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Ketamine triggers a switch in excitatory neuronal activity across neocortex

Nature Neuroscience volume 26, pages 39–52 (2023)Cite this article

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Abstract

The brain can become transiently disconnected from the environment while maintaining vivid, internally generated experiences. This so-called ‘dissociated state’ can occur in pathological conditions and under the influence of psychedelics or the anesthetic ketamine (KET). The cellular and circuit mechanisms producing the dissociative state remain poorly understood. We show in mice that KET causes spontaneously active neurons to become suppressed while previously silent neurons become spontaneously activated. This switch occurs in all cortical layers and different cortical regions, is induced by both systemic and cortical application of KET and is mediated by suppression of parvalbumin and somatostatin interneuron activity and inhibition of NMDA receptors and HCN channels. Combined, our results reveal two largely non-overlapping cortical neuronal populations—one engaged in wakefulness, the other contributing to the KET-induced brain state—and may lay the foundation for understanding how the brain might become disconnected from the surrounding environment while maintaining internal subjective experiences.

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Fig. 1: KET induces a switch in the active population of L2/3 neurons in S1.
Fig. 2: KET induces a neuronal switch in the input and output cortical layers of S1.
Fig. 3: KET induces widespread reorganization of L2/3 activity across the neocortex.
Fig. 4: Local KET delivery in S1 induces a neuronal switch in the active population of L2/3 neurons.
Fig. 5: Systemic and local KET suppresses activity in S1 interneuron subtypes.
Fig. 6: KET-induced suppression of SST or PV interneurons is necessary for the activity switch in L2/3 pyramidal neurons.
Fig. 7: Inhibition of NMDARs or HCN channels drives a neuronal activity switch.
Fig. 8: Combined inhibition of NMDARs and HCN channels recapitulates the effects of KET administration.

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Acknowledgements

We thank W. Gan for insightful discussions, M. Fina for animal management and genotyping and S. Nikonov of the University of Pennsylvania Vision Research Center for technical and imaging support. This work was supported by the University of Pennsylvania Department of Anesthesia Dripps scholarship and FAER MRTG to J.C.; the Training Program in Neuroengineering and Medicine T32NS091006 to A.W.; National Institutes of Health (NIH) R01GM124023-01A1 and 5R01NS113366 to A.P.; NIH R01GM088156 to M.B.K.; and NIH R01EY020765 to D.C.

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Authors and Affiliations

  1. Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Joseph Cichon, Andrzej Z. Wasilczuk, Max B. Kelz & Alex Proekt

  2. Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Joseph Cichon & Diego Contreras

  3. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Loren L. Looger

  4. Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Max B. Kelz & Alex Proekt

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Contributions

J.C. initiated the project. J.C. performed imaging and behavioral experiments. J.C. processed and analyzed the data, with input from A.P., M.B.K. and D.C. A.W. processed and analyzed EEG recordings. J.C., A.P., L.L.L. and M.B.K wrote the paper, with input from D.C.

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Correspondence to Joseph Cichon or Alex Proekt.

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Extended data

Extended Data Fig. 1 Subhypnotic ketamine impairs sensorimotor processing but not gross animal movement.

(a, b) Duration effect of single dose of ketamine (KET; 50 mg/kg: 20 mice, 100 mg/kg: 11 mice) or saline (11 mice) on time for complete removal of adhesive from snout (TAR) (a) and forelimb withdrawal response to air puff (b). (c) Example images of marble burial test under different conditions. Note that saline-injected mice bury a fraction of marbles over 30 min (middle image). (d) Summary of results of marble burial test for 16 mice in each treatment group. Mice injected with KET at either 50 or 100 mg/kg fail to bury a single marble (example image in c). Number of marbles buried within 30 min was 0 in KET (50 or 100 mg/kg) vs. 4.1 ± 1 in saline-injected mice. One-sided Kruskal-Wallis with Dunn’s multiple comparisons test, P < 0.001. (e) Tracking of total distance and average speed for 14 mice over 5 min period before and after KET at 50 mg/kg in a standard mouse cage. Two-sided Wilcoxon rank test, P > 0.05 for both parameters. Exact P values in figure. Error bars show s.e.m.

