Deep posteromedial cortical rhythm in dissociation

Abstract

Advanced imaging methods now allow cell-type-specific recording of neural activity across the mammalian brain, potentially enabling the exploration of how brain-wide dynamical patterns give rise to complex behavioural states^1-12. Dissociation is an altered behavioural state in which the integrity of experience is disrupted, resulting in reproducible cognitive phenomena including the dissociation of stimulus detection from stimulus-related affective responses. Dissociation can occur as a result of trauma, epilepsy or dissociative drug use^13,14, but despite its substantial basic and clinical importance, the underlying neurophysiology of this state is unknown. Here we establish such a dissociation-like state in mice, induced by precisely-dosed administration of ketamine or phencyclidine. Large-scale imaging of neural activity revealed that these dissociative agents elicited a 1-3-Hz rhythm in layer 5 neurons of the retrosplenial cortex. Electrophysiological recording with four simultaneously deployed high-density probes revealed rhythmic coupling of the retrosplenial cortex with anatomically connected components of thalamus circuitry, but uncoupling from most other brain regions was observed-including a notable inverse correlation with frontally projecting thalamic nuclei. In testing for causal significance, we found that rhythmic optogenetic activation of retrosplenial cortex layer 5 neurons recapitulated dissociation-like behavioural effects. Local retrosplenial hyperpolarization-activated cyclic-nucleotide-gated potassium channel 1 (HCN1) pacemakers were required for systemic ketamine to induce this rhythm and to elicit dissociation-like behavioural effects. In a patient with focal epilepsy, simultaneous intracranial stereoencephalography recordings from across the brain revealed a similarly localized rhythm in the homologous deep posteromedial cortex that was temporally correlated with pre-seizure self-reported dissociation, and local brief electrical stimulation of this region elicited dissociative experiences. These results identify the molecular, cellular and physiological properties of a conserved deep posteromedial cortical rhythm that underlies states of dissociation.

Extended Data Fig. 1 ∣. Time course of ketamine-induced retrosplenial rhythm.

a, Prominent 1–3 Hz retrospenial activity after ketamine injection seen in a frequency response from Thy1-GCaMP6s (top) but not control (Thy1–GFP) mice (bottom). b, Expansion of the first 15 min of the recording shown in Fig. 1g. c, Power in 1–3 Hz band in motor (MOT), somatosensory (SS), visual (VIS), posterior parietal (PP) and retrosplenial (RSP) cortices (mean ± s.e.m., n = 6 mice). d, Example 410-nm channel-corrected trace of RSP and SS activity from 3 min before to 12 min after intraperitoneal injection of 50 mg kg⁻¹ ketamine. Vertical lines indicate injection time. e, Magnifications of 30 s of data demonstrating oscillatory rhythm in the RSP but not the SS at ten minutes after injection, or before injection. f, PSD in the RSP during five consecutive days of 50 mg kg⁻¹ ketamine injection (mean ± s.e.m., n = 6 mice). g, Summary of power in the 1–3 Hz band in the RSP during five consecutive days of 50 mg kg⁻¹ ketamine injection (ns, corrected paired t-test comparison with first day, P = 0.10, 0.10, 0.80, 0.75, Hedge’s g = 0.73, 0.49, 0.12, 0.15). h, Direct comparison of oscillation in similarly aged male and female mice. PSD in the RSP and summary of power in the 1–3 Hz band. Both sexes have significant increase in power relative to pre-injection, but the magnitude is not significantly different between male and female mice (P < 0.01, corrected t-test versus 0, and P = 0.21, t-test between groups, Hedge’s g = 0.76). i, Video tracking of mouse facial movements. Facial movements did not show 1–3 Hz features before or after administration of ketamine (paired t-test P = 0.24, Hedge’s g = 0.80). j, Top, correlation of cortical areas imaged with widefield imaging, before and after saline injection (n = 5 mice, corrected paired t-test P = 0.85, 0.51, 0.37, 0.25, Hedge’s g = 0.08, 0.27, 0.36, 0.21). Bottom, correlation of cortical areas imaged with widefield imaging, before and after injection of 50 mg kg⁻¹ ketamine (n = 5 mice, corrected paired t-test P = 0.009, 0.009, 0.009, 0.009, Hedge’s g = 3.88, 3.67, 3.75, 3.39, 3.36). k, Schematic of cranial window preparation and two-photon imaging of superficial neuropil. l, Example fullfield fluorescence trace from the RSP in a ketamine-injected Thy1-GCaMP6s mouse, imaged 30 μm below the cortical surface. m, Example fullfield fluorescence trace from the visual cortex, in the same mouse (same ketamine injection as in l). n, Quantification of oscillation frequency content from neuropil in cortical surface before (blue) and after (red) ketamine injection, n = 4 mice. o, Comparison of mean power in the 1–3 Hz band after ketamine injection; n = 4 mice (one-sided paired t-test, P = 0.035; Hedge’s g = 1.60). p, Quantification of oscillation frequency content before and after injection of 80 mg kg⁻¹ ketamine, n = 5 mice; solid line represents the mean and shading denotes the s.e.m. q, After injection of 80 mg kg⁻¹ ketamine, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 3.74, P = 0.025. Corrected two-sided paired t-tests P = 0.016, 0.034, 0.016, 0.034; Hedge’s g 1.92, 1.90, 1.66, 1.65. r, After injection of 80 mg kg⁻¹ ketamine, example temporal video sequence of 410 nm-corrected fluorescence over 1 s in a Thy1-GCaMP6s mouse. A prominent rhythm is seen in the RSP.

