Endocannabinoids mediate presynaptic inhibition of glutamatergic transmission in rat ventral tegmental area dopamine neurons through activation of CB1 receptors

Abstract

The endogenous cannabinoid system has been shown to play a crucial role in controlling neuronal excitability and synaptic transmission. In this study we investigated the effects of a cannabinoid receptor (CB-R) agonist WIN 55,212-2 (WIN) on excitatory synaptic transmission in the rat ventral tegmental area (VTA). Whole-cell patch clamp recordings were performed from VTA dopamine (DA) neurons in an in vitro slice preparation. WIN reduced both NMDA and AMPA EPSCs, as well as miniature EPSCs (mEPSCs), and increased the paired-pulse ratio, indicating a presynaptic locus of its action. We also found that WIN-induced effects were dose-dependent and mimicked by the CB1-R agonist HU210. Furthermore, two CB1-R antagonists, AM281 and SR141716A, blocked WIN-induced effects, suggesting that WIN modulates excitatory synaptic transmission via activation of CB1-Rs. Our additional finding that both AM281 and SR141716A per se increased NMDA EPSCs suggests that endogenous cannabinoids, released from depolarized postsynaptic neurons, might act retrogradely on presynaptic CB1-Rs to suppress glutamate release. Hence, we report that a type of synaptic modulation, previously termed depolarization-induced suppression of excitation (DSE), is present also in the VTA as a calcium-dependent phenomenon, blocked by both AM281 and SR141716A, and occluded by WIN. Importantly, DSE was partially blocked by the D2DA antagonist eticlopride and enhanced by the D2DA agonist quinpirole without changing the presynaptic cannabinoid sensitivity. These results indicate that the two pathways work in a cooperative manner to release endocannabinoids in the VTA, where they play a role as retrograde messengers for DSE via CB1-Rs.

Figure 1.

WIN induces inhibition of glutamatergic synaptic transmission in rat anterior VTA DA cell. A, A typical whole-cell patch clamp recording showing that bath application of the CB-R agonist WIN 55, 212-2 (WIN; 1 μm) inhibits NMDA EPSCs amplitude (n = 6; p < 0.0001) when cells are held at +40 mV. Representative traces are shown in the inset. The inset shows single NMDA EPSC from a typical experiment, before (black line) and during (dashed line) superfusion of WIN. Calibration: 100 pA, 10 msec. B, WIN reduces NMDA EPSCs amplitude (n = 6; p < 0.0001), when cells are held at +40 mV. All data are normalized to the respective baseline (5 min of baseline). Black bars show time of superfusion of WIN. SEM bars are smaller than symbols in some cases. The inset shows 10-trace averages of NMDA EPSCs before (black line) and during (dashed line) application of WIN. Calibration: 100 pA, 20 msec. C, WIN reduces AMPA EPSC amplitude (n = 7; p < 0.0001) when cells are held at -70 mV. All data are normalized to the respective baseline (5 min of baseline). Black bars show time of superfusion of WIN. SEM bars are smaller than symbols in some cases. Representative traces of AMPA EPSCs before and during application of WIN are shown in the inset. Calibration: 100 pA, 20 msec. D, WIN enhances the paired-pulse ratio of AMPA EPSCs (from EPSC2/EPSC1 = 1.07 ± 0.2 to EPSC2/EPSC1 = 2.06 ± 0.5; n = 7; p < 0.0005). The left-hand graph plots the paired-pulse ratio for each of the experiments in C before (basal) and after (WIN) the application of WIN, whereas the right-hand graph plots the averaged paired-pulse ratio in a bar graph form. Representative traces are shown in the inset. Calibration: 100 pA, 20 msec.

Figure 2.

WIN reduces mEPSCs frequency without affecting the amplitude. A, Representative consecutive 1 sec sweeps from a cell in which mEPSCs were recorded before (left) and after 5 min of application of WIN (right) are shown in the top panel. The running average histogram of mEPSC frequency showing the time course of the decrease produced by WIN (left), and the summary graph histogram (right) of the effect induced by WIN on mEPSCs frequency (from 1.19 ± 0.3-0.4 ± 0.1 Hz; n = 5; p < 0.001) and amplitude (basal: 15.86 ± 1.1 pA; WIN: 14.99 ± 0.88 pA) are shown in the bottom panel. Calibration: 20 pA, 150 msec. B, Cumulative distribution plot of mEPSC interevent intervals of a single neuron (154 events) shows a decreased frequency after 5 min of superfusion with WIN (dashed line, 40 events) compared with the baseline (black line). C, On the contrary, mEPSC amplitude distribution was unchanged (dashed line) when compared with the baseline (black line).