Extended Data Fig. 2 Various ketamine-induced layer 2/3 responses in the forelimb region of primary somatosensory cortex.

(a) Line scatterplots of L2/3 neurons before and after KET at different doses, 50 (49 cells from 4 regions), 100 (37 cells from 3 regions), 150 mg/kg (34 cells from 3 regions) from single animals. Scatterplot of entire L2/3 population for 50 and 100 mg/kg in Fig. 1g. (b) Representative fluorescent traces (left) of individual L2/3 axonal boutons imaged in layer 1 under wakefulness and KET 100 mg/kg. Line scatterplots (right) of individual boutons before and after KET 100 mg/kg reveals emergence of a switch. (c) Difference in calcium activity induced by KET at 10 mg/kg (left, 85 neurons from 3 mice) or 150 mg/kg IP (right, 64 neurons from 2 mice) from baseline wakefulness vs. wakefulness activity. KET at 150 mg/kg (one-sided Pearson correlation: r = −0.62, P = 7.8 × 10−8), but not at 10 mg/kg (r = −0.06, P = 0.56), induced a neuronal switch in L2/3 activity.

Extended Data Fig. 3 GABAergic hypnotic agents induce net suppression of L2/3 pyramidal cell activity.

(a, b) Representative GCaMP6 traces of individual L2/3 neurons under wakefulness and different hypnotic agents (a, sevoflurane (sevo), yellow shaded region; b, midazolam (midaz), green shaded region). (c) (Left) Line scatterplots and (Right) box plots of average spontaneous rate of L2/3 activity before and after volatile (sevo or isoflurane/iso, purple) and midaz hypnosis in S1. Spontaneous activity: Sevo group (71 neurons from 4 mice), awake: 4.0 ± 0.8 vs. Sevo: 1.0 ± 0.4, P = 2.8 × 10−8; Iso group (78 neurons from 7 mice), awake: 2.4 ± 0.5 vs. Iso: 0.1 ± 0.1, P < 1 × 10−15; Midaz group (85 neurons from 3 mice), awake: 2.8 ± 0.6 vs. Midaz: 0.4 ± 0.1, P = 4 × 10−15). (d) Peak ΔF/F0 before and after hypnosis (Sevo: awake: 238 ± 30% vs. Sevo: 47 ± 5%, P = 2.9 × 10−10; Iso: awake: 102 ± 12% vs. Iso: 14 ± 0.5%, P < 1 × 10−15; Midaz: awake: 83 ± 7% vs. Midaz: 30 ± 2%, P = 5.6 × 10−12). Same cells from c. (e) Relative change in L2/3 activity for different hypnotic agents. Unlike KET, GABAergic hypnotics induced a uniform suppression of L2/3 neuronal activity (fraction of cells with reduced activity under GABAergic agents 65–78% vs. KET ~40%). While KET induced substantial activation of the network (~40–45% of cells), GABAergic agents activated fewer than 10% of cells. Box and whisker plot show min to max, centre (median), 25th and 75th percentile box bounds. One-sided Kruskal-Wallis with Dunn’s multiple comparisons test in c, d, ***P < 0.001.

Extended Data Fig. 4 Ketamine-induced activity switch not explained by random changes in activity.

(a–d) Shows joint distribution of activity of L2/3 neurons under normal wakefulness (x-axis) and after ketamine (KET)/saline administration (y-axis). The left column shows the experimentally observed joint probability distribution. Middle column shows the joint distribution expected if activity after KET/saline administration did not depend on baseline activity (Methods). (a) Saline control, (b–d) KET at 10, 50, and 100 mg/kg administered systemically. The right column shows statistical deviations of the observed joint distribution (left column) from one expected if activity during normal wakefulness and after drug administration was independent (middle column). Statistical significance was computed using a two-sided permutation test consisting of shuffling neuron IDs randomly 1000 times. Deviations were statistically significant at P < 0.01 (bootstrap estimates). Red points indicate locations where experimentally observed probability was higher than chance, blue points indicate locations where experimentally observed probability was lower than chance, white points show areas where no statistically significant differences were detected. The regions of statistical deviations directly abutting the x and y axes under KET at 50 and 100 mg/kg indicate that KET preferentially suppresses active neurons and activates a subset of silent neurons.

