A Dissertation Entitled 3,4-Methylenedioxy

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A Dissertation

Entitled

3,4-methylenedioxy-methamphetamine(MDMA)-induced Increases in Hippocampal

Glutamate Concentrations and Its Impact on the Dentate Gyrus

Stuart Collins

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Biomedical Sciences

______Dr. Bryan Yamamoto, Committee Member

______Dr. Nicolas Chiaia, Committee Member

______Dr. Scott Molitor, Committee Member

______Dr. Richard Mooney, Committee Member

______Dr. Joseph Margiotta, Committee Member

______Dr. Patricia R. Komuniecki, Dean

The University of Toledo July 2015

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

3,4-methylenedioxy-methamphetamine(MDMA)-induced increases in hippocampal glutamate concentrations and its impact on the dentate gyrus

Stuart Collins

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences

The University of Toledo

June 2015

Recently it was demonstrated that MDMA causes a long-term decrease in parvalbumin (PV) expressing interneurons in the dentate gyrus which coincides with an increase in hippocampal glutamate. We hypothesized that the hippocampal glutamate increases mediated by MDMA leads to a loss of PV interneurons and reduces inhibition in the dentate gyrus. Accordingly, the decreases in PV interneurons seen 10 days following MDMA exposure were shown to be dependent upon activation of NMDA receptors during MDMA exposure. Additionally, our experiments demonstrated a role for

5HT2A receptors in mediating both the increases in glutamate within the dentate gyrus and PV interneuron decreases caused by MDMA. We also provided evidence suggesting that 5HT2A receptor activation mediates prostaglandin E2 (PGE2) increases and subsequent Prostaglandin E receptor 1 (EP1) receptor activation. EP1 receptor activation was shown to be necessary for MDMA-induced increases in glutamate concentrations and PV interneuron decreases in the dentate gyrus. Decreased parvalbumin immunoreactivity 10 days after MDMA exposure coincided with a decrease in paired- pulse inhibition and afterdischarge threshold. These changes in physiology were

prevented by inhibition of EP1 and 5HT2A receptors during MDMA exposure. These findings suggest that MDMA-induced 5HT increases lead to 5HT2A receptor activation in the dentate gyrus, which elicits PGE2 increases and subsequent increases in glutamate concentrations. Furthermore, we demonstrate that this signaling is also necessary for

MDMA-induced PV interneuron decreases and subsequent changes in dentate gyrus physiology suggestive of a lack of GABAergic inhibition.

Acknowledgements

I would like to thank all of those who encouraged and supported me during my graduate studies. Firstly, I would like to thank my major advisor, Dr. Bryan Yamamoto, whose efforts in advising and supporting my research project were instrumental to the completion of my degree. For his efforts to impart wisdom and instill within me the ability to think critically and conduct scientific research, I will forever be grateful. I would like to thank all of the members of the lab which have helped in numerous ways.

Especially Dr. Nicole Northrup who helped train me in my first days in the lab and was always very kind and helpful. I would also like to thank all of my committee members for their efforts and constructive criticism which was critical in the molding my project.

Special thanks to Dr. Nicolas Chiaia, who was always available to provide insight and guide my understanding of neurophysiology. Lastly, I would like to thank the many faculty members at The University of Toledo for creating an excellent learning atmosphere.

In addition, I would like to thank my family members for their support. To my mother and father who always helped support my passion for knowledge and for inspiring me to work hard. I would also like to thank my wonderful wife and children for all the love that they’ve shared over the years which has been a blessing many times over.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... xi

List of Abbreviations ...... xii

1 General Introduction to MDMA Neurotoxicity and the Role of Parvalbumin

Interneurons in Hippocampal Function ...... 1

1.1 MDMA Use...... 1

1.2 Acute Behavioral effects of MDMA ...... 2

1.3 MDMA Pharmacology ...... 2

1.4 MDMA-induced Neurotoxicity to 5HT Terminals ...... 4

1.5 Mechanisms for MDMA-induced Neurotoxicity ...... 5

1.5.1 Oxidative stress ...... 5

1.5.3 Hyperthermia ...... 7

1.5.3 Inflammation ...... 8

1.5.4 Excitotoxicity...... 10

1.6 MDMA-induced Cell Death ...... 12

1.7 Parvalbumin Interneurons ...... 13

1.7.1 Susceptibility to Excitotoxicity ...... 13

1.7.2 Morphology and Function ...... 14

1.8 Dentate Gyrus...... 16

1.8.1 Sensory Information Processing ...... 16

1.8.2 Gating of Epileptogenesis ...... 17

1.9 Long-term Behavioral Consequences of MDMA Exposure ...... 18

1.10 Summary of Past Findings and Gaps in Knowledge ...... 20

1.11 Hypothesis...... 21

2 MDMA-induced Loss of Parvalbumin Interneurons within the Dentate

Gyrus is Mediated by 5HT2A and NMDA Receptors ...... 23

2.1 Abstract ...... 23

2.2 Introduction ...... 24

2.3 Methods...... 25

2.3.1 Animals ...... 26

2.3.2 Drug Treatments ...... 26

2.3.3 Microdialysis ...... 27

2.3.4 High-performance Liquid Chromatography ...... 27

2.3.5 Immunohistochemistry ...... 28

2.3.6 Stereology ...... 29

2.3.7 Statistical Analysis ...... 29

2.4 Results……………………………………………………………………29

2.4.1 Effects of Systemic Injections of MDMA on Extracellular

Glutamate Concentrations in the Dentate Gyrus …………….….30

vii

2.4.2 NMDA Receptor Inhibition Prevents MDMA-induced PV Cell

Losses………………………………...... 30

2.4.3 MDL100907 Prevents Increased Glutamate Concentrations Caused

by Local Perfusion of MDMA ………………………………. .... 31

2.4.4 Effect of MDL100907 on MDMA-induced PV Cell Losses ...... 31

2.5 Discussion ...... 37

3 MDMA Increases Excitability in the Dentate Gyrus: Role of 5HT2A

Receptor-induced PGE2 Signaling...... 41

3.1 Abstract ...... 41

3.2 Introduction ...... 42

3.3 Methods…...... 44

3.3.1 Animals ...... 44

3.3.2 Drug Treatments ...... 45

3.3.3 Microdialysis ...... 45

3.3.4 High-performance Liquid Chromatography ...... 46

3.3.5 ELISA Analysis of PGE2 Concentrations ...... 47

3.3.6 Immunohistochemistry ...... 47

3.3.7 Stereology ...... 48

3.3.8 Electrophysiology ...... 48

3.3.9 Statistical Analysis ...... 49

3.4 Results……………………………………………………………………50

3.4.1 MDMA-induced Increases in PGE2 Concentrations within the

Dentate Gyrus…………...... 50

viii

3.4.2 EP1 Receptor Inhibition and Local Perfusion of MDMA into the

Dentate Gyrus…………...... 50

3.4.3 Effect of SC-51089 on MDMA-induced PV Cell Losses in the

Dentate Gyrus…………...... 51

3.4.4 Effect of MDMA on Input-output Function in the Dentate

Gyrus…………...... 51

3.4.5 The Effects of MDL100907 and SC-51089 on Decreases in Paired-

pulse Depression in the Dentate Gyrus Caused by

MDMA…………...... 52

3.4.6 Effects of MDL100907 and SC-51089 on MDMA-induced

Reductions in Perforant Path-induced Afterdischarge Threshold in

the Dentate Gyrus…………...... 53

3.5 Discussion ...... 60

4 Discussion of Findings ...... 66

4.1 Summary of Findings ...... 66

4.2 General Discussion ...... 70

References ...... 81

A MDMA-induced Changes in Paired-Pulse Facilitation in the Dentate

Gyrus…………………………………………………………………………...102

A.1 Rationale……...... 102

A.2 Methods ...... 102

A.3 Results...... 104

A.4 Discussion ...... 106

B The Effects of Stress and MDMA on Parvalbumin Interneurons in the

Dentate Gyrus………………………………….………………………………108

B.1 Rationale ...... 108

B.2 Methods ...... 109

B.3 Results...... 111

B.4 Discussion ...... 113

List of Figures

1-1 Hypothetical model of MDMA-induced glutamate increases and subsequent decreases in PV interneurons ...... 22

2-1 Systemic MDMA increases glutamate in the dentate gyrus...... 32

2-2 NMDA receptors mediate MDMA-induced PV cell losses ...... 33

2-3 MDL100907 prevents MDMA-induced glutamate increases ...... 34

2-4 MDL100907 prevents MDMA-induced PV cell decreases………………..…….35

3-1 MDL100907 prevents MDMA-induced PGE2 increases in the dentate gyrus ..... 54

3-2 SC-51089 prevents MDMA-induced glutamate increases in the dentate gyrus .... 55

3-3 SC-51089 prevents MDMA-induced PV-ir interneuron decreases...... 56

3-4 MDMA has no effect on input-output relationship in the dentate gyrus ...... 57

3-5 MDMA-induced reductions in paired-pulse depression in the dentate gyrus...... 58

3-6 MDMA-induced decreases in afterdischarge threshold...... 59

4-1 Theoretical model for MDMA-induced glutamate increases and subsequent PV

interneuron decreases and increased excitability in the dentate gyrus...... 68

4-2 Theoretical model for MDMA-induced cell signaling in the dentate gyrus ...... 69

A-1 MDMA-induced reductions in paired-pulse facilitation in the dentate gyrus. .... 105

B-1 Figure B-1: The Effects of Stress and MDMA on PV Interneurons in the Dentate

Gyrus…...... 112

List of Abbreviations

MDMA...... ±-3,4-methyenedioxymethamphetamine PGE2………………..Prostaglandin E2 EP1………………….Prostaglandin E receptor 1 5HT ...... Serotonin PV ...... Parvalbumin PV-IR ...... Parvalbumin-Immunoreactive GABA ...... gamma-Aminobutyric acid AMPA ...... α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid NMDA ...... N-Methyl-D-aspartate COX ...... Cyclooxygenase PBS ...... Phosphate Buffered Saline SERT...... Serotonin Transporter DG ...... Dentate Gyrus RT ...... Room Temperature

xii

Chapter 1:

General Introduction to MDMA Neurotoxicity and the Role of Parvalbumin Interneurons in Hippocampal Function

1.1 MDMA Use

±-3,4-methyenedioxymethamphetamine (MDMA) is a psychoactive substituted amphetamine which has gained popularity amongst teenagers and young adults. Also referred to as ecstasy or molly, MDMA is commonly taken at night club or rave scenes.

According to 2013 surveys, as high as 12.9 percent of adults aged 19-25 have taken

MDMA in their lifetime (SAMSA 2013). These surveys also put the total estimate of those having taken MDMA in their lifetime at nearly 18 million. Interestingly, surveys from 2012 estimated the total number of individuals having taken MDMA at nearly 1.7 million fewer than in 2013, suggesting a recent gain in popularity of MDMA abuse

(SAMSA 2012).

Despite gaining such widespread popularity over the past three decades, MDMA received little attention when it was first discovered. First synthesized in 1912 by Merck chemist, Anton Köllisch, MDMA was created as an intermediate product of hydrastine production. The first scientific study characterizing the toxicity and behavior effects of 1

MDMA were published in 1973 (Hardman et al., 1973). Shortly thereafter, MDMA was reportedly used as an aid in psychotherapy without the support of FDA approval (Greer and Tolbert, 1986, Pentney, 2001). It was also around the same time that reports spread, depicting the recreational abuse of MDMA. Despite the claims of therapeutic potential by psychiatrists, MDMA was placed on the scheduled list of drugs as a schedule 1 controlled substance.

1.2 Acute Behavioral Effects and Addictive Qualities of MDMA

The acute mood enhancing effects may help explain both the potential for

MDMA to aid in psychotherapy as well as the abuse potential of MDMA. By inducing feelings of increased emotional openness and closeness towards others, MDMA has earned the description as being an entactogen or empathogen. These acute feelings emotional openness and closeness towards others allow for a better emotional bond to occur between a therapist and patient during therapy (Greer and Tolbert, 1986, Pentney,

2001). These same feelings of connectedness are also likely contributors to the recreational use of MDMA, which commonly occurs at dance clubs. Other acute psychological effects of MDMA include feelings of euphoria, elevated self-confidence and heightened sensory awareness (Liechti and Vollenweider, 2001). MDMA is also known to have a number of physical effects which include increased heart rate, blood pressure, vasoconstriction, hyperthermia and dehydration (Hayner and McKinney, 1986,

Buchanan and Brown, 1988).

1.3 MDMA Pharmacology 2

The short-term physical and psychological effects of MDMA are mainly attributable to its effects on the monoaminergic system within the central nervous system and periphery. MDMA has been shown to cause the release of serotonin (5HT) and to a lesser extent dopamine and norepinephrine in rodent brains (Nichols et al., 1982,

Yamamoto and Spanos, 1988, Johnson et al., 1991). The increased brain concentrations of dopamine, caused by MDMA, likely contribute to the euphoric feelings reported by

MDMA abusers (Morland, 2000, Liechti and Vollenweider, 2001). Increased activity of the 5HTergic nervous system within the limbic nervous system and hypothalamus likely contributes to the emotional effects of MDMA through the ability of 5HT to increase oxytocin (Forsling et al., 2002, Thompson et al., 2007). 5HT, through activation of

5HT2A receptors have been suggested to underlie the hallucinogenic properties of

MDMA (Liechti and Vollenweider, 2001). In the periphery, 5HT is a potent vasoconstrictor and is a likely contributor to the vasoactive nature of MDMA (Vitolo et al., 1962, Hayner and McKinney, 1986, Gordon et al., 1991). This vasoconstriction, which limits heat loss and would effectively limit thermoregulation, contributes to the hyperthermic effects of MDMA (Tehan et al., 1993).

Several studies have highlighted the role of monoamine transporters in mediating

MDMA-induced increases in monoamines. Studies have indicated that MDMA inhibits the serotonin transporter (SERT) to a greater extent than either dopamine and norepinephrine transporter (Han and Gu, 2006). By inhibiting these transporters MDMA could prevent the reuptake of monoamines, leading to an increase extracellular concentrations of said monoamines (Steele et al., 1987). Studies investigating the interaction of MDMA and SERT have supported the idea that MDMA, like other 3

substituted amphetamines, are substrates for SERT (Gudelsky and Nash, 1996). Thus, by being taken up into the 5HT terminals, MDMA can cause non-exocytotic release of 5HT.

This process is thought to be mediated by increases in cytosolic 5HT concentrations as

MDMA competes for and inhibits uptake of 5HT into vesicles, ultimately leading to reversal of transport function due to changes in the 5HT gradient. Three key experiments led to this conclusion, the first of which being that the increases in monoamines were not dependent upon neuronal activation or were not blocked by TTX (Gudelsky and Nash,

1996). Second, it was shown that MDMA can mediate the release of 5HT from vesicles preloaded with 5HT, which would theoretically increase cytosolic 5HT concentrations

(Rudnick and Wall, 1992). The third piece of evidence supporting this conclusion is that

MDMA-induced extracellular 5HT increases could be blocked via inhibition of monoamine transporters. For instance, fluoxetine, which is an inhibitor of SERT, prevents MDMA-induced 5HT release in the striatum and the prefrontal cortex as well as brain slices (Bradberry et al., 1990, Gudelsky and Nash, 1996).

MDMA has also been suggested to cause dopamine release in a similar fashion to that of 5HT, but instead by acting on the dopamine transporter (Koch and Galloway,

1997). Additionally MDMA has been shown to mediate dopamine release in the striatum via activation of 5HT2A receptors and subsequent reductions in GABA in the substantia nigra (Gudelsky et al., 1994, Yamamoto et al., 1995). This reduced inhibition in the substantia nigra is thought to increase activity of dopaminergic neurons in this region leading to dopamine release in the striatum.

1.4 MDMA-induced Neurotoxicity to 5HT Terminals 4

In addition to the short-term effects of MDMA on 5HT, MDMA has been shown to cause long lasting depletions in 5HT as well as its major metabolite, 5- hydroxyindoleacetic acid, content in the striatum, hippocampus and cortex (Schmidt et al., 1986, Commins et al., 1987, Schmidt, 1987). Subsequent studies determined that

MDMA causes a decrease in tryptophan hydroxylase and SERT in these brain regions while leaving numbers of 5HT neurons intact, suggesting that this neurotoxicity was specific to 5HT terminals. Battaglia et al. (1988) demonstrated that reductions in 5HT uptake sites in rats following a neurotoxic dose of MDMA are present for at least 6 months, demonstrating the long-lasting effects that MDMA has on serotonergic neurotransmission.

