Chemogenetic Inhibition of Infralimbic Prefrontal Cortex GABA-ergic Parvalbumin-positive Interneurons Attenuates Chronic Stress Adaptions in Male Mice

Hypofunction of the prefrontal cortex (PFC) contributes to stress-related neuropsychiatric illnesses. Mechanisms leading to prefrontal hypoactivity remain to be determined. Prior evidence suggests that enhanced activity of parvalbumin (PV) expressing GABAergic interneurons (INs) play a role in chronic stress related pathologies. In this study, the role of PFC PV INs in stress related phenotypes were explored using Cre inducible inhibitory DREADDs (Designer Receptors Exclusively Activated by Designer Drugs). Mice were first tested in the tail suspension test (TST) to determine the effects of PV IN inhibition during acute stress. Following this, the long term impact of PV IN inhibition during chronic variable stress (CVS) was tested in the forced swim test (FST). Acute PV IN inhibition reduced active (struggling) and increased passive coping behaviors (immobility) in the TST. In contrast, inhibition of PV INs during CVS increased active and reduced passive coping behaviors in the FST. Moreover, chronic inhibition of PV INs attenuated CVS-induced changes in Fos in the prelimbic cortex, basolateral amygdala and ventrolateral periaqueductal gray and also prevented adrenal hypertrophy associated with chronic stress. Our results suggest differential roles of PV INs to acute vs chronic stress, indicative of distinct biological mechanisms underlying acute vs. chronic stress responses. Our results also indicate a role for PV INs in driving chronic stress adaptation and support growing literature evidence suggesting cortical GABAergic interneurons as a therapeutic target in stress related diseases. SIGNIFICANCE Stress related diseases are associated with prefrontal hypoactivity, the mechanism of which is currently not known. In this study we showed that by inhibiting prefrontal Parvalbumin interneurons using DREADDs, we can attenuate some of chronic stress related phenotypes. Additionally, we also showed that modulation of PV IN activity during acute vs chronic stress had opposing effects on stress coping strategies, suggesting different underlying mechanisms behind acute vs chronic stress paradigms. Our findings indicate that GABA-ergic Parvalbumin interneurons may be involved in driving stress related phenotypes and thereby an important target for treatment of stress-related illnesses. Our data suggests that reducing PV IN activity to promote prefrontal output may be an effective treatment strategy for stress related disorders.

Various clinical and preclinical studies implicate altered GABAergic circuitry and prefrontal hypofunction in the generation of depression in humans as well as depression-related behaviors in rodent chronic stress models (Duman, 2014;Luscher et al., 2011;McKlveen et al., 2016;Musazzi et al., 2015;Veeraiah et al., 2014). Recent functional and electrophysiological studies indicate increased infralimbic (IL) PFC ( rodent homolog of the human ventromedial prefrontal cortex) GABAergic transmission (e.g., increased inhibitory synaptic drive and increased expression of GABA-ergic marker) following chronic variable stress (CVS), suggesting that enhanced interneuron (IN) activity may be involved in disruption of prefrontal cortical signaling (McKlveen et al., 2016;Shepard et al., 2016).

GABA-ergic parvalbumin interneurons (PV INs) synapse onto cell bodies of PFC pyramidal
neurons and exert strong control over medial PFC (mPFC) output, maintaining appropriate excitatory/inhibitory (E/I) balance (Cardin et al., 2009;Courtin et al., 2014;Tremblay et al., 2016) and coordinating oscillatory activity (gamma oscillation) required for efficient PFC signaling. Consequently PV INs are well-positioned to play an important role in stress mediated prefrontal dysfunction (DeFelipe et al., 2013;Ferguson and Gao, 2018;Rymar and Sadikot, 2007;Sherwood et al., 2007). Increases in expression of PV and enhancement of glutamatergic transmission onto PV-INs following chronic stress, are associated with prefrontal hypofunction, anxiogenesis and impaired coping behaviors in FST (Page et al., 2018;Shepard et al., 2016;Shepard and Coutellier, 2017). Reduction in PV expression in PFC is also associated with antidepressant efficacy (Ohira et al., 2013;Page and Coutellier, 2019;Zhou et al., 2015). In contrast to chronic, acute inhibition of PV INs in the PFC has opposing effects, resulting in increase in passive coping behavior such as learned helplessness in mice (Perova et al., 2015). This suggests differential role of PV INs in response to acute vs chronic stress, indicative of distinct brain circuitry being involved in modulating acute vs chronic stressmediated phenotypes.
This study was designed to specifically investigate the role of IL PV INs in driving physiological and behavioral manifestations of acute and chronic stress-related phenotypes.
We employed a chemogenetic strategy using DREADDs (Designer receptors Exclusively Activated by Designer Drugs) to specifically inhibit PV INs in the IL mPFC during exposure to acute stress and throughout exposure to CVS. Our results indicate that acute inhibition of PV INs increases passive coping behavior in tail suspension test (TST). In contrast chronic inhibition of IL PV INs reduces passive coping behavior in the forced swim test (FST).

