Amygdala AVPR1A mediates susceptibility to chronic social isolation in female mice

Abstract

Sex differences in responsiveness to social stress in adulthood are highly conserved across species, with females more sensitive to isolation. Here, we show that Arginine vasopressin receptor 1a (AVPR1A) in the central nucleus of the amygdala (CeA) mediates the enhanced susceptibility of females to post-pubertal chronic social isolation stress (CSIS) in mice. Chemogenetic activation of AVPR1A^CeA circuits induces anxiety-related behaviors in both sexes. However, genetic, pharmacological, chemogenetic and optogenetic loss of function approaches support the idea that it is only endogenously engaged in females in the context of CSIS. Using a combination of virus-based tools, we identified a major source of AVP ligand in the posterodorsal part of the medial amygdala (MePD) as well as an important downstream target of AVPR1A^CeA neurons, the dorsolateral striatum (DLS). Loss of function approaches identified three nodes in the circuit that provide sex-specificity in the effects of CSIS on anxiety-related behaviors: 1) ERα signaling in AVP^MePD neurons; 2) engagement of the AVPR1A pathway in the CeA; and 3) number of AVPR1A^CeA projections to the DLS. These data support new therapeutic applications for AVPR1A antagonists in women experiencing social isolation or loneliness.

Fig. 1. Stimulating AVPR1A^CeA neurons promotes anxiety-related behaviors in both sexes.

a Neurons expressing the Avpr1a-Cre::tdTomato lineage trace are localized to the CeA in coronal sections of the amygdala from bregma −1.22 to −1.82. b No sex difference in Avpr1a-Cre::tdTomato lineage traced neurons in the amygdala (P = 0.3802, n = 7 males, n = 10 females). c Experimental design to assess the effect of chemogenetic activation of AVPR1A^CeA neurons in group-housed mice on anxiety-related behaviors. d Schematic of viral injection into the CeA of Avpr1a-Cre mice. e Representative expression of the viral mCherry reporter in the CeA (n = 26). f, g Chemogenetic activation of AVPR1A^CeA neurons increased marble burying (f) and reduced time spent in the open arms of the EPM (g) (marble burying: group P = < 0.0001, sex P = 0.8657, interaction group*sex P = 0.6425; EPM: group P = < 0.0001, sex P = 0.3904, interaction group*sex P = 0.6247, n = 6 control females, n = 7 Gq females, n = 9 control males, n = 8 Gq males). An unpaired two-tailed t-test was used in (b). Two-way ANOVA (with RM for marble burying) and Tukey’s multiple comparisons were used in (f, g). Different letters denote significant differences. n denotes the number of mice in each group. Scale bars represent 100 μm. Schematic in (c) was generated with BioRender icons.

Fig. 2. CSIS increases Avpr1a expression and anxiety-related behaviors in females.

a Schematic of CeA/MePD micropunches analyzed. b Experimental design to assess effects of CSIS vs. group housing from 5 weeks of age for ≥7 weeks in WTs on Avpr1a expression in the CeA/MePD. c CSIS increased Avpr1a in females but not males (group P = 0.0012, sex P = 0.0272, interaction group*sex P = 0.0012, n = 13 control females, n = 14 CSIS females, n = 13 control males, n = 15 CSIS males). d Avpr1a in females remained elevated after re-grouping for 3 weeks (P = 0.0025, n = 9). e Initiating 7 weeks of CSIS at 8 weeks of age increased Avpr1a in females, but exposure to 2 weeks of isolation from 10 weeks of age did not (P = 0.0011, n = 9 control females, n = 10 7-wk SIS females, n = 9 2-wk SIS females). f Experimental design to assess the effect of CSIS vs. group housing on anxiety-related behaviors in WTs. g, h CSIS increased marble burying (g) and time spent in the center of the elevated plus maze (EPM) (h) in females and not males (marble burying: group P = 0.0020, sex P = 0.4645, group*sex P = 0.0969, n = 9–14; EPM: group P = 0.1, sex P = 0.0214, group*sex P = 0.0102, n = 9 control females, n = 14 CSIS females, n = 9 control males, n = 11 CSIS males). i Experimental design to assess effects of re-grouping females for 3 weeks after exposure to CSIS on anxiety-related behaviors. j Re-grouping females did not reduce marble burying (group P = 0.8702, age P = 0.0307, group*age P = 0.9509, n = 6). Two-way ANOVA (with RM for marble burying) and Tukey’s multiple comparisons were used in (c, g, h, j). Two-tailed Mann-Whitney test used in (d). Kruskal-Wallis test and Dunn’s multiple comparisons in (e). Different letters denote significant differences. n denotes the number of mice per group. Schematics in (a), (b), (f) and (i) were generated with BioRender icons.

Fig. 3. AVPR1A in the CeA is necessary for CSIS-induced anxiety-related behaviors in females.