1.5 Mechanisms for MDMA-induced Neurotoxicity

1.5.1 Oxidative Stress

Several studies have suggested that MDMA-induced neurotoxicity to 5HT terminals is mediated by oxidative stress through the generation of reactive oxygen species (ROS). The production of ROS can be potentially damaging to cells due to the highly reactive nature of these molecules, which can oxidize various biological molecules including phospholipids, nucleic acids and amino acids, ultimately impairing the function of these molecules. Neurotoxic doses of MDMA have been shown to cause an acute increase in ROS production within the striatum (Shankaran and Gudelsky, 1999,

Shankaran et al., 2001). Likewise, neurotoxic doses of MDMA have been shown to cause an increase in lipid peroxidation in rat brains (Sprague and Nichols, 1995b, Alves et al., 5

2007). Furthermore the oxidation of tryptophan hydroxylase have been documented in rodent brains following MDMA exposure(Stone et al., 1989). These studies highlight not only potential oxidative stress elicited by MDMA but also pinpoint the 5HTergic nervous system as being impacted by the production of ROS. In support of the role for oxidative stress in mediating 5HT terminal damage, experiments by Shankaran et al. (2001) showed that striatal 5HT depletions caused by MDMA could be prevented by cotreatment with the antioxidant, ascorbic acid, which also prevented MDMA-induced increases in

ROS in the area. Additionally, MDMA has also been shown to increase DNA oxidation in the cortex, hippocampus and striatum, suggesting that non-5HTergic cells may be impacted by increased ROS caused by MDMA (Jeng et al., 2006).

Several sources for the production ROS occurring during MDMA exposure have been proposed. For instance, MDMA has been shown to reduce cytochrome oxidase activity in the striatum, shortly following an acute dose of MDMA (Burrows et al., 2000).

Decreases in cytochrome oxidase activity, either via reduced expression or inhibition, leads to increased ROS production in mitochondria (Gunasekar et al., 1996, Galati et al.,

2009). Metabolism of MDMA has also been suggested to mediate the production of

ROS. High doses of MDMA have been shown to produce significant concentrations of the metabolites; (±)-3,4-Methylenedioxyamphetamine α-Methyldopamine, 3,4-

Dihydroxyamphetamine, N-methyl-α-methyldopamine, and 3,4-

Dihydroxymethamphetamine (Lim and Foltz, 1988, Monks et al., 2004, Pizarro et al.,

2004). Oxidation of these molecules leads to the production of quinone intermediates, which are conjugated cyclic dione molecules that can undergo adduction with nucleophiles like proteins and DNA, thus altering fuction of these molecules (Monks and 6

Lau, 1997). Additionally, in the presence of transition metals like iron, quinones can undergo redox cycling, which leads to the formation of semiquinone radicals and ROS

(Monks and Lau, 1997). Another potential source for ROS caused by MDMA is through the metabolism of 5HT and DA. These neurotransmitters are metabolized via oxidative deamination reactions catalyzed via the enzymes monoamine oxidase A & B (Nagatsu,

2004). This reaction leads to the production of H2O2, which can ultimately form hydroxyl radicals. In support of this notion, experiments performed by Alves et al. (2006) showed that inhibition of monoamine oxidase B reduced the formation of both lipid peroxides and protein carbonyls caused by a high dose of MDMA.

1.5.2 Hyperthermia

Numerous studies have indicated a role of hyperthermia in mediating MDMA- induced neurotoxicity. Malberg and Seiden (1998) showed that MDMA treatments of 20 or 40 mg/kg in lower ambient temperature environments (20-24°C) produced no neurotoxicity. This was in contrast to animals which were treated at slightly higher temperatures (26-30°C), which exhibited significant 5HT depletions in several forebrain regions. Not only did the animals treated at higher ambient temperatures display MDMA- induced neurotoxicity, but they also displayed significant levels of hyperthermia during

MDMA treatment. This findings suggests that MDMA-induced neurotoxicity is dependent upon hyperthermia. Furthermore, it has been demonstrated that MDMA- induced hyperthermia and 5HT neurotoxicity can be enhanced when MDMA exposure takes place at increased ambient temperatures (Sanchez et al., 2004). However, other studies have demonstrated that MDMA-treatment at lower environment temperatures can 7

prevent hyperthermia caused by MDMA without blocking depletions in 5HT in the forebrain of rats. In summary, these findings suggest that MDMA-induced 5HT neurotoxicity may be enhanced by hyperthermia but isn’t necessarily dependent upon its presence.

1.5.3 Inflammation

Neurotoxic doses of MDMA have been shown to cause gliosis, which is an inflammatory response where either astrocytes or microglia become hypertrophic in response to neuronal damage (Gao et al., 2013). In this aroused state, astrocytes and microglia have increased proliferation as well as activity, leading to the production of cytokines which have been demonstrated to cause damage to neurons (Kreutzberg, 1996,

Gao et al., 2013). Experiments by Miller and O’Callaghan (1994), demonstrated the presence of astrogliosis in the striatum of mice treated with MDMA. Their work was followed by several others depicting the presence of increased expression GFAP as well as increased total numbers of astrocytes, in the striatum, hippocampus and cortex of rodents treated with MDMA (Aguirre et al., 1999, Johnson et al., 2004, Khairnar et al.,

2010, Frau et al., 2013, Herndon et al., 2014). In support of a role for astrogliosis in mediating MDMA induced neurotoxicity, Aguirre et al. (1999) demonstrated that both astrogliosis as well as MDMA-induced serotonin depletions could be prevented with the antioxidant, alpha-lipoic acid.

In addition to increased staining and expression of astrocytic markers, several studies have also shown the presence of increased markers for microglia in the striatum and cortex of rodent brains following MDMA exposure (Thomas et al., 2004, Khairnar et 8

al., 2010, Torres et al., 2010). Experiments by Thomas et al. (2004) demonstrated that only amphetamines known to be neurotoxic to monoamine terminals, which includes

MDMA, d-methamphetamine and d-amphetamine, exhibited increased microglial staining in the striatum of mice. Whereas, increased microglial staining was not found in the striatum following exposure to fenfluramine and l-methamphetamine, suggesting that microglia may play a role in terminal damage caused by neurotoxic amphetamines such as MDMA. In agreement with this argument, experiments by Zhang et al. (2006), showed that the known anti-inflammatory drug minocycline prevented MDMA-induced 5HT and

SERT depletions as well as the microglial activation in the frontal cortex and hippocampus.

Although the existence of astrogliosis and microgliosis correlate with MDMA- induced neurotoxicity it remains unclear as to whether this response mediates MDMA- induced neurotoxicity or whether it is a consequence. Microglia, which are the primary phagocytes within the brain have been well established as potential mediators of neuronal damage during pathological situations (Kreutzberg, 1996). Likewise, astrocytes, which typically have a supportive role have been suggested to mediate neuronal damage during conditions of heightened inflammation(Pekny and Pekna, 2014). Both astrocytes and microglia, when activated are known to cause the release of cytokines, which have been suggested to cause neuronal damage. Several studies have demonstrated the presence of increased levels of IL-1β in the brains of rats exposed to neurotoxic doses of MDMA

(Orio et al., 2004, O'Shea et al., 2005, Torres et al., 2011). The potential mechanism through which IL-1β may mediate MDMA-induced neurotoxicity remains unclear, however IL-1β has been shown to cause hyperthermia, enhance monoamine release and 9

promote oxidative stress in rodent brains, all of which are contributing factors to MDMA- induced neurotoxicity (Shintani et al., 1993, Plata-Salaman and Ilyin, 1997, Hsieh et al.,

2014).

1.5.4 Excitotoxicity

Several studies have suggested a potential role for excitotoxicity mediating

MDMA-induced neurotoxicity to 5HT terminals. Through increased activation of glutamate receptors such as NMDA, AMPA or certain metabotropic receptors, glutamate can increase intracellular calcium levels (Miller et al., 1989, Mayer and Miller, 1990).

When intracellular calcium levels reach a certain concentration, pathophysiological processes such as mitochondrial stress and the production of ROS or the activation of intracellular proteolytic enzymes such as calpain can occur (Seubert et al., 1988, Choi,

1994, Nicholls, 2004). These processes can ultimately lead to cellular dysfunction and apoptosis. The NMDA receptor antagonist, dextrorphan, was shown to reduce MDMA- induced decreases in 5HT content in the cortex, hippocampus and striatum(Finnegan et al., 1989). Another study showed that the NMDA receptor antagonist, MK-801, could prevent MDMA-induced decreases in tryptophan hydroxylase activity in the striatum of rats. Experiments by Farfel et al. (1995) showed that MDMA-induced decreases in 5HT could also be prevented with the AMPA receptor antagonist, DNQX, further supporting the role of glutamate receptors in mediating MDMA-induced 5HT depletions. However, additional experiments by Farfel et al. (1995), demonstrated that both NMDA and AMPA receptor antagonists also prevented MDMA-induced hyperthermia. When the treatments took place at higher ambient temperatures, which prevented the inhibitory effects of these 10

glutamate antagonists on MDMA-induced hyperthermia, the protective effects of these drugs on MDMA-induced 5HT depletions were abolished. These experiments suggest that glutamate receptor antagonists protect against MDMA-induced neurotoxicity by reducing the hyperthermia caused by MDMA.

MDMA has been shown to elicit an increase in glutamate concentrations in the dorsal hippocampus (Anneken and Gudelsky, 2012). These MDMA-induced glutamate increases were not found to be present in the striatum or frontal cortex and exhibited a sustained duration in that they were still present 6 hours following 2 i.p. injections of 10 mg/kg. Interestingly, the glutamate increases caused by systemic injections could be mimicked via direct application of MDMA into the hippocampus, suggesting that these effects were mediated via local effects of MDMA. Further experiments showed that these glutamate increases were dependent upon 5HT release and subsequent activation of

5HT2A/2C receptors. Moreover, MDMA induced glutamate increases caused by MDMA could not be blocked via TTX inhibition, suggesting a non-neuronal source of these glutamate increases.

In a more recent article by Anneken et al. (2013), MDMA-induced glutamate increases in the dorsal hippocampus were shown to be blocked by via inhibition of cyclooxygenase (COX). This finding suggests that the inflammatory mediators, prostaglandins may have a role in mediating the glutamate increases caused by MDMA.

The prostaglandin, prostaglandin E2 (PGE2) is known to mediate glutamate release from astrocytes in the hippocampus (Bezzi et al., 1998, Sanzgiri et al., 1999). 5HT2A receptors are known to mediate increases in arachidonate (Felder et al., 1990), which is a substrate for COX-induced prostaglandin production (Weissmann, 1983). Thus, Anneken et al. 11

(2013) proposed that 5HT2A receptor activation mediated by MDMA-induced increases in 5HT may lead to glutamate through the production of prostaglandins.

1.6 MDMA-induced Cell Death

Studies investigating the neurotoxicity to 5HT terminals represent the majority of studies examining MDMA-induced neurotoxicity. However, several studies have suggested that MDMA neurotoxicity extends beyond that of 5HT terminals to include neurons in various regions of the brain. For instance, a dose known to be neurotoxic to

5HT terminals was shown to activate calpain-1 and caspase-3 within neurons of the cortex at 24 hours following MDMA exposure (Warren et al., 2007a). These findings suggest that neurons within the cortex may be undergoing apoptosis shortly after MDMA exposure. Other studies have corroborated such findings, depicting apoptosis in cortical neurons following MDMA exposure (Tamburini et al., 2006, Warren et al., 2007b).

Tamburini et al. (2006) also noted significant increases in activated caspase-3 in the hippocampus of rats, 7 days following MDMA exposure. Increases in cytochrome C were also seen in the hippocampus of rats treated with MDMA(Wang et al., 2009).

Despite the numerous findings suggesting that MDMA causes cell death within cortical and hippocampal regions, few studies have noted significant decreases in any particular subset of neurons within either of these areas. However, growing evidence supports the notion that MDMA may cause damage to GABAergic interneurons in the central nervous system. [3 H]-flunitrazapam binding assays performed on rat brains following MDMA exposure suggests that there may be decreases in GABAergic synapses within the hippocampus and some cortical regions (Armstrong and Noguchi, 12

2004). In support of this notion, Perrine et al. (2010) demonstrated that chronic MDMA exposure results in decreases in GABA concentrations within the hippocampus of rats.

More recently it was discovered that a subset of GABAergic interneurons which express parvalbumin (PV) are decreased in the dentate gyrus (DG) of rodents previously exposed to MDMA (Anneken et al., 2013, Abad et al., 2014). Anneken et al. (2013) showed that these decreases coincided with an acute increase in hippocampal glutamate concentrations. Additional experiments demonstrated that both the acute increases in extracellular glutamate concentrations in the hippocampus and long-term decreases could be prevented by administration of a COX inhibitor during MDMA exposure. These findings suggest a potential role for inflammatory and excitotoxic processes in mediating

MDMA-induced decreases in PV interneurons.

1.7 Parvalbumin Interneurons

1.7.1 Susceptibility to Excitotoxicity

Several papers have alluded to the fact that PV interneurons may be more susceptible to excitotoxicity due to the profile of glutamatergic receptors commonly seen on these neurons. For instance, PV interneurons in the hippocampus have been shown to expression GluR2 lacking AMPA receptors, which are known to be particularly permeable to calcium and thus have been implicated in excitotoxicity (Leranth et al.,

1996). In addition, PV interneurons have been shown to express NMDA and mGlur1/5 receptors, which have also been implicated in excitotoxicity (Kerner et al., 1997, Le

Roux et al., 2013). Thus, collective activation of these receptors by increased glutamate 13

concentrations may promote excitotoxicity to PV interneurons. Studies have reported decreases in PV interneurons within the hippocampus resulting from insults where excitotoxicity is a presumed mechanism (Phillips et al., 1998, Kwon et al., 2000).

Furthermore these studies highlighted the role of NMDA receptors in mediating these decreases in PV interneurons. Additionally, PV interneurons in the hippocampus have been shown to be particularly susceptible to AMPA receptor activation via kainic acid

(Sanon et al., 2005).

1.7.2 Morphology and Function

The parvalbumin subset of interneurons, also referred to as fast-spiking interneurons, represent a well-studied class of GABAergic interneurons which have been characterized as having a key role in circuit functions such as feedforward and feedback inhibition, as well as gamma oscillations (Kneisler and Dingledine, 1995a, b, Bartos et al.,

2002). As such, PV interneuron dysfunction is thought to underlie various diseases of the central nervous system including epilepsy, depression and schizophrenia (DeFelipe et al.,

1993, Beasley and Reynolds, 1997, Zhang et al., 2002, Rajkowska et al., 2007)

The morphology, which gives insight into the function of PV interneurons can help guide understanding of how PV interneurons are involved in such network functions.

The dendritic morphology of PV interneurons is described as being highly branched, with overall lengths being as long as 9 mm in some cases (Gulyas et al., 1999, Kubota et al.,

2011, Tukker et al., 2013). In fact, PV interneurons are known to have the longest dendrites of all interneurons within the hippocampus. Inputs onto these dendrites are rather dense, with the vast majority residing from glutamatergic inputs (Gulyas et al., 14

1999). Thus, PV cells sense and summate inputs from a various amount of sources via dendritic inputs. In addition to receiving numerous inputs on dendritic regions, the somatic and axonal regions of hippocampal PV interneurons are highly innervated (Sik et al., 1995). These inputs, which are located in proximity to higher than normal densities of voltage gated Na channels allow PV interneurons to respond to increased activity of inputs in a relatively short order and with high efficacy (Norenberg et al., 2010).

The axons of PV interneurons, like other interneurons do not cover a large area, however the high number of branches exhibited by PV interneurons is somewhat unique.

These highly arborized axons have an accumulative length of 33 mm and 46 mm in the dentate gyrus and CA1 respectively (Sik et al., 1995, Norenberg et al., 2010). From these axons, extend dense synaptic inputs which mainly innervate principle neurons. In the dentate gyrus, PV interneurons predominately innervate the axo-axonic and somatic regions of granule cells (Ribak et al., 1993, Freund and Buzsaki, 1996). Overall, the massive arborization, density of synapses and precise synaptic location on areas of granule cells where action potentials are generated allows PV interneurons to have strong control over the action potential firing of these cells. This coupled with the fact that PV interneurons receive dense glutamatergic input and are able to respond reliably, would allow for them to have precise control over network activity and have a key role in information processing (Buzsaki and Chrobak, 1995).

One such example of how PV interneurons have been demonstrated to modulate network activity is through their ability to maintain oscillatory activity of networks, which is thought to have a key role in mediating temporal encoding of information

(Buzsaki, 1997, Nyhus and Curran, 2010). This has been demonstrated in optogenetic 15

studies where stimulation of hippocampal PV interneurons at theta frequencies could reliably induce theta spike resonance in hippocampal pyramidal cells (Stark et al., 2013).