Extended Data Fig. 2 ∣. PCP and MK801—but not memantine, saline or LSD—induce RSP-localized rhythms.

a, Quantification of oscillation frequency content before and after PCP injection, n = 5 mice. b, For PCP, example temporal video sequence of 410 nm-corrected fluorescence over 1 s in a Thy1-GCaMP6s mouse showing RSP rhythm. c, Quantification of oscillation frequency content before and after MK801 injection, n = 5 mice. d, For MK801, example temporal video sequence of 410 nm-corrected fluorescence over 1 s in a Thy1-GCaMP6s mouse, showing RSP rhythm. e, Quantification of oscillation frequency content before and after memantine injection, n = 5 mice. f, For memantine, example temporal video sequence of 410 nm-corrected fluorescence across 1 s in a Thy1-GCaMP6s mouse. Here, neither rhythms nor travelling waves are seen. g, Quantification of oscillation frequency content before and after saline injection, n = 5 mice. h, Quantification of mean power in 1–3 Hz band for saline (n = 5 mice), corrected two-sided paired t-tests (*P < 0.05, **P < 0.01, NS P > 0.05; Hedge’s g 0.36, 0.18, 0.14, 0.01). i, Quantification of oscillation frequency content before and after LSD injection, n = 5 mice. j, Quantification of mean power in 1–3 Hz band for LSD (n = 5 mice), corrected two-sided paired t-tests (*P < 0.05, **P < 0.01, NS P > 0.05; Hedge’s g LSD 2.05, 0.62, 1.07, 0.65). k, For LSD, example temporal video sequence of 410 nm-corrected fluorescence across 1 s in a Thy1-GCaMP6s mouse. Here, neither rhythms nor travelling waves are seen.

Extended Data Fig. 3 ∣. Other anaesthetic classes do not induce retrosplenial cortex-localized rhythms.

a, Quantification of oscillation frequency content before and after injection of 1.5 mg kg⁻¹ dexmedetomidine, n = 5 mice. b, After injection of 1.5 mg kg⁻¹ dexmedetomidine, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 2.66, P = 0.07. Corrected two-sided paired t-tests, P = 0.91, 0.27, 0.15, 0.91; Hedge’s g = 0.14, 0.77, 1.01, 0.06. c, Example temporal video sequence of 410 nm-corrected fluorescence after injection of 1.5 mg kg⁻¹ dexmedetomidine over 1 s in a Thy1-GCaMP6s mouse. d, Quantification of oscillation frequency content before and after injection of 0.75 mg kg⁻¹ dexmedetomidine, n = 5 mice. e, After injection of 0.75 mg kg⁻¹ dexmedetomidine, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 2.340, P = 0.10. Corrected two-sided paired t-tests, P = 0.92, 0.92, 0.04, 0.86; Hedge’s g = 0.05, 0.11, 0.54, 0.13. f, Quantification of oscillation frequency content before and after injection of 0.35 mg kg⁻¹ dexmedetomidine, n = 5 mice. g, After injection of 0.35 mg kg⁻¹ dexmedetomidine, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 2.68, P = 0.07. Corrected two-sided paired t-tests, P = 0.29, 0.95, 0.013, 0.95; Hedge’s g = 0.68, 0.05, 0.32, 0.01. h, Quantification of oscillation frequency content before and after injection of 140 mg kg⁻¹ propofol, n = 5 mice. i, After injection of 140 mg kg⁻¹ propofol, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 6.46, P = 0.0027. Corrected two-sided paired t-tests, P = 0.0026, 0.0029, 0.0029, 0.28; Hedge’s g = 4.36, 3.07, 1.75, 0.61. j, Example temporal video sequence of 410 nm-corrected fluorescence after injection of 140 mg kg⁻¹ propofol over 1 s in a Thy1-GCaMP6s mouse. k, Quantification of oscillation frequency content before and after injection of 70 mg kg⁻¹ propofol, n = 5 mice. l, After injection of 70 mg kg⁻¹ propofol, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 1.34, P = 0.30. Corrected two-sided paired t-tests, P = 0.11, 0.11, 0.11, 0.11; Hedge’s g = 0.79, 1.17, 0.37, 0.83. m, Quantification of oscillation frequency content before and after injection of 35 mg kg⁻¹ propofol, n = 5 mice. n, For injection of 35 mg kg⁻¹ propofol, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 2.69, P = 0.07. Corrected two-sided paired t-tests, P = 0.17, 0.07, 0.07, 0.17; Hedge’s g = 0.66, 1.56, 0.63, 0.82. o, Quantification of oscillation frequency content before and after injection of ketamine/xylazine, n = 5 mice. p, After injection of ketamine/xylazine, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 1.977, P = 0.15. Corrected two-sided paired t-tests, P = 0.08, 0.08, 0.82, 0.82; Hedge’s g = 1.44, 2.25, 0.08, 0.13. q, Example temporal video sequence of 410 nm-corrected fluorescence after injection of ketamine/xylazine over 1 s in a Thy1-GCaMP6s mouse.

