Adult Hippocampal Neurogenesis Modulates Fear Learning through Associative and Nonassociative Mechanisms

Abstract

Adult hippocampal neurogenesis is believed to support hippocampus-dependent learning and emotional regulation. These putative functions of adult neurogenesis have typically been studied in isolation, and little is known about how they interact to produce adaptive behavior. We used trace fear conditioning as a model system to elucidate mechanisms through which adult hippocampal neurogenesis modulates processing of aversive experience. To achieve a specific ablation of neurogenesis, we generated transgenic mice that express herpes simplex virus thymidine kinase specifically in neural progenitors and immature neurons. Intracerebroventricular injection of the prodrug ganciclovir caused a robust suppression of neurogenesis without suppressing gliogenesis. Neurogenesis ablation via this method or targeted x-irradiation caused an increase in context conditioning in trace but not delay fear conditioning. Data suggest that this phenotype represents opposing effects of neurogenesis ablation on associative and nonassociative components of fear learning. Arrest of neurogenesis sensitizes mice to nonassociative effects of fear conditioning, as evidenced by increased anxiety-like behavior in the open field after (but not in the absence of) fear conditioning. In addition, arrest of neurogenesis impairs associative trace conditioning, but this impairment can be masked by nonassociative fear. The results suggest that adult neurogenesis modulates emotional learning via two distinct but opposing mechanisms: it supports associative trace conditioning while also buffering against the generalized fear and anxiety caused by fear conditioning.

Significance statement: The role of adult hippocampal neurogenesis in fear learning is controversial, with some studies suggesting neurogenesis is needed for aspects of fear learning and others suggesting it is dispensable. We generated transgenic mice in which neural progenitors can be selectively and inducibly ablated. Our data suggest that adult neurogenesis supports fear learning through two distinct mechanisms: it supports the ability to learn associations between traumatic events (unconditioned stimuli) and predictors (conditioned stimuli) while also buffering against nonassociative, anxiogenic effects of a traumatic experience. As a result, arrest of neurogenesis can enhance or impair learned fear depending on intensity of the traumatic experience and the extent to which it recruits associative versus nonassociative learning.

Keywords: adult neurogenesis; anxiety; dentate gyrus; doublecortin; fear conditioning; trace conditioning.

Figure 1.

HSV-TK expression in DCX-TK transgenic mice. A, Transgenic mice express HSV-TK under control of the dcx gene promoter. HSV-TK catalyzes the formation of GCV-triphosphate (tri-P), which prevents DNA replication and kills dividing cells. B–D, DCX-TK mice expressed HSV-TK in DCX+ cells in the DG (B, C) and around the lateral ventricle (D). E, Percentage of DCX+ cells expressing HSV-TK in the SGZ and SVZ. F, Percentage of HSV-TK+ cells expressing DCX. Scale bars: B, D, 100 μm; C, 10 μm.

Figure 2.

GCV administration to DCX-TK transgenic mice depletes DCX+ immature neurons in the DG but does not deplete putative quiescent DCX+ cells in the piriform cortex. A, DCX-TK and WT mice were treated with GCV or vehicle (PBS) for 2 weeks. Mice were injected with BrdU and killed 1 week later. B, Body mass during the 2 weeks of GCV administration did not differ among DCX-TK/GCV, DCX-TK/PBS, and WT/GCV mice. C, D, Representative images of DCX immunohistochemistry in the DG. E, Immunohistochemistry against HSV-TK in the piriform cortex of DCX-TK transgenic mice. F, Representative image of an HSV-TK+/DCX+ double-labeled cell in the piriform cortex. Most HSV-TK+ cells in the piriform cortex also expressed DCX. G, H, The number of DCX+ cells in anterior and posterior DG was greatly reduced in DCX-TK/GCV mice relative to controls. E, I, In contrast, HSV-TK+ cells in the piriform cortex were not ablated by GCV treatment in DCX-TK mice. Scale bars: C, D, 100 μm; E, F, 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001. CTX, cortex; I.C.V., intracerebroventricular.

Figure 3.

GCV administration to DCX-TK mice suppresses DG neurogenesis but not gliogenesis. A, Representative images of BrdU immunohistochemistry in the DG. B, Examples of BrdU+ cells colabeled with NeuN or GFAP. C, In both anterior and posterior DG, the number of BrdU+ cells was greatly reduced in DCX-TK/GCV mice relative to controls D, E, Quantification of BrdU/GFAP and BrdU/NeuN double-labeled cells. The proportion of BrdU+ cells expressing NeuN was reduced in DCX-TK/GCV mice relative to WT/GCV controls (D). However, the proportion of BrdU+ cells expressing GFAP did not differ between DCX-TK/GCV and WT/GCV mice (E). Scale bars: A, 100 μm; B, 5 μm. *p < 0.05, **p < 0.01.

Figure 4.

Markers of brain inflammation in DCX-TK and WT mice treated with GCV. A, B, Representative images of Iba-1 (A) or GFAP (B) immunohistochemistry in the DG. The insets show the high-magnification images. Tissue samples were collected 1 week after a 2-week GCV treatment. C, D, There was no significant difference in microglial activity DCX-TK/GCV and WT/GCV mice as indicated by Iba-1 intensity (C) and the number of Iba-1 expression cells (D). E, Intensity of the astroglial marker GFAP also did not differ between DCX-TK/GCV and WT/GCV mice. Scale bars: A, B, 100 μm; insets, 10 μm.

Figure 5.