Additionally, PV interneurons of the hippocampus are thought to have a role in gamma oscillations as they have been shown to reliably fire in phase with the peaks of gamma activity (Hajos et al., 2004).

1.8 Dentate Gyrus

1.8.1 Sensory Information Processing

The DG is thought to be the main input center of the hippocampus. The DG receives inputs relaying various modalities of sensory information including vestibular, olfactory, visual and somatosensory (Kesner, 2007). These inputs project from the perirhinal and entorhinal cortex and travel to the DG via the medial and lateral perforant pathways (Witter et al., 1989, Hargreaves et al., 2005). The medial perforant pathway

(MPP) has received a great deal of attention due in part to the robust excitatory effect that it has on the DG granule cells (McNaughton and Barnes, 1977, Crunelli et al., 1983). The

MPP relays spatial information to the DG via activation of NMDA receptors located on granule cells (Crunelli et al., 1983). This information is then relayed to the CA3 via mossy fiber projections from granule cells (Lynch et al., 1973). The relatively convergent projections of granule cells onto CA3 neurons, low excitability of granule cells and strong GABAergic inhibition allows the dentate gyrus to act as a pattern separation generator (Rolls, 1990, Treves and Rolls, 1994). That is, the dentate gyrus receives several inputs relaying various spatial information and separates this information into 16

non-overlapping ensembles within the CA3. This basic network function is thought to be critical for the processing of spatial information and thus the formation and recall of spatial memories.

1.8.1 Gating of Epileptogenesis

The dentate gyrus has been widely studied in the context of its role in modulating seizure activity. The role of the dentate gyrus as an initiating source of spontaneous seizure activity originated due to structural changes seen in epileptic patients. These changes include sprouting of excitatory fibers emanating from mossy cells in the hilus and dispersion of the granule cell layer, both of which coincided with the timing of spontaneous seizures (Tauck and Nadler, 1985, Haas and Frotscher, 2010). Studies on the normal microcircuitry of the dentate gyrus as well as intrinsic properties of granule cells have suggested that the low level of excitability of the dentate gyrus may allow it to serve as a gate for seizure activity by controlling excitation of downstream hippocampal microcircuits (Heinemann et al., 1992). In particular, granule cells were shown to have a relatively low input resistance and hyperpolarized resting membrane potential, which likely contribute to the low probability of action potential firing of these neurons (Mody et al., 1992). In addition, Mody et al (1992) demonstrated the presence of a strong

GABAA receptor mediated depolarizing shunt acting on granule cells. PV interneurons have been suggested to play a large role in this inhibition via inputs to the somatic and axoaxonic regions of granule cells (Freund and Buzsaki, 1996, Miles et al., 1996). This strong GABAergic inhibition within the dentate gyrus is thought to aid in the ability of the dentate gyrus to prevent the spread of excitation through the hippocampal formation 17

(Lothman et al., 1992). In support of this role, numerous studies have noted decreases in

PV interneurons in the dentate gyrus following convulsant-induced seizure models as well as in epileptic patients (Kamphuis et al., 1989, DeFelipe et al., 1993, Arellano et al.,

2004).. The lack of PV interneurons is thought to impair the inhibition of the dentate gyrus, effectively increasing excitability of dentate gyrus granule cells and promoting seizure susceptibility.

1.9 Long-term Behavioral Consequences of MDMA Exposure

The use of MDMA has been associated with a number of neuropsychological impairments and mood changes. In self-report survey data collected from MDMA abusers, it was found that those having abused MDMA report anxiety, depression as well as mood imbalances (Curran and Travill, 1997, Parrott et al., 2002, Parrott et al., 2006).

Several studies have also reported increased levels of anxiety and depression in adult rats treated with MDMA (Morley et al., 2001, Gurtman et al., 2002, Thompson et al., 2004,

Cunningham et al., 2009). Given the known role of 5HT neurotransmission in modulating mood, MDMA-induced 5HT depletions are thought to underlie these mood changes seen following MDMA exposure. Experiments by Gurtman et al. (2002), showed a correlation between 5HT depletions in MDMA treated animals with increased anxiety and depression. Additionally, Thompson et al. (2004) demonstrated the ability of fluoxetine, which is known to enhance 5HT neurotransmission, to reverse MDMA-induced increases in anxiety and depression.

In addition to mood changes caused by MDMA several researchers have reported memory impairments in abstinent MDMA abusers (Krystal et al., 1992, Parrott et al., 18

1998, Wareing et al., 2004, Parrott et al., 2006). These studies found impairments in prospective, verbal as well as spatial memory in MDMA abusers. In addition to these memory impairments, other studies have reported significant impairments in executive functioning and attention, as well as increased impulsivity in those who have abused

MDMA (Morgan, 1998, McCann et al., 1999). It has been suggested that these cognitive impairments may underlie drug addiction processes observed with MDMA. It should be noted that the presence of these symptoms before MDMA use can’t be determined with human studies and thus may precede and contribute to MDMA abuse and addiction.

However, several animal studies have also reported memory impairments caused by

MDMA exposure. Animal studies performed on rats, mice, and rhesus monkeys have all demonstrated long-lasting spatial memory impairments caused by MDMA exposure

(Taffe et al., 2002, Sprague et al., 2003, Busceti et al., 2008, Cunningham et al., 2009). In several papers, these spatial memory deficits were shown to coincide with 5HT depletions in the hippocampus caused by MDMA, suggesting a potential role for 5HT depletions in mediating these memory impairments (Taffe et al., 2002, Sprague et al.,

2003). However, Arias-Cavieres et al. (2010) demonstrated the presence of spatial memory impairments in rats treated with a low non-neurotoxic dose of MDMA, suggesting that alternative processes may be causing the spatial memory impairments.

1.10 Summary of Past Findings and Gaps in Knowledge

The majority of studies regarding the neurotoxicity of MDMA have focused on the neurotoxicity to 5HT terminals. These studies have highlighted oxidative stress, inflammation and excitotoxicty as major processes involved in this neurotoxicity.

Increases in hippocampal glutamate concentrations represent an intriguing aspect of

MDMA-induced changes in neurochemistry, which likely contributes to the neurotoxic effects of MDMA. Past studies have highlighted the potential role for prostanoids in mediating these increases in hippocampal glutamate however little is known about the role of these molecules in mediating hippocampal glutamate and subsequent alterations in hippocampal physiology. A growing number of studies have depicted a potential damage to the GABAergic nervous system and in particular PV interneurons in the dentate gyrus caused by MDMA. It is known that PV interneurons are susceptible to excitotoxicity and thus may be susceptible to the increases in hippocampal glutamate concentrations caused by MDMA. Lastly, the impact that deficits in GABAergic function resulting from the

MDMA-induced decrease in PV interneurons has not been established. Given the role of these cells in modulating complex network functions in the hippocampus and the role of this region in spatial memory, decreases in PV interneuron cells caused by MDMA is a likely contributor to spatial memory deficits seen in rats exposed to MDMA.

1.11 Hypothesis

The overarching hypothesis of this dissertation, as depicted in Figure 1-1, is that

MDMA increases in glutamate concentrations in the DG are mediated via increased activation of 5HT2A receptors and consequent increases in PGE2 signaling.

Subsequently, these increases in glutamate concentrations are responsible for the decreases in PV interneurons in the DG caused by MDMA which potentially leads to a decrease in inhibitory signaling in the region. Experiments in Chapter 1 investigated the role of local 5HT2A receptors in mediating MDMA-induced increases in glutamate neurotransmission in the DG as well as the role of this signaling in causing decreases in

PV interneurons. Chapter 2 features experiments aimed at elucidating the role of PGE2 in mediating glutamate increases as well as the losses in PV interneurons. In addition, these experiments investigated the role of this 5HT2A receptors and EP1 receptors in mediating changes in inhibitory signaling in the DG. Overall, these experiments investigated the potential impacts of MDMA-induced increases in dentate gyrus glutamate concentrations on PV interneurons and physiology of the DG.

Figure 1-1: Hypothetical model of MDMA-induced glutamate increases in the dentate gyrus and subsequent PV decreases. MDMA causes an increase in 5HT2A receptor activation by mediating increases in 5HT in the dentate gyrus. The increased activation of 5HT2a receptors, which is known mediate the production of arachidonate, leads to increases in PGE2 concentrations in the dentate gyrus. PGE2 is known to mediate glutamate release in the hippocampus, which promotes excitotoxicity to PV interneurons through activation of NMDA receptors. The loss of PV interneurons in the dentate gyrus caused by MDMA then leads to a reduction in inhibition, promoting increased excitability of dentate gyrus granule cells.

Chapter 2:

MDMA-induced Loss of Parvalbumin Interneurons within the Dentate Gyrus is Mediated by 5HT2a and NMDA Receptors

(A modified version of this chapter has been published in the European Journal of

Pharmacology)

Collins, S., Gudelsky, G., & Yamamoto, B. (n.d.). MDMA-induced loss of parvalbumin interneurons within the dentate gyrus is mediated by 5HT2A and

NMDA receptors. European Journal of Pharmacology, 95-100

2.1 Abstract

MDMA is a widely abused psychostimulant which causes a rapid and robust release of the monoaminergic neurotransmitters dopamine and serotonin. Recently, it was shown that MDMA increases extracellular glutamate concentrations in the dorsal hippocampus, which is dependent on serotonin release and 5HT2A/2C receptor activation. The increased extracellular glutamate concentration coincides with a loss of parvalbumin-immunoreactive (PV-IR) interneurons of the dentate gyrus region. Given the known susceptibility of PV interneurons to excitotoxicity, we examined whether

MDMA-induced increases in extracellular glutamate in the dentate gyrus are necessary for the loss of PV cells in rats. Extracellular glutamate concentrations increased in the dentate gyrus during systemic and local administration of MDMA. Administration of the

NMDA receptor antagonist, MK-801, during systemic injections of MDMA, prevented the loss of PV-IR interneurons seen 10 days after MDMA exposure. Local administration of MDL100907, a selective 5HT2A receptor antagonist, prevented the increases in glutamate caused by reverse dialysis of MDMA directly into the dentate gyrus and prevented the reduction of PV-IR. These findings provide evidence that MDMA causes decreases in PV within the dentate gyrus through a 5HT2A receptor-mediated increase in glutamate and subsequent NMDA receptor activation.

2.2 Introduction

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a psychostimulant popular amongst both teenagers and younger adults. MDMA targets primarily the serotonin (5HT) neurotransmitter system to produce a rapid acute release of 5HT and a long-term depletion of 5HT resulting from what can be best described as a distal axotomy

(Nichols et al., 1982, Johnson et al., 1986). This persistent decrease in 5HT is observed within the striatum, cortex and hippocampus and may underlie the cognitive deficits evident in human abusers of MDMA (Battaglia et al., 1987, O'Hearn et al., 1988, Parrott et al., 1998).

Several studies support the notion that MDMA-induced neurotoxicity may extend beyond 5ht terminals to include apoptosis within the hippocampus (Tamburini et al.,

2006, Wang et al., 2009, Kermanian et al., 2012, Soleimani Asl et al., 2012). Further 24

evidence for neuronal cell loss caused by MDMA originates from recent reports of a loss of parvalbumin (PV) interneurons in the DG of MDMA-exposed rodents (Anneken et al.,

2013, Abad et al., 2014). These studies concluded that the loss of PV interneurons is associated with MDMA-induced increases in hippocampal glutamate; however, a direct causal role for glutamate receptor activation in mediating this effect remains to be established. Along these lines, PV neurons express mGluR1/5 receptors, GluR2 lacking

AMPA receptors as well as NMDA receptors within the hippocampus such that their coordinate or convergent activation may convey susceptibility to excitotoxicity (Kerner et al., 1997, Catania et al., 1998, Le Roux et al., 2013). In fact, decreases in PV immunoreactivity (PV-IR) are evident after neurotoxic insults in which glutamate neurotransmission is a presumed mechanism (Kwon et al., 1999, Gorter et al., 2001,

Sanon et al., 2005).

The increases in extracellular glutamate concentrations caused by systemic injections of MDMA appear to be mediated by 5HT efflux and prevented by the systemic injections of the non-specific 5HT2 receptor antagonist ketanserin (Anneken and

Gudelsky, 2012, Anneken et al., 2013). It remains to be determined which 5HT2 receptor subtype within the hippocampus mediates the increases in extracellular glutamate concentrations. Therefore, the current study determined if 5HT2a and/or NMDA receptor activation within the DG of the hippocampus during MDMA exposure is responsible for the increases in extracellular glutamate and decreases in PV-IR.

2.3 Methods

2.3.1 Animals

Adult male Sprague-Dawley rats (200-275 g. Harlan Sprague Dawley, In, USA) were used in all experiments. Upon arrival, rats were grouped two rats per cage and allowed one week to acclimate. The rats were kept on a 12/12-hr light dark cycle in a temperature and humidity controlled room with food and water available ad libitum. All procedures were performed in accordance with the NIH Guide for the Care and Use of

Laboratory Animals and in approval with Institutional Animal Care and Use Committee at the University of Toledo.

2.3.2 Drug Treatments

MDMA was obtained from the National Institutes of Drug Abuse (NIDA,

Research Triangle). For PV cell count experiments, rats were injected with 0.9% (1 ml/kg) saline or MDMA (7.5 mg/kg, once every 2 hours X 4 injections). MDL100907

(generous gift from Wyeth) and MK801 (Tocris) were dissolved in saline with a pH 3

(adjusted with HCl) and were given via IP injections (0.1mg/kg), 30 minutes before each administration of MDMA. These doses were based on previous studies where this dose was used (Schreiber et al., 1998, Tutka et al., 2002). Core body temperatures were recorded 1 hour after each injection of saline or MDMA using a rectal probe digital thermometer (Thermalert TH-8, Physitemp Instruments). To control for decreases in

MDMA-induced hyperthermia caused by MK801 or MDL100907, treatments of these groups were performed in an elevated ambient temperature (28.7°C).

2.3.3 Microdialysis

Rats were anesthetized with a xylazine/ketamine hydrochloride mixture (6/70 mg/kg i.p.) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA).

The active membrane (~1mm, 13KDa cutoff) of a microdialysis probe was placed in the

DG of the dorsal hippocampus (coordinates: 3.9 mm rostral from bregma, 2.2 mm lateral from the midline and 4.2 mm ventral). Three additional holes, with stainless steel screws were placed in the skull to act as anchors for the dental cement.

The morning after surgeries, Dulbecco’s phosphate-buffered saline (138 mM

NaCl, 2.1 mM KCl, 0.5 mM MgCl2, 1.5 mM KH2PO4, 8.1 mm NaHPO4, 1.2 mm CaCl2, and 0.5 mM D-glucose, pH 7.4) (Sigma-Aldrich) was perfused at a flow rate of 1.5

µL/min using a model 22 syringe perfusion pump (Harvard Apparatus). After a 1 hour equilibration period, four 30 minute baseline samples were collected. MDMA exposure began immediately following baseline collections and dialysate samples were collected every hour. MDMA exposure consisted of either 4 i.p. injections of 7.5 mg/kg, each

DG. Reverse dialysis of the antagonists (100 nM) MDL100907 was started 30 minutes prior to reverse dialysis of MDMA. This dose was used based on previous findings highlighting selectivity of MDL100907 for 5HT2a receptors as compared to 5HT2c receptors (Kehne et al., 1996). Brains from rats used for microdialysis were sectioned and probe placement was verified.

2.3.4 High-performance Liquid Chromatography

Dialysate samples (20μL) were injected onto a C18 column (150 x 2mm, 3μm particle filter, Phenomenex) and were eluted using a mobile phase which consisted of 0.1

M Na2HPO4, 0.1mM EDTA and 10% methanol (pH 6.4). As previous described, o- pthaldialdehyde was used to derivatize glutamate for electrochemical detection a using a

LC-4C amperometric detector (BAS Inc.) (Donzanti and Yamamoto, 1988).

