GABAergic control of adult hippocampal neurogenesis in relation to behavior indicative of trait anxiety and depression states

Abstract

Stressful experiences in early life are known risk factors for anxiety and depressive illnesses, and they inhibit hippocampal neurogenesis and the expression of GABA(A) receptors in adulthood. Conversely, deficits in GABAergic neurotransmission and reduced neurogenesis are implicated in the etiology of pathological anxiety and diverse mood disorders. Mice that are heterozygous for the gamma2 subunit of GABA(A) receptors exhibit a modest functional deficit in mainly postsynaptic GABA(A) receptors that is associated with a behavioral, cognitive, and pharmacological phenotype indicative of heightened trait anxiety. Here we used cell type-specific and developmentally controlled inactivation of the gamma2 subunit gene to further analyze the mechanism and brain substrate underlying this phenotype. Heterozygous deletion of the gamma2 subunit induced selectively in immature neurons of the embryonic and adult forebrain resulted in reduced adult hippocampal neurogenesis associated with heightened behavioral inhibition to naturally aversive situations, including stressful situations known to be sensitive to antidepressant drug treatment. Reduced adult hippocampal neurogenesis was associated with normal cell proliferation, indicating a selective vulnerability of postmitotic immature neurons to modest functional deficits in gamma2 subunit-containing GABA(A) receptors. In contrast, a comparable forebrain-specific GABA(A) receptor deficit induced selectively in mature neurons during adolescence lacked neurogenic and behavioral consequences. These results suggest that modestly reduced GABA(A) receptor function in immature neurons of the developing and adult brain can serve as a common molecular substrate for deficits in adult neurogenesis and behavior indicative of anxious and depressive-like mood states.

Figure 1.

Lack of Cre-induced recombination in PAV-positive interneurons, a major subpopulation of GABAergic interneurons. Brain sections of Emx1Cre × Z/EG (a–e) and CaMKIICre2834 × Z/EG mice (f–j) were stained for PAV and tested for colocalization of PAV (red) and Cre-mediated GFP (green) expression. Representative images are shown for the hippocampal CA1 region (a, f), dentate gyrus (DG; b, g), neocortex (CTX; c, h), amygdala (d, i), and striatum (e, j). Arrows point to GFP-positive cells that were subject to Cre-induced recombination, and arrowheads point to PAV-positive interneurons. Images represent stacks of confocal images. Note the lack of overlap between PAV- and GFP-positive cell populations. Consistent with published results, GFP-positive cells in Emx1Cre × Z/EG sections suggest recombination in both glia and glutamatergic neurons, whereas Cre-induced expression of GFP in CaMKIICre2834 × Z/EG brain sections is selective for glutamatergic neurons, as expected.

Figure 2.

Quantitation of GABA_AR deficits in Emx1Cre × fγ2/+ and CaMKIICre2834 × fγ2/+ mice by [³H]flumazenil autoradiography and phosphorimage analysis. a, Representative autoradiographs of brain sections from 10-week-old Emx1Cre × fγ2/+ mouse and a fγ2/+ littermate control, together with the density of BZ binding sites determined in different brain regions. The density of [³H]flumazenil binding sites in brain sections of Emx1Cre × fγ2/+ was reduced in neocortex, CA1 region of hippocampus (t₍₃₈₎ = 4.6 and 4.4, respectively, t test), as well as in dentate gyrus (U = 21), striatum (U = 13), and amygdala (U = 10) (Mann–Whitney test) compared with fγ2/+ controls. The number of BZ sites was unchanged in thalamus, globus pallidus, and cerebellum (U > 26). b, Representative sections of CaMKIICre2834 × fγ2/+ and control brains and results of quantitation of [³H]flumazenil binding sites as in a. The density of [³H]flumazenil sites in CaMKIICre2834 × fγ2/+ mice was reduced in neocortex (t₍₃₈₎ = 3.0), CA1 (t₍₃₈₎ = 7.5), dentate gyrus (U = 19), striatum (U = 8.0), and amygdala (U = 8.0) compared with fγ2/+ littermate controls. BZ site densities in thalamus, globus pallidus, and cerebellum were unaltered (U > 31). Results represent means ± SEM (n = 9–20 per genotype; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3.

