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. Author manuscript; available in PMC: 2021 Mar 15.
Published in final edited form as: Biol Psychiatry. 2019 Aug 22;87(6):492–501. doi: 10.1016/j.biopsych.2019.08.006

Androgen-dependent excitability of mouse ventral hippocampal afferents to nucleus accumbens underlies sex-specific susceptibility to stress

Elizabeth S Williams 1,*, Claire E Manning 1,2,*, Andrew L Eagle 1, Ashlyn Swift-Gallant 2,3, Natalia Duque-Wilckens 1,4, Sadhana Chinnusamy 1, Adam Moeser 4, Cynthia Jordan 2, Gina Leinninger 1, AJ Robison 1,2,†
PMCID: PMC7035179  NIHMSID: NIHMS1538026  PMID: 31601425

Abstract

Background:

Depression affects women nearly twice as often as men, but the neurobiological underpinnings of this discrepancy are unclear. Preclinical studies in male mice suggest that activity of ventral hippocampus (vHPC) neurons projecting to nucleus accumbens (NAc) regulates mood-related behavioral responses to stress. Here we sought to characterize this circuit in both sexes and investigate its role in potential sex differences in models of depression.

Methods:

We used male and female adult C57BL/6J mice in the subchronic variable stress (SCVS) model to precipitate female-specific reduction in sucrose preference and performed gonadectomies to test the contributions of gonadal hormones to this stress response. In addition, ex vivo slice electrophysiology of transgenic Cre-inducible Rosa-eGFP-L10a mice in combination with retrograde viral tracing to identify circuits was used to test contributions of gonadal hormones to sex differences in vHPC afferents. Finally, we used an intersecting viral DREADD strategy to directly manipulate vHPC-NAc excitability in awake behaving mice.

Results:

We show a testosterone-dependent lower excitability in male vs female vHPC-NAc neurons as well as corresponding testosterone-dependent male resilience to reduced sucrose preference after SCVS. Importantly, we show that long-term DREADD stimulation of vHPC-NAc neurons causes decreased sucrose preference in males following SCVS, while DREADD inhibition of this circuit prevents this effect in females.

Conclusions:

We demonstrate a circuit-specific sex difference in vHPC-NAc neurons which is dependent upon testosterone and causes susceptibility to stress in females. These data provide a substantive mechanism linking gonadal hormones to cellular excitability and anhedonia, a key feature in depressive states.

Keywords: Sex differences, Stress, Anhedonia, Hippocampus, Nucleus Accumbens, DREADDs, Excitability

Introduction:

Affective disorders such as major depression disproportionately affect women (1, 2), but studies investigating depression-related behaviors in animal models that include both female and male subjects are lacking. There are sex differences in brain regions that regulate reward and motivation following some stress paradigms, including subchronic variable stress (SCVS) (3), chronic mild stress (4) and Peromyscus californicus social defeat stress (5). However, neurophysiological mechanisms causing sex differences in stress responses and depression-related behaviors remain unclear. Numerous studies have demonstrated the importance of the nucleus accumbens (NAc) in regulating depression-like behaviors after stress (6–9), and glutamatergic inputs to NAc from areas such as prefrontal cortex and ventral hippocampus (vHPC) alter NAc activity and ultimately behavioral responses to stress (10, 11). Critically, the vHPC is essential for social and affective memories (12–14), and glutamatergic vHPC afferents to the NAc directly regulate male behavioral responses to chronic social defeat stress (15), making this circuit a potential candidate mediating sex differences in mood-related disorders.

Unfortunately, most circuit-specific animal model studies investigating depression-related behaviors have not included female subjects. This gap in study design exists despite sex differences in several depression-related brain regions, including the hippocampus. For example, hippocampal spine morphology differs between male and female rodents prior to stress, and can exhibit sex-specific responses to stressful events (16), and male and female rodents differ in hippocampal LTP and hippocampus-dependent contextual learning (17). Sex differences in responses to stress are also documented, and one key model in mice is subchronic variable stress (SCVS), after which only female mice exhibit anhedonic behaviors (3, 18) analogous to human depression patients. To this end, gonadal steroid hormone receptors have been identified in the NAc as modulators of female susceptibility to SCVS (19). However, whether vHPC-NAc activity in male and female mice is regulated by gonadal hormones has not been tested. Thus, in this study, we hypothesize that adult circulating testosterone conveys resilience to SCVS in males by reducing vHPC-NAc excitability.

