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

Open AccessPublished:February 12, 2016DOI:https://doi.org/10.1016/j.biopsych.2016.02.009

      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

      Major depressive disorder (MDD) is a leading cause of total disability with limited treatment options that are often ineffective (
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      Stable functioning of neural networks in the face of rapid changes in neural excitability is critically dependent on homeostatic self-tuning mechanisms that, on a slower time course, preserve the balance of excitation and inhibition and the average firing rates of neurons (
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      Mice rendered hemizygous for the γ2 subunit gene (Gabrg2) of GABAARs (γ2+/− mice) have been extensively characterized as a model with construct, face, and predictive validity for anxious MDD (
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      The GABAergic deficit hypothesis of major depressive disorder.
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      Anxiety and depression: Mouse genetics and pharmacological approaches to the role of GABA(A) receptor subtypes.
      ). These mice exhibit a modest impairment of GABAergic transmission characterized by loss of the γ2 subunit in ~15% of GABAARs averaged across brain regions, with the most prominent reductions in neocortex and hippocampus (−25% to −35%) (
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      ). The γ2-lacking GABAARs are functionally impaired as indicated by their reduced channel conductance (12 vs. 28 pS) and failure to accumulate at synapses (
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      ). Behaviorally, γ2+/− mice exhibit signs of heightened anxiety, despair, anhedonia, and constitutive stress axis activation, and all these phenotypes are normalized by long-term treatment with the antidepressant desipramine (
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      • Luscher B.
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      ,
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      • Rudolph U.
      Anxiety and depression: Mouse genetics and pharmacological approaches to the role of GABA(A) receptor subtypes.
      ).
      In this study, we explored the consequences of GABAergic deficits on glutamatergic synapses. We found that γ2+/− mice exhibit reduced cell surface expression and function of NMDARs and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), along with reduced expression of the synaptic adhesion molecule neuroligin 1 (NL1) and defects in the density and function of glutamatergic synapses in the hippocampus and medial prefrontal cortex. Similar defects were observed in γ2+/− cultured neurons. Moreover, treatment of γ2+/− mice (or cultures) with a subanesthetic dose of ketamine resulted in lasting (≥3 days) enhancement and normalization of glutamate receptor (GluR) expression and glutamatergic synapse function. Thus, depression-related brain states of γ2+/− mice involve a homeostatic-like reduction of glutamatergic transmission that can be normalized for a prolonged period by the rapidly acting antidepressant ketamine. Ketamine also potentiated the function of GABAergic synapses but only in anterior cingulate cortex (ACC). These data unite the GABAergic and glutamatergic deficit hypotheses of MDD, suggest that MDD may be caused by aberrant homeostatic plasticity, and provide novel insights into the synaptic mechanisms underlying antidepressant efficacy of ketamine.

      Methods and Materials

      See the more detailed and complete version in Supplemental Methods and Materials.

      Production and Husbandry of Mice

      Two different GABAAR γ2+/− mouse lines were used as part of this study, with virtually identical germline deletions of exon 8 of the Gabrg2 locus. A first line of γ2+/− mice was maintained on a 129X1/SvJ background as previously described (
      • Crestani F.
      • Lorez M.
      • Baer K.
      • Essrich C.
      • Benke D.
      • Laurent J.P.
      • et al.
      Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.
      ,
      • Shen Q.
      • Lal R.
      • Luellen B.A.
      • Earnheart J.C.
      • Andrews A.M.
      • Luscher B.
      Gamma-aminobutyric acid-type A receptor deficits cause hypothalamic-pituitary-adrenal axis hyperactivity and antidepressant drug sensitivity reminiscent of melancholic forms of depression.
      ,
      • Gunther U.
      • Benson J.
      • Benke D.
      • Fritschy J.M.
      • Reyes G.
      • Knoflach F.
      • et al.
      Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.
      ). A second line was generated on the C57BL/6J background by mating γ2f/f mice (
      • Schweizer C.
      • Balsiger S.
      • Bluethmann H.
      • Mansuy M.
      • Fritschy J.M.
      • Mohler H.
      • et al.
      The gamma2 subunit of GABA(A) receptors is required for maintenance of receptors at mature synapses.
      ) with an oocyte-specific Cre line followed by outcrossing of the Cre transgene. Mice used for experimentation were littermates produced by crossing of γ2+/− mice and wild-type (WT) mice. The 129X1/SvJ line was used for preparation and analyses of cortical cultures as well as biochemical and electrophysiologic analyses of brain slices. The C57BL/6J line was used for biochemical and behavioral experimentation involving ketamine treatment.

      Drug Treatments

      For treatment of cultures, the drugs were diluted or dissolved in culture media to the following final concentrations: ketamine, 10 μmol/L (Ketaject; Phoenix Pharmaceutical, Inc., St. Joseph, MO); D-(-)-2-amino-5-phosphonopentanoic acid (D-APV), 100 μmol/L (Sigma-Aldrich, St. Louis, MO); bicuculline (BIC), 20 μmol/L (R&D Systems, Minneapolis, MN); and Ro 25-6981, 10 μmol/L (Sigma-Aldrich). For treatment of mice (8–9 weeks old), ketamine (Ketaject diluted to 1 mg/mL in 0.9% saline) was administered at 10 mg/kg (biochemical and electrophysiologic analyses) or 3 mg/kg (behavioral analyses) (intraperitoneal injection [i.p.]) as previously described (
      • Li N.
      • Lee B.
      • Liu R.J.
      • Banasr M.
      • Dwyer J.M.
      • Iwata M.
      • et al.
      mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists.
      ,
      • Autry A.E.
      • Adachi M.
      • Nosyreva E.
      • Na E.S.
      • Los M.F.
      • Cheng P.F.
      • et al.
      NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses.
      ).

      Cell Surface Biotinylation

      Cortical cultures from γ2+/− and WT mice were generated from embryonic day 14–15 embryos (129X1/SvJ line) and subjected to cell surface biotinylation at 21 days in vitro and purification using NeutrAvidin agarose beads (Thermo Scientific, Rockford, IL) as described previously (
      • Yuan X.
      • Yao J.
      • Norris D.
      • Tran D.D.
      • Bram R.J.
      • Chen G.
      • et al.
      Calcium-modulating cyclophilin ligand regulates membrane trafficking of postsynaptic GABA(A) receptors.
      ). For biotinylation of brain slices, we adapted the protocol of Terunuma et al. (
      • Terunuma M.
      • Xu J.
      • Vithlani M.
      • Sieghart W.
      • Kittler J.
      • Pangalos M.
      • et al.
      Deficits in phosphorylation of GABA(A) receptors by intimately associated protein kinase C activity underlie compromised synaptic inhibition during status epilepticus.
      ). The biotinylated proteins were quantitated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/western blot using an Odyssey CLx infrared imager (LI-COR Biosciences, Lincoln, NE). Amounts of cell surface biotinylated proteins were normalized to amounts of β-tubulin in total extracts quantitated on parallel gels.

