Article Figures & Data
Figures
- Figure 1.
Intranasal delivery of a single subanesthetic dose of ketamine evokes sustained cortical disinhibition, which is reversed by exogenous NRG1. A, B, Schematic of recording electrically evoked IPSCs in L2/3 pyramidal (PYR) neurons of bV1 by preferentially activating L4→L2/3 feedforward projections to L2/3 PV+ interneurons through L4 electrical stimulation. Recorded neurons are filled with biocytin for post hoc verification. C, D, Electrically evoked IPSCs in L2/3 PYRs of bV1 from mice with intranasal delivery of a single subanesthetic dose of ketamine (10 mg/kg, i.n.; 24 and 48 h). For each neuron, the recording is performed through baseline, NRG1 application, and washout. Recordings are from different neurons and different mice at time points that are on different days (24 h and 48 h). C, Representative example traces of IPSCs maintained on the same neurons at baseline, after NRG1 application, and after washout under different treatment conditions. For each trial, electrical stimulation (2 ms, 20 µA) is applied, represented by a black tick beneath one example trace. The arrow indicates the current injection response to monitor access resistance during the experiment. Intranasal ketamine reduces evoked IPSCs to PYR neurons at 24 h and 48 h after ketamine treatment. Ketamine-evoked decreases in IPSCs are reversed with bath NRG1. Acute bath application of NRG1 (5 nM) increases inhibitory input to L2/3 PYRs within 10–20 min, and this is washed out within 20 min. Traces show IPSCs before (top), during NRG1 application (20 min after NRG1 application; middle), and after washout (bottom). In control mice (left column), NRG1 does not affect IPSCs. In mice that received intranasal delivery of ketamine, IPSC amplitudes at baseline are significantly reduced compared with controls for both the 24 and 48 h time points. These reduced inhibitory inputs are increased by NRG1 application (middle row) and this increase can be washed out (bottom row). D, Summary data of IPSC amplitudes at baseline under different treatment conditions (control, 24, and 48 h after intranasal ketamine treatment). Compared with those in control, IPSC amplitudes are significantly reduced after ketamine treatment [control: n = 11 cells (10 cells have washout data) from 10 slices of 5 mice; 24 h: n = 10 cells (all cells have washout data) from 10 slices of 4 mice; 48 h: n = 10 cells (9 cells have washout data) from 10 slices of 4 mice (two-way ANOVA with post hoc Tukey’s HSD: ketamine vs control, 24 h p < 0.0001; 48 h p < 0.0001)] and NRG1 vs baseline comparison at 24 and 48 h (two-way ANOVA with post hoc Tukey’s HSD: ketamine baseline vs NRG1: 24 h p < 0.0001, 48 h p < 0.0001).
- Figure 2.
Intranasal delivery of a single subanesthetic dose of ketamine evokes sustained inhibition of PV+ interneurons, which is reversed by exogenous NRG1. A, B, Schematic of electrically evoked EPSCs in L2/3 PV+ interneurons of bV1 by activating L4→L2/3 projecting excitatory neurons through L4 electrical stimulation. Recorded neurons from PV-Cre;Ai9 mice are filled with biocytin for post hoc verification. C–E, Electrically evoked EPSCs in L2/3 PV+ interneurons of bV1 from mice with intranasal delivery of a single subanesthetic dose of ketamine (10 mg/kg, i.n.; 24 and 48 h). For each neuron, the recording is performed through baseline, NRG1 application, and washout. Recordings are from different neurons and different mice at time points that are on different days (24 h and 48 h). C, Example EPSC traces maintained on the same neurons recorded at baseline, after NRG1 application, and after washout under different treatments. For each trial, electrical stimulation (2 ms, 20 µA) is applied. The