Disinhibition of CA1 pyramidal cells by low-dose ketamine and other antagonists with rapid antidepressant efficacy

Abstract

Low-dose ketamine, an open-channel N-methyl d-aspartate receptor (NMDAR) antagonist, mediates rapid antidepressant effects in humans that are mimicked in preclinical rodent models. Disinhibition of pyramidal cells via decreased output of fast-spiking GABAergic interneurons has been proposed as a key mechanism that triggers the antidepressant response. Unfortunately, to date, disinhibition has not been directly demonstrated. Furthermore, whether disinhibition is a common mechanism shared among other antagonists with rapid antidepressant properties in humans has not been investigated. Using in vitro electrophysiology in acute slices of dorsal hippocampus from adult male Sprague-Dawley rats, we examined the immediate effects of a clinically relevant concentration of ketamine to directly test the disinhibition hypothesis. As a mechanistic comparison, we also tested the effects of the glycine site NMDAR partial agonist/antagonist GLYX-13 (rapastinel), the GluN2B subunit-selective NMDAR antagonist Ro 25-6981, and the muscarinic acetylcholine receptor (mAChR) antagonist scopolamine. Low-dose ketamine, GLYX-13, and scopolamine reduced inhibitory input onto pyramidal cells and increased synaptically driven pyramidal cell excitability measured at the single-cell and population levels. Conversely, Ro 25-6981 increased the strength of inhibitory transmission and did not change pyramidal cell excitability. These results show a decrease in the inhibition/excitation balance that supports disinhibition as a common mechanism shared among those antagonists with rapid antidepressant properties. These data suggest that pyramidal cell disinhibition downstream of NMDAR antagonism could serve as a possible biomarker for the efficacy of rapid antidepressant therapy.

Keywords: GABAergic transmission; disinhibition; excitation/inhibition balance; hippocampal networks; major depression.

Fig. 1.

Ketamine reduces sIPSCs and sEPSCs and disinhibits pyramidal cells. (A) Representative traces of sIPSCs in baseline conditions and in the presence of ketamine. (Scale bars: 100 pA and 5 s; Inset, 100 pA and 500 ms.) (B) Cumulative probability of the IEI showing a significant increase in ketamine (P < 0.0001, n = 10, 1,880 baseline events, 1,250 ketamine events), and there is a trend toward decreased peak amplitude of sIPSCs in the presence of ketamine (P = 0.051, n = 10, 1,880 baseline events, 1,250 ketamine events). (C) Representative traces of sEPSCs in baseline and ketamine conditions. (Scale bars: 20 pA and 3 s; Inset, 20 pA and 500 ms.) (D) Cumulative probability of increased IEI (P < 0.01, n = 8, 1,460 baseline events, 1,440 ketamine events) and decreased peak amplitude (P < 0.001, n = 8, 1,460 baseline events, 1,440 ketamine events) of sEPSCs in the presence of ketamine. (E) Schematic of whole-cell recordings of CA1 pyramidal cells where APs were elicited with a depolarizing current step (1) and electrical stimulation of Schaffer collaterals (2). (Scale bars: 20 mV and 100 ms.) (Inset) Subthreshold EPSP-IPSP sequence from cell 1. The baseline EPSP-IPSP (black) shows a decrease in the IPSP amplitude in the presence of ketamine (red, black arrow). (Scale bars: 2 mV and 50 ms.) (F) Raster plot (Top) and summary plots (Bottom) showing a significant increase in synaptic AP probability with bath application of ketamine (*P < 0.001, n = 7). (G) Raster plot (Top) and summary plots (Bottom) demonstrating no significant change in synaptic AP probability in control cells recorded over 30 min (n = 16). (H) No change in intrinsic properties (AP number generated via direct current injection, AP threshold, or input resistance) in the presence of ketamine. (Scale bars: 20 mV and 100 ms, 2 mV and 100 ms.) (I) No change in intrinsic properties in control recordings. (Scale bars: 20 mV and 100 ms, 2 mV and 100 ms.) All values are mean ± SEM.