      Immunofluorescent Staining of Cortical Cultures

      Immunofluorescent staining of neurons employed glia-free cortical cultures prepared from embryonic day 14–15 embryos as previously described (
      • Alldred M.J.
      • Mulder-Rosi J.
      • Lingenfelter S.E.
      • Chen G.
      • Luscher B.
      Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin.
      ). The cells were fixed, permeabilized, and stained at 21 days in vitro as described (
      • Alldred M.J.
      • Mulder-Rosi J.
      • Lingenfelter S.E.
      • Chen G.
      • Luscher B.
      Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin.
      ) using rabbit anti-MAP2 (1:1000, Ab5622; EMD Millipore Corp., Billerica, MA), guinea pig anti-VGluT1 (1:500, LV1439669; EMD Millipore Corp.), mouse anti-PSD95 (1:1500, No. 28879; EMD Millipore Corp.), mouse anti-gephyrin (1:500, No. 147111; Synaptic Systems GmbH, Goettingen, Germany), and rabbit anti–vesicular GABA transporter (1:1000, No. 131002; Synaptic Systems GmbH). Synaptic immunoreactivities were developed and quantified as described (
      • Alldred M.J.
      • Mulder-Rosi J.
      • Lingenfelter S.E.
      • Chen G.
      • Luscher B.
      Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin.
      ).

      Electrophysiology

      Coronal slices (350 μm) containing the dorsal hippocampus or ACC were prepared using a vibratome (Leica VT1200S; Leica Biosystems, Inc., Buffalo Grove, IL) from 7- to 13-week-old 129X1/SvJ mice (of either sex) in a solution containing 210 mmol/L sucrose, 7 mmol/L D-glucose, 25 mmol/L sodium bicarbonate, 1.25 mmol/L monosodium phosphate, 2.5 mmol/L potassium chloride, 1.3 mmol/L sodium ascorbate, 3 mmol/L sodium pyruvate, 0.5 mmol/L calcium chloride, and 7 mmol/L magnesium chloride, saturated with 95% oxygen/5% carbon dioxide. During recordings, slices were perfused with 50 mmol/L sucrose, 119 mmol/L sodium chloride, 26.2 mmol/L sodium bicarbonate, 11 mmol/L glucose, 2.5 mmol/L potassium chloride, 1 mmol/L monosodium phosphate, 2.5 mmol/L calcium chloride, and 1.3 mmol/L magnesium chloride, saturated with 95% oxygen/5% carbon dioxide, and 0.5 μmol/L tetrodotoxin, 10 μmol/L 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, and 25 μmol/L D-APV for miniature inhibitory postsynaptic currents (mIPSCs), or 50 μmol/L picrotoxin for excitatory postsynaptic currents (EPSCs), or 10 μmol/L N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride and 50 μmol/L picrotoxin were added to perfusate lacking magnesium chloride for NMDAR-mediated EPSCs. Internal solutions consisted of 127 mmol/L cesium chloride, 8 mmol/L sodium chloride, 1 mmol/L calcium chloride, 10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 10 mmol/L ethylene glycol tetraacetate, 0.3 mmol/L guanosine 5′-triphosphate sodium salt, and 2 mmol/L adenosine 5′-triphosphate magnesium salt, pH 7.2 for mIPSCs, or 127 mmol/L cesium methanesulfonate, 8 mmol/L sodium chloride, 1 mmol/L calcium chloride, 10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 10 mmol/L ethylene glycol tetraacetate, 0.3 mmol/L guanosine 5′-triphosphate sodium salt, 2 mmol/L adenosine 5′-triphosphate magnesium salt, 0.1 mmol/L spermine, and 5 mmol/L N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride (QX 314), pH 7.2 for EPSCs. All recordings were obtained at Vh = −70 mV, unless otherwise indicated. EPSCs were evoked using borosilicate glass pipette stimulators positioned ~50–200 μm away from the primary apical dendrite of the recorded cell. Recordings and analyses were performed using pCLAMP version 10 software (Molecular Devices, LLC, Sunnyvale, CA). Only cells with a stable access resistance throughout the recording period were included in the analysis.

      Statistics

      Statistical comparisons were performed using two-tailed Student t tests or analysis of variance (ANOVA) followed by post hoc analyses as detailed in the text and figure legends.

      Results

      GABAergic Deficits of γ2-Deficient Neurons Result in Homeostatic Downregulation of GluRs

      Chronic blockade of GABAARs in cultured neurons with BIC results in homeostatic downregulation of NMDA and AMPAR function (Supplemental Figure S1A) (
      • Watt A.J.
      • van Rossum M.C.
      • MacLeod K.M.
      • Nelson S.B.
      • Turrigiano G.G.
      Activity coregulates quantal AMPA and NMDA currents at neocortical synapses.
      ). Therefore, to begin to test whether similar changes in GluR expression might occur in γ2+/− mice, we began our analyses in γ2−/− and WT cultured cortical neurons (21 days in vitro). As a proxy for NMDARs, we quantitated the cell surface expression of the obligatory subunit GluN1 as well as the GluN2B subunit, which is part of an NMDAR subtype that is implicated in mediating the detrimental effects of excess glutamate (
      • Liu Y.
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      NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo.
      ) and antidepressant effects of ketamine (
      • Miller O.H.
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      • Zhang Y.
      • Delpire E.
      • et al.
      GluN2B-containing NMDA receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine.
      ). In addition, we quantitated the cell surface expression of NL1, a postsynaptic cell adhesion molecule that controls the accumulation of NMDARs at synapses (
      • Budreck E.C.
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      • et al.
      Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling.
      ). Lastly, we surveyed the expression of AMPARs using antibodies for GluA2/3 subunits. Using cell surface biotinylation assays, we found that the plasma membrane accumulation of all four proteins was drastically reduced in γ2−/− compared with WT cultures (Figure 1A). The cell surface expression of the same four proteins also was reduced in γ2+/− compared with WT cultures (Figure 1B), with effect sizes comparable to those in γ2−/− versus WT cultures (Figure 1A) and BIC-treated WT cultures (Supplemental Figure S1A). Total expression of GluN1 and GluA2/3 was unaffected in γ2−/− compared with WT cultures and BIC-treated WT cultures, suggesting that the changes at the cell surface were due to impaired trafficking of receptors (Supplemental Figure S1A, B). Therefore, a modest defect in GABAAR function in γ2+/− cultures (
      • Lorez M.
      • Benke D.
      • Luscher B.
      • Mohler H.
      • Benson J.A.
      Single-channel properties of neuronal GABAA receptors from mice lacking the 2 subunit.
      ,
      • Ren Z.
      • Sahir N.
      • Murakami S.
      • Luellen B.A.
      • Earnheart J.C.
      • Lal R.
      • et al.
      Defects in dendrite and spine maturation and synaptogenesis associated with an anxious-depressive-like phenotype of GABAA receptor-deficient mice.
      ) leads to a profound downregulation of GluRs that is reminiscent of homeostatic scaling down of synapses induced by complete pharmacologic blockade of GABAARs.
      Figure thumbnail gr1
      Figure 1Analyses of cell surface expression of synaptic proteins in cortical cultures. (A) γ2−/− cultures showed significantly reduced surface levels of neuroglobin 1 (NL1), GluN1, GluN2B, and GluA2/3 compared with wild-type (WT) neurons (γ2−/− vs. WT, NL1, p < .001, n = 5–6; GluN1, p < .01, n = 5–7 cultures; GluN2B, p < .01, n = 12–13; GluA2/3, p < .01, n = 3–4, t tests). (B) The same markers were also reduced in γ2+/− cultures (γ2+/− vs. WT, NL1, p < .01, n = 4–5; GluN1, p < .001, n = 12; GluN2B, p < .01, n = 10–11; GluA2/3, p < .05, n = 14–15; t tests). (C) A time course of ketamine treatment of γ2+/− cultures revealed increased GluN1 at 3 hours of ketamine treatment (γ2+/− vs. γ2+/− ketamine, p < .01, n = 9) that remained elevated thereafter (p < .05, n = 6–13 for both 4.5-hour and 6-hour time points, analysis of variance, Dunnett tests). GluA2/3 was significantly increased first at 4.5 hours and remained elevated (γ2+/– vs. γ2+/– ketamine, 4 hours, p < .05, 6 hours, p < .001, n = 11–12, analysis of variance, Dunnett tests). Cell surface GluN1 and GluA2/3 levels showed a greater 3-hour ketamine response for GluN1 than GluA2/3 (F1,48 = 4.78, p < .05, analysis of variance). Data represent means ± SE. *p < .05, **p < .01, ***p < .001. Tub, tubulin.

