results described in this dissertation. First, the benefits and drawbacks of the microelectrode array (MEA) as a technique to measure in vivo neurotransmission will be discussed. Second, this discussion section will 115 evaluate the mesolimbic stimulation assay and its potential as a tool to continue to examine novel alpha7 agonists and PAMs. Finally, the potential for alpha7

PAMs to be effective treatments of the cognitive deficits of schizophrenia will be discussed in light of the results described in this dissertation as well as the limited behavioral data that currently exists.

6.3 Evaluation of the MEA

The MEAs used in this study were built and sold by the Quanteon

Company (Quanteon, LLC, Nicholasville, KY). They exist as a technique to measure in vivo neurotransmission in anesthetized and freely- moving animals as an alternative to older techniques such as microdialysis. The largest benefit of this technique is the MEA’s ability to detect transmitter release with a high degree of temporal (2 readings per second) and spatial (synaptic and extra- synaptic release) resolution. Microdialysis collections, for example, describe transmitter release on the order of minutes and likely only measures extra- synaptic basal fluctuations. Thus, these MEAs provide the ability to detect transient increases in transmitter release that might otherwise go undetected with other methods. This fact is critical given the importance of transient increases in neurotransmitter levels to executive functions such as attention (Parikh et al., 2007). Importantly, this benefit also occurs without the loss of ability to detect absolute basal neurotransmitter levels.

MEAs also produce significantly less damage to the surrounding neurons than microdialysis probes (figures 4, 9, and 14). This is important because

116 neuronal damage can result in inflammatory responses that may artificially elevate glutamate levels (Casamenti et al., 1999; Ward et al., 2009). This would be a major confound since glutamate levels are the principle outcome measure of these experiments.

A major drawback of the MEA is that they are a relatively new technique, used by only a few labs in the world, and are, therefore, not as well validated and characterized as older techniques such as microdialysis. This lack of knowledge about the technique provokes the important question: How reliable is the identification of the signal as glutamate, choline, etc.? The results from several studies collectively support the interpretation that the self-referenced electrochemical signals from the MEA represent the current generated exclusively by the oxidation of glutamate and choline at the surface of the electrode. First, administration of ceftriaxone, a drug that enhances excitatory amino acid transporters (Wei et al., 2012), reduces the amplitude and clearance of a self-referenced glutamate peak stimulated locally (Bortz et al., 2013). TβOA, a drug that suppresses excitatory amino acid transporters (Shimamoto et al.,

1998), dramatically slows the clearance of a locally stimulated, self-referenced glutamate peak (Bortz et al., 2013). Similarly, blockade of the choline transporter by hemicholinium-3 significantly increases the amplitude and lengthens the clearance of the choline signal (Parikh & Sarter, 2006), whereas the addition of the AChE inhibitor neostigmine completely abolishes the detection of endogenous choline by the MEA (Parikh et al, 2008). Second, local infusions of

117 extracellular glutamate or choline standards yield differences among the enzyme- coated and sentinel sites that resemble those displayed in the results. Thus, this question has been sufficiently answered to date.

A second drawback of the MEA is the brevity of their functionality after they have been implanted. This lab has performed successful recordings up to 7 days post-implantation, but frequently the signal begins to deteriorate after 4-5 days. This short timeline limits the use of these MEAs to short-term experiments that can be performed over a couple of days, and makes their use in performing animals difficult due to time-for-training requirements. In addition to this, the

MEAs cannot be re-calibrated once they are implanted. This creates a potential scenario where the sensitivity to glutamate or choline could change day to day without the knowledge of the researcher. This risk has been shown to be relatively low over the first 3 or 4 days as endogenous glutamate and choline recordings evoked by a single dose of NMDA are not different regardless of whether they were evoked on the first (day 2 post-surgery) or third (day 4 post- surgery) day of testing (Bortz et al., unpublished observations). In conclusion, though the MEA carries some small drawbacks, the ability to reliably detect phasic neurotransmitter release in freely- moving animals makes utilizing this technique worthwhile.

6.4 The mesolimbic stimulation assay

The assay used in this study involves stimulating the shell of the nucleus accumbens (NAcSh) with NMDA and measuring glutamate or choline release in

118 the PFC. This is an excellent assay to examine the neurochemical effects of novel alpha7 PAMs due to both reasons that have been previously known and from novel findings presented in this dissertation. First, mesolimbic stimulation results in the endogenous, dose-dependent release of glutamate in the PFC

(Bortz et al., 2014). This glutamate release is phasic (rise times typically 1-2 seconds), which is typical of glutamate peaks shown to be critical for executive functions such as sustained attention (Parikh et al., 2008). Second, mesolimbic stimulation results in the endogenous, dose-dependent release of choline in PFC

(chapter 3). Third, the glutamate release described above was blocked by local application of the alpha7 antagonist MLA, indicating that afferent cholinergic signaling is driving the glutamate release locally via the alpha7 receptor. Fourth, this assay has been shown to be cognitively relevant and a possible model for top-down regulation (St Peters et al., 2011). Finally, the mechanism by which glutamate and choline are evoked in the PFC involves multiple brain regions within a distributed neural system (figure 1) that is implicated in schizophrenia.

Thus, this assay meets the criteria needed to study the neurochemical effects of alpha7 PAMs, and does so in a manner that makes the results more interesting from a therapeutic standpoint. Taken together, this was the best possible assay to perform the first in vivo characterizations of two alpha7 PAMs.

6.5 Unanswered questions and drawbacks

The mesolimbic stimulation assay has a number of unanswered questions and drawbacks as well. The first drawback of this assay is that it is difficult to

119 perform. The NAcSh is a relatively large-spanning brain region, but it is very heterogeneous from anterior to posterior in terms of the behaviors its stimulation elicits (Reynolds & Berridge, 2003). Accordingly, in order to stimulate glutamate and choline release in the PFC, the researcher must implant the infusion cannula in the anterior portion of the NAcSh, a much smaller target. Cannula implantations that are not anterior enough in the NAcSh will not elicit the desired results (Bortz et al., unpublished observations). Additionally, to account for the space needed to implant the MEA, cannulae must be implanted at an angle, thus increasing the difficulty of implantation precision. The difficulty of the procedure leads to a proportion of animals that cannot be used due to incorrect cannula placements, which detracts from the usefulness of the assay.

Second, the exact mechanism by which the glutamate and choline release occurs is still somewhat unknown. It is known that deafferination of the BF neurons that are contacted by the NAc medium spiny neurons (Zaborszky &

Cullinan, 1992) and project to the PFC (Luiten et al., 1987) dramatically attenuates the NAcSh-stimulated ACh release in the PFC (St Peters et al., 2011).

However, it has not been determined that the choline measured by the MEA in these experiments is from the BF, nor that it is the choline that activates alpha7 receptors to produce the glutamate release. Though both are likely, further study is required to confirm that this is the case.

Based on the results displayed in chapter 4, it is clear that the glutamate is stimulated via ACh/choline’s activation of alpha7 receptors locally in the PFC.