2.3.5 Immunohistochemistry

Fixation of the brain was performed by transcardially perfusing 0.1M phosphate- buffered saline (PBS) followed by 4% paraformaldehyde on the 10th day after drug or saline exposure. Brains were cryoprotected, flash frozen and hippocampi sectioned into

50 µm thick slices. Sections were treated with 1% H2O2 for 30 min at room temperature

(RT). The sections were blocked for 2 hr at RT with 10% normal goat serum (Life

Technologies) in 0.1M PBS containing 0.5% Triton-X 100 and Avidin block (4 drops/mL; Vector Laboratories). For PV immunostaining, 50μm thick sections were incubated for 36 hr at 4°C with a mouse monoclonal PV antibody (1:2000; Swant, cat#

PV235) in 0.1 M PBS containing 0.5% Triton-X 100, 1% NGS and Biotin block (4 drops/mL; Vector Laboratories). Sections were then incubated in goat anti-mouse biotinylated secondary antibodies (Millipore, cat# AP124b) for 2hr at RT followed by incubation in avidin-biotin- horseradish peroxidase (Vectastain Elite ABC Kit; Vector

Laboratories) for 2hr at RT. Sections were then developed in diaminobenzidine

(DAB/Metal Concentrate; Pierce) and mounted on glass slides and coverslipped with

Eukitt mounting medium (Sigma- Aldrich). All slides were coded and the code was not broken until the end of quantitative analysis. 28

2.3.6 Stereology

To assess PV-IR GABA interneurons in the dorsal hippocampus, a modified optical fractionator counting technique was used (West et al., 1991, Gundersen et al.,

1999). Neuronal counts were made using a BX51 Olympus microscope equipped with a

DVC camera interfaced with StereoInvestigator 8.21 software (MBF Bioscience).

During quantification, every fourth section for a total of six sections through the dorsal hippocampus was systematically sampled. A border which contained the granule cell layer and hilus region was drawn with a 10x objective and counts were performed using a

20x objective. Tissue shrinkage due to processing prevented the use of guard zones above and below the dissector. PV counts were performed using 100μm X 100μm grid dimensions. Parameters for stereology experiments were determined in order to obtain a

Gunderson coefficient of around 0.1 or below, which allows for an accurate estimate of total PV interneurons within the region.

2.3.7 Statistical Analyses

Stereological neuronal counts were analyzed using a two-way ANOVA to compare the effects of saline or MDMA and an interaction between these effects and that of the antagonists (MK801 or MDL100907). Extracellular glutamate concentrations were compared between groups using a two-way ANOVA with repeated measures. Post-hoc analysis was performed using Tukey’s test. Statistical significance was set at p<0.05

2.4 Results

2.4.1 Effects of Systemic Injections of MDMA on Extracellular Glutamate

Concentrations in the DG

The effects of systemic injections of MDMA (7.5 mg/kg x 4 injections, each injection every 2 hrs) on extracellular glutamate concentrations within the DG were determined via microdialysis. Two-way repeated measures ANOVA revealed a significant effect of MDMA treatment on extracellular glutamate concentrations

(F(1,110)=9.635; p<0.01). Extracellular glutamate concentrations were significantly increased 1 hour after the second injection of MDMA (p<0.05) and at subsequent sample collection times (Figure 2-1).

2.4.2 NMDA Receptor Inhibition Prevents MDMA-induced PV Cell Losses

The role of NMDA receptor activation in mediating MDMA-induced PV deficits within the DG was examined. Representative images (10x magnification) of the DG from treatment groups, 10 days following drug exposure are shown in Figure 2-2A. PV cells within the DG were localized to the granule cell layer and hilus region. MDMA+vehicle treated rats had significantly fewer (30%, p<0.001) PV interneurons in the DG than saline vehicle treated control animals. MDMA+vehicle treated animals had significantly increased core body temperatures (p<0.05) compared to saline+vehicle controls, but were not significantly different than MDMA+MK801 treated animals (mean temperatures were 39.1 oC and 39.2 oC for MDMA+vehicle and MDMA+MK801 respectively (data not shown). As shown in figure 2-2.B, two-way ANOVA revealed a significant effect of

MDMA on PV cells (F(1,27)=22.7; p<0.001) as well as an interaction between MDMA and

MK801 treatment on PV cell counts (F(1,27)=9.283; p<0.01). PV cell counts in the DGof rats treated with MK801 prior to and during MDMA exposure were not significantly different from saline vehicle controls.

2.4.3 MDL100907 Prevents Increased Glutamate Concentrations Caused by Local

Perfusion of MDMA

The effects of local administration of the 5HT2a receptor antagonist MDL100907 on extracellular glutamate increases caused by reverse dialysis of 100 µM MDMA into the DG was determined. As shown in Figure 2-3, reverse dialysis of 100 μM

MDMA+vehicle resulted in a significant increase in extracellular glutamate concentrations compared to aCSF+vehicle (p<0.01). Two-way repeated measures

ANOVA on MDMA+vehicle and aCSF+vehicle groups revealed a significant effect of

MDMA treatment (F(1,100)=6.68; p<0.05) and time(F(6,100)=2.27; p<0.05). A significant effect of MDL100907 treatment (F(1,107)=4.95; p<0.05) was revealed when two-way repeated measures ANOVA was performed on MDMA+vehicle and

MDMA+MDL10097 groups. Additionally, extracellular glutamate concentrations were not significantly different between MDMA+MDL100907 and aCSF+vehicle groups.

2.4.4 Effect of MDL100907 on MDMA-induced PV Cell Losses

The ability of MDL100907 to prevent MDMA-induced decreases in PV cell counts was determined. As shown in Figure 2-4, MDMA+vehicle treated animals had significantly fewer PV interneurons in the DG compared with saline+vehicle treated rats

(p<0.05). A two-way ANOVA analysis revealed a main effect of MDMA (F(1,21)=9.26; 31

p<0.01) as well as an interaction between MDMA and MDL100907 (F(1,21)=11.5; p<0.01) on the decreases in the number of PV-positive cells. MDL100907 prior to and during MDMA exposure blocked the decrease in PV cell counts and were not significantly different from saline vehicle controls. Core body temperatures of

MDMA+MDL100907 treated rats did not differ significantly from those of

MDMA+vehicle treated rats (mean temperatures were 39.1 oC and 39 oC. for

MDMA+vehicle and MDMA+MDL100907, respectively).

Figure 2-1: Systemic MDMA increases glutamate in the DG. Rats received MDMA (7.5mg/kg every 2h X 4 i.p. injections) or saline (1mL/kg, every 2h). n=8-12 per group. Two-way repeated measures ANOVA revealed a significant main effect of MDMA treatment (p<0.005) *=statistically different from saline treated controls (p<0.05).

Figure 2-2: Effect of MK801 on MDMA-induced PV-ir interneuron decreases in the DG. Rats were treated with MK801 (0.1mg/kg, i.p.) 30 minutes prior to each MDMA (7.5mg/kg every 2h X 4 i.p injections) or saline injection. Ten days after exposure, stereologic assessment of DG PV-IR interneurons was performed. A) Representative photomicrographs of PV-IR for treatment groups with PV positive cells labeled with black arrows. B) Quatitative assessment of PV-IR (n=5-8 per group). MDMA+vehicle significantly decreased PV-IR neurons in the DG compared to saline+vehicle treatments (p<0.001). MK801 pretreatment prevented decreases in PV cell counts caused by MDMA (p<0.005). *=statistically different from saline+vehicle controls (p<0.05). 34

Figure 2-3: MDL100907 prevents MDMA-induced glutamate increases. Microdialysis measurements of extracellular glutamate concentrations were performed in the DG MDL100907 (100nM) or vehicle was reverse dialysed 30 minutes prior and during MDMA. n=6-11 per group. Two-way repeated measures ANOVA revealed a significant main effect of MDMA (p<0.05). MDL100907 prevented MDMA-induced increases in glutamate (p<0.05). * denotes values statistically different from aCSF+vehicle controls (p<0.05).

Figure 2-4: MDL100907 prevents MDMA-induced PV cell decreases. Rats were treated with MDL100907 (0.1mg/kg) 30 minutes prior to each MDMA (7.5mg/kg every 2h X 4 injections) or saline injection. Ten days following exposure, stereologic assessment of dentate PV-ir interneurons was performed. PV counts for saline+vehicle and MDMA+vehicle groups are repeated from Figure 2. n=6-8 per group. MDMA+vehicle significantly reduced PV-IR neurons in the DG compared to saline+vehicle treatments (p<0.001). MDL100907 pretreatment prevented MDMA- induced decreases in PV neurons (p<0.001). *=statistically different from saline+vehicle controls (p<0.05).

2.5 Discussion

Our findings support a role for glutamate within the DG in mediating the long- term loss of PV interneurons caused by MDMA. It has been reported that systemic injections of MDMA as well as reverse dialysis of 100 μM MDMA increased glutamate concentrations within the hippocampus; however these experiments did not localize this effect to the DG where PV cell losses occur (Anneken and Gudelsky, 2012, Anneken et al., 2013). We found that both the systemic injections of 7.5mg/kg MDMA (1 injection given every 2 hr for a total of 4 injections) as well as the direct administration of 100 μM

MDMA into the DG produced an increase in extracellular glutamate concentrations within the DG. Therefore, these findings support the idea that the loss of PV interneurons in the DG is a result of local increases in glutamate during MDMA exposure.

PV interneurons within the hippocampus are known to express NMDA receptors

(Le Roux et al., 2013). Antagonism of NMDA receptors during MDMA exposure prevented the deficits in PV interneurons observed 10 days following MDMA exposure

(Figure 2). This effect was not dependent on the ability of MK801 to reduce MDMA- induced hyperthermia (Colado et al., 1998) since the increases in body temperatures between MDMA and MDMA+MK801 groups were similar. Our findings that MK801 pretreatment is able to prevent MDMA-induced decreases in PV cell numbers support the conclusion that increases in glutamate during MDMA exposure play a role in the PV cell losses through increased activation of NMDA receptors. Other studies have reported deficits in PV interneurons within the hippocampus resulting from neurotoxic insults where excitotoxicity is a presumed mechanism (Phillips et al., 1998, Kwon et al., 2000). 37

These studies showed that these PV deficits could be prevented through inactivation of

NMDA receptors. Because NMDA receptor activation plays a role in mediating excitotoxicity, it is possible that MDMA-induced glutamate increases are excitotoxic through activation of NMDA receptors on PV interneurons.

Anneken and Gudelsky (2012) concluded that the increases in hippocampal glutamate concentrations were the result of MDMA-induced increases in 5ht and subsequent 5HT2 receptor activation. In support of this conclusion, we found that

MDL100907, an antagonist with high selectivity for 5HT2A receptors, prevented

MDMA-induced glutamate increases in the DG. Immunoreactivity of 5HT2A receptors is known to be relatively dense in the DG, in particular the hilus region bordering the granule cell layer, and is similar to the densities of PV expressing interneurons in the region (Figure 2A) (Luttgen et al., 2004). The relative density of 5HT innervation follows a similar pattern to that of 5HT2A receptor expression (Moore and Halaris, 1975). Thus,

MDMA causes the release of 5HT to presumably increase 5HT2A receptor activation within the DG.

5HT2A receptors within the DG are known to be expressed by astrocytes and granule cells within the DG (Xu and Pandey, 2000). MDMA-induced 5HT and activation of 5HT2A receptors would increase downstream signaling cascades including the activation of phospholipases. 5HT2A receptors in comparison to 5HT2A receptors are known to preferentially activate phospholipase A2 and subsequent arachidonate production (Felder et al., 1990, Berg et al., 1998). Arachidonate production could thus contribute to cyclooxygenase 2 (COX2) mediated synthesis of prostaglandins such as

PGE2, to increase glutamate release from astrocytes and neurons and cause excitotoxicity 38

(Bezzi et al., 1998, Lin et al., 2014b). It was previously reported that MDMA-induced increases in glutamate within the hippocampus could be prevented via inhibition of

COX2 (Anneken et al., 2013) and supports the interpretation that MDMA-induced increases in glutamate (Figures 1 and 3) are mediated via activation of 5HT2A receptors

(see Figure 3) with subsequent production of prostaglandins.

As mentioned, the localization of 5HT innervations and 5HT2A receptors is particularly dense within the inner portions of the granule cell layer and hilus region

(Luttgen et al., 2004). This corresponds with the pattern of PV interneuron localization within the DG and suggests that PV cells in this region have an increased likelihood of being impacted by increased 5HT2A receptor activation and subsequent increases in glutamate caused by MDMA. In support of this idea, we found that the 5HT2A receptor antagonist MDL100907 prevents MDMA-induced decreases in PV cells observed 10 days later (Figure 4). The PV decreases are not mediated by the reduction in MDMA- induced hyperthermia since the average core body temperatures were similar between

MDMA+vehicle and MDMA+MDL100907 treated rats.

The loss of PV interneurons following MDMA exposure may help explain a number of findings that suggest MDMA exposure may impair inhibition within the hippocampus and promote a loss of hippocampal function. PV interneurons within the

DG are known to synapse directly onto the somatic region of granule cells, which allows them to exert strong inhibitory control of action potential firing (Freund and Buzsaki,

1996, Miles et al., 1996). For this reason, a dysfunction in PV interneuron activity has been implicated in epileptogenesis. Along these lines, MDMA exposure in rodents has been shown to increase susceptibility to kainate induced seizures which correlates with a 39

loss of PV interneurons within the DG(Giorgi et al., 2005, Abad et al., 2014). In addition,

PV interneurons play a role in feed-forward and feed-back inhibition within the DG and thereby are integral components of information processing within the hippocampus

(Kneisler and Dingledine, 1995b, a). Thus, a loss of PV interneurons could potentially impair hippocampus-mediated information processing. In fact, several studies have reported spatial memory impairments typically associated with dysfunction of hippocampal based cognitive processes in both adolescent and adult rats treated with

MDMA (Robinson et al., 1993, Sprague et al., 2003, Williams et al., 2003, Vorhees et al.,

2004, Skelton et al., 2006, Arias-Cavieres et al., 2010). Further studies are needed to determine the extent to which GABAergic function is impaired in the hippocampus after

MDMA and whether these impairments underlie changes in hippocampus-mediated cognitive function caused by exposure to the drug.

In conclusion, MDMA-induced decreases in PV interneurons are mediated by the

5HT2A receptor, increases in glutamate neurotransmission and subsequent activation of

NMDA receptors within the DG during MDMA exposure. These effects of MDMA on

PV interneurons within the hippocampus may have a significant impact on hippocampal physiology and explain spatial memory deficits as well as seizure susceptibility in animals treated with MDMA or human MDMA abusers.

Chapter 3:

MDMA Increases Excitability in the Dentate Gyrus: Role of 5HT2A Receptor-induced PGE2 Signaling

(A modified version of this chapter has been submitted for peer review in the

Journal of Neurochemistry)

3.1 Abstract

MDMA is a widely abused psychostimulant which causes release of serotonin through its actions on the serotonin transporter. Recently, it was reported that MDMA increases extracellular glutamate concentrations in the dentate gyrus, which is dependent on activation of local 5HT2A receptors. We examined the role of prostaglandin signaling in mediating the effects of 5HT2A receptor activation on the increases in extracellular glutamate in the dentate gyrus and the subsequent long-term loss of parvalbumin interneurons caused by MDMA. Local administration of MDMA into the dentate gyrus of rats caused an increase in PGE2 which was prevented by coadministration of

MDL100907, a selective 5HT2A receptor antagonist. MDMA-induced increases in extracellular glutamate were inhibited by local administration of sc51089, an inhibitor of the EP1 prostaglandin receptor. Systemic administration of SC-51089 during injections

of MDMA prevented the decreases in parvalbumin interneurons observed 10 days later.

The loss of parvalbumin immunoreactivity after MDMA exposure coincided with a decrease in paired-pulse inhibition and afterdischarge threshold. These changes in physiology were prevented by inhibition of EP1 and 5HT2A receptors during MDMA exposure. In all, these findings support a role for 5HT2A receptors in mediating MDMA- induced PGE2 signaling and subsequent increases in glutamate. This signaling appears to mediate parvalbumin cell losses as well as physiologic changes reminiscent of reduced inhibition within dentate gyrus.

3.2 Introduction

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a widely abused psychostimulant in the substituted amphetamine class of drugs. Acutely, MDMA causes the release of serotonin (5HT) via reversal of the serotonin transporter (Nichols et al.,

1982). In addition to the acute effects of MDMA on 5HT, it has been well documented that MDMA can cause neurotoxicity to 5HT terminals (Schmidt et al., 1986, Schmidt,

1987, Battaglia et al., 1988). Given that the abuse of MDMA has been associated with long-lasting psychiatric effects such as depression and anxiety as well as detrimental effects on learning and memory, it has been suggested that these long-term effects are attributed to the neurotoxic effects of MDMA on the 5HTergic nervous system (Curran and Travill, 1997, Parrott et al., 1998, Parrott et al., 2001).

A growing number of studies support the notion that the GABAergic nervous system within the hippocampus may be impacted by MDMA exposure. Chronic MDMA administration was shown to reduce the binding of [3 H]-flunitrazapam in the 42

hippocampus, suggesting that GABAergic synapses are reduced following MDMA exposure (Armstrong and Noguchi, 2004). In agreement with this finding, Perrine et al.