Normal anxiety-related behavior of pseudo-wt mice. a, In the free-choice exploration test, fγ2/+ mice were indistinguishable from wt littermates (+/+) with respect to the number of retractions from entering a novel unit, the total time spent in the novel units, and the number of familiar units visited (for the three variables, t₍₃₆₎ < 2.00, Student's t test; n = 19 per group). b, In the same test, Emx1Cre mice were indistinguishable from fγ2/+ littermates (for the three variables, t₍₃₆₎ < 1.00; n = 18–20). c, In the light–dark choice test, Emx1Cre mice did not differ from fγ2/+ controls with respect to total time spent in the lit area and the number of light–dark transitions (t₍₃₇₎ < 1.00; n = 19–20).

Figure 4.

GABA_ARs act during development to control trait anxiety. a–c, Heightened anxiety-related behavior of Emx1Cre × fγ2/+ mice. In the free-choice exploration test (a), Emx1Cre × fγ2/+ mice made more retractions from entering a novel unit after the removal of the partition (U = 10.5; p < 0.01; n = 8–10 per genotype), and, once they entered, they spent less time in the novel compartment (U = 8; p < 0.01) than fγ2/+ littermate controls. The mean number of familiar units visited was similar in the two groups (U = 28.5; NS). In the light–dark choice test (b), the mean time spent in the lit area was significantly lower in Emx1Cre × fγ2/+ (n = 9) than in fγ2/+ littermate control mice (n = 22) (p < 0.05, least significant difference test after unweighted mean genotype × area ANOVA with area as within-subject factor, F_(1,29) = 4.0; p = 0.05). The group difference was not significant for the mean number of light–dark transitions (t_(13.17) = 1.8; p < 0.1, t test with separate variances). In the elevated plus maze (c), the mean proportion of both entries and time spent on the open arms over 5 min were decreased in Emx1Cre × fγ2/+ compared with littermate control mice (U = 19.5 and 20, respectively; p < 0.01; n = 10–14 per genotype). d–f, The anxiety-related behavior of CaMKIICre2834 × fγ2/+ mice and littermate fγ2/+ controls was tested under the same conditions. In the free-choice exploration test (d), CaMKIICre2834 × fγ2/+ mice behaved as controls with respect to mean number of retractions (t_(36.99) = 1.5; n = 22–30 per genotype) and mean time spent in the novel compartment (t_(45.58) = 0.3). In the light–dark choice test (e), CaMKIICre2834 × fγ2/+ mice were indistinguishable from controls for the mean time spent in the lit area and the mean number of light–dark transitions (U > 34; n = 10 per genotype). In the elevated plus maze (f), CaMKIICre2834 × fγ2/+ mice did not differ from controls with respect to the mean proportion of entries and time spent on the open arms and the number of closed arm entries (t₍₃₄₎ ≤ 0.7; n = 18 per genotype). All experiments were performed with females reared in group-housed cages. Data are representative of two to four experiments each. Values shown represent group means ± SEM. g–i, The anxiety-related behavior of 4- to 6-month-old CaMKIICre2834 × fγ2/+ mice was assessed as in d–f. In the free-choice exploration test (g), 6-month-old CaMKIICre2834 × fγ2/+ were indistinguishable from fγ2/+ littermate controls with respect to the number of retractions from entering a novel unit, the total time spent in the novel units, and the number of familiar units visited (for the three variables, U > 60; n = 9–16 per genotype). Behavior in the light–dark choice test (h) of CaMKIICre2834 × fγ2/+ mice (n = 7) assessed at 4 months of age, mice did not differ from fγ2/+ (n = 21) with respect to both the total time spent in the lit area and the number of light–dark transitions (U > 45). Similarly, behavior in the elevated plus maze (i) of 4-month-old CaMKIICre2834 × fγ2/+ mice (n = 7) was indistinguishable from fγ2/+ littermates (n = 21) with respect to the number of open and closed arm entries and time spent on the open arms (U > 38). Data represent means ± SEM.

Figure 5.

