Disinhibition of somatostatin-positive GABAergic interneurons results in an anxiolytic and antidepressant-like brain state

Abstract

Major depressive disorder (MDD) is associated with reduced concentrations of γ-aminobutyric acid (GABA) that are normalized by antidepressant therapies. Moreover, depressive-like phenotypes of GABA_A receptor mutant mice can be reversed by treatment with conventional antidepressants drugs, as well as by subanesthetic doses of ketamine. Thus GABAergic deficits may causally contribute to depressive disorders, while antidepressant therapies may enhance GABAergic synaptic transmission. Here we tested the hypothesis that sustained enhancement of GABAergic transmission alone is sufficient to elicit antidepressant-like behavior, using disinhibition of GABAergic interneurons. We focused on somatostatin-positive (SST⁺) GABAergic interneurons because of evidence that their function is compromised in MDD. To disinhibit SST⁺ interneurons, we inactivated the γ2 subunit gene of GABA_A receptors selectively in these neurons (SSTCre:γ2^f/f mice). Loss of inhibitory synaptic input resulted in increased excitability of SST⁺ interneurons. In turn, pyramidal cell targets of SST⁺ neurons showed an increased frequency of spontaneous inhibitory postsynaptic currents. The behavior of SSTCre:γ2^f/f mice mimicked the effects of anxiolytic and antidepressant drugs in a number of behavioral tests, without affecting performance in a spatial learning- and memory-dependent task. Finally, brain extracts of SSTCre:γ2^f/f mice showed decreased phosphorylation of the eukaryotic elongation factor eEF2, reminiscent of the effects of ketamine. Importantly, these effects occurred without altered activity of the mammalian target of rapamycin pathway nor did they involve altered expression of SST. However, they were associated with reduced Ca²⁺/calmodulin-dependent auto-phosphorylation of eEF2 kinase, which controls the activity of eEF2 as its single target. Thus enhancing GABAergic inhibitory synaptic inputs from SST⁺ interneurons to pyramidal cells and corresponding chronic reductions in the synaptic excitation:inhibition ratio represents a novel strategy for antidepressant therapies that reproduces behavioral and biochemical end points of rapidly acting antidepressants.

Figure 1

Deletion of postsynaptic γ-aminobutyric acid type A receptors (GABA_ARs) and gephyrin from somatostatin-positive (SST⁺) neurons of SSTCre:γ2^f/f mice. (a) Strategy for γ2 subunit knockout-mediated disinhibition of SST⁺ interneurons. Loss of synaptic GABA_ARs removes inhibitory input (IN) to SST⁺ neurons and increases excitability of these neurons. Increased excitability of SST⁺ neurons strengthens inhibitory synaptic inputs to apical dendrites and spines of pyramidal cells (PN). (b) Schematic of Cre-mediated inactivation of the ‘floxed' γ2 locus. (c) Representative micrographs of the soma of an SST⁺ neuron from a SSTCre: γ2^f/+:LSL-YFP control mouse (left column) compared with a SST⁺ neuron from a SSTCre:γ2^f/f:LSL-YFP mutant animal, immunostained for the γ2 subunit (top row, green), gephyrin (second row; red) and yellow fluorescent protein (YFP; third row; blue) with merged images showing colocalization of γ2 and gephyrin in yellow in the bottom row. Note the drastic reduction in punctate staining for both the γ2 subunit and gephyrin, indicative of loss of functional synapses. Residual staining for γ2 is likely attributable to dendrites of Cre-lacking neighboring neurons. (d) Quantification of puncta densities overlapping with YFP⁺ cell somata (puncta per μm²) in S. pyramidale and S. radiale of the hippocampus. Densities for both proteins were significantly reduced in both areas (P<0.001, respectively). ***P<0.001, Mann–Whitney, n=30–40 cells, 2 mice/genotype.