      Ketamine-Induced Reversal of GluR Deficits

      We hypothesized that downregulation of GluRs was related to the depressive-like brain state of γ2+/− mice (
      • Earnheart J.C.
      • Schweizer C.
      • Crestani F.
      • Iwasato T.
      • Itohara S.
      • Mohler H.
      • et al.
      GABAergic control of adult hippocampal neurogenesis in relation to behavior indicative of trait anxiety and depression states.
      ,
      • Shen Q.
      • Lal R.
      • Luellen B.A.
      • Earnheart J.C.
      • Andrews A.M.
      • Luscher B.
      Gamma-aminobutyric acid-type A receptor deficits cause hypothalamic-pituitary-adrenal axis hyperactivity and antidepressant drug sensitivity reminiscent of melancholic forms of depression.
      ) and that it should be reversible by antidepressant concentrations of ketamine. We treated γ2+/− cultures with ketamine (10 µmol/L) for variable amounts of time and found that GluN1 cell surface expression was increased significantly within 3 hours of treatment (Figure 1C). Cell surface AMPARs were increased similarly but with about a 1.5-hour delay. Ketamine also increased cell surface expression of NL1, with a time course similar to that of GluN1 (Supplemental Figure S2A), which is consistent with a role of NL1 in cell surface trafficking of NMDARs (
      • Budreck E.C.
      • Kwon O.B.
      • Jung J.H.
      • Baudouin S.
      • Thommen A.
      • Kim H.S.
      • et al.
      Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling.
      ). The effect of 10 µmol/L ketamine on cell surface GluRs of γ2+/− cultures was reproduced by the GluN2B specific antagonist Ro 25-6981 (10 µmol/L) and the competitive NMDAR antagonist APV (100 µmol/L) (Supplemental Figure S2B, C).

      Glutamatergic Synapse Density Is Reduced by GABAAR Deficits and Normalized by Ketamine Treatment

      To address whether GABAAR deficits and ketamine impact the density of glutamatergic synapses, we immunostained cultured cortical neurons for the presynaptic and postsynaptic markers vGluT1 and PSD95. The density of punctate immunoreactivity for both markers and their colocalization along dendrites was reduced in γ2+/− compared with WT neurons (Figure 2AE). Treatment of γ2+/− cultures with ketamine (10 µmol/L, 6 hours) normalized the expression and colocalization of vGluT1 and PSD95 to WT levels (Figure 2AE), whereas the size of immunoreactive puncta was unaffected by genotype and drug treatment (Figure 2F). Thus, GABAAR deficit–induced reductions in GluR and NL1 cell surface expression and their normalization by ketamine correlate with changes in the number of synapses, rather than a change in protein accumulation at individual synapses.
      Figure thumbnail gr2
      Figure 2Analyses of glutamatergic synapses of γ2+/− cortical cultures by immunostaining. (A–C) Representative micrographs of neurons cultured from wild-type (WT) (A) and γ2+/− (B, C) embryos (20–21 days in vitro) subjected to treatment with ketamine (Ket) (C) and immunostained for the dendritic markers MAP2 (blue) (A1–C1), PSD95 (green) (A2–C2), and vGluT1 (red) (A3–C3). Boxed dendritic segments including merged images are shown enlarged to the right of the panels (A4–C4). Scale bar = 16.7 μm. (D) The density of punctate dendritic immunoreactivity of teardrop-shaped cells for PSD95 and VGluT1 was significantly reduced in γ2+/− compared with WT cultures (PSD95 [F2,33 = 4.11], VGluT1 [F2,33 = 5.17, p < .05 for both proteins, analysis of variance]; WT vs. γ2+/−, p < .05, n = 21–23 cells for both PSD95 and VGluT1, Tukey test). Puncta densities γ2+/− cultures treated with ketamine were reversed to WT levels (γ2+/− vs. γ2+/− ketamine, p < .05, n = 21–23, for PSD95 and VGluT1, Tukey 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 (F2,33 = 8.71, p < .01, analysis of variance; γ2+/− vs. WT and γ2+/− vs. γ2+/− ketamine, p < .01, n = 21–23 for both comparisons, Tukey test). (F) Puncta sizes were unaltered across conditions (PSD95 [F2,33 = 5.05], VGluT1 [F2,33 = 1.392, p = not significant, analysis of variance]). Data represent means ± SE. *p < .05, **p < .01, Tukey tests.

      GABAAR γ2+/- Model Shows Increased Sensitivity to Anxiolytic-like and Antidepressant-like Behavioral Effects of Ketamine