Additionally, PV IN inhibition blocks CVS induced adrenal hypertrophy and reduces stress-
induced Fos expression in stress-related regions downstream of the IL. These data suggest that IL PV INs play a role in driving physiological and neuronal adaptations associated with both acute and chronic stress related phenotypes.

Mice
Male breeders from BL6 PV-Cre knock-in homozygous mice line (Pvalb tm1(cre)Arbr , JAX stock# 017320, Jackson Laboratories Maine, USA) were bred with WT C57BL/6J females ( JAX stock# 000664) to generate an in-house colony of heterozygous PV-Cre C57BL/6J at the University of Cincinnati animal housing facility. Mice were maintained under standard conditions (12/12h light/dark cycle, 22 +/-1°C, food and water ad libitum; 4 mice per cage) in accordance with the [Author University] Institutional Animal Care and Use Committee, which specifically approved all acute and chronic stress regimens employed in this proposal. All protocols conformed to the Society's Policies on the Use of Animals in Neuroscience Research. All experiments were performed on adult male mice (~7.5 months of age at surgery).

Stereotaxic Viral Vector Injection with AAV vectors
PV-Cre mice were anaesthetized with isoflourane, scalp shaved and placed in the stereotax frame. The incision site was disinfected using chlorohexidine and 70% ethanol. An incision at the midline was made using a single-edged blade. Cre-dependent AAV2 virus vectors AAV2hsyn-DIO-hM4D(Gi)-mCherry (Gift from Bryan Roth; Addgene viral prep # 44362-AAV2) and AAV2-hsyn-DIO-mcherry (Gift from Bryan Roth; Addgene viral prep # 50459-AAV2) were injected bilaterally at a volume of 300nL (~10 12 genome copies/ml) into the IL mPFC. The coordinates used were as follows: (anterior/posterior range defined as +1.8 mm anterior to bregma, medial-lateral range defined as +/-0.2 mm lateral to the midsagittal suture; dorsalventral range defined as −2.9 mm ventral to skull (Paxinos and Watson, 2007). Viruses were infused using a 2ul hamilton syringe at a rate of 60nl/min for 5 minutes. Following infusion, the injector was kept at the site for 8 minutes to allow for the virus to diffuse. The injection site was covered with gel foam and incision site sutured. 2.5mg/kg Meloxicam was administered for 3 days following surgery. Behavioral studies and stress protocols were initiated 3 weeks post injection to allow sufficient time for viral expression. Diagrammatic representation of experimental timeline is outlined in Figure.1

Drug administration
Clozapine N-oxide (CNO, NIMH Chemical synthesis and Drug Supply Program) was used as the DREADD actuator to activate the inhibitory DREADD. CNO was dissolved in 5% dimethyl sulfoxide (Sigma) and then diluted with 0.9% saline and administered intraperitoneally at a dose of 1mg/kg twice a day, 30 minutes before start of each stressor. All animals received chronic injection of CNO for 14 days. A maximum time period of 6 hours was given between stressors, to allow sufficient time for CNO to be cleared from the body (Jendryka et al., 2019;MacLaren et al., 2016). Each individual stress session did not last for more than 2 hours in order to ensure that CNO was on-board throughout the stressor. To control for a potential effect of chronic exposure to CNO on our behavioral assays, a separate group of C57BL/6J mice (n=4) was chronically injected with saline for 14-days (no CVS) and then behaviorally tested in the FST in parallel with the rest of the animals (Figure 4-2).

