Sex-Specific Timelines for Adaptations of Prefrontal Parvalbumin Neurons in Response to Stress and Changes in Anxiety- and Depressive-Like Behaviors

Cellular changes in female and male prefrontal PV⁺ neurons after 4 and 8 weeks of UCMS are indicative of sex-specific patterns of stress-induced increases in their activity. a, Left, Representative picture of immunofluorescent signal of PV (green), ΔFosB (red), and their colocalization in the PFC of mice. Right, Experimental timeline. b–d, PV/ΔFosB immunohistochemistry analyses. b, Increased percentage of prefrontal PV⁺ neurons expressing the marker of chronic activity, ΔFosB, in the whole mPFC after 4 and 8 weeks of UCMS in females [UCMS (4 weeks) vs controls, ***p = 0.0002; UCMS (8 weeks) vs controls, ***p = 0.0003], and after 8 weeks of UCMS in males [UCMS (8 weeks) vs controls, **p = 0.0022]. c, d, This is paralleled by subregion analyses wherein the PrL (c) UCMS did not change the percentage of PV⁺ neurons expressing ΔFosB in males but increased it in females [control vs UCMS (8 weeks) **p = 0.0046], and in the IL (d) 8 weeks of UCMS significantly increased percentage of PV⁺ neurons expressing ΔFosB in both males and females (males, **p = 0.0014; females, **p = 0.0024), while 4 weeks of UCMS increased this number in females (**p = 0.0020). We also observed significant sex differences, particularly after 4 weeks of UCMS, when females displayed more PV neurons expressing FosB than males in the whole PFC, PrL region, and IL region (whole PFC, ^ap = 0.0015; PrL, ^ap = 0.0085; IL, ^ap = 0.0167). N = 4–5/group [female: c, N = 4; UCMS (4 weeks), N = 4; UCMS (8 weeks), N = 4; male: c, N = 5; UCMS (4 weeks), N = 4; UCMS (8 weeks), N = 4]. Data are presented as the mean ± SEM.

Activation of prefrontal PV⁺ neurons in male mice affects the expression of anxiety-like and depressive-like behaviors under baseline and stressful conditions. Top left, Representative image of mCherry-DREADD virus expression in the PrL and IL regions of the PFC. Top right, Schematic representation of experimental design. a–d, In the EPM test, UCMS interacted with the chronic activation of prefrontal PV⁺ neurons to modulate the time spent in the open arms (a; *p = 0.0441; **p = 0.0052), but no other significant interactions were observed. e–h, In the OF test, UCMS decreased time in the center (e; *p = 0.011), increased thigmotaxis ratio (g; *p = 0.0244), and increased overall activity (h; **p = 0.0019). i, j, In the marble-burying test, a significant UCMS effect was driven by a higher number of marbles buried by UCMS/hM3DGq mice compared with their nonstressed controls (**p = 0.0038; i); a similar effect was observed in the splash test (j), with decreased time spent grooming by UCMS/hM3DGq mice compared with their nonstressed controls (**p = 0.0088). k, In the CPA test, significant effects of stress and virus were detected; Tukey’s post hoc analyses revealed that the aversion score of mice from the UCMS/hM3DGq was lower than that of control mice (*p = 0.021). N = 8–11/group (C/mCherry, N = 8; UCMS/mCherry, N = 11; C/hM3DGq, N = 8; UCMS/hM3DGq, N = 9). Data are presented as the mean ± SEM.

Altered neuronal excitability patterns of IL layer II/III PV⁺ neurons after 4 and 8 weeks of UCMS. a, Representative images of recorded neurons (left, position in slice; center, fluorescence indicating PV presence; right, IR-DIC image of patched neuron) and schematic of experimental design. b–d, Representative traces of PV⁺ neuron firing in male neurons showing the characteristic nonadapting firing patterns of PV⁺ neurons (control) at threshold (i.e., 50 pA, red) and suprathreshold (i.e., 400 pA, black) current injection (b) and altered firing patterns after 4 weeks [UCMS (4 weeks)] and 8 weeks [UCMS (8 weeks)] of UCMS (c, d). e, Male PV⁺ neurons showed no significant differences in the number of APs after either 4 or 8 weeks of UCMS at threshold current injections, but a significant reduction in the number of events at suprathreshold step currents (i.e., from 250 pA) after only 8 weeks of UCMS [#p < 0.001, control vs UCMS (8 weeks), Tukey’s post hoc test; exact p values: control vs UCMS (8 weeks): 250 pA, p = 0.0017; 300 pA, p = 0.0003; 350 pA, p = 0.0003; 400 pA, p = 0.0001]. f–h, Representative traces of PV⁺ neuron firing in female neurons showing characteristic PV⁺ neuron firing patterns in controls (f), as above, and alterations after UCMS (4 weeks) and UCMS (8 weeks; g, h). i, Female PV⁺ neurons showed maladaptive firing patterns with increased numbers of APs at lower current injections [*p < 0.05, Control vs UCMS (4 weeks), Tukey’s post hoc test; exact p values: control vs UCMS (4 weeks): 50 pA, p = 0.0016; 100 pA, p = 0.0009; 150 pA, p = 0.036], but a reduced number of events at higher, suprathreshold current injections [**p < 0.001, control vs UCMS (4 weeks); #p < 0.001, control vs UCMS (8 weeks), Tukey’s post hoc tests; exact p values: control vs UCMS (4 weeks): 300 pA, p = 0.036; 350 pA, p = 0.0059; 400 pA, p = 0.012; control vs UCMS (8 weeks): 350 pA, p = 0.021; 400 pA, p = 0.017]. j, Total number of APs fired across the 500 ms stimulus duration for all stimulus intensities decreased after 8 weeks of UCMS, regardless of sex [@p < 0.05, Control vs UCMS (8 weeks), Tukey’s post hoc test for main effect of UCMS; exact p value: p = 0.0090]. k, RMP was significantly increased after UCMS (8 weeks), independent of sex [@p < 0.05, control vs UCMS (8 weeks), Tukey’s post hoc test for main effect of UCMS, exact p = 0.0080]. l, Input resistance was significantly increased after UCMS (4 weeks), independent of sex [@p < 0.05, control vs UCMS (4 weeks), Tukey’s post hoc test for main effect of UCMS; exact p = 0.015]. Data in histograms are presented as the mean ± SEM for control (C), 4 weeks of UCMS (4 weeks) and 8 weeks of UCMS (8 weeks) by sex (male, green; female, orange). N = 4–6 mice/group, n = 11–19 recorded cells/group [females: C: N = 6, n = 19; 4 weeks: N = 5, n = 17; 8 weeks: N = 5, n = 15; males: C: N = 4, n = 14; 4 weeks: N = 4, n = 11; 8 weeks: N = 5, n = 17). Individual mice contributed 2–6 cells to the experiment (mean n per N, 3.23; median n per N, 3).