In vivo metabotropic glutamate receptor 5 (mGluR5) antagonism prevents cocaine-induced disruption of postsynaptically maintained mGluR5-dependent long-term depression

Abstract

Metabotropic glutamate receptor 5 (mGluR5) plays a critical role in psychostimulant-induced behavior, yet it is unclear whether mGluR5 is activated by psychostimulant administration, or whether its role is constitutive. We previously reported that activation of mGluR5 with the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) can induce a long-term depression (DHPG-LTD) of glutamatergic transmission in the bed nucleus of the stria terminalis (BNST), and that ex vivo induction of this LTD is disrupted by repeated in vivo administration of cocaine. Here we demonstrate that DHPG-LTD is not maintained by alterations in glutamate release, and that postsynaptic endocytosis is necessary. Furthermore, we find that a single administration of cocaine produces a transient disruption of DHPG-LTD, and the duration of this disruption was increased by repeated days of cocaine administration. The disruption produced by cocaine was not permanent, because DHPG-LTD could be induced 10 d after cocaine administration. To test the role of mGluR5 in vivo in the cocaine-induced disruption of DHPG-LTD, we injected mice with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine before cocaine. mGluR5 antagonism during in vivo cocaine administration rescued subsequent ex vivo induction of DHPG-LTD. The effects of in vivo cocaine could be mimicked by application of cocaine to BNST-containing slices, suggesting that the actions of cocaine are local. Thus, using a novel strategy of in vivo antagonist-induced rescue of ex vivo agonist effects for the same receptor, we provide evidence suggesting that mGluR5 activation is actively recruited by in vivo cocaine.

Figure 1.

mGluR5-LTD in the BNST. A, Group I mGluR LTD in the BNST is dependent on mGluR5 function. Average time courses of normalized EPSC amplitudes evoked by local stimulation in the BNST in acute slice preparations are shown. DHPG (100 μm) was applied for 5 min as indicated. ○, mGluR5 knock-out; ■, wild-type littermates. B, Mouse brain coronal slices containing the dorsal BNST (top) or a portion of the cerebellum (bottom) stained with DRAQ nuclear stain (left) or anti-mGluR5 (right) show a specific mGluR5 signal in the BNST. AC, Anterior commissure; Str, striatum; AL, ansiform lobule; F1, fissure 1. C, Schematic depicting the region contained in the micrographs (red squares). Dorsal BNST in blue (top), cerebellum (bottom). D–F, Mouse brain coronal slices were stained with anti-mGluR5 (D; red) or the dendritic marker anti-MAP2 (E; green) (F; merge). Scale bar, 20 μm.

Figure 2.

A, B, Normalized time course of effects of DHPG on EPSC amplitude in external ACSF containing 1 mm Ca²⁺/2.8 mm Mg²⁺ (A) or 1 mm Ca²⁺/1 mm Mg²⁺ (B). C, Summary graph of effects of varying extracellular cations on average basal PPR (EPSC₂/EPSC₁). *p < 0.05. D, Varying extracellular cations did not affect PPR normalized to baseline 30 min after DHPG washout. E, Representative experiment depicting time course of γ-DGG-induced depression of EPSCs before and after DHPG application. F, Percentage of inhibition by γ-DGG is unchanged before and after DHPG application. Error bars indicate SEM.

Figure 3.

DHPG decreased the frequency but not the amplitude of BNST mEPSCs. A, Sample current traces of mEPSCs in a BNST neuron before DHPG application (basal) and at 30 min after (late; LTD) DHPG. Calibration: 20 pA, 512 ms. B, Cumulative probability plot of DHPG induced effects on mEPSC inter-event interval within the basal (3–5 min, open squares) and late (LTD; min 35–37, closed circles) time periods (n = 11; p < 0.05, Kolmogorov–Smirnov test). C, Cumulative probability of effects of DHPG on mEPSC amplitude within the same current recordings as shown in B. Inset, Representative traces before DHPG application and during DHPG-mediated LTD. Inset calibration, 5 pA. Error bars indicate SEM.