Extended Data Fig. 4 ∣. A sedative and an analgesic do not induce the retrosplenial rhythm.

a, Quantification of oscillation frequency content before and after injection of 2 mg kg⁻¹ diazepam, n = 5 mice. b, After injection of 2 mg kg⁻¹ diazepam, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 39, P < 0.001. Corrected two-sided paired t-tests, P = 0.013, 0.028, 0.67, 0.013; Hedge’s g = 3.10, 2.07, 0.16, 1.27. c, Example temporal video sequence of 410 nm-corrected fluorescence after injection of 2 mg kg⁻¹ diazepam over 1 s in a Thy1-GCaMP6s mouse. d, Quantification of oscillation frequency content before and 2 h after injection of buprenorphine SR, n = 5 mice. e, After injection of buprenorphine SR, comparison of mean power in the 1–3 Hz band, n = 5 mice. One-way ANOVA with repeated measures, F_4,16 = 1.55, P = 0.23. Corrected two-sided paired t-tests, P = 0.51, 0.33, 0.33, 0.33; Hedge’s g = 0.17, 0.51, 0.33, 0.38. f, Example temporal video sequence of 410 nm-corrected fluorescence after injection of buprenorphine SR, over 1 s in a Thy1-GCaMP6s mouse.

Extended Data Fig. 5 ∣. Additional information for two photon imaging, TRAP and 32-contact electrophysiology experiments.

a, Schematic of two-photon, head-fixed imaging. GCaMP6m was expressed in layer-specific transgenic mice, and a cranial window was placed over the RSP. b, Epifluorescence images of coronal sections showing GCaMP6m expression restricted to either layer 2/3 or layer 5 neurons in the RSP. Scale bar, 100 μm. c, Single-cell activity traces of all recorded layer 2/3 retrosplenial neurons before ketamine injection, from one mouse. d, Quantification of oscillation frequency content in single layer 2/3 cells before and after ketamine injection, data show average and s.e.m. from n = 5 mice. e, Single-cell activity traces of all recorded layer 5 retrosplenial neurons before ketamine injection from one mouse. f, Quantification of oscillation frequency content in single layer 5 cells before and after ketamine injection, data show average and s.e.m. from n = 5 mice. g, Fluorescence images showing tdTomato expression after recombination of TRAP2;Ai14 mice, after saline injections. Scale bar, 1 mm. h, Magnified example image of the RSP after ketamine injection. Dense labelling (tdTomato⁺ neurons are black cells) in deep layers is observed. i, Quantification of tdTomato⁺ induction in the RSP in TRAP2;Ai14 mice after saline (blue, n = 6 coronal sections) or ketamine (red, n = 8 coronal sections) injections. Two-sided unpaired t-test, layer 2/3, P = 0.42; layer 5, P < 0.0004; Hedge’s g=2.42. j, Left, histogram of spike rate during ON periods for mouse 3. Right, summary plot for three mice. k, Left, histogram of spike rate during OFF periods for mouse 3. Right, summary plot for three mice. l, Quantification of the percentage of recorded units that participated in each burst. Left, histogram of per cent units participating in a burst from mouse 3 (n = 249 bursts). Right, summary plot for three mice. m, Quantification of the number of spikes in each burst. Left, histogram from mouse 3. Right, summary plot for three mice. n, Quantification of the percentage of bursts that each neuron participates in. Left, histogram from mouse 3. Right, summary plot for three mice. o, For all 3 mice, correlation of rank sequence across all bursts over 2 min, compared to shuffling of unit IDs. Two-sided paired t-test, P < 0.0001 for all mice. The box denotes the mean and the bars the s.e.m. Hedge’s g = 2.30, 2.25, 3.93. p, Temporal organization of firing across bursts for each mouse. Left, rank of each neuron’s first spike in burst (averaged across bursts for each neuron). Right, rank of each neurons first spike with shuffling of unit IDs.

Extended Data Fig. 6 ∣. Brain-wide single-cell firing rates and spectral features.

a, Recording locations overlaid on coronal sections from the Allen Mouse Brain Atlas. b, Change in firing rate per brain region between 8–12 min before and 8–12 min after ketamine injection, each grey dot is an individual cell. Cells from all 4 sessions are included. c, Nucleus accumbens spike rasters before (top) and after (bottom) ketamine injection from a single session. d, PSD plots for cortical and subcortical regions in Fig. 2d-f; before (blue) and after (red) ketamine injection. e, PSD plots for additional subcortical regions; before (blue) and after (red) ketamine injection. f, Average regional power in the 1–3Hz band for cortical and subcortical regions from d. Each recording is represented as a single line. The total number of recorded units is indicated. g, Average regional power in the 1–3Hz band for additional subcortical regions from e. Each recording is represented as a single line. The total number of recorded units is indicated.

Extended Data Fig. 7 ∣. Brain-wide functional correlations with retrosplenial cortex and thalamic rhythms.

a, Before ketamine infusion, correlation with the average RSP trace for every recorded cell (grey dot) categorized by region. b, After ketamine infusion, correlation with the average RSP trace for every recorded cell (grey dot) categorized by region. c, For every neuron, change in correlation with the mean RSP trace. d, For every region, magnitude of correlation with the mean RSP trace. e, For every region, change in the correlation with mean RSP trace. Regional averages (bar) and s.e.m (line) are shown. f, For mouse 1, raster plot of spike trains from simultaneously recorded single units in somatosensory cortex (SS), anteromedial thalamus (AM), anteroventral thalamus (AV), lateral dorsal thalamus (LD) and retrosplenial cortex (RSP) before and after ketamine injection. g, For mouse 2, raster plot of spike trains from simultaneously recorded single units in somatosensory cortex (SS), anteromedial thalamus (AM), anteroventral thalamus (AV), lateral dorsal thalamus (LD) and retrosplenial cortex (RSP) before and after ketamine injection. h, Z-scored mean activity traces from three simultaneously recorded thalamic nuclei and retrosplenial cortex (green) corresponding to rasters in f. i, Histological assessment registered to the Allen Mouse Brain Atlas (2004). Electrode insertions revealed the location of mouse 2’s thalamic probe at the edge of the anteromedial thalamus, a possible explanation for this recording’s fewer AM neurons and weaker anti-correlation with RSP. Right, a coronal image from the Allen Mouse Brain Atlas shows the locations of thalamic nuclei.

