Ketamine disinhibits dendrites and enhances calcium signals in prefrontal dendritic spines - PubMed (original) (raw)

Fig. 1. Effects of subanesthetic ketamine on the activity of pyramidal neurons and SST interneurons in vivo.

a Schematic and timeline of the experiments. b Coronal histological section, showing the extent of AAV-mediated expression of GCaMP6f in the mouse medial prefrontal cortex. Cg1, cingulate cortex. M2, secondary motor cortex. PrL, prelimbic cortex. c Example motion traces from a head-fixed animal during a two-photon imaging session before and after saline injection (black), and another session before and after ketamine (10 mg/kg) injection (red). Gray shading denotes the 5 min during which the injection was made and imaging was done to verify that the field of view had not shifted. d Time-averaged motion (mean ± s.e.m.) for epochs including pre-injection, 5–30 min post-injection, and 30–60 min post-injection for saline (left) and ketamine (right). For saline injection (black), there were no detectable differences between epochs (pre-injection vs. 5–30 min post-injection: P = 0.6; pre-injection vs. 30–60 min post-injection: P = 0.1; Wilcoxon signed rank test). For ketamine (red), hyperlocomotion was detected transiently following injection (pre-injection vs. 5–30 min post-injection: P = 0.06; pre-injection vs. 30–60 min post-injection: P = 1; Wilcoxon signed rank test). Each point is an imaging session. n = 5 animals each for saline and ketamine. e Schematic of imaging location, and an in vivo two-photon image of GCaMP6s-expressing pyramidal neurons in Cg1/M2. Inset, magnified view of neuronal cell bodies. f The normalized difference in the rate of spontaneous calcium events of pyramidal neurons. Normalized difference was calculated as post-injection minus pre-injection values normalized by the pre-injection value (ketamine (10 mg/kg): 23.7 ± 2.1%, saline: 9.4 ± 1.9%, mean ± s.e.m.; P = 3 × 10−8, two-sample _t_-test). For ketamine, n = 613 cells from 5 animals. For saline, n = 681 cells from 5 animals. g Each row shows time-lapse fluorescence transients from the same pyramidal cell in the pre-injectiion (left) and post-injection (right) periods. Two example cells were plotted for saline injection (black), and two other examples were plotted for ketamine (10 mg/kg) injection (pre-injection: black; post-injection: red). h–j Same as (e–g) for GCaMP6s-expressing SST interneurons in Cg1/M2 of SST-IRES-Cre animals (ketamine (10 mg/kg): −12 ± 3%, saline: 13 ± 6%, mean ± s.e.m.; P = 1 × 10−4, two-sample _t_-test). For ketamine, n = 198 cells from 5 animals. For saline, n = 179 cells from 5 animals. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

Fig. 2. Ketamine induces opposing effects on dendrite-targeting SST axons and apical dendrites of pyramidal neurons.

a Schematic of imaging location, and an in vivo two-photon image of GCaMP6s-expressing SST axons in superficial layers of Cg1/M2 of SST-IRES-Cre animals. Inset, magnified view of a SST axonal segment. b The normalized difference in the rate of spontaneous calcium events for SST axons in superficial layers. Normalized difference was calculated as post-injection minus pre-injection values normalized by the pre-injection value (ketamine (10 mg/kg): −9 ± 3%, saline: 12 ± 7%, mean ± s.e.m.; P = 0.002, two-sample t-test). For ketamine, n = 269 boutons from 5 animals. For saline, n = 214 boutons from 5 animals. c Left, schematic of imaging location. Right, each row shows time-lapse fluorescence transients from the same dendritic spine in the pre-injection (left) and post-injection (right) periods. Two example spines were plotted for saline injection (black) and two other examples were plotted for ketamine (10 mg/kg) injection (pre-injection: black; post-injection: red). Locations of the spines in the in vivo two-photon images are indicated by white arrows. d Same as (b) but for dendritic spines in superficial layers of Cg1/M2 (ketamine (10 mg/kg): 43.42 ± 0.01%, saline: 4.34 ± 0.01%, mean ± s.e.m.; P = 0.02, two-sample _t_-test). For ketamine, n = 280 dendritic spines from 5 animals. For saline, n = 231 dendritic spines from 5 animals. e The normalized difference in amplitude (ketamine (10 mg/kg): 5 ± 4%, saline: −4 ± 1%, mean ± s.e.m.; P = 0.03, two-sample _t_-test), and frequency of binned calcium events (ketamine (10 mg/kg): 16 ± 4%; saline: 4 ± 2%; P = 0.008, two-sample _t_-test). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