2010 showed that GABA concentrations within the hippocampus are reduced following chronic MDMA exposure. More recent findings demonstrated that MDMA exposure causes a long-term decrease in parvalbumin (PV) expressing interneurons within the dentate gyrus (Anneken et al., 2013, Abad et al., 2014, Collins et al., 2015).

PV interneurons are a subset of interneurons known to mediate fast GABAergic neurotransmission within the dentate gyrus. These cells receive dense glutamatergic innervations from dentate granule cells and are thought to be critical for feed-back inhibition (Ribak, 1992, Kneisler and Dingledine, 1995a, Blasco-Ibanez et al., 2000). By primarily innervating the somatic and axoaxonic regions of granule cells, PV interneurons exhibit strong control over action potential firing of these cells (Ribak et al.,

1993, Freund and Buzsaki, 1996). This control of action potential firing is thought to be an important component in the ability of PV interneurons to synchronize principal cell activity (Bartos et al., 2002). Along these lines, decreases in hippocampal PV interneurons have been reported in both epileptic and schizophrenic patients, which is thought to underlie both cognition dysfunction present in both diseases as well as the imbalance between inhibitory and excitatory function (DeFelipe et al., 1993, Beasley and

Reynolds, 1997, Zhang et al., 2002, Arellano et al., 2004).

PV interneurons have been theorized to be susceptible to excitotoxicity as demonstrated by decreases in PV immunoreactivity (PV-IR) following neurotoxic insults in which glutamate neurotransmission is a presumed mechanism (Kwon et al., 1999,

Gorter et al., 2001, Moga et al., 2002, Sanon et al., 2005). More recently, we 43

demonstrated that MDMA-induced decreases in PV interneurons were dependent upon activation of NMDA receptors, implicating a role for glutamate in mediating a decrease in these neurons (Collins et al., 2015). Furthermore, MDMA causes an increase in extracellular glutamate concentrations within the hippocampus via increases in serotonin and subsequent activation of 5HT2 receptors (Anneken and Gudelsky, 2012). More recently it was demonstrated that these increases in glutamate were dependent upon cyclooxygenase-2 (COX2) activity, suggesting a role for prostaglandins in mediating these effects (Anneken et al., 2013). Prostaglandins such as PGE2 have been shown to mediate glutamate release in the hippocampus (Bezzi et al., 1998, Sanzgiri et al., 1999).

Furthermore, several studies have suggested that Prostaglandin E receptor 1 (EP1) receptors, which are activated by PGE2, can play a role in excitotoxicity (Ahmad et al.,

2006, Ahmad et al., 2008, Mohan et al., 2013).

The goal of this study was to determine whether MDMA mediates increases in

PGE2 in the dentate gyrus and whether this was dependent upon activation of 5HT2A receptors. We also investigated the potential role of PGE2 signaling in mediating both the increases in extracellular glutamate, the decreases in PV-IR as well as the long-term effects of these acute changes produced by MDMA on paired-pulse depression and afterdischarge threshold in the dentate gyrus.

3.3 Methods

3.3.1 Animals

Adult male Sprague-Dawley rats (200-275 g. Harlan Sprague Dawley, In, USA) were used. Prior to experimentation, rats were allowed at least 5 days to acclimate. The rats were kept on a 12/12-hr light dark cycle in a temperature and humidity controlled room with food and water available ad libitum. Experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Toledo and were based on NIH Guide for the Care and Use of Laboratory Animals.

3.3.2 Drug Treatments

MDMA was obtained from the National Institutes of Drug Abuse (NIDA,

Research Triangle). Physiological saline or MDMA (7.5 mg/kg) was injected once every

2 hours for a total of 4 i.p. injections. Systemic injections (20 μg/kg, i.p.) of the EP1 receptor antagonist, SC-51089, (Tocris, dissolved in .05% DMSO) were given 30 minutes before each administration of MDMA. This dose was based on previous studies depicting the neuroprotective effects of this drug (Abe et al., 2009). Core body temperatures were recorded 1 hour after each injection of saline or MDMA using a rectal probe digital thermometer (Thermalert TH-8, Physitemp Instruments).

3.3.3 Microdialysis

Rats were anesthetized with a xylazine/ketamine hydrochloride mixture (6/70 mg/kg i.p.) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA).

The exposed dialysis membrane (~1mm, 13KDa cutoff) of the microdialysis probe was placed in the dentate gyrus of the dorsal hippocampus (coordinates: 3.9 mm rostral from bregma, 2.2 mm lateral from the midline and 4.2 mm ventral). Three additional screws 45

were placed in the skull and dental cement was used to anchor the microdialysis probe to the skull.

The morning after surgeries, Dulbecco’s phosphate-buffered saline (138 mM

NaCl, 2.1 mM KCl, 0.5 mM MgCl2, 1.5 mM KH2PO4, 8.1 mm NaHPO4, 1.2 mm CaCl2, and 0.5 mM D-glucose, pH 7.4) (Sigma-Aldrich) was perfused at a flow rate of 1.5

µl/min using a model 22 syringe perfusion pump (Harvard Apparatus). An equilibration period of 1 hour was followed by four 30 minute baseline samples. Reverse dialysis of

collections. Reverse dialysis of the selective 5HT2A antagonist MDL100907 (100 nM) or

These doses were used based on previous findings highlighting selectivity or efficacy of these antagonists for their respective receptors (Kehne et al., 1996, Jones et al., 2009).

Brains were sectioned and microdialysis probe placements were verified.

3.3.4 High-performance Liquid Chromatography Analysis of Extracellular

Glutamate

Dialysate samples (20μl) were injected onto a C18 column (150 x 2mm, 3μm particle filter, Phenomenex) and glutamate eluted using a mobile phase which consisted of 0.1 M Na2HPO4, 0.1mM EDTA and 10% MeOH (pH 6.4). As previously described, glutamate was derivatized using o-pthaldialdehyde and electrochemical detection was done using a LC-4C amperometric detector (BAS bioanalytical)(Donzanti and

Yamamoto, 1988).

3.3.5 ELISA Analysis of PGE2 Concentrations

Concentrations of extracellular PGE2 in dentate gyrus dialysate samples were measured utilizing a chemiluminescence ELISA kit (Arbor Assays). 50 µl of dialysate sample was diluted using 50µl Dulbecco's phosphate-buffered saline.

Chemiluminescence was measured using a CLARIOstar microplate reader (BMG

Labtech). Concentrations in diluted dialysate samples were calculated using the standard curve generated from standards prepared in Dulbecco's phosphate-buffered saline.

3.3.6 Immunohistochemistry

Phosphate-buffered saline (0.1 M PBS) was perfused intracardially followed by

4% paraformaldehyde on the 10th day after drug or saline exposure. Brains were cryoprotected, flash frozen and the dorsal hippocampus was sectioned into 50 µm thick slices. Background peroxidase activity was reduced by incubating in 1% H2O2, at room temperature for 30 minutes. The sections were blocked for 2 hr at room temperature with

10% normal goat serum (Life Technologies) in 0.1M PBS containing 0.5% Triton-X 100 and Avidin block (4 drops/mL; Vector Laboratories). For PV immunostaining, 50μm thick sections were incubated for 36 hr at 4°C with a mouse monoclonal PV antibody

(1:2000; Swant, cat# PV235) in 0.1 M PBS containing 0.5% Triton-X 100, 1% NGS and

Biotin block (4 drops/ml; Vector Laboratories). Sections were then incubated in goat anti-mouse biotinylated secondary antibodies (Millipore, cat# AP124b) for 2hr at room temperature followed by incubation in avidin-biotin- horseradish peroxidase (Vectastain

Elite ABC Kit; Vector Laboratories) for 2hr at room temperature. Sections were then developed in diaminobenzidine (DAB/Metal Concentrate; Pierce) and mounted on glass 47

slides and coverslipped with Eukitt mounting medium (Sigma- Aldrich). The experimenter was blind to all treatment groups such that a non-experimenter coded all slides and the code was not broken until the end of quantitative analysis.

3.3.7 Stereology

Quantification of PV-IR GABA interneurons in the dorsal hippocampus was made using a modified optical fractionator counting technique . Neuronal counts were made using a BX51 Olympus microscope equipped with a DVC camera interfaced with

StereoInvestigator 8.21 software (MBF Bioscience). Every fourth section for a total of six sections through the dorsal hippocampus was sampled. The hilus was outlined under a

10x objective and counts were performed using a 20x objective. Grids of 100μm X

100μm were used during counting. A Gunderson coefficient of around 0.1 or below were obtained using these stereology experiments, which allows for an accurate estimate of total PV interneurons within the region.

3.3.8 Electrophysiology

On the 10th day after MDMA/saline exposure, rats were anesthetized with an initial injection of ketamine-xylazine anesthesia (20 mg/kg, 6 mg/kg, i.p., respectively).

A nose cone containing gauze moistened with isoflurane was used to insure an anesthetic state during tracheotomy. For the remainder of electrophysiology experiments, rats were anesthetized with only isoflurane (.6% ± .2%, .5 L/min). Heart rate was kept constant

(220-240 BPM) to ensure stable levels of anesthesia. Extracellular field potentials within the upper blade of the dentate gyrus (-4.0 mm A.P., 2.0 mm M.L, 3.2 mm D.V. bregma) 48

were monitored using teflon coated tungsten electrodes (.7-1 Mῼ). Stimulation of the perforant path (-7.0 mm A.P., 3.5 mm M.L., 3.5 mm D.V. bregma) was done using a concentric bipolar metal electrode. Stimuli were generated using an A-M systems isolated pulse stimulator. Waveforms were amplified using a BAK electronic amplifier and digitized with Digidata 1322A (Axon. Instruments, Inc.) and stored for analysis using pCLAMP9 software (Molecular Devices). Population spike amplitude (PS) was measured by drawing a tangent to the positive going peaks and from this tangent measuring the amplitude of the negative going peak (PS). Input-output curves were determined using

0.1 ms pulses stepped by 1V intensities (.125 Hz), beginning at 3V. Paired-pulse experiments were performed at stimulus intensities resulting in 60% max PS responses as determined by input-output curves. After-discharge threshold was determined using 1ms stimuli given at 10 Hz for 10 seconds. Stimuli were given at 2 minute intervals with each stimulus exceeding the previous stimulus intensity by .1V. After-discharges were defined as spontaneous discharges lasting at least 5 seconds.

3.3.9 Statistical Analyses

Stereological counts, PGE2 measurements and electrophysiology experiments were analyzed using a two-way ANOVA to compare the effects of saline or MDMA and determine an interaction with SC51089 and MDL100907. Extracellular glutamate concentrations were compared between groups using a two-way ANOVA with repeated measures. Post-hoc analysis was performed using Tukey’s test. Statistical significance was set at p<0.05.

3.4 Results

3.4.1 MDMA-induced Increases in PGE2 Concentrations within the Dentate Gyrus

PGE2 concentrations in the dentate gyrus were measured in dialysate samples in which 100 µM MDMA or aCSF was reverse dialyzed directly into the dentate gyrus. As shown in Fig. 3-1, reverse dialysis of MDMA+Vehicle resulted in a significant increase in PGE2 concentrations compared to aCSF+vehicle in the 0-2 and 4-6 hour collection samples. PGE2 concentrations from the MDMA+MDL100907 group were significantly reduced compared to the MDMA+Vehicle group (p<0.05). Furthermore, the PGE2 concentrations from the MDMA+MDL100907 treated group were not significantly different than the aCSF+Vehicle treated group. Two-way ANOVA analysis revealed a significant main-effect of MDMA on PGE2 concentrations at the 0-2 and 4-6 hour time points (F(1,29)=5.385 (0-2 hr), F(1,29)=6.542 (4-6 hr) p<0.05).

3.4.2 EP1 Receptor Inhibition and Local Perfusion of MDMA into the Dentate

Gyrus

The potential role of local EP1 receptor activation in mediating increases in extracellular glutamate concentrations caused by reverse dialysis of 100 µM MDMA into the dentate gyrus was determined. SC-51089 was reverse dialyzed 30 minutes prior to and during MDMA administration. Reverse dialysis of MDMA+vehicle resulted in a significant increase in extracellular glutamate concentrations compared to aCSF+vehicle

(p<0.05, Fig.3-2). A two-way repeated measures ANOVA comparing MDMA+vehicle and aCSF+vehicle groups revealed a significant effect of MDMA treatment 50

(F(1,84)=5.379; p<0.05) and time (F(6,84)=4.489; p<0.05) as well as a significant interaction

(F(6,84)=3.369; p<0.05). A significant effect of SC-51089 treatment (F(1,113)=5.424; p<0.05) was also revealed when comparing MDMA+vehicle and MDMA+SC-51089 treated groups. Extracellular glutamate concentrations were not significantly different between MDMA+SC-51089 and aCSF+vehicle groups.

3.4.3 Effect of SC-51089 on MDMA-induced PV Cell Losses

The ability of SC-51089 coadministration to prevent MDMA-induced decreases in PV cell counts was determined. As shown in Fig. 3-3, MDMA+vehicle treated animals had significantly fewer PV interneurons in the dentate gyrus compared with saline+vehicle treated rats (p<0.05). Two-way ANOVA analysis revealed a main effect of MDMA (F(1,22)=9.384; p<0.05) as well as an interaction between MDMA and SC-

51089 (F(1,22)=5.892; p<0.05) on the number of PV-positive cells. MDMA+vehicle treated rats had significantly fewer parvalbumin cells than MDMA+SC-51089 rats, which were not significantly different from saline vehicle controls. The body temperatures of

MDMA+SC-51089 treated rats did not differ significantly from those of MDMA+vehicle

(mean temperatures were 38.8 oC and 38.6 oC. for MDMA+vehicle and MDMA+SC-

51089, respectively).

3.4.4 Effect of MDMA on Input-output function in the Dentate Gyrus

To determine whether MDMA has any effect on the excitability of dentate gyrus granule cells, we monitored the PS amplitude of granule cells in response to perforant path stimulation. These experiments were performed 10 days following MDMA 51

exposure, which corresponds to the time when MDMA-induced PV interneuron decreases are evident. Fig. 3-4.A shows a representative trace of an evoked response in the dentate gyrus, noting the PS, which is an indication action potential firing in granule cells. Input-output curves, as shown in Fig. 3-4.B, were calculated in order to determine baseline synaptic transmission in the dentate gyrus. There was no significant change in input-output function mediated by MDMA treatment.

3.4.5 The Effects of MDL100907 and SC-51089 on Decreases in Paired-pulse

Depression in the Dentate Gyrus Caused by MDMA

To determine potential changes in GABAergic inhibition caused by MDMA, field potential recordings were performed in the dentate gyrus 10 days after MDMA exposure.

Perforant path induced paired-pulse responses were monitored at interstimulus intervals known to mediate paired-pulse depression (20-80 ms). As shown in Fig. 3-5.B & 3-5.C,

MDMA+Vehicle treated rats exhibited a significant reduction in paired-pulse depression at 40, 50 and 65 ms interstimulus intervals compared to Saline+Vehicle treated rats

(p<0.05). Two-way ANOVA analysis revealed a significant main-effect of MDMA at these intervals (F(1,24)=4.616 (40ms), F(1,24)=8.906 (50ms), F(1,24)=5.740 (65ms); p<0.05)

This analysis also revealed a significant interaction of MDL100907 and SC-51089 and

MDMA treatment at the 40 and 50 ms interstimulus intervals (F(1,24)=6.686 (SC-51089,

40ms), F(1,24)=4.902 (SC51089, 50ms) , F(1,24)=6.272 (MDL100907, 40ms), F(1,24)=4.945

(MDL100907, 50ms); p<0.05). Furthermore, there was no difference between paired- pulse depression at any interstimulus intervals between either MDL100907+MDMA or

SC-51089+MDMA treated rats and Saline+Vehicle treated rats. 52

3.4.6 Effects of MDL100907 and SC-51089 on MDMA-induced Reductions in

Perforant Path-induced Afterdischarge Threshold in the Dentate Gyrus

Potential changes in the excitatory/inhibitory balance in the dentate gyrus caused by MDMA were determined by measuring perforant path stimulus intensities needed to drive spontaneous afterdischarges in the dentate gyrus, 10 days after MDMA exposure.

These experiments revealed a significant reduction in the stimulus intensity required to drive spontaneous afterdischarges in the dentate gyrus of MDMA+Vehicle treated rats compared to Saline+Vehicle treated rats (p<0.05). Two-way ANOVA analysis revealed a significant main effect of MDMA treatment on reducing afterdischarge threshold

(F(1,24)=15.187; p<0.05). Further analysis revealed a significant interaction between either

MDL100907 or SC-51089 treatment and MDMA treatment (F(1,24)=9.188 (SC-51089),

F(1,23)=4.663 (MDL100907); p<0.05). There was no significant difference in the afterdischarge threshold between either MDL100907+MDMA or SC-51089 treated rats and Saline+Vehicle treated rats.