arrow indicates the current injection response to monitor access resistance during the experiment. Intranasal ketamine reduces evoked EPSCs in PV+ interneurons at 24 h and 48 h after ketamine treatment. Ketamine-evoked decreases in EPSCs are reversed with NRG1. Acute bath application of NRG1 (5 nM) increases excitatory input to L2/3 PV+ interneurons within 10–20 min, and this is washed out within 20 min. Traces show EPSCs before (top), during NRG1 application (20 min after NRG1 application; middle), and after washout (bottom). In control mice (left column), NRG1 does not affect EPSCs. In mice that received intranasal delivery of ketamine, EPSC amplitudes at baseline are significantly reduced compared with controls for both the 24 and 48 h time points. Reduced excitatory input is increased by NRG1 application (middle row) and this increase can be washed out (bottom row). D, Summary data of EPSC amplitudes at baseline under the different treatments (control, 24 and 48 h after intranasal ketamine treatment). Compared with those in control, EPSC amplitudes are significantly reduced after ketamine treatment [control: n = 10 cells (9 cells have washout data) from 10 slices of 4 mice; 24 h: n = 10 cells (all cells have washout data) from 10 slices of 4 mice; 48 h: n = 10 cells (all cells have washout data) from 10 slices of 4 mice; two-way ANOVA with post hoc Tukey’s HSD: ketamine vs control, 24 h p < 0.0001; 48 h p = 0.0001] and NRG1 vs baseline comparison at 24 and 48 h (two-way ANOVA with post hoc Tukey’s HSD: ketamine baseline vs NRG1, 24 h p < 0.0001, and 48 h p < 0.0001).
- Figure 3.
Both subcutaneous and intranasal delivery of ketamine evoke disinhibition of PYRs in L2/3 of bV1, which is reversed by NRG1. A, B, There is similarity of NRG1 effects on IPSCs in PYRs (NRG1/baseline ratio) for intranasal delivery of a single subanesthetic dose of ketamine (one-way ANOVA with post hoc Tukey’s HSD: NRG1/baseline ratio compared with control, 24 h p = 6.747 × 10−5, and at 48 h p = 2.904 × 10−7) and for subcutaneous delivery of a single subanesthetic dose of ketamine (Kruskal–Wallis test with post hoc Dunn's test: NRG1/baseline ratio compared with control, 24 h p = 9.55 × 10−5, and at 48 h p = 6.21 × 10−5).
- Figure 4.
Intranasal delivery of ketamine increases pCREB in excitatory neurons. A, Left, Confocal images of genetically labeled PV+ interneurons (red), pCREB immunostaining (green), and their overlay in bV1 in 2-month-old PV-Cre;Ai9 mice treated with saline; scale bar, 200 µm. The white box indicates the area of bV1 digitally enlarged (A, right). Right; scale bar, 50 µm. White arrowheads indicate that PV+ interneurons have very low pCREB. B, C, Confocal images of genetically labeled PV+ interneurons (red), pCREB immunostaining (green) and their overlay in bV1 in 2-month-old PV-Cre;Ai9 mice 24 h after a single subcutaneous (B) or intranasal (C) delivery of subanesthetic ketamine, respectively (10 mg/kg; s.c. or i.n.). D, Quantification of the increase in pCREB immunoreactivity of non-PV+ interneurons, putative excitatory neurons in L2/3 of bV1 at 24 h after ketamine treatment [Kruskal–Wallis test: overall p < 0.00001, with post hoc Dunn's test (adjusted for multiple comparisons): subcutaneous ketamine vs saline p < 0.00001, intranasal ketamine vs saline p < 0.00001, mean ± SEM]. The overall normalized values (all groups normalized such that the mean of the combined sal group is equal to 1) of putative excitatory neurons pooled from different mice were grouped in accordance with different treatments (n = 9 mice with 220 cells for saline groups, n = 3 mice and 120 cells for subcutaneous ketamine, and n = 4 mice and 120 cells for intranasal ketamine).
- Figure 5.
