(R)-ketamine restores anterior insular cortex activity and cognitive deficits in social isolation-reared mice

Abstract

Chronic social isolation increases the risk of mental health problems, including cognitive impairments and depression. While subanesthetic ketamine is considered effective for cognitive impairments in patients with depression, the neural mechanisms underlying its effects are not well understood. Here we identified unique activation of the anterior insular cortex (aIC) as a characteristic feature in brain-wide regions of mice reared in social isolation and treated with (R)-ketamine, a ketamine enantiomer. Using fiber photometry recording on freely moving mice, we found that social isolation attenuates aIC neuronal activation upon social contact and that (R)-ketamine, but not (S)-ketamine, is able to counteracts this reduction. (R)-ketamine facilitated social cognition in social isolation-reared mice during the social memory test. aIC inactivation offset the effect of (R)-ketamine on social memory. Our results suggest that (R)-ketamine has promising potential as an effective intervention for social cognitive deficits by restoring aIC function.

Fig. 1. Insular cortex activation as a key feature in (R)-ketamine-treated brain.

a Experimental timeline and schematic of the social isolation rearing and brain-wide IEG mapping after the forced swim test using Arc-dVenus mice. FST, forced swim test. b Immobility time of social isolation-reared Arc-dVenus mice treated with indicated drugs in the FST (for saline, 10 mg/kg (R)-ketamine, and 10 mg/kg (S)-ketamine; n = 5, in each group). Statistical analysis was performed using Levene’s test and one-way ANOVA, followed by Tukey’s multiple comparison test. Levene’s test: p = 0.223; one-way ANOVA: F(2, 12) = 8.129, p = 0.0059. *p < 0.05, **p < 0.01. (R)-ket, (R)-ketamine; (S)-ket, (S)-ketamine. c The box-whisker plot (center: median; box: 25–75th percentiles; whiskers: minimum to maximum) displays the z-score values for the number of dVenus-positive cells in each brain region. The abbreviations for the brain areas are provided in table S1. d The formula shows the transformation of classifier weights into corresponding activation patterns (left). The absolute values of the activation patterns of brain regions are plotted in the two indicated classifications. e Representative coronal images of the anterior insular cortex (aIC) of Arc-dVenus mice treated with indicated drugs (top). Scale bar, 500 µm. The anteroposterior distribution of dVenus-positive cells in the IC is shown in the bottom panel. mIC, the middle IC; pIC, the posterior IC. Statistical analysis was performed using Two-way ANOVA, followed by Tukey’s multiple comparison test: spatial distribution, F(9, 120) = 4.378, p < 0.0001; treatment, F(2, 120) = 14.12, p < 0.001; interaction, F(18, 120) = 1.191, p = 0.2791. **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are presented as the mean ± standard error of the mean (s.e.m.).

Fig. 2. Attenuation of (R)-ketamine’s effect on immobility time via aIC inhibition.

a Experimental timeline and schematic of chemogenetic manipulation of the aIC in social isolation-reared mice and a schematic and representative image illustrating the selective transduction of hM4Di-mCherry at the injection site (highlighted in red). CNO, clozapine-N-oxide. b Immobility time of hM4Di-mCherry-expressing mice treated with 10 mg/kg (R)-ketamine or 20 mg/kg (S)-ketamine in the FST. Statistical analysis was performed using Levene’s test and one-way ANOVA, followed by Bonferroni’s multiple comparison test. Levene’s test: p = 0.153 (vehicle), p = 0.012 (CNO); one-way ANOVA: Vehicle, F(2, 47) = 6.198, p = 0.0041; CNO, F(2, 43) = 6.044, p = 0.0049. *p < 0.05, **p < 0.01. c Immobility time of hM3Dq-mCherry- or mCherry-expressing mice treated with vehicle or CNO in the FST. The number in parentheses indicates the number of mice used in the experiment. Statistical analysis was performed using Levene’s test and unpaired t test. Levene’s test: p = 0.744 (mCherry), p = 0.828 (hM3Dq). *p < 0.05. Data are presented as the mean ± s.e.m.