Materials and Methods

Animals.

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Michigan State University and performed in accordance with AAALAC and NIH guidelines. Experiments were performed on adult C57BL6/J mice purchased from Jackson Labs or transgenic colony mice (Cre-inducible RosaeGFP-L10a). Mice were provided with ad libitum access to water and standard lab diet.

Subchronic Variable Stress.

SCVS was performed according to previously published protocols (3, 18). Briefly, group-housed mice were exposed to a stressor every day for six days under white light conditions. Stressors were administered daily in the following order on consecutive days: group foot shock of 10 mice with 100 random foot shocks over an hour at 0.45mA (Shock Box H13-15, Coulbourn Instruments, Holliston, MA), one hour tail suspension, and one hour restraint stress; sequence repeated once for six days total stress. Restraint tubes were manufactured in-house by drilling air holes in 50mL falcon tubes.

DREADD activation.

Clozapine N-oxide (CNO; Fischer Scientific NC1044836) was diluted in vehicle solution: 5%DMSO and 95% 0.9% saline. CNO or vehicle was administered i.p. throughout DREADD SCVS experiments at 0.3mg/kg daily an hour prior to the stressor or behavior.

Behavior.

Following SCVS, animals were tested using sucrose preference, splash test, novelty surpressed feeding (NSF), elevated plus maze (EPM), and social interaction (SI), all of which are described in detail in the extended methods.

Immunohistochemistry.

35 μm sections were stained using the following primary antibodies: Goat anti-GFP (Abcam; ab5450 1:1000) and Rabbit anti-Androgen Receptor (Abcam; ab52615 1:1000); and secondary antibodies: Alexa-Flour 488 anti-goat IgG (Jackson; 705-545-147 1:200) and Cy3 anti-rabbit IgG (Jackson; 711-165-152 1:200).

Statistical Analysis.

Statistical analysis was performed with PRISM software 7.0 (Graphpad). All error bars represent the mean +/− SEM. See Supplemental Information for more detail and for group sizes and comparisons.

Results

Female mice have increased vHPC-NAc circuit excitability and are selectively susceptible to SCVS.

We observed that female mice are selectively vulnerable to reduced sucrose preference after SCVS, in agreement with prior studies (3, 18, 20). Following either the 6-day SCVS battery of stressors (foot shock, tail suspension, and restraint; Fig. 1a) or 6 days of control handling, stressed female mice had a significant reduction in free-choice preference for sucrose solution over water alone compared to controls (Fig 1b), with a significant interaction between stress and sex. Although animals still displayed a preference for sucrose, this reduction in preference can be linked to anhedonia (9, 21, 22), modelling a core aspect of enhanced female depression vulnerability. We also used a variety of other assays to measure anxiety, autogrooming, and social withdrawal in the same mice (Fig. S1a–e), but we did not observe the sex-specific vulnerability to stress in any measure other than sucrose preference. Therefore, we focused on sucrose preference for the remainder of the current study, though we report all other measures throughout.

Figure 1|. Selective female susceptibility to SCVS and to vHPC-NAc hyper-excitability.

Figure 1|

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(A) Schematic depicting stressors used in SCVS (top) and experimental time course of stress and sucrose preference (bottom). (B) Only female mice displayed reduced sucrose preference after SCVS. (C) Schematic depicting retrograde Cre viral vector strategy and electrophysiology time course (left) and L10-GFP vHPC stained with anti-GFP demonstrating vHPC-NAc projections (right). (D, top) Action potential (AP) number across sequential depolarizing current steps (25-300 pA) for male (n=20 cells from n=6 animals) and female (n=19 cells from n=5 animals) vHPC-NAc projections. Females showed significantly higher AP number at all steps ≥ 125 pA (inset: sum of AP number across all current steps). (D, bottom) Representative traces for male and female vHPC-NAc groups, 200 pA step. (E, top) AP number across sequential depolarizing steps for male (n=22 cells from n=7 animals) and female (n=12 cells from n=4 animals) vHPC-BLA projections. Male and female AP number did not differ at any current step. Inset: sum of AP number across all current steps. (E, bottom) Representative traces for male and female vHPC-BLA groups, 200 pA step. DG: Dentate Gyrus; VTA: Ventral Tegmental Area; Sub: Subiculum.