Tail suspension test (TST)
The TST (Can et al., 2012) was used as the first stressor in the CVS group to observe acute effects of inhibiting PV INs on passive coping behavior. The TST procedure was conducted as previously described (Can et al., 2012). Mice were suspended 55 cm above ground using a 17cm long tape that was attached to a suspension bar, for a total time period of 6 minutes.
Sessions were video-recorded from the side to allow full body visualization of mice behaviorsactive coping (struggling) behavior, which comprised of strong shaking of the body and movement of all 4 limbs, and passive coping (immobility) behavior which comprised of not making any active limb movements. Latency to reach immobility was also measured. Behaviors were quantified by an experimenter blinded to the group assignments using behavioral scoring software Kinoscope 3.0.4. Behaviors during the 6 minute block were reported.

Forced swim test (FST)
Following completion of the CVS procedure, FST was conducted as previously described (Ghosal et al., 2017;Wohleb et al., 2016). Mice were placed in a clear cylinder ( 2L glass beaker) filled with water (24±1°C, 18 cm depth) for a period of 10 minutes. Sessions were videorecorded from the side to allow full body visualization for total immobility duration, which comprised of not making any active movements or floating in the water without struggling, and total swimming duration, which comprised of moving limbs in an active manner and making circular movements around the cylinder. Behaviors were quantified by an experimenter blinded to the group assignments using behavioral scoring software Kinoscope 3.0.4. Behaviors during the 10 minute block were reported.

Euthanasia and tissue collection
Mice were euthanized with an overdose of sodium pentobarbital 120 minutes after FST, and transcardially perfused with 0.9% saline followed 4% paraformaldehyde in 0.01 M phosphate buffer (PBS), pH 7.4. Brains were removed and post-fixed in 4% paraformaldehyde at 4°C for 24 hours, then transferred to 30% sucrose in 0.01MPBS at 4°C until processed. Thymi and adrenal glands were collected, cleaned and weighed from all animals.
Immunohistochemistry was performed at room temperature and 0.01M PBS was used to rinse brain slices before each treatment described below.
Neuronal activation was measured using Fos as a marker. Free floating sections were incubated in 1% Sodium Borohydride for 20 minutes and then in 3% hydrogen peroxide in PBS for 20 minutes. After that, slices were incubated in blocking solution (NGS, 0.3% TritonX-100, 0.2% bovine serum albumin (BSA) in 0.01M PBS) for 1 hour. Sections were then incubated with Fos rabbit polyclonal antibody (1:200, Santa Cruz, sc-52) in blocking solution overnight and was followed by incubation in secondary antibody (biotinylated goat anti-rabbit, (1:400; Vector Laboratories, BA1000) in blocking solution for 1 hour the next day. Sections were then treated with avidin-biotin horseradish peroxidase complex (1:800 in 0.01M PBS; Vector Laboratories, PK6100) for 1 hour and then developed with an 8 minute incubation in DAB-Nickel solution: 10mg 3,3′-diaminobenzidine (DAB) tablet (Sigma, DF905), 0.5 ml of a 2% aqueous nickel sulfate solution, 20ul of 30% hydorgen peroxide in 50ml of 0.01M PBS. Sections were mounted on superfrost slides (Fisherbrand, Fisher), allowed to dry, dehydrated with xylene, and then coverslipped with DPX mounting medium (Sigma).
Images were acquired using microscope Carl Zeiss Imager Z1 at a 5X objective. For analysis, we counted minimum of 3 bilateral sections per brain region/animal covering the prelimbic cortex (PrL) (Bregma 2.80mm to 1.98mm), basolateral amygdala (BLA) (Bregma -1.06 to -1.58) and ventrolateral periaqueductal gray (vlPAG) ( Bregma -4.16 to -4.36) as defined in the Franklin and Paxinos mouse brain atlas (3 rd Edition) (Franklin and Paxinos, 2008). The number of Fos positive nuclei was counted using a semi-automated analysis macro in the Image J software package (National Institutes of Health, Bethesda, MD). The macro was generated using the "Analyze Particle" tool, with a defined common level of background intensity, nuclei circularity and size (previously validated manually). The relative density of the population of immunopositive cells was calculated by dividing the number of Fos positive cells by the respective brain area.