Fig. 3. Effect of ketamine on calcium dynamics for dendritic spines in the primary motor cortex.

a Left, coronal histological section, showing the extent of AAV-mediated expression of GCaMP6f in the primary motor cortex, M1. Right, schematic of imaging location. b The normalized difference in the rate of spontaneous calcium events for apical dendritic spines in M1. Normalized difference was calculated as post-injection minus pre-injection values normalized by the pre-injection value (ketamine (10 mg/kg): −7 ± 2%, mean ± s.e.m.; saline: −2 ± 2% for saline; P = 0.05, two-sample _t_-test). For ketamine, n = 124 dendritic spines from 3 animals. For saline, n = 120 dendritic spines from 3 animals. c The normalized difference in amplitude (ketamine (10 mg/kg): −7 ± 2%; saline: −5 ± 2%; P = 0.004, two-sample _t_-test) and frequency of binned calcium events (2 ± 2% for ketamine; −1 ± 1% for saline; P = 0.3, two-sample _t_-test). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

Fig. 4. Ketamine increases synaptically evoked calcium responses in dendritic spines.

a Schematic of experimental setup and imaging location. Right, an in vivo two-photon image of GCaMP6f-expressing apical dendritic spines in the superficial layer of Cg1/M2. b Protocol for the RSC stimulation. c Fluorescence transients in dendritic spines in Cg1/M2 in response to electrical stimulation of the retrosplenial cortex (RSC). Each trace shows a single 32-pulse-stimulation trial during pre-injection (left) or post-injection (right) period. One example spine was plotted for saline injection (black), and another example spine was plotted for ketamine injection (red). Dashed line, time of stimulation onset. d Trial-averaged spine calcium responses as a function of the number of stimulation pulses applied in a trial, for pre-injection (dashed line) vs. post-injection period (solid line) and for saline (black line) vs. ketamine (10 mg.kg) injection (red line). A three-way mixed ANOVA was performed with drug (saline, ketamine) as a between-subjects factor, and stimulation levels (1, 2, 4, 8, 16, 32, 64) and epoch (pre-injection, post-injection) as within-subjects factors. A three-way interaction was significant (F(6,2364) = 5.6, P = 9 × 10−6). A subsequent two-way mixed ANOVA was performed separately on the saline and ketamine datasets, which showed a significant two-way interaction between epoch (pre vs. post) and stimulation levels for ketamine (F(6,1146) = 6.6, P = 7 × 10−7), but not for saline (F(6,1218) = 20.0, P = 0.06). Post-hoc Tukey-Kramer test on the ketamine dataset indicated significantly elevated spine responses for post-injection vs. pre-injection for stimulation pulse numbers of 4 (P = 0.01), 8 (P = 0.007), 16 (P = 0.008), 32 (P = 0.02), and 64 (P = 9 × 10−5). Line, mean ± s.e.m. For saline, n = 204 dendritic spines from 4 animals. For ketamine, n = 192 dendritic spines from 4 animals. Refer to Supplementary Fig. 5f for full distribution. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Error bars, ±s.e.m.