This sex difference in susceptibility to SCVS suggests an underlying difference in physiology between male and female mice. Glutamatergic vHPC-NAc projections have been linked to stress susceptibility and reward in several elegant studies (11, 15). To explore the vHPC-NAc circuit and its role in mediating stress-dependent changes in sucrose preference, we employed whole cell patch clamp electrophysiology and a circuit-specific retrograde HSV-Cre labeling strategy (23) to record from vHPC-NAc cells in both male and female mice. Transgenic mice expressing the Cre-inducible L10-GFP (24) were injected in NAc with a retrograde herpes simplex virus (HSV) expressing Cre recombinase (Fig 1c, left), leading to GFP expression in all neurons projecting to or within NAc. After 21 days to allow full retrograde expression, brain slices containing vHPC were prepared for whole-cell recordings from GFP-positive vHPC neurons projecting to NAc. In hippocampus, only pyramidal neurons of the ventral CA1/subiculum region were found to project to NAc using this method (Fig 1c, right). Female vHPC-NAc neurons were more excitable than those of male mice, as indicated by an elevated number of action potentials elicited by increasing depolarizing current injections (Fig 1d top, example traces for each group below). No differences in resting membrane potential (VM, Fig S2a) or membrane resistance (RM, Fig S2b) were observed between male and female vHPC-NAc neurons. Despite the apparent heightened excitability of female vHPC-NAc neurons, input resistance (Rin) was significantly lower in these neurons compared to male vHPC-NAc neurons (Fig S2c). Membrane capacitance was significantly higher in female vHPC-NAc neurons (Fig S2d), despite the female neurons’ exaggerated response to injected current compared to male neurons. Rheobase was not significantly different between male and female vHPC-NAc neurons (Fig S2e). Action potential threshold voltage was significantly lower in female neurons (Fig S2f), also indicating a heightened excitability in vHPC-NAc projections in female mice. There was no difference in sag ratio between male and female vHPC-NAc neurons (Fig S2g), indicating no difference in H-current between sexes. Frequency of spontaneous excitatory postsynaptic currents (sEPSCs) did not differ between male and female vHPC-NAc neurons (Fig S2h), but sEPSC amplitude was significantly decreased in female mice (Fig S2i).

To determine whether this sex difference in excitability is specific to vHPC-NAc neurons, we injected retrograde HSV-Cre into BLA of a separate cohort of male and female mice. Whole-cell recordings obtained from vHPC-BLA projections revealed no significant difference in excitability of female vHPC-NAc neurons compared to male (Fig 1e top, example traces for each group below). There were no differences between male and female vHPC-BLA neuron membrane properties in any measure (VM, RM, Rin, CM, and rheobase; Fig S3a–e). No difference was observed between sexes in AP voltage threshold (Fig S3f), supporting similar male and female excitability in this circuit. Sag ratio was observed to be lower in female vHPC-BLA neurons (Fig S3g), indicating a possible sex difference in after-hyperpolarization current in this circuit. Spontaneous EPSCs did not differ in frequency or amplitude between male and female vHPC-BLA projections (Fig S3h–j).

Overall, these findings demonstrate that female vHPC-NAc, but not vHPC-BLA, neurons have increased excitability compared to males, suggesting a circuit-specific physiological difference that could underlie the sex differences in the susceptibility to reduced sucrose preference following SCVS.

Adult testosterone is necessary for male resilience to reduced sucrose preference after SCVS and reduced excitability of vHPC-NAc neurons.

