Environmental enrichment requires adult neurogenesis to facilitate the recovery from psychosocial stress

Abstract

The subgranular zone of the adult hippocampal dentate gyrus contains a pool of neural stem cells that continuously divide and differentiate into functional granule cells. It has been shown that production of new hippocampal neurons is necessary for amelioration of stress-induced behavioral changes by antidepressants in animal models of depression. The survival of newly born hippocampal neurons is decreased by chronic psychosocial stress and increased by exposure to enriched environments. These observations suggest the existence of a link between hippocampal neurogenesis, stress-induced behavioral changes, and the beneficial effects of enriched environment. To show causality, we subjected transgenic mice with conditionally suppressed neurogenesis to psychosocial stress followed by environmental enrichment. First, we showed that repeated social defeat coupled with chronic exposure to an aggressor produces robust and quantifiable indices of submissive and depressive-like behaviors; second, subsequent exposure to an enriched environment led to extinction of the submissive phenotype, while animals exposed to an impoverished environment retained the submissive phenotype; and third, enrichment was not effective in reversing the submissive and depressive-like behaviors in transgenic mice lacking neurogenesis. Our data show two main findings. First, living in an enriched environment is highly effective in extinguishing submissive behavioral traits developed during chronic social stress, and second, these effects are critically dependent on adult neurogenesis, indicating that beneficial behavioral adaptations are dependent on intact adult neurogenesis.

Figure 1

(a) Immunohistochemistry against HSV-tk and GFAP in the hippocampal dentate gyrus (DG) of adult hGFAPtk mice reveals HSV-tk expression in GFAP⁺ cells. (b–d) The DG of control mice contains many doublecortin (DCX) positive neuroblasts (aqua) while these cells are virtually abolished (F_8,8=428.2, Student's t-test P<0.0001) in hGFAPtk mice treated with VGCV for 3 weeks (NG-, 26.67±7.688, n=9, gray bar; Controls, 1577±159.1, n=9, white bar) . Staining for Iba1 (purple) reveals no increase in activated microglia. Scale bars=30 μm. (e) Hematoxylin and Eosin (H&E) staining shows no indication of inflammation in the hippocampus of NG- mice treated with VGCV for >8 weeks. (f) Analysis of cell proliferation and short-term survival in the adult CNS of Ctrl and NG- mice. After >8 weeks of VGCV treatment mice were injected twice daily for 5 days with BrdU (200 mg kg^–1 body weight) and perfused 24 h after the last injection. BrdU⁺ cells were counted in the hippocampal DG and Cornu Ammonis regions (CA3 and CA1), in the hypothalamus (Hy) including the paraventricular nucleus (PVN), as well as in medio-prefrontal cortical regions (mPFCx), motor cortex (mCx) and the subventricular region of the lateral ventricles (SVZ). A decrease in BrdU⁺ cells was only detected in the DG (F_3,5=12.27, Student's t-test P=0.0155) and the SVZ (F_3,5=1.691, Student's t-test P<0.001). (Control, n=4, white bars, NG- n=6, black bars) Note that NG- mice still show a high rate of cell proliferation in the DG and SVZ. This is most likely due to the generally high proliferative capacity in these regions including cell types of non-neuronal lineages that do not undergo differentiation into DCX⁺ positive neuroblasts such as microglial cells, endothelial cells as well as oligodendrocyte progenitor cells.

Figure 2

Control and NG- mice show no differences in baseline physiology, no changes in baseline behavioral tests for locomotion, anxiety and depressive-like behavior, and no difference in olfactory habituation/dishabituation. hGFAPtk mice treated for 8 weeks with VGCV (NG-) do not show any differences in body weights (a), motor strength in the wire hang test (b) or novel cage stress-induced hyperthermia (c). NG- mice do not differ from controls in activity in or habituation to an open field (d), show no differences in anxiety or exploration in the open field or light–dark box test (d and e) and have normal saccharine preference (f). NG- mice do not differ from controls in an olfactory habituation dishabituation test for standard odors (g) nor for odors carrying a social connotation (h). Cotton tips dipped in different odors; water (w1-3), banana (b1-3), almond (a1-3) and imitation almond (ia1-3) were introduced to the animals homecage three consecutive times for 3 min. Both NG- and control mice spend the same amount of time sniffing and habituating to the different odors (g). Similarly, no difference was found between control and NG- mice sniffing and habituating to cotton tips dipped in urine from female animals in non-estrous (NE) or estrous (E) phase of their estrous cycle as well as urine from dominant (Dom) CD-1 animals and subordinate (Sub) C57Bl/6J animals (h).