Fig. 2.

GLYX-13 mimics ketamine by decreasing sIPSCs and sEPSCs and disinhibiting pyramidal cells. (A) Representative traces demonstrating a decrease in sIPSC frequency and amplitude in the presence of GLYX-13. (Scale bars: 100 pA and 5 s; Inset, 100 pA and 500 ms.) (B) Cumulative probability shows a significant increase in sIPSC IEI in GLYX-13 (P < 0.0001, n = 6, 2,350 baseline events, 2,175 GLYX-13 events) and a decrease in peak amplitude with bath application of GLYX-13 (P < 0.0001, n = 6, 2,350 baseline events, 2,175 GLYX-13 events). (C) Representative traces of sEPSCs in baseline conditions and in the presence of GLYX-13. (Scale bars: 25 pA and 3 s; Inset, 25 pA and 500 ms.) (D) Cumulative probability of the IEI increasing (P < 0.01, n = 8, 1,900 baseline events, 1,700 GLYX-13 events) and peak amplitude of sEPSCs decreasing (P < 0.0001, n = 8, 1,900 baseline events, 1,700 GLYX-13 events) in the presence of GLYX-13. (E) Schematic of whole-cell recordings of CA1 pyramidal cells where APs were elicited with a depolarizing current step (1) and an electrical stimulation of CA3 synapses (2). (Scale bars: 20 mV and 100 ms.) (Inset) Subthreshold EPSP-IPSP sequence from cell 2. The baseline EPSP-IPSP (black) shows a decrease in the IPSP amplitude with bath application of GLYX-13 (red, black arrow). (Scale bars: 2 mV and 50 ms.) (F) Raster plot (Left) and summary plots (Right) showing a significant increase in synaptic AP probability with bath application of GLYX-13 (*P < 0.001, n = 10). (G) No significant change is observed in the AP number generated via direct current injection, AP threshold, or input resistance measured in from the depolarizing and hyperpolarizing current steps (n = 10). (Scale bars: 20 mV and 100 ms, 2 mV and 100 ms.) All values are mean ± SEM.

Fig. 3.

Ro 25-6981 increases sIPSC frequency and amplitude, decreases sEPSC amplitude, and does not significantly alter synaptic AP probability. (A) Representative traces showing an increase in sIPSC frequency in the presence of Ro 25-6981. (Scale bars: 100 pA and 5 s; Inset, 100 pA and 500 ms.) (B) Cumulative probability shows a decrease in sIPSC IEI and increase in sIPSC amplitude during bath application of Ro 25-6981 (IEI: P < 0.0001, peak amplitude: P < 0.0001, n = 6, 3,900 baseline events, 4,400 Ro 25-6981 events). (C) Representative traces showing sEPSCs in baseline conditions and in the presence of Ro 25-6981. (Scale bars: 20 pA and 3 s; Inset, 20 pA and 500 ms.) (D) Cumulative probability shows no change in sEPSC IEI (P = 0.18, n = 5, 1,500 baseline events, 1,300 Ro 25-6981 events) but a significant decrease in sEPSC peak amplitude (P < 0.0001, n = 5, 1,500 baseline events, 1,300 Ro 25-6981 events). (E) Schematic of whole-cell recordings of CA1 pyramidal cells where APs were elicited with a depolarizing current step (1) and an electrical stimulation of CA3 synapses (2). (Scale bars: 20 mV and 100 ms.) (Inset) Representative traces in baseline (black) and Ro 25-6981 (red) from cell 5 in a raster plot demonstrating enhanced inhibition in the EPSP-IPSP sequence (black arrow). (Scale bars: 2 mV and 50 ms.) (F) Raster plot and summary plots displaying no significant change in synaptic AP probability in the presence of Ro 25-6981 (n = 10). (G). No change in the measured intrinsic proprieties (AP number generated via direct current injection, AP threshold, and input resistance) is observed (n = 10). (Scale bars: 20 mV and 100 ms, 2 mV and 100 ms.) All values are mean ± SEM.