Figure 2

Recordings from somatostatin-positive (SST⁺) neurons. (a–f) Spontaneous inhibitory synaptic current (sIPSC), miniature inhibitory synaptic current (mIPSC) and spontaneous excitatory postsynaptic current (sEPSC) recordings from SST⁺ neurons of SSTCre:γ2^f/f:LSL-YFP mutants and SSTCre:γ2^f/+:LSL-YFP control mice in hippocampus CA1 (a–c) and L2/3 cingulate cortex (d–f). Representative traces are shown on top of the summary statistics. Note the significant reductions in both sIPSC and mIPSC frequencies and amplitudes recorded from CA1 (a, b) and L2/3 (d, e) SST⁺ neurons of mutant vs control mice (P<0.05 for all eight measures, n=6 and 8 cells, one cell/slice, three mice/genotype). sEPSCs recorded from SST⁺ neurons were unaffected by genotype independent of brain area (c, f, CA1, L2/3 frequency, P, NS (non-significant); amplitude, P, NS; n=6 and 8 cells and slices, 3 mice/genotype). (g–n) Current injection data from SST⁺ neurons in CA1 and L2/3 cingulate cortex of SSTCre:γ2^f/f:LSL-YFP mutants and controls. Representative traces are shown in panels (g) (CA1) and (k) (L2/3) with summary data in panels (h–j) (CA1) and (l–n) (L2/3). Note the increased number of action potentials recorded from SST⁺ neurons in mutant vs control mice in both CA1 and L2/3 (h, l) (P<0.05 for both comparisons, Bolzman fit, W50, t-tests). The input resistance (R_in; j, n) was significantly reduced in SSTCre:γ2^f/f:LSL-YFP mutant vs control mice in both brain areas (R_in, CA1, P<0.05; L2/3, P<0.01, n=9 and 10), while the resting membrane potentials (RMP; j, n) of SST⁺ cells were unaffected by genotype independent of brain area (RMP, P, NS). Data represent means±s.e. *P<0.05, **P<0.01, t-tests.

Figure 3

Recordings from pyramidal cells. (a–h) Spontaneous inhibitory synaptic current (sIPSC), miniature inhibitory synaptic current (mIPSC), tonic inhibition and spontaneous excitatory postsynaptic current (sEPSC) recordings from pyramidal neurons of SSTCre:γ2^f/f compared with control mice (SSTCre:γ2^f/+, γ2^f/f and γ2^+/+) in CA1 hippocampus (a–d) and L2/3 cingulate cortex (e–h). Representative traces are shown on top of the bar graph summary statistics. Note the significant increases in sIPSC frequency and amplitude recorded from neurons of SSTCre:γ2^f/f mice in both brain areas (a, e, P<0.05 for all four measures; n=8 and 15 neurons, recording 1 neuron/slice, from 4 mice/genotype). By contrast, mIPSCs showed a moderate increase in amplitude for SSTCre:γ2^f/f mice in CA1 only (b, f) (CA1 amplitude, P<0.05, frequency, P, NS (non-significant); for CA1 frequency, L2/3 amplitude and L2/3 frequency). Tonic inhibition (c, g) was unaffected by genotype (P, NS; for all comparisons, n=8 and 11 neurons and slices, 3 mice/genotype). Similarly, the sEPSCs (d, h) did not differ between genotypes independent of brain area (P, NS; all comparisons, n=8 and 15 neurons and slices, 4 mice/genotype). Data represent means±s.e. *P<0.05, t-tests.

Figure 4

SSTCre:γ2^f/f mice show an anxiolytic- and antidepressant-like behavioral phenotype. (a) Elevated Plus Maze: Percentage of open arm entries of SSTCre:γ2^f/f mutants was increased compared with γ2^f/f controls (sexes combined (M&F), F_{(1, 60)}=6.34, P<0.05, n=29 and 35) with similar trends in females (P=0.06, n=14 and 16) and males (P=0.08, n=15 and 19). The percentage of time spent in open arms was increased in mutants vs controls in both sexes (F_{(1, 57)}=19.16, P<0.001; male: P<0.05, n=14 and 19; female: P<0.001, n=16 and 12). The number of closed arm entries did not differ between genotypes (P>0.05, NS (non-significant), t-test). (b) Novelty Suppressed Feeding Test: SSTCre:γ2^f/f mutants showed a reduced latency to feed vs controls (F_{(1, 62)}=6.4, P=0.014), with significant effects in females (P<0.05, n=16) and a tendency in the same direction in males (P, NS; n=15 and 19). (c) Forced Swim Test: SSTCre:γ2^f/f mice of both sexes showed increased latencies to their first bout of immobility (F_{(1, 62)}=15.41, P=0.0002; female: P<0.01, n=16; male: P<0.05, n=15 and 19) and a reduced total time immobile vs controls (F_{(1, 62)}=15.98, P<0.0001; female: P<0.05; male: P<0.01). The average swim speed during the first minute did not differ between genotypes (M&F: P, NS; n=32 and 34, t-test). (d) Learned Helplessness Test: Male SSTCre:γ2^f/f mice showed fewer escape failures than controls (P<0.05, n=17 and 19, Mann–Whitney). Data represent means±s.e. *P<0.05, **P<0.01, ***P<0.001, analyses of variance and posthoc t-tests, t-tests or Mann–Whitney.

