DELETION OF GLUTAMATE RECEPTOR TRAFFICKING PROTEINS IN THE MEDIAL PREFRONTAL CORTEX AND THEIR SEX-SPECIFIC EFFECTS ON COCAINE ADDICTION
A Dissertation Submitted to the Temple University Graduate Board
In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY
by Megan Marie Wickens May 2020
Examining Committee Members:
Dr. Lisa Briand, Advisory Chair, Temple University Psychology Department Dr. Debra Bangasser, Temple University Psychology Department Dr. Vinay Parikh, Temple University Psychology Department Dr. Mathieu Wimmer, Temple University Psychology Department Dr. Vishnu Murty, Temple University Psychology Department Dr. Scott Rawls, Temple University Pharmacology Department ABSTRACT
Dysregulation of glutamatergic signaling mechanisms is a component of many
psychiatric diseases. A number of these diseases exhibit a bias toward one sex, yet the ways
in which glutamate is affected by or modulates this bias is poorly understood. In cocaine
addiction, women progress from initial use of the drug to substance use disorder faster than
men, and have more difficulty remaining abstinent. The same is true in female rodents. We
used a mouse model of cocaine self-administration to study the role of glutamate receptor
trafficking proteins in cocaine addiction-like behavior in males and females. In the first set
of experiments, mice received a conditional knockout of glutamate receptor interacting
protein 1 (GRIP1) in the medial prefrontal cortex (mPFC). This led to an increase in
motivation for cocaine as well as enhanced likelihood of relapse behavior, as measured by
a progressive ratio schedule and cue-induced reinstatement, respectively. No sex
differences were seen after prefrontal deletion of GRIP1. The next set of experiments used
the same behavioral paradigm, but mice received a conditional knockout of protein
interacting with C kinase 1 (PICK1) in the mPFC. PICK1 and GRIP1 are both involved in
the activity dependent trafficking of the GluA2-containing AMPA receptor, but while
GRIP1 maintains these receptors in the synapse, PICK1 internalizes them in response to a
stimulus such as drug experience. The prefrontal deletion of PICK1 was predicted to
decrease cue-reinstatement responding, and this was observed in the male mice. The female
mice displayed an increase in cue-induced reinstatement responding, similar to the effects
seen by prefrontal GRIP1 deletion. Sex differences in PICK1 have not previously been
described in the literature. Our results suggest that PICK1 is involved in different
baseline processes in females, and merit further study. The final set of experiments
ii considered the interaction of gonadal hormones and PICK1 in males. Bilateral gonadectomy or sham surgery was combined with prefrontal PICK1 knockout to determine if circulating gonadal hormones could explain the results in males. After gonadectomy or sham surgery, there was no significant effect of prefrontal PICK1 deletion on cue-induced reinstatement. These results do not fully explain the sex difference observed in intact mice. Together, these studies suggest that baseline sex differences exist in PICK1-mediated mechanisms of cocaine reinstatement and that these differences are not due to the influence of gonadal hormones alone.
iii
ACKNOWLEDGMENTS
This dissertation would not have been possible without the enormous and continued support of many people. First and foremost, I would like to thank my advisor Dr. Lisa
Briand. I have learned so much over the past five years and I do not think I would be where
I am today if it were not for her continued support. Dr. Briand encourages us to strive for the highest scientific standards, not only in the lab, but at conferences, seminars, and outreach events as well. It is thanks to her that I can say I truly feel like a scientist.
I would also like to thank my committee, Dr. Debra Bangasser, Dr. Vinay Parikh,
Dr. Mathieu Wimmer, Dr. Scott Rawls, and Dr. Vishnu Murty. You have provided invaluable feedback and advice and I am grateful for the time you took to help me progress through graduate school.
To the current and former members of Briand lab, I cannot thank you enough. I truly could not have done this without you. And to Sam, Evie, and Corey, thanks for keeping me in good spirits as I made these final edits to the document.
Of course, I cannot forget to thank my family for their support over the last few years. Whether it was Dad driving 1000 miles in one weekend to help me move, or Mom listening to my practice poster presentations, or Aunt Kim inviting me over to dinner and even paying the toll over the bridge for me – I really cannot thank you enough.
Lastly, I would be remiss if I did not thank Jim, Meg, and Rachel of Breaking Point
Fitness. Your welcoming community became my “home away from home” and I can’t believe how lucky I am to have been able to join you all for the last three years.
iv TABLE OF CONTENTS
Page
ABSTRACT ...... ii
ACKNOWLEDGMENTS ...... iv
LIST OF TABLES………………………………………………………………………..ix
LIST OF FIGURES ...... x
CHAPTER 1
1. SEX DIFFERENCES IN PSYCHIATRIC DISEASE: A FOCUS ON THE
GLUTAMATE SYSTEM ...... 1
The Glutamate System ...... 2
Sex Differences in the Glutamate System ...... 3
Baseline Differences ...... 3
Changes Across the Lifespan ...... 5
Sex Differences in Glutamate System in Disease ...... 7
Alzheimer’s Disease ...... 7
Major Depressive Disorder ...... 10
Schizophrenia ...... 13
Autism Spectrum Disorder...... 18
Attention Deficit Hyperactivity Disorder (ADHD) ...... 20
Conclusion ...... 22
v
CHAPTER 2
2. GLUTAMATE RECEPTOR INTERACTING PROTEIN ACTS WITHIN THE PREFRONTAL CORTEX TO BLUNT COCAINE SEEKING ...... 27
Methods...... 29
Subjects ...... 29
Prefrontal Microinjections and Adeno-Associated Virus Constructs ...... 30
Operant Food Training ...... 30
Jugular Catheterization Surgery ...... 31
Cocaine Self-Administration...... 31
Operant Set Shifting Task ...... 32
Western Blot ...... 33
Electrophysiology ...... 34
Statistical Analysis ...... 36
Results ...... 36
Viral Mediated Deletion of GRIP1 in the Medial Prefrontal Cortex ...... 36
Prefrontal GRIP1 Knockout Does Not Affect Fixed Ratio Self-
Administration of Sucrose or Cocaine ...... 37
Prefrontal GRIP1 Knockout Enhances Responding for Cocaine but
not Sucrose on a Progressive Ratio Schedule ...... 41
Prefrontal GRIP1 Knockout Enhances Responding for Cocaine but
not Sucrose During Cue-Reinstatement ...... 41
Prefrontal GRIP1 Knockout Does Not Lead to Deficits in
Cognitive Flexibility ...... 42
vi Prefrontal GRIP1 Knockout Alters Glutamate Transmission in the
PFC and the NAc ...... 45
Discussion ...... 46
GRIP1 Knockout in the Prefrontal Cortex Increases Motivation for
Cocaine and Potentiates Cocaine Seeking in Both Males and
Females ...... 46
Prefrontal GRIP1 Does Not Play a Role in Natural Reward Taking
or Seeking ...... 48
Knocking Out GRIP1 in the PFC Does Not Alter Cognitive
Function ...... 49
Altering AMPA Trafficking in the PFC has Downstream Effects
on Accumbal Physiology ...... 50
Conclusion ...... 52
CHAPTER 3
3. SEX-SPECIFIC EFFECTS OF PREFRONTAL PICK1 KNOCKOUT ON
COCAINE REINSTATEMENT ...... 53
Methods ...... 55
Subjects ...... 55
Prefrontal Microinjections and Adeno-Associated Virus Constructs ...... 56
Experiment I ...... 56
Experiment II ...... 58
Results ...... 60
Viral Mediated Deletion of PICK1 in the Medial Prefrontal Cortex ...... 60
vii Prefrontal PICK1 Knockout Does Not Affect Food Training or
Cocaine Self-Administration in Intact Mice ...... 62
Prefrontal PICK1 Knockout Has a Sex-Specific Effect on Cue-
Induced Reinstatement in Intact Mice...... 62
Gonadectomy Reversed the Effect of Prefrontal PICK1 Knockout
on Cue-Induced Reinstatement of Cocaine Seeking in Males ...... 65
Discussion ...... 68
PICK1 Knockout in the Prefrontal Cortex Dampens Cocaine
Seeking in Males ...... 68
PICK1 Knockout in the Prefrontal Cortex Leads to a Sex-Specific
Effect in Cocaine Seeking ...... 69
Gonadectomy Eliminates PICK1 Knockout Effect on Cocaine
Cue-Induced Reinstatement in Males ...... 70
Prefrontal PICK1 Knockout Does Not Play a Role in Fixed Ratio
Measures of Natural Reward or Cocaine Self-Administration ...... 71
Conclusions ...... 72
4. CONCLUSION ...... 73
REFERENCES CITED ...... 79
viii LIST OF TABLES Table Page
1. Summary of Changes in Glutamatergic Measures ...... 24
2. Self-Administration Measures by Sex and GRIP1 Knockout ...... 40
ix LIST OF FIGURES Figure Page
1. Significant Viral Mediated Prefrontal GRIP1 Knockout ...... 38
2. No Difference Between GFP Controls and Cre GRIP1 Knockout During Food Self-Administration Training ...... 39
3. No Differences Between GFP Controls and Cre GRIP1 Knockout During Cocaine Self-Administration ...... 39
4. Prefrontal GRIP1 Knockout Increases Progressive Ratio Breakpoint and Reinstatement Responding for Cocaine But Not Sucrose ...... 43
5. Prefrontal GRIP1 Knockout Does Not Impact Cognitive Flexibility ...... 44
6. Prefrontal GRIP1 Knockout Enhances Signaling Within the PFC But Only Affects Accumbal Signaling After Cocaine Experience ...... 45
7. Significant Viral-Mediated Prefrontal PICK1 Knockout ...... 61
8. PICK1 Knockout in the mPFC Does Not Alter Operant Learning During Food Self-Administration in Male or Female Mice ...... 63
9. PICK1 Knockout in the mPFC Does Not Alter Cocaine Self-Administration on a Fixed Ratio Schedule of Reinforcement ...... 64
10. Prefrontal PICK1 Knockout Leads to Sex-Specific Effects on Cue-Induced Reinstatement of Cocaine Seeking… ...... 66
11. Gonadectomy Did Not Alter the Effect of mPFC Knockout on Food or Cocaine Self-Administration ...... 67
12. Gonadectomy Eliminates the Effect of Prefrontal PICK1 Knockout on Cue-Induced Reinstatement ...... 67
x CHAPTER 1
SEX DIFFERENCES IN PSYCHIATRIC DISEASE: A FOCUS ON THE
GLUTAMATE SYSTEM
Accumulating data indicate that disruptions in glutamate neurotransmission are a common underlying pathology in multiple psychiatric diseases including Alzheimer's disease (AD), major depressive disorder (MDD), schizophrenia (SCZ), autism spectrum disorder (ASD), and attention deficit hyperactivity disorder (ADHD) (Counts, Che,
Ginsberg, & Mufson, 2011; Gray, Hyde, Deep-Soboslay, Kleinman, & Sodhi, 2015; Magri et al., 2008; Shimmura et al., 2011; Sokolow et al., 2012). Furthermore, these diseases all exhibit a sex bias, with increased prevalence of ASD and SCZ in men and increased prevalence of MD and AD in women (Fombonne, 2005; Markham, 2012; Mielke,
Vemuri, & Rocca, 2014; Noble, 2005). Although little work has been done to elucidate baseline sex differences in the glutamate system, it is clear from work in these disease populations that sex differences must be considered. To promote a better understanding of these sex biases in disease along with sex differences in treatment response, we must first gain a better understanding of sex differences in the glutamate system. To date, very little work has been done to elucidate these differences. This review will focus on the sex differences in the glutamate system that have been revealed in clinical populations and preclinical studies of glutamatergic sex differences, highlighting how much more work is needed to obtain a clear picture of how sex differences in the glutamate system contribute to disease.
1 The Glutamate System
Glutamate is the primary excitatory neurotransmitter in the brain, and it is essential for normal brain development and plasticity. Glutamate receptors come in two types, ionotropic ligand-gated ion channels and metabotropic G-protein coupled receptors. These receptor subtypes can be even further subdivided. Currently there are 8 identified metabotropic glutamate receptors: mGluR1-8, and 3 identified ionotropic glutamate receptor subtypes: a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, N-methyl-D-aspartate (NMDA) receptors, and kainate receptors. These receptor subtypes can be further divided based upon their subunit composition. AMPA receptors may be calcium-permeable or calcium-impermeable, depending on the absence or presence of the GluA2 subunit, respectively (Hanley, 2014). Heteromeric, GluA2-containing receptors are calcium impermeable; homomeric GluA1-only receptors are calcium permeable and exhibit a higher conductance upon activation (Dong et al., 1997). NMDA receptors are composed of two GluN1 and two GluN2 (or rarely GluN3 subunits). The four subtypes of GluN2 subunits (GluN2A-2D) confer functional diversity with each GluN2 subunit exhibiting unique biophysical, pharmacological and signaling properties (Ferreira et al., 2017;
Paoletti & Neyton, 2007; Sanz-Clemente, Nicoll, & Roche, 2013; Wyllie, Livesey, &
Hardingham, 2013). After being cleared from the synapse by excitatory amino acid transporters (EEATs), glutamate is converted to glutamine. As the levels of both glutamate and glutamine can be measured using proton magnetic resonance spectroscopy
(MRS) in humans, many studies have examined these amino acids as potential biomarkers for psychiatric disease (Chiu et al., 2018; Sheikh-Bahaei et al., 2018;
2 Shimmura et al., 2011).
Sex Differences in the Glutamate System
Baseline Differences
The little work that has been done in humans to elucidate sex differences in the glutamate system has led to somewhat mixed results. MRS studies have demonstrated a slight increase in glutamate concentration within the parietal gray matter of men compared to women, while no differences were detected in the frontal gray or white matter or the basal ganglia (Sailasuta, Ernst, & Chang, 2008). However, when looking more carefully at specific brain regions, women seem to exhibit higher levels of glutamate compared to men. Specifically, women exhibit increased glutamate levels in the striatum and cerebellum compared to men (Zahr et al., 2013). There also appears to be increases in glutamate within the sensorimotor cortex and anterior cingulate cortex (ACC) of women
(Grachev & Apkarian, 2000). Along with these studies examining glutamate within the brain, studies have also shown sex differences in serum glutamate concentration (Stover
& Kempski, 2005; Teichberg, Cohen-Kashi-Malina, Cooper, & Zlotnik, 2009). In contrast to the majority of studies examining glutamate in the brain, studies in blood have revealed higher glutamate concentrations in men compared to women (Zlotnik et al.,
2011). As glutamate is present in many tissues in the body, these differences in serum glutamate may not reflect changes within the central nervous system (Shulman et al.,
2006).
Sex differences in the glutamate system are more readily examined in rodent models.
