A neuronal coping mechanism linking stress-induced anxiety to motivation for reward

Abstract

Stress coping involves innate and active motivational behaviors that reduce anxiety under stressful situations. However, the neuronal bases directly linking stress, anxiety, and motivation are largely unknown. Here, we show that acute stressors activate mouse GABAergic neurons in the interpeduncular nucleus (IPN). Stress-coping behavior including self-grooming and reward behavior including sucrose consumption inherently reduced IPN GABAergic neuron activity. Optogenetic silencing of IPN GABAergic neuron activation during acute stress episodes mimicked coping strategies and alleviated anxiety-like behavior. In a mouse model of stress-enhanced motivation for sucrose seeking, photoinhibition of IPN GABAergic neurons reduced stress-induced motivation for sucrose, whereas photoactivation of IPN GABAergic neurons or excitatory inputs from medial habenula potentiated sucrose seeking. Single-cell sequencing, fiber photometry, and optogenetic experiments revealed that stress-activated IPN GABAergic neurons that drive motivated sucrose seeking express somatostatin. Together, these data suggest that stress induces innate behaviors and motivates reward seeking to oppose IPN neuronal activation as an anxiolytic stress-coping mechanism.

Fig. 1.. Restraint stress increases IPN neuronal activity.

(A) Experimental design for recording GCaMP activity from IPN neurons in GAD2-Cre mice during no-stress and stress conditions. (B) Strategy for measuring plasma CORT. ELISA, enzyme-linked immunosorbent assay. (C) Plasma CORT measurements taken from no-stress (control) and restraint-stressed (5 min) mice (n = 6). (D) Recording protocol to determine changes in GCaMP fluorescence before, during, and after restraint stress. (E) Representative GCaMP recordings from pre-stress (left), stress (middle), and post-stress (right) conditions. Analysis of area under the curve (AUC) (F), maximum dF/F₀ (G), and mean peak amplitude (H) from GCaMP recordings (n = 20). (I) Representative images of IPN c-fos expression from no-stress (left) and restraint-stressed (right) mice (n = 3; bregma, −3.4 mm). (J) Analysis of c-fos expression across treatment groups expressed as number of neurons/mm² of IPN tissue. IPR, IPN rostral; IPL, IPN lateral; IPI, IPN intermediate; IPC, IPN caudal. Scale bars, 100 μm. Data presented as means ± SEM. Unpaired t tests or repeated-measures one-way ANOVAs with Tukey’s posttests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. Optical inhibition of IPN GAD2⁺ neurons reduces anxiety-like behavior following stress.

(A) NpHR or eYFP was injected into the IPN of GAD2-Cre mice. (B) Under no stress or following 5 min of restraint stress, NpHR and eYFP mice spent 5 min exploring the elevated plus maze (EPM) followed by 10 min exploring the open-field arena during light delivery into the IPN. (C) Time spent in open arms (top) and total arm entries (bottom) of the EPM (n= 7 to 9 per group). (D) Representative heatmaps showing location of eYFP and NpHR mice under no stress (left) and following restraint stress (right) in the open-field arena. (E) Time spent in the center and periphery (F) of the open-field arena (n = 6 to 8 per group). (G) Total distance traveled in the open-field arena. Scale bars, 100 μm. Data presented as means ± SEM. Two-way ANOVAs with Bonferroni’s posttests. *P < 0.05 and **P < 0.01.

Fig. 3.. Post-stress self-grooming behavior decreases activity of IPN GAD2⁺ neurons and increases activity of VTA DAT⁺ neurons.

(A) Experimental strategy for recording GCaMP activity from IPN neurons in GAD2-Cre mice during self-grooming behavior under no stress or stress conditions. (B) Time spent grooming before and following restraint stress (n = 20). (C) Representative GCaMP expression and recording location in the IPN (D) with GCaMP fluorescence and time-locked grooming bouts depicted in pink-shaded areas under no stress. (E) Z-score of GCaMP signal before and after onset of grooming bouts from all mice (n = 12) with arrowhead and blue area indicating onset of grooming. (F) Heatmap showing z-score of GCaMP signal before and after onset of grooming bouts from individual mice (one mouse per column). (G) Comparative analysis of z-scores before versus after onset of grooming bouts calculated from the average per mouse. (H) Restraint stress significantly reduced the latency to groom compared to the no-stress condition. (I) Representative photometry recording following restraint stress with time-locked grooming bouts depicted in pink-shaded areas. (J) Z-score of GCaMP signal before and after onset of grooming bouts from all mice (n = 20). (K) Heatmap showing z-score of GCaMP signal before and after onset of grooming bouts from individual mice. (L) Comparative analysis of z-scores before versus after onset of grooming bouts. (M) Strategy for recording GCaMP activity from VTA neurons in DAT-Cre mice (n = 11). (N) Representative GCaMP expression and fiber location in the VTA (O) with photometry recording and time-locked grooming bouts depicted in blue-shaded areas. (P) Z-score of GCaMP signal before and after onset of grooming bouts with arrowhead and pink area indicating the grooming onset. (Q) Heatmap and comparative analysis (R) of z-scores before versus after onset of grooming bouts in. Scale bars, 100 μm. Data presented as means ± SEM. Unpaired or paired two-tailed t tests. **P < 0.01 and ****P < 0.0001.

