Dorsal CA1 NECTIN3 Reduction Mediates Early-Life Stress-Induced Object Recognition Memory Deficits in Adolescent Female Mice

Abstract

Early-life stress (ES) leads to cognitive dysfunction in female adolescents, but the underlying neural mechanisms remain elusive. Recent evidence suggests that the cell adhesion molecules NECTIN1 and NECTIN3 play a role in cognition and ES-related cognitive deficits in male rodents. In this study, we aimed to investigate whether and how nectins contribute to ES-induced cognitive dysfunction in female adolescents. Applying the well-established limited bedding and nesting material paradigm, we found that ES impairs recognition memory, suppresses prefrontal NECTIN1 and hippocampal NECTIN3 expression, and upregulates corticotropin-releasing hormone (Crh) and its receptor 1 (Crhr1) mRNA levels in the hippocampus of adolescent female mice. Genetic experiments revealed that the reduction of dorsal CA1 (dCA1) NECTIN3 mediates ES-induced object recognition memory deficits, as knocking down dCA1 NECTIN3 impaired animals' performance in the novel object recognition task, while overexpression of dCA1 NECTIN3 successfully reversed the ES-induced deficits. Notably, prefrontal NECTIN1 knockdown did not result in significant cognitive impairments. Furthermore, acute systemic administration of antalarmin, a CRHR1 antagonist, upregulated hippocampal NECTIN3 levels and rescued object and spatial memory deficits in stressed mice. Our findings underscore the critical role of dCA1 NECTIN3 in mediating ES-induced object recognition memory deficits in adolescent female mice, highlighting it as a potential therapeutic target for stress-related psychiatric disorders in women.

Keywords: Cognition; Early-life stress; Female; Hippocampus; NECTIN3.

Fig. 1

Schematic of the experimental design. AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein; ES, early-life stress; EPM, elevated plus maze; GFP, green fluorescent protein; IHC, immunohistochemistry; i.p., intraperitoneal injection; LDB, light-dark box; mPFC, medial prefrontal cortex; NOR, novel object recognition test; OF, open field test; PND, postnatal day; SOR, spatial object recognition test; SPT, sucrose preference test; FST, forced swim test; TST, tail suspension test; TOM, temporal order memory test; WB, Western blot; Y-maze, Y-maze spontaneous alternation test.

Fig. 2

Early-life stress selectively impairs cognition in adolescent female mice. A Effect of early-life stress on body weight in female mice at PND9 and PND35. B, C Effect of early-life stress on Crh and Crhr1 mRNA levels in the mPFC (B) and dorsal hippocampus (C) of adolescent female mice. D–G Cognitive behavioral tests and results. The left panels in D–G depict schematics of the behavioral tasks. D In the novel object recognition test, control mice distinguish the novel object from the familiar one, whereas stressed mice do not, showing significantly poorer performance compared to controls. E In the spatial object recognition test, stressed mice fail to distinguish the displaced object from the stationary one, exhibiting a lower discrimination index than control mice. F In the temporal order memory test, stressed mice are unable to distinguish the remote object from the recent one, performing significantly worse than control mice. G Early-life stress has no significant impact on performance in the Y-maze spontaneous alternation test. H–J Anxiety-like behavioral tests and results. The left panels in H–J depict schematics of the behavioral tasks. Early-life stress does not significantly affect anxiety-like behaviors in the open field test (H) or light-dark box test (I). In the elevated plus maze test (J), while there is no significant change in the time spent in the open arms, stressed mice exhibit an increased latency to enter the open arms. K–N Depression-like behavioral tests and results. The left panels in K–N depict schematics of the behavioral tasks. Social interaction time in the social approach test (K), immobility time in the tail suspension test (L) and forced swim test (M), and sucrose preference in the sucrose preference test (N) are not significantly affected by early-life stress. Data are presented as the mean ± SEM. ^*P <0.05, ^**P <0.01, ^***P <0.001, comparisons between control (CT) and ES groups; ^#P <0.05, ^###P <0.001, one-sample t-test. AAR, alternative arm return; Crh, corticotropin-releasing hormone; Crhr1, corticotropin-releasing hormone receptor 1; CT, control; ES, early-life stress; PND, postnatal day; SAR, same arm return. See also Fig. S1.

Fig. 3

Early-life stress down-regulates prefrontal NECTIN1 and hippocampal NECTIN3 expression in adolescent female mice. A, B Western blots and analysis reveal that early-life stress significantly decreases NECTIN1 expression (A), but not NECTIN3 expression (B), in the mPFC of adolescent female mice. C, D Immunohistochemistry confirms that early-life stress reduces NECTIN1 (C), but not NECTIN3 (D), immunoreactivity in the mPFC of adolescent female mice. Representative images display NECTIN1 and NECTIN3 immunostaining in the mPFC of control and stressed mice. Scale bars, 100 µm and 10 µm. E, F Western blots and analysis show a reduction in NECTIN3 (F), but not NECTIN1 (E), protein levels in the dorsal hippocampus. Representative immunoblots are shown. G, H Representative images of NECTIN1 (G) and NECTIN3 (H) expression in the dorsal hippocampus of control and stressed mice. Immunostaining analysis demonstrates that ES selectively reduces NECTIN3 expression, but not NECTIN1 expression, in the dorsal hippocampus of adolescent female mice. Scale bars, 100 µm and 20 µm. Data are presented as the mean ± SEM. ^*P <0.05, ^**P <0.01, comparisons between control (CT) and early-life stress (ES) groups; ^&&P <0.01, stress effect by two-way ANOVA. Cg, cingulate cortex; CT, control; DG, dentate gyrus; dHP, dorsal hippocampus; ES, early-life stress; IL, infralimbic cortex; mPFC, medial prefrontal cortex; PrL, prelimbic cortex. See also Figs. S2 and S3.