Several brain regions in rodents show sex differences in glutamate concentrations,
3 including higher glutamate in the lateral hypothalamus and habenula of males and higher glutamate in the medial preoptic area of females (Frankfurt, Fuchs, & Wuttke, 1984). Along with these overall sex differences in glutamate levels there are also changes in glutamate concentration across the estrous cycle (Frankfurt et al., 1984). These changes are brain region specific, with higher levels observed in the lateral septum during proestrus— the phase of the cycle where ovarian hormones are highest—compared to estrus; in the medial septum and diagonal band of Broca during proestrus compared to diestrus; and lower in the anterior hypothalamic area during proestrus compared to diestrus (Frankfurt et al.,
1984).
Sex differences are also observed in synaptic glutamate signaling. Under basal conditions, female rats show larger hippocampal AMPA receptor synaptic responses, possibly due to enhanced phosphorylation of the GluA2 subunit (Monfort, Gomez-
Gimenez, Llansola, & Felipo, 2015). However, this enhanced glutamate signaling may occlude further plasticity. Female rats show a reduction in the magnitude of tetanus- induced long-term potentiation (LTP) compared to male rats and this reduction is associated with a decrease in tetanus- induced phosphorylation of GluA1 (Monfort et al.,
2015). As the phosphorylation of GluA1 AMPA subunits is involved in the insertion of
GluA1-containing AMPA receptors into the synapse, this could reflect a mechanism for this diminished synaptic plasticity (Man, 2011). Along with these alterations in AMPA receptor signaling, sex differences also exist in NMDA receptor signaling. For example,
NMDA antagonism increases prefrontal dopamine in male rats but decreases levels in females (Locklear, Cohen, Jone, & Kritzer, 2016). This may reflect a leftward shift in the
4 dose response curve since females seem to be more sensitive to NMDA receptor manipulations. Female rats are more sensitive to excitotoxic damage following administration of an NMDA receptor antagonist, MK-801 (Wozniak et al., 1998) and exhibit a greater behavioral response to ketamine, an NDMA receptor antagonist
(McDougall, Moran, Baum, Apodaca, & Real, 2017). This increase in NMDA sensitivity may be the result of increased receptor expression as female rats exhibit higher levels of both NR1 and NR2B NMDA subunits (Wang et al., 2015). Along with these changes in ionotropic glutamate signaling, there also appear to be basal sex differences in the metabotropic glutamate receptor system, with female rats exhibiting higher levels of mGluR2/3 and mGluR5 within the hippocampus along with increased mGluR5 in the prefrontal cortex (Wang et al., 2015). Steroid hormones may influence this overall increase in glutamatergic transmission. The neurosteroid, 17β-estradiol (E2) is known to potentiate excitatory transmission by increasing the probability of glutamate release in females (Smejkalova & Woolley, 2010).
Changes Across the Lifespan
While relatively subtle sex differences in glutamate exist in healthy younger individuals, more dramatic sex differences seem to emerge with age. When examining glutamate levels in the brain across the lifespan, men exhibit a clear decline in glutamate from age 21 to age
70 within the basal ganglia and the parietal gray matter that is not present in women
(Sailasuta et al., 2008). However, in the ACC, women show a more pronounced age-related decline (between ages 19 and 56) in glutamate levels compared to men (Hadel, Wirth,
Rapp, Gallinat, & Schubert, 2013). Healthy men have been shown to have higher levels of
5 glutamine (Gln) in the ACC, compared to healthy women (Tayoshi et al., 2009). In serum, women exhibit an increase in glutamate concentration as they age (from age 20 to 80), whereas men do not (Kouchiwa et al., 2012). Along with these age-related changes in glutamate levels, there appear to be changes in glutamate receptors as well. Over the course of aging (age 25 vs. age 70), men exhibit an increase in the distribution of mGluR1 in the cerebellum, parietal cortex, putamen, amygdala, and hippocampus (Sakata et al., 2017).
Women do not show these aging-related differences in mGluR1 distribution (Sakata et al.,
2017). Postmortem tissue analysis has demonstrated that glutamate-related gene expression, including genes that code for glutamate receptors and trafficking proteins, decrease over the first 50 years of life within the prefrontal cortex (Choi, Zepp, Higgs,
Weickert, & Webster, 2009). However, no studies have yet been adequately powered to detect normal sex differences in these effects nor have more advanced ages been examined.
Nevertheless, studies on aging and disease provide us with some insight into potential differences.
Similar to the changes in the glutamate system that occur across the lifespan in humans, rodents also exhibit developmental changes in glutamate. Glutamate concentrations rise over the first 3 months of life in both male and female mice (Kulak, Duarte, Do, & Gruetter,
2010). These changes in the glutamate system do not stop when animals reach adulthood.
Glutamate concentrations decrease over the course of aging in the hippocampus, cortex, and striatum (Duarte, Do, & Gruetter, 2014). Although there were no sex differences in the total glutamate concentrations, the authors report a significant interaction between age and brain region in the ratio of glutamine/glutamate, which may reflect differences in
6 glutamatergic transmission between neurons and glial cells (Duarte et al., 2014). Decreased levels of GluA1, GluN2A, and GluN2B glutamate receptor subunit levels over the course of aging (6 mo vs. 24 mos) have been correlated with poorer cognitive performance in male rats, but these studies have not been done in females (Ménard et al., 2015).
Taken together, although much more work is needed to fully understand sex differences in the glutamate system, there appears to be an overall increase in glutamate transmission in females. This increase may be subtle in young adulthood but during aging glutamate transmission decreases in males and the sex difference is amplified. These alterations in glutamate transmission at different ages could contribute to sex differences in incidence, symptomology, and treatment response for many psychiatric diseases. However, much more work is needed to examine differences within the glutamate system in different brain regions in males and females and determine whether there is in fact an overall increase in glutamate tone in females or if the differences are more subtle.
Sex Differences in the Glutamate System in Disease
Alzheimer's Disease
Alzheimer's disease (AD) is the leading cause of dementia and it is more likely to affect women than men, with nearly two-thirds of AD cases being women (Mielke et al., 2014).
AD is characterized by accumulation of amyloid beta (Aβ) oligomers that are able to block glutamate uptake, leading to increased glutamate levels (Domingues, Almeida, da
Cruz e Silva, Oliveira, & Rego, 2007; Mattson et al., 1992). This increased glutamate can lead to excitotoxicity and neurodegeneration. Dampening glutamate transmission can be helpful in the treatment of AD, as the non-competitive NMDA receptor antagonist
7 memantine shows efficacy in the management of moderate-to-severe AD (Reisberg et al.,
2003; Winblad, Jones, Wirth, Stöffler, & Möbius, 2007). This increase in glutamate levels could more severely impact women with AD as they exhibit lower levels of GluA2- containing AMPA receptor subunits during late mild cognitive impairment compared to men at the same point in the progression of AD (Counts et al., 2011). Reduced levels of
GluA2- containing AMPA receptor subunits could result in a greater proportion of
GluA2-lacking, Ca2+-permeable AMPA receptors, and thus, increased vulnerability to excitotoxicity due to increased calcium conductance (Counts et al., 2011). To date, there are no studies that have examined whether glutamatergic drug treatments for AD exhibit similar effectiveness in men and women (Canevelli et al., 2017). Future work examining these sex differences in treatment response could provide insight into mechanistic differences in AD progression in men and women.
Just as sex differences are seen in patients with AD, sex differences are observed in AD phenotypes in mouse models of the disease. In the triple transgenic mouse model of AD
(3xTg- AD), impairments in spatial memory and inhibitory avoidance tasks appear earlier in female mice than male mice (Clinton et al., 2007). Among 3xTgAD mice, both males and females show deficits in working memory, short-term memory, and increased anxiety- like behavior by 12 months of age, though female mutants show additional impairments in reference memory (Blázquez, Cañete, Tobeña, Giménez-Llort, &
Fernández-Teruel, 2014). This same early onset of cognitive deficits is also seen in two other mouse models of AD, tTa:APPsi mice, in which amyloid precursor protein (APP) expression is driven by the tetracycline transactivator (Melnikova et al., 2016) and
8 APP(SW) mice which overexpress human APP (King et al., 1999). Furthermore, female mice exhibit greater deficits in cognitive function following overexpression of corticotropin releasing factor (CRF) in the presence of human APP compared to males
(Bangasser et al., 2017). These differences in behavioral phenotypes are accompanied by differences in pathology. In another AD mouse model, the APP/PS1 transgenic line, female mice show an increase in plasma levels of amyloid protein with age, while males do not (Ordóñez-Gutiérrez, Antón, & Wandosell, 2015). Female APP/PS1 mice also exhibit higher levels of parenchymal Aβ in the hippocampus, along with higher levels of phosphorylated tau and proinflammatory cytokines compared to male mutant mice (Jiao et al., 2016).
Building upon the work done in clinical studies, preclinical mouse models have found a role for glutamate in AD symptomatology. Learning deficits and amyloid plaque formation are among the AD symptoms implicated by disruptions in the glutamatergic system. Rats given a competitive NMDA receptor blocker showed deficits in reversal learning, yet no changes in the initial acquisition of a spatial memory task (J. Zhang, Li,
Xu, & Yang, 2014), suggesting that NMDA receptors are at least partially involved in the learning deficits associated with AD. NMDA receptors have also been examined in mouse models. Treatment with memantine decreases amyloid plaque formation in
APP/PS1-21 mice (Scholtzova et al., 2008). However, when treated with memantine,
APP/PS1-21 mice performed similarly to WT controls in the object recognition test
(Scholtzova et al., 2008). GluCEST and 1H MRS imaging of the APP- PS1 mouse model showed decreased glutamate levels throughout the brain (compared to WT controls), but
9 the largest difference was observed in the hippocampus (Haris et al., 2013). This suggests that glutamate, and especially NMDA receptors, may be involved in the pathogenesis of
AD (Monfort et al., 2015).
Furthermore, glutamatergic sex differences have been observed in preclinical models of
AD. Reductions in glutamate within the dorsal hippocampus are seen only in male
McGill- R-Thy1- APP rats and not females (Nilsen, Melø, Witter, & Sonnewald, 2014).
Sex differences in AD development could be due to an interaction of glutamatergic systems with sex hormones. Estrogen is thought to play a protective role against cognitive impairments in female, and potentially male, rodents (J. C. Carroll et al., 2007;
Frye, Rhodes, & Dudek, 2005; Li et al., 2004). It is hypothesized that estrogen is an underlying factor of sex differences in cognitive deficits following stress in rodents
(Luine, Beck, Bowman, Frankfurt, & Maclusky, 2007). After repeated stress, female rats show normal PFC glutamatergic transmission (Wei et al., 2014), suggesting that estrogen may be protective of PFC-mediated functioning. Furthermore, E2 treatment ameliorates
Aβ- induced deficits in synaptic plasticity (Logan, Sarkar, Zhang, & Simpkins, 2011).
However, as women age, their estrogen levels decline and this decline in estrogen may increase vulnerability (Barron & Pike, 2012). To date, the studies done in mouse and rat models of AD have not taken declining estrogen levels into account.
Major Depressive Disorder
Women are nearly twice as likely as men to develop MDD and among those diagnosed with MDD, women experience more severe symptoms than men (Kornstein et al., 1995).
Although the efficacy of SSRIs has focused the depression field on the serotonergic system,
10 recent work on the efficacy of ketamine in treating MDD has led to increased interest in the glutamatergic system (Berman et al., 2000). Individuals with MDD have lower levels of both glutamate and glutamine in several brain regions including the ACC, dorsolateral prefrontal cortex (dlPFC), dorsomedial amygdala, and hippocampus (Auer et al., 2000;
Block et al., 2009; Michael et al., 2003a, 2003b). While the majority of studies have found this relationship, a few studies have not detected differences in glutamate metabolites
(either glutamate or glutamine) in MDD (Binesh, Kumar, Hwang, Mintz, & Thomas, 2004;
Milne, MacQueen, Yucel, Soreni, & Hall, 2009; Price et al., 2009). It is possible that some of the disparities in findings regarding glutamate levels and MDD are due to inconsistencies among participants between studies i.e., the ratio of men to women in the study and whether women were pre- or post- menopause (Gray et al., 2015). However, to date, none of these studies have examined potential sex differences in glutamate metabolite levels in MDD patients. Women with MDD have been shown to have higher levels of glutamate receptor gene expression postmortem, particularly in both AMPA and NMDA receptor subunit expression Additionally, women with postpartum depression exhibit an increase in prefrontal glutamate compared to healthy controls (McEwen et al., 2012). Thus, there is evidence for increased dysregulation in the glutamate system in women with MDD.
Similar to what has been seen in the clinical population, increased activity in the glutamatergic system has been connected to depression-like behavior in preclinical models.
Male rats from the Flinders sensitive line (FSL), a model of depression, exhibit increased glutamatergic synaptic transmission in the hippocampus compared to controls (Gómez-
Galán, De Bundel, Van Eeckhaut, Smolders, & Lindskog, 2013). However, female FSL
11 rats exhibit higher levels of glutamate within the PFC compared to their male FSL counterparts (Kokras, Antoniou, Polissidis, & Papadopoulou-Daifoti, 2009). Female rats also exhibit an increase in glutamate in the PFC in response to acute stress whereas males do not (Kokras et al., 2018). Furthermore, antidepressant administration increases cortical glutamate levels in both male and female FSL rats, while only increasing hippocampal glutamate in females (Kokras et al., 2009). Female rats expressing learned helplessness behavior similarly would have increased glutamate, because they exhibit decreased glutamate uptake in the hippocampus, cortex, and striatum (Almeida et al., 2010).
Furthermore, genetic alterations in the glutamate system can lead to depressive symptoms.
Decreasing levels of vesicular glutamate transporter-1 with a heterozygous knockout
(VGLUT1+/−) leads to depressive-like behavior in mice (Tordera et al., 2011). However, chronic mild stress, another model of depression, leads to increased VGLUT1 levels in the hippocampus suggesting that bidirectional dysregulation of the glutamate system can be associated with depressive phenotypes (Garcia-Garcia et al., 2009). Along with these broad differences in the glutamate system, preclinical models have revealed sex-specific alterations in the glutamate system in models of depression. Following prenatal chronic mild stress, male rats displayed higher expression of mGluR2/3, mGluR5, and NR1 in the prefrontal cortex; while female rats did not (Wang et al., 2015). Neonatal NMDA receptor blockade increases both physiological stress responsivity, CORT response, and anxiety- like behavior in the elevated plus maze in adult male mice, while female mice exhibit reduced anxiety-like behavior following the same treatment (Amani et al., 2013).
Although the glutamate system of males appears more vulnerable to manipulations early
12 in life, in adulthood, female mice are more sensitive to the antidepressant effects of ketamine, an NMDA receptor antagonist (Carrier & Kabbaj, 2013). Female mice exhibit a decrease in immobility in the forced swim test as well as an antidepressant response in the novelty suppressed feeding test at doses of ketamine that have no effect in males
(Carrier & Kabbaj, 2013). These studies suggest that adult female mice have increased glutamate tone on NMDA receptors that may be leading to increased anxiety and depressive-like behaviors. This increased NMDA receptor tone may be responsible for the increased hippocampal dendritic spine density in females at baseline (Shors,
Falduto, & Leuner, 2004; Woolley, Gould, Frankfurt, & McEwen, 1990). This idea is supported by work demonstrating that male and female rats exhibit opposite spine density changes in response to acute stress and these different responses are mediated by NMDA receptor activation (Shors et al., 2004). Further, this could provide a mechanism by which females are hyper-responsive to anxiety provoking stimuli in their environment.