To investigate the role of adult sex hormones in susceptibility to SCVS, male mice were orchidectomized (ORCH), allowed to recover for 10 or 28 days, and exposed to SCVS (Fig 2a–b). Ten days of recovery resulted in main effects of ORCH and stress on sucrose preference without interaction between the two (Fig 2a), suggesting that SCVS this close to the stressful surgery (sham or ORCH) was sufficient to render male mice susceptible to reduced sucrose preference following SCVS. In contrast, twenty-eight days following ORCH, male mice displayed reduced sucrose preference following SCVS, while those undergoing sham surgeries remained resilient to the decrease in sucrose preference (Fig 2b), with an interaction between the two variables. There were no differences between sham and ORCH groups in any other behavioral measure at either time point (Fig S4a–d). These data thus suggest that long-term reduction in androgen signaling is sufficient for reduced sucrose preference following SCVS in male mice.

Figure 2|. Orchidectomy induces susceptibility to SCVS and increases vHPC-NAc excitability in male mice.

Figure 2|

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(A, top) Schematic depicting experimental time course of surgery, stress, and measurement of sucrose preference for short time course orchidectomy experiment. (A, bottom) Short time course sham vs orchidectomized male mice showed no interaction between control and stress. (B, top) Schematic depicting experimental time course of surgery, stress, and measurement of sucrose preference for long time course orchidectomy experiment. (B, bottom) Orchidectomized male mice showed significant reduction in sucrose preference compared to non-stress controls, while sham male sucrose preference was unaffected by SCVS. (C) Schematic depicting retrograde Cre viral strategy for labeling vHPC-NAc projections (left) and experimental time course for surgery and recording (right). (D, left) Action potential number across sequential depolarizing current steps (25-300 pA) for sham male (n=11 cells from n=3 animals) and orchiectomized male (n=31 cells from n=6 animals) vHPC-NAc projections. Orchiectomized male projections showed significantly higher AP number at all steps > 25 pA (inset: sum of AP number across all current steps). (D, right) Representative traces for sham and ORCH vHPC-NAc projections, 200 pA step.

To determine whether the sex difference we observed in vHPC-NAc excitability was also dependent on adult sex hormones, we compared vHPC-NAc excitability in ORCH vs. sham male and OVX vs. sham female mice at 10 days post-gonadectomy. Retrograde Cre was injected into NAc, 10 days following intracranial injection gonads were removed, and 10 days following gonadectomy, vHPC-NAc activity was recorded (Fig 2c). ORCH significantly increased the excitability of male vHPC-NAc neurons compared to that of sham controls (Fig 2d left, example traces for each group right). In contrast, OVX had no effect on the excitability of female vHPC-NAc neurons (Fig S5a). ORCH had no effect on VM, RM, Rin, or CM (Fig S6a–d), but did increase rheobase (Fig S6e), with no change in AP voltage threshold (Fig S6f). There was no observed difference between sham and ORCH animals in sag ratio (Fig S6g), and ORCH did not affect sEPSC frequency or amplitude (Fig S6h–j). OVX did not affect membrane properties (Fig Sb-g) or sEPSC frequency or amplitude (Fig S5i–k).

These data suggest that reduction of circulating androgens causes a change in vHPC-NAc excitability that may require time-dependent restructuring of vHPC-NAc neurons over weeks, and this change in physiology may cause susceptibility to reduced sucrose preference following SCVS similar to that seen in female mice.

Androgen receptor antagonism increases male vHPC-NAc excitability.

We next questioned whether androgen receptors (ARs) are directly involved in the regulation of vHPC-NAc physiology. To verify that ARs are present on vHPC-NAc projection neurons, we used double-label immunofluorescence to stain for AR and GFP in vHPC of L10-GFP reporter mice injected with retrograde HSV-Cre in NAc. We found that vHPC-NAc projection neurons, as well as many of the surrounding pyramidal cells in vHPC CA1, do indeed express AR (Fig 3a). To test whether ARs are directly involved in the regulation of vHPC-NAc neuronal excitability, the AR antagonist flutamide was used in conjunction with slice electrophysiology to determine effects on projection physiology. Either vehicle (DMSO) or flutamide + picrotoxin-spiked aCSF solutions were used in the slice incubation chamber as well as the main bath to record from vHPC-NAc cells in L10-GFP mice injected with retrograde Cre virus at NAc (Fig 3b). Those cells exposed to flutamide + picrotoxin aCSF were found to have increased excitability compared to those treated with vehicle aCSF, as indicated by a significantly elevated total number of spikes across all current injections (Fig 3b, inset), although no significant differences were revealed at any current injections (Fig 3b). Vehicle- and flutamide + picrotoxin-treated male vHPC-NAc neurons did not differ in any other electrophysiological measure (Fig S7a–j). These data support AR activation, possibly through stimulation by androgens as suggested by our orchidectomy findings, as a potential regulator of excitability of male vHPC-NAc neurons that could mediate resilience to reduced sucrose preference following SCVS.