Extended Data Fig. 8 ∣. Brain-wide single-cell functional connectivity matrices.

a, Correlation matrix between individual cells, grouped by brain region, before ketamine infusion for recording session 1. b, Correlation matrix between individual cells, grouped by brain region, after ketamine infusion for recording session 1. c, Change in regional correlation for recording session 1. d, Correlation matrix between individual cells, grouped by brain region, before ketamine infusion for recording session 2. e, Correlation matrix between individual cells, grouped by brain region, after ketamine infusion for recording session 2. f, Change in regional correlation for recording session 2. g, Correlation matrix between individual cells, grouped by brain region, before ketamine infusion for recording session 3. h, Correlation matrix between individual cells, grouped by brain region, after ketamine infusion for recording session 3. i, Change in regional correlation for recording session 3.

Extended Data Fig. 9 ∣. Additional characterization of dissociation-like phenotype elicited by ketamine and PCP.

a, Hot-plate test. Time to jump in ketamine-injected mice. The maximum allowable test time was 90 s. One-way ANOVA with repeated measures: jump, 1 × 10⁻¹². Horizontal dotted line indicates the experimental time limit; mice that did not jump by 90 s were removed and the experiment ended. Corrected two-sided unpaired t-tests, *P < 0.05, **P < 0.01, ***P < 0.001. n = 5 mice in each group. For each dose in order, Glass’s Δ effect size for jump = 0.55, 1.9, Inf, Inf. b, Rate of rears in hot-plate test. One-way ANOVA with repeated measures, P < 1 × 10⁻⁴. Two-sided unpaired t-tests. For each dose in order, Glass’s Δ effect sizes = −0.82, −0.42, −24.6, −24.6. c, Latency to first flick, lick and rear in hot-plate test. The horizontal dotted line indicates the experimental time limit. Hedge’s g effect sizes for flick = 0.16, 0.34, −0.30, −0.17. Glass’s Δ effect size for lick = 0.66, 0.17, 1.86, 7.62. Glass’s Δ effect size for rear = 0.47, 0.85, Inf, 10.5. d, Tail suspension test. Percentage of time spent struggling. One-way ANOVA with repeated measures, F_4,20 = 9.36, P = 0.0002, n = 5 mice for each group. Glass’s Δ effect sizes = −0.30, 0.024, −2.49, −585. e, Time spent interacting with an intruder mouse in 2 min. One-way ANOVA with repeated measures, F_4,20 = 15.8; P < 0.0001, n = 5 mice for each group. Glass’s Δ effect sizes = −0.27, −0.67, −44.7, −79.5. f, Number of mice that corrected postural inversion within 5 s. Mice dosed with 50 mg kg⁻¹ ketamine or less reflexively righted. Mice injected with 200 mg kg⁻¹ ketamine did not right. n = 5 for each group. g, Hot-plate test, time to jump. The maximum allowable time is indicated by a dashed horizontal line (90 s); mice that did not jump by 90 s were removed and the experiment ended. For all statistical comparisons, each experimental group was compared with the saline (control) group via corrected Mann–Whitney U-test. *P < 0.05, **P < 0.01, ***P < 0.001; for each drug in order, Glass’s Δ effect size for jump = Inf, 4.97, 0.35, −1.69, 0.60. h, Rearing rate was significantly decreased for the PCP group and increased for the LSD group. Glass’s Δ = −10.9, −0.52, 0.34, 1.00, −0.10. i, Latencies to expression of affective (licking) and escape (rear) behaviours were significantly longer for the PCP mice. The maximum allowable time is indicated by dashed horizontal line (90 s). Hedge’s g effect sizes for flick = −0.78, 1.87, 1.07, −0.24, 1.05. Glass’s Δ effect size for lick = 1.14, 0.75, 0.072, 0.96, 0.52. Glass’s Δ effect size for rear = 12.4, 0.33, −0.63, −1.92, 0.39. j, Tail suspension test. Significantly decreased struggling time was observed in mice treated with PCP and diazepam compared with saline-injected mice (Mann–Whitney U-test with FDR correction, **P < 0.01). Glass’s Δ effect sizes = −2.51, 0.074, −0.17, 0.58, −6.50. k, Social interaction. Significantly decreased social interaction time was observed in mice treated with PCP and buprenorphine (Mann–Whitney U-test with FDR,**P < 0.01). Hedge’s g effect sizes = −2.10, −2.13, −0.068, 1.88, −1.23. I, Righting reflex, showing the number of mice that right for each treatment group. For each group, all mice successfully corrected postural inversion. m, Open field test. Example traces of body position after treatment with each drug (2 example mice shown). Grey line represents full 5-min session, black line represents 20 s of tracking (from 2 min to 2 min 20 s).

