Bidirectional Homeostatic Regulation of a Depression-Related Brain State by Gamma-Aminobutyric Acidergic Deficits and Ketamine Treatment

Abstract

Background: Major depressive disorder is increasingly recognized to involve functional deficits in both gamma-aminobutyric acid (GABA)ergic and glutamatergic synaptic transmission. To elucidate the relationship between these phenotypes, we used GABAA receptor γ2 subunit heterozygous (γ2(+/-)) mice, which we previously characterized as a model animal with construct, face, and predictive validity for major depressive disorder.

Methods: To assess possible consequences of GABAergic deficits on glutamatergic transmission, we quantitated the cell surface expression of N-methyl-D-aspartate (NMDA)-type and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors and the function of synapses in the hippocampus and medial prefrontal cortex of γ2(+/-) mice. We also analyzed the effects of an acute dose of the experimental antidepressant ketamine on all these parameters in γ2(+/-) versus wild-type mice.

Results: Modest defects in GABAergic synaptic transmission of γ2(+/-) mice resulted in a strikingly prominent homeostatic-like reduction in the cell surface expression of NMDA-type and AMPA-type glutamate receptors, along with prominent functional impairment of glutamatergic synapses in the hippocampus and medial prefrontal cortex. A single subanesthetic dose of ketamine normalized glutamate receptor expression and synaptic function of γ2(+/-) mice to wild-type levels for a prolonged period, along with antidepressant-like behavioral consequences selectively in γ2(+/-) mice. The GABAergic synapses of γ2(+/-) mice were potentiated by ketamine in parallel but only in the medial prefrontal cortex.

Conclusions: Depressive-like brain states that are caused by GABAergic deficits involve a homeostatic-like reduction of glutamatergic transmission that is reversible by an acute, subanesthetic dose of ketamine, along with regionally selective potentiation of GABAergic synapses. The data merge the GABAergic and glutamatergic deficit hypotheses of major depressive disorder.

Keywords: Antidepressant drug mechanisms; GABA; Glutamate; Homeostatic synaptic plasticity; Major depressive disorder; Neuroligin.

Figure 1. Analyses of cell surface expression of synaptic proteins in cortical cultures

A γ2^−/− cultures showed significantly reduced surface levels of NL1, GluN1, GluN2B and GluA2/3, compared to WT neurons (γ2^−/− vs. WT: NL1, P < 0.001, n = 5–6; GluN1: P < 0.01, n = 5–7 cultures; GluN2B, P < 0.01, n = 12–13; GluA2/3, P < 0.01, n = 3–4, t-tests). B. The same markers were also reduced in γ2^+/− cultures (γ2^+/− vs. WT: NL1, P < 0.01, n = 4–5; GluN1, P < 0.001, n = 12; GluN2B, P < 0.01, n = 10–11; GluA2/3, P < 0.05, n = 14–15; t-tests). C. A time course of ketamine treatment of γ2^+/− cultures revealed increased GluN1 at 3 h of ketamine treatment (γ2^+/− vs. γ2^+/− Ket, P < 0.01, n = 9) and remained elevated thereafter (P < 0.05, n = 6–13 for both the 4.5 and 6 h time points, ANOVA, Dunnett’s tests). GluA2/3 was significantly increased first at 4.5 h and then remained elevated (γ2^+/− vs. γ2^+/− Ket, 4h, P < 0.05, 6h, P < 0.001, n = 11–12, ANOVA, Dunnett’s tests). Cell surface GluN1 and GluA2/3 levels showed a greater 3h ketamine response for GluN1 than GluA2/3 [F_(1,48) = 4.78, P < 0.05, ANOVA]. Data represent means ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2. Analyses of glutamatergic synapses of γ2^+/− cortical cultures by immuno-staining

