Metabotropic glutamate receptor I (mGluR1) antagonism impairs cocaine-induced conditioned place preference via inhibition of protein synthesis

Abstract

Antagonism of group I metabotropic glutamate receptors (mGluR1 and mGluR5) reduces behavioral effects of drugs of abuse, including cocaine. However, the underlying mechanisms remain poorly understood. Activation of mGluR5 increases protein synthesis at synapses. Although mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome, it remains unknown whether group I mGluR-mediated protein synthesis is involved in any behavioral effects of drugs of abuse. We report that group I mGluR agonist DHPG induced more pronounced initial depression of inhibitory postsynaptic currents (IPSCs) followed by modest long-term depression (I-LTD) in dopamine neurons of rat ventral tegmental area (VTA) through the activation of mGluR1. The early component of DHPG-induced depression of IPSCs was mediated by the cannabinoid CB1 receptors, while DHPG-induced I-LTD was dependent on protein synthesis. Western blotting analysis indicates that mGluR1 was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. We also show that cocaine conditioning activated translation machinery in the VTA via an mGluR1-dependent mechanism. Furthermore, intra-VTA microinjections of mGluR1 antagonist JNJ16259685 and protein synthesis inhibitor cycloheximide significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP) and activation of translation elongation factors. Taken together, these results suggest that mGluR1 antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine.

Figure 1

DHPG-induced depression of IPSCs in VTA dopamine neurons is mediated mainly by mGluR1. (a) Bath application of group I mGluR agonist DHPG (100 μM, 10 min) induced long-term depression (LTD) of evoked IPSCs (n=7, p<0.01 vs baseline). (b) The mGluR1-selective antagonist LY367385 (100 μM) blocked DHPG-induced depression of IPSCs (n=7, p<0.05 vs control). (c) Another mGluR1-selective antagonist, JNJ16259685 (100 nM) blocked DHPG-induced depression of IPSCs (n=6, p<0.05 vs control). (d) The mGluR5-selective antagonist MTEP (10 μM) did not affect DHPG-induced depression of IPSCs (n=8, p<0.05 vs control). Error bars indicate SEM.

Figure 2

The early component of DHPG-induced depression of IPSCs is CB₁ receptor-dependent. (a) The CB₁ receptor antagonist AM251 (2 μM) attenuated the early component of DHPG-induced depression of IPSCs (n=6, p<0.05 vs control, n=6) but did not affect DHPG I-LTD (p>0.05). The amplitude of the first IPSCs was normalized. (b) DHPG increased the PPR during the early component of DHPG-induced depression of IPSCs (p<0.05 vs control) but did not affect the PPR during I-LTD (p>0.05). Error bars indicate SEM.

Figure 3

Protein synthesis is required for DHPG-induced I-LTD in the VTA. The protein synthesis inhibitor anisomycin (30 μM, n=8, p<0.05 vs control) or cycloheximide (80 μM, n=6, p<0.05 vs control) blocked DHPG-induced I-LTD but did not significantly affect the early component of DHPG-induced depression of IPSCs (p<0.05 vs control). Error bars indicate SEM.

Figure 4

DHPG-induced I-LTD requires the activation of ERK1/2 and mTOR signaling pathways. (a) The MEK inhibitor U0126 (20 μM) exerted only a modest effect on DHPG-induced I-LTD that did not reach statistical significance (n=8, p>0.05 vs control). U0124 (20 μM), an inactive analog of U0126, had no effect on DHPG I-LTD (n=7, p>0.05 vs control). (b) The mTOR inhibitor rapamycin (Rapa, 100 nM) also had no significant effect on DHPG-induced I-LTD (n=7, p>0.05 vs control), whereas co-application of rapamycin (100 nM) and U0126 (20 μM) blocked DHPG-induced I-LTD (n=8, p<0.05 vs control). Error bars indicate SEM.

Figure 5

DHPG induced ERK and mTOR phosphorylation and activation in the VTA via mGluR1. (a) DHPG (100 μM, 10 min) increased p-ERK1/2 levels in VTA homogenates compared, this effect was blocked by the JNJ16259685 (JNJ, 100 nM, ***p<0.001, Tukey post hoc test). (b) DHPG also increased p-mTOR levels in VTA homogenates compared with control, this effect was blocked by JNJ16259685 (100 nM, ***p<0.001). Each group of data was obtained from 6 to 7 VTA slices prepared from 3 to 4 rats. Error bars indicate SEM.

Figure 6

Intra-VTA infusions of the mGluR1 antagonist JNJ16259685 or protein synthesis inhibitor cycloheximide during the conditioning phase attenuated the acquisition of CPP to cocaine. (a) Timeline of drug treatment and behavioral paradigm. Groups of rats received saline and cocaine place conditioning once daily for 8 days. Vehicle (Veh), JNJ 16259685 (JNJ) or cycloheximide (Cyc) was bilaterally infused into the VTA 30 min prior to each saline- or cocaine-pairing. (b) Pre-test indicates that rats did not exhibit baseline bias in place preference in all groups (n=7–10, p>0.05). (c) Intra-VTA infusions of JNJ16259685 or cycloheximide significantly attenuated CPP in cocaine-conditioned rats but did not affect CPP scores in saline-conditioned rats (n=7–10, *p<0.05, ***p<0.001, Tukey post hoc test). (d) Intra-VTA infusions of JNJ16259685 or cycloheximide did not significantly affect locomotor activity in cocaine- or saline-conditioned rats (p>0.05, n=7–10). Error bars indicate SEM.

Figure 7

Cocaine CPP activates ERK and mTOR signaling pathways and increases the synthesis of the 5′TOP-encoded protein eEF1A through mGluR1 activation. (a, b) p-ERK1/2 (a) and mTOR levels (b) in the VTA were significantly increased in cocaine-conditioned rats compared with those in saline-conditioned rats, and the increase was blocked by intra-VTA infusions of JNJ16259685 (JNJ) (***p<0.001, Tukey post hoc test). (c, d, e) p-p70S6K (c), p-S6 (d), and p-eIF4E (e) levels in the VTA were significantly increased in cocaine-conditioned rats compared with those in saline-conditioned rats, and these increases were blocked by intra-VTA infusions of JNJ16259685 during the conditioning phase (**p<0.01, ***p<0.001). (f) eEF1A was significantly increased in cocaine-conditioned rats compared with that in saline-conditioned rats (***p<0.001), and this increase was blocked by intra-VTA infusions of JNJ16259685 or cycloheximide (Cyc) during the conditioning phase (***p<0.001). Western blotting was performed on VTA samples collected ∼1 h after the CPP tests. n=6 rats each group. Error bars indicate SEM.

Figure 8

Histological verification of intra-VTA cannula placements. (a) Cresyl Violet-stained coronal section of typical bilateral intra-VTA cannula placements. (b) Representative sites of cannula tips in the VTA from data shown in Figures 6 and 7.

Figure 9

Intra-SN infusions of JNJ16259685 or cycloheximide did not affect cocaine CPP. (a) Timeline of drug treatment and behavioral paradigm. (b) Pre-test indicated that rats did not exhibit baseline, non-conditioning place preference (n=7–8, p>0.05). Intra-SN infusions of JNJ16259685 (JNJ) or cycloheximide (Cyc) did not affect CPP to cocaine (n=7–8, p>0.05). The group for intra-VTA infusions of vehicle (Veh) was obtained from Figure 6 and was re-plotted here for the purpose of comparison. Error bars indicate SEM. (c) Representative sites of cannula tips in the SN.