Schizophrenia
In contrast to AD and MDD, SCZ is more prevalent in men, with a male to female ratio of ~1.4:1.0 (Castle, Wessely, & Murray, 1993). Furthermore, men exhibit an earlier age of onset, greater symptom severity, and poorer response to treatment (Abel, Drake, &
Goldstein, 2010). Although there are many factors contributing to these sex differences, differences in the glutamatergic system are a critical component. Impairments in the glutamatergic system contribute to the pathophysiology of SCZ. (Coyle, Tsai, & Goff,
2002; Goff & Coyle, 2001; Javitt, 2007; Olney & Farber, 1995; Tsai & Coyle, 2002).
However, this contribution appears to be different in men and women. For example,
13 polymorphisms in different glutamate related genes increase the risk for SCZ in males and females. Multiple single-nucleotide polymorphisms (SNPs) in an X-linked gene coding for the AMPA receptor subunit 3, GRIA3, confer increased risk for the development of SCZ in females only (Magri et al., 2008). On the other hand, SNPs in the SAP97 gene that encodes a scaffolding protein involved in membrane targeting of glutamate receptors, is associated with an increased risk of SCZ in males but not females (Uezato et al., 2012).
Along with differences in genetic contributions, sex differences in glutamate related protein expression and metabolites have been found. Glutamine synthetase, an enzyme involved in the maintenance of glutamate levels, is upregulated in women with SCZ but not men (Martins-de-Souza et al., 2010). Additionally, women with SCZ exhibit higher levels of NMDA receptor density compared to men with SCZ (Nudmamud-Thanoi &
Reynolds, 2004). NMDA receptor hypofunction is hypothesized to contribute to the pathophysiology of SCZ, therefore increased NMDA receptor density in women with SCZ could be protective and contribute to sex differences in symptomology (Coyle et al.,
2002; Leung & Chue, 2000). Examinations of sex differences in preclinical models of
SCZ are few and far between. Much of the research on SCZ has focused on behavioral endophenotypes. Prepulse inhibition of startle (PPI), the reduction of startle produced by a prepulse stimulus, is diminished in patients with SCZ and can be easily modeled in animals (Swerdlow & Geyer, 1998). Female rats exhibit higher levels of PPI compared to males at baseline (Gogos, Kwek, & van den Buuse, 2012; Nozari, Shabani, Farhangi,
Mazhari, & Atapour, 2015; X. Zhang et al., 2015). NMDA receptor antagonist, MK-801, decreases PPI in both intact and gonadectomized male mice whereas female mice only
14 exhibit this decrease following ovariectomy (van den Buuse, Low, Kwek, Martin, &
Gogos, 2017). This suggests that circulating hormones protect females against NMDA receptor mediated disruption of PPI. In support of this, estradiol treatment following ovariectomy blunts the ability of MK-801 to disrupt PPI (Gogos et al., 2012). Higher doses of MK-801 are able to disrupt PPI in females suggesting that NMDA receptors are still involved in the response in both sexes (Nozari et al., 2015).
In contrast to these static models of behavioral endophenotypes, developmental animal models of SCZ, such as the neonatal hippocampal lesion (nVHL) model, mimic the developmental progression of the disorder. The initial studies describing the nVHL model and the majority of those since then have utilized only the male pups, eliminating the ability to determine whether any sex differences exist (Chambers, Moore, McEvoy, & Levin,
1996; Flores, Barbeau, Quirion, & Srivastava, 1996; Goto & O'Donnell, 2002; Lipska,
Jaskiw, & Weinberger, 1993). An analysis of the literature revealed three papers that examined both males and females after nVHL. Overall, many of the behavioral effects of nVHL are similar in males and females, including deficits in working memory and increased locomotor response to novelty, MK-801 and amphetamine (Beninger et al., 2009;
Bychkov, Ahmed, & Gurevich, 2011). However, following nVHL, male mice exhibit hyperactivity in response to apomorphine, a non-selective dopamine agonist, whereas females do not (Bychkov et al., 2011). Further, following nVHL only male mice exhibit a decrease in phosphorylated extracellular signal-related kinase (pERK), mitrogen activated protein kinase (pMAPK), glycogen synthase kinase 3β (pGSK-3β), and protein kinase B (pAkt) in the accumbens and pERK within the PFC (Bychkov et al., 2011). In
15 contrast, only female mice exhibit a decrease in pAkt and pMAPK in the dorsal striatum following nVHL (Bychkov et al., 2011). Along with these behavioral and molecular sex differences, there are also sex differences in the response to antipsychotics following nVHL. Clozapine can worsen working memory deficits in male nVHL mice whereas a floor effect may limit its effects in female nVHL mice (Levin & Christopher, 2006).
However, control females are vulnerable to the memory dampening effects of clozapine whereas males are not (Levin & Christopher, 2006). Along with the nVHL model, neonatal administration of an NMDA receptor antagonist also induces SCZ-like behavior
(Stefani & Moghaddam, 2005). However, the SCZ-like phenotypes are influenced by sex and hormonal status, with males and diestrous females exhibiting more consistent endophenotypes compared to proestrous females (Célia Moreira Borella et al., 2016).
This could reflect a protective effect of estrogens, as levels of estradiol are highest during proestrus.
Studies utilizing mutant mice have also revealed sex differences that may be relevant to
SCZ. The gene neuregulin1 (NRG1) confers an increased risk of SCZ and mutations in
NRG1 lead to SCZ-like endophenotypes in mice (Gerlai, Pisacane, & Erickson, 2000; Li et al., 2004; Stefansson et al., 2003; Stefansson et al., 2002; Stefansson, Steinthorsdottir,
Thorgeirsson, Gulcher, & Stefansson, 2004). However, there are sex differences in these phenotypes. While male neuregulin deficient mice exhibit deficits in object recognition memory and both contextual and cued fear conditioning, female Nrg1+/− mice do not exhibit any cognitive deficits (Pei, Liu, & Lai, 2014). Additionally, male Nrg1+/− mice exhibited a decrease in the GABAergic markers, GAD67 and parvalbumin, while females
16 did not (Pei et al., 2014). Although both male and female NRG1 mutant mice exhibit an increase in exploratory behavior, the specific elements of this behavior differed between males and female mutants (O'Tuathaigh et al., 2006).
Abnormalities in glutamatergic functioning have been associated with SCZ-like symptoms in animals. NMDA receptor hypofunction has been repeatedly cited as a component of
SCZ and D-serine, an NMDA receptor co-agonist, may have therapeutic effects (Labrie,
Wong, & Roder, 2012). Accordingly, disrupting the glutamate system in a variety of ways, including neonatal NMDA antagonism (Stefani & Moghaddam, 2005) or deletion of
AMPA GluA1 subunits (Procaccini, Maksimovic, Aitta-Aho, Korpi, & Linden, 2013), can lead to behavioral symptoms of the disease. Neonatal VHL rats also display disruptions in glutamate signaling, with reduced glutamate release in the PFC (Beninger et al., 2009).
Furthermore, PCP and MK-801, NMDA receptor antagonists, have long been used to model the cognitive symptoms of SCZ (Moghaddam & Jackson, 2003). Perinatal treatment with PCP leads to deficits in spatial reference memory in male rats but not females
(Andersen & Pouzet, 2004). Furthermore, these deficits were alleviated by treatment with
D-serine, an NMDA co-agonist, suggesting that males may be more sensitive to disruptions of NMDA function than females (Andersen & Pouzet, 2004). Copy number variants (CNV) in the synaptic scaffolding molecular (S-SCAM), which controls synaptic AMPA receptor levels, have been linked to risk for SCZ. Transgenic mice with S-SCAM CNVs exhibit behaviors consistent with positive, negative, and cognitive symptoms of SCZ, as well as cellular and morphological abnormalities (N. Zhang et al., 2015). These mice also mimic the human condition because although both males and females show SCZ-like symptoms,
17 male S-SCAM Tg mice generally exhibit more severe symptoms (N. Zhang et al., 2015).
Taken together these findings suggest that increased glutamatergic tone in females may be protective and lead to differences in symptomology.
Autism Spectrum Disorder
Similar to the sex bias seen in SCZ, ASD is more common in boys, affecting nearly four times as many boys as it does girls (Elsabbagh et al., 2012; Fombonne, 2009). Individuals with ASD have decreased levels of glutamate metabolites in the basal ganglia and ACC and these decreases are correlated with severity of ASD symptoms (Horder et al., 2013;
Tebartz van Elst et al., 2014). In contrast to these decreases in glutamate metabolites in the brain, children with ASD have increased levels of glutamate in plasma and these levels also correlate with symptom severity (Cai, Ding, Zhang, Xue, & Wang, 2016). Despite the clear sex bias in the disease, to date, no studies have examined sex differences in metabolite levels
(Ford & Crewther, 2016).
Similar to animal models of SCZ, animal models of autism focus on endophenotypes. In particular, autism-like behaviors in rodents have focused on deficits in social behavior.
Healthy juvenile male mice exhibit more social exploratory behavior compared to juvenile females (Karlsson, Haziri, Hansson, Kettunen, & Westberg, 2015; Netser, Haskal,
Magalnik, & Wagner, 2017). Following prenatal valproic acid (VPA) treatment, an animal model of autism, male mice show impairments in social behavior in adulthood, while female mice do not (Kim et al., 2013). The prenatal VPA model also leads to male-specific deficits in sensorimotor gating, another phenotype of ASD (Anshu et al., 2017). Similar male-specific effects are seen in the telomerase reverse transcriptase overexpressing mice
18 (TERT-tg). Male TERT-tg mice exhibit impaired social behavior, increased anxiety-like behavior, and lowered seizure threshold, while female TERT-tg mice do not (Kim et al.,
2017). Maternal immune challenge also leads to male-specific deficits in social behavior in the contactin-associated protein-like 2 (Cntnap2) mouse model of ASD (Schaafsma et al., 2017). Individuals with ASD exhibit a decrease in striatal activation in response to social and non-social rewards (Scott-Van Zeeland, Dapretto, Ghahremani, Poldrack, &
Bookheimer, 2010). Male-specific deficits in reward learning are seen following 16p11.2 hemideletion, a gene that is disrupted in ASD (Grissom et al., 2018; Weiss et al., 2008).
Consistent with what has been seen in patients with ASD, preclinical studies demonstrate a clear role for the glutamate system in ASD-like behaviors. Extracellular glutamate concentrations in the lateral septum (LS) increase during social play for both male and female juvenile rats (Bredewold, Schiavo, van der Hart, Verreij, & Veenema, 2015). In a mouse model of a common CNV found in ASD, ubiquitin protein ligase Ube3a, shows deficits in social interaction, impaired communication, and increased incidence of repetitive behaviors are accompanied by impaired glutamate synaptic transmission in male and female mice (Smith et al., 2011). A similar relationship is seen in both male and female
Shank2 knockout mice. These mice show reduced social interaction and communication, impaired spatial learning and memory, and increased anxiety-like behavior, which are accompanied by reductions in NMDA receptor function (Won et al., 2012). Furthermore, restoring NMDA receptor function with D-cycloserine reversed the decreased sociability phenotype (Won et al., 2012). However, disruption of Shank3, another gene implicated in human ASD patients, leads to more pronounced reductions in glutamate transmission in
19 male knockout mice and only juvenile males exhibit deficits in social behavior (Yang et al., 2012). As male mice exhibit higher levels of glutamate induced by social play compared to females, there may be sex differences in sensitivity to perturbations in the glutamate system (Bredewold et al., 2015). Furthermore, the increase in glutamatergic tone may be protective in females.
Attention Deficit Hyperactivity Disorder (ADHD)
Attention deficit hyperactivity disorder (ADHD) also shows a strong male bias, affecting nearly 3 times as many boys as girls (Cuffe, Moore, & McKeown, 2005). Traditionally mechanistic work on ADHD has focused on catecholamine function due to the therapeutic efficacy of stimulants. However, more recently the focus has shifted to the glutamate system due to data from genetic screenings implicating CNVs and SNPs in multiple glutamate receptor subtypes (Elia et al., 2011; Lesch et al., 2008; Mick, Neale,
Middleton, McGough, & Faraone, 2008; Turic et al., 2004; Turic et al., 2005).
Furthermore, MRS imaging studies show increased glutamatergic tone in both the frontal cortex and striatum of ADHD patients and this is normalized by pharmacological treatment (Carrey et al., 2003; MacMaster, Carrey, Sparkes, & Kusumakar, 2003). While no studies have examined male and female ADHD patients and made direct comparisons, female ADHD patients exhibit a positive correlation between ACC glutamate concentration and impulsivity (Ende et al., 2016). Glutamate may play a role in not only the pathology associated with ADHD but also the treatment response. Polymorphisms in
NDMA receptor subunit genes predict better methylphenidate treatment response in children with ADHD (Kim et al., 2016). Notably, while studies discussed above
20 controlled for sex, none of the published clinical studies have examined the influence of sex as an independent variable.
The majority of the work examining animal models of ADHD have either utilized only male mice to assess phenotypes (Archer et al., 1988; Kuwagata, Muneoka, Ogawa,
Takigawa, & Nagao, 2004; Kuwagata & Nagao, 1998; Mergy et al., 2014) or in many cases where males and females were used data were collapsed preventing any examination of possible sex differences (Dell'Anna, Calzolari, Molinari, Iuvone, & Calimici, 1991;
Pappas, Gallivan, Dugas, Saari, & Ings, 1980; Row, Kheirandish, Neville, & Gozal, 2002;
Shaywitz, Gordon, Klopper, & Zelterman, 1977; Shaywitz, Yager, & Klopper, 1976).
However, some studies utilizing the spontaneous hypertensive rat (SHR) model of ADHD have reported sex differences in behavioral phenotypes. Notably, while both male and female SHRs showed hyperactivity and sustained attention deficits, male SHRs exhibit greater impulsivity (Berger & Sagvolden, 1998). While there is evidence that male SHRs perform better on conditioned association tasks than female SHRs, this seems to reflect an increase in performance compared to controls in the males rather than a decrement in females (Bucci et al., 2008). Direct comparisons between controls and SHR males and females revealed attention deficits in male SHR rats that were not present in female SHRs, while both sexes exhibited increased inhibitory control and hyperactivity (Bayless, Darling,
Stout, & Daniel, 2012). Along with these differences in behavioral phenotypes, animal models have also revealed sex differences in treatment response. Omega-3 polyunsaturated fatty acid supplementation lead to improved reinforcement-controlled attention in male
SHRs while not affecting female SHRs (Dervola et al., 2012). These findings may be
21 explained by the effects of sex hormones on fatty acid metabolism, particularly the low level of alpha-linolenic acid to docosahexaenoic acid metabolism in males (Dervola et al.,
2012).