Extended Data Fig. 10 ∣. Rhythmic optogenetic behaviour, alternative cortical stimulation locations, and behavioural controls.

a, Frequency content of rhythmic (magenta) and random (purple) illumination patterns. b, Hot-plate test: time to jump. The maximum allowable time is indicated by dashed horizontal line (90 s). Time-to-jump: YFP/rhythmic P = 0.020, YFP/random P = 0.87, rhythmic/random P = 0.054, corrected two-sided unpaired t-tests, n = 8 YFP, 7 rhythmic, 7 random mice. Hedge’s g effect sizes for jump = 1.58, −0.79, −1.2). c, Rate of rears, two-sided unpaired t-test, YFP/rhythmic P = 0.020, YFP/random P = 0.27, rhythmic/random P = 0.44. Hedge’s g effect sizes = −1.57, −0.68, −0.40. d, Latency to first withdrawal, lick and rear. The maximum allowable time is indicated by the dashed horizontal line (90 s). Two-sided unpaired t-tests, corrected P values for foot flick (>0.5 for all comparisons); foot lick: YFP/rhythmic P = 0.087, YFP/random P = 0.40, rhythmic/random P = 0.12; rear: P = 0.071, 0.42, 0.071; Hedge’s g effect sizes for flick = −0.0084, −0.43, −0.66; lick = 1.20, 0.42, −0.97; and rear = 1.07, −0.42, −1.2. e, Tail suspension test. Two-sided paired t-test for rhythmic/random, corrected P = 0.038; Two-sided unpaired t-tests: YFP/rhythmic corrected P = 0.31 and YFP/rand corrected P = 0.95. Hedge’s g effect sizes: YFP/rhythmic −0.61, rhythmic/random −0.58, YFP/random 0.032. f, Social interaction. Two-sided paired t-test for rhythmic/random, corrected P = 0.21; Two-sided unpaired t-tests: YFP/rhythmic corrected P = 0.21 and YFP/random corrected P = 0.39. Hedge’s g effect sizes: YFP/rhythmic = −0.85, rhythmic/random = 0.60, YFP/random = −0.42. g, Righting reflex, percentage of mice that right. In each group, all mice successfully corrected postural inversion. h, Average velocity in the open field test during optogenetic stimulation, comparing average light off and light on conditions within individual mice. Paired t-test, eNPAC P = 0.49, YFP P = 0.40. i, Time in centre in the open field test, comparing average light off and light on conditions within individual mice. Paired t-test, eNPAC P = 0.06, YFP P = 0.25. j, Velocity for each 3 min epoch. eNPAC (magenta) and YFP (grey). s.e.m. is shown. k, Time in centre for each 3 min epoch. eNPAC (magenta) and YFP (grey). s.e.m. is shown. l, Hot-plate test. Rate of jumps (P = 0.85), rearing (P = 0.03) and latencies. Two-sided paired t-test, n = 8 control mice and n = 6 S1 eNPAC mice. Hedge’s g effect size for jump = 0.09, rear = −1.27, latency to flick = −0.41, latency to lick = 0.78, latency to rear = 0.52. m, Tail suspension test. Percentage of time spent struggling for S1 eNPAC mice, with and without illumination, two-sided paired t-test, P = 0.73. Change in struggling time, P = 0.73. Hedge’s g = 0.11. n, Social interaction. Interaction time significantly decreased with illumination in S1 eNPAC mice, two-sided paired t-test, P = 0.015; percentage change with illumination. Hedge’s g = −0.29. o, Righting reflex for S1 YFP and ENPAC mice. p, Hot-plate test without ketamine. For rate of reflexive withdrawals (one-way ANOVA, F_2,18 = 1.01, P = 0.38), paw-licks (one-way ANOVA, F_2,18 = 2.2, P = 0.14), jump (one-way ANOVA, F_2,18 = 0.55, P = 0.58), rearing (one-way ANOVA, F_2,18 = 0.09, P = 0.91) and latencies (one-way ANOVA for each behaviour, P > 0.05). Controls are pooled from saline injected mice. q, Tail suspension test. Percentage of time spent struggling. One-way ANOVA, F_2,20 = 0.31, P = 0.74. r, Social interaction. Interaction time was similar across groups; one-way ANOVA, F_2,19 = 0.31, P = 0.74. s, Righting reflex for GRIN1-knockout and HCN1-knockout mice.

Extended Data Fig. 11 ∣. Additional information for long-range input and gene knockout experiments.