Figure 5

SSTCre:γ2^f/f mice show biochemical changes indicative of reduced intracellular Ca²⁺ signaling and increased dendritic translational elongation. (a–c) Representative western blottings and summary statistics for phospho-eukaryotic translational elongation factor 2 (p-eEF2), phospho-mammalian target of rapamycin (p-mTOR) and p-S6K in the hippocampus (Hipp) and medial prefrontal cortex (mPFC) normalized to levels of the respective total proteins in the same samples. Western blottings show a reduction of p-eEF2^T56/total eEF2 in the Hipp and mPFC of SSTCre:γ2^f/f vs γ2^f/f control mice (P<0.05, n=10 and 11, for both hipp and mPFC, t-tests) (a). The ratios of p-mTOR^S2448/mTOR (n=6 and 6; both brain regions) (b) and of p-S6K^T389/S6K (c) were unaffected by genotype (P, NS (non-significant); all four comparisons, n=6 and 7, both brain regions, Mann–Whitney). (d) Representative western blottings and summary statistics of phosphorylation of eEF2 kinase (eEF2K) at T348 and S500, normalized to total eEF2K. A two-way analysis of variance for auto-phosphorylation at T348 revealed significant main effects for genotype (F_(1,22)=10.50, P<0.01), brain region (F_(1,22)=10.35, P<0.01) and interaction among the two (F_(1,22)=7.69, P<0.05). Posthoc tests showed significantly reduced eEF2K^T348 auto-phosphorylation in the hippocampus of SSTCre:γ2^f/f vs γ2^f/f controls (P<0.01, n=5 and 8) with a tendency in the same direction in mPFC (P, NS; n=5 and 7, t-tests). By contrast, no genotype-dependent changes were evident for eEF2K^S500 (P, NS; for both brain regions; n=5 and 6 (hipp) and 6 and 8 (mPFC), Mann–Whitney). (e) Schematic of signaling cascades converging on the phospho-state and activity of eEF2K as a target downstream of (i) mTOR- and S6K-mediated inhibitory phosphorylation, (ii) excitation–inhibition (E:I) ratio and calmodulin (CaM)-dependent auto-phosphorylation at T348 (activating, black arrow) and (iii) Ca²⁺-insensitive activating protein kinase A-mediated phosphorylation at S500. Small color-coded arrows illustrate previously reported alterations in phospho-state induced by ketamine (blue^, ) and 5-HT2C antagonists (green) and reported here owing to a reduced synaptic E:I ratio (red, unaltered phospho-states are indicated by a horizontal dash). The unaltered phospho-states of mTOR, S6K and eEF2K^S500 of SSTCre:γ2^f/f mice indicate that reduced phosphorylation of eEF2 involves reduced E:I ratio and reduced CaM-mediated auto-phosphorylation of eEF2K at T348. (f) Somatostatin (SST) mRNA levels quantitated by reverse transcriptase–PCR were reduced in hippocampus (2^−ΔΔCt, P<0.05) with a trend in the same direction in mPFC (P, NS; n=5 and 6, Mann–Whitney). (g) SST protein levels quantitated by enzyme-linked immunosorbent assay were not measurably affected by genotype, independent of brain region (P, NS; n=5 and 6 (hipp), 5 and 5 (mPFC), Mann–Whitney). Data represent means±s.e. *P<0.05, **P<0.01, t-tests or Mann–Whitney.