      Assuming that ketamine-induced surface expression of GluRs was related to its antidepressant activity, we predicted that ketamine exerts increased antidepressant behavioral effects in γ2+/− compared with WT mice. However, preliminary experiments designed to address behavioral effects of ketamine revealed, inexplicably, that γ2+/− and WT mice maintained on a 129X1/SvJ strain background failed to show antidepressant-like behavioral responses to ketamine (not shown), reminiscent of other mouse strains that are insensitive to ketamine (
      • Sato Y.
      • Seo N.
      • Kobayashi E.
      Genetic background differences between FVB and C57BL/6 mice affect hypnotic susceptibility to pentobarbital, ketamine and nitrous oxide, but not isoflurane.
      ,
      • Bechtholt-Gompf A.J.
      • Smith K.L.
      • John C.S.
      • Kang H.H.
      • Carlezon Jr, W.A.
      • Cohen B.M.
      • et al.
      CD-1 and Balb/cJ mice do not show enduring antidepressant-like effects of ketamine in tests of acute antidepressant efficacy.
      ). Therefore, we re-derived γ2+/− mice on a C57BL/6J genetic background, which has been widely used for studies of ketamine. The anxiety-like phenotype of γ2+/− mice in the elevated plus maze was fully normalized to WT levels 8 hours after a single dose of ketamine (3 mg/kg, i.p.), without effects in WT mice (Figure 3A). Moreover, ketamine had antidepressant-like consequences in the forced swim test selectively in γ2+/− mice but not WT mice (Figure 3B). Thus, γ2+/− mice are more sensitive than WT mice to the anxiolytic-like and antidepressant-like behavioral effects of ketamine.
      Figure thumbnail gr3
      Figure 3Gamma-aminobutyric acid A receptor γ2+/− mice exhibit increased behavioral sensitivity to anxiolytic-like and antidepressant-like effects of ketamine (Ket). Separate groups of gamma-aminobutyric acid A receptor γ2+/− mice and wild-type (WT) littermates were injected with vehicle (saline) or ketamine (3 mg/kg, i.p.) and subjected to behavioral testing 8 hours after treatment. (A–C) In the elevated plus maze, the percentage of time spent on open arms (A) showed a significant overall treatment effect (F1,43 = 8.17, p = .006, two-way analysis of variance [ANOVA]) and a strong trend toward a genotype × treatment interaction (F1,43 = 3.81, p = .057). Post hoc analyses revealed an anxiety-like reduction in the percentage time spent on open arms in vehicle-treated γ2+/− vs. WT mice (p < .05, n = 12–16) consistent with the phenotype previously reported for γ2+/− mice on the 129X1/SvJ background (
      • Crestani F.
      • Lorez M.
      • Baer K.
      • Essrich C.
      • Benke D.
      • Laurent J.P.
      • et al.
      Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.
      ,
      • Earnheart J.C.
      • Schweizer C.
      • Crestani F.
      • Iwasato T.
      • Itohara S.
      • Mohler H.
      • et al.
      GABAergic control of adult hippocampal neurogenesis in relation to behavior indicative of trait anxiety and depression states.
      ). Ketamine treatment resulted in an anxiolytic-like increase in the percentage of time on open arms of γ2+/− mice (γ2+/− ketamine vs. vehicle, p < .001, n = 12–14) but not WT mice (WT ketamine vs. vehicle, p > .05, n = 13–16, Fisher test). Similarly, analyses of percentage of open arm entries (B) showed a significant overall treatment effect (F1,51 = 7.71, p < .008, two-way ANOVA) and a significant anxiolytic-like effect of ketamine selectively in γ2+/- mice (γ2+/− ketamine vs. vehicle, p < .01, n = 12–14) but not WT mice (WT ketamine vs. vehicle, p = not significant, n = 14–16, Fisher tests). The four groups were indistinguishable with respect to closed arm entries (C) (genotype: F1,51 = 0.52, p = not significant, treatment: F1,51 = 0.71, p = not significant, two-way ANOVA. (D, E) In the forced swim test, the latency to first immobility (D) showed an overall genotype effect (F1,90 = 19.78, p < .001, n = 18–27, two-way ANOVA) and a genotype × treatment interaction (F1,90 = 4.45, p = .038). Post hoc group comparisons showed a reduced latency to first immobility specifically in vehicle-treated γ2+/− vs. WT mice (γ2+/− vehicle vs. WT vehicle, p < .001, n = 18–25; WT ketamine vs. vehicle, p > .05, n = 20–21, t tests) and an increased latency to first immobility in ketamine-treated γ2+/− vs. vehicle-treated γ2+/− mice (γ2+/− vehicle vs. γ2+/− ketamine, p < .01, n = 18–23, t test), consistent with the depressive-like phenotype previously reported for γ2+/− mice on the 129X1/SvJ background (
      • Earnheart J.C.
      • Schweizer C.
      • Crestani F.
      • Iwasato T.
      • Itohara S.
      • Mohler H.
      • et al.
      GABAergic control of adult hippocampal neurogenesis in relation to behavior indicative of trait anxiety and depression states.
      ,
      • Shen Q.
      • Lal R.
      • Luellen B.A.
      • Earnheart J.C.
      • Andrews A.M.
      • Luscher B.
      Gamma-aminobutyric acid-type A receptor deficits cause hypothalamic-pituitary-adrenal axis hyperactivity and antidepressant drug sensitivity reminiscent of melancholic forms of depression.
      ) and with ketamine-induced changes in glutamate receptor 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 (F1,90 = 9.32, p. < .030, n = 18–27, two-way ANOVA) and an antidepressant-like drug effect specifically in ketamine-treated vs. vehicle-treated γ2+/− mice but not WT mice (γ2+/− ketamine vs. vehicle, p < .01, n = 18–25; WT ketamine vs. vehicle, p > .05, n = 20–21, Fisher tests).

      GABAAR Deficits of γ2+/− Mice Result in Downregulation of GluRs In Vivo

      To begin to assess the fate of glutamatergic synapses in vivo, we quantified the cell surface expression of GluRs in acute brain slices of the hippocampus and the ACC/prelimbic cortex (PLC), which are among the brain areas with the greatest GABAAR deficit in γ2+/− mice (
      • Crestani F.
      • Lorez M.
      • Baer K.
      • Essrich C.
      • Benke D.
      • Laurent J.P.
      • et al.
      Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.
      ) and essential for antidepressant drug–induced behavioral effects in rodents (
      • Samuels B.A.
      • Hen R.
      Neurogenesis and affective disorders.
      ,
      • Chang C.H.
      • Chen M.C.
      • Lu J.
      Effect of antidepressant drugs on the vmPFC-limbic circuitry.
      ). The cell surface AMPARs and NMDARs were drastically reduced in γ2+/− mice compared with WT mice in both hippocampus and ACC/PLC (Figure 4A).
      Figure thumbnail gr4
      Figure 4Downregulation of glutamate receptors in γ2+/− vs. wild-type (WT) mice is reversed by ketamine (Ket) treatment. (A) The cell surface expression of GluN1 and GluA2/3 was reduced in γ2+/− vs. WT mice both in hippocampus (GluN1, p < .001; GluA2/3, p < .05, n = 12–13 mice for both comparisons) and in anterior cingulate cortex/prelimbic cortex (ACC/PLC) (p < .05, n = 12–13, for both comparisons). GluN2B cell surface expression was reduced in hippocampus of γ2+/− mice (γ2+/− vs. WT, p < .05, n = 6–7) and trended lower in ACC/PLC (p = .15, n = 6–7). (B) In γ2+/− mice analyzed 24 hours after an acute dose of ketamine (10 mg/kg, i.p.), the cell surface expression of N-methyl-D-aspartate receptors (NMDARs) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in hippocampus was increased compared with vehicle-treated (Veh) γ2+/− control mice (p < .05, n = 7–11, for GluN1, GluN2B, and GluA2/3 comparisons) to levels comparable to WT mice in (A) (γ2+/− plus ketamine vs. WT, p = not significant, n = 9–12). In ACC/PLC, ketamine treatment of γ2+/− mice resulted in increased expression of NMDARs (γ2+/− ketamine vs. vehicle, GluN1, p < .05) but not AMPARs (GluA2/3, p = not significant, n = 8–9 for both comparisons). Cell surface GluN2B levels trended in the same direction as GluN1, but the effect was not significant (p = .12, n = 3). (C) In hippocampus of γ2+/− mice analyzed 3 days after ketamine treatment, the cell surface expression of NMDA and AMPARs remained increased compared with control mice (p < .05, n = 10–12, for both GluN1 and GluA2/3). By contrast, in ACC/PLC, the GluN1 cell surface level had returned to baseline (p = not significant, n = 8–9), but the GluA2/3 cell surface expression showed a strong trend of an increase (p = .08, n = 6/group) that was not yet evident at 1 day after treatment (B). (D) In WT mice analyzed 24 hours after ketamine treatment, the expression of NMDA and AMPARs was unchanged compared with vehicle controls in hippocampus and ACC/PLC (WT vehicle vs. ketamine, p = not significant, 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 (). Data represent means ± SE. *p < .05, ***p < .001, t tests. Tub, tubulin.