Figure 3|. vHPC-NAc excitability and susceptibility to SCVS-induced anhedonia are dependent on adult testosterone.

Figure 3|

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(A) Representative AR staining of vHPC slices from a male L10-GFP mouse; top left panel shows L10-GFP staining, top right panel shows AR staining, and the bottom panel shows a merged image demonstrating AR expression on vHPC-NAc projection cells. All images at 100x magnification. (B, top) Representative traces for vehicle-and flutamide-treated vHPC-NAc neurons, 200 pA step. (B, bottom) Action potential number across sequential depolarizing current steps (25-300 pA) for vehicle- (n=20 cells from n=7 animals) and flutamide-treated (n=16 cells from n=6 animals) vHPC-NAc projections. Total number of spikes for flutamide-treated cells vs vehicle-treated cells (inset) was increased. (C, top) Schematic depicting experimental time course of surgery, stress, and measurement of sucrose preference. (C, bottom) OVX females with blank pellet implants maintained a reduction in sucrose preference following SCVS, while OVX female mice exposed to testosterone showed no reduction in sucrose preference following SCVS, indicating resilience. (D, top) Representative traces for OVX + BLANK and OVX + T vHPC-NAc projections, 200 pA step. (D, bottom) Action potential number across sequential depolarizing current steps (25-300 pA) for OVX + BLANK (n=11 cells from n=3 animals) and OVX + T (n=19 cells from n=5 animals) vHPC-NAc projections. OVX + T projections showed significantly lower AP number at all steps > 25 pA (inset: sum of AP number across all current steps).

Exogenous testosterone in female mice ameliorates susceptibility to SCVS and decreases vHPC-NAc excitability.

To determine whether exogenous testosterone could protect female mice against reduced sucrose preference following SCVS, we implanted 6mm Silastic blank or testosterone pellets in adult female mice simultaneously with ovariectomy (OVX). Following OVX and implantation, 28 days were allowed for healing and equilibration of hormones. Mice were then exposed to SCVS, and behavioral assessment began immediately following the last day of SCVS (Fig 3c, top). Female mice implanted with blank pellets showed reduced sucrose preference after SCVS and those implanted with testosterone pellets showed no effect of stress on sucrose preference, indicating resilience similar to that of male mice. (Fig 3c, bottom), although the interaction between stress and treatment did not reach significance (p=0.058). Several other behaviors differed between blank and testosterone pellet-implanted female mice, including a main effect of testosterone on NSF and an interaction between stress and testosterone status on EPM (Fig S8a–e). Taken together, these data suggest a direct protective effect of testosterone or its derivatives against reduced sucrose preference following SCVS.

To investigate whether exogenous testosterone could also reduce excitability of vHPC-NAc neurons in female mice, we injected L10-GFP female mice with retrograde HSV-Cre in NAc. One week later, OVX and pellet implantation were performed in a single procedure. Two weeks following implantation, we performed whole-cell slice physiology on vHPC-NAc projection neurons. OVX mice implanted with testosterone pellets exhibited significantly decreased excitability in vHPC-NAc neurons compared to OVX mice implanted with blank pellets (Fig 3d bottom, example traces for each group top). No differences in VM, RM, Rin, or CM were observed (Fig. S9a–d). Mice implanted with testosterone pellets had an increased rheobase compared to those implanted with blank pellets (Fig. S9e), but no difference was observed between groups in the measure of AP voltage threshold (Fig. S9f). Sag ratio was equivalent between testosterone and blank pellet groups (Fig. S9g), and no differences were observed between sEPSC amplitude or frequency when comparing OVX + Blank and OVX + T groups (Fig. S9h–j). These results suggest that exogenous testosterone or its derivatives reduce excitability of female vHPC-NAc neurons and drive resilience to reduced sucrose preference following SCVS in females.

vHPC-NAc excitability directly mediates susceptibility to reduced sucrose preference following SCVS.