Just as alterations in the glutamate system have been implicated in human ADHD patients, animal models of ADHD also exhibit aberrant glutamatergic signaling. SHRs exhibit higher levels of glutamate-evoked norepinephrine release and slower AMPA receptor internalization within the hippocampus compared to controls (Howells & Russell, 2008).
Given that there is evidence for increased extracellular glutamate within the hippocampus, these downstream effects could be even greater than they appear (Sterley, Howells,
Dimatelis, & Russell, 2016). This increase in extracellular glutamate may occur outside the hippocampus as well. SHR males have heightened levels of evoked glutamate release in the PFC and striatum compared to controls (E. M. Miller, Pomerleau, Huettl, Gerhardt, &
Glaser, 2014). Furthermore, manipulations of the glutamate system can lead to ADHD-like behaviors. Infusion of the NMDA antagonist, 3-(R)-2-carboxypiperazin-4-propyl-1- phosphonic acid, into the mPFC of rats leads to increased impulsivity and compulsivity
(Pozzi et al., 2011). Despite this link between glutamate and ADHD behavioral phenotypes and the observed sex differences in preclinical models, differential roles of glutamate or alterations in the glutamate system have not been examined in sex-specific manner.
Conclusion
These studies clearly demonstrate a role for dysregulation in the glutamate system in sex biased psychiatric diseases. The little data that are available suggest that females have increased glutamatergic tone compared to males and this can increase vulnerability in some
22 cases and be protective in others. However, very little work has been done to elucidate potential sex differences in the glutamate system either at baseline or in the disease state
(see Table 1). Although more imaging and postmortem tissue analysis in clinical populations would be insightful, a basic understanding of sex differences in glutamate signaling is needed. To achieve this, more preclinical studies aimed at determining sex differences are warranted. After a fundamental understanding of baseline differences is reached, examination of how dysfunction in the glutamate system can contribute to psychiatric disease would be more informative. As the majority of preclinical work has been done either only in male rodents or studies that have been underpowered to examine sex differences, much of what we know about glutamate system function and psychiatric disease may only apply to males. The examination of how glutamate dysfunction differentially affects males and females could lead to novel avenues for therapeutic development in these sex biased diseases.
In this dissertation, we will examine glutamatergic trafficking proteins and their influence on cocaine addiction-like behaviors in male and female mice. Sex differences in addiction are present across multiple drug classes (Becker & Hu, 2008). In cocaine- abusing inpatients, women report more craving in response to cocaine cues than men
(Robbins, Ehrman, Childress, & O'Brien, 1999), and have longer periods of both abstinence and relapse (Gallop et al., 2007). In general, women progress from first use to addiction faster than men, and often have more difficulty quitting (Becker & Hu, 2008;
M. E. Carroll, Lynch, Roth, Morgan, & Cosgrove, 2004).
23
Table 1. Summary of Changes in Glutamatergic Measures
2
4
The reasons for this sex difference are unclear, though it is likely due to several factors, such as increased sensitivity to the effects of drugs and the effects of estrogens (M. E.
Carroll et al., 2004). However, little work has focused on sex differences in relation to non- reproductive behaviors (McCarthy, 2008). This dissertation work will be valuable in developing a more complete understanding of the role of glutamatergic alterations in addiction and their interaction with biological sex.
Alterations in AMPA receptor subunits are thought to underlie some of the behavioral effects of drug administration. For example, increased levels of GluA2-lacking AMPA receptors within the ventral tegmental area are hypothesized to underlie sensitization to drugs of abuse (Carlezon & Nestler, 2002). Even after a single experimenter administered injection of cocaine, AMPA receptor transmission in the VTA is enhanced for up to 5 days (Ungless, Whistler, Malenka, & Bonci, 2001). It has been established that within the nucleus accumbens, AMPA receptor subunit expression is altered during incubation of cocaine craving (Conrad et al., 2008).
In this region, GluA1 levels are enhanced after 45 days of withdrawal from cocaine self- administration, whereas GluA2 levels remain unchanged (Conrad et al., 2008). This is likely due to the increased expression of GluA1 subunits, because an increase in the surface to intracellular ratio of GluA1 subunits is also observed (Conrad et al., 2008).
Further, existing preclinical literature shows a connection between estradiol and AMPA receptors. The activation of estrogen receptors leads to downstream glutamate release and enhanced dendritic spine formation (Schwarz, Liang, Thompson, & McCarthy, 2008).
The surface expression of GluA1 and GluA2 subunits is controlled in part by the trafficking 25 proteins GRIP1 and PICK1. GRIP1 binds to the GluA2 subunit of AMPA receptors and acts to maintain the receptor in the synaptic membrane. Upon phosphorylation, GRIP1 dissociates and PICK1 binds instead, internalizing the receptor so that it is no longer active at the synapse (Lu & Ziff, 2005). This process maintains the number of GluA2-containing, heteromeric AMPA receptors at the synapse, but does not affect the GluA1-lacking, homomeric AMPA receptors. Therefore, mediating the amount of GRIP1/PICK1 availability is thought to affect the ratio of GluA2-containing to GluA2-lacking AMPA receptors by increasing or decreasing the number of receptors containing the GluA2 subunit.
In the first set of experiments, we altered AMPA receptor trafficking through prefrontal knockout of GRIP1 (aim 1) or PICK1 (aim 2). Prefrontal GRIP1 knockout increased motivation for cocaine, as measured by a progressive ratio schedule, and enhanced cue- induced reinstatement. Prefrontal PICK1 knockout led to unexpected sex-specific effects.
During cue reinstatement, male PICK1 KO mice displayed attenuated reinstatement compared to controls. In contrast, female PICK1 KO mice displayed enhanced cue reinstatement. These findings prompted us to further explore potential sex differences in cocaine self- administration; the final set of experiments (aim 3) involved prefrontal PICK1 knockout in combination with male gonadectomy to remove circulating gonadal hormones.
Overall, these experiments fill a gap in existing literature; limited research has considered how glutamatergic systems may differ in males and females. These experiments provide a starting point for researchers to consider how males and females are differentially affected by drug of abuse throughout the cycle of addiction.
26 CHAPTER 2
GLUTAMATE RECEPTOR INTERACTING PROTEIN ACTS WITHIN THE
PREFRONTAL CORTEX TO BLUNT COCAINE SEEKING
Disruptions within glutamatergic pathways may underlie the development of uncontrollable drug seeking and relapse. Much work has focused on the role of the glutamatergic pathway from the medial prefrontal cortex (mPFC) to the nucleus accumbens in cocaine seeking (McFarland, Lapish, & Kalivas, 2003; Moorman, James, McGlinchey,
& Aston-Jones, 2015; Park et al., 2002; Stefanik, Kupchik, & Kalivas, 2016; Stefanik et al., 2013). After extended drug exposure, deficits in accumbal glutamate reuptake lead to reduced plasticity associated with addiction (Kalivas, 2009). Alterations in glutamate signaling within the PFC also play a role in addiction-like behaviors. Abnormalities in prefrontal functioning lead to compulsive drug taking and impairments in the executive function needed for a number of self-regulatory behaviors (Goldstein & Volkow, 2011).
Cocaine self-administration leads to both a decrease in basal glutamate levels within the mPFC and a decrease in cocaine-evoked glutamate release (Ben-Shahar et al., 2012).
This decrease in release is accompanied by an increase in glutamate receptor expression, specifically GluN2B expression in the mPFC during withdrawal. Increased GluN2B expression is seen with or without cue-induced drug seeking, suggesting that the elevated
GluN2B levels are the result of cocaine experience and/or withdrawal, and not other prefrontal mediated processes such as motivation (Szumlinski et al., 2016). These alterations in glutamate signaling have functional consequences such as increasing glutamate levels within the infralimbic cortex leading to attenuation of incubation of
27 cocaine craving (Shin et al., 2018). While it is clear that dysfunction in glutamate signaling in the PFC plays a role in drug-associated behaviors, the mechanisms underlying these alterations in glutamate signaling within the PFC remain largely unexplored.
Glutamate Receptor Interacting Protein (GRIP) is a scaffolding protein that regulates the trafficking of GluA2-containing AMPA receptors in and out of the cell membrane (Dong et al., 1997). As such, GRIP has been shown to be involved in activity-dependent synaptic plasticity throughout the brain (Summa, Di Prisco, Grilli, Marchi, & Pittaluga, 2011;
Takamiya, Mao, Huganir, & Linden, 2008; Xue, Zhang, Chen, Lin, & Shi, 2010). Although the effects of GRIP on receptor trafficking have been characterized, its role in behavior is less clear. There is some evidence of a role for GRIP in social behavior (Mejias et al., 2011).
Additionally, there has been some research on the role of GRIP in addictive phenotypes.
Exposure to drug-paired cues decreases GRIP expression in the nucleus accumbens core
(Liang et al., 2017). Furthermore, GRIP deletion from the nucleus accumbens enhances cue-induced cocaine seeking (Briand, Kimmey, Ortinski, Huganir, & Pierce, 2014) and impairs the ability of calpain to disrupt reconsolidation of cocaine conditioned reward
(Liang et al., 2017). However, little is known on the behavioral effects of GRIP in the prefrontal cortex. In this experiment, we sought to elucidate the effects of prefrontal
GRIP knockout on addiction- like behaviors. There are two forms of GRIP, GRIP1 and
GRIP2, and both regulate activity dependent AMPA receptor internalization and recycling (Mao, Takamiya, Thomas, Lin, & Huganir, 2010). Although GRIP1 and GRIP2 are homologous and highly conserved, GRIP1 can completely rescue function in GRIP2
KO mice, whereas GRIP2 can only partially restore GRIP1 function (Tan, Queenan, &
28 Huganir, 2015). Therefore, to examine the influence of GRIP function, we utilized a mouse with a floxed GRIP1 gene on a background of GRIP2 knockout. In the absence of cre recombinase, the GRIP1 performs all GRIP functions, thereby rendering this mouse indistinguishable from wildtype. However, the GRIP2 knockout background is needed in order to prevent GRIP2 from rescuing the function of GRIP1 following the inducible knockout. Based on the role of prefrontal glutamate signaling and addiction, we hypothesized that prefrontal GRIP1 knockout would lead to the development of an addictive phenotype in mice. We found that PFC GRIP1 knockout led to increased motivation for cocaine and increased cue-induced cocaine seeking, without altering motivation or seeking for sucrose.
Methods
Subjects
Mice homozygous for the Cre/lox-conditional allele of GRIP1 (flox/flox) and GRIP2 knockout (−/−) were bred on a C57bl/6J background. Male and female mice (2–6-months old, age matched across group) were housed individually following stereotaxic surgery and during experimental paradigms. All animals were housed in a temperature- and humidity- controlled animal care facility with a 12-h light/dark cycle (lights on at 0700 hours). All procedures were approved by the Temple University Animal Care and Use Committee.
Cocaine was obtained from the National Institute on Drug Abuse Drug Supply Program
(Bethesda, MD) and dissolved in sterile 0.9% saline.
29 Prefrontal Microinjections and Adeno-Associated Virus Constructs
The adeno-associated virus (AAV) expressing Cre recombinase (AAV2/9.CMV.PI.CRE, titer 2.84 × 1013 vgc/μl) and the AAV expressing green fluorescent protein (eGFP)
(AAV2/9.CMV.eGFP, titer 3.74 × 1013 vgc/μl) were generated by the University of
Pennsylvania Vector Core. GRIP1 flox/flox mice (6-8 weeks) were anesthetized with isoflurane and 0.4µl of the viral construct (Cre or GFP) was injected bilaterally into the prefrontal cortex through a 30-gauge needle at a rate of 0.1 μl/min. Stereotaxic coordinates for the prefrontal cortex are (from Bregma) anterior-posterior 2.4, lateral +/- 0.3, dorso- ventral -2.3. Following recovery, mice remained in the home cage for 6 weeks prior to behavioral testing. The procedures involving the AAV viruses have all been approved by the Temple University Institutional Biosafety committee. Knockout was confirmed via western blot, and animals removed from study if knockout was less than a 30% decrease from average GFP control levels (n=2).
Operant Food Training
Before catheterization, mice were trained to perform an operant response for sucrose pellets. The mice were placed in operant chambers (Med-Associates) and trained to spin a wheel manipulandum to receive a sucrose pellet, with one-quarter spin measured as a single active response. Mice performed 5 days of FR1 responding followed by 5 days of FR5 responding, and a single day of a progressive ratio schedule (5*EXP(0.2*P)-5, where
P=previous ratio; eg. 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40…). A compound cue stimulus consisting of a cue light above the active wheel, a 2900-Hz tone, and house light off was concurrent with each pellet administration, followed by an additional 8 s time-out when
30 responding had no programmed consequences and the house light remained off. Mice were allowed to self-administer a maximum of 50 pellets per 60 min operant session. During the food training phase, mice were food restricted to ∼90% of their free-feeding weight. Mice returned to ad libitum food access 3 days following the start of the cocaine self- administration phase.
Jugular Catheterization Surgery
Prior to surgery, mice were anesthetized with 80 mg/kg ketamine and 12 mg/kg xylazine.
An indwelling silastic catheter was placed into the right jugular vein and sutured in place.
The catheter was then threaded subcutaneously over the shoulder blade and was routed to a mesh backmount platform (Strategic Applications, Inc) that secured the placement.
Catheters were flushed daily with 0.1 ml of an antibiotic (Timentin, 0.93 mg/ml) dissolved in heparinized saline. The catheters were sealed with plastic obturators when not in use.
Cocaine Self-Administration
Mice were tested for cocaine self-administration behavior in 2-hour sessions in the same chamber used for sucrose pellet self-administration. During testing, responding on the wheel now delivered an intravenous cocaine injection (0.6 mg/kg/infusion), paired with the same compound cue, under the same schedule as the food training. Following 10 days of cocaine self-administration on an FR1 schedule, mice underwent one day of cocaine self- administration on a progressive ratio schedule (5*EXP(0.2*P)-5, where P=previous ratio; eg. 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40…) in which the same compound cue was presented. Breakpoint criteria were defined as failure to acquire an infusion of cocaine within 1800 seconds of the last infusion. The following day, mice began extinction training, in which cocaine-seeking behavior was extinguished by replacing the cocaine 31 with 0.9% saline. During this time the light and tone cues paired with cocaine delivery were not present. Daily 2-h extinction sessions continued until animals met the extinction criterion of less than 25% of their self-administration responding (average of last 3 days). Twenty-four hours following meeting the extinction criterion, animals underwent a cue-induced reinstatement session. During the cue-induced reinstatement session, the light and tone cues were presented non-contingently for 20 seconds every 2 minutes during the first 10 minutes of the session. After this time period, the cues were presented contingent with operant responding, just as was done during the cocaine self- administration phase. During the reinstatement session, animals received saline infusions following responses on the active wheel.