A–C. Representative micrographs of neurons cultured form WT (A) and γ2^+/− (B, C) embryos (DIV 20–21) subjected to treatment with ketamine (Ket) (C) and immunostained for the dendritic marker MAP2 (A1, B1, C1, blue), PSD95 (A2, B2, C2, green) and vGluT1 (A3, B3, C3, red). Boxed dendritic segments including merged images are shown enlarged to the right of the panels (A4, B4, C4). Scale bar, 16.7 μm. D. The density of punctate dendritic immunoreactivity of tear-drop-shaped cells for PSD and VGluT1 was significantly reduced in γ2^+/− vs. WT cultures [PSD95, F_(2,33) = 4.11, VGluT1, F_(2,33) = 5.17, P < 0.05 for both proteins, ANOVA; WT vs. γ2^+/−, P < 0.05, n = 21–23 cells for both PSD95 and VGluT1, Tukey’s test]. Puncta densities γ2^+/− cultures treated with ketamine were reversed to WT levels (γ2^+/− vs. γ2^+/− Ket, P < 0.05, n = 21–23, for PSD95 and VGluT1, Tukey’s test). E. The fraction of VGluT1 puncta colocalized with PSD95 was reduced in γ2^+/− vs. WT cultures and restored by ketamine treatment of γ2^+/− cultures [F_(2,33) = 8.71, P < 0.01, ANOVA. γ2^+/− vs. WT and γ2^+/− vs. γ2^+/− Ket, P < 0.01, n = 21–23 for both comparisons, Tukey’s test). F. Puncta sizes were unaltered across conditions [PSD95, F_(2,33) = 5.05; VGluT1, F_(2,33) = 1.392, ANOVA, P, n.s.]. Data represent means ± SE. *, P < 0.05; **, P < 0.01, Tukey’s tests.

Figure 3. GABA_AR γ2^+/− mice exhibit increased behavioral sensitivity to anxiolytic- and antidepressant-like effects of ketamine

Separate groups of GABA_AR γ2^+/− mice and WT littermates were injected with vehicle (saline) or ketamine (3 mg/kg, i.p.) and subjected to behavioral testing 8 h after treatment. A-C. In the Elevated Plus Maze, the % time spent on open arms (A) showed a significant overall treatment effect [F(1,43) = 8.17, P = 0.006, Two-way ANOVA] and a strong trend towards a genotype x treatment interaction [F(1,43) = 3.81, P = 0.057]. Posthoc analyses revealed an anxiety-like reduction in the % time spent on open arms in vehicle treated γ2^+/− vs. WT mice (P < 0.05, n = 12-16) consistent with the phenotype previously reported for γ2^+/− mice on the 129X1/SvJ background (34, 37). Ketamine treatment resulted in an anxiolytic-like increase in the % time on open arms in γ2^+/− [γ2^+/− Ket vs. Veh, P < 0.001, n = 12-14) but not WT mice (WT Ket vs. Veh, P > 0.05, n = 13-16, Fisher’s test). Similarly, analyses of % open arm entries (B) showed a significant overall treatment effect [F_(1,51) = 7.71, P = 0.008 Two-way ANOVA] and a significant anxiolytic-like effect of ketamine selectively in γ2^+/− (γ2^+/− Ket vs. Veh, P < 0.01, n = 12-14) but not WT mice (WT Ket vs. Veh. P > 0.05, n = 14-16, Fisher tests). The four groups were indistinguishable with respect to closed arm entries (C) [genotype: F_(1,51) = 0.52, P = 0.473, treatment: F_(1,51) = 0.71, P = 0.404, Two-way ANOVA]. D, E. In the Forced Swim Test, the latency to first immobility (D) showed an overall genotype effect [F(1,90) = 19.78, P < 0.001, n = 18-27, Two-way ANOVA] and a genotype x treatment interaction [F(1,90) = 4.45, P = 0.038]. Posthoc group comparisons showed a reduced Latency to First Immobility specifically in vehicle-treated γ2^+/− vs. WT mice (γ2^+/− Veh vs. WT Veh. P < 0.001, n = 18-25; WT Ket vs. Veh, P > 0.05, n = 20-21, t-tests) and an increased Latency to First Immobility in ketamine-treated γ2^+/− vs. vehicle-treated γ2^+/− mice (γ2^+/− Veh vs. γ2^+/− Ket. P < 0.01, n = 18-23, t-test), consistent with the depressive-like phenotype previously reported for γ2^+/− mice on the 129X1/SvJ background (37, 38) and with ketamine-induced changes in GluR expression and synapse function that were limited to or greater in γ2^+/− vs. WT mice. Measurements of total time spent immobile (E) showed an overall treatment effect [F(1,90) = 9.32, P = 0.003, n = 18-27, Two-way ANOVA] and an antidepressant-like drug effect specifically in ketamine vs. vehicle treated γ2^+/− but not WT mice (γ2^+/− Ket vs. Veh, P < 0.01, n = 18-25; WT Ket vs. Veh, P > 0.05, n = 20-21, Fisher’s tests).