Figure 3.

Cannabinoid-induced inhibition of glutamatergic synaptic transmission involves activation of CB1-Rs. A, Concentration-response relationship for percentage decrease in AMPA (open circles) and NMDA (closed circles) EPSCs size produced by WIN (0.1-3 μm). Each point shows the mean ± SEM of responses of different neurons (n = 5-7). B, Concentration-response relationship for percentage decrease in AMPA (open circles) and NMDA (closed circles) EPSCs amplitude produced by HU210 (0.1-3 μm). Each point shows the mean ± SEM of responses of different neurons (n = 5). C, Coapplication of WIN with the selective CB1-R antagonist AM281 (500 nm) abolishes WIN-induced effect on NMDA EPSCs (n = 5; p < 0.001). D, Coapplication of WIN with the CB1-R antagonist SR141716A (SR; 1 μm) readily blocked the WIN-induced inhibition of NMDA EPSCs (n = 8; p = 0.001). E, Coapplication of WIN with the selective CB1-R antagonist AM281 (500 nm) abolishes WIN-induced effect on AMPA EPSCs (n = 5; p < 0.001). F, Coapplication of WIN with the CB1-R antagonist SR141716A (SR; 1 μm) readily prevented the WIN-induced inhibition of AMPA EPSCs (n = 5; p = 0.001). All data are normalized to the respective baseline (5 min of baseline). Black bars show the time of superfusion of the drugs.

Figure 4.

Endocannabinoids modulate excitatory synaptic transmission in rat anterior VTA. A, Superfusion of AM281 (500 nm) by itself produces a significant increase of NMDA EPSCs (n = 6; p < 0.005). All data are normalized to the respective baseline (5 min of baseline). Black bars show the time of superfusion of the drug. The inset shows 10-trace averages of NMDA EPSCs before (black line) and during application of AM281 (gray line). Calibration: 100 pA, 20 msec. B, Bath application of SR (1 μm) per se significantly increased NMDA EPSCs (n = 8; p < 0.0001). All data are normalized to the respective baseline (5 min of baseline). Black bars show the time of superfusion of the drug. SEM bars are smaller than symbols in some cases. The inset shows representative traces of NMDA EPSCs before (black line) and during application of SR (gray line). Calibration: 100 pA, 20 msec. C, Bath application of the selective VR1 antagonist capsazepine (CPZ; 10 μm, open circles) produced a significant decrease of NMDA EPSCs on its own (n = 5; p = 0.0002). Coapplication (closed circles) of the endocannabinoid reuptake inhibitor AM404 (10 μm) with CPZ further decreased NMDA EPSCs (n = 5; p = 0.0005). All data are normalized to the respective baseline (5 min of baseline). Black bars show the time of superfusion of the drugs. The inset shows 10-trace averages of NMDA EPSCs before (black line) and during application of CPZ (dashed line) and CPZ with AM404 (gray line). Calibration: 100 pA, 20 msec. D, Superfusion of methanandamide (mAEA; 2 μm) did not significantly affect NMDA EPSCs (n = 5; p > 0.05). All data are normalized to the respective baseline (5 min of baseline). Black bars show the time of superfusion of the drug. The inset shows 10-trace averages of NMDA EPSCs before (black line) and during application of mAEA (gray line). Calibration: 100 pA, 20 msec. E, Coapplication of mAEA (2 μm) with CPZ (10 μm) significantly decreased NMDA EPSCs (n = 5; p < 0.0002). All data are normalized to the respective baseline (5 min of baseline). Black bars show the time of superfusion of the drugs. SEM bars are smaller than symbols in some cases. The inset shows representative traces of NMDA EPSCs before (black line) and during application of mAEA with CPZ (gray line). Calibration: 100 pA, 20 msec. F, The VR1 agonist capsaicin (CPS; 1 μm, open circles) induces a robust increase of NMDA EPSCs (n = 5; p < 0.0001), that was prevented by coapplication of CPZ (10 μm; n = 5; p < 0.0001; closed circles). All data are normalized to the respective baseline (5 min of baseline). Black bars show the time of superfusion of the drugs. SEM bars are smaller than symbols in some cases. The inset shows representative traces of NMDA EPSCs before (black line) and during application of CPS (dashed line) and CPS with CPZ (gray line). Calibration: 100 pA, 20 msec.

Figure 5.