Operant Set Shifting Task
Mice were run in a cognitive flexibility task as described in Parikh et al., 2016. A standard mouse operant conditioning chamber (MED Associates), containing grid floor, houselight, two large cue lights, central port with fluid dipper, and retractable levers was used. Mice were first trained on an FR1 schedule to acquire the lever press response, which provided
10µl of .066% saccharin solution. Once mice had completed a minimum of 30 lever presses within a 30-minute session, they began a pretraining phase. During pretraining, one of the levers (left or right of the central port) was extended for 10 seconds. Pressing the lever resulted in presentation of the reward and retraction of the lever. After the lever press or omission, an ITI of 9 ± 3 seconds began. Once mice met criteria (30 rewards and ≤ 20% omissions for 3 consecutive days), they were moved on to the visual discrimination phase.
In this phase, both levers were presented for 5 seconds and mice were required to press the
32 one underneath the illuminated cue light in order to receive a reward. After 3 consecutive days of 80% correct responses, mice were moved on to the set shifting phase. During this phase, mice were assigned to a “right lever” or “left lever” condition in a counterbalanced manner. The lever condition denoted which lever (right or left) was the active lever, and mice were required to press that lever to receive a reward, regardless of the location of the illuminated cue light. Once mice again were responding at least 80% correct for three consecutive days, they were moved on to the final stage, reversal. During reversal, mice were assigned the opposite lever condition as their set shift assignment. Once they had responded at 80% correct for three consecutive days, they were removed from the task and water returned ad libitum.
Western Blot
GRIP1 levels in the prefrontal cortex were measured using a western blot, as described in
Briand et al., 2014. Briefly, animals were decapitated, and the prefrontal cortex dissected using a brain block (Braintree Scientific). Protein quantification was performed using a
Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 µg) were loaded into each well of a Tris-glycine gel (Lonza) and transferred to nitrocellulose membranes (Immobilon). Membranes were blocked with Li-Cor blocking buffer and allowed to incubate in primary antibody solution (GRIP1, 1:2000 (BD Biosciences) and
GAPDH, 1:5000 (Cell Signaling)) for 24 hours at 4°C. Membranes were then incubated with fluorescent secondary antibodies (1:20,000; IR-dye 680 or IR-dye 800, Li-Cor) and imaged on an Odyssey fluorescent scanner (Li-Cor). Western blots were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the percent knockout
33 calculated as a fraction of the average of the GRIP1 levels in GFP-infused mice.
Electrophysiology
Slice Preparation. Following prefrontal injection of AAV-Cre or GFP, naïve mice were cervically dislocated and decapitated. The brain was removed and coronal slices of prefrontal cortex and nucleus accumbens were cut with a Vibratome (VT1000S, Leica
Microsystems) in an ice-cold artificial cerebrospinal fluid solution (ACSF), in which NaCl was replaced by an equiosmolar concentration of sucrose. ACSF consisted of 130 mM
NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 1 mM MgCl2, and 2 mM CaCl2 (pH 7.2–7.4 when saturated with 95% O2/5% CO2). Slices were incubated in ACSF at 32–34 °C for 25 min and kept at 22–25 °C thereafter, until transfer to the recording chamber. The osmolarity of all extracellular solutions was 300–315 mOsm.
Slices were viewed using infrared differential interference contrast optics under an upright microscope (Slice Scope Pro, Scientifica) with a 40 × water-immersion objective.
Recordings. The recording chamber was continuously perfused (1–2 ml/min) with oxygenated ACSF heated to 32±1 °C using an automatic temperature controller (Warner
Instruments). Picrotoxin (100 μM) was added to all solutions to block the GABAA receptor-mediated currents. Recording pipettes were pulled from borosilicate glass capillaries (World Precision Instruments) to a resistance of 4–7 MΩ when filled with the intracellular solution (whole-cell recordings) or to a resistance of 1–2 MΩ when filled with extracellular solution (field recordings). All recordings were conducted with a
MultiClamp700B amplifier (Molecular Devices).
34 Whole-cell recordings. Intracellular solution contained (in mM): 100 CsCH3O3S, 50 CsCl,
3 KCl, 0.2 BAPTA, 10 HEPES, 1 MgCl2, 2.5 phosphocreatine-2Na, 2 Mg-ATP, 0.25 GTP-
Tris, 1 QX-314 (pH 7.2–7.3 with CsOH, osmolarity 280–290 mOsm). All sEPSC recordings were conducted in whole-cell voltage-clamp mode (Vh = −70 mV). Currents were low-pass filtered at 2 kHz and digitized at 20 kHz using a Digidata 1440A acquisition board and pClamp10 software (both from Molecular Devices). Access resistance (10–30
MΩ) was monitored throughout the recordings by injection of 10 mV hyperpolarizing pulses and data were discarded if access resistance changed by >25% over the course of data acquisition. sEPSCs were detected using an automated sliding-template-based algorithm in pClamp 10. This method compares the shape of the detected current to that of a template and has been shown to detect events with amplitude of at least 3 times the square deviation of the noise (Clements & Bekkers, 1997). All detected events were verified by visual confirmation of a fast rise time and slower exponential decay to baseline. Mean sEPSC amplitude was analyzed from an average sEPSCs trace computed from a minimum of 150 individual sEPSCs. Mean sEPSC frequencies were analyzed from 180-s long trace segments. Evoked responses were triggered by 100 μs constant-current pulses generated by an A310 Accupulser (World Precision Instruments) and delivered at 0.1 Hz via a bipolar tungsten stimulation electrode positioned within 100 μm of the recorded cell. The amplitude of the current pulses was controlled by a stimulus isolator (WPI Linear Stimulus
Isolator A395) and was adjusted to elicit monosynaptic responses in the range of 100–
300 pA (the required stimulus intensity ranged from 15 to 80 μA). For all measures, cells from at least 3 animals, within each group, were used. Recordings were taken from cells
35 within the accumbens core. Field Recordings. A bipolar tungsten stimulating electrode was placed within 100-300 µm from the recording electrode and used to stimulate excitatory afferents at 0.1 Hz. The field recordings were performed within the core of the nucleus accumbens. The amplitude of current pulses was set at the intensity required to evoke a 70% maximal response.
Stimulations were applied as paired pulses (interval 20-420 ms) at 0.06Hz. The initial slope of sEPSPs was used as a measure of synaptic response.
Statistical Analysis
All self-administration experiments were analyzed with two-way ANOVAs with viral injection and day as the independent variables and pellets/responses/infusions as the dependent variable. Sidak’s post hoc comparisons were made when main effects or interactions were detected (p < 0.05). The protein quantification, progressive ratio, extinction responding and days to criterion, cue-induced reinstatement responding, and cognitive flexibility data were analyzed using unpaired t-tests with viral injection as the independent variable. The sEPSC data were also analyzed using an unpaired t-test with viral injection as the independent variable. The paired pulse recordings were analyzed using a two-way ANOVA with viral injection and interpulse interval as the independent variables.
Results
Viral Mediated Deletion of GRIP1 in the Medial Prefrontal Cortex
Mice used in this experiment were GRIP1-floxed mice bred on a GRIP2-null background, as the elimination of both GRIP isoforms is necessary (Takamiya et al,
2008). Six weeks following the injection of AAV-Cre into the mPFC, we elicited a significant knockout in GRIP1 levels in this region compared to AAV-GFP injected controls [t(35) = 4.79, p < 0.0001; Fig. 1]. The available antibodies only allow us to
36 quantify the extent of the knockout using western blot techniques; this does not allow us to differentiate between GRIP1 knockout in prefrontal cell bodies versus GRIP1 knockout in terminals of neurons projecting to the PFC. However, although AAV9 preferentially targets neurons, the lack of complete knockout may be due to glial expression of GRIP1. Additionally, we confirmed that AAV-Cre injection into the mPFC does not affect GRIP1 expression in the nucleus accumbens [t(29) = 0.337, p =.74; Fig.
1f].
Prefrontal GRIP1 Knockout Does Not Affect Fixed Ratio Self-Administration of Sucrose
or Cocaine
Six weeks after viral injections, GFP controls and Cre GRIP1 knockout mice underwent ten days of sucrose self-administration to acquire the operant response. An ANOVA for training day and viral injection revealed that there were no differences in number of pellets received [F(1, 83)=0.37, p=0.54; Fig. 2a], number of responses on the active response wheel [F(1, 83)=1.37, p=0.25; Fig. 2b], nor in percent of responses on the active response wheel between the two groups [F(1, 75)=2.27, p= 0.14; Fig. 2c]. After acquiring the operant response for food, mice received jugular catheterization surgery and began the cocaine self- administration phase.
37
Figure 1. Significant viral mediated prefrontal GRIP1 knockout. Green areas indicate the location of the bilateral injections of 0.4 μg of GFP or AAV-Cre recombinase into the medial prefrontal cortex (a). Coronal section of the mouse brain showing the viral expression of GFP within the mPFC (b). Quantification of western blot reveals a significant decrease in GRIP1 protein within the mPFC following AAV-Cre injection, as normalized to glyceraldehyde 3-phosphate dehydrogenase [GAPDH; t(35)=4.79, ***p<.0001, n=15-24; c]. Representative western blots showed GRIP1 knockout in the mPFC (d). There was no effect of AAV-Cre injection on either GAPDH expression in the PFC (e) or GRIP1 expression in the nucleus accumbens (NAc; f). Bars represent average ± SEM.
38
Figure 2. No difference between GFP controls and Cre GRIP1 knockout during food self- administration training. Over the 10 days of food self-administration, there were no significant differences between GFP control mice (n=39) and Cre GRIP1 KO mice (n=46) in the number of pellets consumed (a), number of responses on the active wheel (b), or percent of active responses during food training (c). Boxes represent average ± SEM.
Figure 3. No differences between GFP controls and Cre GRIP1 knockout during cocaine self-administration. Over 10 days of cocaine self-administration, there were no significant differences between GFP control mice and Cre GRIP1 KO mice in the number of cocaine infusions or the number of responses on the active wheel (a, b; n=27-34). Boxes represent average ± SEM.
39
Table 2. Self-administration measures by sex and GRIP1 knockout.
40
Again, ANOVA revealed that there were no differences between controls and prefrontal
GRIP1 knockout mice in number of infusions received [F(1,59)=0.01, p=0.91; Fig. 3a], number of responses on the active response wheel [F(1,59)=1.11, p=0.30; Fig. 3b], nor in percent of active wheel responses [F(1,57)=0.21, p=0.65]. As no effect of sex was found for either food or cocaine self-administration (Table 2), sex is collapsed across groups
Prefrontal GRIP1 Knockout Enhances Responding for Cocaine but not Sucrose on a
Progressive Ratio Schedule
After 10 days of fixed ratio cocaine self-administration, GFP control and Cre GRIP1 knockout mice ran on a progressive ratio schedule of reinforcement to assess their willingness to work for cocaine. An unpaired t-test showed that Cre GRIP1 knockout mice exhibited a higher breakpoint compared to the GFP control mice [t(38)=2.18, p=0.04; Fig.
4a]. In contrast, GFP control and Cre GRIP1 knockout mice exhibit similar willingness to work for sucrose on a PR schedule [t(48)=0.33, p=0.75; Fig. 4b]. Sex is collapsed across groups as no sex differences were observed (Table 2).
Prefrontal GRIP1 Knockout Enhances Responding for Cocaine but not Sucrose During
Cue-Reinstatement
Following the cocaine self-administration phase, a subset of mice began extinction training.
No differences were seen in the responding on the first day of extinction [GFP Control=
206.21±60.94; Cre GRIP1 KO= 293.67±91.18; t(40)=0.83, p=0.41] or in the days to reach the extinction criterion [GFP Control=6.63±0.78; Cre GRIP1 KO=6.07±0.95; t(31)=0.46, p=0.65]. However, during the cue-induced reinstatement session, mPFC GRIP1 knockout
41 mice exhibit significantly greater responding compared to GFP control mice [t(31) = 2.27, p = 0.03; Fig. 4c], indicating higher cue-derived cocaine seeking. In a separate cohort of mice, we examined extinction and cue-induced reinstatement of food seeking. Similar to what was seen with extinction of cocaine seeking, we did not see any effect of mPFC
GRIP1 knockout on extinction of food seeking (GFP Control= 483.69±66.44; Cre GRIP1
KO= 481.79±113.15; t(25)=0.01, p=0.99].
Additionally, we did not see a viral-mediated increase in cue-induced food seeking following mPFC GRIP1 knockout [t(24) = 0.61, p = 0.30; Fig. 4d]. There were no sex differences in these behavioral measures (Table 2), so sex is collapsed across groups.
Prefrontal GRIP1 Knockout Does Not Lead to Deficits in Cognitive Flexibility
To determine whether the increased cocaine seeking during reinstatement was due to deficits in cognitive flexibility, we ran a separate cohort of mice on a cognitive flexibility task (Parikh, Cole, Patel, Poole, & Gould, 2016). There were no differences between GFP controls and Cre GRIP1 KO on the number of trials to reach visual discrimination criteria
[t(81)=1.74, p=0.09]. We did not detect any effect of mPFC GRIP1 knockout on the trials to criterion [t(84)=1.47, p=0.15; Fig. 5a] or errors to criterion [t(84)=0.24, p=0.81; Fig.
5b] on the set-shift phase of the task. We also did not see any differences between the groups during the reversal phase of the task in either the trials to criterion [t(84)=0.70, p=.49; Fig. 5c] or the errors to criterion [t(84)=0.25, p=.81; Fig. 5d]. As no sex differences were observed (Table 2), sex is collapsed across groups for analysis.
42
Figure 4. Prefrontal GRIP1 knockout increases progressive ratio breakpoint and reinstatement responding for cocaine but not sucrose. Following prefrontal GRIP1 knockout, mice exhibited an increased breakpoint on a progressive ratio schedule for cocaine [a; t(38) = 2.18, *p = 0.04; n=18-24] but not for food (b; n=24-26). Breakpoint is defined as the final ratio the mice achieved before timing out of the session, i.e. the number active responses required to move onto the next step. Further, PFC GRIP1 knockout mice exhibit greater cocaine seeking during cue-induced reinstatement [c; t(31) = 2.27, *p = 0.03; n=14-20] but not greater reinstatement of food seeking (d; n=13-14). Bars represent average ± SEM.
43
Figure 5. Prefrontal GRIP1 knockout does not impact cognitive flexibility. No differences in were seen following prefrontal GRIP1 knockout in set- shifting performance, either trials to criterion or errors to criterion (a, b; n=40-45). Further, no differences were seen between the groups in trials to criterion or errors to criterion in the reversal learning task (c,d; n=40-45). Bars represent average ± SEM.