Fig. 4.

Scopolamine reduces sIPSC frequency and amplitude, increases sEPSC amplitude, and disinhibits CA1 pyramidal cells. (A) Representative traces showing scopolamine decreases sIPSC frequency and peak amplitude compared with baseline. (Scale bars: 100 pA and 5 s; Inset, 100 pA and 500 ms.) (B) Cumulative probability showing a significant increase in the sIPSC IEI in the presence of scopolamine (P < 0.05, n = 7, 2,700 baseline events, 2,500 scopolamine events) and a decrease in sIPSC peak amplitude (P < 0.0001, n = 7, 2,700 baseline events, 2,500 scopolamine events). (C) Representative traces from baseline conditions and in the presence of scopolamine. (Scale bars: 20 pA and 3 s; Inset, 20 pA and 500 ms.) (D) Cumulative probability showing no change in sEPSC IEI (P = 0.29, n = 5, 1,300 baseline events, 1,300 scopolamine events) and increased peak amplitude with bath application of scopolamine (P < 0.0001, n = 5, 1,300 baseline events, 1,300 scopolamine events). (E) Schematic of whole-cell recordings of CA1 pyramidal cells showing the effects of scopolamine on intrinsically (1) and synaptically (2) driven APs. (Scale bars: 20 mV and 100 ms.) (Inset) Representative subthreshold EPSP-IPSP traces from cell 12 in the raster plot; compared with the baseline trace (black), scopolamine decreases the IPSP magnitude (red trace, black arrow). (Scale bars: 2 mV and 50 ms.) (F) Raster plot and summary plots showing a significant increase in synaptic AP probability in the presence of scopolamine (*P < 0.05, n = 13). (G) No change in the measured intrinsic properties (Direct AP number, AP threshold, and input resistance) is observed (n = 13). (Scale bars: 20 mV and 100 ms, 4 mV and 100 ms.) All values are mean ± SEM.

Fig. 5.

Summary of the effects of rapid antidepressants on synaptically driven APs. Ketamine, GLYX-13, and scopolamine significantly increase the synaptic AP probability compared with baseline (ketamine and GLYX-13, P < 0.001 and scopolamine, P < 0.05), indicating pyramidal cells are disinhibited. Conversely, the GluN2B subunit-selective NMDAR antagonist Ro 25-6981 does not alter synaptically driven pyramidal cell excitability. Replotted from Figs. 1–4. Asterisks indicate a significant difference from baseline and drug for each inhibitor. All values are mean ± SEM.

Fig. 6.

Ketamine and scopolamine, but not GLYX-13 or Ro 25-6981, increase excitability in a neural population. In extracellular recordings, bath application of ketamine (A; *P < 0.05, n = 6) and scopolamine (B; *P < 0.05, n = 10) significantly increases pyramidal cell excitability, while there is no effect with GLYX-13 (C, n = 12) or Ro 25-6981 (D, n = 7). n.s., not significant. In control experiments, switching between two containers of aCSF had no effect on excitability (E, n = 37). (Scale bars: 1 mV and 10 ms.) All values are mean ± SEM.

Fig. 7.

Graphical representation of the disinhibition elicited by NMDAR and muscarinic receptor antagonists. (A) Ketamine, GLYX-13, and scopolamine reduce inhibitory input onto pyramidal cells (black arrow heads). This leads to a decrease in the IPSP (arrow, green trace) in the EPSP-IPSP sequence, which shifts the I/E balance toward excitation, leading to a greater probability for synaptic excitatory input to elicit APs. (B) Ro 25-6981 increases inhibitory input onto pyramidal cells, leading to an enhanced IPSP (arrow, green trace) in the EPSP-IPSP sequence and a shift in the I/E balance away from excitation. This is possibly due to only a subset of interneurons in hippocampus having a large number of GluN2B-containing NMDARs. Therefore, Ro 25-6981 may decrease output of a subset of interneurons (black arrow heads), which likely results in disinhibition of other interneurons and more inhibitory drive onto pyramidal cells (green arrowhead).