To determine whether the excitability of vHPC-NAc neurons is directly responsible for susceptibility to reduced sucrose preference following SCVS, we employed an intersecting viral DREADD strategy. In wild-type mice, retrograde HSV-Cre was injected into NAc, and a Cre-dependent AAV-mCherry-hM3Dq (Gq-coupled DREADD in male mice) or AAV-mCherry-hM4Di (Gi-coupled DREADD in female mice) was injected into vHPC (Fig 4a, top). Using this strategy, only vHPC-NAc neurons transduced by both viruses were altered by systemic CNO administration. We verified the efficacy of the DREADDs using slice electrophysiology in vHPC guided by the mCherry tag and recording before and after CNO wash-on (timeline Fig 4a, bottom). We recorded from neurons expressing mCherry in vHPC allowing us to visualize the presence of Gq- or Gi-coupled DREADD expression in male and female mice, respectively. These neurons in male mice demonstrated an increase in excitability following CNO wash-on, with an increase in number of spikes at 150 pA injected current, as well as a decrease in rheobase (example neuron depicted in Fig 4b). Female mCherry+ vHPC-NAc neurons demonstrated a decrease in excitability following CNO wash-on, with a decrease in the number of spikes at 150 pA injected current, as well as an increase in rheobase (example neuron depicted in Fig 4c).

Figure 4|. vHPC-NAc activity directly mediates susceptibility to SCVS-induced anhedonia.

Figure 4|

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(A, top) Schematic depicting intersecting viral DREADD strategy for circuit-specific manipulation of vHPC-NAc excitability and experimental time course. AAV encoding Cre-inducible DREADD (either Gq- or Gi-coupled) was injected in vHPC, and retrograde HSV-Cre was injected in NAc, causing circuit-specific receptor expression. (A, bottom) Schematic depicting time course for DREADD recordings. Cells were first recorded in regular aCSF, then CNO-containing aCSF was washed on and the same cell was recorded 8-10 minutes later. (B and C) Whole-cell slice electrophysiology demonstrating CNO activating Gq-(B) and Gicoupled (C) receptors. Example traces before (grey) and after (yellow) CNO application along with rheobase at time of each recording. (D, top) Experimental timeline of DREADD surgery, retrograde Cre surgery, SCVS, and subsequent behavioral assessment for short (behavior-only) DREADD experiment for male mice. (D, bottom) Male mice expressing excitatory Gq-coupled DREADD in vHPC-NAc projections and exposed to CNO only during behavior assessment did not show any change in sucrose preference following SCVS. (E, top) Experimental timeline for long (CNO during both SCVS and behavior assessment) DREADD experiment for male mice. (E, bottom) Male mice expressing excitatory Gq-coupled DREADD in vHPC-NAc projections and exposed to CNO during both SCVS and behavior assessment experienced an overall reduction in sucrose preference and a further reduction in sucrose preference following SCVS. (F, top) Experimental timeline for female mice. (F, bottom) Female mice expressing inhibitory Gi-coupled DREADD in vHPC-NAc projections and exposed to activating CNO showed no change in sucrose preference following SCVS.

Three weeks after surgery, male mice expressing the excitatory Gq-coupled DREADD in vHPC-NAc were exposed to SCVS followed by CNO or saline administration via i.p. injection each day of behavioral testing (Fig 4d, top). Intriguingly, CNO failed to directly evoke changes in sucrose preference (Fig 4d, bottom), indicating that short-term increased activity of the circuit does not cause reduced sucrose preference following SCVS. A second cohort of males expressing Gq-coupled DREADD in vHPC-NAc was exposed to SCVS with CNO or saline exposure throughout both stress and behavioral testing (Fig 4e, top). These long-term CNO-treated male mice showed a decrease in sucrose preference overall, as well as a further SCVS-induced decrease in sucrose preference (Fig 4e, bottom). There was a significant interaction between stress and CNO treatment in the measure of SI ratio (Fig S10a), but no interaction between stress and CNO treatment in EPM open arm time (Fig S10b). Because CNO can be metabolized into clozapine, which could itself affect animal behavior, we also performed a control experiment in which male mice without DREADD expression were stressed and given CNO using the same protocol. Here, we observed no change in sucrose preference following SCVS (Fig S10g), indicating that CNO itself has no effect on this behavior. These data suggest that short-term artificial stimulation of the vHPC-NAc circuit does not directly cause reduced sucrose preference in stressed or non-stressed animals. However, prolonged increase in excitability of this circuit induces reduced sucrose preference at baseline, and if these changes in excitability are paired with stress, the male mice display a further reduction in sucrose preference, indicating susceptibility.

