Group II Metabotropic Glutamate Receptors Mediate Presynaptic Inhibition of Excitatory Transmission in Pyramidal Neurons of the Human Cerebral Cortex

Abstract

Group II metabotropic glutamate receptor (mGluR) ligands are potential novel drugs for neurological and psychiatric disorders, but little is known about the effects of these compounds at synapses of the human cerebral cortex. Investigating the effects of neuropsychiatric drugs in human brain tissue with preserved synaptic circuits might accelerate the development of more potent and selective pharmacological treatments. We have studied the effects of group II mGluR activation on excitatory synaptic transmission recorded from pyramidal neurons of cortical layers 2-3 in acute slices derived from surgically removed cortical tissue of people with epilepsy or tumors. The application of a selective group II mGluR agonist, LY354740 (0.1-1 μM) inhibited the amplitude and frequency of action potential-dependent spontaneous excitatory postsynaptic currents (sEPSCs). This effect was prevented by the application of a group II/III mGluR antagonist, CPPG (0.1 mM). Furthermore, LY354740 inhibited the frequency, but not the amplitude, of action potential-independent miniature EPSCs (mEPSCs) recorded in pyramidal neurons. Finally, LY354740 did slightly reduce cells' input resistance without altering the holding current of the neurons recorded in voltage clamp at -90 mV. Our results suggest that group II mGluRs are mainly auto-receptors that inhibit the release of glutamate onto pyramidal neurons in layers 2-3 in the human cerebral cortex, thereby regulating network excitability. We have demonstrated the effect of a group II mGluR ligand at human cortical synapses, revealing mechanisms by which these drugs could exert pro-cognitive effects and treat human neuropsychiatric disorders.

Keywords: EPSC; cognitive enhancer; epilepsy; glutamatergic; human cortex; mGluR; presynaptic receptor; transmitter release.

FIGURE 1

Features of a human cortical pyramidal cell. (A) Confocal microscopic image of a biocytin-filled human cortical pyramidal cell in layer 3 (patient/cell code: H26) showing spiny dendrites, a prominent apical dendrite and axon (a) descending toward the white matter; maximum intensity projection of a z-stack of ∼24 μm thickness; 1.8 μm optical slice thickness; interval 0.9 μm; 25 slices). (B) Voltage responses of the cell shown in (A) recorded in current clamp mode to hyperpolarizing (–50 pA) and depolarizing (+400 pA) current steps (holding potential: –80 mV). (C) Representative traces of spontaneous EPSCs recorded in voltage clamp mode at –90 mV; traces are low-pass filtered at 1 kHz and notch filtered at 50 Hz (width: 0.05 Hz). (A–C) Same cell.

FIGURE 2

Activation of group II mGluRs depresses excitatory synaptic transmission in human cortical pyramidal cells. (A) Representative traces in voltage clamp mode (–90 mV) during baseline and application of the group II mGluR agonist LY354740 (0.1 μM) to a pyramidal cell. (B) Cumulative probability distributions of the inter-sEPSC intervals and sEPSC amplitudes for the cell shown in (A). LY354740 significantly reduces sEPSC frequency (left, p = 0.0001, Kolmogorov–Smirnov test), and non-significantly decreases sEPSC amplitude (right, p = 0.056, Kolmogorov–Smirnov test). (C) Event time histograms (bin size: 10 ms, cell in A,B) showing the effect of LY354740 application on sEPSC frequency (left) and amplitude (right). (D) Baseline normalized effects of LY354740 (0.1 or 1 μM) on individual cells (see Table 2) show significant reduction of sEPSC frequency (left, p = 0.003 Kruskal–Wallis test; baseline vs. LY354740 p < 0.01, baseline vs. washout p < 0.05, LY354740 vs. washout p > 0.05, Dunn’s post hoc test, n = 8) and sEPSC amplitude (right, p = 0.003 Kruskal–Wallis test; baseline vs. LY354740 p < 0.01, baseline vs. washout p < 0.05, LY354740 vs. washout p > 0.05, Dunn’s post hoc test, n = 8). In some cells, washout could not be analyzed due to changes in series resistance (>20% baseline). Numbers denote codes of individual cells (see Table 2). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001.