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This concept is further supported by the demonstration that locally applied alpha7 agonists evoke glutamate release in PFC (Konradsson-Geuken et al., 2009;

Bortz et al., 2013), and that α-bungarotoxin applied directly to the PFC via reverse dialysis blocks mesolimbically- stimulated glutamate release as measured by microdialysis (Valentini et al., in preparation). However, the origination of the glutamate has yet to be determined. As described in the chapter 4 discussion, it is unlikely to be from the MDthal because nicotinic receptors on neuron terminals from this region have been shown to be mainly alpha4beta2 receptors (Gioanni et al., 1999; Lambe et al., 2003); however, further study is needed to determine the glutamate’s exact source.

Third, the amount of time between mesolimbic stimulation and the detection of choline and glutamate increases in the PFC is much longer than expected. The hypothesized polysynaptic mechanisms described in chapters 3 and 4 would, likely, take only milliseconds to produce glutamate/choline release in the PFC, rather than the 30-60 seconds that was reported in these experiments. Therefore, there is clearly an additional aspect to the mechanism by which mesolimbic stimulation leads to glutamate and choline release in PFC that is not yet understood. One possible explanation for this delay is that it is the time necessary for the NMDA to diffuse away from the NAcSh and directly contact the BF. This may seem reasonable, initially, because the BF is quite close, anatomically, to the NAcSh, and because the ACh signal has been shown to originate from this region (St Peters et al., 2011). However, direct stimulation

121 of the BF with NMDA does not result in ACh release in the PFC of rats unless they are performing a behavioral task (Fadel et al., 2001), and the rats in the experiments in this dissertation were not. Therefore, it is unlikely that the NMDA is diffusing to the BF. Additionally, when NMDA is delivered into the core of the

NAc, a region that is directly adjacent to the NAcSh, the rats were significantly more active and no glutamate peaks were observed in the PFC (Bortz et al., unpublished observations). This also argues against the NMDA diffusing to any other region since contact with the region directly adjacent to the NAcSh produced a completely different effect, both behaviorally and chemically.

A second possible explanation is that the PFC glutamate peaks are secondary to some sort of behavioral activation produced by the NMDA. To determine if this hypothesis was viable, behavioral activation was closely observed during every NMDA infusion. The behavioral observations were in distinct opposition to this hypothesis because rats frequently did not move at all after the NMDA infusion took place. Also, as was just mentioned, NMDA infusion into the core of the NAc produced significant behavioral activation, but was not accompanied by any glutamate peaks.

A final possible explanation is that NMDA infused into the NAcSh is stimulating receptors on local interneurons, in addition to the medium spiny (MS) projection neurons. If this were true, the activation of the local interneurons would inhibit the MS projection neurons, putting them into a hyperpolarized state. Thus, the delay of the effect could simply be the time required for these negative feedback loops to expire, freeing the MS projection neurons from their

122 hyperpolarization, and allowing the signal to be transferred to the BF. This seems to be the most reasonable hypothesis as the other hypotheses have been momentarily ruled out. However, to date, the answer to his question has yet to be determined, and, thus, presents a minor drawback of the mesolimbic stimulation assay.

Fourth, the use of NMDA as the stimulus can be troubling as high concentrations of this drug are also used to produce excitotoxic lesions. NMDA is used to activate NMDA receptors in the NAcSh, thus mimicking glutamatergic inputs; however, high doses of NMDA could produce cell death, which might confound the results. The doses used in this study were determined to not cause deleterious, confounding effects for a number of reasons. First, the doses used in this experiment were the same doses that demonstrated cognitive benefit when used by other labs (St Peters et al., 2011). Second, in all studies using this assay, dose order is counterbalanced, with all doses and/or drug combinations occurring on each of the possible testing days. This allows for the ability to analyze order of dose administration to determine if there was any indication of a decrease in peak amplitude, within one dose/drug combination, if that dose was given on the final testing day as opposed to the initial testing day, and no decreases in glutamate peak amplitudes were found. Admittedly, these two pieces of information do not prove that excitotoxicty did not occur, but it does indicate there was not enough to affect peak amplitude or inhibit the benefit to sustained attention. Finally, a representative brain stained with Flouro-Jade, a

123 stain that reliably highlights neuronal degeneration (Schmued et al., 1997), demonstrated minimal neuronal degeneration around the ventral termination of the cannula track. This provides the most convincing evidence that excessive excitotoxic damage was not caused by the NMDA infusions (Bortz et al., unpublished observations; figure 21). Therefore, even though the use of NMDA as the stimulus could be a confound due to its potential excitotoxic effects, this evidence supports the notion that the doses used in the present study were not high enough to create this problem. Taken together, though this assay does present with some unanswered questions and drawbacks, it was still determined to be the best possible assay to perform the first in vivo characterizations of the two alpha7 PAMs.

6.6 Evaluation of alpha7 positive allosteric modulators as treatments for schizophrenia

The use of PAMs to treat the cognitive deficits of schizophrenia is a concept that has begun to draw more and more interest in recent years. The promise of the alpha7 receptor as a treatment target is well established at this juncture, but direct agonists have yet to meet expectations. PAMs represent a promising alternative because they hold at least three potential advantages over direct agonists. The first is that they increase endogenous ligand efficacy, and possibly their potency, without activating the receptor themselves (Gronlien et al.,

2007; Ng et al., 2007; Williams et al., 2011a). This allows for a synergistic rather than competitive effect between the exogenous drug and endogenous ligand,

124 which increases the likelihood of a therapeutic effect by maintaining the temporally-precise pattern of natural transmitter signaling (reviewed in Williams et al., 2011b; Uteshev, 2014). The second advantage is related to the first.

Nicotinic receptors, including the alpha7, desensitize with prolonged presence of an agonist. This creates two problems. First, if agonist is introduced into the body and desensitizes alpha7 receptors, and then an endogenous signal comes through, there will be fewer receptors available to be activated. Thus, the agonist actually carries some competitive/antagonistic properties in reference to endogenous signaling, similar to the problem indicated in advantage point number one (reviewed in Williams et al., 2011b). The second problem is that the desensitization caused by prolonged agonist exposure is likely to eventually result in tolerance of the patient, requiring higher and higher doses (Umana et al.,

2013). Therefore, the second advantage held by PAMs is twofold. Since they do not activate the receptor, they do not increase the number of receptors in a desensitized state, and some PAMs (type II) actually block or reverse receptor desensitization, thus decreasing the likelihood of tolerance development

(Gronlien et al., 2007; reviewed in Williams et al., 2011b; Uteshev, 2014). The third advantage held by PAMs is that allosteric binding sites tend to be less evolutionarily conserved than orthosteric binding sites (Yang et al., 2012). This is evidenced both by the multitude of specific allosteric binding sites on alpha7 receptors (see chapter 5 introduction), as well as by the fact that the orthosteric bind site for alpha7 receptors shares many similarities with the orthosteric sites of other ligand-gated ion channels, such as 5-HT3 receptors (Papke et al., 2004).

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Thus, the third advantage, in essence, is that developers have a greater likelihood of generating PAMs that are highly selective for the alpha7 receptor.