      Subanesthetic Dose of Ketamine Given to γ2+/− Mice Normalizes NMDAR Cell Surface Expression In Vivo

      To examine whether ketamine affected expression of GluR in vivo, we treated γ2+/− mice with ketamine (C57BL/6J, strain, 10 mg/kg, i.p.) and 24 hours later harvested hippocampal and ACC/PLC brain slices for quantification of cell surface proteins. Ketamine treatment of γ2+/− mice resulted in significant upregulation and normalization of cell surface NMDARs in both hippocampus and ACC/PLC (Figure 4B). Expression of the GluN2B subunit appeared increased to similar levels as GluN1, although the effect was more variable and significant only in hippocampus. Total expression of NMDARs remained unaltered, indicating that ketamine acted posttranslationally to normalize impaired cell surface accumulation of NMDARs. In contrast to NMDARs, cell surface AMPARs of γ2+/− mice were upregulated by ketamine only in hippocampus and were unaffected in ACC/PLC (however, see below for effects of ketamine at 3 days after treatment; Figure 4C). Moreover, this drug effect in hippocampus involved increased total expression of AMPARs, rather than merely a change in cell surface accumulation (Figure 4B). Comparing the hippocampal cell surface expression of GluRs of ketamine-treated γ2+/− mice and drug-naïve WT mice (Figure 4A, B, data normalized to values of drug-naïve γ2+/− mice) indicated that ketamine fully restored the expression of GluRs from γ2+/− to WT levels (γ2+/− plus ketamine vs. WT, p = not significant, n = 9–12, for both GluN1 and GluA2/3, ANOVA, Tukey test).
      Given the lasting therapeutic effects of ketamine in patients (
      • Zarate Jr, C.A.
      • Singh J.B.
      • Carlson P.J.
      • Brutsche N.E.
      • Ameli R.
      • Luckenbaugh D.A.
      • et al.
      A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression.
      ), we further assessed whether the drug effects on GluR expression seen 1 day after treatment remained measurable 3 days after treatment. In hippocampus, both GluN1 and GluA2/3 remained significantly elevated compared with vehicle-treated γ2+/− mice 3 days after treatment (Figure4C). By contrast, in prefrontal cortex, the effects of ketamine on GluN1 were no longer detectable. Instead, there was a strong trend for upregulation of GluA2/3 that was not observed 1 day after treatment (Figure 4B, C). Thus, a subanesthetic dose of ketamine can reverse homeostatic downregulation of glutamatergic synapses in the hippocampus induced by impaired GABAergic transmission for at least 3 days after treatment. Moreover, ketamine-induced augmentation of AMPAR expression in prefrontal cortex may be delayed relative to that of NMDAR and slower than in hippocampus.
      Ketamine had no effect on GluR cell surface expression in WT mice, independent of brain region analyzed (1-day treatment) (Figure 4D) and consistent with the selective behavioral effects of ketamine in γ2+/− but not WT mice described in Figure 3. Downregulation of GluRs was evident in γ2+/− mice of both genetic backgrounds, C57BL/6J (Figure 4A) and 129X1/SvJ (Supplemental Figure S1C), suggesting that differential behavioral sensitivity of the two genetic backgrounds to the effects of ketamine was due to strain differences acting downstream of altered GluR expression.

      GABAAR Deficits Reduce the Number of Functional Glutamatergic Synapses In Vivo and This Defect Is Reversed by a Single Dose of Ketamine