Fig. 5. GluN2B expression in SST interneurons mediates effect of ketamine on dendritic spine calcium.

a Schematic of AAV1-CMV-dsRed-pSico-GluN2BshRNA (standard AAV cassettes not drawn) for Cre-dependent expression of shRNA against GluN2B. b Top, timeline for experiments involving selective knockdown of GluN2B expression in SST interneurons (GluN2B-SST KD). Bottom, in vivo two-photon image of ubiquitous dsRed expression (left), Cre-dependent GCaMP6s expression (middle), and a composite image (right). Arrowheads, cells that expressed both dsRed and GCaMP6s. c The rate of spontaneous calcium events for GCaMP6s-expressing SST interneuron cell bodies with or without GluN2B-SST KD (GluN2B-SST KD: 1.5 ± 0.2 Hz, control SST-IRES-Cre animals with no KD: 1.9 ± 0.1 Hz, mean ± s.e.m.; P = 0.04, two-sample t-test). For GluN2B-SST KD, n = 58 cells from 3 animals. For control animals, n = 72 cells from 3 animals. d The normalized difference in the number of spontaneous calcium events GCaMP6s-expressing SST interneuron cell bodies, for GluN2B-SST KD with ketamine or saline injection. Normalized difference was calculated as post-injection minus pre-injection values normalized by the pre-injection value (ketamine (10 mg/kg) with GluN2B-SST KD: 2 ± 4%, saline with GluN2B-SST KD: 7 ± 6%, mean ± s.e.m.; P = 0.46, two-sample _t_-test). For ketamine, n = 58 cells from 3 animals. For saline, n = 39 cells from 3 animals. e Same as (c) for GCaMP6f-expressing apical dendritic spines (GluN2B-SST KD: 0.85 ± 0.03 Hz, mean ± s.e.m., control SST-IRES-Cre animals: 0.73 ± 0.04 Hz, mean ± s.e.m.; P = 0.01, two-sample t-test). For GluN2B-SST KD, n = 277 spines from 4 animals. For control animals, n = 118 spines from 3 animals. f Same as (d) for GCaMP6f-expressing apical dendritic spines (ketamine (10 mg/kg) with GluN2B-SST KD: −4 ± 1%, saline with GluN2B-SST KD: −5 ± 2%, mean ± s.e.m.; P = 0.8, two-sample _t_-test). For ketamine, n = 169 spines from 4 animals. For saline, n = 171 spines from 4 animals. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

Fig. 6. Chronic downregulation of GluN2B in prefrontal cortical SST interneurons and its behavioral consequences.

a Coronal histological section, showing the extent of dsRed expression from bilateral injection of AAV1-CMV-dsRed-pSico-GluN2BshRNA into Cg1/M2 of a SST-IRES-Cre animal. b Fraction of time spent freezing during CS+ and trace period (if any), subtracted by that during CS− and trace period (if any), on the testing day. Freezing behavior for control SST-IRES-Cre animals in delay fear conditioning with no trace period (saline: 47 ± 6%, ketamine (10 mg/kg): 50 ± 5%, mean ± s.e.m.; P = 0.5, Wilcoxon rank-sum test; n = 8 and 12 animals for saline and ketamine, respectively), in trace fear conditioning (saline: 41 ± 7%, ketamine (10 mg/kg): 2 ± 4%, mean ± s.e.m.; P = 6 × 10−6, Wilcoxon rank-sum test; n = 8 animals each for saline and ketamine), and for GluN2B-SST KD animals in trace fear conditioning (saline: 33 ± 7%, ketamine (10 mg/kg): 43 ± 6%, mean ± s.e.m.; P = 0.2, Wilcoxon rank-sum test; n = 8 animals each for saline and ketamine). c Pre-pulse inhibition as a measure of sensorimotor gating. For control SST-IRES-Cre animals, a two-way within-subjects ANOVA with pre-pulse intensity (3, 6, and 9 dB) and drug (saline, ketamine (40 mg/kg)) as within-subjects factors found significant main effects of pre-pulse intensity (F(2,12) = 27.5, P = 4 × 10−6) and drug (F(1,24) = 2.0, P = 2 × 10−4), but a non-significant interaction (F(2,24) 1.8, P = 0.18). Post-hoc Tukey-Kramer’s tests for saline vs. ketamine were significant at 3 dB (40 ± 7%, 5 ± 6%, mean ± s.e.m., P = 0.01), at 6 dB (59 ± 5%, 17 ± 8%, mean ± s.e.m., P = 9 × 10−4), at 9 dB (67 ± 4%, 38 ± 6%, mean ± s.e.m., P = 0.003). n = 13 animals each for saline and ketamine. For GluN2B-SST KD animals, two-way within-subjects ANOVA revealed a significant main effect of pre-pulse intensity (F(2,24) = 6.3, P = 1.7 × 10−4), but non-significant drug effect (F(1,12) = 4.0, P = 0.93) or two-way interaction (F(2,24) = 1.4, P = 0.26). Saline vs. ketamine yielded no difference at 3 dB (32 ± 9%, 39 ± 6%, mean ± s.e.m.), at 6 dB (58 ± 6%, 56 ± 6%, mean ± s.e.m.), and at 9 dB (67 ± 6%, 63 ± 6%, mean ± s.e.m.). n = 13 animals each for saline and ketamine. d Open-field locomotor activity. Each trace comes from a single animal. For control SST-IRES-Cre animals, a two-way ANOVA was performed with epoch (pre-injection, 5–30 min post-injection, and 30–60 min post-injection) and drug (saline, ketamine (10 mg/kg)) as within-subjects factors. There were significant main effects of epoch (F(2,22) = 21.0, P = 7 × 10−5), drug (F(1,11) = 7.1, P = 0.02), and interaction (F(2,22) = 28.5, P = 7 × 10−5). Post-hoc Tukey-Kramer tests for saline vs. ketamine were significant for 5–30 min post-injection (P = 0.001), but non-significant for pre-injection (P = 0.07) and 30–60 min post-injection (P = 0.58). n = 12 animals each for saline and ketamine. For GluN2B-SST KD animals, two-way ANOVA revealed significant main effects of epoch (F(2,22) = 14.1, P = 0.002), drug (F(1,11) = 15.6, P = 0.002), and interaction (F(2,22) = 13.6, P = 0.003). Post-hoc Tukey-Kramer tests for saline vs. ketamine were significant for 5–30 min post-injection epoch (P = 0.003) and 30–60 min post-injection (P = 0.001), but non-significant for pre-injection (P = 1.0). n = 13 animals each for saline and ketamine. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Error bars, ± s.e.m.