a Schematic of intra-CeA injections in Avpr1a^Flox/Flox mice used to generate conditional knockouts (cKOs). b Representative GFP expression in the CeA. c Similar number of GFP-expressing CeA neurons between control and Cre viruses and between sexes (group P = 0.6584, sex P = 0.6804, interaction group*sex P = 0.5333, n = 4 females, n = 5 males). d Diminished Avpr1a in the CeA/MePD in Avpr1a^Flox/Flox injected with Cre versus control virus (P = < 0.0001, n = 6 control, n = 5 Cre). e Experimental design to assess CSIS-induced anxiety-related behaviors in cKOs. f, g Decreased marble burying (f) and increased time in the open arm of the EPM (g) in females but not males (marble burying: group P = 0.0006, sex P = 0.6300, interaction group*sex P = 0.0005, n = 11 control females, n = 10 Cre females, n = 8 control males, n = 10 Cre males; EPM: group P = 0.0283, sex P = 0.4419, interaction group*sex P = 0.0430, n = 7 control females, n = 8 Cre females, n = 8 control males, n = 8 Cre males). h Experimental design to assess CSIS-induced anxiety-related behaviors following pharmacological blockade of AVPR1A. i, j SRX246 reduced marble burying (i) and increased time in the open arm of the EPM (j) in females and not males exposed to CSIS, while SRX49059 had no effect (marble burying: group P = 0.0003, sex P = 0.6348, interaction group*sex P = 0.2733, n = 10 females, n = 12 males; EPM: group P = 0.0397, sex P = 0.1204, interaction group*sex P = 0.046, n = 11 vehicle-injected females, n = 11 SRX246-injected females, n = 10 vehicle-injected males, n = 11 SRX246-injected males). Two-way ANOVA used in (c). Two-way ANOVA (with RM for marble burying) and Tukey’s multiple comparisons used in (f–g) and (i, j). Unpaired two-tailed t-test used in (d). Different letters denote significant differences. n denotes the number of mice per group. Scale bar represents 100 μm. Schematics in (e) and (h) generated with BioRender icons.

Fig. 4. AVPR1A^CeA → DLS circuits mediate behavioral adaptations to CSIS in females.

a Schematic of viral injection for anterograde tracing from AVPR1A^CeA neurons. b Representative expression of the viral mCherry reporter in the CeA (n = 7). c Overview of AVPR1A^CeA neuronal projections in females, with colors corresponding to the heatmap shown in Supplementary Fig. 6. d DLS is the only region with a higher density of synaptic boutons from AVPR1A^CeA neuronal projections in females compared to males (P = 0.04818, n = 2 female NAcc, n = 3 female SON, SubB, LPOA, mfb, BNST, n = 4 female MeA, DLS, BMA, ns, n = 3 males). e, f Representative expression of the viral mCherry reporter in the DLS in females (e) and males (f). g Schematic of viral injections for rabies tracing of AVPR1A^CeA → DLS collateral projections. h Representative expression of the viral mCherry reporter in the DLS (n = 5). i Overview of AVPR1A^CeA neuronal projections with collaterals to the DLS. j Experimental design to assess the effect of optogenetic inhibition of AVPR1A^CeA → DLS projections on CSIS-induced anxiety-related behaviors. k Schematic of viral injection into the CeA and fiber placement in the DLS of Avpr1a-Cre females. l Representative expression of the viral EYFP reporter in the CeA (n = 13). m, n Exposure to light with a wavelength of 590 nm to inhibit AVPR1A^CeA → DLS projections expressing halorhodopsin (NpHR), but not control virus, reduced marble burying (m) and increased time spent in the open arms of the EPM (n) in females exposed to CSIS (marble burying: Ctrl P = 1, NpHR P = 0.0355; EPM: P = 0.0205, n = 6 controls, n = 7 NpHR). Data are presented as means +/− SEM in (d). Multiple unpaired t-tests were used in (d). Paired Wilcoxon signed rank test with continuity correction was used in (m). Unpaired two-tailed t-test was used in (n). n denotes the number of mice in each group. Scale bars represent 100 μm. Schematics in (a), (g), (j) and (k) were generated using BioRender icons.

Fig. 5. ERα neurons in the MePD are a source of AVP.

a Schematic of intra-CeA injections in Avp-Flp::GFP mice to identify sources of AVP. b Co-expression of the viral mCherry reporter in GFP-labeled AVP^MePD neurons (n = 2). c GFP+ fibers from AVP^MePD neurons project toward tdTomato+ AVPR1A^CeA neurons in Avpr1a-Cre::tdTomato::Avp-Flp::GFP mice. d Experimental design to assess effects of MePD Avp knockdown (KD) on CSIS-induced anxiety-related behaviors. e Schematic of intra-MePD injections in WTs. f Reduced Avp in the CeA/MePD (P = 0.0219, n = 4). g, h Avp KD in the MePD reduced marble burying (g) and increased time in the open arms of the EPM (h) in females but not males exposed to CSIS (marble burying: group P = < 0.0001, sex P = 0.1257, interaction group*sex P = 0.0877; EPM: group P = 0.0045, sex P = 0.2467, interaction group*sex P = 0.1309, n = 11 control females, n = 12 KD females, n = 11 control males, n = 9 KD males). i Experimental design to assess effects of Esr1 deletion from AVP^MePD neurons on CSIS-induced anxiety-related behaviors. j Schematic of viral injection in Esr1^Flox/Flox::Avp-Flp::GFP females. k Reduced Esr1 in Avp-expressing MePD neurons in Cre-injected females (n = 4 Cre, n = 3 control). l, m Esr1 knockout from AVP^MePD neurons reduced marble burying (l) and increased time in the open arms of the EPM (m) in females and not males (marble burying: group P = 0.1335, sex P = 0.1243, interaction group*sex P = 0.0381, n = 9 control females, n = 8 Cre females, n = 9 control males, n = 5 Cre males; EPM: group P = 0.0543, sex P = 0.5661, interaction group*sex P = 0.1454, n = 8 females and control males, n = 5 for Cre males). Unpaired two-tailed t-test used in (f). Two-way ANOVA (with RM for marble burying) and Tukey’s multiple comparisons used in (g), (h), (l) and (m). Different letters denote significant differences. n denotes the number of mice per group. Scale bars represent 100 μm in (b) and (c), and 25 μm in (k). Schematics in (a), (d) and (I) were generated with BioRender icons.