Delivery of subanesthetic ketamine affects behavioral activity. A, B, Summary data of distance traveled by mice during a 1 h epoch, at 1 h or 24 h after intranasal or subcutaneous delivery of a single subanesthetic dose of ketamine compared with saline treatment [distance traveled at 1 h: saline (i.n.), n = 8; ketamine (i.n.), n = 8; saline (s.c), n = 8; ketamine (s.c), n = 8 (two-way ANOVA: saline vs ketamine, p = 5.814 × 10−5; intranasal vs subcutaneous, p = 0.1261); distance traveled at 24 h: saline (i.n.), n = 4; ketamine (i.n.), n = 8; saline (s.c), n = 4; ketamine (s.c), n = 8 (two-way ANOVA: saline vs ketamine, p = 0.03112; intranasal vs subcutaneous, p = 0.5562); mean ± SEM]. C, D, Summary data of motionless epochs by mice during the 1 h experiment at 1 h or 24 h after intranasal or subcutaneous delivery of ketamine compared with saline treatment [distance traveled at 1 h: saline (i.n.), n = 8; ketamine (i.n.), n = 8; saline (s.c), n = 8; ketamine (s.c), n = 8 (two-way ANOVA: saline vs ketamine, p = 2.351 × 10−4; intranasal vs subcutaneous, p = 0.7992); distance traveled at 24 h: saline (i.n.), n = 4; ketamine (i.n.), n = 8; saline (s.c), n = 4; ketamine (s.c), n = 8 (two-way ANOVA: saline vs ketamine, p = 0.01564; intranasal vs subcutaneous, p = 0.878); mean ± SEM]. E, Representative animal location heatmaps (top) and trace (bottom) across all treatment conditions for the 1 h time point.
- Figure 6.
Schematic of intranasal delivery of ketamine and PV+ interneuron-mediated cortical disinhibition. A, A diagram showing intranasal delivery of ketamine in mouse. B, A diagram showing disinhibition in visual cortex after intranasal delivery of ketamine and the central role of PV+ interneurons (arrow-up indicates decreases in inhibition for PYRs, and arrow-down indicates decreases in excitation for PV+ interneurons).
Tables
- Table 1.
Numbers of mice, slices, and cells for pCREB immunostaining of bV1 L2/3 at the 24 h time point after either subcutaneous or intranasal administration of ketamine
Mouse # Averaged CTCF per mouse Number of slices Number of cells Subcutaneous saline Mouse 1 4,279.90 ± 296.00 2 N = 40 Mouse 2 6,787.21 ± 368.34 2 N = 40 Mouse 3 6,068.20 ± 295.05 2 N = 40 Intranasal saline Mouse 1 5,734.64 ± 278.95 2 N = 40 Mouse 2 7,408.55 ± 314.74 2 N = 40 Mouse 3 3,553.15 ± 220.02 1 N = 20 Subcutaneous ketamine Mouse 1 9,977.01 ± 848.55 2 N = 40 Mouse 2 10,440.35 ± 958.19 2 N = 40 Mouse 3 16,292.58 ± 788.86 2 N = 40 Intranasal ketamine Mouse 1 10,796.62 ± 475.20 2 N = 40 Mouse 2 12,963.79 ± 702.20 2 N = 40 Mouse 3 14,399.56 ± 870.46 2 N = 40 Mouse 4 11,286.86 ± 505.04 2 N = 40 The average corrected total cell fluorescence (CTCF) is shown for each mouse.
Subcutaneous vs intranasal ketamine 1 h dis 24 h dis 1 h freezing 24 h freezing Cohen's D 0.78 0.32 0.25 0.08 Change (%) 17.01 −3.59 11.84 −4.67 Power (n = 8) 0.31 0.09 0.08 0.05 n (power = 80%) 27.00 150.00 253.00 2,302.00 The first row titled Cohen's D is the effect size for each comparison. The second row is the change in percentage. The third row is the power with the Cohen's D in the first row for n = 8 per group. The last row is the sample size per group required to reach 80% power.
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