a, Percentage of ipsilateral inputs from non-retrosplenial regions across the whole brain (n = 2 mice). The bar denotes the mean. b, Top, example confocal image showing eNPHR3.0–YFP expression. Bottom, localizations of the fibre tip in opsin-expressing mice, each colour is an individual mouse. Scale bars, 500 μm. c, Frequency content of example recording in a dorsal thalamus NpHR3.0 mouse. Each time window (pre, stim, post) consists of 2 min. d, Density of GRIN1 (left) and HCN1 (right) cells in the RSP. GRIN1-expressing neurons were more dense in superficial than in deep layers (paired t-test, P = 0.023; Hedge’s g = −1.94), but HCN1-expressing neurons were mostly found in deep layers (paired t-test, P = 0.0066; Hedge’s g = 2.71). e, Confocal images showing expression of AAVdj-Ef1a-DIO-GCaMP6m and GRIN1 in control (left) and GRIN1-floxed (right) deep RSP. f, Quantification of gene disruption effectiveness in photometry experiments. Co-localization of GRIN1 and HCN1 with GCaMP6m was greatly reduced. g, Quantification of oscillation frequency content (PSD) before (blue) and after (red) ketamine injection, n = 7 mice in each group. h, Confocal images showing distribution of GRIN1⁺ neurons in wild-type and GRIN-knockout mice. i, Confocal images showing distribution of HCN1⁺ neurons in wild-type and HCN1-knockout mice. j, Quantification of gene disruption for behavioural experiments. Density of GRIN1⁺ cells (unpaired t-test, P = 0.0031; Hedge’s g = −2.22) and HCN1⁺ cells (unpaired t-test, P < 1 × 10⁻⁴, Hedge’s g = −4.64) were significantly reduced by injections of AAVdj-Ef1a-Cre (12 injections per mouse). k, Hot-plate test. No mice jumped before the maximum allowable time, indicated by the dashed horizontal line. Rate of rears was not significantly different (corrected Mann–Whitney U-test P > 0.1) for either group. Average latency to first lick was lower for HCN1-knockout mice. l, Tail suspension test. Significantly increased struggling time was observed in HCN1 and GRIN1 mice (corrected Mann–Whitney U-test, **P < 0.001. Glass’s Δ effect sizes = 1.07, 1.13. m, Social interaction. Significantly increased social interaction time was observed in HCN1 and GRIN1 mice (corrected Mann–Whitney U-test,**P < 0.01). Glass’s Δ effect sizes = 0.94, 1.39. n, Righting reflex, fraction of mice that right. For each group, all mice successfully corrected postural inversion.

Extended Data Fig. 12 ∣. Additional information on human iEEG recordings.

a, 3D rendering of electrode locations for right and left hemispheres. b, Screenshot of raw clinical iEEG recordings (no processing). The RC electrode is in the right PMC, with RC1 being the deepest contact. Vertical lines are 1 s. Note rhythm in contacts in right deep posteromedial cortex RC3–4 through RC8–9. One second is highlighted in blue showing an approximately 3 Hz rhythm. c, Frequency spectrum of iEEG traces from frontal lobe regions, parietal, temporal lobe cortical regions, and subcortical areas during aura (red) or non-seizure (blue) epochs. d, Quantification of power in the 3–4 Hz band across all contacts during the seizure aura-period (n = 2 seizures, 10 s for each aura). Deep posteromedial regions on both hemispheres exhibit the high power. Contacts that cross threshold (mean + 2.5 × s.d.) are coloured. e, Location of electrodes that elicited a dissociative experience (Fig. 5) that were not located in a spontaneously oscillating region of the cortex.

Extended Data Fig. 13 ∣. Locations of electrode contacts for human iEEG recordings.

MRI images show anatomical locations of electrode contacts displayed in Fig. 5.

Extended Data Fig. 14 ∣. Summary of behavioural effects in the rodent model.

Overview of effects from all rodent behaviour experiments (corresponding to Figs. 3, 4).

Fig. 1 ∣. Multiregional widefield imaging of cortical activity reveals ketamine-induced retrosplenial rhythm.

a, Schematic of the widefield macroscopy setup. b, Left, transparent skull preparation; right, 488-nm-excited fluorescence image from a Thy1-GCaMP6s mouse. A, anterior; P, posterior. Scale bar, 1 mm. c, Neocortex atlas for alignment. MOT, motor; SS, somatosensory; PP, parietal; VIS, visual; RSP, retrosplenial. d, Cortical activity, visualized by a video sequence of 410-nm-corrected fluorescence over 1 s, in a Thy1-GCaMP6s mouse 10 minutes after i.p. injection of 50 mg kg⁻¹ ketamine. RSP activation is observed at 0.13 s and 0.8 s; deactivation at 0.54 s. dF/F scale is normalized by median fluorescence intensity. e, Top, fluorescence traces from five regions before (blue) and 10 min after (red) injection of 50 mg kg⁻¹ ketamine. Shown are the 10-s, 410-nm-corrected data. Bottom, corresponding power spectral density (PSD) plots, obtained at minutes 10–11 post-injection (data are mean ± s.e.m.; n = 5 mice). f, Change in power of the 1–3-Hz rhythm after injection of ketamine, n = 5 mice, each black line represents a mouse. One-way ANOVA, repeated measures, F_4,16 = 25.4, P < 1 × 10⁻⁴. Corrected two-sided paired t-tests, **P < 0.01. Hedge’s g effect sizes = 3.96, 3.90, 3.76, 3.62. g, Power at 1–3 Hz in the RSP over 55 min, obtained from widefield imaging of GCaMP6s, with 50 mg kg⁻¹ ketamine injected intraperitoneally 5 min into recording. The thick trace shows the mean for n = 6 mice. h, Change in power of the 1–3-Hz rhythm after administration of PCP, MK801 and memantine. The mean power at 1–3 Hz is shown (n = 5 mice per drug). Corrected two-sided paired t-tests, *P < 0.05, **P < 0.01, NS (not significant) P > 0.05. Hedge’s g effect sizes: PCP = 4.23, 4.57, 5.14, 4.87; MK801 = 1.80, 1.79, 2.16, 1.28, 1.56; memantine = 1.00, 0.96, 0.27, 0.82. i, Left, single-cell activity traces obtained from two-photon microscopy from layer 2/3 or layer 5 RSP after ketamine injection. Right, mean power of the 1–3-Hz rhythm across cells before and after ketamine administration. Layer 2/3, n = 5 mice, two-sided paired t-test, P = 0.74, Hedge’s g effect size = −0.46. Layer 5, n = 5 mice, two-sided paired t-test, P < 0.01, Hedge’s g effect size = 3.16. j, tdTomato fluorescence after recombination of TRAP2;Ai14 mice, after injection of 50 mg kg⁻¹ ketamine, showing dense labelling of layer 5 cells. Dark cells are those expressing tdTomato. Scale bar, 1 mm.