Fig. 4

Knockdown of prefrontal NECTIN1 does not affect object recognition memory or working memory in adolescent female mice. A Left panel, schematic of AAV microinjection into the mPFC; right panel, representative image showing region-specific expression of EGFP in the mPFC. Scale bar, 200 µm. B Representative images of NECTIN1 and EGFP expression in the mPFC of EGFP and N1-KD mice. Scale bar, 10 µm. Immunostaining confirms knockdown-induced reduction in NECTIN1 expression in the mPFC. C Western blot analysis confirmed the knockdown-induced reduction in NECTIN1 expression in the mPFC. D–G Prefrontal NECTIN1 knockdown does not affect performance in the novel object recognition task (D), spatial object recognition task (E), temporal order memory task (F), or the Y-maze spontaneous alternation task (G). Data are presented as the mean ± SEM. ^*P <0.05, ^**P <0.01, comparisons between EGFP and N1-KD groups; ^###P <0.001, one-sample t-test. AAR, alternative arm return; EGFP, enhanced green fluorescent protein; N1-KD, Nectin1-knockdown; PND, postnatal day; SAR, same arm return. See also Fig. S4.

Fig. 5

NECTIN3 knockdown in the dCA1 replicates early-life stress-induced novel object recognition memory impairment in adolescent female mice. A–G Effects of NECTIN3 knockdown in the dCA1 on cognitive behavior. A Left panel, schematic of AAV microinjection into the dCA1; right panel, an image showing AAV spread, visualized by GFP expression in the dCA1 (scale bar, 200 µm). B Representative images showing GFP and NECTIN3 immunostaining in the dCA1 of GFP and N3-KD mice. Scale bar, 20 µm. N3-KD mice exhibited reduced NECTIN3 immunostaining compared with GFP mice. C Hippocampal NECTIN3 levels were markedly reduced in N3-KD mice compared to GFP controls. D In the novel object recognition task, GFP mice distinguished the novel object from the familiar one, while N3-KD mice failed to do so, performing significantly worse than GFP controls. NECTIN3 knockdown did not affect performance in the spatial object recognition task (E), temporal order memory task (F), or the Y-maze spontaneous alternation task (G). Data are presented as the mean ± SEM. ^*P <0.05, ^***P <0.001, unpaired t-test; ^#P <0.05, ^##P <0.01, ^###P <0.001, one-sample t-test. AAR, alternative arm return; GFP, green fluorescent protein; N3-KD, Nectin3-knockdown; PND, postnatal day; SAR, same arm return. See also Fig. S5.

Fig. 6

NECTIN3 overexpression in the dCA1 alleviates early-life stress-induced novel object recognition memory deficits in adolescent female mice. A–C Hippocampal NECTIN3 overexpression rescues ES-induced novel object recognition memory deficits in adolescent female mice. A Left panel, schematic of AAV microinjection into the dCA1; right panel, representative image showing region-specific EGFP expression in the dCA1 (scale bar, 200 µm). B Representative images showing NECTIN3 and EGFP expression in the dCA1 across the four groups of mice. Scale bar, 10 µm. Immunostaining analysis confirmed that viral injection significantly increased NECTIN3 levels in the dCA1. C In the novel object recognition task, overexpression of NECTIN3 in the dorsal CA1 reversed ES-induced novel object recognition memory deficits in adolescent female mice. Data are presented as the mean ± SEM. ^*P <0.05, ^***P <0.001, Bonferroni’s post hoc test; ^$$$P <0.001, viral effect by two-way ANOVA. AAR, alternative arm return; CT, control; EGFP, enhanced green fluorescent protein; ES, early-life stress; N3-OE, NECTIN3-overexpression; PND, postnatal day. See also Fig. S5.

Fig. 7

Acute Antalarmin treatment partially attenuates early-life stress-induced cognitive impairments in adolescent female mice. A Hippocampal Nectin3 levels are significantly higher in the ES-Anta group than in the ES-Veh group. B In the novel object recognition task, stressed mice treated with antalarmin successfully distinguished the novel object from the familiar one, whereas stressed mice treated with the vehicle failed to do so. C In the spatial object recognition task, mice in the ES-Anta group successfully identified the displaced object, unlike mice in the ES-Veh group. D In the temporal order memory task, both groups fail to recognize the remote object. Data are presented as the mean ± SEM. ^*P <0.05, ^**P <0.01, unpaired t-test; ^#P <0.05, ^##P <0.01, one-sample t-test. Anta, antalarmin; dHP, dorsal hippocampus; ES, early-life stress; PND, postnatal day; Veh, vehicle. See also Fig. S6.