      We next assessed functional defects in glutamatergic transmission using voltage clamp recordings of CA1 pyramidal cells in hippocampal slices. The frequency of spontaneous EPSCs (sEPSCs) was drastically reduced in γ2+/− compared with WT cells, whereas the amplitude was unaffected (Figure 5AC). Ketamine administered to mice 24 hours before recordings normalized the sEPSC frequency of γ2+/− mice to WT levels and had no effect in WT mice (Figure 5C). Neither ketamine nor genotype affected the amplitude of sEPSCs (Figure 5B). Consistent with results obtained by immunostaining of cultures (Figure 2), these findings suggest that GABAAR deficits and ketamine affect glutamatergic transmission through a change in the number of functional synapses, rather than through changes in the abundance of AMPARs at synapses.
      Figure thumbnail gr5
      Figure 5Downregulation of glutamatergic synaptic inputs to γ2+/− CA1 pyramidal neurons is reversed by ketamine (Ket). (A–C) Spontaneous excitatory postsynaptic current recordings from CA1 pyramidal neurons of γ2+/− and wild-type (WT) mice injected 24 hours earlier with saline (vehicle [Veh]) or ketamine. Representative traces are shown in (A), with summary quantification of spontaneous excitatory postsynaptic current amplitude in (B) and frequency in (C). Note the significant decrease in frequency of spontaneous excitatory postsynaptic current (sEPSC) recorded from vehicle-treated γ2+/− vs. WT mice that was normalized by ketamine treatment (genotype × treatment interaction for sEPSC frequency) (F1,86 = 4.473, p = .037, two-way analysis of variance [ANOVA]; p < .01, n = 12–41 cells, for both group comparisons, Tukey test). (D–G) Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) excitatory postsynaptic currents (EPSCs) recorded from CA1 pyramidal cells evoked by stimulation of the Schaffer collateral (D, E) or temporoammonic path (F, G). Schematics with sites of stimulation and recording and average traces at progressively larger stimulation intensities are shown in (D) and (F). Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) responses were reduced in γ2+/− vs. WT mice at Schaffer collateral (E) and temporoammonic (G) synapses. Moreover, ketamine treatment restored synaptic responses of γ2+/− mice to WT levels. (E) #WT vehicle vs. γ2+/− vehicle (F1,138 = 5.391, p = .022, two-way ANOVA; p < .05, n = 9–17 cells, Tukey test); (G) ###WT vehicle vs. γ2+/− vehicle (F1,102 = 23.78, p < .001, two-way ANOVA, p < .05, Tukey test). (H–K) N-methyl-D-aspartate receptor (NMDAR) EPSCs recorded from CA1 pyramidal cells evoked by stimulation of the Schaffer collateral (H, I) or temporoammonic 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 with WT mice at both Schaffer collateral (I) and temporoammonic (K) synapses. Ketamine treatment restored the synaptic NMDAR responses of γ2+/− mice to WT levels. (I) #WT vehicle vs. γ2+/− vehicle (F1,89 = 4.952, p = .029, two-way ANOVA); ###γ2+/− vehicle vs. γ2+/− ketamine (F1,95 = 12.31, p < .001, two-way ANOVA); (K) ###WT vehicle vs. γ2+/− vehicle (F1,90 = 15.55, p < .001, two-way ANOVA); ###γ2+/− vehicle vs. γ2+/− ketamine (F1,96 = 12.55, p < .001, two-way ANOVA; n = 8–9 cells for all groups in (I) and (K). Data represent means ± SE. *p < .05, **p < .01. DG, dentate gyrus.
      Chronic stress has been shown to preferentially affect temporoammonic (TA) synapses onto CA1 pyramidal neurons rather than Schaffer collateral (SC) synapses (
      • Kallarackal A.J.
      • Kvarta M.D.
      • Cammarata E.
      • Jaberi L.
      • Cai X.
      • Bailey A.M.
      • et al.
      Chronic stress induces a selective decrease in AMPA receptor-mediated synaptic excitation at hippocampal temporoammonic-CA1 synapses.
      ). To further characterize the synaptic deficits of γ2+/− CA1 pyramidal cells, we recorded AMPAR and NMDAR EPSCs evoked by selective stimulation of either the SC or the TA pathway. The AMPAR EPSC amplitudes were significantly reduced in γ2+/− compared with WT slices, with a more pronounced effect at TA synapses (Figure 5DG). Moreover, ketamine treatment of γ2+/− mice normalized SC-evoked AMPAR EPSCs to WT levels, without corresponding effects in WT mice (Figure 5E). At TA synapses, ketamine potentiated the AMPAR responses, with greater effects in γ2+/− than WT mice (WT ketamine as % WT vehicle vs. γ2+/− ketamine as % γ2+/− vehicle [F1,163 = 17.73, p < .0001, two-way ANOVA]) (Figure 5F, G). Similar to AMPAR EPSCs, NMDAR currents of SC-CA1 and TA-CA1 synapses of γ2+/− mice were impaired at baseline and restored to WT levels by ketamine pretreatment (Figure 5HK).
      To further examine the idea that the reduced sEPSC frequency of γ2+/− neurons reflected a reduction in the density of synapses (Figure 5A, B), we assessed the synaptic release probability by measuring paired pulse ratios from SC and TA path-stimulated pyramidal cells (Supplemental Figure S3). The paired pulse ratios were unaffected by genotype for both types of synapses and increased by ketamine selectively at TA-CA1 synapses. Thus, genotype-dependent and treatment-dependent alterations of glutamatergic transmission primarily reflect changes in synapse number rather than release probability. Furthermore, neither genotype nor ketamine treatment affected the rectification of AMPAR responses evoked by SC or TA pathway stimulation (Supplemental Figure S4), indicating that the calcium permeability of AMPARs does not play a significant role in the forms of plasticity examined here.

      Ketamine Enhances GABAergic Synaptic Inhibition in Frontal Cortex

      Ketamine has potent seizure-suppressing effects in animal models of epilepsy and in patients (
      • Schneider P.G.
      • Rodriguez de Lores Arnaiz G.
      Ketamine prevents seizures and reverses changes in muscarinic receptor induced by bicuculline in rats.
      ,
      • Chen J.W.
      • Wasterlain C.G.
      Status epilepticus: Pathophysiology and management in adults.
      ,
      • Synowiec A.S.
      • Singh D.S.
      • Yenugadhati V.
      • Valeriano J.P.
      • Schramke C.J.
      • Kelly K.M.
      Ketamine use in the treatment of refractory status epilepticus.
      ). Therefore, we wondered whether ketamine-induced potentiation of glutamatergic transmission was accompanied by an increase in GABAergic inhibition. Comparison of γ2+/− and WT cultured cortical neurons by immunofluorescent staining for the presynaptic and postsynaptic markers vesicular GABA transporter and gephyrin revealed a significant reduction in the size and density of punctate gephyrin immunoreactivity in γ2+/− versus WT cultures, with both of these defects fully reversed after 6 hours of treatment of γ2+/− cultures with 10 μmol/L ketamine (Figure 6AG). A modest but significant increase in the density of gephyrin puncta was also observed in WT cultures (Figure 6F). Vesicular GABA transporter staining of γ2+/− cultures was increased by ketamine in parallel with gephyrin (Figure 6G), suggesting that ketamine potentiated the postsynaptic apparatus, while increasing the number of GABAergic synapses in γ2+/− cultures. Consistent with this interpretation, the colocalization of vesicular GABA transporter and gephyrin was unaffected by genotype or ketamine treatment (Figure 6H). As expected, the amplitude of mIPSCs recorded from CA1 and L2/3 ACC pyramidal cells of γ2+/− brain slices was reduced compared with WT, whereas the frequency was unaltered (Figure 6IL). Therefore, γ2+/− neurons display a defect in GABAergic inhibition that is not compensated for by any process resembling homeostatic scaling up of inhibitory synaptic strength seen after prolonged treatment of cultured neurons with BIC. We found that ketamine had no effect on mIPSCs recorded from CA1 neurons of γ2+/− mice (Figure 6I, J). However, ketamine fully restored the amplitude of mIPSCs recorded from γ2+/− L2/3 ACC pyramidal cells to WT levels, along with a prominent increase in the frequency of mIPSCs observed selectively in γ2+/− mice but not WT mice (Figure 6K, L). Ketamine did not affect the amplitude or frequency of mIPSCs of WT mice, consistent with our other observations that showed that ketamine effects on GluR expression, synapse density, and emotional behavior were enhanced or specific for γ2+/− mice representing the pathologic condition. Glutamatergic synapses of L2/3 pyramidal cells were downregulated in γ2+/− mice and restored by ketamine administration to WT levels (Supplemental Figure S5A, B), similar to results obtained with CA1 pyramidal cells. Thus, GABAergic synapses in ACC of γ2+/− mice are potentiated by ketamine both presynaptically and postsynaptically, in concert with restoration of glutamatergic synapses and antidepressant behavioral effects.
      Figure thumbnail gr6
      Figure 6Characterization of gamma-aminobutyric acid (GABA)ergic synapse deficits and their reversal by ketamine (Ket) in γ2+/− cultured cortical neurons and CA1 and L2/3 anterior cingulate cortex of γ2+/− mice. (A–H) Cortical cultures (21 days in vitro) prepared from wild-type (WT) (A, C) and γ2+/− (B, D) embryos were either untreated (A, B) or treated with ketamine (C, D) (10 μmol/L, 6 hours) and subjected to immunofluorescent staining for gephyrin (green) (A1–D1) and vesicular GABA transporter (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 (F3,73 = 5.3, p < .01, analysis of variance [ANOVA]; γ2+/− vs. WT, p = .05; γ2+/− vs. γ2+/− ketamine, p < .01, WT vs. WT ketamine, p = not significant, n = 18–21, Bonferroni). (F) The density of punctate gephyrin staining per 40 μm was significantly reduced in γ2+/− vs. WT cultures (F3,73 = 64.9, p < .001, ANOVA; γ2+/− vs. WT, p < .05, n = 18–21, Tukey test) and increased by ketamine selectively in γ2+/− cultures (γ2+/− vs. γ2+/− ketamine, p < .001, WT vs. WT ketamine, p = not significant, n = 18–21, both comparisons, Tukey test). (G) The density of vesicular GABA transporter puncta along 40-μm segments of dendrite was increased by ketamine independent of genotype (F1,73 = 16.4, p < .001, ANOVA; WT vs. WT ketamine, p < .01, γ2+/− vs. γ2+/− ketamine, p < .05, n = 18–21, t tests). (H) The fraction of gephyrin puncta colocalized with vesicular GABA transporter was unaltered across conditions (F3,73 = 1.99, p = .12, ANOVA). (I–L) Representative traces of miniature inhibitory postsynaptic current (mIPSC) recordings from CA1 (I) and L2/3 anterior cingulate cortex pyramidal neurons (K) of WT and γ2+/− mice injected with saline (vehicle) or ketamine 24 hours before recording. (J) Quantification of mISPC amplitude and frequency for CA1 neurons showed a significant decrease in amplitude in γ2+/− vs. WT mice, regardless of treatment (mIPSC amplitude by genotype [F1,45 = 18.02, p = .001, two-way ANOVA], p < .05, n = 10–13 cells, Tukey test). (L) Quantification of mIPSC amplitude and frequency for anterior cingulate cortex neurons showed a significant decrease in amplitude in γ2+/− vs. WT mice that was reversed by ketamine treatment of γ2+/− mice (mIPSC amplitude, genotype × treatment interaction [F1,25 = 7.879, p < .01, n = 6–8 cells, two-way ANOVA]; treatment comparison [F1,25 = 9.388, p = .005, Bonferroni]). Data represent means ± SE. *p < .05, **p < .01, ***p < .001.

