Presynaptic mGluRs Control the Duration of Endocannabinoid-Mediated DSI

Abstract

GABA synapses in the brain undergo depolarization-induced suppression of inhibition (DSI) that requires activation of presynaptic cannabinoid type 1 receptors (CB₁Rs). The brevity of DSI, lasting ∼1 min in most brain regions, has been ascribed to the transient production of 2-arachidonoylglycerol (2-AG). Here, we propose that the duration of DSI is controlled by heterologous interactions between presynaptic mGluRs and CB₁Rs. By examining GABA synapses on parvocellular corticotropin-releasing hormone-expressing neurons in the paraventricular nucleus of the hypothalamus (PVN) of male and female mice, we show that DSI decays quickly in experimental conditions in which both GABA and glutamate are released from adjacent nerve terminals. Pharmacological inhibition of group I mGluRs prolongs DSI, whereas prior activation of mGluRs inhibits DSI, collectively suggesting that group I mGluRs quench presynaptic CB₁R signaling. When photostimulation of genetically identified terminals is used to release only GABA, CB₁R-dependent DSI persists for many minutes. Under the same conditions, activation of group I mGluRs reestablishes classical, transient DSI. The long-lasting DSI observed when GABA synapses are independently recruited functionally uncouples inhibitory input to PVN neurons. These observations suggest that heterologous interactions between mGluRs and CB₁Rs control the temporal window of DSI at GABA synapses, providing evidence for a powerful new way to affect functional circuit connectivity in the brain.SIGNIFICANCE STATEMENT Postsynaptic depolarization liberates endocannabinoids, resulting in a rapid and transient decrease in release probability at GABA synapses. We discovered that mGluRs control the duration of depolarization-induced suppression of inhibition (DSI), most likely through heterologous desensitization of cannabinoid type 1 receptors by presynaptic mGluR₅ By shortening the duration of DSI, mGluRs control the temporal window for retrograde signaling at GABA synapses. Physiological or pathological changes that affect glutamate spillover may profoundly affect network excitability by shifting the duration of cannabinoid inhibition at GABA synapses.

Keywords: CB1; DSI; endocannabinoid; heterologous desensitization; hypothalamus; mGluR.

Figure 1.

Inhibition of mGluR₅ prolongs DSI. A, Top, Representative traces of eIPSCs from baseline (1, gray) immediately after postsynaptic depolarization (2, red) and 3 min later (3, blue) corresponding to the indicated regions below. Inset, Schematic of experimental setup. Bottom, Time course of eIPSC amplitude of 10 cells from 9 animals (8 male, 1 female) following repeated postsynaptic depolarization (PD) to +20 mV for 5 s (vertical dashed line) (PD1: F_(2,9) = 17.06, p = 0.0001, PD2: F_(2,9) = 34.15, p = 0.0001). Significance is reported with respect to the preceding shaded baseline region. B, Summary data corresponding to shaded regions in A showing that sIPSC frequency undergoes a transient reduction that returns to baseline following PD (F_(2,9) = 7.89, p = 0.0035), whereas sIPSC amplitude (C) is unaffected (F_(2,9) = 2.13 p = 0.15). D, Top, Sample eIPSC traces in the presence of MTEP (10 μm). Inset, Experimental setup. Bottom, Time course showing eIPSC amplitude of 8 cells from 7 mice (4 male, 3 female) undergoes a transient and persistent reduction in the presence of a mGluR₅ antagonist (PD1: F_(2,7) = 17.81, p = 0.0009, PD2: F_(2,7) = 6.85, p = 0.018). E, MTEP causes a transient and persistent reduction in sIPSC frequency (F_(2,7) = 23.33, p = 0.0001), whereas sIPSC amplitude (F) is unaltered (F_(2,7) = 1.43, p = 0.27). Scale bars, 100 pA, 20 ms. Post hoc versus baseline p-values are shown as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as mean ± SEM.

Figure 2.

No role for astrocytic or postsynaptic GPCR signaling in LDSI. A, Left, Schematic showing postsynaptic loading of GDPβs. Right, Time course summary of 7 cells from 6 animals (4 male, 2 female) showing rapid recovery from DSI in neurons with impaired GPCR signaling (PD1: F_(2,6) = 22.89, p = 0.0001, PD2: F_(2,6) = 23.87, p = 0.0012). B, Left, Schematic showing astrocytes patched and filled with an internal solution that clamps Ca²⁺ transients and prevents GPCR activation. Following astrocytic loading, an adjacent neuron is patched and eIPSCs are elicited. Right, Time course summary of 6 cells from 5 animals (4 male, 1 female) showing that PNCs within the field of a patched astrocyte exhibit rapid recovery from DSI (PD1: F_(2,5) = 13.61, p = 0.0040, PD2: F_(2,5) = 5.23, p = 0.028). Post hoc versus baseline p-values are shown as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as mean ± SEM.

Figure 3.

Group I mGluR activation quenches DSI. A, Temporal schematic of experiment detailing timing of recordings and drug application. B, Summary time course (left) and graphs (bottom) showing eIPSCs of 11 cells from 9 mice (3 male, 6 female) undergo DSI (F_(2,10) = 19.59, p = 0.001). Top, Experimental setup. C, Following DHPG washout in the same cells, DSI was absent in both the summary time course (left) and graphs (bottom) (F_(2,10) = 2.21, p = 0.14). Top, Experimental setup. Post hoc versus baseline p-values are shown as follows: ***p < 0.001. Data are shown as mean ± SEM.

Figure 4.