Fig. 2 ∣. Retrosplenial in vivo electrophysiology.

a, Top, spike-train rasters from all simultaneously recorded single units from the deep RSP in a head-fixed mouse after injection of a sub-anaesthetic dose of 50 mg kg⁻¹ ketamine. Bottom, average firing rate (FR) of the units shown above (in Hz, 10-ms bin). The pink boxes indicate ON-states for bursts (see Methods). b, Timeline for a multi-Neuropixel recording session. Ketamine was injected i.p. 20 min into the recording session through a previously implanted cannula. c, 3D-reconstruction showing the insertion of Neuropixel probes (16 probe insertions are shown in total for 4 sessions). The different areas of the brain are shown by the changing colours along each probe. Four Neuropixels were implanted into each mouse and recorded simultaneously. d, Average change in firing rate per region between 8–12 min before and 8–12 min after ketamine injection. Cells from all four sessions were included. The number of units for each region is shown along the x axis, after the region name. Data are mean ± s.e.m. Corrected two-sided t-test versus 0, *P < 0.05, **P < 0.01, ***P < 0.001. Taenia t., taenia tecta; dors. pedunc., dorsal peduncular; ant. olfact. n., anterior olfactory nucleus; caudate put., caudate putamen; nuc. accumbens, nucleus accumbens; reticular nuc., reticular nucleus; mid. retic. nuc., midbrain reticular nucleus; super. col., superior colliculi; red nuc., red nucleus. e, Average RSP correlation (mean regional activity) before and after ketamine injection. The number of units for each region is shown along the x axis after the region names, which are stated as abbreviated forms of the names in d. Data are mean ± s.e.m. Corrected two-sided t-test versus 0, *P < 0.05, **P < 0.01, ***P < 0.001. f, Correlation matrix between cells, grouped by region before and after ketamine injection; obtained from 4-Neuropixel recordings in mouse. g, Z-scored mean activity traces (corresponding to rasters in Extended Data Fig. 7f) of three simultaneously recorded thalamic nuclei and RSP (green).

Fig. 3 ∣. Ketamine- and optogenetically elicited dissociation-like behavioural phenotype.

a, Stimulus detection (reflexive paw-flick) and affective/emotional response (protective paw-licking) during the hot-plate test. b, Rate of reflexive paw flicks (left) and licks (right) in ketamine-injected mice. One-way ANOVA with repeated measures: flick, P = 0.38; lick, P = 5 × 10⁻⁴. Corrected two-sided unpaired t-tests. n = 5 mice per group. For each dose in order, Hedge’s g effect sizes for flick: 0.16, 0.26, 0.77, 0.77; and lick: −0.028, 0.53, −1.43, −2.19. c, Left, fibre photometry traces from RSP: the rhythm was observed at 25 mg kg⁻¹ but not at 13 mg kg⁻¹. Right, 10 min after injection, the power at each dose across several mice (n = 5 mice for 13 mg kg⁻¹; n = 3 mice for 25 mg kg⁻¹; bars show mean values). Unpaired-t-test, P = 0.008, Glass’s Δ = 3.2. d, Mean power at 1–3 Hz as assessed by widefield imaging for different doses of ketamine, with 13 and 25 mg kg⁻¹ doses statistically compared. n = 4 mice per dose, two-sided unpaired t-test, *P = 0.027. e, Results of the hot-plate test after administration of drugs from other classes. Each experimental group is compared with the saline (control) group via a corrected Mann–Whitney U-test. For each drug in order, Hedge’s g effect sizes: flick 3.10, −2.97, −2.19, 1.14, −2.16; Glass’s Δ: lick −15.2, −2.89, 0.093, −0.40, −0.90. Corrected P values versus saline: flick 0.0001, 0.0001, 0.0008, 0.0297, 0.0008; lick 1.9 × 10⁻⁵, 0.0092, 0.8146, 0.5761, 0.1395. Bup., buprenorphine; lido., lidocaine; diaz., diazepam. **P < 0.01, ***P < 0.001. f, Left, average velocity of mice in the open-field test over a period of 5 min. Ketamine, LSD and diazepam decreased velocity, whereas buprenorphine increased velocity compared with controls. Each experimental group was compared with saline (control) via a corrected Mann–Whitney U-test. Hedge’s g effect sizes: −6.14, 1.02, 4.98, 0.20, −2.34, −4.77. Corrected P values versus saline: 0.1021, 2.5 × 10⁻⁵, 0.7206, 0.0020, 2.5 × 10⁻⁵, 9.1× 10⁻⁶. Right, traces of body position during the open-field test after administration of saline or the stated dissociative drugs. The grey line shows the position of the mouse over the full 5-min session, whereas the black line shows the position over a 20-s tracking period, beginning in minute 2. g, Viral injections, illumination pattern and behavioural arms. AA V1-nEF-DIO-eNpHR3.0-p2a-ChR2-YFP (eNPAC) was injected bilaterally into the RSP of Rpb4-cre mice. Illumination was with 20 Hz blue light and constant yellow light, alternating every 250 ms. Results of the hot-plate test after illumination in the RSP and the SS: for RSP, left to right: paw-flick: YFP versus rhythmic, P = 0.53; YFP versus random, P = 0.48; rhythmic versus random, P = 0.43. Paw-lick: YFP versus rhythmic, *P = 0.034; YFP versus random, P = 0.38; rhythmic versus random, P = 0.20. Corrected two-sided unpaired t-tests, n = 8 YFP, n = 7 rhythmic, n = 7 random mice. Hedge’s g effect sizes for flick: −0.31, 0.50, 0.79; lick −1.43, −0.45, 0.80. For SS, left to right: paw-flick: P = 0.50; paw-licks: P = 0.71; Hedge’s g effect size for flick: −0.35; lick: −0.19. Two-sided paired t-test, n = 8 control, n = 6 SS eNPAC mice.