Figure 4.

Maintenance of mGluR5 LTD is dependent on postsynaptic endocytosis machinery. A, Average time course of effects of 100 μm DHPG on normalized EPSC amplitudes from BNST neurons perfused with 2 mm dynamin-inhibitory peptide. B, Time course of effects of 100 μm DHPG on EPSCs from BNST neurons postsynaptically perfused with either jasplakinolide (2 μm) (○) or vehicle (DMSO) (■). n = 7 (1 cell was excluded from this analysis because DHPG produced a depression of the EPSC twice the normal observed amplitude in naive neurons). Error bars indicate SEM.

Figure 5.

Repeated exposure to cocaine attenuates group I mGluR LTD in the BNST. A–E, Average time courses of normalized EPSC amplitudes evoked by local stimulation in the BNST in slices taken from saline and cocaine (20 mg/kg)-treated mice. DHPG (100 μm) was applied for 5 min as indicated. Specific mouse treatments were as follows: A, mice killed and slices prepared 24 h after 10 d of intraperitoneal saline injections; B–D, mice killed and slices prepared 24 h after 3, 5, and 10 d of intraperitoneal cocaine injections, respectively; E, mice killed and slices prepared 10 d after 10 d of intraperitoneal cocaine or saline injections. F, Summary graph in which values represent percentage change in EPSC ± SEM, 30 min after DHPG washout. *p < 0.05 (Bonferroni and Tukey test after one-way ANOVA). Error bars indicate SEM.

Figure 6.

A single cocaine exposure transiently attenuates group I mGluR LTD in the BNST. A–C, Average time courses of normalized EPSC amplitudes evoked by local stimulation in the BNST in slices taken from saline and cocaine (20 mg/kg)-treated mice 30 min (A, B) or 4 h (C) after injection. DHPG (100 μm) was applied for 5 min as indicated.

Figure 7.

Cocaine has local effects on mGluR5 signaling in BNST. Summary graph representing percentage change in mEPSC frequency after bath application of 30 μm cocaine (Coc) and 30 min after subsequent DHPG (100 μm) application.

Figure 8.

Repeated cocaine-induced changes in group I mGluR LTD are dependent on mGluR5 function in vivo. A–D, Average time courses of normalized EPSC amplitudes evoked by local stimulation in the BNST in acute slice preparations. DHPG (100 μm) was applied for 5 min as indicated. In each case, mice underwent 10 d of intraperitoneal injections and were killed 24 h after the last injection. Specific daily mouse treatments were as follows: A, intraperitoneal vehicle followed 30 min later by intraperitoneal saline; B, intraperitoneal vehicle followed 30 min later by intraperitoneal cocaine (20 mg/kg); C, intraperitoneal MPEP (10 mg/kg) followed 30 min later by intraperitoneal saline; D, intraperitoneal MPEP (10 mg/kg) followed 30 min later by intraperitoneal cocaine (20 mg/kg). E, Summary graph in which values represent percentage change in EPSC ± SEM, 30 min after DHPG washout. *p < 0.05 (Bonferroni and Tukey test after one-way ANOVA). Error bars indicate SEM.

Figure 9.

A single cocaine exposure alters mGluR5 LTD in BNST slices in an mGluR5-dependent manner on an acute (30 min after cocaine) time scale. A–B, Average time courses of normalized EPSC amplitudes evoked by local stimulation in the BNST in acute slice preparations. DHPG (100 μm) was applied for 5 min as indicated. A, Intraperitoneal MPEP (10 mg/kg) followed 30 min later by intraperitoneal cocaine (Coc) (20 mg/kg). In B, mice were conditioned as in A, and MPEP (10 μm) was then applied to the slice 10 min before DHPG application. C, Summary graph in which values represent percentage change in EPSC ± SEM, 30 min after DHPG washout. *p < 0.05 (Bonferroni and Tukey test after one-way ANOVA). Error bars indicate SEM. Values taken from time courses in A and B and Figure 6, A and B.