Based on these advantages, interest in PAMs as alternatives to direct agonists is high. However, all of the advantages reviewed above are based on in vitro studies, computer docking simulations, and theoretical evidence. Very few behavioral studies have been performed, none of which were on developmental schizophrenia models, and no studies have attempted to confirm that the abovementioned advantages exist in vivo. The data presented in this dissertation, for the first time in vivo, does confirm that PAMs potentiate neurotransmitter release (glutamate release in PFC), and that this potentiation interacts with and is dependent upon endogenous afferent signaling. Ultimately, these data set the stage for PAMs to be tested for efficacy in PFC glutamate- mediated executive function deficits in developmental animal models of schizophrenia by presenting a possible mechanism of action for their efficacy in such models.

6.7 Type I versus type II positive allosteric modulators

An important discussion that has been raised by those who have reviewed the in vitro and initial behavioral data of alpha7 PAMs is whether type I or type II

PAMs are more likely to be efficacious at providing cognitive-enhancement. As mentioned previously, both type I and II PAMs increase peak current response in the presence of an agonist in vitro (Gronlien et al., 2007); however, type II PAMs also slow peak decay kinetics through the ability to both block and reverse

126 receptor desensitization (Gronlien et al., 2007; Thomsen & Mikkelsen, 2012).

When considering this divergence in mechanism, the first point of discussion within this debate is on safety profile. Because type II PAMs block/reverse receptor desensitization, more receptors are active for longer than with type I

PAMs, which presents the possibility for excess calcium-induced excitotoxicity and cell death. This would be a major concern for type II PAMs if it were true, but studies have shown mixed results. One study performed by Ng et al., (2007) showed that 24-hour exposure to the type II PAM PNU120596 reduced the viability of SH-SY5Y cells in an MLA-sensitive manner, but their type I PAM,

AVL-3288, did not. Another study with a similar paradigm showed toxicity with

PNU120596, but not a different type II PAM, JNJ-1930942, in alpha7-expressing

GH4CI cells (Dinklo et al., 2011). Finally, a third study in undifferentiated PC12 cells showed no toxicity with any of the PAMs tested, which included PNU120596 and another type II PAM (Hu et al., 2009). In addition, two caveats to the mechanism of type II PAMs have been recently discovered that may naturally increase their safety profile. The first is that prolonged high levels of PNU120596 and agonist have been shown to stabalize a further desensitized state that is insensitive to the reversal properties of the type II PAM (Williams et al., 2011a).

This more stable desensitized state has been demonstrated as level of PAM or agonist is increased beyond a certain threshhold. Second, a study performed by

Kalappa & Uteshev (2013) demonsrated that the prolonged channel opening produced by PNU120596 left it more suseptible to voltage-dependent inhibition by positively charged molecules. This voltage-dependent open channel block

127 was shown to reduce the potentiation by PNU120596 (Kalappa & Uteshev,

2013). Thus, both of these indirect effects of the type II PAM, PNU120596, may naturally counteract an excess calcium-induced excitotoxicity.

In terms of behavioral data, both type I and II PAMs have shown efficacy in several behavioral tasks. For example, both types were able to reverse deficits in auditory gating in a suseptible mouse model (DBA/2, Ng et al., 2007; Faghih et al., 2008; Dinklo et al., 2011); as well as improve cognitive abilities such as spatial learning (Ng et al., 2007; Pandya & Yakel, 2013) and memory (see chapter 5 introduction for complete review, Ng et al., 2007; Timmermann et al.,

2007). Few studies, however, have been performed that compare the difference between types in terms of behavioral efficacy, and only one has published any differences. This study, performed by Thomsen et al. (2011), revealed that the type I PAM, AVL3288, improved short-term memory (social recognition task) 24 hours after 7 consecutive days of administration, whereas PNU120596 did not.

Neither PAM improved short-term memory after a single injection or immediately after the 7-day administration (Thomsen et al., 2011). Interestingly, PNU120596 also blocked agonist-induced receptor upregulation and AVL3288 did not (BTX binding assay, Thomsen & Mikkelsen, 2012). On the other hand, PNU120596 increased agonist-induced ERK1/2 phosphorylation and calcium release to detectable levels in PC12 cells, whereas type I PAMs, in general, did not (Hu et al., 2009). Both calcium release and ERK1/2 phosphorylation are associated with plasticity-dependent learning (Kushner et al., 2005). Additionally, there is no correlation between BTX binding and short-term memory, suggesting that the

128 differential effects of PAM types on receptor upregulation reported by Thomsen &

Mikkelsen (2012) may not translate into a difference in cognitive-enhancing abilities between the two types (Thomsen et al., 2011).

Only two alpha7 PAMs have been examined in clinical trials to date. JNJ-

39393406, a type II PAM, was tested in a phase I trial to examine its safety profile and to determine if it showed any ability to reverse sensory gating deficits in patients (Winterer et al., 2013). Though the drug proved safe, it provided no improvement in sensory gating deficits in patients (Winterer et al., 2013). More recently, a phase I clinical trial has begun with the type I PAM, AVL3288, to determine its pharmacokinetic and safety profile after oral administration. This trial has not concluded yet, but if positive data is reported, then these results would provide one piece of evidence supporting type I over type II PAMs.

The data presented in this dissertation also draws comparisons between type I and II PAMs, but in terms of potentiation of glutamate release in PFC.

PNU120596 potentiated mesolimbically-stimulated glutamate release in PFC by

~200% at the most optimal dose combination. AVL3288’s maximum degree of potentiation was ~80%. As mentioned previously, a potentiation of PFC glutamate release, while not a behavioral measure itself, would likely translate into cognitive-enhancement based on its well-established importance to executive functions (Stefani & Moghaddam, 2005; Aultman & Moghaddam, 2001;

Parikh et al., 2008). However, it is not known whether 80 or 200% potentiation would be more cognitively beneficial because even modest increases in cholinergic transmission may be sufficient to produce cognitive improvement.

129

Importantly, AVL3288’s highest degree of potentiation was seen at higher endogenous choline levels (0.30 g NMDA) than PNU120596’s (0.05 g NMDA), which could be significant since these drugs may be used to treat disorders where endogenous ACh/choline transmission is reduced or irregular. Again, however, AVL3288 produced a reliable, significant potentiation of PFC glutamate at the low dose of AVL3288 and low dose of NMDA (0.05 g; ~25%); and, as previously mentioned, it is not known what degree of glutamate potentiation would produce the most cognitive benefit. Therefore, AVL3288’s modest potentiation at the low AVL3288, low NMDA dose combination could end up being quite effective. Thus to summarize, neither the data presented in this dissertation nor any of the in vitro or behavioral data reported by others to date clearly answer the question of whether type I or II PAMs will be more effective cognitive-enhancers. Data from the AVL3288 phase I trial may begin to answer this question (trial ends May, 2015), but future comparative studies performed in developmental animals models is the best way to answer the question of which type is safer and more effective.

