Group 1 mGlu-family proteins promote neuroadaptation to ethanol and withdrawal-associated hippocampal damage

Abstract

Background: Group 1 mGlu-family proteins (i.e., mGlu) consist of mGlu1 and mGlu5 and their activity may influence voluntary ethanol intake. The present studies sought to examine the influence of these receptors on the development of ethanol dependence using in vitro and in vivo models of chronic, intermittent ethanol (CIE).

Methods: Rat hippocampal explants were exposed to CIE with or without the addition of mGlu1 antagonist (7-hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; 0.5, 1, and 3μM) or mGlu5 antagonist (E)-2-methyl-6-styryl-pyridine (SIB-1893; 20, 100, and 200μM) to assess sparing of withdrawal-induced cytotoxicity. In a separate study, adult male rats were administered CIE with or without the addition of oral administration of group 1 mGlu antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 3mg/kg). Blood ethanol levels (BELs) were determined at 0930h on Day 2 of Weeks 1, 2, and 3. Withdrawal behavior was monitored during Day 6 of the third consecutive withdrawal.

Results: CIE produced significant hippocampal cytotoxicity. These effects were attenuated by co-exposure to CPCCOEt (3μM) with ethanol in the CA3. By contrast, these effects were blocked by SIB-1893 (20μM) in each primary cell layer. Oral administration of MPEP with ethanol significantly attenuated behavioral effects of subsequent withdrawal and reduced BELs.

Conclusions: These data demonstrate that ethanol activates group 1 mGlu-family proteins to promote withdrawal-associated cytotoxicity in vitro and physical dependence in vivo. These findings suggest that group 1 mGlu-family proteins may be therapeutic targets for treatment of alcohol use disorders.

Keywords: CIE; Chronic intermittent ethanol; Metabotropic glutamate receptor; NeuN; Neuron specific nuclear protein; mGlu.

Figure 1

Effects of mGlu1 antagonist CPCCOEt (0.5–3 μM) on NeuN immunofluorescence in the rat hippocampus. Data are presented as percent control of the mean +/− the SEM. ** = statistical significance (p <0.05) compared to control hippocampi; # = statistical significance (p <0.05) compared to ethanol hippocampi. N = 12 for control hippocampi; N = 17 for hippocampi exposed to ethanol; N = 13 for hippocampi co-exposed to ethanol and 0.5 μM CPCCOEt; N = 14 for hippocampi co-exposed to ethanol and 1.0 μM CPCCOEt; N = 16 for hippocampi co-exposed to ethanol and 3.0 μM CPCCOEt for each primary cell layer of the hippocampal formation. Representative images depict hippocampi exposed to ethanol-naïve media (control) or ethanol media (50 mM), or hippocampi co-exposed to 3.0 μM CPCCOEt and ethanol (50 mM).

Figure 2

Effects of mGlu5 antagonist SIB-1893 on NeuN immunofluorescence in the primary cell layers of the hippocampal formation. Data are presented as percent control of the mean +/− the SEM. ** = statistical significance (p <0.05) compared to control hippocampi; # = statistical significance (p <0.05) compared to ethanol hippocampi. N = 25 for control hippocampi; N = 28 for hippocampi exposed to ethanol; N = 23 for hippocampi co-exposed to ethanol and 20 μM SIB-1893; N = 27 for hippocampi co-exposed to ethanol and 100 μM SIB-1893; N = 24 for hippocampi co-exposed to ethanol and 200 μM SIB-1893 for each hippocampal subregion. Representative images depict hippocampi exposed to ethanol-naïve media (control) or ethanol media (50 mM), or hippocampi co-exposed to 20 μM SIB-1893 and ethanol (50 mM).

Figure 3

Body weight data were obtained daily on Week 1, 2, and 3. Data points show the range and means for subjects exposed to experimental groups. ** = statistical significance (p <0.05) compared to subjects administered an isocaloric control diet. N=7 for control subjects; N=8 for control with MPEP; N=6 for ethanol subjects; N=8 for subjects administered ethanol and MPEP

Figure 4

Withdrawal behavior was assessed during withdrawal on Day 6 of Week 3. Data points show mean scores in withdrawal behavior observed during the third consecutive withdrawal from CIE. # = statistical drug by diet interaction (p <0.001). N=7 for control subjects; N=8 for control with MPEP; N=6 for ethanol subjects; N=8 for subjects administered ethanol and MPEP.

Figure 5

Frequency of ethanol withdrawal behaviors observed in the present study in ethanol-dependent subjects that received oral administration of ethanol with or without the addition of MPEP (3 mg/kg).

Figure 6

Peak BELs were assessed 90 minutes post-ethanol administration on Day 2 of Week 1, 2, and 3. Data points show mean score BELs for subjects exposed to ethanol with or without MPEP. * = statistical main effect of week (p <0.05). # = statistical main effect of MPEP on BELs in ethanol-dependent rats. N=4 for ethanol subjects; N=5 for ethanol with MPEP.