      Discussion

      We have shown in the present study that the depression-related brain state of γ2+/− mice induced by GABAergic deficit involves a homeostatic-like reduction of glutamatergic transmission. A modest reduction in GABAergic transmission in γ2+/− mice, consisting of ~20% reduction in the mIPSC amplitude of principal cells, leads to a robust ~30%–50% reduction in cell surface NMDA and AMPARs and ~50% reduction in sEPSC frequency. Hyperexcitability of cultured neurons induced by chronic BIC treatment is known to result in homeostatic scaling down of glutamatergic synapses in concert with scaling up of inhibitory synapses (
      • Watt A.J.
      • van Rossum M.C.
      • MacLeod K.M.
      • Nelson S.B.
      • Turrigiano G.G.
      Activity coregulates quantal AMPA and NMDA currents at neocortical synapses.
      ,
      • Peng Y.R.
      • Zeng S.Y.
      • Song H.L.
      • Li M.Y.
      • Yamada M.K.
      • Yu X.
      Postsynaptic spiking homeostatically induces cell-autonomous regulation of inhibitory inputs via retrograde signaling.
      ) and increased expression of postsynaptic GABAARs (
      • Rannals M.D.
      • Kapur J.
      Homeostatic strengthening of inhibitory synapses is mediated by the accumulation of GABA(A) receptors.
      ). Compared with complete (but transient) blockade of GABAARs by BIC treatment of cultures, γ2+/− mice and cultures display only a modest reduction of GABAAR function. However, in contrast to in BIC-treated cultures, inhibitory synapses are not strengthened in γ2+/− mice, possibly as a result of the limiting amounts of γ2-GABAARs. It is conceivable that homeostatic mechanisms of γ2+/− mice that serve to restore the balance of excitation and inhibition ended up exacerbating downregulation of glutamatergic transmission. Similar homeostatic mechanisms are likely to operate under conditions of chronic or repeated stress, which are used to model depression in rodents, involve downregulation of AMPARs and NMDARs (
      • Thompson S.M.
      • Kallarackal A.J.
      • Kvarta M.D.
      • Van Dyke A.M.
      • LeGates T.A.
      • Cai X.
      An excitatory synapse hypothesis of depression.
      ), result in increased behavioral responsiveness to ketamine (
      • Garcia L.S.
      • Comim C.M.
      • Valvassori S.S.
      • Reus G.Z.
      • Stertz L.
      • Kapczinski F.
      • et al.
      Ketamine treatment reverses behavioral and physiological alterations induced by chronic mild stress in rats.
      ), and hence involve changes in glutamatergic transmission comparable to changes observed in the γ2+/− model. Chronic stress also increases the chloride reversal potential, which renders GABAergic inhibition ineffective or excitatory (
      • Hewitt S.A.
      • Bains J.S.
      Brain-derived neurotrophic factor silences GABA synapses onto hypothalamic neuroendocrine cells through a postsynaptic dynamin-mediated mechanism.
      ,
      • Wake H.
      • Watanabe M.
      • Moorhouse A.J.
      • Kanematsu T.
      • Horibe S.
      • Matsukawa N.
      • et al.
      Early changes in KCC2 phosphorylation in response to neuronal stress result in functional downregulation.
      ,
      • MacKenzie G.
      • Maguire J.
      Chronic stress shifts the GABA reversal potential in the hippocampus and increases seizure susceptibility.
      ). Thus, stress-induced downregulation of GluRs may be a consequence of excessive excitatory drive and impaired upregulation of inhibition analogous to mechanisms observed in the γ2+/− model. Our findings merge the GABAergic (
      • Luscher B.
      • Shen Q.
      • Sahir N.
      The GABAergic deficit hypothesis of major depressive disorder.
      ,
      • Levinson A.J.
      • Fitzgerald P.B.
      • Favalli G.
      • Blumberger D.M.
      • Daigle M.
      • Daskalakis Z.J.
      Evidence of cortical inhibitory deficits in major depressive disorder.
      ) and glutamatergic (
      • Feyissa A.M.
      • Chandran A.
      • Stockmeier C.A.
      • Karolewicz B.
      Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression.
      ,
      • Thompson S.M.
      • Kallarackal A.J.
      • Kvarta M.D.
      • Van Dyke A.M.
      • LeGates T.A.
      • Cai X.
      An excitatory synapse hypothesis of depression.
      ) deficit hypotheses of MDD and provide a novel mechanism for how these two neurotransmitter systems interact in the etiopathology of MDD.
      Second, we showed that the GABAergic deficit–induced adaptations of glutamatergic synapses in γ2+/− mice are reversed by an acute subanesthetic dose of ketamine. Normalization of GluR expression and synapse density was also observed in γ2+/− cultures in the continuous presence of ketamine or APV or Ro 25-6981, whereas WT cultures were unaffected by ketamine or Ro 25-6981. These observations suggest that downregulation of NMDARs in γ2+/− cultures and mice involves excessive or untimely activation of NMDARs, most likely as a result of chronically increased release of glutamate. Evidence indicates that the initial effect of a subanesthetic dose of ketamine is to block a subpopulation of NMDARs that are active at rest (
      • Autry A.E.
      • Adachi M.
      • Nosyreva E.
      • Na E.S.
      • Los M.F.
      • Cheng P.F.
      • et al.
      NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses.
      ), followed by increased expression of AMPARs (
      • Li N.
      • Lee B.
      • Liu R.J.
      • Banasr M.
      • Dwyer J.M.
      • Iwata M.
      • et al.
      mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists.
      ). Our data extend these findings and show that initial antagonistic effects of ketamine on NMDARs are followed by restoration of previously compromised expression and function of postsynaptic NMDARs, in addition to increased expression and function of AMPARs. Moreover, the effects of ketamine were greatly enhanced or specific for γ2+/− mice compared with WT mice (i.e., under conditions representing the pathologic condition).