Female mice expressing inhibitory Gi-coupled DREADD in vHPC-NAc were exposed to SCVS while CNO or saline was administered via i.p. injection each day of stress and throughout behavioral testing (Fig 4f top). Saline-treated female mice showed a decrease in sucrose preference following SCVS that reached p=0.0518, while CNO-treated female mice showed an amelioration of this susceptibility following stress (Fig 4f, bottom). There was no significant interaction between stress and CNO treatment in the measures of SI ratio (Fig S10c) or EPM open arm time (Fig S10d). Taken together with the male DREADD data above, these findings indicate that prolonged, but not acute, alterations in vHPC-NAc neuronal excitability can directly drive or prevent susceptibility to reduced sucrose preference following SCVS.

Discussion

In the current study, we use mouse behavior, slice electrophysiology, and intersecting DREADD viral manipulation of circuit function to define a novel neurophysiological mechanism for sex differences in anhedonic responses to stress. Exploring sex as a biological variable in preclinical models of mood disorders is essential, as there is an enigmatic sex difference in human depression diagnostic rates. Women are more likely than men to be diagnosed with depression across the lifespan (25), and these diagnoses often surround changes to the reproductive cycle such as puberty, menstruation, postpartum, and menopause (26, 27). Moreover, meta-analyses of studies totaling nearly one thousand subjects demonstrate that adult (18-60yrs) but not aged (>60yrs) males with reduced testosterone are more likely to suffer depression and their symptoms, including anhedonia, can be significantly relieved by testosterone supplement (28). However, exploring the circuit and molecular underpinnings of sex differences in depression has been difficult, in part due to many preclinical behavioral models of depression and stress-induced behaviors relying upon intrasexual aggression, leaving female models understudied (9, 29, 30). In the last decade, there has been a proliferation of models attempting to address this disparity (31–34), including SCVS (3, 18). Previous reports show that SCVS induced female-specific deficits in time grooming after splash test and in latency to eat in NSF as well as reduced sucrose preference (18), but we did not observe statistically significant effects in any test but sucrose preference. A number of factors could contribute to this discrepancy: housing conditions differ between institutes, experimenters handle mice differently, and although both studies used mice from Jackson Labs, they occurred nearly five years apart, and thus vendor conditions could have changed. Nevertheless, both studies report female-specific vulnerability to some behavioral effects of SCVS and highlight the role of limbic brain regions, like vHPC and NAc, in this phenomenon.

Previous studies have highlighted the importance of the NAc in the regulation of SCVS-induced behaviors, including processes such as transcriptional and epigenetic regulation, and many of these studies have implicated steroid hormones and their receptors (18, 19, 35). Furthermore, recent work demonstrates that stress-induced changes in females may be presynaptic, either through glutamatergic inputs onto NAc medium spiny neurons or in other reward-related circuits (3, 36). This is concordant with previous work in males suggesting the strength of vHPC glutamatergic synapses in the NAc underlies susceptibility to chronic social defeat stress (15). These studies, along with human studies that demonstrate a decreased hippocampal volume in patients with MDD (37), suggest that the vHPC may be a mediator of sex differences in stress outcomes and depression-related behaviors, especially through its projections to the NAc. Additionally, circulating hormones dramatically affect vHPC CA1 structure and function (38–40) as well as social and hippocampal-dependent learning (41, 42).