6.8 Positive allosteric modulator drawbacks

Regardless of which PAM type proves to be the better choice for pharmaceutical development, another, bigger problem exists in the preclinical data supporting the use of PAMs for schizophrenia treatment -- the lack of support in anything other than pharmacological models of schizophrenia. Two of the several disorders and conditions that could benefit from alpha7 receptor

130

PAMs as treatments are schizophrenia and Alzheimer’s, and both of these disorders feature reductions in endogenous cholinergic signaling (Wallace &

Porter, 2011; Wallace & Bertrand, 2013). This is important for two reasons. First,

PAMs require sufficient endogenous signaling to be effective, as indicated by this dissertation. Second, the pharmacological models previously used to substantiate the claim that PAMs can reverse cognitive deficits involve the use of drugs that naturally elevate endogenous ACh/choline levels in the brain (MK801, phencyclidine, scopolamine, amphetamine; Bymaster et al., 1993; Arnold et al.,

2001; Del Arco et al., 2007). A true test of whether a PAM would be an effective schizophrenia or Alzheimer’s treatment could only come with a model that also exhibits reduced or irregular cholinergic functioning, such as aged animals

(Callahan et al., 2013) or the embryonic kynurenic acid model (Zmarowski et al.,

2009; Pershing et al., 2015).

This critical point is emphasized in a study done by Callahan et al. (2013), where the ability of PNU120596 to improve various learning and memory deficits in aged rats and monkeys was assessed. The concepts that cholinergic neurons are compromised with age and loss of cholinergic tone correlates with cognitive decline are well established in the literature (see Bartus, 2000; Terry &

Buccafusco, 2003 for review). Thus, the use of aged rats and monkeys in this study is an appropriate test of PAMs’ efficacy, per the qualifications stated above.

In the study, it was determined that PNU120596 alone did not improve performance on a spatial learning and memory task (water maze) in aged rats, nor did it improve performance on a working memory task (delayed match to

131 sample) in aged Rhesus monkeys (Callahan et al., 2013). Thus, doses of

PNU120596 (0.1-10 mg/kg) that are in the range of those that potentiated glutamate release in the PFC under conditions of stimulated choline release (3 and 9 mg/kg) did not improve cognition in animals with reduced cholinergic tone.

While this is initially concerning, several points maintain that the potential of PAMs still exists. First, it is possible that the tasks used in the Callahan et al.

(2013) study engaged cholinergic systems differently than other tasks that have been shown to evoke increases in basal ACh levels (sustained attention, St

Peters et al., 2011), thus preventing the usefulness of the PAM. This is not a great reply to the problem described above because working memory and spatial learning are both among the cognitive deficits seen in schizophrenia; however, a partial alleviation of symptoms may still be valuable. Second, no type I PAMs nor any other type II PAMs were examined in the Callahan et al. (2013) study.

Therefore, the negative PNU120596 results could be a factor of this drug or just type II PAMs, making further study of other PAMs, and comparisons between type I and II PAMs, a requirement before judgment is passed. Finally and most importantly, even if PAMs were not successful at treating the cognitive deficits of schizophrenia alone, there would still be a great value in adding them as adjunctive therapies to reduce agonist doses. This would be valuable because the therapeutic threshold of some current cholinergic-mediated medications is marked by side effects. For example, acetylcholinesterase inhibitors (AChEI) are currently prescribed for Alzheimer’s, but therapeutic doses can produce nausea, vomiting, and diarrhea (Maelicke & Albuquerque, 2000). Similarly, the

132 alpha4beta2 agonist ABT-594 is being investigated for use for neuropathic pain, but therapeutic doses produce problematic increases in heart rate and body temperature (Lee et al., 2011). Therapeutic doses of alpha7 receptor agonists are likely to result in receptor desensitization and tachyphylaxis. On the other hand, the addition of PNU120596 to donepezil (AChEI) reduced the amount of drug needed to produce learning and memory enhancement in both aged rodents and monkeys in the Callahan et al. (2013) study described above. The same was shown when an alpha4beta2 PAM was added to ABT-594. The combination of sub-threshold agonist dose and PAM produced the desired analgesic response without the autonomic side effects (Lee et al., 2011). Finally, based on type II PAMs’ ability to block receptor desensitization, their addition to a low dose of alpha7 agonist may prevent the side effects associated with desensitization and dose increases due to tachyphylaxis.

In conclusion, though some questions remain to be answered, the use of

PAMs has a great deal of potential in treating the cognitive deficits of schizophrenia, as well as other disorders. The initiation of the phase I clinical trial with AVL3288 demonstrates that, though novel, the use of PAMs to treat schizophrenia is valid and has been recognized as a promising avenue of treatment. Data from this clinical trial, if positive, will go a long way in furthering the investigation of alpha7 PAMs, but it will not answer all the questions that need to be answered. Comparative studies in developmental models of schizophrenia to both confirm the ability of PAMs to be cognitive-enhancers as

133 well as determine which of the two types is more efficacious is still a great need, and these experiments must be performed for the true potential of these novel drugs to be realized.

6.9 Conclusions

This dissertation sought to characterize the ability of two novel alpha7

PAMs to potentiate glutamate release in vivo in the PFC as a function of varying local choline levels. To do so required a detection method with the ability to measure phasic glutamate release in vivo, as well as an assay where glutamate release was stimulated endogenously based on afferent activity at the alpha7 receptor. The experiments in this dissertation reported that the mesolimbic stimulation assay produced dose-dependent increases in choline release in PFC, similar to previous reports on PFC glutamate release, and that the glutamate release was driven by local cholinergic activity at the alpha7 receptor. Next, it reported that both a type I and a type II PAM were able to potentiate evoked glutamate release in the PFC. Importantly, this potentiation was dependent upon and varied with levels of afferent signaling. Finally, it revealed interesting and important differences in the potentiation profile of the type I versus the type II

PAM, which may prove useful in discriminating between the two in future studies.

This data is incredibly valuable because it is the first study to examine the neurochemical effects of alpha7 PAMs in vivo. Thus, it will provide the framework for the future development of alpha7 PAMs, and, possibly, lead to the discovery

134 of the first cognition-enhancing therapy able to improve executive functioning in schizophrenic patients.

135

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APPENDIX A

TABLES AND FIGURES

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Table 1. Means and standard errors for chapter 4 This table presents the means and standard errors (Mean + SEM) for the experiments in chapter 4. Basal glutamate levels (M), total peak amplitude (M), Latency to glutamate peak onset (sec), time to clear 50% of the glutamate peak (T50; sec), time to clear 80% of the glutamate peak (T80; sec), and glutamate peak duration (sec) are included in this table.

Table 1.

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Table 2. Means and standard errors for chapter 5 This table presents the means and standard errors (Mean + SEM) for the experiments in chapter 5. Basal glutamate levels (M), total peak amplitude (M), Latency to glutamate peak onset (sec), time to clear 50% of the glutamate peak (T50; sec), time to clear 80% of the glutamate peak (T80; sec), glutamate peak duration (sec), and percent change from 5% DMSO vehicle (percent) are included in this table. Means and standard errors were not included for the animals that received aCSF infusions into their NAcSh because no infusions produced any detectable change in glutamate in any animal tested.

Table 2.