Statistical Analysis
The experiment was setup as a 2X2 study design, with stress (CVS or No CVS) and DREADD (hM4Di or control) as factors with a sample size of n=10 per group (See Figure 1 for experimental design and timeline). Statistical analyses for FST and Fos protein quantification were performed using a two-way ANOVA with stress (No CVS, CVS) and DREADD (Control, hM4Di) as main factors. TST data was analyzed using Student's t-test. FST measurements over time were done using two-way repeated measure ANOVA with stress (No CVS, CVS) and DREADD (Control, hM4Di) as main factors analyzed over time. Tukey's post-hoc test was performed in cases with significant interaction between factors. Because specific hypotheses were formed a priori on the effects of CVS within groups, planned comparisons using Fisher's least significant difference (LSD) were performed in cases with no significant interaction effect.
Data were analyzed by STATISTICA 7.0 (Statsoft, Inc.,Tulsa, USA) and Graph Pad Prism 8.1.2 (GraphPad Software, La Jolla California USA). Outliers were detected using the Grubbs' test (GraphPad Software) and removed from analysis. After exclusion of outliers, all data was assessed for normal distribution (Shapiro-Wilk) and appropriate parametric and /or nonparametric tests used. Data are presented as mean ± SEM with statistical significance set at P ≤ 0.05. See Table 1 for details regarding data structure and type of test used. Superscript letters listed with p-values correspond to the statistical tests shown in Table 1.

Selective Targeting of PV INs achieved using DREADDs
The AAV virus constructs used in this experiment are shown in Figure 2A. Following Cre recombination, the viral construct expresses inhibitory DREADD sequence hM4Di along with a fluorescent report (mCherry), allowing visualization of cells undergoing recombination. Control virus was a Cre-inducible mCherry lacking the DREADD construct. Figure 2B depicts stereotaxic injection site in the IL and also demonstrates that the expression of the DREADD was restricted to the IL. The Cre dependency and specific targeting of PV INs was verified by immunostaining. Cell-type specificity was conferred by expression of Cre recombinase specifically in PV INs (the hSyn promoter drives expression in all transformed cells). Figure 2C, demonstrates that hM4Di-mCherry expression was restricted to PV INs, confirmed by colocalization of red mCherry with green PV immunostaining.

Acute inhibition of PV INs: TST
The behavioral consequences of acute inhibition of PV INs in the IL were tested by using the TST as the first stressor in the CVS paradigm ( Figure 3). Animals were dosed with 1mg/kg CNO 30 minutes prior to the start of TST. We observed significant changes in coping behavior following acute inhibition of PV INs in the IL. Compared with control mice, mice expressing hM4Di showed significant reduction in struggling duration (t= 2.7, df=18, p=0.02 a ; Figure 3A), significant increase in immobility duration (t=2.9, df=18, p=0.009 b ; Figure. 3B) and decreased latency to immobility (t=2.5, df=17, p=0.02 c ; Figure. 3C) respectively.

Chronic Inhibition of PV INs during CVS: FST
Animals were tested for coping behaviors in the FST the day following cessation of chronic stress. Our purpose was to test whether inhibition of PV INs during the chronic stress regimen, could block the aggregate effect of repeated stress on subsequent coping behavior (FST used as a novel stressor) and brain activation patterns (Fos expression). All subjects received the same viral and CNO treatments, and because CNO was only administered during CVS, any phenotypes observed during FST were interpreted as reflecting an impact of PV IN manipulation on stress coping behavior. We observed significant differences in stress coping behaviors following chronic inhibition of PV INs during CVS compared with the CVS control group.
Specifically, chronic PV IN inhibition during stress increased active coping (swimming) and reduced passive coping (immobility) behaviors in the FST. Two-way ANOVA of total swimming duration showed a significant main effect of stress [F(1,35) =11.7; p=0.002 d ; Figure 4A

Chronic Inhibition of PV INs: Impact on Fos induction by FST
To test for Fos activation, animals were perfused after FST and brains were collected to analyze neuronal activation in brain regions typically activated by stress. We observed significant p<0.001 i , Figure 5B] on Fos expression in the IL. There was a significant main effect of stress in the BLA [F(1,12)= 12.4; p=0.004 j ; Figure 5C] and vlPAG [F(1,12)= 16.4; p=0.004 k ; Figure 5D] as well, with planned comparisons revealing significant reduction in Fos expression in the CVS control group (p=0.008 and p=0.005 in BLA and vl PAG respectively) that was prevented by chronic PV IN inhibition in the CVS hM4Di group (p=0.75 and p=0.6 in BLA and vl PAG respectively) . Analysis of Fos protein expression in the lateral septum, bed nucleus of the stria terminalis (BNST) and dorsolateral PAG (dlPAG) showed no significant treatment effects (Table   5-1).