Extended Data Fig. 5 Layer 4 and 5 calcium responses under various conditions.

(a, b) Representative GCaMP6 traces of individual neurons (a, top for L4, b, bottom for L5) under wakefulness and following saline injection (left, purple shaded region) and KET at 100 mg/kg (right, red shaded region). (c) Line scatterplots (left) and average spontaneous activity (right) of individual L4 and L5 activity before and after saline. Saline did not significantly increase calcium activity compared to wakefulness. L4 (105 neurons from 9 mice), awake: 3.8 ± 0.6 vs. saline: 3.9 ± 0.5, P > 0.99. L5 (97 neurons from 8 mice), awake: 3.4 ± 0.7 vs. saline: 2.9 ± 0.7, P = 0.63. (d) Peak ΔF/F0 in saline-treated mice (same cells from c). L4, awake: 98 ± 7% vs. saline: 90 ± 6%, P = 0.30. L5, awake: 112 ± 12% vs. saline: 95 ± 7%, P = 0.30. Box and whisker plot show min to max, centre (median), 25th and 75th percentile box bounds. One-sided Kruskal-Wallis with Dunn’s multiple comparisons test in c, d.

Extended Data Fig. 6 Ketamine at 100 mg/kg induces a neuronal switch in L2/3 activity across the neocortex.

(a) Two-photon images of L2/3 neurons from various cortical regions in Thy1-GCaMP6f mice. Scale bar: 20 µm. (b) Cartoon of mouse brain in center of panel demarcates imaging locations (teal boxes) across the neocortex. Surrounding traces are representative L2/3 neurons from these regions under wakefulness and KET 100 mg/kg. (c) The effect of KET was negatively correlated with the activity in wakefulness across all imaged cortical regions. M2 (65 from 4 mice; r = −0.64, P = 1.1 × 10−8), M1 (140 from 4 mice; r = −0.56, P = 3.6 × 10−13), vibrissal (v) S1 (65 from 2 mice; r = −0.50, P = 2.5 × 10−5), and V1 (120 from 4 mice; r = −0.55, P = 2.2 × 10−10). (d) L2/3 activity switch index across saline and KET-injected mice for various regions. M2 (P = 0.001), M1 (P = 2.7 × 10−10), vS1 (P = 0.003), and V1 (P = 1.6 × 10−8). Error bars show s.e.m. One-sided Pearson correlations in c, One-sided Kruskal-Wallis with Dunn’s multiple comparisons test in d, ***P < 0.001.

Extended Data Fig. 7 Labelling different inhibitory interneurons subtypes in the somatosensory cortex with GCaMP6 and their responses to ketamine at 100 mg/kg.

(a) Example two-photon imaging fields of interneuron subtypes (SST, pink; PV cells, blue; VIP cells, green) expressing GCaMP6f in the S1. Interneurons outlined with white circles. Scale bar: 20 µm. Representative images carried out on at least 3 animals per group. (b) Pie graphs show the portion of cells increasing (green)/decreasing (red)/no change (black) following saline administration for each interneuron subtypes. (c, d) Comparison between spontaneous rate of activity (c) and peak signals (d) across interneuron subtypes following KET at 100 mg/kg. SST: 82 cells from 6 mice, PV: 84 cells from 4 mice, VIP: 85 mice from 5 mice (cell numbers per subtype italicized in c). Note the strong reduction of spontaneous interneuron activity and peak signals (One-sided Kruskal-Wallis with Dunn’s multiple comparisons for c: P < 0.05 for SST and P < 0.001 for all other comparisons; d: P < 0.001 for all comparisons). Exact P values in figure. Error bars show s.e.m.

Extended Data Fig. 8 Systemic administration of CNO does not impair ketamine’s neuronal activity switch in L2/3 neurons of S1.