Fig. 7. Acute suppression of SST interneuron activity in Cg1/M2 occludes the behavioral effects of ketamine.

a Timeline for experiments involving use of Cre-dependent expression of designer receptors exclusively activated by designer drugs (DREADD) in SST-IRES-Cre animals. b Fraction of time spent freezing during CS+ and trace period, subtracted by that during CS− and trace period on the testing day. For hM4D(Gi) + Vehicle condition, ketamine (10 mg/kg) treatment caused an impairment in freezing compared to saline (saline: 44 ± 7%, ketamine: 4 ± 2%, mean ± s.e.m.; P = 2 × 10−4, Wilcoxon rank-sum test; n = 10 animals for saline and 9 for ketamine). For hM4D(Gi) + CNO condition, there was impairment in freezing in saline-treated animals with no further difference relative to ketamine-treated (10 mg/kg) animals (saline: 5 ± 2%, ketamine: 3 ± 2%, mean ± s.e.m.; P = 0.78, Wilcoxon rank-sum test; n = 9 animals for saline and 10 for ketamine). There was no effect of CNO on ketamine-induced (10 mg/kg) impairment as tested in the mCherry + CNO condition (saline: 39 ± 5%, ketamine: 1.5 ± 1%, mean ± s.e.m.; P = 2 × 10−4, Wilcoxon rank-sum test; n = 10 animals each for saline and ketamine). c Pre-pulse inhibition as a measure of sensorimotor gating. For hM4D(Gi) + Vehicle condition, a two-way within-subjects ANOVA with pre-pulse intensity (3, 6, and 9 dB) and drug (saline, ketamine (40 mg/kg)) as within-subjects factors found significant main effects of pre-pulse intensity (F(2,30) = 30.6, P = 6 × 10−7) and drug (F(1,15) = 21.9, P = 3 × 10−4), and a significant interaction (F(2,30) = 4.0, P = 0.03). Post-hoc Tukey-Kramer’s tests for saline vs. ketamine were significant at 3 dB (22 ± 4%, 6 ± 4%, mean ± s.e.m., P = 0.01), at 6 dB (42 ± 6%, 12 ± 5%, mean ± s.e.m., P = 1 × 10−4), at 9 dB (51 ± 5%, 23 ± 6%, mean ± s.e.m., P = 0.002). n = 16 animals each for saline and ketamine. For hM4D(Gi) + CNO condition, a two-way within-subjects ANOVA found a significant main effect of pre-pulse intensity (F(2,26) = 11.9, P = 2 × 10−4) but a non-significant main effect of drug (F(1,13) = 4.1 P = 0.06) and interaction (F(2,26) = 1.2, P = 0.3). Post-hoc Tukey-Kramer’s tests for saline vs. ketamine were not significant at 3 dB (−14 ± 8%, 1 ± 4%, mean ± s.e.m., P = 0.06), at 6 dB (8 ± 3%, 10 ± 6%, mean ± s.e.m., P = 0.71), at 9 dB (17 ± 5%, 26 ± 4%, mean ± s.e.m., P = 0.16). n = 14 animals each for saline and ketamine. For mCherry + CNO condition, a two-way within-subjects ANOVA found significant main effects of pre-pulse intensity (F(2,28) = 30.4, P = 9 × 10−8) and drug (F(1,14) = 56.0, P = 3 × 10−5), and a non-significant interaction (F(2,28) = 0.5, P = 0.62. Post-hoc Tukey-Kramer’s tests for saline vs. ketamine were significant at 3 dB (27 ± 4%, −3 ± 5%, mean ± s.e.m., P = 5 × 10−4), at 6 dB (49 ± 4%, 12 ± 5%, mean ± s.e.m., P = 1 × 10−5), at 9 dB (58 ± 5%, 24 ± 7%, mean ± s.e.m., P = 1 × 10−5). n = 16 animals each for saline and ketamine. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

