Ongoing neurogenesis in the adult dentate gyrus mediates behavioral responses to ambiguous threat cues

Abstract

Fear learning is highly adaptive if utilized in appropriate situations but can lead to generalized anxiety if applied too widely. A role of predictive cues in inhibiting fear generalization has been suggested by stress and fear learning studies, but the effects of partially predictive cues (ambiguous cues) and the neuronal populations responsible for linking the predictive ability of cues and generalization of fear responses are unknown. Here, we show that inhibition of adult neurogenesis in the mouse dentate gyrus decreases hippocampal network activation and reduces defensive behavior to ambiguous threat cues but has neither of these effects if the same negative experience is reliably predicted. Additionally, we find that this ambiguity related to negative events determines their effect on fear generalization, that is, how the events affect future behavior under novel conditions. Both new neurons and glucocorticoid hormones are required for the enhancement of fear generalization following an unpredictably cued threat. Thus, adult neurogenesis plays a central role in the adaptive changes resulting from experience involving unpredictable or ambiguous threat cues, optimizing behavior in novel and uncertain situations.

Fig 1. Behavioral response to ambiguous conditioned fear cues is decreased in adult neurogenesis-deficient mice.

(A) Examples of conditioned fear training and testing protocols. (B) In a cued fear conditioning task, a perfectly predictive tone cue (Reliable) elicited similar freezing in transgenic (TK) mice (n = 8), which lack adult neurogenesis, and wild-type (WT) controls (n = 6) (*, main effect of tone versus baseline F_1,12 = 48.6, p < 0.0001; no other significant effects). (C) A tone that coterminated with a shock only 50% of the time (ambiguous) increased freezing in both WT (n = 7) and TK mice (n = 9; main effect of tone, F_1,14 = 55.9, p < 0.0001; post hoc tests show tone greater than baseline, p < 0.005, in both genotypes). However, the tone increased freezing more in WT mice relative to TK mice (tone x genotype interaction, F_1,14 = 5.0, p = 0.04; †, post hoc testing indicates p < 0.05 for TK versus WT freezing during the tone). (D) Freezing responses to the reliably predictive tone cue (averaged across six trials for each session) were virtually identical in WT (n = 7) and TK (n = 8) mice during all extinction days. (E) After reliable cue training with a weak shock (0.3 mA compared to 0.5 mA in earlier experiments), WT (n = 11) and TK (n = 13) mice showed increased freezing to the tone (main effect of tone F_1,22 = 13.7, p = .001) but equivalent freezing across genotype (main effect of genotype F_1,22 = 0.007, p = .93), suggesting equivalent learning with reliable cues even with a weaker shock training protocol. (F) After fear conditioning, a reliable tone cue increased the startle response similarly in mice of both genotypes (*, main effect of tone F_1,20 = 4.7, p = 0.04, main effect of genotype F_1,20 = 0.016, p = .94; n = 11 for both groups). (G) An ambiguous cue increased startle in WT mice (n = 11) but not TK mice (n = 10) (tone x genotype interaction F_1,19 = 4.5, p = 0.047; †, post hoc testing indicates p < 0.05 versus WT at the same time point). Data are represented as mean ± standard error of the mean (SEM). The numerical data used in all figures can be found in S1 Data.

Fig 2. Activation of hippocampal granule and pyramidal neurons by ambiguously conditioned cues is altered in mice lacking adult neurogenesis.

(A) Confocal image of the hippocampus shows neurons that were active (Fos+, red) and inactive (blue counterstain) during fear conditioning in the dentate gyrus (DG), CA3, and CA1. (B) Two hours after the third session of tone-shock pairings, TK mice (n = 8) had fewer Fos+ cells than wild-type (WT) mice (n = 7) in the ambiguous cue condition but not the reliable cue condition (WT, TK n = 6, 5) across all hippocampal regions (cue type x genotype interaction F_1,22 = 6.3, p = 0.020; †, post hoc testing indicates p < 0.05 versus WT in the same condition/region). (C) Confocal image of Fos immunostaining (red) in BrdU+ (green) cell in the granule cell layer (gcl) shows a 4-wk-old granule neuron active during fear conditioning. (D) Adult-born granule neurons, labeled with BrdU, in WT mice were similarly activated by reliable (r) and ambiguous (a) cue training (t₁₀ = 1.1, p = 0.32); TK mice had no new neurons. (E) Confocal image of the amygdala shows Fos staining in the lateral/basolateral (LA/BLA) and central (CeA) nuclei of the amygdala. (F) The number of Fos+ LA/BLA cells was lower in the ambiguous cue condition relative to the reliable cue condition but there was no effect of genotype (main effect of predictor type F_1,22 = 5.0, p = 0.0363; main effect of genotype F_1,22 = 0.09, p = .7628; WT, TK n = 6, 5). No significant differences were observed across cue type or genotype in the CeA (all effects F_1,22 < 1.67, p > 0.2). Data are represented as mean ± standard error of the mean (SEM). The numerical data used in all figures can be found in S1 Data.

Fig 3. Fear conditioning effects on future behavior depend on cue reliability, adult neurogenesis, and adrenal hormones.

(A) Fear conditioning and anxiodepressive behavior testing timeline. (B) Latency to eat in the novelty-suppressed feeding (NSF) test was increased by reliably cued fear conditioning in TK mice (n = 20), but not wild-type (WT) mice (n = 19), relative to unshocked mice of both genotypes (WT, TK n = 19, 19; training effect F_1,73 = 4.1, p = 0.048; genotype effect F_1,73 = 3.0, p = 0.086; interaction F_1,73 = 2.0, p = 0.165; †, post hoc testing indicates p < 0.05 versus unshocked condition. (C) NSF latency was increased by ambiguous cue training, relative to reliable cue training, in WT mice but not TK mice. WT mice had longer latencies than TK mice after ambiguous cue training but had shorter latencies than TK mice after reliable cue training (predictor type x genotype interaction F_1,77 = 12.3, p = 0.0008; †, post hoc testing indicates p < 0.05 versus WT in the same condition; WT n = 20, 21; TK n = 20, 20 for reliable, ambiguous). (D) When mice were adrenalectomized, latency to eat was longer in TK mice than WT mice regardless of cue type (*, genotype main effect F_1,25 = 11.5, p = 0.002; †, post hoc testing indicates p < 0.05; ††, p < 0.1 versus WT in the same condition; WT n = 7,7; TK n = 9, 6 for reliable, ambiguous). Data are represented as mean ± standard error of the mean (SEM). The numerical data used in all figures can be found in S1 Data.