Figure 4. Downregulation of glutamate receptors in γ2^+/− vs. WT mice is reversed by ketamine treatment

A. The cell surface expression of GluN1 and GluA2/3 was reduced in γ2^+/− vs. WT mice both in hippocampus (GluN1, P < 0.001; GluA2/3, P < 0.05, n = 12–13 mice for both comparisons) and ACC/PLC (P < 0.05, n = 12–13, for both comparisons). GluN2B cell surface expression was reduced in hippocampus of γ2^+/− mice (γ2^+/− vs. WT, P < 0.05, n = 6–7) and trended lower in ACC/PLC (P = 0.15, n = 6–7). B. In γ2^+/− animals analyzed 24h after an acute dose of ketamine (10 mg/kg, i.p.) the cell surface expression of NMDARs and AMPARs in hippocampus was increased compared to vehicle-treated γ2^+/− controls (P < 0.05, n = 7–11, for the GluN1, GluN2B and GluA2/3 comparisons) to levels comparable to those of WT mice in (A) (γ2^+/−+Ket vs. WT, P, n.s., n = 9–12). In ACC/PLC, ketamine treatment of γ2^+/− mice resulted in increased expression of NMDARs (γ2^+/− Ket vs. Veh: GluN1, P < 0.05) but not AMPARs (GluA2/3, P, n.s., n = 8–9 for both comparisons). Cell surface GluN2B levels trended in the same direction as GluN1 but the effect was not significant (P = 0.12, n = 3). C. In hippocampus of γ2^+/− animals analyzed three days after ketamine treatment the cell surface expression of NMDARs and AMPARs remained increased compared to controls (P < 0.05, n = 10–12, for both GluN1 and GluA2/3). By contrast, in ACC/PLC, the GluN1 cell surface level had returned to base line (P, n.s., n = 8–9) but the GluA2/3 cell surface expression showed a strong trend of an increase (P = 0.08, n = 6/group) that was not yet evident at one day after treatment (B). D. In WT mice analyzed 24 h after ketamine treatment the expression of NMDARs and AMPARs was unchanged compared to vehicle controls in both hippocampus and ACC/PLC (WT Veh vs. Ket: P, n.s., n = 5–6, for all four comparisons). Data are from mice maintained on a C57BL/6J background. The genotype differences were reproduced with 129X1/SvJ mice (Fig. S1C in Supplement 1). Data represent means ± SE. *, P < 0.05; ***, P < 0.001, t-tests.

Figure 5. Downregulation of glutamatergic synaptic inputs to γ2^+/− CA1 pyramidal neurons is reversed by ketamine

A–C. sEPSC recordings from CA1 pyramidal neurons of γ2^+/− and WT mice injected 24 h earlier with saline (Veh) or ketamine (Ket). Representative traces are shown in (A), with summary quantification of sEPSC amplitude in (B) and frequency in (C). Note the significant decrease in frequency of sEPSC recorded from vehicle-treated γ2^+/− vs. WT mice that was normalized by ketamine treatment (genotype x treatment interaction for sEPSC frequency, F_(1,86) = 4.473, P = 0.037, Two-way ANOVA; P < 0.01, n = 12–41 cells, for both group comparisons, Tukey’s test). D–G. AMPAR EPSCs recorded from CA1 pyramidal cells evoked by stimulation of the SC (D, E) or TA path (F, G). Schematics with sites of stimulation and recording and average traces at progressively larger stimulation intensities are shown in (D) and (F). AMPAR responses were reduced in γ2^+/− vs. WT mice at SC (E) and TA (G) synapses. Moreover, ketamine treatment restored synaptic responses of γ2^+/− mice to WT levels [(E) # WT Veh vs. γ2^+/− Veh F_(1,138) = 5.391, P = 0.022, Two-way ANOVA; P < 0.05, n = 9–17 cells, Tukey’s test; (G) ### WT Veh vs. γ2^+/− Veh, F_(1,102) = 23.78, P < 0.001, Two-way ANOVA, P < 0.05, Tukey’s test). H–K. NMDAR EPSCs recorded from CA1 pyramidal cells evoked by stimulation of the SC (H, I) or TA path (J, K). Schematics with sites of stimulation and recordings, along with average sample traces at progressively larger stimulation intensities are shown in (H) and (J), with summary quantification in (I) and (K). NMDAR responses in γ2^+/− mice were reduced compared to WT at both SC (I) and TA (K) synapses. Ketamine treatment restored the synaptic NMDAR responses of γ2^+/− mice to WT levels [(I) # WT Veh vs. γ2^+/− Veh, F_(1,89) = 4.952, P = 0.029, Two-way ANOVA; ### γ2^+/− Veh vs. γ2^+/− Ket, F_(1,95) = 12.31, P < 0.001, Two-way ANOVA; (K) ### WT Veh vs. γ2^+/− Veh F_(1,90) = 15.55, P < 0.001, Two-way ANOVA; ### γ2^+/− Veh vs. γ2^+/− Ket F_(1,96) = 12.55, P < 0.001, Two-way ANOVA; n = 8–9 cells for all groups in (I) and (K)]. Data represent means ± SE. *, P < 0.05; **, P < 0.01.