NMDA (0.05 µg) NMDA (0.30 µg)

Dose of AVL3288 (mg/kg) 0 (5% DMSO vehicle) 1.00 3.00 0 (5% DMSO vehicle) 1.00 3.00

Basal Glutamate (µM) 0.79 + 0.25 0.69 + 0.19 0.96 + 0.21 0.59 + 0.32 0.51 + 0.28 0.62 + 0.37

Peak Amplitude (µM) 3.38 + 1.00 3.92 + 1.02 1.97 + 0.73 6.09 + 1.55 10.06 + 2.06 2.86 +1.32

Latency to Peak Onset (Sec) 62.83 + 17.89 44.67 + 7.39 48.00 + 18.19 70.50 + 20.85 41.60 + 9.84 60.40 + 14.91

T50 (Sec) 8.17 + 3.88 3.50 + 0.67 4.00 + 0.77 13.50 + 6.62 4.20 + 1.02 3.40 + 0.51

T80 (Sec) 15.33 +5.73 16.00 + 6.56 11.40 + 2.60 19.50 + 6.86 11.60 + 2.44 6.20 + 1.39

Peak Duration (Sec) 49.83 + 9.64 37.33 + 9.89 37.20+ 8.33 54.33 + 5.85 53.80 + 4.32 45.80 + 7.33 Percent Change from 5% DMSO n/a 24.12 + 5.77 (-5.48 + 38.37) n/a 84.70 + 34.78 (-64.24 + 14.16) vehicle

NMDA (0.05 µg) NMDA (0.30 µg)

Dose of PNU120596 (mg/kg) 0 (5% DMSO vehicle) 3.00 9.00 0 (5% DMSO vehicle) 3.00 9.00

Basal Glutamate (µM) 0.61 + 0.18 0.56 + 0.20 0.56 + 0.19 0.95 + 0.34 0.92 + 0.34 0.76 + 0.32

Peak Amplitude (µM) 2.17 + 0.49 2.60 + 0.65 6.65 + 1.56 10.12 + 2.18 10.73 + 2.43 11.91 + 3.73

Latency to Peak Onset (Sec) 48.00 + 2.71 53.50 + 9.65 53.33 + 8.23 51.83+ 7.12 42.33 + 8.41 55.50 + 12.89

T50 (Sec) 8.17 + 1.28 6.33 + 2.46 6.33 + 0.95 4.67 + 1.02 7.50 + 3.36 3.83 + 0.87

T80 (Sec) 15.00 + 2.13 14.33 + 3.88 15.33 + 2.43 11.67+3.56 8.00 + 1.39 7.33 + 1.45

Peak Duration (Sec) 50.00 + 6.57 51.33 + 8.19 56.50 + 8.09 46.67 + 6.62 47.33 + 7.57 48.00 + 7.30 Percent Change from 5% DMSO n/a 42.33+39.42 211.95+50.80 n/a 6.67+11.91 37.57 + 36.38 vehicle

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Figure 1. Schematic representation of the distributed neural system believed to control executive functioning. This system includes the prefrontal cortex (PFC), ventral tegmental area (VTA), basolateral amygdala (BLA), ventral hippocampus (VH), nucleus accumbens (NAc) core and shell, basal forebrain (BF), ventral pallidum (VP), and medial dorsal thalamus (MDthal). This diagram is not meant to be comprehensive as all of the brain regions pictured have many other afferent and efferent connections. There are also many other brain regions and neurotransmitter systems involved in cognition that are not pictured here. Therefore, this is simply a representation of some of the more predominant regions and their projection pathways meant to illustrate the complex interplay between cortical and sub-cortical regions in executive functioning.

Figure 1.

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Figure 2. Diagram describing MEA signal transduction scheme and organization. This diagram describes the mechanism by which the microelectrode array (MEA) detects and isolates the choline and glutamate signal, as well as the organization of the neurotransmitter-sensitive and sentinel channels on the MEA tip. Glutamate is detected in vivo when a glutamate molecule contacts the surface of a platinum channel of the MEA that has been coated with glutamate oxidase. Glutamate is converted to hydrogen peroxide and alpha ketoglutarate by the enzyme. Then the hydrogen peroxide is reduced to 2 electrons by the 0.7 volt applied potential. This current is amplified and converted to a concentration by a slope that is determined during in vitro calibration, and it is specific to each individual MEA. Thus, the unique slope, which describes each MEA’s individual sensitivity to glutamate, allows for a glutamate concentration measurement that is directly proportional to the number of glutamate molecules that contact the platinum surface. Choline is detected in a similar fashion, only using choline oxidase instead of glutamate oxidase. The sentinel channels are not coated with enzyme, so they are sensitive to everything the enzyme channels are sensitive to, except for glutamate or choline. This allows for the background signal to be subtracted, isolating the glutamate or choline signal. The two sets of platinum recording sites are situated very near each other on the tip of the MEA (< 90 µm), allowing for the detection and isolation of glutamate and choline within a single brain subregion.

Figure 2.

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Figure 3. Representative in vitro calibration of the choline MEA. This calibration is conducted immediately before implantation into the prefrontal cortex. The red tracing is the choline-sensitive (Cholox) recording channel and the adjacent green tracing is the sentinel background channel. Arrows correspond to the addition of various substances into the stirred calibration beaker. Current (nAmp) is depicted along the vertical axis and time (second) along the horizontal axis. The successive additions of choline (20 µM/aliquots) produce a linear increase in choline signal. Expectedly, there were no changes in choline-related current on the sentinel channel. Important for self-referencing, the calibration also reveals equivalent high sensitivities on both channels to the reporting molecule H2O2. The effectiveness of the m-PD barrier is evident by the lack of increase in current depicted following the addition of large concentrations of potential in vivo interferents ascorbic acid (AA) and dopamine (DA) to the beaker.

Figure 3.

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Figure 4. Representative photomicrograph of the regions within the PFC and the NAcSh where the choline MEA and infusion cannula were situated. The first panel depicts a graphical representation of the medial PFC and indicates that choline MEAs were placed at the border between the prelimbic and infralimbic cortices and medial of the forceps minor. Directly adjacent is a photomicrograph from a representative animal showing that the ventral termination of the MEA was in this location, and that the MEA produced very minor tissue disruption. Similarly, the bottom depicts a graphical representation of the NAcSh and a photomicrograph from a representative animal showing the ventral termination of the infusion cannula within the NAcSh. Any animal with MEA or cannula placements found outside the desired brain regions were disqualified from analysis.

Figure 4.