Figure 3

(a) Timeline shows BrdU injections for studying survival of newborn neurons under different housing conditions. Animals were injected with BrdU (200 mg kg^–1) 1 × per day for 5 days, 1 week before being placed in either an enriched environment (EE) or an impoverished environment (IE) for 3 weeks before perfusion. In the social conflict (SC) condition, animals were injected 1 × per day for 5 days, 2 weeks before being placed in SC for 2 weeks before perfusion. (b) Newborn neurons were analyzed using double labeling for BrdU (green) and the neuronal marker, NeuN (red). Almost all BrdU-labeled cells also expressed NeuN. (c) Animals living in EE displayed significantly increased survival of newborn neurons as compared with either animals in IE or SC (EE, 2838±562.8, n=6, white bars; IE, 1291±164.8, n=5, gray bars; SC, 572.0±45.18, n=6, dark gray bars; F_2,14=10.98, P=0.0014; Tukey-Kramer post hoc test for EE vs IE P<0.05, for EE vs SC P<0.01). (d) Timeline shows BrdU injections for studying proliferation of newborn neurons under differential housing conditions. Animals were given a single injection of BrdU 3 h before being euthanized after having lived in either EE, IE or SC housing conditions for 2–3 weeks (EE, 1715±530.5, n=8, white bars; IE, 1578±634.2, n=8, gray bars; SC, 1709.0±499.2, n=8, dark gray bars).

Figure 4

Diagram depicting experimental groups and study design (a). An initial cohort consisting of control and NG- mice were first housed for 14 days under social conflict (SC) conditions during which time aggressive encounters were scored for 5 min day^–1. Following SC induction, all animals were removed from the SC living condition and divided into four groups: 1. Control animals living under EE conditions, 2. NG- animals living under EE conditions, 3. Control animals living under IE conditions and 4. NG- animals living under IE conditions. Following 3 weeks of either the EE or IE condition, all groups were re-exposed to SC for 14 days during which aggressive encounters were scored for 5 min day^–1. In addition, all groups underwent a behavioral battery during the final week of the SC condition (a). SC induction period (b–d): Chronic psychosocial stress and daily social defeat induce subordinate/submissive phenotype in Ctrl and NG- mice. The induction of submissive behavior was determined by; (b) percentage of conflicts won, (c) the number of approaches by the intruder mouse towards the resident dominant CD-1 mouse and (d) time spent immobile. As NG- (n=22) and control (n=22) mice accumulated social conflict defeats, aggressive behaviors declined and immobility increased. Ctrl and NG- mice showed no significant differences in the development of submissive behavior. SC Re-exposure (e–g): In previously submissive male mice, hippocampal neurogenesis and environmental enrichment opposes the development of subordinate phenotype during re-exposure to chronic psychosocial stress. Compared with all other groups, EE Ctrl mice showed; (d) a significant increase in percentage of conflicts won (significant effect of group [F_3,41=73.66; P<0.001], day [F_13,29=2.581; P<0.02] and a significant interaction between group × day [F_13,29=2.053; P<0.01], (e) a significant increase in approaches towards resident CD-1 mouse (significant effect of group [F_3,41=22.54; P<0.001], and (f) a significant decrease in time spent immobile during conflict sessions (significant effect of group [F_3,41=25.302; P<0.001], day [F_13,29=2.966; P<0.01]. ^*indicates significant difference (P<0.05) between EE Ctrl and all other treatment groups. ^**indicates significant difference (P<0.05) between EE Ctrl and IE Ctrl/IE NG- groups. ^***indicates significant difference (P<0.05) between Ctrl and NG- groups. N=11 per group. Data are expressed as mean±s.e.m. (h) EE exposed groups show higher levels of exploration in the first 15 min in a novel environment compared with all other treatment groups (significant effect of treatment: F_3,19=5.268, P<0.01). n=6 per group. (i) Only animals living in EE with intact neurogenesis showed preference for saccharine (values above 50% [horizontal line] indicate preference for saccharine solution above chance) (significant effect of treatment: F_3,38=3.539, P=0.0235). n=10–11 per group. (j) In the light–dark box test, animals that had lived in EE with intact neurogenesis spend less time in the dark compartment compared with all other groups (significant effect of treatment: F_3,39=12.41, P<0.0001). n=10–11 per group. Results are expressed as mean±s.e.m. ^*indicates significantly different (P<0.05) from all other treatment groups. One-way ANOVA followed by Newman Keuls post hoc test.

Figure 5

Social exploration of an aggressor mouse is restored by EE only when neurogenesis is intact. Mice were tested in two social interaction tests. In the first, mice were allowed to explore an open field containing two wire cups, one empty and one containing an unfamiliar mouse. In the second test the unfamiliar mouse was replaced with the known aggressor from the SC living conditions. (a–d) No difference in behavior was observed between treatment groups when mice were exposed to an unfamiliar mouse. All groups preferred (Interaction Quotient Duration on Mouse [s]/Duration on Object [s]≅2) interacting with an unfamiliar mouse compared with interacting with an inanimate object. (e–h) However, when the aggressor was presented EE NG- mice as well as both IE groups spend significantly less time with the mouse than the EE Ctrl group (significant effect of treatment; F_3,41=5.097, P<0.005, in f) The interaction quotient was also significantly higher in EE Ctrl as compared with all other groups (significant effect of treatment; F_3,41=7.553, P<0.001, in h). Results are expressed as mean±s.e.m. n=11; ^*indicates significantly different (P<0.05) from all other treatment groups. One-way ANOVA followed by Tukey-Kramer post hoc test.