Figure 6. Characterization of GABAergic synapse deficits and their reversal by ketamine in γ2^+/− cultured cortical neurons, and CA1 and L2/3 ACC of γ2^+/− mice

A–H. Cortical cultures (DIV21) prepared from WT (A, C) and γ2^+/− (B, D) embryos were either untreated (A, B) or treated with ketamine (C, D) (10 μM, 6 h) and subjected to immunofluorescent staining for gephyrin (green, A1–D1) and VGAT (red, A2–D2). Colocalization in merged images is shown in yellow (A3–D3) with enlarged dendritic segments depicted to the right. Scale bar, 16.7 μm. E. Quantitation of punctate immunoreactivity in dendritic segments of pyramidal neurons showed that the size of gephyrin clusters was reduced in γ2^+/− vs. WT neurons and restored to WT levels by ketamine treatment [F_{(3, 73)} = 5.3, P < 0.01, ANOVA, γ2^+/− vs. WT, P = 0.05; γ2^+/− vs. γ2^+/− Ket, P < 0.01, WT vs. WT Ket, P, n.s., n = 18–21, Bonferroni]. F. The density of punctate gephyrin staining per 40 μm was significantly reduced in γ2^+/− vs. WT cultures [F_{(3, 73)} = 64.9, P < 0.001, ANOVA; γ2^+/− vs. WT, P < 0.05, n = 18–21, Tukey’s test] and increased by ketamine selectively in γ2^+/− cultures (γ2^+/− vs. γ2^+/− Ket, P < 0.001, WT vs. WT Ket, P, n.s., n = 18–21, both comparisons, Tukey’s test). G. The density of VGAT puncta along 40 μm segments of dendrite was increased by ketamine independent of genotype [F_(1/73) = 16.4, P < 0.001, ANOVA; WT vs. WT Ket, P < 0.01, γ2^+/− vs. γ2^+/− Ket, P < 0.05, n = 18–21, t-tests). H. The fraction of gephyrin puncta colocalized with VGAT was unaltered across conditions [F_(3,73) = 1.99, P = 0.12, ANOVA]. I–L. Representative traces of mIPSC recordings from CA1 (I) and L2/3 ACC pyramidal neurons (K) of WT and γ2^+/− mice injected with saline (Veh) or Ket 24 h prior to recording. (J) Quantification of mIPSC amplitude and frequency for CA1 neurons showed a significant decrease in amplitude in γ2^+/− vs. WT mice, regardless of treatment (mIPSC amplitude by genotype, F_(1,45) = 18.02, P = 0.001, Two-way ANOVA, P < 0.05, n = 10–13 cells; Tukey’s test). (L) Quantification of mIPSC amplitude and frequency for ACC neurons showed a significant decrease in amplitude in γ2^+/− vs. WT mice that was reversed by ketamine treatment of γ2^+/− mice (mIPSC amplitude, genotype x treatment interaction, F_(1,25) = 7.879, P < 0.01, n = 6–8 cells; Two-way ANOVA; treatment comparison F_(1,25) = 9.388, P = 0.005, Bonferroni). Data represent means ± SE. * P < 0.05, ** P < 0.01; *** P < 0.001.