44 Prefrontal GRIP1 Knockout Alters Glutamate Transmission in the PFC and the NAc
Six to eight weeks after viral-mediated knockout of GRIP1 in the prefrontal cortex, we examined spontaneous excitatory transmission in drug-naïve mice. Following mPFC
GRIP1 knockout we see an increase in the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) compared to GFP-injected controls [t(15)=3.24, p<0.01;
Fig. 6a]. No differences were seen between the groups in sEPSC frequency [t(15) = 0.91, p = 0.38; Fig. 6b]. To determine whether these physiological effects within the PFC altered downstream transmission in the nucleus accumbens, we examined paired-pulse ratio (PPR) in both cocaine-experienced and naïve mice. Medial PFC GRIP1 knockout led to a decrease in PPR in the nucleus accumbens regardless of drug history [F(1,33)=4.35, p=0.04; Fig.
6c]. No sex differences were observed.
Figure 6. Prefrontal GRIP1 knockout enhances signaling within the PFC but only affects accumbal signaling after cocaine experience. Quantification of sEPSC amplitude reveals an increase in prefrontal GRIP1 knockout mice compared to GFP controls [a; t(15)=3.24, **p<0.01; n=15-20]. No differences were seen between the groups in sEPSC frequency (b; n=15-19). Prefrontal GRIP1 knockout led to a decrease in the paired-pulse ratio (40ms IPI) in both naïve and cocaine-experienced mice [c; F(1,33)=4.35, p=0.04; n=12-13]. Bars represent average ± SEM.
45 Discussion
Overall, we find that the scaffolding protein, GRIP1, plays a critical role within the prefrontal cortex in mediating cocaine seeking. Our data demonstrate that knockout of prefrontal GRIP1 increases motivation for cocaine and cocaine seeking during cue-induced reinstatement, while not affecting sucrose seeking or consumption. Furthermore, these alterations are not simply the result of alterations in cognitive function. Prefrontal GRIP1 knockout does not alter set-shifting or reversal learning. Electrophysiological recordings demonstrate that GRIP1 knockout leads to increased sEPSC amplitude within the PFC and downstream alterations in presynaptic transmission in the NAc.
GRIP1 Knockout in the Prefrontal Cortex Increases Motivation for Cocaine and
Potentiates Cocaine Seeking in Both Males and Females
The glutamatergic projection from the PFC to the NAc is critically involved in the reinstatement of drug seeking in part due to its role in consolidation of cue-driven reward memory (Berke & Hyman, 2000; Kalivas, Volkow, & Seamans, 2005). Inactivation of the medial PFC disrupts reinstatement of drug seeking (Martin-Garcia et al., 2014;
McLaughlin & See, 2003; Palombo et al., 2017; Rocha & Kalivas, 2010; Zavala, Weber,
Rice, Alleweireldt, & Neisewander, 2003). However, the mechanisms underlying this involvement are less well understood. The current study demonstrates that glutamate trafficking within the PFC, specifically mediated by GRIP1 function, plays a role in cocaine seeking.
46 Our findings build upon previous work showing cocaine-induced alterations in glutamate signaling in the prefrontal cortex. Prefrontal glutamatergic transmission, and especially the pathway between the PFC and nucleus accumbens, is involved in reinstatement of cocaine- seeking (McFarland et al., 2003). Within the PFC itself, glutamate release plays a role in protracted withdrawal from cocaine and the development of incubation of craving (Shin et al., 2018). An increase in prefrontal glutamate is also seen during the first day of withdrawal after chronic cocaine treatment (Williams & Steketee, 2004). Within 24 hours of cocaine self-administration, prefrontal glutamate levels are decreased (Ben-Shahar et al.,
2012). However, while basal glutamatergic activity within the PFC is decreased, burst firing in response to cocaine is increased in drug-experienced rats (Sun & Rebec, 2006).
This increase, not seen in drug-naïve rats, suggests a mechanism by which cocaine usurps prefrontal circuits during addiction (Sun & Rebec, 2006).
After repeated cocaine experience, the rat prefrontal cortex displays enhanced neuronal excitability for at least three weeks (Nasif, Sidiropoulou, Hu, & White, 2005). These changes may be due to differing contributions of GluA2-containing and GluA2-lacking
AMPA receptors, as seen after conditioned place preference for cocaine (Pena-Bravo,
Reichel, & Lavin, 2017). Specifically, increased GluA1 levels have been observed in the vmPFC after cocaine extinction in rats (Nic Dhonnchadha et al., 2013). Moreover, these molecular changes are hypothesized to underlie cocaine withdrawal symptoms (Nasif et al., 2005). Our findings align with this interpretation and suggest that GRIP1 KO has similar effects in mice. Prefrontal GRIP1 KO lead to enhanced prefrontal sEPSC amplitude; in conjunction with downstream changes in accumbal paired pulse ratio this
47 suggests a functional increase in PFC activity after GRIP1 KO. Without GRIP1 to maintain
GluA2-containing AMPA receptors to the synaptic membrane, we hypothesize that this increased activity is due to a relative increase in the amount of GluA2-lacking AMPA receptors at the membrane. Because GRIP1 KO was also shown to potentiate cocaine seeking, we believe this behavioral effect may be explained in part by the enhancement of PFC activity and its downstream effects on glutamatergic drive in in the nucleus accumbens.
Prefrontal GRIP1 Does Not Play a Role in Natural Reward Taking or Seeking
We did not find any effect of prefrontal GRIP1 knockout on natural reward taking or seeking. This is consistent with past work showing that glutamatergic afferents from the
PFC are active during drug, but not food, reinstatement (McFarland et al., 2003). Moreover, experimental manipulation of AMPA receptors has consistently failed to affect food seeking (Anderson et al., 2008; Briand et al., 2014; Famous et al., 2008). Therefore, we must conclude that glutamatergic plasticity within the PFC is more sensitive to drug use than to natural reward. Given that specific neuronal ensembles within the vmPFC are responsible for encoding food reward and extinction of food seeking, we may have seen different results if we had manipulated GRIP1 expression after the formation of these memories (Warren et al., 2016). However, it is also possible that we would have found different results had we examined a high fat food reward that can lead to more compulsive food seeking (Decarie-Spain et al., 2018; Ghitza, Gray, Epstein, Rice, & Shaham, 2006;
Johnson & Kenny, 2010).
48 The differing effects between drug seeking and natural reward are also seen in the extracellular accumbal glutamate levels of rats trained to self-administer cocaine
(McFarland et al., 2003). Extracellular glutamate levels are increased after cocaine self- administration but not food self-administration. Additionally, food self-administration has been shown to create a reversible potentiation of glutamatergic signaling within the VTA, whereas cocaine self-administration leads to VTA potentiation stable for at least 3 weeks
(B. T. Chen et al., 2008). Thus, our data is congruent with the existing literature on the effects of glutamate during drug versus natural reward.
Knocking Out GRIP1 in the PFC Does Not Alter Cognitive Function
The PFC is involved in executive behavior, including whether a rodent should engage in or suppress an action based on context (Moorman & Aston-Jones, 2015). Prefrontal lesions impair set-shifting in mice as well as rats (Bissonette et al., 2008). Additionally, lesions of the mPFC disrupt the formation of an attentional set (Bissonette et al., 2008), impair both sustained attention and response inhibition (Broersen & Uylings, 1999), and impair performance on a delay discounting task in both rodents (Déziel & Tasker, 2017) and humans (Bechara, Tranel, & Damasio, 2000). More specifically, glutamate within the PFC is critical for maintenance of these cognitive functions. Disrupting either AMPA receptor or NMDA receptor function in the PFC leads to deficits in behavioral flexibility in male rats retrieving a food reward from a T-maze (Stefani, Groth, & Moghaddam, 2003).
Similarly, AMPA antagonists injected into the mPFC of male rats causes deficits in extradimensional shifting when digging for a food reward in pots containing two different odors and digging mediums (Jett, Bulin, Hatherall, McCartney, & Morilak, 2017).
49 Given the role of the PFC in cognitive function (E. K. Miller, 2000), it is perhaps surprising that the current study did not find any effects of prefrontal GRIP1 knockout on strategy set- shifting, reversal learning or the ability of mice to learn an operant task. However, prefrontal GRIP1 knockout did not lead to an overall disruption in glutamate signaling in the PFC. To the contrary, we saw increased AMPA transmission. Therefore, our findings are consistent with previous work highlighting the role of the PFC in cognition. In rats, microinfusion of an AMPA receptor positive allosteric modulator into the prelimbic cortex has been shown to enhance cognition on an odor-reward association task (Yefimenko,
Portero- Tresserra, Martí-Nicolovius, Guillazo-Blanch, & Vale-Martínez, 2013). Likewise, injection of an AMPA receptor antagonist into the mPFC of rats impairs discrimination learning and set-shifting due to general learning deficits (Stefani & Moghaddam, 2006).
Consistent with our findings, an increase in prefrontal AMPA transmission would not be predicted to inhibit cognitive learning on an operant task.
Altering AMPA Trafficking in the PFC has Downstream Effects on Accumbal Physiology
Disrupting GRIP1 function leads to a decrease in the anchoring of GluA2-containing
AMPA receptors to the synapse (Mejias et al., 2011). Therefore, one might expect a decrease in prefrontal glutamate transmission following site-specific GRIP1 deletion. In contrast, we found that prefrontal GRIP1 knockout led to an increase in sEPSC amplitude, measured in layer 5 of the PFC. While GluA2-containing AMPA receptors are the primary subtype, GluA2- lacking AMPA receptors are also present in the PFC. In fact, cocaine exposure can lead to an increase in the contribution of GluA2-lacking AMPA receptors in the prefrontal cortex (Pena-Bravo et al., 2017). GluA2-lacking AMPA receptors are
50 calcium permeable and therefore exhibit a higher conductance than the GluA2-containing
AMPA receptors. By knocking out GRIP1 in the PFC and disrupting the insertion of GluA2- containing AMPA receptors into the membrane, it’s possible that GluA2-lacking AMPA were preferentially inserted in the synapse, leading to an enhanced sEPSC amplitude. As the sEPSC recordings are influenced by not only AMPA-mediated currents but also spontaneous action potential firing, it is possible that the effects we see on sEPSC amplitude are mediated by other glutamate receptor subtypes (i.e. NMDA receptors).
However, as GRIP1 has not been demonstrated to be involved in NMDA receptor trafficking but plays an established role in AMPA receptor trafficking, the effects we see are likely due to differences in AMPA receptor signaling.
As cocaine increases excitability in the prefrontal cortex (Nasif et al., 2005), the increased sEPSC amplitude following GRIP1 knockout could contribute to the increased cocaine seeking. The current study also found that prefrontal GRIP1 knockout led to a decrease in paired pulse ratio within the nucleus accumbens of both naïve and cocaine- experienced mice. The decrease in paired-pulse ratio suggests that prefrontal GRIP1 knockout leads to an increase in glutamate release probability in the nucleus accumbens
(Fioravante & Regehr, 2011; Regehr, 2012). This increase in release probability could lead to an increase in cue-evoked glutamate release in the nucleus accumbens, perhaps driving the increases in reinstatement behavior.
51 Conclusion
In the current study, we have shown that conditional deletion of GRIP1 in the mPFC leads to a specific increase in cocaine seeking and motivation for cocaine in both male and female mice. Disrupting GRIP1 in the mPFC does not alter intake or seeking of natural rewards nor does it affect cognitive flexibility. Furthermore, GRIP1 knockout leads to an increase in AMPA transmission in the mPFC as well as alterations in glutamate transmission downstream in the nucleus accumbens. These results suggest that pharmacotherapies aimed at augmenting the interaction between GRIP1 and GluA2 could be effective in treating cocaine use disorder.
52 CHAPTER 3
SEX-SPECIFIC EFFECTS OF PREFRONTAL PICK1 KNOCKOUT ON COCAINE
REINSTATEMENT
Substance use disorder affects nearly 20 million people in the United States, 966,000 of whom primarily abuse cocaine (Administration, 2018). In 2017, the United States
Department of State expressed concern over the rapidly increasing amount of export- quality cocaine produced in several South American countries, from 915 metric tons in
2009 to 1,930 metric tons in 2017 (State, 2019). These figures are particularly troublesome given the lack of pharmacological treatments for cocaine abuse. Therefore, it is of utmost importance that the molecular mechanisms underlying cocaine abuse are identified.
Dysregulated glutamate levels within the prefrontal cortex (PFC) have been shown to enhance cocaine use in animal models of addiction. Memory of cocaine-associated contexts involves prefrontal glutamatergic activity in mice (T. Zhang et al., 2019). In rats, incubation of craving after cocaine self-administration is associated with elevated glutamate release in the ventromedial PFC (Koya et al., 2009). The elevated glutamate release within the PFC impacts downstream regions such as the nucleus accumbens (Shin et al., 2016). This process is hypothesized to lead to the development of addiction in both rats and humans
(Kalivas et al., 2005; Park et al., 2002).
The trafficking of glutamate receptors into and out of the synaptic membrane ensures appropriate levels of glutamate transmission in healthy individuals. However, cocaine experience alters the balance of glutamate receptors at the synapse. During extinction of cocaine seeking, elevated levels of the GluA1 subunit are observed in the PFC
53 (Ghasemzadeh et al., 2011) and nucleus accumbens (Sutton et al., 2003). Work in the nucleus accumbens has also found an increase in calcium permeable AMPA receptors, which are comprised of the GluA1 subunit (McCutcheon, Wang, Tseng, Wolf, & Marinelli,
2011). Notably, this was true only after self-administration of cocaine, not experimenter- administered IP injections. Although these findings were described as a possible cause of extinction (Sutton et al., 2003), this phenomenon has also been hypothesized to lead to incubation of craving (Conrad et al., 2008) and therefore represents a potential mechanism driving relapse to cocaine use.
The expression of AMPA receptors is dependent upon several scaffolding proteins. For
GluA2-containing AMPA receptors, the two main proteins involved are glutamate receptor interacting protein (GRIP) and protein interacting with C-kinase (PICK). While GRIP is responsible for anchoring GluA2-containing AMPA receptors at the synapse, PICK is involved in their removal (Lu & Ziff, 2005). These two proteins work to regulate the activity-dependent expression of GluA2-containing AMPA receptors and are affected by interactions with several signaling pathways. Testosterone represents one such interaction.
Testosterone has been shown to upregulate AMPA receptor subunits in the rat hypothalamus (Diano, Naftolin, & Horvath, 1997a). Androgens have also been shown to increase glutamate synthesis and GluA1 subunit expression within the hypothalamus of male hamsters (Fischer, Ricci, & Melloni, 2007). However, the functional effects of these cellular adaptations remain unclear. Higher peripheral testosterone levels are associated with decreased behavioral effects of cocaine: in rats, gonadectomy reduced cocaine- induced stereotypies (R. Chen, Osterhaus, McKerchar, & Fowler, 2003), and
54 administration of testosterone to gonadectomized males rescued behavioral sensitization to cocaine (Menéndez-Delmestre & Segarra, 2011). The effect of testosterone on cocaine related behaviors seems to be stronger in adolescent male rats than in adults (Minerly et al., 2010). However, the connection between testosterone’s influence on the glutamatergic system and cocaine addiction must be further studied.
