Dorsal CA3 overactivation mediates witnessing stress-induced recognition memory deficits in adolescent male mice

Abstract

Witnessing violent or traumatic events is common during childhood and adolescence and could cause detrimental effects such as increased risks of psychiatric disorders. This stressor could be modeled in adolescent laboratory animals using the chronic witnessing social defeat (CWSD) paradigm, but the behavioral consequences of CWSD in adolescent animals remain to be validated for cognitive, anxiety-like, and depression-like behaviors and, more importantly, the underlying neural mechanisms remain to be uncovered. In this study, we first established the CWSD model in adolescent male mice and found that CWSD impaired cognitive function and increased anxiety levels and that these behavioral deficits persisted into adulthood. Based on the dorsal-ventral functional division in hippocampus, we employed immediate early gene c-fos immunostaining after behavioral tasks and found that CWSD-induced cognition deficits were associated with dorsal CA3 overactivation and anxiety-like behaviors were associated with ventral CA3 activity reduction. Indeed, chemogenetic activation and inhibition of dorsal CA3 neurons mimicked and reversed CWSD-induced recognition memory deficits (not anxiety-like behaviors), respectively, whereas both inhibition and activation of ventral CA3 neurons increased anxiety-like behaviors in adolescent mice. Finally, chronic administration of vortioxetine (a novel multimodal antidepressant) successfully restored the overactivation of dorsal CA3 neurons and the cognitive deficits in CWSD mice. Together, our findings suggest that dorsal CA3 overactivation mediates CWSD-induced recognition memory deficits in adolescent male mice, shedding light on the pathophysiology of adolescent CWSD-induced adverse effects and providing preclinical evidence for early treatment of stress-induced cognitive deficits.

Fig. 1. Effects of chronic witnessing social defeat stress on cognition and anxiety-like behaviors in adolescent mice.

A The experimental timeline of the cognitive and anxiety-like behavioral tests after chronic witnessing social defeat stress (CWSD). B During the 10-day stress exposure, CWSD mice exhibited less weight gain than control mice. C CWSD mice showed reduced social interaction with CD-1 mice. CWSD mice showed significantly lower discrimination index than control mice in novel object recognition (D) and spatial object recognition (E) tests. Left, representative trajectory maps for CT and CWSD mice during the test phase in each task; warm colors represent more time. F CWSD mice had a lower spontaneous alternation ratio in the Y-maze test than control mice. Left, diagram of Y-maze apparatus; middle, spontaneous alternation ratio (SA); right, percentage of erroneous responses: alternative arm return (AAR) and same arm return (SAR). G In the open field test, CWSD mice spent less time in the center zone and traveled significantly less than control mice. Left, representative motion path in the open field test; middle, time spent in center zone; right, total distance traveled in 10 min. H In the light-dark box test, CWSD mice spent significantly less time in the light box. Left, representative motion path in the light chamber of the light-dark box; right, time spent in the light chamber. I In the elevated-plus maze test, CWSD did not affect the time spent in the open arm. Left, representative trajectory map in the elevated plus maze test; warm colors represent more time; right, time spent in open arms. AAR alternative arm return, CT control, CWSD chronic witnessing social defeat stress, EPM elevated plus maze, LDB light-dark box, NOR novel object recognition, OFT open field test, PND postnatal day, SA spontaneous alteration, SAR same arm return, SOR spatial object recognition. ^* p < 0.05, ^** p < 0.01^{, ***} p < 0.001; ^### p < 0.001.

Fig. 2. Neural alterations in dorsal and ventral CA3 in adolescent mice exposed to chronic witnessing social defeat stress (CWSD).

CWSD increased the number of c-fos⁺ neurons in dCA3 (not vCA3) after the spatial object recognition test (A) and decreased the number of c-fos⁺ neurons in vCA3 (not in dCA3) after the light-dark box test (B). Representative images showing the expression of c-fos and DAPI in the dCA3 (upper) and vCA3 (lower) of control and CWSD mice. Scale bar, 30 µm. CWSD significantly upregulated VGluT1 and VGAT expression in dCA3 (C), and significantly downregulated the VGluT1/VGAT ratio in vCA3 (D). Upper, representative images show the expression of VGluT1 and VGAT in dCA3 and vCA3 in CT and CWSD groups. Scale bar, 50 µm or 10 µm. Lower, the relative optical density of VGluT1 (left), VGAT (middle), and their ratio (right) in dCA3 and vCA3 in both groups. Adolescent CWSD reduced the length and complexity of apical dendrites of pyramidal neurons in dCA3 (E–G) and vCA3 (I–K). E, I representative tracings of neurons from the two groups. Scale bar, 50 µm. Adolescent CWSD reduced the spine density on apical dendrites in dCA3 (H) and vCA3 (L), especially in thin and stubby spines. The total spine density was calculated as the sum of three spine types. Scale bar, 10 µm. CT control, CWSD chronic witnessing social defeat stress, dCA3 dorsal CA3, LDB light-dark box, SOR spatial object recognition, vCA3 ventral CA3, VGAT vesicle GABA transporter, VGluT1 vesicle glutamate transporter-1. ^* p < 0.05, ^** p < 0.01^{, ***} p < 0.001; ^& p < 0.05, ^&&& p < 0.001.

Fig. 3. Dorsal CA3 neuronal activity mediates witnessing stress-induced recognition memory deficits in adolescent mice.

