Hippocampal neurogenesis mediates sex-specific effects of social isolation and exercise on fear extinction in adolescence

Abstract

Impaired extinction of conditioned fear is associated with anxiety disorders. Common lifestyle factors, like isolation stress and exercise, may alter the ability to extinguish fear. However, the effect of and interplay between these factors on adolescent fear extinction, and the relevant underlying neural mechanisms are unknown. Here we examined the effects of periadolescent social isolation and physical activity on adolescent fear extinction in rats and explored neurogenesis as a potential mechanism. Isolation stress impaired extinction recall in male adolescents, an effect prevented by exercise. Extinction recall in female adolescents was unaffected by isolation stress. However, exercise disrupted extinction recall in isolated females. Extinction recall in isolated females was positively correlated to the number of immature neurons in the ventral hippocampus, suggesting that exercise affected extinction recall via neurogenesis in females. Pharmacologically suppressing cellular proliferation in isolated adolescents using temozolomide blocked the effect of exercise on extinction recall in both sexes. Together, these findings highlight sex-specific outcomes of isolation stress and exercise on adolescent brain and behavior, and highlights neurogenesis as a potential mechanism underlying lifestyle effects on adolescent fear extinction.

Keywords: Adolescence; Exercise; Neurogenesis; Sex differences; Stress.

Graphical abstract

Fig. 1

Social isolation stress and exercise differentially alter extinction in adolescent males and females. a. Timeline: male and female P21 rats were reared in different conditions and underwent behavioral testing P43–P48. b. Weekly weights were unaffected by rearing conditions. Males were heavier than females at weeks 1–4. Females ran more than males at week 3 for c. grouped and week 4 for d. isolated rats. e & i. Isolated females, but not males, had a transient delay in acquiring CS-elicited freezing during conditioning. f, g, j & k. Exercise transiently delayed acquisition of extinction only in grouped females in extinction session 1 and 2. Extinction 2 was transiently delayed in isolated males. h. Isolated males froze more compared to grouped males at test. Exercise mitigated this effect. l. Exercise increased freezing only in isolated females. Values are means ± SEM. For weight and fear behaviors, n = 7–11 per group per sex. For running, grouped n = 3–4 boxes per sex and individual n = 9–10 boxes per sex. *Post hoc effect of Sex (p < 0.05). ^Post hoc effect of Environment (p < 0.05). ^#Post hoc effect of Exercise (p < 0.05). ^&Significant post hoc difference compared to all other groups (ps < 0.05). g, gram; km, kilometers; P, postnatal day; BL, baseline.

Fig. 2

Representative images of the dentate gyrus showing Ki-67⁺ cells (red), DCX⁺ cells (green), and double labelled Ki-67⁺DCX⁺ cells (yellow). Scale bar = 25 μm dHPC, dorsal hippocampus; vHPC, ventral hippocampus. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Voluntary wheel running increases hippocampal neurogenesis depending on isolation stress in males but not in females a, b, d & e. Exercise increased Ki-67⁺ and DCX⁺ cells in dHPC and vHPC in all groups except in isolated males. c. Pearson's correlation (r) values between average % CS-elicited freezing at Test and Ki-67⁺ or DCX⁺ cell counts. A signficant positive association exists in the vHPC of isolated female adolescents only. Color corresponds to the r value. f. As an example, individual data points displayed for the correlations between average % CS-elicited freezing at Test and vHPC DCX⁺ cell counts in isolated adolescents, showing that there is a signficant correlation in isolated females. ^#Exercise main or post hoc effect (p < 0.05). *Significant correlation (p < 0.05). Values are means ± SEM. n = 4–5 per group per sex. For correlations, n's were pooled across running condition. dHPC, dorsal hippocampus; vHPC, ventral hippocampus. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Social isolation stress and restricted voluntary wheel running activity alters extinction of learned fear in adolescent females. a. Timeline: females were reared in different conditions and underwent behavioral testing P43-48. b. Weekly weights of female rats were not affected by Environment and Restricted Exercise factors. Weekly mean daily running wheel activity was stable across weeks for c. grouped and d. isolated. e. During conditioning, isolated females had a transient delay in acquiring CS-elicited freezing compared to grouped females. f & g. All rats simarly decreased their CS-elicited freezing across extinction sessions 1 and 2 regardless of rearing condition. h. Restricted exercise impaired extinction recall in isolated females. Values are means ± SEM. For weight and fear behaviors, n = 7–9 per group per sex. For running data, grouped n = 3 boxes and individual n = 8 boxes per sex. ^Post hoc effect of Environment (p < 0.05). ^#Post hoc effect of Exercise (p < 0.05). g, gram; km, kilometers; P, postnatal day; BL, baseline.

Fig. 5

a, b,d & e. Restricted exercise increased Ki-67⁺ and DCX⁺ cells in dHPC and vHPC regardless of isolation stress. c. Pearson's correlation (r) values between average % CS-elicited freezing at Test and Ki-67⁺ or DCX⁺ cell counts. Color corresponds to the r value. f. A signficant positive correlation was observed between average % CS-elicited freezing at Test and vHPC DCX⁺ cell counts in isolated, but not grouped, females, showing that there is a signficant correlation in isolated females. ^#Exercise main effect (p < 0.05). *Significant correlation (p < 0.05). Values are means ± SEM. n = 3 per group per sex. For correlations, n's were pooled across running condition. dHPC, dorsal hippocampus; vHPC, ventral hippocampus. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

TMZ-treatment blocks the effect of exercise on fear extinction recall in isolated adolescent male and female rats. a. Timeline: isolated adolescents received either TMZ or VEH treatment and underwent behavioral testing P43-8. b. TMZ-treatment reduced weight only at week 2 only in males. c. Females ran more than males at week 2,3 and 4. d & h. Rats similarly acquired conditioned fear. e & i. Extinction 1 was comparable across groups. f & j. TMZ-treatment increased CS-elicited freezing at extinction 2 block 2 only in male adolescents. g. VEH-treated sedentary males froze more than their exercising counterparts when isolation-reared. TMZ-treatment blocked this effect of exercise. k. VEH-treated sedentary females froze less than their exercising counterparts when isolation-reared. TMZ-treatment blocked the effect of exercise. Values are means ± SEM. For running, weight and fear behaviors, n = 7–11 per group per sex. *Post hoc effect of Sex (p < 0.05), ⁺Post hoc effect of Drug (p < 0.05) ^#Post hoc effect of Exercise (p < 0.05). g, gram; km, kilometers; P, postnatal day; BL, baseline.

Fig. 7

Pharmacological supression of neurogenesis blocks the effect of exercise on extinction recall in male and female isolated adolescents. a. Representative images of the dHPC and vHPC dentate gyrus after VEH- or TMZ-treatment showing Ki-67⁺(red), DCX⁺ (green). Scale bar = 25 μm b-e. TMZ-treatment reduced Ki-67⁺ and DCX⁺ cells in the dHPC and vHPC in all groups. In females, TMZ prevented the exercise-induced increase in neurogenesis except for dHPC Ki-67⁺. f & g. Average % CS-elicited freezing at test and vHPC DCX⁺ cell counts were significantly and positively correlated for VEH-treated females. There were no other correlations. ⁺Main effect of Drug (p < 0.05). ^#Main or post hoc effect of Exercise (p < 0.05). *Significant correlation (p < 0.05). Values are means ± SEM. n = 3 per group per sex. For correlations, n's were pooled across running condition. dHPC, dorsal hippocampus; vHPC, ventral hippocampus; h, hilus; GC, granule cell layer. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)