Physiological impact of chronic stress
Adrenal and thymus weights were used to assess somatic effects of CVS. Adrenal hypertrophy and/or thymic atrophy are often observed following chronic stress and are used as indicators of repeated/chronic hypothalamic pituitary adrenal (HPA) axis activation. Here, there was a main effect of stress [F(1,35)=11.2; p=0.002 l ; Figure 6A] and a significant stress x DREADD  Figure   6B] as well, however with no effect of DREADD [F(1,35)=0.2; p=0.7] or stress X DREADD interaction were observed [F(1,35)=57; p=0.1]. Finally, decreased body weight gain is observed following chronic mild stress exposure in rodents (Ghosal et al., 2017). Two-way repeated Our data suggest that PV INs play an important role in chronic stress-mediated inhibition of the IL. GABAergic PV INs are well positioned to provide strong, fast-spiking inhibitory signals to pyramidal projection neurons in the PFC and reduce network excitability, and therefore could be contributing to chronic stress-mediated hypoactivity (Safari et al., 2017;Tremblay et al., 2016;Winkelmann et al., 2014). Chronic inhibition of IL PV INs during CVS resulted in increased active and decreased passive coping behaviors in FST. A switch to active coping can be interpreted as an adaptive strategy to deal with chronic stress, and drugs that are effective antidepressants in humans typically promote active coping styles and reduce passive coping in the FST in mice (Martí and Armario, 1993;Porsolt et al., 1977). GABA receptor antagonists have been shown to have antidepressant and anxiolytic properties (Bhutada et al., 2010;Mehta et al., 2017;SAMAD et al., 2018;Zanos et al., 2017). It is known that antidepressants such as fluoxetine and ketamine reduce PV expression in the PFC (Ohira et al., 2013;Page and Coutellier, 2019;Zhou et al., 2015). Moreover, preventing the reduction in PV IN activity leads to loss of antidepressant efficacy, further suggesting that reduced activity of PV INs might be playing a role in therapeutic efficacy of antidepressants (Page and Coutellier, 2019;Zhou et al., 2015). Therefore based on prior studies and our findings, inhibition of PV INs during chronic stress may lead to more adaptive stress coping strategies and reverse some of the behavioral deficits associated with chronic stress. It is important to note that the effects observed in FST are specifically due to changes in coping strategies due to PV IN inhibition and not due to any changes in locomotor activity (Figure 4-1). Additionally, we did not detect any effects on FST due to chronic dosing of CNO alone, suggesting that repeated CNO dosing did not alter our behavior ( Figure 4-2).
Our experiments revealed that inhibition of PV IN in the IL during stress can prevent chronic stress-induced decreases in Fos expression in key stress regulatory regions such as the PrL, BLA and vlPAG following FST (Berton et al., 2007;Keedwell et al., 2005;Vialou et al., 2014).
Since we cannot verify direct PV IN modulation of IL projections to these regions, we cannot preclude the possibility that reversal of CVS-related inhibition of Fos induction is due to actions of the IL through other projection systems. Nonetheless, the data suggest that PV IN inhibition reduces inhibitory effects of CVS on IL outflow, permitting drive of downstream structures known to participate in physiological reactivity and stress coping behavior (Maier and Watkins, 2010). Notably, this includes the neighboring PrL, which is not targeted by our DREADD injections and thus has Fos excitability modulated by cortico-cortical connections.
Involvement of the PrL is consistent with its prominent role in mediation of coping behavior (Fiore et al., 2015;Johnson et al., 2019;Molendijk and de Kloet, 2019). As part of our design, we assessed the impact of IL PV IN inhibition acutely following the first stressor in our CVS regimen, the TST, which allows for a behavioral readout (duration of struggling, immobility and latency to immobility). Acute inhibition of IL PV INs resulted in decreased active coping (struggling) and increased passive coping (immobility) in the TST. Our data are consistent with a prior study indicating that reduced excitatory synaptic drive onto PV INs is linked with increase stress susceptibility and enhanced helplessness behavior (Perova et al., 2015). These data indicate that PV INs may play a role in driving active coping responses, when an animal with no history of prior stress is exposed to a novel acute stressor such as the TST. Together, these studies suggest that activation of PV INs is required for coping responses to acute stress.  (Seybold et al., 2012). Our data suggests distinct neuronal ensembles and brain circuitry may be involved in modulating acute vs chronic stress mediated behavioral outcomes. It is also possible that chronic stress may result in plastic changes in the same neuronal ensemble recruited by acute stress, leading to differences in stress response. However, it is not known what specific plasticity in the neural network underlies the emergence of opposing phenotypes following chronic stress and further studies are needed to investigate the mechanisms.