Figure 3-1: MDL100907 prevents MDMA-induced PGE2 increases in the dentate gyrus. PGE2 concentrations were measured in dialysate samples from the dentate gyrus MDL100907 (100nM) or vehicle was reverse dialysed 30 minutes prior to and during MDMA. n=6-11 per group. Two-way repeated measures ANOVA revealed a significant main effect of MDMA (p<0.05). MDL100907 prevented MDMA-induced increases in PGE2 (p<0.05). * denotes values statistically different from aCSF+vehicle controls (p<0.05).

Figure 3-2: SC-51089 prevents MDMA-induced glutamate increases in the dentate gyrus. Extracellular glutamate concentrations were measured via microdialysis in the dentate gyrus d bar). SC-51089 (100nM) or vehicle was reverse dialysed 30 minutes prior and during MDMA. n=6-11 per group. Two-way repeated measures ANOVA revealed a significant main effect of MDMA (p<0.05). SC51089 prevented MDMA-induced increases in glutamate (p<0.05). * denotes values statistically different from aCSF+vehicle controls (p<0.05).

Figure 3-3: SC-51089 prevents MDMA-induced PV-ir interneuron decreases. Rats were treated with SC- 51089 (20 μg/kg, i.p.) 30 minutes prior to each MDMA (7.5mg/kg every 2h X 4 i.p injections) or saline injection. Stereologic cell counts of PV-IR interneurons was performed 10 days after drug exposure. A) Representative staining of PV-IR for treatment groups with PV positive cells labeled with black arrows. B) Quantitative assessment of PV-IR (n=6-7 per group). MDMA+vehicle treatment resulted in a significant decrease in PV-IR neurons in the dentate gyrus compared to saline+vehicle treatments (p<0.05). SC-51089 pretreatment prevented MDMA-induced decreases in PV-IR (p<0.05). *=statistically different from saline+vehicle controls (p<0.05).

Figure 3-4: MDMA has no effect on input-output relationship in the dentate gyrus. 10 days after MDMA (7.5mg/kg every 2h X 4 i.p injections) or saline injections local field potentials in the dentate gyrus were recorded. A) Representative trace from electrophysiology recordings depicting a population response recorded in the granular cell layer in response to perforant path stimulation. Population spikes (PS) were measured as the negative peak amplitude drawn from a line tangent to the two positive peaks. B) Input-output curves depicting PS amplitudes as a function of stimulus intensity. No significant difference in input-output curves was noted between MDMA and Saline treated rats. n=6-7 per group

Figure 3-5: MDMA-induced reductions in paired-pulse depression in the dentate gyrus. Rats were treated with MDMA (7.5mg/kg every 2h X 4 i.p injections) 10 days prior to electrophysiology recordings. MDL100907 or SC-51089 were given 30 minutes prior to each injection of MDMA. A) Representative traces from a MDMA + vehicle and Saline + Vehicle treated rat at 50 ms inter-pulse interval. B) Summary of the paired-pulse responses (PS2/PS1) for MDMA + vehicle and Saline + Vehicle treated rats at varying interstimulus intervals of 20-80 ms. C) Summary of 40, 50 and 65 ms interstimulus interval paired-pulse responses for all treatment groups. Paired-pulse depression was significantly reduced in MDMA + vehicle treated rats compared to Saline + Vehicle treated rats. MDL100907 and SC-51089 pretreatment prevented MDMA-induced decreases in paired-pulse depression. n=6-7 per group *=statistically different from saline+vehicle controls (p<0.05).

Figure 3-6: MDMA-induced decreases in afterdischarge threshold. Afterdischarge thresholds were performed in the dentate gyrus of anesthetized animals, 10 days following drug treatments. MDL100907 or SC-51089 were given 30 minutes prior to each injection of MDMA. A) Example extracellular field potential recording in the dentate gyrus displaying afterdischarges following threshold electrical stimulation of the perforant path. B) Mean thresholds for inducing afterdischarges. MDMA + vehicle treated rats had significantly reduced afterdischarge thresholds compared to saline + vehicle controls. MDL100907 and SC-51089 pretreatment prevented MDMA-induced decreases in afterdischarge threshold. n=6-7 per group *=statistically different from saline+vehicle controls (p<0.05). 59

3.5 Discussion

The findings of the current dissertation demonstrate that the acute effects of

MDMA on glutamatergic neurotransmission mediate long-term changes in hippocampal function. These findings support a link between 5HT2A receptor activation and prostaglandin signaling in mediating the increases in extracellular glutamate concentrations within the dentate gyrus caused by MDMA. This signaling induced by

MDMA mediates the long-term changes in PV interneurons within the dentate gyrus and reduced inhibition within the region.

Prior studies have shown that systemic neurotoxic doses of MDMA increase extracellular glutamate concentrations within the hippocampus via increases in 5HT release (Anneken and Gudelsky, 2012) and 5HT2A activation within the dentate gyrus

(Collins et al., 2015). However, it was unclear whether this was a direct effect of the local activation of 5HT2A receptors on glutamate terminals by MDMA or an indirect effect of 5HT2A receptor activation. Moreover, previous findings by Anneken et al

(2013) demonstrated that MDMA-induced increases in glutamate could be prevented by inhibition of COX2, suggesting a potential role for prostaglandins in mediating the increases in glutamate (Anneken et al., 2013) but the relationship if any, between prostaglandins, 5HT2A receptors, glutamate, and the loss of PVir remained to be established. As an initial step in testing this relationship, the current findings show that the local perfusion of MDMA into the dentate gyrus caused an increase in the extracellular concentrations of prostaglandin, PGE2 (Fig. 1). Consistent with the current findings, activation of 5HT2A receptors have been shown to mediate increases in arachidonate, primarily via increases in activity of phospholipase A2 (Felder et al., 1990). 60

Moreover, several studies have demonstrated the ability of PGE2 to mediate glutamate release in the hippocampus (Bezzi et al., 1998, Sanzgiri et al., 1999). It remains to be determined if the increases in PGE2 are mediated via 5HT2A receptor-induced increases in phospholipase A2 activity and subsequent arachidonate production.

In line with the observations that MDMA induced increases in glutamate are related to the loss of PVir, several studies have highlighted the role of PGE2 in mediating excitotoxicity (Kim et al., 2001, Gendron et al., 2005, Ahmad et al., 2006, Ikeda-Matsuo et al., 2010). More recently, studies have demonstrated a potential excitotoxic role of

PGE2-induced activation of EP1 receptors (Ahmad et al., 2008, Mohan et al., 2013). The current findings now demonstrate a relation between PGE2 and glutamate such that the reverse dialysis of SC-51089 into the dentate gyrus prevented the increases in glutamate caused by the local administration of MDMA (Fig. 2). Although the location of EP1 receptors involved in MDMA-induced glutamate release is not known, previous studies have implicated astrocytes as a likely source of PGE2-induced glutamate increases in the hippocampus (Bezzi et al., 1998, Sanzgiri et al., 1999). These studies linked increases in calcium within astrocytes to glutamate release caused by PGE2. Thus, EP1 receptors, which are known to mediate increases in intracellular calcium and are found on astrocytes may be mediating glutamate release from astrocytes via increases in intracellular calcium (Fiebich et al., 2001, Zonta et al., 2003, Rojas et al., 2014).

Although PGE2 has been shown to mediate K+-induced glutamate release from cortical nerve terminals via calcium increases induced by EP2 receptor signaling (Lin et al.,

2014b), glutamate increases caused by MDMA are not action potential dependent

(Anneken and Gudelsky, 2012). Therefore, it is likely that MDMA-induced glutamate increases within the dentate gyrus are by astrocytes.

Previous reports have shown that the decrease in PV interneurons in the hippocampus following MDMA could be prevented by inhibition of COX-2 during

MDMA exposure (Anneken et al., 2013). More recently, we showed that the decreases in

PV immunoreactivity were dependent upon activation of NMDA receptors (Collins et al.,

2015) but the relationship between the long-term decreases in PV interneurons and the observed changes in extracellular glutamate and EP1 receptor activation remained to be determined. As seen in Figure 3, SC-51089 administration 30 minutes prior to each

MDMA injection prevented the loss of PV immunoreactivity observed 10 days after

MDMA exposure. SC-51089 treatments had no effect on MDMA-induced hyperthermia.

These data provides additional evidence supporting the role of prostaglandins in mediating decreases in PV cells. Furthermore, as SC-51089 also prevented the increases in glutamate, the results indicate that the loss of PV interneurons within the dentate gyrus are mediated through increases in glutamate as a consequence of PGE2-induced activation of EP1 receptors.

PV interneurons receive glutamatergic efferents from both granule cells and the perforant path (Kneisler and Dingledine, 1995b, a). Upon stimulation of the perforant path, PV interneurons provide strong mediated inhibition of dentate gyrus granule cells via inhibitory synapses onto the somatic and axoaxonic regions of granule cells (Freund and Buzsaki, 1996, Miles et al., 1996). Thus deficits in PV interneuron function may significantly alter the feed-forward and feedback inhibition in the dentate gyrus. As shown in Figure 4b, MDMA did not produce a shift in the input-output curves produced 62

by MDMA suggesting that baseline neurotransmission is not altered. To further gauge whether inhibition within the dentate gyrus is affected, the paired-pulse experiments were performed and showed a significant decrease in paired-pulse inhibition at the 40, 50 and

65 ms intervals of MDMA treated rats (Fig. 4b & 4c). Paired-pulse depression in dentate gyrus has been main attributed to GABAergic inhibition (Sloviter, 1991, DiScenna and

Teyler, 1994). Thus, these findings support the interpretation that MDMA-induced changes in paired-pulse inhibition is mediated by decreases in PV interneurons. It is possible that these changes may be mediated by intrinsic changes in granule cell excitability or presynaptic perforant path input. Regardless, the decrease in paired-pulse inhibition was prevented with SC-51089 or MDL100907 during MDMA treatments, and extends the relationship between EP1, 5HT2A receptors and extracellular glutamate to the long-term physiological functioning of the dentate gyrus.

To further investigate changes in excitatory/inhibitory signaling in the dentate gyrus caused by MDMA, we measured afterdischarge threshold in the dentate gyrus 10 days following MDMA exposure. MDMA treatment caused a significant reduction in the threshold stimulus intensity needed to drive afterdischarges in the dentate gyrus (Fig. 6).

Furthermore, the changes in afterdischarge threshold could be prevented by inhibition of

5HT2A and EP1 during MDMA exposure, suggesting that decreases in PV interneurons may play a role in the afterdischarge threshold changes caused by MDMA. Several studies and reviews have highlighted the dentate gyrus as being involved in the generation of seizure activity (Lothman et al., 1992, Scharfman, 1994). GABAergic inhibition mediated by PV interneurons is known to reduce action potential firing of granule cells and has been suggested to play a central role in regulating seizure activity 63

(Freund and Buzsaki, 1996, Miles et al., 1996). The current findings support a change in the excitatory/inhibitory balance within the dentate gyrus which is likely mediated by decreases in PV interneuron function and explain previous findings that MDMA treated mice are more susceptible to kainic acid induced seizures (Giorgi et al., 2005, Abad et al.,

2014).

The dentate gyrus plays a key role in the formation of episodic memories. It receives inputs conveying different modalities of sensory information but the most recognized inputs are those which relay spatial information (Hargreaves et al., 2005).

Thus, damage to the dentate gyrus by MDMA may impair spatial memory performance.

In fact, several studies have reported significant impairments in spatial memory performance in rodents treated with MDMA (Robinson et al., 1993, Sprague et al., 2003,

Williams et al., 2003, Vorhees et al., 2004, Skelton et al., 2006, Arias-Cavieres et al.,

2010). Our results demonstrate changes in physiology within the dentate gyrus which likely alter the processing of spatial information by this region and explain the spatial memory deficits caused by MDMA. Further studies are warranted to further characterize the potential impacts that MDMA-induced changes in GABAergic inhibition may have on dentate gyrus function and resulting behavioral changes.

In conclusion, our findings highlight a potential novel signaling mechanism mediated by MDMA in which 5HT2A receptor activation elicits PGE2 signaling, ultimately leading to increases in glutamatergic neurotransmission. This signaling appears to mediate both the changes in PV interneurons as well as changes in inhibition within the dentate gyrus. The significance of these findings are evident in light of

previous studies highlighting spatial memory deficits as well as seizure susceptibility seen in animals treated with MDMA as well as human MDMA users.

Chapter 4:

Summary and Discussion of Findings

4.1 Summary of Findings

This dissertation investigated the mechanism through which MDMA mediates an increase in hippocampal glutamate concentrations and whether these processes lead to decreases in PV interneurons and changes in inhibition of the dentate gyrus. We found that extracellular glutamate concentrations increased in the dentate gyrus during systemic and local administration of MDMA. Local administration of either a 5HT2A or EP1 receptor antagonist prevented the increases in glutamate caused by reverse dialysis of

MDMA. Additionally, it was shown that local administration of MDMA into the dentate gyrus caused an increase in PGE2 concentrations, which were mediated by activation of

5HT2A receptors. Administration of the NMDA receptor antagonist, MK-801, during systemic injections of MDMA, prevented the loss of PV-IR interneurons seen 10 days after MDMA exposure. PV interneuron decreases could also be prevented by inhibiting

5HT2A or EP1 receptors during MDMA administration. Decreased parvalbumin immunoreactivity 10 days after MDMA exposure coincided with a decrease in paired- pulse inhibition and afterdischarge threshold. These changes were prevented by inhibition

of EP1 and 5HT2A receptors during MDMA exposure. In all, these findings support a role for 5HT2A receptors in mediating MDMA-induced PGE2 signaling and subsequent increases in glutamate. This signaling appears to mediate parvalbumin cell losses as well as physiologic changes reminiscent of reduced inhibition within dentate gyrus.

Figure 4-1: Theoretical model for MDMA-induced glutamate increases and subsequent PV interneuron decreases and increased excitability in the dentate gyrus. MDMA causes an increase in 5HT2A receptor activation by mediating increases in 5HT in the dentate gyrus. The increased activation of 5HT2A receptors during MDMA promotes the production of PGE2 and subsequent activation of EP1 receptors in the dentate gyrus. EP1 receptors mediate glutamate increases in the dentate gyrus caused by MDMA, which promotes excitotoxicity to PV interneurons through activation of NMDA receptors. The loss of PV interneurons in the dentate gyrus caused by MDMA then leads to a reduction in inhibition as explained by a reduction in paired-pulse depression and afterdischarge threshold in the dentate gyrus.

Figure 4-2: Theoretical model for MDMA-induced glutamate increases and subsequent PV interneuron excitotoxicity. Figure 4-2.1 depicts the 5HT2A receptor activation mediated by MDMA- induced 5HT increases. Activation of these receptors, which are found on astrocytes and granule cells, leads to activation of PLA2 and subsequent production of PGE2. Figure 4-2.2 illustrates the binding of PGE2 to EP1 receptors on either astrocytes or neurons, which is known to mediate intracellular Ca2+ increases via an IP3 dependent process. Intracellular Ca2+ mediated by PGE2 has been shown to cause glutamate release from both astrocytes and neurons (Fig.4-2.3). This glutamate can activate NMDA receptors on PV interneurons, leading to increased intracellular calcium levels and excitotoxicity.

4.2 Discussion of Findings

The findings of this dissertation substantiate a link between 5HTergic signaling and prostaglandin signaling in mediating increased extracellular glutamate concentrations within the dentate gyrus during MDMA exposure. These findings support those of

Anneken et al. (2012), which demonstrated the role of MDMA-induced 5HT release and subsequent 5HT2A/2C receptor activation in mediating increases in hippocampal glutamate concentrations. Additionally, we provide evidence that MDMA-induced increases in glutamate concentrations can be seen in the dentate gyrus alone (Chapter 2).

This was shown to occur either by systemic injections of MDMA, or by reverse dialysis of MDMA directly into the dentate gyrus. Experiments in Chapter 2 also show that the glutamate increases mediated by direct infusion of MDMA into the dentate gyrus can be inhibited by antagonizing 5HT2A receptors specifically in the dentate gyrus.

By localizing the effects of MDMA on hippocampal glutamate concentrations to the dentate gyrus it provides additional support for idea that MDMA-induced decreases in

PV interneurons within the dentate gyrus are caused by these increases in glutamate. This idea was proposed by Anneken et al. (2013), following experiments which noted that increases in extracellular glutamate as well as long-term decreases in PV interneurons could both be prevented via treatment with the COX inhibitor, ketoprofen. In experiments highlighted in Chapter 2, we provided additional support for the role of glutamate in mediating the decreases in PV interneurons caused by a binge dose regimen of MDMA.