Fig. 4 ∣. RSP HCN1 channels are required for ketamine to induce deep rhythm and dissociation-like behaviour.

a, Possible mechanisms underlying RSP rhythm. b, Confocal images showing the monosynaptic inputs to the layer 5 RSP, from left to right: RSP, cingulate cortex, subiculum and dorsal thalamus cells. Green, GFP; blue, DAPI. Scale bars, 1 mm. c, RSP recording during subcortical inhibition. Left, AAVdj-CaMKIIa-eNpHR3.0-YFP was injected bilaterally into the dorsal thalamus or subiculum, and GCaMP6m was expressed in the RSP. Right, relative power of the 1–3-Hz rhythm before, during and after optogenetic inhibition over 2-min periods beginning 10 min after ketamine injection. Data are normalized by PSD during the pre-period, and then linearly corrected to account for the decrease in oscillation power across the 6-min recording. Thalamus inhibition increased oscillation power (two-sided paired t-test, YFP P = 0.057, n = 5 mice; subiculum P = 0.91, n = 5 mice; thalamus P = 0.003, n = 7 mice). Hedge’s g effect sizes: YFP 0.54; subiculum 0.065; thalamus 2.90. d, Confocal microscopy images of immunohistochemistry with antibodies against GRIN1 (left) or HCN1 (right) in wild-type (WT) mice, to reveal channel expression. The HCN1 image reveals low HCN1⁺ density in the superficial RSP. cc, corpus callosum. Scale bars, 75 μm. e, RSP recording after local knockout. Left, photometry experiments in the RSP. AAVs expressing Cre-recombinase and Cre-dependent GCaMP6 were injected to induce local disruption of NMDA-receptor or HCN1-channel expression. Middle, photometry traces of the control, GRIN1 and HCN1 knockout (KO) experiments. Right, reduced ketamine-induced oscillation power was observed in GRIN1- and HCN1-knockout mice. Corrected two-sided paired t-test for within-mouse comparison and corrected two-sided independent t-test for between genotype comparisons: NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, n = 7 mice per group. Hedge’s g effect sizes: WT-pre/WT-post 3.22; GRIN1-pre/GRIN1-post 2.03; HCN1-pre/HCN1-post 1.02; WT-post/HCN1-post −2.75; WT-post/GRIN1-post −2.26; HCN1-post/GRIN1-post 1.24; WT-pre/HCN1-pre 0.318; WT-pre/GRIN1-pre 0.322; HCN1-pre/GRIN1-pre 0.09. f, Results of the hot-plate test on knockout mice after the administration of ketamine. Reflexive paw-flicks (left; corrected Mann–Whitney U-test, P > 0.1) and affective paw-licks (corrected Mann–Whitney U-test, WT versus GRIN1, P = 0.56; WT versus HCN1, ***P < 0.001). Hedge’s g effect-sizes for flick: 0.77, 0.070; and lick: 0.40, 2.46.

Fig. 5 ∣. Human PMC rhythm and self-reported dissociation.

a, Simultaneous iEEG and 3D-electrode locations in a patient, 10 s before seizure onset. Locations are coded as follows (using RC3 as an example): R (right hemisphere) C (electrode letter) 3 (contact number) followed by anatomical region (for example, posteromedial). The rhythm was observed in the PMC in both hemispheres across contacts (magenta) and not in other regions (black). Contacts on each electrode are represented by dots. b, Comments from the patient describing their pre-seizure aura experience. Notably, consistently described symptoms of dissociation¹³ can involve reproducible perceptions of depersonalization (a feeling of being an outside observer of one’s body and/or thoughts) and derealization (feeling of being detached from the surroundings). For the interview transcript, see Supplementary Note 1. c, Simultaneously recorded iEEG traces during the pre-seizure period, taken from a different seizure to that shown in a. Traces from one contact per electrode are shown in black, and the trace from the deep PMC contact is shown in magenta. Seizure onset was determined by an epileptologist and is marked with a vertical line. d, iEEG spectra from the deep and the superficial PMC during aura (red) or non-seizure (blue) periods. Mean ± s.e.m. in right (top) and left (bottom) regions across aura periods. e, Power of the 3–4 Hz rhythm during aura (red) or non-seizure/aura period (blue). Mean across channels in each region; the aura periods are shown as a black dot. f, Left, locations of electrodes in the brain of the patient (coloured dots). Electrodes in the deep PMC are accentuated with blue (right) and grey (left) indicators. Comments describing the patient’s experience during electrical stimulation of the deep PMC are also shown. Right, the percentage of times that aura was reported for each sham or electrical stimulation (≥6 mA).