A developmental deficit in γ2 subunit-containing GABA_ARs results in greater inhibition in two behavioral assays sensitive to antidepressant drug treatment. The three different mouse lines (γ2^+/−, Emx1Cre × fγ2/+, and CaMKIICre2834 × fγ2/+) together with their respective littermate controls (wt and fγ2/+ mice, respectively) were subjected to the modified forced swim (Lucki, 1997) (a–c) and novelty-suppressed feeding (Santarelli et al., 2003) (d–f) tests. a, In the forced swim test, γ2^+/− mice started floating significantly earlier and spent more time immobile than wt controls (U = 13.0 and 20.0, respectively; p < 0.05; n = 9 per genotype). b, Tested under the same conditions, Emx1Cre × fγ2/+ mice did not differ significantly from fγ2/+ with respect to mean time to first immobility (U = 26) but exhibited an increase in the mean time spent immobile compared with controls (U = 2.5; n = 6–14; p < 0.001). c, CaMKIICre2834 × fγ2/+ mice were indistinguishable from fγ2/+ littermates for both parameters. d, In the novelty-suppressed feeding test, γ2^+/− mice showed a heightened mean latency to initiate feeding compared with wt (U = 19; p < 0.05; n = 9 per genotype). e, Likewise, the mean latency to initiate feeding was significantly increased in Emx1Cre × fγ2/+ compared with controls (U = 3.0; n = 6–14; p < 0.001). f, In the novelty-suppressed feeding test, CaMKIICre2834 × fγ2/+ mice were indistinguishable from fγ2/+ littermate controls (U > 33; n = 8–12 per genotype). Values represent group means ± SEM.

Figure 6.

A developmental but not adult deficit in γ2 subunit-containing GABA_ARs leads to a reduction in adult-born hippocampal neurons. a–d, Hippocampal brain sections of mice labeled at 8 weeks of age and harvested 4 weeks later were stained using antibodies specific for BrdU (red) and the pan-neural marker NeuN (green). a, A representative confocal image of a section through the dentate gyrus shows a BrdU-positive neuron (arrow) and an adult-born non-neural cell (arrowhead). Quantitation of total BrdU-positive cells and BrdU/NeuN double-positive neurons in the subgranule and granule cell layer of serial hippocampal sections of γ2^+/− (b), Emx1Cre x fγ2/+ (c), and CaMKIICre2834 × fγ2/+ mice (d) compared with littermate controls (n = 5–6 mice per genotype in each experiment) revealed a profound reduction in BrdU/NeuN double-positive neurons relative to littermate controls in γ2^+/− (54.9 ± 8.5% of wt; U = 3.5; p < 0.05) and Emx1Cre × fγ2/+ mice (58.5 ± 13.5% of fγ2/+; U = 2.0; p < 0.05), which suffer from a GABA_AR deficit throughout brain development. In contrast, the proportion of adult-born neurons in the hippocampus of CaMKIICre2834 × fγ2 mice was not different from that of littermate controls (U = 12.0; NS). The number of total BrdU-positive cells did not differ significantly from controls in any of the mutant mouse lines analyzed (γ2^+/−, 88.2 ± 18.4% of wt; Emx1Cre × fγ2/+, 82.4 ± 12.8% of fγ2/+ and CaMKIICre2834 × fγ2/+, 113.5 ± 6.2% of fγ2/+; U > 6.5 for all three groups). e, The number of proliferating cells in the subgranule cell layer of mice labeled with BrdU 24 h before harvesting of brains was not different in γ2^+/− compared with wt mice (γ2^+/−, 92.2 ± 5.1% of wt; n = 5 per genotype; U = 9.5; NS). Data indicate means ± SEM.

Figure 7.

Differential Emx1Cre and CaMKIICre2834-mediated recombination in immature neurons of the subgranular and subventricular zones. Coronal brain sections of 6-week-old Emx1Cre × Z/EG (a-a″, d, e) and CaMKIICre2834 × Z/EG mice (b–b″, f, g) through the hippocampus (a–a″, b–b″) or lateral ventricles (d--g) were stained with antibody for DCX (red) and analyzed for colocalization with Cre-induced GFP (green) by confocal microscopy. c, Schematic drawing indicating the location (red squares) of medial (d, f) and dorsal (e, g) sections of the periventricular zone. Note the efficient recombination in immature (DCX-positive, arrows) granule cells in the subgranule cell layer of Emx1Cre × Z/EG mice (a–a″), whereas DCX-positive cells in corresponding sections of CaMKIICre2834 × Z/EG mice lack GFP (b–b″, arrowheads). Similarly, DCX/GFP double-positive neurons were evident in the subventricular zone of Emx1Cre × Z/EG mice (d, e, arrows) but absent in corresponding sections of CaMKIICre2834 × Z/EG mice (f, g). Merged green and red images (a″, b″, d–g) include orthogonal views of x–z and y–z planes to confirm colocalization of GFP and DCX in sections of Emx1Cre × Z/EG mice. GFP-positive cells are more abundant in Emx1Cre × Z/EG than CaMKIICre2834 × Z/EG mice, consistent with Cre-mediated recombination extending to glia in Emx1Cre × Z/EG but not in CaMKIICre2834 × Z/EG mice. Scale bars, 20 μm.