These experiments demonstrate that MDMA-induced decreases in PV interneurons could be prevented when the NMDA receptor antagonist, MK801, was administered during

MDMA exposure. Results from Chapter 2 also show that inhibition of 5HT2A receptors during MDMA admnistration prevents the decreases in PV interneurons seen in the dentate gyrus. It is likely that this effect is mediated by the ability of MDL100907 to prevent glutamate increases in the dentate gyrus caused by MDMA. Several studies have suggested that PV interneurons may be more susceptible to excitotoxicity due to the expression of glutamate receptors, such as NMDA receptors, which are known to mediate excitotoxicity (Leranth et al., 1996, Kerner et al., 1997, Le Roux et al., 2013). In addition, other studies have noted decreases in PV interneurons following neurotoxic insults mediated by excitotoxicity (Phillips et al., 1998, Kwon et al., 2000). Thus, our findings suggest that PV interneuron decreases caused by MDMA is the result of increases in hippocampal glutamate and is potentially mediated by excitotoxicity.

Previous studies have demonstrated relatively high densities of 5HT and 5HT2A receptors in the dentate gyrus (Moore and Halaris, 1975, Luttgen et al., 2004).

Interestingly, the localization of both 5HT and 5HT2A receptors are greatest in the border between the granule cell layer and the hilus region, which coincides with where we saw the greatest density of PV interneurons. This highlights the potential sensitivity of the dentate gyrus and regions where PV interneurons are located to MDMA-induced 5HT increases. This sensitivity may explain why experiments by our lab as well as other have seen only decreases in PV interneurons within the dentate gyrus (Anneken et al., 2013).

For instance, we did not see decreases in PV interneurons in the CA3 or CA1 regions of the hippocampus 10 days following the same dose which causes decreases in the dentate gyrus (data not shown). Additionally, we did not see decreases in PV interneurons in mPFC (data not shown). These findings suggest that the PV interneurons of the dentate 71

gyrus may be more susceptible to the effects of MDMA, which could be the result of higher density of 5HT and 5HT2A receptors in this region. 5HT2A receptors within the

DG are located on astrocytes and granule cells within the DG (Xu and Pandey, 2000).

Activation of 5HT2A receptors on glial cells has been shown to cause glutamate increases via a Ca2+ dependent mechanism (Meller et al., 2002). However, it remains unclear as to how 5HT2A receptor activation on glia may promote increases in intracellular calcium levels, leading to glutamate release.

Activation of 5HT2A receptors is known to activate phospholipase A2, which mediates arachidonate production (Felder et al., 1990, Berg et al., 1998). Thus, it is likely that MDMA causes increases in arachidnonate by increasing 5HT concentrations and subsequent activation of 5HT2A receptors in the dentate gyrus. Arachidonate is converted to the prostaglandin PGH2, which is a precursor to many other prostaglandins.

Experiments by Anneken et al. (2013), showed that MDMA-induced acute increase in glutamate concentrations within the hippocampus could be prevented by inhibition of

COX, supporting a role for prostaglandins in mediating these glutamate increases. The prostaglandin PGE2 had been demonstrated to have a role in mediating excitotoxicity

(Kim et al., 2001, Gendron et al., 2005, Ahmad et al., 2006, Ikeda-Matsuo et al., 2010).

Furthermore, PGE2 has been shown to mediate glutamate release from both astrocytes and neurons in cultures and hippocampal slices (Bezzi et al., 1998, Sanzgiri et al., 1999,

Lin et al., 2014b). Experiments in Chapter 3 showed that local perfusion of MDMA into the dentate gyrus causes a significant increases in extracellular concentrations of PGE2.

Additionally, these PGE2 increases could be inhibited when MDL100907, the 5HT2A antagonist, was coperfused with MDMA. Because 5HT2A receptors were also shown to 72

mediate MDMA-induced glutamate increases, these findings suggest that MDMA- induced glutamate increases are mediated by 5HT2A receptor induced PGE2 production.

However, it should be noted that activation of NMDA receptors has been shown to mediate the release of both PGE2 as well as glutamate from astrocytes. These findings suggest a potential feed-forward type signaling in astrocytes in which glutamate receptor activation mediates glutamate release. Thus, future studies are warranted in order to determine whether this signaling is necessary for MDMA-induced glutamate increases in the hippocampus.

Experiments by Bezzi et al. (1998) demonstrated that the PGE2-induced glutamate release from astrocytes was dependent upon increases in intracellular Ca2+.

Although this paper did not examine the mechanism through which PGE2 mediates intracellular Ca2+ increases, EP1 receptors represent a potential process through which this occurs. These receptors, which are activated by PGE2, have been shown to mediate increases in intracellular concentrations of Ca2+ via an IP3 dependent mechanism

(Fiebich et al., 2001, Zonta et al., 2003, Rojas et al., 2014). As we demonstrate in Chapter

3, glutamate increases mediated by reverse dialysis of MDMA directly into the dentate gyrus could be prevented when SC51089, the EP1 receptor antagonist was reverse dialyzed with MDMA. This finding together with those from Anneken et al.(2013), showing a role for COX in mediating MDMA-induced glutamate release, suggest that the glutamate increases in the hippocampus, caused by MDMA, are mediated by PGE2 increases and subsequent activation of EP1 receptors. In addition, we demonstrate that

PV decreases caused by MDMA are prevented when SC51089 was given prior to each

MDMA injection, which further supports the role of PGE2 in mediating these decreases. 73

In addition, this finding further supports the role of MDMA-induced glutamate increases in mediating decreases in PV interneurons. Although the location of the EP1 receptors mediating these glutamate increases is unknown, it has been demonstrated that astrocytes express EP1 receptors in rat brains (Fiebich et al., 2001). Furthermore, Anneken and

Gudelsky (2012) demonstrated that MDMA-induced hippocampal glutamate increases were not action potential mediated, suggesting a non-neuronal source. Thus, we propose that the hippocampal glutamate increases mediated by MDMA originate from astrocytes.

Astrocytes have been shown to mediate glutamate release via several mechanisms. Astrocytes have been shown to express all of the machinery for vesicular mediated glutamate release (Montana et al., 2004). For example, it has been demonstrated that astrocytes express soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) complexes, which would enable these cells to regulate exocytosis (Parpura et al., 1995, Jeftinija et al., 1997, Hepp et al., 1999, Montana et al.,

2004). Additionally astrocytes express small vesicles, which contain VGLUT1/2 and these vesicles can undergo exocytosis upon stimulation by increases in intracellular Ca2+ concentrations (Bezzi et al., 2004). Thus, MDMA-induced PGE2 increases may stimulate vesicular release of glutamate via activation of EP1 receptors and subsequent increase in intracellular Ca2+ concentrations.

Astrocytes have also been shown to release glutamate via several transporters and channels. For instance, it has been suggested that astrocytes can release glutamate via reversal of glutamate transporters (Swanson et al., 1995, Zeevalk et al., 1998, Seki et al.,

1999). This has been proposed to occur during ischemia or situations where metabolism is compromised. Compromised metabolism could also cause the release of glutamate 74

from astrocytes via transport through the cystine-glutamate antiporter or release from volume regulated anion channels (VRAC). Cystine-glutamate antiporters function to provide intracellular cysteine for the production of the antioxidant glutathione(McBean,

2002). This is done by importing cystine molecules while exporting glutamate. Thus, under circumstances where astrocytes are metabolically compromised, activity of this antiporter may increase, leading to increased transport of glutamate outside of astrocytes.

VRACs are channels that are glutamate permeable channels whose activity is increased by hypo-osmotic situations (Mongin and Orlov, 2001). Under circumstances where metabolism is compromised, such as ischemia, which promotes swelling of astrocytes,

VRACs may be opened, promoting the release of glutamate (Kimelberg et al., 1990,

Kimelberg, 2005). Thus, release of glutamate from astrocytes via several transporters could be linked to oedema or increased oxidative stress known to occur during MDMA exposure (Sprague and Nichols, 1995a, Finch et al., 1996, Wilkins, 1996, Shankaran et al., 2001).

As mentioned, previous studies have highlighted the ability of either 5HT through

5HT2A receptors or PGE2 in mediating glutamate increases in the brain (Bezzi et al.,

1998, Meller et al., 2002, Lin et al., 2014a). The findings of Chapter 2 and 3 suggest a novel link between 5HT2A receptor activation and prostaglandin signaling in mediating the increases in extracellular glutamate concentrations within the dentate gyrus caused by

MDMA. Whether this signaling has any relevance for normal brain physiology remains an intriguing possibility. Furthermore, there exists the possibility that other 5HTergic agonists such as those commonly used to treat mood disorders, may target this type of signaling. Accordingly, the dentate gyrus may represent a highly sensitive area to 5HT- 75

induced PGE2 signaling due to the high density of 5HT and 5HT2A receptors in this region (Moore and Halaris, 1975, Luttgen et al., 2004). As we demonstrate, the dentate gyrus does appear to undergo long-lasting changes following MDMA exposure, in the form of PV interneuron decreases.

Although the findings of Chapters 2 & 3 argue for a role of MDMA-induced glutamate increases in mediating long-term decreases in PV interneurons, suggesting that excitotoxicity is involved, our findings don’t conclusively rule out other possible mechanisms for the decreases in PV interneuron counts seen following MDMA exposure.

Additionally, we were unable to co-localize either cleaved caspase-3 or Fluorojade B staining with PV-IR at 2, 6, 12, 24, 36 or 48 hour time points following MDMA exposure

(data not shown). These findings suggest that PV interneurons may not be undergoing apoptosis following MDMA exposure. However, it still could be that these experiments missed a critical period for PV interneuron apoptosis. Furthermore, the results of these experiments rely on the assumption that PV interneurons undergoing apoptosis would still express PV. Another possible explanation for the decrease in PV cell counts following MDMA exposure is that the expression of PV following MDMA is reduced to a level which is below the threshold for detection by immunohistochemistry. It should be noted that we did not see a reduction in PV expression in the dentate gyrus, 10 days following MDMA exposure (data not shown). Because these experiments take into account the accumulative expression of PV within all cells, these experiments still do not rule out the possibility that the reduced PV interneuron cell counts is a result of reduced

PV expression. Interestingly, it should be noted that PV expression has been shown to have a positive correlation with activity and thus any reductions in PV expression caused 76

by MDMA may represent a reduction in PV interneuron activity (Philpot et al., 1997,

Bender et al., 2000, Patz et al., 2004).

PV interneurons of the dentate gyrus receive glutamatergic efferents from both granule cells and the perforant path (Kneisler and Dingledine, 1995b, a). The primary synaptic target of PV interneurons are granule cells, thus providing both feedforward and feedback inhibition. We hypothesized that the MDMA-induced decreases in PV interneurons and/or their function would lead to a significant reduction in inhibition within the dentate gyrus, potentially leading to impaired hippocampal function. Although we did not see a decrease in input-output function of the dentate gyrus, suggesting that baseline neurotransmission is not altered, we did see a significant reduction in paired- pulse inhibition. Paired-pulse depression in dentate gyrus has been mainly attributed to

GABAergic inhibition (Sloviter, 1991, DiScenna and Teyler, 1994). Therefore, these findings support the idea that GABAergic inhibition in the dentate gyrus is reduced following MDMA, which could potentially be a result of reduced PV interneurons and their function. However, it should be noted that the MDMA-induced decreases in paired- pulse depression could be mediated by presynaptic changes. For instance Ca2+ transients have been noted to play a role in modulating paired-pulse responses by facilitating the release of neurotransmitter (Zucker, 1989). Thus, MDMA may cause long-lasting synaptic changes in the perforant path which enhances Ca2+ transients, leading to an increased glutamate release and subsequent reduction in paired-pulse depression.

Additionally, MDMA may result in an increased ability of dentate gyrus granule cells to fire successive action potentials. This would increase the chance that any given granule cell would fire an action potential at P2, which would ultimately reduce paired-pulse 77

depression. However, we also saw that treatment with either SC-51089 or MDL100907 during MDMA exposure, prevented MDMA-induced decreases in paired-pulse depression. Because both of these antagonists were also shown to prevent PV interneuron deficits caused by MDMA, this supports the notion that these changes are the result of decreases in these neurons. Future experiments are warranted to fully characterize the extent through which MDMA may alter neurotransmission in the dentate gyrus and in particular GABAergic inhibition.

The dentate gyrus is thought to act as a gate for excitatory neurotransmission entering the hippocampus, preventing epileptogenesis (Heinemann et al., 1992). This is in part due to the reduced intrinsic excitability seen in dentate gyrus granule cells (Mody et al., 1992). Studies have also suggested that strong GABAergic inhibition also plays a key role in reducing the spread of excitatory neurotransmission through the dentate gyrus to downstream areas in the hippocampus formation (Lothman et al., 1992). Likewise, numerous studies have seen decreases in PV interneurons in the dentate gyrus following convulsant-induced seizure models as well as in epileptic patients (Kamphuis et al., 1989,

DeFelipe et al., 1993, Arellano et al., 2004). Previous studies have noted decreases in kainic acid induced seizure threshold in MDMA treated mice, suggesting an altered balance of excitation/inhibition in the brains of these animals. We theorized that this may be the result of reduced inhibition in the dentate gyrus due to a reduction in PV interneurons in this region. As shown in Chapter 3, we saw a decrease in the threshold stimulus needed to drive afterdischarges in the dentate gyrus of MDMA treated animals.

The results could be explained by a reduction in GABAergic inhibition in the dentate gyrus. In light of the prominent role of the dentate gyrus in gating epileptogenesis, the 78

reduced afterdischarge threshold in MDMA treated animals may also explain the increased susceptibility of MDMA treated mice. As shown in Chapter 3, the decreases in afterdischarge threshold in the dentate gyrus of MDMA treated animals could be prevented with MDL100907 or SC-51089 treatments during MDMA exposure. These results suggest that PV interneuron decreases caused by MDMA may mediate these changes in afterdischarge threshold.

The findings of this dissertation suggest that the acute effects of MDMA on hippocampal glutamatergic neurotransmission mediate long-term changes in hippocampal physiology. These changes in hippocampal physiology were evidenced by both a reduction in paired-pulse inhibition as well as reduction in afterdischarge threshold. Both of these changes could be potentially explained by a reduction in

GABAergic inhibition within the dentate gyrus resulting from a decrease in PV interneurons. Given the known role of PV interneurons in complex network functions thought to underlie information processing in the hippocampus, these deficits likely impact hippocampal function (Kneisler and Dingledine, 1995a, b, Bartos et al., 2002).

This could help explain the numerous studies depicting spatial memory impairments, which is indicative of hippocampal dysfunction, in adolescent and adult rats treated with

MDMA (Robinson et al., 1993, Sprague et al., 2003, Williams et al., 2003, Vorhees et al.,

2004, Skelton et al., 2006, Arias-Cavieres et al., 2010). Additionally, MDMA-induced deficits in GABAergic inhibition mediated by PV interneurons could explain the many reports of cognitive deficits reported in MDMA abusers (Krystal et al., 1992, Parrott et al.,

1998, Wareing et al., 2004, Parrott et al., 2006). Additionally, given the known role of the dentate gyrus preventing epileptogenesis, these findings could help explain previous 79

findings suggesting an increased susceptibility to kainic acid seizures in mice following

MDMA exposure (Giorgi et al., 2005, Abad et al., 2014). To our knowledge, no literature exists suggesting that MDMA abusers exhibit epilepsy. However, given the findings of this dissertation as well as several other suggesting long-lasting changes in GABAergic neurotransmission in rodents having been treated with MDMA, this may warrant future examination.

In conclusion, our findings highlight a potential novel signaling mechanism mediated by MDMA in which 5HT2A receptor activation elicits PGE2 signaling, ultimately leading to increases in glutamatergic neurotransmission within the hippocampus. Our findings suggest that this signaling induced by MDMA mediates the long-term decreases in PV interneurons within the dentate gyrus and reduced inhibition.

The effects of MDMA on PV interneurons within the hippocampus may have a significant impact on hippocampal physiology and explain spatial memory deficits as well as seizure susceptibility in animals treated with MDMA or human MDMA abusers.

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Appendix A

MDMA-induced Changes in Paired-pulse Facilitation in the Dentate Gyrus

A.1 Rationale

The experiments shown in Chapter 3 revealed a significant reduction in paired- pulse depression in the dentate gyrus of rats treated with MDMA 10 days prior. Paired- pulse depression in dentate gyrus has been mainly attributed to GABAergic inhibition

(Sloviter, 1991, DiScenna and Teyler, 1994). This effect was also shown to correspond with a decrease PV interneurons seen in the dentate gyrus following MDMA exposure

(Chapter 2 & 3). Furthermore, we noted that the changes in paired-pulse depression could be prevented by treatment with either SC-51089 or MDL100907 during MDMA exposure, which also blocked the decreases in PV interneuron losses. To further investigate the changes in neurotransmission in the dentate gyrus during MDMA exposure, we investigated whether there were changes in in paired-pulse facilitation in the dentate gyrus following MDMA exposure.