Repeated inactivation of PV
There are few caveats to the present study that must be considered in the interpretation of the data. In this study we did not observe alterations in stress coping strategies in FST in our CVS group compared to controls. This is due to high rates of immobility in our control animals.
Typically immobility duration in control mice should be around 60% (Wohleb et al., 2016) , but in our control mice immobility was around 73%. It is known that body weight and age of rodents has a significant effect on behavior in the FST (Bogdanova et al., 2013;Hryhorczuk et al., 2013). It is possible that the high body weight and age of our mice resulted in a predominantly floating behavior leading to a ceiling effect, reducing the window to detect an increase in immobility typically observed after CVS exposure. Our control No CVS rats also received chronic injections. Chronic injection stress increases glutamate levels in the brain (Moghaddam and Bolinao, 1994) and might be acting as a stressor which may explain greater immobility in our controls. Nevertheless, we do see CVS effects in body and organ weights and in reduced Fos activation in stress regulatory brain regions demonstrating physiological effects of CVS in our study. This study was conducted only in male mice, to further prior research that showed alteration in inhibitory synaptic drive in the IL of males (McKlveen et al., 2019(McKlveen et al., , 2016. Because parvalbumin interneuron modulation may have sex specific effects (Shepard et al., 2016), it would be important to examine the effects of PV IN modulation during stress in females. Finally, in this study we euthanized animals after exposure to one behavioral paradigm FST in order to get anatomical Fos expression dataset to a novel stressor following CVS. Chronic stress can be characterized by cellular and behavioral changes spanning multiple interconnected neural network adaptations which were not explored in our current study. In order to get a clear representation of how PV IN modulation during stress is affecting emotionality, additional behaviors may be worth exploring in follow-up experiments.
Taken together, our data is consistent with a causal role of IL PV INs in initiating and coordinating coping strategies and physiological outcomes in response to stress. Chronic stress-mediated hypoactivity and aberrant behavioral responses may be mediated partly via plastic changes in PV IN function and may play a role of stress-related pathologies (e.g., depression and PTSD). Our data indicate that chemogenetic inhibition of PV INs during chronic stress, which reduces PV-initiated inhibition in the context of each individual stressor experience, may block or attenuate inhibition of glutamatergic neurons. In this case, maintenance of glutamatergic excitability is sufficient to attenuate some (but not all) behavioral and physiological consequences of chronic stress exposure, including decreased passive (immobility) and increased active coping behaviors (swimming) in the FST, preventing CVS effects on reduction in neuronal Fos activity and in preventing adrenal hypertrophy . Taken together, our findings suggest that reducing the activity of PV INs in the PFC during chronic stress may facilitate output of prefrontal neurons and could provide therapeutic benefits for stress related disorders.
In conclusion, this study provides support that PV INs play a role in chronic stress mediated coping behaviors and physiological phenotypes. Furthermore, the study adds to the current knowledge regarding possible mechanisms of prefrontal hypofrontality, and how PV INs may be involved in driving chronic stress related pathologies. The study also highlights opposing effects of acute and chronic PV IN inhibition indicating different underlying mechanisms involved in acute vs chronic stress paradigms. Overall, this study shows that reducing PV IN activity to promote prefrontal output may be an effective treatment strategy for stress related illnesses.

Declaration of interest
The authors declare no conflicts of interest.

Author contributions
NN designed and performed all experiments, performed all statistical analysis for this study,        in FST. Chronic CNO administration has no effect on immobility duration in forced swim test.
Values represent mean ± SEM, n = 4 per group (p>0.05).      Table depicts Fos protein expression in lateral septum, anterior and ventral bed nucleus of stria terminals (BNST) and dorsolateral periaqueductal gray (PAG). No significant treatment effects of PV IN inhibition was observed in any of the above mentioned brain regions. Values represent mean ± SEM, n = 9-10 per group.