Previous work in our lab demonstrated that disrupting glutamate receptor interacting protein 1 (GRIP1) in the medial PFC (mPFC) potentiates cocaine reinstatement in both male and female mice. In the current experiment, we aimed to determine if disrupting PICK1 in the mPFC would lead to blunted reinstatement of cocaine seeking. Under normal circumstances, GRIP1 and PICK1 act to balance the number of AMPA receptors available at the synapse. Following chronic cocaine use, changes in levels of AMPA receptors may represent a maladaptive form of learning (Jones & Bonci, 2005). Therefore, this study aimed to examine a potential mechanism to disrupt GluA2-containing AMPA receptor trafficking and alter cocaine seeking behavior in male and female mice.
Methods
Subjects
Mice homozygous for the Cre/lox-conditional allele of PICK1 (flox/flox) were bred on a
C57bl/6J background. Adult male and female mice (2–6-months old, age matched across group) were group housed until three days before the first day of food training, at which time they were single housed and began food deprivation. Food deprivation (receiving 1 standard rodent chow pellet/day) lasted until the third day of cocaine self-administration.
The single housing condition lasted throughout the duration of the experiment. All animals
55 were housed in a temperature- and humidity-controlled animal care facility with a 12-h light/dark cycle (lights on at 0700 hours). All procedures were approved by the Temple
University Animal Care and Use Committee. Cocaine was obtained from the National
Institute on Drug Abuse Drug Supply Program (Bethesda, MD) and dissolved in sterile
0.9% saline.
Prefrontal Microinjections and Adeno-Associated Virus Constructs
The adeno-associated virus (AAV) expressing Cre recombinase (AAV2/9.CMV.PI.CRE, titer 2.84 × 1013 vgc/μl) and the AAV expressing green fluorescent protein (eGFP)
(AAV2/9.CMV.eGFP, titer 3.74 × 1013 vgc/μl) were generated by Addgene. PICK1 flox/flox mice (6-8 weeks) were anesthetized with isoflurane and 0.4µl of the viral construct (Cre or GFP) was injected bilaterally into the prefrontal cortex through a 30- gauge needle at a rate of 0.1 μl/min. Stereotaxic coordinates for the prefrontal cortex are
(from Bregma) anterior-posterior 2.4, lateral +/- 0.3, dorso-ventral -2.3. Following recovery, mice remained in the home cage for 6 weeks prior to behavioral testing. The procedures involving the AAV viruses have all been approved by the Temple University
Institutional Biosafety committee. Knockout was confirmed via western blot, and animals removed from study if knockout was less than a 30% decrease from average GFP control levels.
Experiment I
Operant Food Training
Before catheterization, mice were trained to perform an operant response for sucrose pellets. The mice were placed in operant chambers (Med-Associates) and trained to spin a
56 wheel manipulandum to receive a sucrose pellet, with one-quarter spin measured as a single active response. Mice performed 5 days of FR1 responding followed by 5 days of FR5 responding. A compound cue stimulus consisting of a cue light above the active wheel, a
2900-Hz tone, and house light off was concurrent with each pellet administration, followed by an additional 8 s time-out when responding had no programmed consequences and the house light remained off. Mice were allowed to self-administer a maximum of 50 pellets per 60 min operant session. During the food training phase, mice were food restricted to
>90% of their free-feeding weight. Mice returned to ad libitum food access 3 days following the start of the cocaine self-administration phase.
Jugular Catheterization Surgery
Prior to surgery, mice were anesthetized with 80 mg/kg ketamine and 12 mg/kg xylazine.
An indwelling silastic catheter was placed into the right jugular vein and sutured in place.
The catheter was then threaded subcutaneously over the shoulder blade and was routed to a mesh backmount platform (Strategic Applications, Inc) that secured the placement.
Catheters were flushed daily with 0.1 ml of an antibiotic (Timentin, 0.93 mg/ml) dissolved in heparinized saline. The catheters were sealed with plastic obturators when not in use.
Cocaine Self-Administration
Mice were tested for cocaine self-administration behavior in 2-hour sessions in the same chamber used for sucrose pellet self-administration. During testing, responding on the wheel now delivered an intravenous cocaine injection (0.6 mg/kg/infusion), paired with the same compound cue, under the same schedule as the food training. After the cocaine self- administration phase, mice began extinction training, in which cocaine-seeking behavior
57 was extinguished by replacing the cocaine with 0.9% saline. During this time the light and tone cues paired with cocaine delivery were not present. Daily 2-h extinction sessions continued until animals met the extinction criterion of less than 25% of their self- administration responding (average of last 3 days). Twenty-four hours following meeting the extinction criterion, animals underwent a cue-induced reinstatement session. During the cue-induced reinstatement session, the light and tone cues were presented non- contingently for 20 seconds every 2 minutes during the first 10 minutes of the session.
After this time period, the cues were presented contingent with operant responding, just as was done during the cocaine self-administration phase. During the reinstatement session, animals received saline infusions following responses on the active wheel.
Experiment II
Subjects
As the goal of this experiment was to examine the relationship between mPFC PICK1
KO and androgens, only male mice were used in these studies.
Gonadectomy
Five weeks prior to food training, mice underwent orchiectomy or sham surgery. Mice were anesthetized with .80 mg/kg ketamine and 12 mg/kg xylazine via an IP injection. The abdominal area surrounding the incision site was soaked in a 70% ethanol solution before making an incision into the skin, which was pulled out of the way and held in place using a pair of hemostats. Next, a small incision into the peritoneum was made along the midline and a pair of blunt forceps was used to gently pull the fat pad surrounding the testes to the outside of the body. The fat pad was sutured below the caudal epididymis, and the region
58 above the suture (including the reproductive structure) was removed. This process was repeated bilaterally. The peritoneum and skin were then sutured, and a drop of betadine surgical scrub was applied to promote healing. Sham animals received incisions into the skin and peritoneum only. All animals were placed under a heat lamp until awake and mobile. For the week following gonadectomy or sham surgery, animals were monitored to ensure proper healing. Meloxicam (2.0 mg/kg, subcutaneous injection) was administered for the first three days following surgery, and triple antibiotic ointment applied to the surgical site until the skin had fully healed.
Operant Food Training, Jugular Catheterization, and Cocaine Self-Administration
See methods for Experiment I.
Western Blot
PICK1 levels in the prefrontal cortex were measured using a western blot, as described in
Briand et al., 2014. This method was used for animals in both experiment 1 and 2. Briefly, animals were decapitated, and the prefrontal cortex dissected using a brain block (Braintree
Scientific). Protein quantification was performed using a Pierce BCA Protein Assay Kit
(Thermo Scientific). Equal amounts of protein (30 µg) were loaded into each well of a Tris- glycine gel (Lonza) and transferred to nitrocellulose membranes (Immobilon). Membranes were blocked with Li-Cor blocking buffer and allowed to incubate in primary antibody solution (PICK1, 1:2000 (NeuroMab) and GAPDH, 1:5000 (Cell Signaling)) for 24 hours at 4°C. Membranes were then incubated with fluorescent secondary antibodies (1:20,000;
IR-dye 680 or IR-dye 800, Li-Cor) and imaged on an Odyssey fluorescent scanner (Li-
Cor). Western blots were normalized to glyceraldehyde 3-phosphate dehydrogenase
59 (GAPDH), and the percent knockout calculated as a fraction of the average of the PICK1 levels in GFP-infused mice.
Statistical analysis
In experiment 1, all self-administration experiments were analyzed with two-way
ANOVAs with viral injection and day as the independent variables and pellets/responses/infusions as the dependent variable. Experiment 2 was analyzed in the same manner, though groups were separated into “sham control” or “gonadectomy.”
Sidak’s post hoc comparisons were made when main effects or interactions were detected
(p < 0.05). The protein quantification, progressive ratio, extinction responding and days to criterion, and cue-induced reinstatement responding data were analyzed using unpaired t- tests with viral injection as the independent variable.
Results
Viral Mediated Deletion of PICK1 in the Medial Prefrontal Cortex
Six weeks following the injection of AAV-Cre into the mPFC of PICK1 floxed mice, we demonstrate a significant knockout in PICK1 levels in this region compared to AAV-GFP injected controls [Fig. 7; t(16)=2.40, p=0.03. The available antibodies only allow us to quantify the extent of the knockout using western blot techniques; this does not allow us to differentiate between GRIP1 knockout in prefrontal cell bodies versus GRIP1 knockout in terminals of neurons projecting to the PFC. Although AAV9 preferentially targets neurons, the lack of complete knockout may be due to glial expression of PICK1 (Lorgen, Egbenya,
Hammer, & Davanger, 2017).
60
Figure 7. Significant viral-mediated prefrontal PICK1 knockout. Male and female mice (N= 26) were injected bilaterally with 0.4 µg of GFP or AAV-Cre recombinase into the medial prefrontal cortex (A). Western blots showed significant prefrontal PICK1 knockdown [B, C; t(16) = 2.40, *p = 0.03].
61 Prefrontal PICK1 Knockout Does Not Affect Food Training or Cocaine Self-
Administration in Intact Mice
Six weeks following the viral injections, GFP controls and Cre PICK1 knockout mice were given 10 days of operant training to acquire food self-administration. Both groups acquired the operant response for food, exhibiting a significant increase in the number of pellets earned over the 10 sessions [males: F(9,441)=31.6, p<0.01; females: F(9,450)=23.6, p<0.01]. However, there was no effect of PICK1 deletion on the number of pellets received
[males: F(1,49)=2.84, p=0.09; females: F(1,50)=0.11, p=0.74], number of active responses
[males: F(1,47) = 0.38, p = 0.54; females: F(1,50)=0.69, p=0.41], or percent active responding [males: F(1,49)=2.39, p = 0.13; females: F(1,50)<0.01, p=0.99; Fig. 8]. During the cocaine self-administration phase, there were no differences between GFP controls and
Cre PICK1 knockouts in number of infusions received [males: F(1, 39) = 0.08, p = 0.77; females: F(1,33)=0.22, p=0.64] or the number of active responses [males: F(1, 39) =0.33, p = 0.57; females: F(1,34)=0.95, p=0.34; Fig. 9].
Prefrontal PICK1 Knockout Has a Sex-Specific Effect on Cue-Induced Reinstatement in
Intact Mice
Following the cocaine self-administration phase, a subset of mice began extinction training.
No differences were seen between the GFP controls and PICK1 knockouts in the days to reach the extinction criteria [F(1, 52) = 1.29, p = 0.26], nor was there an effect of sex
[F(1,52) = 1.20, p = 0.28; Fig 4a]. However, during cue-induced reinstatement of cocaine seeking we found a sex specific effect of PICK1 knockout. In male mice, Cre PICK1 knockouts exhibited a significant decrease in cocaine seeking compared to GFP controls
62
Figure 8. PICK1 knockout in the mPFC does not alter operant learning during food self-administration in male or female mice. There were no significant effects of prefrontal PICK1 KO among males or females during food self-administration, as measured by the number of sucrose pellets received (A, B), the number of active responses (C, D), and the percent active responses in comparison to all responses (E, F) during a one-hour testing session (N= 50-53).
63
Figure 9. PICK1 knockout in the mPFC does not alter cocaine self-administration on a fixed ratio schedule of reinforcement. There were no significant effects of prefrontal PICK1 KO among males or females during the self-administration of cocaine, as measured by the number of cocaine infusions received during a two-hour period (A, B) or the number of active responses for cocaine during that period (C, d; N= 32-48).
64 [Interaction, F(1,20)=4.82, p=.04, Sidak post-hoc GFP control reinstatement vs. Cre
PICK1 KO reinstatement, p=.0085; Fig. 10b]. In contrast, female Cre PICK1 knockouts exhibit an increase in cue-induced cocaine seeking during the reinstatement test
[Interaction, F(1,19)=5.01, p=.038, Sidak post-hock GFP control reinstatement vs. Cre
PICK1 KO reinstatement, p=.0069; Fig. 10c]
Gonadectomy Reversed the Effect of Prefrontal PICK1 Knockout on Cue-Induced
Reinstatement of Cocaine Seeking in Males
Male gonadectomy did not alter the total number of food pellets earned over the 10 food self-administration sessions [effect of GDX: F(1,40)=0.003, p=0.96; effect of virus:
F(1,40)=0.0007, p=.98; Fig 11a]. Similarly, there was no effect of gonadectomy or PICK1 knockout in the total number cocaine infusions received over the 10 days of cocaine self- administration [effect of GDX: F(1,37)=0.11, p=.74; effect of virus: F(1,37)=0.12, p=73;
Fig. 11b]. Neither prefrontal PICK1 KO or gonadectomy influenced the number of days required to meet extinction criteria among gonadectomized males [effect of GDX:
F(1,26)=0.48, p=.49; effect of virus: F(1,26)=0.71, p=0.41 Fig. 12a]. In contrast, there was a significant interaction between prefrontal PICK1 KO and gonadectomy during cue- reinstatement (F(1,26)=4.44, p=0.45; Fig. 12b]
65
Figure 10. Prefrontal PICK1 knockout leads to sex-specific effects on cue-induced reinstatement of cocaine seeking. Male and female mice showed no effect of prefrontal PICK1 KO on the time to reach extinction criterion (A). However, PICK1 knockout in the mPFC of male mice led to a significant decrease in active responses during cue-induced reinstatement [B, F(1,20)= 4.44, *p<0.01]. In contrast, PICK1 knockout in the mPFC of female mice led to an increase in active responses during cue-induced reinstatement [C, interaction: F(1,19)= 4.87, *p<0.01, N= 25-31].
66
Figure 11. Gonadectomy did not alter the effect of mPFC PICK1 knockout on food or cocaine self- administration. Among gonadectomized and intact male mice, there were no differences during food self-administration for number of pellets received during a one-hour period (A). During cocaine self- administration, there were no differences in the number of infusions received during a two-hour period (B) (N= 8-12).
Figure 12. Gonadectomy eliminates the effect of prefrontal PICK1 knockout on cue-induced reinstatement. There was no effect of PICK1 KO or gonadectomy on active responses performed on the last day of extinction [A; F(1,27)= 3.48, p=0.07]. There was a significant interaction between gonadectomy and prefrontal PICK1 KO on responses during the cue-reinstatement session [B; F(1,27)= 4.31, *p<0.05] (N= 6-9).
67 Discussion
Overall, we find that the AMPA receptor scaffolding protein, PICK1, within the PFC plays a sex-specific role in mediating cocaine seeking. Our data demonstrate that knockout of prefrontal PICK1 decreases cue-induced cocaine seeking in males while not affecting food or cocaine self-administration. In contrast, prefrontal PICK1 knockout increases cue- induced cocaine seeking in females. The effects of prefrontal PICK1 knockout in males are mediated in part by gonadal hormones, as gonadectomizing males made them respond more like females to the PICK1 manipulation.
PICK1 Knockout in the Prefrontal Cortex Dampens Cocaine Seeking in Males
The scaffolding proteins GRIP1 and PICK1 bind to the GluA2 subunit of the AMPA receptor (Lu & Ziff, 2005). GRIP1 maintains the GluA2-containing receptor within the synapse, whereas PICK1 internalizes the receptor after phosphorylation (Lu & Ziff, 2005).
