Continuous neurogenesis in the adult forebrain is required for innate olfactory responses

Abstract

Although the functional significance of adult neurogenesis in hippocampal-dependent learning and memory has been well documented, the role of such neurogenesis in olfactory activity is rather obscure. To understand the significance of adult neurogenesis in olfactory functions, we genetically ablated newly born neurons by using tamoxifen-treated Nestin-CreER(T2);neuron-specific enolase-diphtheria toxin fragment A (NSE-DTA) mice. In these mice, tamoxifen-inducible Cre recombinase allows the NSE (Eno2) gene to drive DTA expression in differentiating neurons, leading to the efficient ablation of newly born neurons in the forebrain. These mutant mice were capable of discriminating odors as competently as control mice. Strikingly, although control and mutant mice frequently showed freezing behaviors to a fox scent, a predator odor, mutant mice approached this odor when they were conditioned to associate the odor with a reward, whereas control mice did not approach the odor. Furthermore, although mutant males and females showed normal social recognition behaviors to other mice of a different sex, mutant males displayed deficits in male-male aggression and male sexual behaviors toward females, whereas mutant females displayed deficits in fertility and nurturing, indicating that sex-specific activities, which are known to depend on olfaction, are impaired. These results suggest that continuous neurogenesis is required for predator avoidance and sex-specific responses that are olfaction dependent and innately programmed.

Fig. 1.

Ablation of newly born neuron decreased the number of granule cells in the accessory olfactory bulb. (A and B) Experimental design. (C–L) Histology of the accessory olfactory bulb in oil- (control) and tamoxifen-treated (mutant) Nesin-CreER^T2;NSE-DTA mice. Sections were stained for NenN. (M) Quantification of the number of NeuN⁺ granule cells in the accessory olfactory bulb of oil- (control) and tamoxifen-treated (mutant) Nesin-CreER^T2;NSE-DTA mice. The average of three independent samples with a SD was shown at each time point. *P < 0.05, **P < 0.01, ***P < 0.001, t test. (Scale bar, 50 μm.)

Fig. 2.

Ablation of newly born neurons affected aversive responses to a predator odor in odor–reward association tests. At 2 or 10 mo after tamoxifen treatment, NSE-DTA (control, n = 6) and Nestin-CreER^T2;NSE-DTA (mutant, n = 6) mice were subjected to odor tests. (A) Habituation–dishabituation tests. Control and mutant mice were exposed to oil and TMT. When exposed to TMT, control and mutant mice frequently showed freezing behaviors and did not approach this odor. ns, not significant, t test. (B) Control and mutant mice were trained for 4 d to associate the reward (sugar grains) with TMT. On day 4, TMT with a sugar reward was presented under the bedding. A control mouse frequently showed freezing behaviors, whereas a mutant mouse was digging at the site of TMT. (C) Odor–reward association tests after 4 d of training. Mutant mice spent significantly more time digging at the site of TMT, a sugar-associated odor, than at the site of eugenol, a nonsugar-associated odor. By contrast, control mice did not approach either site. Mean digging time ± SEM during a 4-min test period is shown as a bar graph. ns, not significant; *P < 0.05, t test. Mutant behaviors were similar, irrespective of duration of inhibition of neurogenesis (A and C, at 2 or 10 mo after tamoxifen treatment).

Fig. 3.

Ablation of newly born neurons affected male-specific activities. At 2 or 10 mo after tamoxifen treatment, NSE-DTA (control, n = 13) and Nestin-CreER^T2;NSE-DTA (mutant, n = 14) mice, as well as wild-type mice (n = 10), were subjected to behavior tests. (A and B) Male–male aggression behavior tests. (C and D) Mounting behaviors of wild-type, control, and mutant males toward wild-type females. (E) Proportions of vaginal plug formation in wild-type females within 1 wk. The average with a SE (A–D) or the proportions (E) are shown. *P < 0.05, **P < 0.01, one-way ANOVA.

Fig. 4.

Ablation of newly born neurons affected female-specific activities. At 2 or 10 mo after tamoxifen treatment, NSE-DTA (control, n = 13) and Nestin-CreER^T2;NSE-DTA (mutant, n = 14) mice, as well as wild-type mice (n = 10), were subjected to behavior tests. (A and B) Mounting behaviors of wild-type males toward wild-type, control, and mutant females. (C) Proportions of vaginal plug formation by wild-type males within 1 wk. (D) Proportions of delivery by females that had vaginal plugs. The average with a SE (A and B) or the proportions (C and D) are shown. (E) Control females retrieved their offspring within 5 min, whereas mutant females treated with tamoxifen at 2 or 10 mo before did not. (F) The stomachs of the offspring of control females were full of milk, but this was not observed in the offspring of mutant females treated with tamoxifen at 2 or 10 mo before. (G) Proportions of survival of pups after postnatal day 1 (P1). ns, not significant; ***P < 0.001, one-way ANOVA.