Fig. 4.. Optogenetic manipulation of IPN GAD2⁺ neuron activity influences self-grooming behavior.

(A) Grooming behavior was measured in NpHR or eYFP mice (n = 6 to 9 per group) during light-off and light-on conditions after stress and in ChR2 or eYFP mice (n = 6 to 7 per group) during delivery of blue light into the IPN. (B) Time spent grooming in NpHR and eYFP mice. (C) Time spent grooming in ChR2 and eYPF mice. Scale bars, 100 μm. Data presented as means ± SEM. Two-way ANOVAs with Bonferroni’s posttests or unpaired t test. **P < 0.01 and ***P < 0.001.

Fig. 5.. Responses of IPN GAD2⁺ neurons during sucrose consumption in mice without food restriction, during food restriction and following restraint stress.

(A) GCaMP fluorescence of IPN neurons was recorded from GAD2-Cre mice without food restriction, during food restriction (B) and following restraint stress (C). [(A) to (C), middle panels] Representative GCaMP recordings with time-locked periods of sucrose consumption depicted in blue-shaded areas. [(A) to (C), right panels] Z-score of GCaMP signal before and after onset of sucrose consumption bouts from all mice (n = 6) under each condition. (D) Event plot showing z-scores across treatments. (E) Comparative analysis of sucrose responses across conditions. Data presented as means ± SEM. Repeated-measures one-way ANOVAs with Tukey’s posttests. *P < 0.05 and **P < 0.01.

Fig. 6.. Optical silencing of IPN GAD2⁺ neurons blocks stress-induced increases in motivated sucrose seeking, and optical activation of IPN GAD2⁺ neurons and MHb-IPN inputs enhances motivated sucrose seeking.

(A) Experimental timeline for sucrose drinking in the dark, operant training, and PR testing of sucrose motivation under no-stress (NS) or stress (S) conditions. (B) Motivated sucrose-seeking (PR) sessions were conducted during light-off and light-on conditions in GAD2-Cre mice injected with NpHR or eYFP. (C) Sucrose breakpoint measured in NpHR mice without stress and following restraint stress in the absence or presence of light delivered to the IPN (n = 9). (D) Sucrose breakpoint measured in NpHR mice with no stress in light-off and light-on conditions (n = 7). (E) Sucrose breakpoint measured in eYFP mice following stress with and without light (n = 6). (F) Motivated sucrose-seeking sessions were conducted during light-off and light-on conditions in the IPN of GAD2-Cre mice injected with ChR2 or eYFP. (G) Sucrose breakpoint measured in ChR2 and eYFP mice in light-off and light-on conditions (n = 6 to 8 per group). (H) PR sessions were performed in ChAT-Cre mice with ChR2 or eYFP injected into the MHb and during no-light or light delivery into the IPN with or without stress exposure. (I) Sucrose breakpoints measured during no-light or light stimulation of MHb-IPN terminals in ChAT-Cre ChR2-expressing mice with and without stress (n = 10). (J) Sucrose breakpoints measured during light-off and light-on conditions in ChAT-Cre eYFP-expressing mice with and without stress (n = 7). Scale bars, 100 μm. Data presented as means ± SEM. Paired t test, repeated-measures one-way ANOVAs with Tukey’s posttests, or repeated measures two-way ANOVAs with Bonferroni’s posttests. *P < 0.05 and **P < 0.01.

Fig. 7.. A subpopulation IPN GABAergic neurons containing SST is activated by restraint stress and control stress-mediated increases in motivated sucrose seeking.

(A) Experimental design for scRNA-seq of IPN neurons from no-stress and stressed mice. (B) UMAP of IPN neuronal populations. (C) Comparison of c-fos expression across neuronal subclusters from control (no restraint) and restraint-stressed mice (n = 5). (D) Volcano plot identifying enriched SST expression within the neuronal cluster exhibiting increased c-fos expression in stressed mice. (E) Experimental strategy for recording GCaMP signal from IPN neurons of SST-Cre mice. (F) Representative GCaMP recordings from SST-Cre mice (n = 10) during no-stress (left), stress (middle), and post-stress (right) conditions. Analysis of AUC (G), maximum dF/F₀ (H), and mean peak amplitude (I) from GCaMP recordings. (J) PR sessions were performed during light-off and light-on conditions in SST-Cre mice injected with NpHR or eYFP. (K) Sucrose breakpoint measured in NpHR mice (n = 8) without stress and following restraint stress in the absence or presence of light delivered to the IPN. (L) Sucrose breakpoint measured in eYFP mice (n = 7) with no stress and following restraint stress during light-off and light-on conditions. Scale bars, 100 μm. Data presented as means ± SEM. Wilcoxon rank sum test, unpaired t test, or repeated-measures one-way ANOVAs with Tukey’s posttests. *P < 0.05 and **P < 0.01.