Fig. 3. Boost of aIC neuronal responses to social investigation by (R)-ketamine.

a Experimental timeline and schematic of fiber photometry recording in social isolation- or group- reared mice during the three-chamber test. Neuronal activity of the aIC in social isolation-reared mice was observed during nonsequential interactions with a novel mouse or object in the 1st-3-chamber test with saline treatment (b, d, and f) and the 2nd-3-chamber test with drug treatment (c, e, and g for saline, 20 mg/kg (R)-ketamine, and 20 mg/kg (S)-ketamine; n = 5, 7, and 7, respectively). The right graphs show the quantification of social (orange) or object (gray) contact-evoked normalized GCaMP6f signals (mean evoked z-score of 0‒3 s at post-contact subtracted by the mean baseline z-score of −3‒0 s at pre-contact). Statistical analysis was performed using paired t test. *p < 0.05. Neuronal activity of the aIC in group-reared mice was observed in nonsequential interactions with a novel mouse or object in the 1st-3-chamber test (h) and 2nd-3-chamber test (i) with saline treatment (n = 7). The right graphs show the quantification of social (orange) or object (gray) contact-evoked normalized GCaMP6f signals (mean evoked z-score of 0‒3 s at post-contact subtracted by the mean baseline z-score of −3–0 s at pre-contact). Dashed lines indicate the onset of contact. Statistical analysis was performed using paired t test. *p < 0.05. Data are presented as the mean ± s.e.m.

Fig. 4. Restoration of social cognitive deficits through (R)-ketamine-induced activation of the aIC.

a Experimental timeline and schematic of the social memory test using social isolation reared-mice with or without chemogenetic aIC inhibition and representative image of hM4Di-mCherry transduction at the injection site (highlighted in red). b Difference of investigation time to a familiar mouse between 1st trial and indicated trial (2nd to 4th trial) and to a novel mouse (5th trial) in the 1st social memory test 24 h after vehicle and saline treatments. c–e Difference of investigation time to a familiar mouse between 6th trial and indicated trial (2nd to 4th trial) and to a novel mouse (10th trial) in the 2nd social memory test 24 h after vehicle and saline or 20 mg/kg (R)-ketamine treatments (for saline and (R)-ketamine; n = 14 and 16, respectively). Statistical analysis was performed using two-way repeated measured ANOVA, followed by Holm-Sidak multiple comparison test (c): time, F(4, 112) = 7.023, p < 0.0001; treatment, F(1, 28) = 1.57, p = 0.2206; interaction, F(4, 112) = 3.36, p = 0.0123, and using Levene’s test and unpaired t test (d, e). Levene’s test: p = 0.207 (d), p = 0.179 (e); unpaired t test, *p < 0.05. f Immobility time of hM4Di-mCherry-expressing mice in the FST 48 h after drug treatments. Statistical analysis was performed using Levene’s test and unpaired t test (f). Levene’s test: p = 0.707; unpaired t test, *p < 0.05. g Difference of investigation time to a familiar mouse between 1st trial and indicated trial (2nd to 4th trial) and to a novel mouse (5th trial) in the 1st social memory test 24 h after vehicle and saline treatments. h–j Difference of investigation time to a familiar mouse between 6th trial and indicated trial (2nd to 4th trial) and to a novel mouse (10th trial) in the 2nd social memory test 24 h after CNO and saline or 20 mg/kg (R)-ketamine treatments (for saline and (R)-ketamine; n = 15 and 16, respectively). Statistical analysis was performed using two-way repeated measured ANOVA: time, F(4, 116) = 15.03, p < 0.0001; treatment, F(1, 29) = 0.004067, p = 0.9496; interaction, F(4, 116) = 1.352, p = 0.2550, and using Levene’s test and unpaired t test (i, j). Levene’s test: p = 0.276 (i), p = 0.221 (j); unpaired t test, *p < 0.05. k Immobility time of hM4Di-mCherry-expressing mice in the FST 48 h after drug treatments. Statistical analysis was performed using Levene’s test and unpaired t test. Levene’s test: p = 0.441. Black lines, mouse treated with saline in both 1st and 2nd social memory test; Orange line, mouse treated with saline in 1st and (R)-ketamine in 2nd social memory test. Data are presented as the mean ± s.e.m.