Optically evoked GABA release in the PVN of vGAT-ChR2 BNST_Fu mice. A, Left, Sagittal illustration denoting the projection from the BNST_Fu to the PVN, targeted for stereotaxic viral injection. Right, Coronal map through the level of the BNST, indicating the location of the BNST_Fu. Inset, Confocal image (10× magnification) showing eYFP expression in the BNST_Fu of a vGAT-IRES-Cre mouse. B, Injection maps outlining BNST_Fu targeting. C, eYFP-positive neurons from the BNST_Fu were patched and characterized by their membrane voltage response before being exposed to varying durations of 473 nm light (D). BNST_Fu neurons exhibited a characteristic steady-state current in response to a sustained exposure to blue light. E, Confocal image (20× magnification) depicting ChR2-eYFP-positive BNST_Fu afferent fibers innervating the PVN. Inset, Representative trace showing a PNC membrane voltage response. F, Synaptic currents evoked by 473 nm light of varying durations. A 2 ms pulse was found to elicit synchronous release and was used for all other experiments. G, oPSCs were completely inhibited in a single cell by the application of the selective GABA_A antagonist GABAzine (100 μm). Scale bars: A, E, 100 μm; C–F, 50 mV/pA, 20 ms.

Figure 5.

oIPSCs exhibit LDSI. A, Experimental setup of optical fiber placement for BNST_Fu stimulation. B, Representative oIPSC traces (top) and amplitude time course (bottom) from an individual cell following a single postsynaptic depolarization. C, Summary time course showing that LDSI in 9 cells from 7 mice (6 male, 1 female) persists for many minutes following a single postsynaptic depolarization (F_(2,8) = 13.52, p = 0.0012). D, Top, Traces of oIPSCs corresponding to shaded regions below. Bottom, Summary time course showing that LDSI in 11 cells from 11 mice (2 male, 9 female) is not enhanced by repeated postsynaptic steps (PD1: F_(2,10) = 18.60, p = 0.0001, PD2: F_(2,10) = 10.70, p = 0.0016). E, Summary graphs data corresponding to shaded regions in D showing DSI (F_(2,10) = 39.38, p = 0.0001) and LDSI (F_(2,10) = 7.94, p = 0.0055) values compared with the initial baseline indicating that LDSI2 remains significantly depressed compared with the initial baseline. Scale bars, 100 pA, 20 ms. Post hoc versus baseline p-values are shown as follows: *p < 0.05, ***p < 0.001. Data are shown as mean ± SEM.

Figure 6.

LDSI is an eCB-dependent process that affects GABA synapses globally. A, Top, Traces from recordings in the presence of AM251 (5 μm). Inset, Schematic describing experimental setup. Bottom, Summary time course of 10 cells from 7 mice (4 male, 3 female) showing that oIPSCs do not undergo DSI or LDSI with AM251 (PD1: F_(2,9) = 1.89, p = 0.19, PD2: F_(2,9) = 1.57, p = 0.24). B, Summary graphs showing that repeated postsynaptic depolarization affects neither DSI (F_(2,9) = 1.03, p = 0.35) nor LDSI (F_(2,9) = 1.92, p = 0.19). C, Representative traces of control sIPSC events during the baseline (1) and LDSI (2) time points shown in D. D, Time course summary of sIPSC frequency in control depresses following repeated postsynaptic depolarization (gray, PD1: F_(2,10) = 33.59, p = 0.0001, PD2: F_(2,10) = 11.44, p = 0.0025), but is unaltered in AM251 (orange, PD1: F_(2,9) = 1.73, p = 0.21, PD2: F_(2,9) = 0.54, p = 0.57). E, Summary graphs showing that, compared with the initial baseline, postsynaptic depolarization causes phasic (F_(2,10) = 72.66, p = 0.0001) and persistent (F_(2,10) = 9.52, p = 0.0039) depression of sIPSC frequency in control. Scale bars: A, 100 pA, 20 ms; C, 25 pA, 200 ms. Post hoc versus baseline p-values are shown as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as mean ± SEM.

Figure 7.

mGluR₅ coactivation curtails optically induced LDSI. A, Top, Representative traces during DHPG application (50 μm). Inset, Experimental setup. Bottom, Summary time course of 6 cells from 6 mice (2 male, 4 female) showing postsynaptic depolarizations in DHPG repeatedly elicit DSI, but not LDSI (PD1: F_(2,5) = 19.59, p = 0.0010, PD2: F_(2,5) = 16.63, p = 0.0038). B, C, Summary graphs of a phasic suppression of sIPSC frequency (F_(2,5) = 10.30, p = 0.0037; B), whereas sIPSC amplitude is unaffected by postsynaptic depolarization (F_(2,5) = 0.29, p = 0.67; C). Scale bars, 100 pA, 20 ms. Post hoc versus baseline p-values are shown as follows: *p < 0.05, **p < 0.01. Data are shown as mean ± SEM.

Figure 8.

LDSI impairs afferent control of firing. A, Spiking is reliably inhibited by a single oIPSC from BNST_Fu afferents (blue line), as shown in the current-clamp trace (top) and summary graph (bottom). B, Temporal schematic of experiment detailing timing of recordings, optical stimulation, and postsynaptic depolarization (square pulse). C, Action potentials in one cell are inhibited by optical stimulation at 10 Hz (dashed blue lines) before LDSI. D, Inhibition is less reliable following LDSI induction. E, Summary data of the mean changes in firing of 6 cells from 4 mice (4 male) during the 1 s, 10 Hz stimulation for both conditions (F_(3,5) = 9.89, p = 0.0048). Scale bars: A, 50 mV, 20 ms; C, D, 50 mV, 200 ms. Post hoc versus baseline p-values are shown as follows: *p < 0.05. Data are shown as mean ± SEM.