(a) Coronal slices (left) of S1 region from interneuron-specific Cre-positive mice expressing DREADD variant hM3D(Gq)-mCherry. Scale bar: 100 µm. (Right) Number of hM3D(Gq)-mCherry positive cells in a 1 mm2 region of forelimb S1 (slice thickness 30 µm). 15 (SST), 16 (PV), and 16 (VIP) S1 slices from 4 mice in each group. Box and whisker plot show min to max, centre (median), 25th and 75th percentile box bounds. (b) Coronal slice of S1 region from mouse expressing AAV-hSynapsin-1-tdTomato. Scale bar: 100 µm. (c) Representative traces of L2/3 pyramidal cells under wakefulness, CNO, then KET at 50 mg/kg in mice expressing AAV-hSynapsin-1-tdTomato. (d) L2/3 cell activity in the setting of CNO vs. its response to KET at 50 mg/kg in mice expressing AAV-hSyn1-tdTomato (97 neurons from 3 mice). KET at 50 mg/kg induced a strong neuronal switch (Pearson correlation r = −0.68, P = 2 × 10−14) and a significant elevation in activity switch index (KET 4.9 ± 0.5) whereas CNO injection did not (1.8 ± 0.2, P > 0.05). (e) Spontaneous rate of activity of L2/3 pyramidal neurons before/baseline (BL), after interneuron subtype activation (CNO activation of hM3D(Gq)) and following KET treatment at 50 mg/kg. Same cells from Fig. 6d. One-sided Kruskal-Wallis, P < 0.001 with Dunn’s multiple comparisons and P values listed in graph. Representative images and traces carried out on at least 3 animals per group. Error bars show s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Fig. 9 Ketamine-induced neuronal switch attenuated in the presence of local midazolam to S1.

(a) Representative GCaMP6 traces of L2/3 pyramidal neurons under wakefulness and local (L-) midazolam at 100 (79 neurons from 3 mice) and 500 (125 neurons from 3 mice) µM. (b) L-midazolam reduced the normalized calcium response as compared to wakefulness indicating the likely facilitation of spontaneous GABAA receptor activity. Same cells from a. Error bars show s.e.m. (c) Scatterplot of L2/3 activity in the presence of L-midazolam vs. KET at 50 mg/kg. KET-induced neuronal switch was attenuated in the presence of L-midazolam (100 µM: r = 0.06, P = 0.58; 500 µM: r = −0.14, P = 0.13). Two-sided Wilcoxon matched-pairs signed-rank test in b, Pearson correlations in c. ***P < 0.001.

Extended Data Fig. 10 Layer 2/3 calcium responses under various drug conditions.

(a) Representative GCaMP6 traces of individual L2/3 neurons from S1 under wakefulness and following local (nifedipine at 100 µM and mecamylamine at 100 µM) or systemic (fentanyl at 0.3 mg/kg IP and ephedrine at 20 mg/kg IP) drug injection (color shaded regions). These potential KET molecular targets were ineffective inducers of an activity switch in L2/3 neurons (see Fig. 7). (b) Experimental design (top) for sequential local (L-) blockade of NMDA-R and AMPA-R in vivo. (Bottom) Representative GCaMP6 traces of L2/3 pyramidal neurons under wakefulness followed over 40 min followed by sequential delivery of L-MK801 at 100 µM and then DNQX at 100 µM (73 cells from 2 mice). (c) Compared to baseline recording in wakefulness, L-MK801 significantly reduced (61%) the L2/3 activity. The remaining cohort of active cells in the presence of L-MK801 could be completely silenced in the presence of L-DNQX. Same cells from b. One-sided Kruskal-Wallis with Dunn’s multiple comparisons test in c. (d, e) Scatter plots of L2/3 activity modulated by either L-MK801 (73 neurons from 2 mice, one-sided Pearson correlation r = −0.57, P = 1.6 × 10−7) or systemic KET at 50 mg/kg (94 neurons from 3 mice, r = −0.86, P = < 1 × 10−15) vs. L-DNQX effect. L-DNQX blocks newly activated cells induced by KET. Error bars show s.e.m. ***P < 0.001.

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Supplementary Figs. 1–3

Reporting Summary

Supplementary Video

Mice held in tail suspension exhibit escape-like behaviors (pre-KET). Post-KET (50 mg kg−1), mice no longer attempt to escape, and truncal movement is minimal. Meanwhile, KET induces a sustained vertical head twitch response.

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Cichon, J., Wasilczuk, A.Z., Looger, L.L. et al. Ketamine triggers a switch in excitatory neuronal activity across neocortex. Nat Neurosci 26, 39–52 (2023). https://doi.org/10.1038/s41593-022-01203-5

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