Effects of DCX-TK-mediated arrest of adult neurogenesis on trace and delay fear conditioning. A, DCX-TK and WT mice were trained in delay or trace fear conditioning 1 week after a 2 week GCV treatment. B, During delay training, WT mice displayed a more rapid acquisition of freezing behavior than DCX-TK mice. C, In contrast, during trace training DCX-TK mice displayed a more rapid acquisition of freezing behavior. However, freezing levels in the final minute of the training session (after the final shock) were equivalent between the two genotypes in both Delay and Trace conditioning. D, E, Freezing during the context test. In trace (E) but not delay (D) conditioning, DCX-TK/GCV mice displayed significantly higher freezing than WT/GCV mice. F–I, Freezing in response to the tone CS in a novel context. H, I, Mean freezing during the baseline (20 s before tone presentation), during tone presentation, and during the 20 s after tone presentation. In delay conditioning (H), WT/GCV and DCX-TK/GCV displayed freezing in response to the tone, and there was no effect of genotype on freezing. Similarly, in trace conditioning (I), mice displayed freezing in response to the tone, and there was no genotype effect. *p < 0.05, ***p < 0.001. I.C.V., intracerebroventricular.

Figure 6.

Shock reactivity during trace fear conditioning. A, The number of crossings of the conditioning chamber did not differ between genotypes. B, The two genotypes showed similar behaviors during the shock-induced activity burst.

Figure 7.

Trace fear conditioning in vehicle-treated DCX-TK and WT mice. DCX-TK or WT mice were given trace conditioning 1 week after the 2 week PBS treatment. A, In the training session there was no effect of genotype on freezing. B, Freezing during the test of context-elicited fear. There was no effect of genotype on freezing. C, Freezing during the test for tone-elicited fear in a novel context. D, Mean freezing during the tone test. Both genotypes displayed freezing in response to the tone, and there was no effect of genotype on freezing; ***p < 0.001.

Figure 8.

Effect of x-irradiation-induced ablation of adult neurogenesis on delay and trace fear conditioning. A, Fear conditioning was conducted 6 weeks after the first of three doses of x-irradiation. B, C, DCX immunohistochemistry confirmed that DCX+ immature neurons were greatly reduced in x-irradiated mice. D, Freezing as a function of time during the tone test session. E, Mean freezing during the baseline period (20 s before presentation of the first tone), the tone presentations, and the 20 s following each tone presentation. In both delay and trace training, there was a significant effect of period but no effect of x-irradiation treatment or the interaction. F, Consistent with the DCX-TK experiments, in the context test, there was an effect of neurogenesis ablation in trace conditioning but not delay conditioning. In trace conditioning, x-irradiated mice displayed more context-elicited freezing than sham controls, but in delay conditioning the groups did not differ; *p < 0.05, **p < 0.01.

Figure 9.

Evidence for nonassociative tone-elicited freezing. DCX-TK/GCV and WT/GCV mice received shock-alone (“foreground”) contextual fear conditioning. A, B, There was no effect of genotype on freezing during the training session (A) or the context test (B). C, During the tone test in a novel context, both DCX-TK/GCV and WT/GCV mice displayed freezing in response to the tone. D, Mean freezing during the post-tone period exceeded that during the baseline and tone periods. DCX-TK/GCV mice significantly froze more than WT/GCV mice in general. E, F, Further analysis as a function of trial revealed that the DCX-TK/GCV mice significantly froze more than WT/GCV mice during the tone period (E). In the trace periods (F), there was a Genotype × Trial interaction, but the genotype effect did not reach significance; *p < 0.05; ***p < 0.001.

Figure 10.

Effect of fear conditioning (FC) on behavior in the open field. Separate groups of DCX-TK/GCV and WT/GCV mice were tested in the open field without prior fear conditioning or 3 d after fear conditioning. A, E, Occupancy plots for representative individual mice. A–D, Among mice without prior fear conditioning, there was no effect of genotype on open field behavior. E–H, In contrast, after fear conditioning, DCX-TK/GCV mice displayed reduced center time (F), center distance (G), and marginal distance (H) as compared with WT/GCV mice; *p < 0.05, **p < 0.01.

Figure 11.

Effect of fear conditioning on behavior in the elevated plus maze. DCX-TK/GCV and WT/GCV mice were tested in the elevated plus maze before fear conditioning (FC) and after fear conditioning. A–D, Before fear conditioning, there was no effect of genotype on activity in the open or closed arms. A, C, However, after fear conditioning, mice lacking neurogenesis exhibited less time (A) and traveled distance on the open arms (C); *p < 0.05.

Figure 12.

Effect of DCX-TK-mediated ablation in an alternate trace fear-conditioning procedure that minimized nonassociative tone freezing. DCX-TK and WT mice were treated with GCV for 2 weeks and subjected to trace fear conditioning either 3 weeks (A–E) or 6 weeks (F–M) after the start of GCV. A, F, Freezing during the test of context-elicited fear. There was no effect of genotype in 3 week (A) or 6 week (F) experiments. B–E, G-J, Freezing during the test for tone-elicited fear in a novel context. The freezing responses were analyzed as a function of trial during the CS, post-CS, and ITI periods. In the 3 week conditioning, DCX-TK mice displayed reduced fear during the CS and post-CS periods (C, D). In the 6 week condition, DCX-TK mice displayed reduced fear during the final CS presentation (H) and during the ITI periods (J). K–M, Depletion of DCX+ immature neurons in DCX-TK mice 6 weeks after the start of GCV. K, Representative images of DCX immunohistochemistry in the DG. The number of DCX+ cells in anterior (L) and posterior DG (M) was greatly reduced in DCX-TK/GCV mice relative to WT/GCV mice. Scale bars: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001.