GABAergic control of critical developmental periods for anxiety- and depression-related behavior in mice

Abstract

Vulnerability for anxiety and depressive disorders is thought to have origins in early life and is increasingly recognized to involve deficits in GABAergic neurotransmission. Mice that were rendered heterozygous for the γ2 subunit gene of GABA(A) receptors (GABA(A)Rs) show behavioral, cognitive, neuroendocrine and pharmacologic features expected of a mouse model of melancholic anxious depression, including reduced survival of adult-born hippocampal neurons. Here we embarked on elucidating the developmental substrate underlying this phenotype, focusing on the Elevated Plus Maze and Forced Swim Test as relevant behavioral paradigms. In a first series of experiments using hemizygous tamoxifen-induced genetic inactivation of a floxed γ2 genomic locus we show that reducing the gene dosage at postnatal days (P)13/14 but not P27/28 results in altered behavior in both of these tests in adulthood, reminiscent of the anxious-depressive phenotype previously described for global heterozygous mice. However, in contrast to global heterozygous mice, the behavioral changes induced by γ2 subunit knockdown at P13/14 occurred without changes in adult hippocampal neurogenesis, indicating that altered neurogenesis is not an absolute prerequisite for anxiety- and depression-related behavior in this model. In a separate series of experiments using a pharmacological approach, acute but transient potentiation of GABA(A)Rs with diazepam uncovered distinct developmental vulnerabilities for altered behavior in the Elevated Plus Maze and Forced Swim Test, respectively. Specifically, diazepam given during P10-16 but not during later weeks resulted in increased anxiety-like behavior in adulthood, while diazepam administered during P29-35 but not earlier nor later resulted in increased immobility behavior in adulthood. We conclude that anxiety-like behavior in the Elevated Plus Maze and behavioral despair-like immobility in the Forced Swim Test are controlled by separate postnatal critical periods characterized by distinct developmental sensitivity to manipulation of GABAergic transmission via γ2 subunit-containing GABA(A)Rs.

Figure 1. Behavioral effects of tamoxifen in the EPMT and FST independent of target gene recombination.

TAM was injected into CAGGCre-ER™, fγ2/+ and WT littermates at P13/14 or P27/28 to test for TAM- and Cre-induced behavioral effects at 8–10 weeks of age that occurred independently of recombination of the fγ2 locus. a) In the EPMT, the CAGGCre-ER™ and fγ2/+ mice visited open arms and stayed in the open arms as much as WT mice. b) In the FST, the behavior of fγ2/+ mice was indistinguishable from WT mice. CAGGCre-ER™ mice showed a normal latency to immobility and reduced immobility duration compared to TAM-treated fγ2/+ and WT mice. All values represent group means ± SEM. *p<0.05, Dunnett’s test.

Figure 2. Developmental control of anxious depressive behavior induced by tamoxifen-induced Cre-mediated recombination of the fγ2 locus.

a) Experimental time line. Littermates representing all three genotypes were injected with TAM at P13/14 or P27/28, followed by behavioral testing starting after 8 weeks of age. b) In the EPMT, CAGGCre-ER™ X fγ2/+ mice treated with TAM at P13/14 visited the open arm less often than identically treated fγ2/+ and CAGGCre-ER™ controls. Moreover, P13/14 TAM-treated CAGGCre-ER™ X fγ2/+ mice spent less time on the open arms than identically treated fγ2/+ controls, while the number of closed arm entries was unaffected. c) CAGGCre-ER™ X fγ2/+ mice treated with TAM at P27/P28 were indistinguishable from identically treated fγ2/+ and CAGGCre-ER™ littermate controls with respect to % open arm entries, % time spent on open arms, and number of closed arm entries. d) In the FST, the latency of P13/14 TAM-treated CAGGCre-ER™ X fγ2/+ mice to float was reduced compared to identically treated CAGGCre-ER™ and fγ2/+ littermate controls. Moreover, P13/14 TAM-treated CAGGCre-ER™ X fγ2/+ mice showed increased immobility compared to identically treated CAGGCre-ER™ controls. e) P27/28 TAM-treated CAGGCre-ER™ X fγ2/+ mice were indistinguishable from identically treated fγ2/+ and CAGGCre-ER™ littermates with respect to both latency to immobility and immobility duration. f) In the OFT, the distance traveled of P13/14 TAM-treated CAGGCre-ER™ X fγ2/+ mice over a period of 15 min was indistinguishable from that of identically treated CAGGCre-ER™ and fγ2/+ littermates. All values represent group means ± SEM. *p<0.05, **p<0.01, ***p<0.001, Dunnett’s tests.

Figure 3. Quantitation of hippocampal neurogenesis and analyses of recombination in neural progenitors.

a) P13/14 TAM-treated CAGGCre-ER™ X fγ2/+ mice and littermate controls (CAGGCre-ER™ and fγ2/+) were injected with BrdU at P63 and transcardially perfused at P91. The number of BrdU-positive or BrdU/NeuN doubly positive hippocampal granule cells quantitated in serial sections was indistinguishable in TAM treated CAGGCre-ER™ X fγ2/+ mice compared to identically treated littermate controls (mixed CAGGCre-ER™ and fγ2/+ genotypes). b) Cre-mediated recombination in adult-born hippocampal neurons of P13/14 TAM-treated CAGGCre-ER™ X ROSA26-YFP mice was analyzed at 6 weeks of age. The micrographs show a representative section immunostained for YFP (upper left panel, green), DCX (upper right, red), with merged images showing YFP/DCX-doubly positive cells (lower left) and the outlines of the granule cell layer stained with the nuclear stain, DAPI (lower right). Arrows point to DCX positive immature neurons lacking YFP that therefore were not subject to Cre-mediated recombination. The arrowhead points to a rare DCX/YFP positive immature neuron that was subject to Cre-mediated recombination. Scale bar, 50 µm.

Figure 4. Behavioral consequences of diazepam treatment during one- or two-week postnatal developmental temporal windows.

a) Schematic showing the schedule of DZP treatment and behavioral testing of the five cohorts of mice analyzed. DZP was administered during P10-16, P14-28, P22-28, P29-35 or P50-56, followed by behavioral testing between 8 and 11 weeks of age or, in the case of P29-35 and P50-56, between 4 and 9 weeks after the end of DZP treatment. b–f) In the EPMT, the percentage of open arm entries of P10-16 DZP- vs. vehicle-treated mice was reduced (b). Similarly, the percentage of open arm entries trended lower in P14-28 DZP-treated vs. vehicle treated mice (c). However, the number of closed arm entries was unaffected for both treatment periods, consistent with unaltered locomotion. By contrast, DZP treatment during P22-28, P29-35 and P50-56 was without long-term behavioral consequences in this test (d-f). g–k) When tested in the FST, the mice treated with DZP during P10-16, P14-28, P22-28 and P50-56 showed normal behavior compared to vehicle treated controls (g-i, k). By contrast, mice treated with DZP during P29-35 showed a reduced latency to immobility and increased immobility duration (j). l) Increased immobility of P29-35 DZP-treated mice observed in the FST was confirmed by increased immobility duration in the TST, although the latency to assume an immobile position was unchanged. m–o) The distance traveled of P10-16, P29-35 and P50-56 DZP-treated mice in a 15 min OFT was unaltered compared to controls, showing that locomotion was unaffected. *p<0.05, **p<0.01, ***p<0.001, t-tests.