Fig. 8. Elevated prefrontal cortical functional connectivity depends on dendritic inhibition.

a Left, schematic of experimental setup. Right, spectrogram of an example local field potential (LFP) recording in Cg1/M2 of a SST-IRES-Cre animal. Dashed lines indicate the upper and lower bounds of the frequency range used to calculate the integrated gamma band signal, which is plotted below the spectrogram. b Integrated gamma band signals from LFPs recorded simultaneously from Cg1/M2 (solid line) and RSC (dashed line) of a SST-IRES-Cre animal. Example signals were plotted before (black) and after (red) ketamine (10 mg/kg) injection. c Network-scale effects of prefrontal cortical SST interneuron manipulations (SST-IRES-Cre animals with or without GluN2B-SST KD). Functional connectivity between Cg1/M2 and RSC was quantified by determining the correlation and coherence between integrated gamma band signals of the two brain regions. At baseline prior to injection, control and GluN2B-SST KD animals had significantly different correlation coefficients (controls: 0.48 ± 0.08, GluN2B-SST KD: 0.72 ± 0.05, mean ± s.e.m.; P = 0.03, Wilcoxon rank sum test) and coherence magnitude (controls: 0.53 ± 0.07, GluN2B-SST KD: 0.73 ± 0.05, mean ± s.e.m.; P = 0.04, Wilcoxon rank sum test). n = 8 animals each for controls and GluN2B-SST KD. d Functional connectivity between Cg1/M2 and RSC after ketamine (10 mg/kg) or saline for SST-IRES-Cre animals with or without GluN2B-SST KD. For correlation, a two-way mixed ANOVA was performed with treatment (GluN2B-SST KD, no KD) as the between factor, and drug (saline, ketamine) as the within factor. The interaction effect was significant (F(1,14) = 6.2, P = 0.03). Post-hoc Tukey-Kramer test showed a significant ketamine-saline difference in the no KD group (P = 0.003), but not in the GluN2B-SST KD group (P = 0.9). For coherence, the interaction effect was significant (F(1,14) = 7.1, P = 0.02). Post-hoc Tukey-Kramer test showed a significant ketamine-saline difference in the no KD group (P = 0.003), but not in the GluN2B-SST KD group (P = 0.8). n = 8 animals each for controls and GluN2B-SST KD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.