Endocannabinoids mediate depolarization-induced suppression of excitatory synaptic transmission in rat anterior VTA. A, DSE protocol. Under voltage-clamp mode, a 10 sec voltage step (from -70 to +40 mV) was applied to induce transient suppression of EPSCs. Representative traces for each condition from a single experiment are shown. B, Transient suppression of EPSCs induced by depolarization of VTA DA neuron (n = 5; p < 0.0001). The VTA DA neurons were depolarized from -70 to +40 mV for 10 sec under voltage-clamp mode. Each point is the average of the mean EPSCs for the 10 sec period from five different cells. C, DSE increases paired-pulse ratio of AMPA EPSCs (n = 5; p < 0.05). The left-hand graph plots the paired-pulse ratio for each of the experiments in B before (basal) and 5-15 sec after (DSE) the depolarizing step, whereas the right-hand graph plots the averaged paired-pulse ratio in a bar graph form. D, In the presence of either AM281 (500 nm, open triangles) or SR (1 μm, closed circles) the depolarizing voltage pulse caused no changes of EPSCs size (n = 5 for each condition; p < 0.0001). The VTA DA neurons were depolarized from -70 to +40 mV for 10 sec under voltage-clamp mode. Each point is the average of the mean EPSCs for the 10 sec period from five different cells for each condition. E, Time course of the transient suppression of EPSCs induced by depolarization of VTA DA neuron (n = 5; p < 0.0001; open circles) and occlusion of DSE induced by WIN (n = 5; p = 0.0005; closed squares). In the presence of WIN (1 μm; closed squares) the depolarizing voltage pulse caused no changes of EPSCs (n = 5; p = 0.0005). The VTA DA neurons were depolarized from -70 to +40 mV for 10 sec under voltage-clamp mode. Each point is the average of the mean EPSCs for the 10 sec period from five different cells for each condition. F, The magnitude of EPSCs inhibition induced by DSE is plotted as a function of the inhibition induced by AM404 for the five cells in B and Figure 3C, respectively. The data are fit by linear regression with r² = 0.98 (p < 0.001).

Figure 6.

DSE in VTA DA neurons is a presynaptic phenomenon mediated by a postsynaptic excitation. A, Induction of DSE depends on duration of depolarization. Example of DSE by various depolarizing pulses with durations of 0.5-10 sec. The postsynaptic neuron was depolarized at the times indicated by arrows. Representative traces of EPSCs acquired at the indicated points are shown in the inset. Calibration: 10 msec, 100 pA. B, Averaged data for DSE induced by depolarizing pulses with a duration of 0.5, 1, 3, 5, and 10 sec are plotted in the top panel. The relationship between the depolarizing pulse duration and the relative amplitude of EPSCs obtained after 5-15 sec after the end of depolarization is plotted in the bottom panel. The amplitude was normalized to the averaged value (dotted line) before depolarization. Each symbol represents the averaged value obtained from different cells (n = 6-10; ^*p = 0.001; ^**p < 0.0001). C, DSE in the VTA requires endocannabinoids and Ca²⁺, as well as DA. AM281 blocks DSE by antagonism, and WIN by occlusion. DSE is abolished by filling cells with BAPTA (15 mm), and partially blocked by the D₂DA antagonist eticlopride. Magnitude of EPSCs amplitude after the depolarizing pulse for all conditions plotted as the percentage of baseline before the pulse (control, n = 5; WIN, n = 5; AM281, n = 5; BAPTA; n = 9, Eticlopride, n = 7). D, Effect of eticlopride on DSE. The graph plots individual data obtained before (control) and during application of eticlopride. DSE was induced by 10 sec depolarization.

Figure 7.

Facilitation of DSE by quinpirole. A, A representative experiment demonstrating a dose-dependent enhancement of DSE by quinpirole (quin). DSE was induced by 3 sec depolarization. EPSC traces acquired before (black line) and after depolarization (dashed line) have been superimposed for each DSE trial and are shown in the inset. Calibration: 10 msec, 100 pA. B, Summary graph showing averaged data for the enhancement of DSE by quinpirole (n = 5) and blockade by the CB1-R antagonist AM281 (n = 5; p = 0.0006). Quinpirole concentrations are shown below the columns. DSE was induced by either 5 or 3 sec (four columns on the left, and first one from the right) depolarization. The asterisk represents statistically significant difference from the control (3 sec depolarization, 0 quinpirole; ^*p = 0.001). C, Dose-dependent suppression of EPSCs by WIN. The graph plots the effect of WIN on EPSC amplitude in absence (open columns) and presence (shaded) of 1 μm quinpirole (n = 5). D, Relative amplitudes of EPSCs are plotted against the concentration of WIN. Each symbol represents the averaged value from different cells (n = 5-7). Horizontal dotted lines indicate the levels of suppression induced by 1 μm quinpirole, 3 sec depolarization, and 1 μm quinpirole + 3 sec depolarization.