A The experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after hM3D(Gq) viral injection. B Left panel, an image showing region-specific expression of mcherry in dCA3; right panel, a schematic of activation virus (AAV-Gq) microinjection into the dCA3 of adolescent mice. Scale bar, 500 µm. C Representative images show the expression of DAPI, c-fos, and mcherry in the dCA3 of control and Gq mice. Immunostaining analyses confirmed that Gq-induced an increase in the expression of c-fos in the dCA3. Scale bar, 20 µm. Gq mice exhibited impaired recognition memory in the novel object recognition task (D), in the spatial object recognition task (E), and in the Y-maze spontaneous alternation (F). G In the open field test, the two groups spent similar time in the central zone, despite Gq mice traveling a greater total distance. H The experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after hM4D(Gi) viral injection. I Left panel, an image shows region-specific expression of mcherry in the dCA3; right panel, schematic of inhibition virus (AAV-Gi) microinjection into the dCA3 of adolescent mice. Scale bar, 500 µm. J Representative images show the expression of DAPI, c-fos, and mcherry in the dCA3 of four groups. Scale bar, 20 µm. K During the spatial object recognition test, CWSD increased the number of c-fos⁺ neurons in dCA3, while Gi virus reduced it. CWSD-induced deficits of novel object recognition memory (L) and spatial object recognition memory (M) were reversed by inhibition of dCA3 neurons. N In the Y-maze test, CWSD-induced spatial working memory deficits were not reversed by inhibition of dCA3 neurons. O In the open field test, CWSD-induced higher anxiety levels were not relieved by inhibition of dCA3 neurons. CNO Clozapine N-oxide, CT control, CWSD chronic witnessing social defeat stress, NOR novel object recognition, OFT open field test, PND postnatal day, SA spontaneous alteration, SOR spatial object recognition. ^* p < 0.05, ^** p < 0.01^{, ***} p < 0.001; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001; ^& p < 0.05, ^&&& p < 0.001; ⁺⁺ p < 0.01.

Fig. 4. Inhibition or activation of vCA3 neuronal activity increases anxiety in adolescent male mice.

A The experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after hM4D(Gi) viral injection. B Left panel, an image shows region-specific expression of mcherry in vCA3; right panel, schematic of activation virus (AAV-Gi) microinjection into the vCA3 of adolescent mice. Scale bar, 500 µm. C Representative images show the expression of DAPI, c-fos, and mcherry in the vCA3 of control and Gi mice. Immunostaining analyses confirmed Gi-induced reduction the number of c-fos⁺ neurons in the vCA3. Scale bar, 20 µm. D In the open field test, inhibition of vCA3 neurons decreased the time spent in the central zone. E Compared with control mice, Gi mice spent less time in the light box in the light-dark box (LDB) test. F In the elevated-plus maze test, inhibition of vCA3 neurons did not affect the time spent in the open arm. G In the spatial object recognition test, the two groups showed comparable spatial memory. H The experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after hM3D(Gq) viral injection. I Left panel, an image showing region-specific expression of mcherry in vCA3; right panel, schematic of inhibition virus (AAV-Gq) microinjection into the vCA3 of adolescent mice. Scale bar, 500 µm. J Representative images show the expression of DAPI, c-fos, and mcherry in the vCA3 of four groups. Scale bar, 20 µm. K During the LDB test, CWSD decreased the number of c-fos⁺ neurons in vCA3, while the two Gq groups exhibited increased number of c-fos⁺ neurons. L Activation of vCA3 neurons decreased the time spent in the center zone, increased the total distance traveled, and did not reverse CWSD-induced higher anxiety levels in the open field test. M In the LDB test, activation of vCA3 neurons significantly reduced the time spent in the light box, increased the latency to the light box, and did not reverse CWSD-induced higher anxiety levels in adolescent mice. N Activation of vCA3 neurons did not restore CWSD-induced spatial memory loss, but instead impaired spatial memory. CNO Clozapine N-oxide, CT control, CWSD chronic witnessing social defeat stress, EPM elevated plus maze, LDB light-dark box, OFT open field test, PND postnatal day, SOR spatial object recognition. ^* p < 0.05, ^** p < 0.01^{, ***} p < 0.001; ^## p < 0.01, ^### p < 0.001; ^& p < 0.05, ^&&& p < 0.001; ⁺ p < 0.05, ⁺⁺⁺ p < 0.001.

Fig. 5. Chronic systemic administration of vortioxetine reverses CWSD-induced recognition memory deficits and dCA3 neuronal over-activation.

A The experimental timeline of the vortioxetine injection and behavioral tests after stress exposure. B Vortioxetine did not reverse CWSD-induced social avoidance. CWSD-induced deficits of novel object recognition memory (C) and spatial object recognition memory (D) were reversed by chronic vortioxetine administration, while spatial working memory deficits were not restored (E). F In the LDB test, CWSD-induced higher anxiety levels were not alleviated by vortioxetine. G, H Vortioxetine reversed CWSD-induced dCA3 neuronal over-activation during the spatial object recognition test, not vCA3 neuronal de-activation during the LDB test. Representative images show the expression of c-fos and DAPI in the dCA3 in the four groups of animals. Scale bar, 30 µm. CT control, CWSD chronic witnessing social defeat stress, LDB light-dark box, NOR novel object recognition, PND postnatal day, SA spontaneous alteration, SOR spatial object recognition, Veh vehicle, Vort vortioxetine. ^* p < 0.05, ^** p < 0.01^{, ***} p < 0.001; ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001.