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Figure 5. Representative tracings describing the dose-dependent release of Choline following NMDA stimulation of the NAcSh. These representative MEA tracings were recorded from the PFC following the infusion of three different doses of NMDA (0.05, 0.15, 0.30 µg in 0.5 µL) into intact animals. The black tracing represent the signal from the choline-sensitive channel (Cholox), whereas the green tracings represent the signal from their adjacent sentinel channels. The red tracings represents the signal derived from subtracting the second channel from the first, thereby isolating the signal obtained exclusively from the oxidation of extracellular choline. Changes in the vertical axis are transformed from current to changes in concentration (µM), whereas the horizontal axis reflects the passage of time (sec). Each tracing represents different, counterbalanced experiment sessions separated by at least 24 hours (time cut). Baselines are shown uniformly for ease of comparison (see chapter 3 results for actual basal glutamate values). While aCSF infused into the NAcSh (infusions indicated by black arrows) resulted in no change in choline levels, the low dose of NMDA (0.05 µg) resulted in a robust, multi-phasic increase in PFC choline (1.03 µM) that occurred 71 seconds post injection and persisted for 46 seconds. The middle and high doses of NMDA (0.15 and 0.30 µg) also resulted in PFC choline release, but they occurred more quickly post- infusion (40 and 33 seconds, respectively) and were higher in total amplitude (1.85 and 1.73 µM, respectively). The high dose of NMDA also produced a choline peak that lasted longer (2,175 seconds) than either the low (46 seconds) or middle (116 seconds) dose due to a protracted clearance of the final 20% of the choline peak (high dose T80= 95 seconds).

167

Figure 5.

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Figure 6. Group data describing the dose-dependent release of Choline following NMDA stimulation of the NAcSh This group data represents the maximum peak amplitude (µM), latency to peak onset (sec), and peak duration (sec; Mean + SEM) of prefrontal choline release following the infusion of three different doses of NMDA (0.05, 0.15, 0.30 µg in 0.5 µL) into intact animals. Each rat (N=6) received all three doses of NMDA in separate, counterbalanced recording sessions, and all dependent measures were taken after self-referencing. Artificial CSF vehicle infusions were administered 10 minutes before every NMDA infusion as per the procedure introduced in the methods section. No aCSF vehicle infusions for any animal at any dose produced a detectable change in choline from basal values. *The middle and high dose of NMDA (0.15 and 0.30 µg) produced significantly more PFC choline release that occurred significantly faster than the low dose (0.05 µg; p < .05). # The high dose of NMDA produced choline peaks that were significantly longer in duration than either the low or middle dose (p < .05).

169

Figure 6.

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Figure 7. Representative in vitro calibration of the glutamate MEA. This representative in vitro calibration of the MEA was conducted immediately before implantation into the prefrontal cortex. The black tracing is the glutamate- sensitive (Gluox) recording channel and the adjacent blue tracing is the sentinel background channel. Arrows correspond to the addition of various substances into the stirred calibration beaker. Current (nAmp) is depicted along the vertical axis and time (second) along the horizontal axis. The successive additions of glutamate (20 µM/aliquots) produce a linear increase in glutamate signal. Expectedly, there were no changes in glutamate-related current on the sentinel channel. Important for self-referencing, the calibration also reveals equivalent high sensitivities on both channels to the reporting molecule H2O2. The effectiveness of the m-PD barrier is evident by the lack of increase in current depicted following the addition of large concentrations of potential in vivo interferents ascorbic acid (AA) and dopamine (DA) to the beaker.

Figure 7.

171

Figure 8. Expanded view of MEA to identify cannula termination zone. This expanded view of the tip of the cannula/MEA complex demonstrates where the PFC cannulae terminated in relation to the surface of the MEA. Cannulae for intra-PFC infusions were secured to the MEA with wax so that they would be approximately 40-70 m from the surface and at a 20 degree angle, as displayed above. Cannulae were situated to deliver MLA and DHβE at the center of the four platinum recording sites (right graphic, indicated by red circle) to allow for minor drug diffusion.

Figure 8.

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Figure 9. Representative photomicrograph of the regions within the PFC and the NAcSh where the choline MEA and two infusion cannulae were situated. This photomicrograph and associated graphical representation indicates the regions within the PFC and the NAcSh where the glutamate MEA and infusion cannulae were situated. The cartoon on the right illustrates the desired location for both the MEA/cannula in the medial PFC (PreL, prelimbic; InfL, infralimbic cortices) and the cannula in the NAcSh. Black arrows in the cartoon on the right indicate the positioning of each item in the photomicrograph on the left for ease of comparison. In the photomicrograph on the left, the blue arrow indicates the ventral termination of the MEA, the red arrows indicate the ventral termination of the guide cannulae, and the purple arrows indicate the protrusion of the infusion cannulae through the bottom of the guides. Comparison of the black arrows on the right and the colored arrows on the left confirms that all three items are within their desired locations.

Figure 9.

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Figure 10. Representative tracings describing the dose-dependent inhibition of glutamate by MLA following NMDA stimulation of the NAcSh. These are representative MEA tracings recorded from the PFC when NMDA (0.15 µg in 0.5 µL) was infused into the NAcSh two minutes after each of three doses of MLA (saline, 3.38, 6.75 µg in 0.5 µL) were infused into the PFC of intact animals. The red tracing represents the signal from the glutamate-sensitive channel (Gluox), whereas the blue tracing represents the signal from its adjacent sentinel channel. The green tracing represents the signal derived from subtracting the second channel from the first, thereby isolating the signal obtained exclusively from the oxidation of extracellular glutamate. Changes in the vertical axis are transformed from current to changes in concentration (µM), whereas the horizontal axis reflects the passage of time (sec). Each tracing represents different, counterbalanced experiment sessions separated by at least 24 hours, and the timing of drug infusions relative to glutamate peaks are indicated with black arrows. Baselines are shown uniformly for ease of comparison (See Table 1 for actual basal glutamate values). NMDA (0.15 µg) delivered to the NAcSh two minutes after saline was infused into the PFC (top green tracing) resulted in a robust, sharp peaked increase in PFC glutamate (3.90 µM) that occurred 65 seconds post injection and persisted for 47 seconds. However, the low (3.38 µg; middle green tracing) and high (6.75 µg; bottom green tracing) doses of MLA, when infused into the PFC two minutes before NMDA, produced a dose-dependent inhibition of the glutamate release (1.89 and 0.00 µM, respectively). Glutamate standards (500 µM) were infused into the PFC following MLA-induced blockades to ensure proper functioning of the MEA (bottom green tracing, far right).

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Figure 10.

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Figure 11. Group data describing the dose-dependent inhibition of glutamate by MLA following NMDA stimulation of the NAcSh. This group data represents the maximum peak amplitude (Mean + SEM) of prefrontal glutamate release (µM) following the infusion of NMDA (0.15 µg in 0.5 µL) into the NAcSh two minutes after each of three different doses of MLA (saline, 3.38, 6.75 µg in 0.5 µL) infused into the PFC of intact animals. Each rat (N=6) received all three doses of MLA in separate, counterbalanced recording sessions, and all maximum amplitudes were taken after self-referencing. Artificial CSF vehicle infusions were administered 10 minutes before every NMDA infusion as per the procedure introduced in the methods section. No aCSF vehicle infusions for any animal at any dose produced a detectable change in glutamate from basal values. *The high dose of MLA (6.75 µg) into the PFC significantly attenuated the prefrontal glutamate increases following NMDA infusion into the NAcSh (0.15; p < .05). The low dose of MLA (3.38 µg) did not significantly attenuate glutamate release, though there was a clear trend towards significance (P= .064).

Figure 11.