      Third, we showed that in concert with enhanced glutamatergic transmission, ketamine potentiates the presynaptic and postsynaptic function of GABAergic inhibitory synapses of L2/3 ACC pyramidal cells of γ2+/− mice. This drug effect occurred despite the impairment of inhibitory synapses that is evident in untreated γ2+/− mice. An enduring potentiation of synaptic inhibition is consistent with the known seizure-suppressing effects of ketamine (
      • Schneider P.G.
      • Rodriguez de Lores Arnaiz G.
      Ketamine prevents seizures and reverses changes in muscarinic receptor induced by bicuculline in rats.
      ,
      • Chen J.W.
      • Wasterlain C.G.
      Status epilepticus: Pathophysiology and management in adults.
      ,
      • Synowiec A.S.
      • Singh D.S.
      • Yenugadhati V.
      • Valeriano J.P.
      • Schramke C.J.
      • Kelly K.M.
      Ketamine use in the treatment of refractory status epilepticus.
      ).
      Fourth, downregulation of cell surface NMDARs in γ2+/− neurons and its reversal by ketamine were paralleled by corresponding changes in the cell surface expression of NL1, a synaptogenic cell adhesion protein that contributes to activity-dependent balancing of glutamatergic and GABAergic synaptic transmission (
      • Chubykin A.A.
      • Atasoy D.
      • Etherton M.R.
      • Brose N.
      • Kavalali E.T.
      • Gibson J.R.
      • et al.
      Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.
      ) and controls the synaptic accumulation of NMDARs (
      • Budreck E.C.
      • Kwon O.B.
      • Jung J.H.
      • Baudouin S.
      • Thommen A.
      • Kim H.S.
      • et al.
      Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling.
      ). Thus, aberrant trafficking of NL1 may play a key role in maladaptations observed in γ2+/− mice and their reversal by ketamine.
      In conclusion, GABAAR deficit–induced downregulation of cell surface GluRs is reminiscent of chronic stress–induced downregulation of AMPARs and NMDARs in medial prefrontal cortex (
      • Gourley S.L.
      • Kedves A.T.
      • Olausson P.
      • Taylor J.R.
      A history of corticosterone exposure regulates fear extinction and cortical NR2B, GluR2/3, and BDNF.
      ,
      • Yuen E.Y.
      • Wei J.
      • Liu W.
      • Zhong P.
      • Li X.
      • Yan Z.
      Repeated stress causes cognitive impairment by suppressing glutamate receptor expression and function in prefrontal cortex.
      ,
      • Li N.
      • Liu R.J.
      • Dwyer J.M.
      • Banasr M.
      • Lee B.
      • Son H.
      • et al.
      Glutamate N-methyl-D-aspartate receptor antagonists rapidly reverse behavioral and synaptic deficits caused by chronic stress exposure.
      ) and downregulation of AMPARs selectively at TA-CA1 synapses (
      • Kallarackal A.J.
      • Kvarta M.D.
      • Cammarata E.
      • Jaberi L.
      • Cai X.
      • Bailey A.M.
      • et al.
      Chronic stress induces a selective decrease in AMPA receptor-mediated synaptic excitation at hippocampal temporoammonic-CA1 synapses.
      ,
      • Cai X.
      • Kallarackal A.J.
      • Kvarta M.D.
      • Goluskin S.
      • Gaylor K.
      • Bailey A.M.
      • et al.
      Local potentiation of excitatory synapses by serotonin and its alteration in rodent models of depression.
      ). Similar to selective stress-induced impairment of TA-CA1 synapses, the functional deficit in γ2+/− mice was greater at TA-CA1 than at SC-CA1 synapses (TA-CA1 γ2+/− as % WT vs. SC-CA1 γ2+/− as % WT [F1,120 = 5.714, p = .0184, two-way ANOVA]) (Figure 5E vs. G). The TA-CA1 synapses map to distal apical dendrites of CA1 pyramidal cells that are targets of somatostatin-positive oriens-lacunosum moleculare cells (
      • Klausberger T.
      GABAergic interneurons targeting dendrites of pyramidal cells in the CA1 area of the hippocampus.
      ). Somatostatin interneurons are highly sensitive to stress and functionally impaired in patients with MDD (
      • Lin L.C.
      • Sibille E.
      Somatostatin, neuronal vulnerability and behavioral emotionality.
      ). Thus, endogenous or stress-induced GABAergic deficits may be key for stress-induced downregulation of CA1 pyramidal neuron glutamatergic synapses. Elucidating the signaling pathways underlying homeostatic plasticity of GABAergic and glutamatergic synaptic transmission should lead to novel approaches for the treatment of MDD.

      Acknowledgments and Disclosures

      This work was supported by the National Institutes of Mental Health Grant Nos. MH089111 and MH099851 (to BL) and Canadian Institutes of Health Research Grant No. MOP136881 and Natural Sciences and Engineering Research Council Grant No. DG391521 (to DS). The contents of this article are solely the responsibility of the authors and do not necessarily represent the views of the funding agencies.
      We thank Yao Guo for technical assistance and Dr. Qiuying Shen for expert advice on statistical analyses.
      ZR, HP, DS, and BL conceived and designed the experiments; ZR, HP, SJJ, MS, and TF performed the experiments; ZR, HP, SJJ, MS, and TF analyzed the data; ZR, HP, and BL wrote the manuscript; and SJJ, TF, and DS critically read the manuscript.
      The authors report no biomedical financial interests or potential conflicts of interest.

      Appendix A. Supplementary materials

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      Linked Article

      • Ketamine for Depression: An Update
        Biological PsychiatryVol. 80Issue 6
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          A decade has now passed since research into the antidepressant effects of ketamine began in earnest, after the clinical trial reported by Zarate et al. in 2006 (1). In that proof-of-concept study, 18 medication-free patients with treatment-resistant major depressive disorder (TRD) showed a large reduction in core depressive symptoms within hours of receiving a single low-dose 0.5 mg/kg intravenous infusion of ketamine as measured by the 21-item Hamilton Depression Rating Scale compared with saline placebo.
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