Together, these proteins regulate the GluA2-containing AMPA receptors available at the synapse. Previous work has shown that knockout of prefrontal GRIP1 potentiates cocaine seeking (Wickens, Deutschmann, McGrath, Parikh, & Briand, 2019). Therefore, we hypothesized that disrupting PICK1 function in this region would have the opposite effect.
This is exactly what we found in the male mice. As prefrontal GRIP1 knockout leads to an increase in AMPA-mediated glutamate transmission (Wickens et al., 2019), we believe our
PICK1 manipulation leads to a decrease in AMPA-mediated transmission due to a decrease in the contribution of GluA1-lacking AMPA receptors. This would lead to a net decrease in
AMPA transmission. This is consistent with work in male rats demonstrating that inactivation of the mPFC disrupts reinstatement of cocaine seeking (Martin-Garcia et al.,
68 2014; McLaughlin & See, 2003; Palombo et al., 2017; Rocha & Kalivas, 2010; Zavala et al., 2003).
PICK1 Knockout in the Prefrontal Cortex Leads to a Sex-Specific Effect in Cocaine
Seeking
In contrast to what we found in male mice, female mice exhibited an increase in cue- induced reinstatement following prefrontal deletion of PICK1. These findings suggest that
PICK1 plays a sex-specific role in glutamate receptor trafficking. As we previously demonstrated that knocking out GRIP1 from the PFC leads to similar effects on cocaine seeking in male and female mice (Wickens et al., 2019), this would suggest something unique to PICK1 rather than an overall sex difference in the role of PFC glutamate trafficking in cocaine seeking. The GluA2 subunit is only one of over forty proteins that bind to PICK1 (Xu & Xia, 2006). Therefore, PICK1 may exhibit a different binding profile in the PFC of male and female mice.
One such protein that PICK1 binds to is the dopamine transporter (DAT) (Xu & Xia, 2006), the primary target of cocaine. Colocalization of PICK1 and DAT increases dopamine uptake in vitro (Torres et al., 2001), thereby attenuating the effects of dopamine at the synapse. If PICK1 binds with greater affinity to the DAT in female mice compared to male mice, then disrupting PICK1 function in females could lead to alterations in dopamine transmission. While male PICK1 KO mice do not show evidence for alterations in surface expression of DAT (Jensen et al., 2018), these studies have not been done in female PICK1 knockout mice. As dopamine antagonist administration into the PFC dampens cue-induced cocaine seeking on a second order schedule (Di Pietro, Mashhoon, Heaney, Yager, &
69 Kantak, 2008), it is plausible that PICK1 increases cocaine seeking via dopaminergic mechanisms.
Gonadectomy Eliminates PICK1 Knockout Effect on Cocaine Cue-Induced
Reinstatement in Males
Although studies on the effects of androgens on impulsive behaviors shows an association with drug addiction (Fattore & Melis, 2016), studies involving rodent self-administration are severely lacking. Our data show that males receiving sham surgery looked similar to intact animals, exhibit lower levels of cue-induced reinstatement responding after prefrontal PICK1 KO. After removal of circulating gonadal hormones, males with prefrontal PICK1 KO not only did not exhibit this decrease but rather showed a nonsignificant trend towards increased responding during cue-induced reinstatement. This indicates that the effect of PICK1 knockout was dependent upon gonadal hormones. As gonadectomy did not alter cue-induced reinstatement in GFP control mice, the effects in the PICK1 knockout mice were not due to a generalized effect of gonadectomy on cocaine seeking.
Androgen and glutamate receptor colocalization has previously been described in several brain regions (Diano, Naftolin, & Horvath, 1997b), though the impact on substance use disorder has not been explored. Gonadectomy of male rats does not change the levels of the GluA2 subunit in the caudate putamen or thalamus (D'Souza, Harlan, & Garcia, 2003).
Likewise, our results did not show reinstatement effects after gonadectomy alone, suggesting that circulating androgens do not directly impact the expression of the GluA2 subunit. We propose that the loss of PICK1 leads to an increase in the ratio of GluA2-
70 containing to GluA2-lacking AMPA receptors, thereby dampening glutamate transmission.
Androgen receptor antagonism can lead to an increase in glutamate-evoked intracellular calcium (Foradori, Werner, Sandau, Clapp, & Handa, 2007). Therefore, it is possible that gonadectomy is potentiating the effects of the calcium-permeable GluA2-lacking AMPA receptors thereby preventing this decrease in net glutamate transmission.
Prefrontal PICK1 Knockout Does Not Play a Role in Fixed Ratio Measures of Natural
Reward or Cocaine Self-Administration
We did not find any effect of prefrontal PICK1 KO on food or cocaine taking. This is consistent with previous work in our lab showing that prefrontal GRIP1 knockout had no effect on fixed ratio measures of reward administration (Wickens et al., 2019), and with existing literature showing no effect of PICK1 inhibition on natural reward (McFarland et al., 2003; Turner et al., 2020).
Of note, we did not observe sex-specific differences in the acquisition of food or cocaine taking, though it has been well established that females acquire drug seeking behavior at a faster rate than males (Becker & Hu, 2008; M. E. Carroll et al., 2004; Jackson, Robinson,
& Becker, 2006). However, this effect is not seen in every cohort of animals due to differences in cocaine dose, time of day, and reinforcement schedule (Baird & Gauvin,
2000; Caine et al., 2004). It is possible that the parameters of our self-administration setup minimize sex differences during the acquisition phase, or that females must begin self- administration at a specific point in the estrus cycle (likely estrus, see (Lynch, Arizzi, &
Carroll, 2000)) to show robust differences. Additionally, gonadectomy did not alter food
71 training of cocaine self-administration in male animals. Testosterone in male rats is required for cocaine sensitization (Menéndez- Delmestre & Segarra, 2011), particularly during adolescence (Parylak, Caster, Walker, & Kuhn, 2008). However, operant learning during adulthood is primarily dependent upon changes during adolescence (Dalla & Shors,
2009; Parylak et al., 2008). As all the animals described in the current experiment were adults, no deficits in operant learning were expected.
Conclusions
In the current study, we have shown that conditional deletion of PICK1 in the mPFC leads to sex-specific effects during cue-induced reinstatement to cocaine. Male mice showed decreased reinstatement while females increased their reinstatement responding. The sex- specific effect was not fully explained by circulating testosterone in the males. Further study will be needed to parse apart effects in the females and to develop an understanding of interactions between gonadal hormones and prefrontal addiction circuitry. In the future, this could represent a novel avenue for treatment of psychostimulant addiction. However, currently these are the first results to show that PICK1 may be playing a different role in males and females, which highlights the importance of considering sex differences even in basic biological processes.
72 CONCLUSION
In order to understand the dramatic behavioral effects caused by substance use disorder, we must first understand how drugs impact the brain. The neurotransmitter glutamate is vital to the maintenance of addiction-like behaviors (Kalivas & Volkow, 2005).
Glutamatergic dysregulation is a component in a wide variety of psychiatric disorders, many of which exhibit a bias toward one sex. The bias may present as an increased frequency or enhanced severity of the disease. The introduction to this dissertation provides an overview on what is known about the role of glutamate in Alzheimer’s disease and major depressive disorder (which present a female bias in frequency), as well as schizophrenia, autism spectrum disorder, and attention deficit hyperactivity disorder (which present a male bias in frequency). In chapters two and three, cocaine addiction is the main focus.
The first set of experiments was designed to determine the role of GRIP1 in the mPFC on addiction-like behaviors in mice. The mPFC was chosen as a region of interest due to its high amount of glutamatergic activity. In addition to receiving a number of glutamatergic inputs, this region sends glutamatergic efferents to a number of areas heavily involved in addiction, such as the nucleus accumbens (Russo & Nestler, 2013). The scaffolding protein
GRIP1 plays a key role in regulating the strength of these glutamatergic projections through its binding to the GluA2 subunit of the AMPA receptor. When bound to GRIP1, the GluA2 containing AMPA receptor remains in the synaptic membrane. We used a conditional knockout model to remove GRIP1 from the mPFC of male and female mice. Without
GRIP1, the GluA2-containing AMPA receptors cannot remain in the synaptic membrane and are internalized – this allows calcium-permeable, GluA2-lacking AMPA receptors to
73 exist in the membrane in their place. The end result of this process is a stronger glutamatergic signal in the postsynaptic neuron. This manipulation led to increased active responses during progressive ratio responding for cocaine and increased cue-induced reinstatement in both male and female mice. Therefore, prefrontal GRIP1 acts as a deterrent against uncontrolled motivation for cocaine use and against relapse to use in both males and females. This is likely due to its role in maintaining an appropriate amount of glutamatergic signaling through its control over GluA2-containing AMPA receptor expression. Support for this theory comes from our electrophysiological data showing increased amplitude of sEPSCs in the mPFC after prefrontal GRIP1 knockout.
The next experiments were designed to build off the results of knocking out GRIP1. Still using the mPFC, we performed a conditional knockout of PICK1 instead. Whereas GRIP1 maintains GluA2-containing AMPA receptors in the synaptic membrane, PICK1 internalizes them. Our deletion of PICK1 allowed GluA2-containing receptors to remain at the synapse. This manipulation resulted in an unexpected sex difference in cue-induced reinstatement. The males with PICK1 KO attenuated their reinstatement responding, consistent with the proposed mechanism of the GRIP1 KO results. However, the females with PICK1 KO still showed enhanced cue-induced reinstatement, despite the opposing effects of PICK1 and GRIP1. A sex-specific effect of PICK1 has not previously been described in the literature, yet these results suggest a baseline sex difference in PICK1 signaling pathways.
Given the sex difference we observed after prefrontal PICK1 KO, we next set out to determine if the sex specific effects were influenced by gonadal hormones. We found that
74 following gonadectomy, male mice with a prefrontal PICK1 KO no longer demonstrated a decrease in cue-induced reinstatement of cocaine seeking. This suggests that androgens influence PICK1 dependent processes or are involved in the compensatory changes following PICK1 deletion. Androgen receptors colocalize with glutamate receptors (Diano et al., 1997b) and gonadectomy can lead to alterations in glutamate transmission (Nestor et al., 2016; Pouliot, Handa, & Beck, 1996). However, it is not clear what role androgens might play in glutamate receptor trafficking. We propose a model in which removal of
PICK1 prevents the insertion of GluA2-lacking AMPA receptors because the GluA2- containing AMPA receptors are not cycled out of the membrane. This leads to a net decrease in glutamatergic tone because GluA2-containing AMPA receptors are not calcium permeable. Decreases in intracellular calcium are observed in hippocampal cell culture after glutamate is administered with androgen receptor antagonism (Zup, Edwards, &
McCarthy, 2014). However, this is due to changes in intracellular calcium reserves
(Foradori et al., 2007; Zup et al., 2014) and not calcium permeability of membrane bound receptors. If the same processes are engaged in vivo, males receiving both gonadectomy and PICK1 deletion would display attenuated levels of intracellular calcium availability in the postsynaptic glutamatergic cells. The prefrontal PICK1 deletion would decrease calcium from entering the cell via AMPA receptors while the gonadectomy would decrease intracellular calcium from being released due to androgen receptor inactivation. This is not consistent with our model, as the decrease in intracellular calcium after both gonadectomy and PICK1 deletion would be expected to attenuate cue-induced reinstatement yet we observed an increase in reinstatement after gonadectomy with PICK1 deletion. The role of
75 androgens in modulating PICK1 processes likely involves additional signaling pathways.
Androgens are known to play a role in glutamate-mediated dopamine release in the PFC
(Aubele & Kritzer, 2012; van Haaren, van Hest, & Heinsbroek, 1990). Intact male rats display attenuated dopamine release in the presence of an AMPA antagonist (Aubele &
Kritzer, 2012). After gonadectomy, prefrontal dopamine release shows no change from baseline in response to an AMPA antagonist (Aubele & Kritzer, 2012), suggesting that androgens may modulate prefrontal dopamine release after glutamatergic insult. The effect of AMPA receptor antagonism in males was negated by gonadectomy (Aubele & Kritzer,
2012), just as the PICK1 male mice did not show a main effect of reinstatement after gonadectomy. Together, these findings support the idea that androgens are required for compensatory changes in response to dysregulated AMPA signaling. The importance of androgens in mediating plasticity after AMPA receptor insult is observed in males, but the role of gonadal hormones in females remains unexplored. Estradiol has been hypothesized to increase susceptibility to addiction-like behaviors (Becker & Hu, 2008; Ramôa, Doyle,
Naim, & Lynch, 2013). More work is necessary to determine if this susceptibility is enough to explain the sex differences in prefrontal PICK1 related pathways.
The existence of a sex difference after prefrontal PICK1 deletion implies that the process of GluA2-containing AMPA receptor trafficking is different in females. Additional pathways or mechanisms are involved, and these interactions are poorly understood.
Females may use different compensatory mechanisms after insult to the prefrontal glutamatergic system, such as a reliance on non-activity dependent trafficking proteins, increased activity in downstream regions of the reward pathway, or enhanced glutamate
76 release from the damaged area. Examining these possibilities presents the opportunity to create a more complete model of the development of substance use disorder.
Women – and female rodents – experience a more rapid development of substance use disorder and poorer treatment outcomes (Becker & Hu, 2008; Hu & Becker, 2003), yet there is no one unifying theory that explains how this sex difference might arise. Our data suggest that mechanisms underlying AMPA receptor trafficking may be sex-specific.
One way this might be possible is if females rely to a greater extent on basal trafficking of AMPA receptors while males rely on activity dependent trafficking in response to drugs of abuse. In this case, females may experience enhanced glutamate release in the event of damage to proteins like PICK1. They would then display greater addiction-like behavior because the GluA2-containing AMPA receptor can still be replaced by a GluA2-lacking
AMPA receptor through non-PICK1 dependent mechanisms. It is also possible that female gonadal hormones themselves enhance activity in brain regions associated with addiction, however, this may be specific to the phase of addiction (Larson, Roth, Anker, & Carroll,
2005).
In conclusion, prefrontal glutamate receptor trafficking is involved in reinstatement to cocaine but not the initial acquisition of cocaine self-administration. This is consistent with literature describing the role of prefrontal glutamate release in reinstatement (McFarland et al., 2003; Park et al., 2002; Van den Oever, Spijker, Smit, & De Vries, 2010). GRIP1 and
PICK1 show some overlapping effects on cue-induced reinstatement to cocaine after their deletion. PICK1, but not GRIP1, displays sex specific effects on reinstatement. Prefrontal
77 knockout of PICK1 enhances reinstatement in females while attenuating reinstatement in males, but this effect was eliminated by gonadectomy. Therefore, circulating androgens are thought to play a role in PFC-dependent regulation of drug seeking. These are the first results describing sex differences in PICK1 and its interaction with circulating androgens.
There is still much to learn about the biological mechanisms underlying substance use disorder, but this work provides important information which should be considered when designing further studies.
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