A.2 Methods

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Adult male Sprague-Dawley rats (200-275 g. Harlan Sprague Dawley, In, USA) were used. Prior to experimentation, rats were allowed at least 5 days to acclimate.

MDMA was obtained from the National Institutes of Drug Abuse (NIDA, Research

Triangle). Rats were injected with 0.9% (1 ml/kg) saline or MDMA (7.5 mg/kg, once every 2 hours X 4 injections). Systemic injections (20 μg/kg, i.p.) of the EP1 receptor antagonist, SC-51089, (Tocris, 20 μg/kg, i.p.) or the 5HT2A receptor antagonist,

MDL100907 (Wyeth, 0.1 mg/kg) were given 30 minutes before each administration of

MDMA. These doses were based on previous studies where this dose was used

(Schreiber et al., 1998, Abe et al., 2009).

On the 10th day after MDMA/saline exposure, rats were anesthetized with an initial injection of ketamine-xylazine anesthesia (20 mg/kg, 6 mg/kg, i.p., respectively).

(220-240 BPM) to ensure stable levels of anesthesia. Extracellular field potentials within the upper blade of the dentate gyrus (-4.0 mm A.P., 2.0 mm M.L, 3.2 mm D.V. bregma) were monitored using teflon coated tungsten electrodes (.7-1 Mῼ). Stimulation of the perforant path (-7.0 mm A.P., 3.5 mm M.L., 3.5 mm D.V. bregma) was done using a concentric bipolar metal electrode. Stimuli were generated using an A-M systems isolated pulse stimulator. Waveforms were amplified using a BAK electronic amplifier and digitized with Digidata 1322A (Axon. Instruments, Inc.) and stored for analysis using pCLAMP9 software (Molecular Devices). Population spike amplitude (PS) was measured by drawing a tangent to the positive going peaks and from this tangent measuring the 103

amplitude of the negative going peak (PS). Input-output curves were determined using

Experiments were analyzed using a two-way ANOVA to compare the effects of saline or MDMA and determine an interaction with SC51089 and MDL100907. Post-hoc analysis was performed using Tukey’s test. Statistical significance was set at p<0.05.

A.3 Results

As shown in Fig. A-1, MDMA+Vehicle treated rats exhibited a significant reduction in paired-pulse facilitation at 120, 140 and 160 ms interstimulus intervals compared to

Saline+Vehicle treated rats (p<0.05). Two-way ANOVA analysis revealed a significant main-effect of MDMA at these intervals (F(1,23)=3.936 (120ms), F(1,23)=4.87 (140ms),

F(1,23)=12.147 (160ms); p<0.05) This analysis also revealed a significant interaction of

MDL100907 and SC-51089 and MDMA treatment at the 120 ms interstimulus intervals

(F(1,24)=5.236 (MDL100907), F(1,24)=4.151 (SC51089); p<0.05). Furthermore, there was no difference between paired-pulse facilitation at any interstimulus intervals between either MDL100907+MDMA or SC-51089+MDMA treated rats and Saline+Vehicle treated rats.

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Figure A-1: MDMA-induced reductions in paired-pulse facilitation in the dentate gyrus. Rats were treated with MDMA (7.5mg/kg every 2h X 4 i.p injections) 10 days prior to electrophysiology recordings. MDL100907 or SC-51089 were given 30 minutes prior to each injection of MDMA. Summary of 120, 140 and 160 ms interstimulus interval paired-pulse responses for all treatment groups. Paired-pulse facilitation was significantly reduced in MDMA + vehicle treated rats compared to Saline + Vehicle treated rats. MDL100907 and SC-51089 pretreatment prevented MDMA-induced decreases in paired-pulse depression. n=6-7 per group *=statistically significant from saline+vehicle controls (p<0.05).

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A.4 Discussion

Our findings demonstrate a significant reduction in paired-pulse facilitation (PPF) in the dentate gyrus of rats, 10 days following MDMA exposure (7.5 mg/kg x 4, i.p given every other hour). These reductions in PPF coincide with a reduction in PV interneurons in the dentate gyrus (Chapters 2 & 3). Furthermore, as we demonstrate, inhibition of

5HT2A or EP1 receptors during MDMA exposure, which was shown to prevent PV interneuron decreases (Chapters 2 & 3), also prevented the decreases in PPF. Previous studies have suggested that PPF in the dentate gyrus is mediated by a GABAB receptors

(Lambert and Wilson, 1994, Brucato et al., 1995). GABAB receptors are known to be located on presynaptic terminals of GABAergic interneurons in the dentate gyrus and thus it has been suggested that PPF is mediated by disinhibition mediated by activation of these GABAB receptors (Lambert and Wilson, 1994, Jiang et al., 2000). The decreases in

PV interneurons caused by MDMA could lead to less GABAergic inhibition and a reduction in activation of GABAB receptors. This could reduce the disinhibition resulting from P1 and reducing PPF.

Although our findings suggest that MDMA-induced changes in PPF results from a change in GABAergic inhibition within the dentate gyrus, changes in presynaptic physiology may confound this interpretation. At neuronal synapses in other brain regions,

PPF is thought to be mediated by presynaptic mechanisms, through a Ca2+ dependent mechanism (Zucker, 1989). Thus, an alternative explanation is that MDMA exposure results in a reduced Ca2+ transient in perforant path synapses, leading to reduced PPF.

Another possible explanation is that MDMA is increasing the release probability of perforant path synapses, which has been shown reduce PPF (Millar et al., 2002, Zucker 106

and Regehr, 2002). However, we would expect that an increase in release probability at perforant path synapses would likely result in a leftward shift in the input-output function of the dentate gyrus input-output. As seen in Chapter 3, we saw no such change in input- output function in MDMA treated animals. Another possibility is that the decreases in

PPF caused by MDMA may result from a change in excitability in dentate gyrus granule cells. Population spikes are thought to be a result of such action potentials and thus a reduced probability of firing in granule cells would likely lead to reduced PPF.

Regardless, these changes in short-term plasticity likely contribute to a significant change in function in the dentate gyrus. In light of the role of the dentate gyrus in spatial information processing(Rolls, 1990), these changes likely contribute to MDMA-induced deficits in spatial memory seen in rodents (Taffe et al., 2002, Sprague et al., 2003,

Busceti et al., 2008, Cunningham et al., 2009). Future studies are needed, which fully characterize the effects of MDMA on dentate gyrus physiology.

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Appendix B

The Effects of Stress and MDMA on Parvalbumin Interneurons in the Dentate Gyrus

B.1 Rationale

Exposure to stress is thought to precipitate psychostimulant drug use in humans (Sinha,

2008). Furthermore in rodent models of drug taking, chronic stress has been shown to enhance self-administration of psychostimulants (Piazza and Le Moal, 1998). In addition to the potential enhancement of rewarding effects of psychostimulants, chronic stress has been shown to enhance the neurotoxic effects of MDMA and methamphetamine on monoaminergic terminals (Matuszewich and Yamamoto, 2004, Raudensky and

Yamamoto, 2007, Tata and Yamamoto, 2007, Johnson and Yamamoto, 2010, Northrop and Yamamoto, 2013). This enhancing effect of stress on MDMA was shown to be mediated through inflammatory and excitotoxic mechanisms. Other studies have shown that stress can prime the brain for excitotoxic insults in part by increasing inflammatory markers such as prostaglandins (Tata and Yamamoto, 2008, Zoppi et al., 2011).

The role of prostaglandins in mediating the loss of PV interneurons caused by MDMA was first highlighted by Anneken et al. (2013), which showed that the decrease in these neurons could be prevented by inhibition of COX during MDMA exposure. The 108

experiments shown in Chapter 3 provide evidence for the pontential role of PGE2 in mediating the MDMA-induced decreases in PV interneurons in the dentate gyrus through activation of EP1 receptors. This effect is potentially mediated by the role of EP1 receptors in mediating MDMA-induced glutamate increases in the dentate gyrus.

Additionally, it was shown that chronic stress can enhance the hippocampal glutamate concentrations caused by methamphetamine (Tata and Yamamoto, 2008). Thus, we sought to determine whether chronic unpredictable stress prior to MDMA would enhance

PV decreases.

B.2 Methods

Adult male Sprague-Dawley rats (200-275 g. Harlan Sprague Dawley, In, USA) were used. Prior to experimentation, rats were allowed at least 5 days to acclimate. For 10 days prior to MDMA or saline administration, rats were either handled or exposed to 10 days of various stressors as previously described (Cunningham et al., 2009). The procedure was as follows: Day 1: 1000 30-min cage rotation, 1600 isolation overnight;

Day 2: 1000 3 hr lights off, 1800 food and water deprivation overnight; Day 3: 1000 30- min restraint, 1800 lights on overnight; Day 4: 1100 wet bedding, 1500 social stress; Day

5: 0900 60-min restraint, 1800 food and water deprivation overnight; Day 6: 1500 2 hr lights off, 1700 isolation overnight; Day 7: 1000 60-min cage agitation, 1800 60-min restraint stress; Day 8: 1100 wet bedding, 1800 food and water deprivation overnight;

Day 9: 0900 3 hr lights off, 1800 lights on overnight; Day 10: 1000 30-min cage agitation, 1800 isolation overnight. MDMA was obtained from the National Institutes of

Drug Abuse (NIDA, Research Triangle). Rats were injected i.p. with 0.9% (1 ml/kg) 109

saline or MDMA (7.5 mg/kg, once every 2 hours X 4 injections) on the morning following completion of 10 day stressors.

On the 10th day after MDMA/saline exposure, rats were anesthetized with an injection of ketamine-xylazine anesthesia (60 mg/kg, 18 mg/kg, i.p., respectively) and phosphate-buffered saline (0.1 M PBS) was perfused intracardially followed by 4% paraformaldehyde. Brains were cryoprotected, flash frozen and the dorsal hippocampus was sectioned into 50 µm thick slices. Background peroxidase activity was reduced by incubating in 1% H2O2, at room temperature for 30 minutes. The sections were blocked for 2 hr at room temperature with 10% normal goat serum (Life Technologies) in 0.1M

PBS containing 0.5% Triton-X 100 and Avidin block (4 drops/mL; Vector Laboratories).

For PV immunostaining, 50μm thick sections were incubated for 36 hr at 4°C with a mouse monoclonal PV antibody (1:2000; Swant, cat# PV235) in 0.1 M PBS containing

0.5% Triton-X 100, 1% NGS and Biotin block (4 drops/ml; Vector Laboratories).

Sections were then incubated in goat anti-mouse biotinylated secondary antibodies

(Millipore, cat# AP124b) for 2hr at room temperature followed by incubation in avidin- biotin- horseradish peroxidase (Vectastain Elite ABC Kit; Vector Laboratories) for 2hr at room temperature. Sections were then developed in diaminobenzidine (DAB/Metal

Concentrate; Pierce) and mounted on glass slides and coverslipped with Eukitt mounting medium (Sigma- Aldrich). The experimenter was blind to all treatment groups such that a non-experimenter coded all slides and the code was not broken until the end of quantitative analysis.

Quantification of PV-IR GABA interneurons in the dorsal hippocampus was made using a modified optical fractionator stereologic counting technique. Counts were 110

made using a BX51 Olympus microscope equipped with a DVC camera interfaced with

StereoInvestigator 8.21 software (MBF Bioscience). Every fourth section for a total of six sections through the dorsal hippocampus was sampled. Cell counts were made using a

20x objective. Grids sizes were set at 100μm X 100μm during counting. A Gunderson coefficient of 0.1 or below were obtained using these stereology experiments, which allows for an accurate estimate of total PV interneurons within the region.

Experiments were analyzed using a two-way ANOVA to compare the effects of saline or MDMA and determine an interaction with stress exposure. Post-hoc analysis was performed using Tukey’s test. Statistical significance was set at p<0.05.

B.3 Results

As shown in Fig. B-1, either MDMA or stress resulted in a significant reduction in PV interneurons in the dentate gyrus (p<0.05). Two-way ANOVA analysis revealed a significant main-effect of MDMA or stress (F(1,22)=9.384; p<0.05). Two-way ANOVA analysis revealed no interaction between MDMA and stress exposure (p>0.05).

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Figure B-1: The Effects of Stress and MDMA on PV Interneurons in the Dentate Gyrus. Rats were exposed to a chronic unpredictable stress paradigm for 10 days prior to treatment with MDMA (7.5mg/kg every 2h X 4 i.p injections) or .9% saline (.1 mL/kg). PV-IR cell counts in the dentate gyrus were performed 10 days following MDMA exposure. PV-IR cells were significantly reduced by either stress or MDMA exposure. The combination of stress and MDMA did not produce a significant enhancement of PV interneuron decreases in the dentate gyrus n=6- 8 per group *=statistically significant from No Stress + Saline controls (p<0.05).

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B.4 Discussion

We investigated the effects of stress and MDMA alone or in combination on PV interneurons in the dentate gyrus. As previously shown, MDMA given every other hour resulted in a significant decrease in PV interneurons in the dentate gyrus (Anneken et al.

2013, Abad et al. 2014, Collins et al. 2015). Additionally, 10 days of chronic unpredictable stress was shown to cause a significant reduction in PV interneurons in the dentate gyrus. In fact several others have reported significant decreases in PV interneurons in the dentate gyrus following chronic stress exposure (Czeh et al., 2005, Hu et al., 2010, Steullet et al., 2010). The mechanism through which stress reduces PV interneurons is largely unknown. Experiments by Czeh et al. (2005) demonstrated that

PV decreases caused by chronic stress could be prevented by inhibition of substance P receptors. Previous studies have noted increases in glutamate concentrations in the hippocampus during exposure to an acute stressor (Gilad et al., 1990, Moghaddam, 1993,

Lowy et al., 1995). Given the potential susceptibility of PV interneurons to excitotoxicity, it is possible that stress may be reducing PV interneurons through an excitotoxic mechanism.

Interestingly, the number of PV-IR neurons in the dentate gyrus of Stress +

Saline, No Stress + MDMA and Stress + MDMA rats were similar. These findings suggest that either exposure to stress inhibits the acute effects of MDMA which mediate decreases in PV interneurons or that there is a ceiling effect of either stress or MDMA on

PV decreases in the dentate gyrus. The first potential explanation seems unlikely given the previous findings suggesting that chronic stress exposure enhances the neurotoxic effects of a subsequent MDMA or methamphetamine (Matuszewich and Yamamoto, 113

2004, Raudensky and Yamamoto, 2007, Tata and Yamamoto, 2007, Johnson and

Yamamoto, 2010, Northrop and Yamamoto, 2013). Additionally, it was shown that chronic stress enhances the hippocampal glutamate increases caused by methamphetamine (Tata and Yamamoto, 2008). Given the role of glutamate receptors in mediating PV decreases, the lack of an effect of MDMA on PV interneurons following

MDMA exposure suggests that stress may inhibit hippocampal glutamate increases caused by MDMA, which seems unlikely given those findings by Tata and Yamamoto

(2008). Although methamphetamine and MDMA share similar acute effects in terms of mediating 5HT release, it is unknown whether they also share a similar mechanism through which they mediate glutamate release. Thus, future studies are warranted to determine the effects of chronic stress exposure on MDMA-induced glutamate increases in the hippocampus.

Another potential explanation for the lack of an additive effect of stress and

MDMA on PV interneuron decreases in the dentate gyrus is that there exist subpopulations of PV interneurons in the dentate gyrus that are more or less susceptible to neurotoxicity. Thus, the more susceptible PV interneurons would undergo apoptosis during stress exposure and the remaining less susceptible PV interneurons would not be affected by MDMA, creating a ceiling effect. Interestingly, it has been demonstrated that there are different subpopulations of PV interneurons in the hippocampus (Klausberger et al., 2005). These subpopulations of PV interneurons can be classified based on their morphology (bistratified or basket) or by their axonal targets (axoaxonic or somatic).

Differences between these subpopulations of PV interneurons in terms of receptor expression or intrinsic characteristics, which may convey susceptibility to excitotoxicity 114

is largely unknown. However, based on their morphology and axonal targets it has been suggested that these interneurons play unique roles in network functions involved in information processing (Klausberger and Somogyi, 2008). Thus, decreases in the function of PV interneurons may lead to different changes in network function and information processing depending on which PV interneuron subpopulations are reduced.

Further studies are warranted to determine the morphological and regional characteristics of PV interneuron decreases caused by MDMA.

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