FIGURE 3

An antagonist of group II/III mGluRs prevents depression of excitatory synaptic transmission by LY354740. (A) Representative traces in voltage clamp mode (–90 mV) during baseline and application of LY354740 (1 μM) in the presence of the group II/III mGluR antagonist CPPG (0.1 mM) in a neuron. (B) Cumulative probability distributions of the inter-sEPSC intervals and sEPSC amplitudes for the cell shown in (A). LY354740 does not reduce sEPSC frequency (p = 0.967, Kolmogorov–Smirnov test) or amplitude (p = 0.999, Kolmogorov–Smirnov test). (C) Event time histograms (bin size: 10 ms, cell in A,B) showing the effect of LY354740 application on sEPSC frequency (left) and amplitude (right) in presence of CPPG. (D), on average, LY354740 (1 μM) does not significantly change sEPSC frequency (left, p = 0.312 Wilcoxon test, n = 6) or sEPSC amplitude (p > 0.999 Wilcoxon test, n = 6). Numbers denote codes of individual cells (see Table 2).

FIGURE 4

Activation of group II mGluRs does not lead to detectable inward currents in human cortical pyramidal cells. (A) Representative trace in voltage clamp mode (–90 mV) during baseline and application of LY354740 (1 μM). The trace was processed with a low pass filter (5 Hz stop band), a notch filter (50 Hz) and boxcar averaging (window of 50 data points; original sampling rate: 20 kHz) to remove synaptic events and isolate the holding current. (B) LY354740 (1 μM) does not significantly affect the holding current (p = 0.175, Wilcoxon test, n = 11). Numbers denote codes of individual cells (see Table 2). (C) Baseline normalized effects of LY354740 (0.1 or 1 μM) on individual cells (see Table 2) show significant reduction of R_in (median change –5%, IQR: 3–10%, p = 0.04 Kruskal–Wallis test; baseline vs. LY354740 p < 0.05, baseline vs. washout p > 0.05, LY354740 vs. washout p > 0.05, Dunn’s post hoc test, n = 11). (D) On average, LY354740 (1 μM) does not significantly impact pyramidal cells’ R_in when CPPG (0.1 mM) is pre-applied (p = 0.7, Wilcoxon test, n = 6). (E) Boxplot showing significantly bigger reduction of R_in between application of LY354740 only and LY354740 with CPPG (p = 0.03, Mann–Whitney test). In some cells, washout could not be analyzed due to changes in series resistance (>20% baseline). Numbers denote codes of individual cells (see Table 2). ^∗p < 0.05.

FIGURE 5

Activation of group II mGluRs depresses excitatory synaptic transmission via a presynaptic effect. (A) Representative traces in voltage clamp mode (–90 mV) from a neuron during baseline and application of the group II mGluR agonist LY354740 (1 μM) in the presence of 1 μM tetrodotoxin; mEPSCs are marked with arrows. (B) Cumulative probability distributions of the inter-mEPSC intervals and mEPSC amplitudes for the cell shown in (A). LY354740 significantly reduces mEPSC frequency (left, p < 0.0001, Kolmogorov–Smirnov test) but not mEPSC amplitude (right, p = 0.999, Kolmogorov–Smirnov test). (C) Event time histograms (bin size: 10 ms, cell in A,B) showing the effect of LY354740 application on mEPSC frequency (left) and amplitude (right). (D) Baseline normalized effect of (0.1 μM, n = 3 cells, or 1 μM, n = 6 cells) on individual cells. LY354740 significantly reduces mEPSC frequency (left, p = 0.019 Kruskal–Wallis test; baseline vs. LY354740 p < 0.05, baseline vs. washout p > 0.05, LY354740 vs. washout p > 0.05, Dunn’s post hoc test, n = 9) but not mEPSC amplitude (right, p = 0.239 Kruskal–Wallis test; n = 9). In some cells, washout could not be analyzed due to change in series resistance (>20% baseline). Numbers denote codes of individual cells (see Table 2). ^∗p < 0.05, ^∗∗∗∗p < 0.0001.