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Figure 12. Representative tracings describing the inhibition of glutamate by MLA, but not DHE, following NMDA stimulation of the NAcSh. These are representative MEA tracings, recorded from the PFC, comparing the difference between NMDA (0.15 µg in 0.5 µL) infusions into the NAcSh after PFC infusions of DHE (6.75 µg) or MLA (6.75 µg). Only the self-referenced currents, those derived from subtracting a sentinel channel from a Gluox channel, are depicted in this figure. Changes in the vertical axis depict changes in concentration (µM), whereas the horizontal axis reflects the passage of time (sec). The tracings divided by a time cut represent different, counterbalanced experiment sessions separated by 24 hours. The different colors of tracing represent different animals. Arrows indicate the timing of each drug infusion, and baselines are shown uniformly for ease of comparison (See Table 1 for actual basal glutamate values). NMDA (0.15 µg) delivered to the NAcSh two minutes after saline was infused into the PFC resulted in similar increases in PFC glutamate in both animals (top, 4.49 µM; bottom, 3.90 µM). The high dose of DHE (6.75 µg; top), when infused into the PFC two minutes before NMDA, produced a very minor attenuation of the PFC glutamate amplitude (3.81 µM). This is in stark contrast to the complete blockade of the glutamate signal produced by the high dose of MLA (bottom). Time scale bars under each glutamate peak demonstrate the similar peak onset and duration times between all animals in this experiment.

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Figure 12.

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Figure 13. Group data describing the inhibition of glutamate by MLA, but not DHE, following NMDA stimulation of the NAcSh. This group data compares the maximum peak amplitude (Mean + SEM) of prefrontal glutamate release (µM) following the infusion of NMDA (0.15 µg in 0.5 µL) into the NAcSh two minutes after each of three different doses of MLA or DHE (saline, 3.38, 6.75 µg in 0.5 µL) into the PFC of intact animals. Each rat (N=6) received all three doses of MLA or DHE in separate, counterbalanced recording sessions and all maximum amplitudes were taken after self- referencing. *The high dose of MLA (6.75 µg) into the PFC significantly attenuated the prefrontal glutamate increases following NMDA infusion into the NAcSh (0.15; p < .05), whereas DHE, at both doses, produced only very minor, nonsignificant attenuations in the PFC glutamate signal (p > .5).

Figure 13.

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Figure 14. Representative photomicrograph of the regions within the PFC and the NAcSh where the glutamate MEA and infusion cannula were situated. This photomicrograph and associated graphical representation indicate the regions within the PFC and the NAcSh where the glutamate MEA and infusion cannula, respectively, were situated. The first panel depicts a graphical representation of the medial PFC and indicates that glutamate MEAs were placed at the border between the prelimbic and infralimbic cortices and medial of the forceps minor. Directly adjacent is a photomicrograph from a representative animal showing that the ventral termination of the MEA was in this location, and that the MEA produced very minor tissue disruption. Similarly, the bottom depicts a graphical representation of the NAcSh and a photomicrograph from a representative animal showing the ventral termination of the infusion cannula within the NAcSh. Any animal with MEA or cannula placements found outside the desired brain regions were disqualified from analysis.

Figure 14.

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Figure 15. Representative tracings comparing the difference in degree of potentiation by AVL3288 (1mg/kg) following NMDA stimulation of the NAcSh. These were representative MEA tracings, recorded from the PFC of two different animals (top and bottom), comparing the difference in degree of potentiation by 1 mg/kg AVL3288 (IP injection) as a function of the level of NMDA stimulation (NAcSh infusion; 0.05 or 0.30 µg in 0.5 µL). Only the self-referenced currents are depicted in this figure. Changes in the vertical axis depict changes in concentration (µM), whereas the horizontal axis reflects the passage of time (sec). Each tracing is divided twice by a time cut. The first, smaller time cut represents the 40 minutes between when AVL3288 was injected IP and NMDA infused into the NAcSh. The second, larger time cut indicates different, counterbalanced experiment sessions separated by 24 hours. Arrows mark the timing of each drug infusion, and baselines are shown uniformly for ease of comparison (See Table 2 for actual basal glutamate values). The low dose of NMDA (0.05 µg), after an IP injection of vehicle (5% DMSO), resulted in a typical increase in PFC glutamate (2.83 µM) that occurred 35 seconds post injection and persisted for 32 seconds (top). However, when the low dose of NMDA (0.05 µg) was delivered after the low dose of AVL3288 (1 mg/kg) the resultant glutamate release was potentiated (3.46 µM; 22.3% increase). Peak onset (45 seconds) and duration (35 seconds) were not affected by the presence of AVL3288. When the high dose of NMDA (0.30 µg) was delivered into the NAcSh after an IP injection of vehicle (5% DMSO; bottom), the resultant glutamate release was similar to the low dose of NMDA, but the total glutamate amplitude was higher (5.15 µM). In this animal, an IP injection of AVL3288 (1mg/kg) resulted in a large potentiation of glutamate release (15.59 µM; 202.7% increase). Peak onset (25 seconds) and duration (57 seconds) after the vehicle injection were similar to after AVL3288 injection (28 and 51 seconds, respectively).

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Figure 15.

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Figure 16. Representative tracings comparing the difference in degree of potentiation by AVL3288 (3mg/kg) following NMDA stimulation of the NAcSh. These are representative MEA tracings, recorded from the PFC of two different animals (top and bottom), comparing the difference in degree of potentiation by 3 mg/kg AVL3288 (IP injection) as a function of the level of NMDA stimulation (NAcSh infusion; 0.05 or 0.30 µg in 0.5 µL). Only the self-referenced currents are depicted in this figure. Changes in the vertical axis depict changes in concentration (µM), whereas the horizontal axis reflects the passage of time (sec). Each tracing is divided twice by a time cut. The first, smaller time cut represents the 40 minutes between when AVL3288 was injected IP and NMDA infused into the NAcSh. The second, larger time cut indicates different, counterbalanced experiment sessions separated by 24 hours. Arrows mark the timing of each drug infusion, and baselines are shown uniformly for ease of comparison (See Table 2 for actual basal glutamate values). In sharp contrast to the potentiation produced by 1 mg/kg AVL3288, the high dose of AVL3288 (3 mg/kg) lead to an inhibition of glutamate after both the low and high doses of NMDA. The high dose of AVL3288 (3 mg/kg) injected prior to the low dose of NMDA (0.05 µg) attenuated the total glutamate amplitude (3.76 µM) as compared to the low dose of NMDA after a vehicle injection (5.23 µM; 28.1% decrease). Peak onset (69 seconds) and duration (36 seconds) after the vehicle injection were similar to after AVL3288 injection (36 and 32 seconds, respectively). Similarly, the high dose of AVL3288 injected prior to the high dose of NMDA (0.30 µg) also attenuated the total glutamate amplitude (7.16 µM) as compared to the high dose of NMDA after a vehicle injection (9.44 µM; 24.2% decrease). Again, peak onset (30 seconds) and duration (40 seconds) after the vehicle injection was similar to after AVL3288 injection (35 and 36 seconds, respectively).

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Figure 16.