vHPC-NAc afferents have been previously implicated in modulating social behaviors (43, 44). Here, we demonstrate vHPC-NAc projection-specific regulation of the likewise positively-valenced measure of sucrose preference, a well-validated measure of anhedonia (9, 45, 46). We show that reduced sucrose preference after SCVS is female-specific and that this parallels a heightened vHPC-NAc excitability. This sex difference appears to be dependent on circulating gonadal hormones, as orchidectomy in male mice feminizes the vHPC-NAc excitability and SCVS-induced anhedonia, and testosterone in female mice decreased vHPC-NAc excitability and prevents anhedonia. Our gonadal hormone manipulations were systemic and may therefore act in a non-cell autonomous manner, perhaps exerting their effects at the level of adjacent circuitry or via support cells such as glia. In addition, androgens may be aromatized to estrogens in the brain. However, as we show that vHPC-NAc neurons express AR, their excitability is unchanged following ovariectomy, and bath application of an AR antagonist increases vHPC-NAc excitability, we hypothesize that direct activation of androgen receptors mediates these effects, potentially within the vHPC-NAc cells themselves.

The basal sex differences we demonstrate in vHPC-NAc excitability and behavioral responses to stress are in agreement with work in males that highlights the activity of vHPC-NAc afferents as a driver of behavioral responses to chronic social defeat stress (11, 15). We found that 10 days DREADD-mediated activation of vHPC-NAc decreased baseline sucrose-preference and promoted susceptibility to anhedonia following SCVS. Intriguingly, recent reports suggest a chronic multimodal stress model reduces vHPC synaptic strength onto D1 medium spiny neurons of the NAc in stress-susceptible males (11, 15). This discordance may be due to our study’s inability to distinguish between projections to D1 vs D2 NAc MSNs or due to changes in other reward related circuits in response to chronic activity of vHPC-NAc. Indeed, prolonged changes in activity may lead to neuroadaptations, including changes in gene expression and synaptic strength, to drive differential responses to stress, both within vHPC-NAc neurons and adjacent circuitry. This notion is supported by demonstrations that long-term changes in the strength of vHPC synapses onto NAc medium spiny neurons can alter motivated I behaviors in multiple contexts including chronic stress and drugs of abuse (11, 15). Moreover, the effects of DREADD-mediated stimulation or inhibition on the excitability of neurons over the time courses used in this study are not understood. Although we interpret our behavioral effects as stemming from long-term changes in excitability, it is possible that G-protein-mediated signaling separate from or in addition to altered excitability may be driving the changes we observe in sucrose preference. However, as we demonstrate that higher vHPC-NAc excitability is concordant with lower sucrose preference in many different conditions (i.e., the two sexes, orchidectomy and ovariectomy, and with artificial testosterone treatment), the most parsimonious explanation for our DREADD results is that altered activity of this circuit is driving the changes in sucrose preference.

As our short time courses of orchidectomy or vHPC-NAc activity manipulation did not elicit the same robust behavioral effects as prolonged manipulations of the circuit, we hypothesize that vHPC-NAc neuronal excitability influences long-term neuroadaptations that cause stress-induced anhedonia. Uncovering these mechanisms underlying the basal sex differences in vHPC-NAc excitability, such as the precise mechanism of androgen action and the influence of circuit excitability on long-term neuroadaptations, will be a critical goal of future studies. The preclinical study presented here uncovers a basal sex difference in excitability of a specific brain circuit driven by adult testosterone that is causally linked to susceptibility to anhedonia-like behavior, a key behavioral phenotype of mood disorders. This discovery may in part explain the clinical observation that women are more than twice as likely as men to experience depressive disorders, paving the way for future study of sex-specific treatment of depression.

Supplementary Material

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KEY RESOURCE TABLE

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Antibody Goat anti-GFP Abcam ab5450
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Acknowledgments

The authors thank Kenneth Moon for excellent technical assistance. AJR acknowledges support from the National Institutes of Mental Health (MH111604), the National Institutes of Neurological Disease and Stroke (NS085171), the National Institutes of Drug Abuse (DA040621, and DA040621-03S1) and the Avielle Foundation.

Footnotes

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Financial Disclosures

The authors report no biomedical financial interests or potential conflicts of interests.

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NIHMS1538026-supplement-2.pdf (2.3MB, pdf)

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