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Figure 17. Group data comparing the difference in degree of potentiation by AVL3288 following NMDA stimulation of the NAcSh. This group data compares the percent change (degree of potentiation/inhibition; Mean + SEM) of prefrontal glutamate release (µM) produced by AVL3288 (1 or 3 mg/kg), relative to 5% DMSO vehicle, after the infusion of each of three doses of NMDA (aCSF, 0.05, 0.30 µg in 0.5 µL). Each rat (aCSF, N=6; 0.05, N=6; 0.30, N=5) received all three doses of AVL3288 in separate, counterbalanced recording sessions, but only one dose level of NMDA. A histogram depicting mean choline values (the endogenous ligand) for each dose level of the NMDA are set in the upper left corner of the graphic. This is meant to aid in the visualization of the clear interaction that occurred between the amount of the endogenous ligand present in the PFC and the degree of AVL3288-mediated potentiation. a The low dose of AVL3288 potentiated glutamate release in the PFC significantly more after NMDA (both 0.05 and 0.30 µg) than after aCSF. b When the high dose of NMDA (0.30 µg) was infused into the NAcSh, the low dose of AVL3288 potentiated glutamate release significantly more than after an injection of 5% DMSO vehicle or c 3 mg/kg AVL3288. d When the high dose of NMDA (0.30 µg) was infused into the NAcSh, the high dose of AVL3288 produced a significant attenuation of PFC glutamate release, compared to an injection of 5% DMSO vehicle (all p’s < .05).

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Figure 17.

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Figure 18. Representative tracings comparing the difference in degree of potentiation by PNU120596 (3mg/kg) following NMDA stimulation of the NAcSh. These are representative MEA tracings, recorded from the PFC of two different animals (top and bottom), comparing the difference in degree of potentiation by 3 mg/kg PNU120596 (IP injection) as a function of the level of NMDA stimulation (NAcSh infusion; 0.05 or 0.30 µg in 0.5 µL). Only the self-referenced currents are depicted in this figure. Changes in the vertical axis depict changes in concentration (µM), whereas the horizontal axis reflects the passage of time (sec). Each tracing is divided twice by a time cut. The first, smaller time cut represents the 40 minutes between when PNU120596 was injected IP and NMDA infused into the NAcSh. The second, larger time cut indicates different, counterbalanced experiment sessions separated by 24 hours. Arrows mark the timing of each drug infusion, and baselines are shown uniformly for ease of comparison (See Table 2 for actual basal glutamate values). The low dose of NMDA (0.05 µg), after an IP injection of vehicle (5% DMSO), resulted in a typical increase in PFC glutamate (3.24 µM) that occurred 53 seconds post injection and persisted for 73 seconds (top). However, when the low dose of NMDA (0.05 µg) was delivered after the low dose of PNU12596 (3 mg/kg) the resultant glutamate release was potentiated (4.32 µM; 33.3% increase). Peak onset (42 seconds) and duration (70 seconds) were not affected by the presence of PNU120596. When the high dose of NMDA (0.30 µg) was delivered into the NAcSh after an IP injection of vehicle (5% DMSO; bottom), the resultant glutamate release was similar to the low dose of NMDA, but the total glutamate amplitude was much higher (12.07 µM). In this animal, an IP injection of PNU120596 (3 mg/kg) resulted in a similarly sized potentiation of glutamate release (16.22 µM; 34.4 % increase). Again, peak onset (77 seconds) and duration (22 seconds) after the vehicle injection was similar to after PNU120596 injection (72 and 28 seconds, respectively).

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Figure 18.

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Figure 19. Representative tracings comparing the difference in degree of potentiation by PNU120596 (9mg/kg) following NMDA stimulation of the NAcSh. These are representative MEA tracings, recorded from the PFC of two different animals (top and bottom), comparing the difference in degree of potentiation by 9 mg/kg PNU120596 (IP injection) as a function of the level of NMDA stimulation (NAcSh infusion; 0.05 or 0.30 µg in 0.5 µL). Only the self-referenced currents are depicted in this figure. Changes in the vertical axis depict changes in concentration (µM), whereas the horizontal axis reflects the passage of time (sec). Each tracing is divided twice by a time cut. The first, smaller time cut represents the 40 minutes between when PNU120596 was injected IP and NMDA infused into the NAcSh. The second, larger time cut indicates different, counterbalanced experiment sessions separated by 24 hours. Arrows mark the timing of each drug infusion, and baselines are shown uniformly for ease of comparison (See Table 2 for actual basal glutamate values). In contrast to the moderate potentiation produced by 3 mg/kg PNU120596, the high dose of PNU120596 (9 mg/kg) produced a large, significant potentiation of PFC glutamate release after the low dose of NMDA. The top tracing demonstrates that the high dose of PNU120596 (9 mg/kg) injected prior to the low dose of NMDA (0.05 µg) significantly increased the amount of glutamate released (4.27 µM) compared to the low dose of NMDA after a vehicle injection (1.72 µM; 148.3% increase). Peak onset (48 seconds) and duration (37 seconds) after the vehicle injection was similar to after PNU120596 injection (42 and 29 seconds, respectively). On the other hand, the high dose of PNU120596 (9 mg/kg) injected prior to the high dose of NMDA (0.30 µg) did not potentiate glutamate release (7.68 µM) compared to the high dose of NMDA after a vehicle injection (8.43 µM; 8.9% decrease). Again, peak onset (52 seconds) and duration (60 seconds) after the vehicle injection was similar to after PNU120596 injection (61 and 53 seconds, respectively).

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Figure 19.

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Figure 20. Group data comparing the difference in degree of potentiation by PNU120596 following NMDA stimulation of the NAcSh. This group data compares the percent change (degree of potentiation; Mean + SEM) of prefrontal glutamate release (µM) produced by PNU120596 (3 or 9 mg/kg), relative to 5% DMSO vehicle, after the infusion of each of three doses of NMDA (aCSF, 0.05, 0.30 µg in 0.5 µL). Each rat (aCSF, N=6; 0.05, N=6; 0.30, N=6) received all three doses of PNU120596 in separate, counterbalanced recording sessions, but only one dose level of NMDA. A histogram depicting mean choline values (the endogenous ligand) for each dose level of the NMDA are set in the upper left corner of the graphic. This is meant to aid in the visualization of the clear interaction that occurred between the amount of the endogenous ligand present in the PFC and the degree of PNU120596-mediated potentiation. The high dose of PNU120596 (9 mg/kg) potentiated glutamate release in the PFC significantly more after the low dose of NMDA (0.05 µg) than after a aCSF or b the high dose of NMDA (0.30 µg). When the low dose of NMDA was infused into the NAcSh, the high dose of PNU120596 potentiated glutamate release significantly more than after an injection of c 5% DMSO vehicle or d 3 mg/kg PNU120596 (all p’s < .05).

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Figure 20.

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Figure 21. Flouro-Jade stain of an NMDA infusion cannula track. Flouro-Jade stains degenerating neurons, thus this stain is used to determine whether or not the doses of NMDA used for this experiment are producing excitotoxicity. Some decaying cells are stained (indicated by arrows); however, this amount of cell death is probably due to the lowering of the cannula and not excitotoxicity.

Figure 21.

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