Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA
* Author to whom correspondence should be addressed at: Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA. Tel.: +1 252 744 2758; Fax: +1 252 744 3203; E-mail: mcmillenb{at}mail.ecu.edu
(Received 18 March 2005; first review notified 31 May 2005; in final revised form 29 July 2005; accepted 29 July 2005)
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ABSTRACT |
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INTRODUCTION |
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The mGlu5 metabotropic glutamate receptor is in the Group I mGlu receptor family that activates phospholipase C through Gq/II and releases calcium from internal stores (reviewed by Conn and Pinn, 1997
). This receptor appears to be located post-synaptically in dopaminergic terminal areas (Romano et al., 1995
) and would have an effect similar to the NMDA glutamate receptor in that intracellular Ca2+ concentrations would rise after stimulation of either receptor. The NMDA and mGlu5 glutamate receptors have many important interactions with dopaminergic function and can change motor behaviour (Breysse et al., 2002
), fear and anxiety responses (Spooren et al., 2000
; Schulz et al., 2001
), nicotine self-administration (Paterson et al., 2003
), prevent neurotoxicity of amphetamines (McMillen et al., 1992
), and other forms of neurotoxicity (Bruno et al., 2000
) among a long list of effects on the central nervous system.
Antagonists at different acceptor sites on the NMDA receptor reduce the volitional consumption of ethanol by genetic drinking rats (McMillen et al., 2004). Both the NMDA and mGlu5 glutamate receptors increase intracellular Ca2+. This suggests that an antagonist of the mGlu5 Type 1 group receptor should reduce the consumption of ethanol. The following experiments tested the ability of a highly preferential mGlu5 receptor non-competitive antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP; Spooren et al., 2001
), for its ability to reduce the volitional consumption by the Myers high ethanol-preferring (mHEP) rat in a 24 h access paradigm. The data were compared to the ability of MPEP to alter the activity of the Sprague-Dawley rat in the elevated plus-maze, a measure of potential anti-anxiety activity (File, 1990
).
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MATERIALS AND METHODS |
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At 60 days of age, each male mHEP rat was placed in a suspended stainless steel cage with three drinking tubes mounted on the front. After a 1-day adaptation, one tube was filled with water, one with 3% ethanol (v/v), and one was left empty. Each day, the volumes of fluids and food consumed, and body weights were recorded and positions of the bottles rotated. The concentration of ethanol was increased daily: 5, 7, 9, 11, 13, 15, 20, 25, and 30%. The concentration of ethanol that resulted in maximal drinking with a proportion for consumption of ml ethanol to total ml fluids closest to 0.5 was selected as each rat's maximally preferred concentration. This concentration was then provided to each rat for the remainder of the experiment and drinking allowed to stabilize before initiating drug injections.
Experimental procedures
The elevated plus-maze consisted of four arms 50 cm long and 10 cm wide with two arms open and two arms that had 40 cm high walls. The maze was mounted 60 cm above the floor under fluorescent lighting. Each rat was handled daily for 5 min for three consecutive days. On the fourth day, doses of 0, 0.1, 1.0, or 3.0 mg/kg MPEP were injected subcutaneously (s.c.) into each rat 60 min before placement in the central region of the maze and faced towards one of the open arms. These doses of MPEP, as the free base, were chosen based on previous experiments (Spooren et al., 2000; Kuhn et al., 2002
) and the drug was dissolved in normal saline. Movement was recorded for 5 min with the computer program BEHAVIOR (Prof. L. W. Means, Department of Psychology, ECU). Latency to first open arm entry, frequency of open arm entry, duration on open arms, and total frequency of arm entries were recorded and analysed by ANOVA followed by Dunnett's t-test (Zar, 1984
).
For the experiment on MPEP and the consumption of ethanol, a 4-day baseline period preceded 3 days of s.c. injections 2 h before and 2 h after lights out and then a 4-day post-treatment period. Doses of MPEP or vehicle (saline) were administered in a counter-balanced design starting with either 0.1 or 1.0 mg/kg. Once drinking had stabilized at baseline levels, a different dose of drug was injected. Over the series of injections, four rats did not return to baseline and were discontinued from the experiment. The data were averaged for each period for each rat and then grouped and analysed by one-way repeated measures ANOVA and Tukey/Kramer procedure used post hoc (Zar, 1984). Statistical analyses were performed with GB-STAT, Dynamic Microystems, Silver Spring, MD.
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RESULTS |
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DISCUSSION |
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In contrast to the elevated plus-maze results, MPEP had a clear doseresponse effect on the consumption of ethanol. Although a handling effect due to the injection of saline vehicle occurred (Table 2), the lowest dose of MPEP did not have a significant effect. The percent decline in consumption was much greater after the 1.0 and 3.0 mg/kg doses of MPEP. That the proportion of total fluids consumed as solutions of ethanol declined, demonstrates an effect on the selection of ethanol rather than a general decline in fluid intake. This result indicates a pharmacological interaction that has caused the rats to accept less ethanol.
These effects, including that of vehicle, are similar to that reported for antagonists of the NMDA glutamate receptor (McMillen et al., 2004). Different laboratories have obtained different results with MPEP in different animal models. McGeehan and Olive (2003)
used mice for drug-induced conditioned place preference. They demonstrated an effect on cocaine-induced conditioning, but conditioning induced by either morphine or ethanol was unaffected. This is in contrast to the present results, which examined the selection and consumption of ethanol in a more naturalistic setting with 24 h access to solutions. Backstrom et al. (2004)
used conditioning cues to reinstate lever pressing for ethanol after a period of extinction and found that MPEP would reduce reinstatement of ethanol responding. They also reported that MPEP would blunt the deprivation-induced increased response for ethanol. In contrast, MPEP was reported to reduce lever pressing for intravenous self-administration of nicotine and cocaine, but did not alter the ability of these drugs to lower thresholds for intracranial self-stimulation. Clearly, more research is needed with both MPEP and other mGlu5 receptor antagonists in order to understand the behavioural impact of antagonists at this receptor.
The present results are in harmony with previous results on NMDA glutamate receptor antagonists. Drugs that prevent binding of agonists at each of the three different sites on the NMDA receptor to reduce its activation by glutamate all caused the selection and the consumption of ethanol solutions to decline (McMillen et al., 2004). Both the NMDA receptor and the mGlu5 receptor, through very different mechanisms, lead to increased intracellular concentrations of Ca2+. Both receptors may be located on neurons that are also post-synaptic to the limbic dopaminergic projection (Romano et al., 1995
; see reviews by Baker et al., 2003
; Witkin et al., 2003
). Whether or not these interactions are at the dopaminergic neurons or at the cells post-synaptic to the dopaminergic projections is not clear. It is known that MPEP, in doses from 1.0 to 9.0 mg/kg intraperitoneal, will reduce nicotine and cocaine self-administration by the rat (Paterson et al., 2003
; Kenny et al., 2003
), but will not prevent the enhancement of intracranial self-stimulation by these drugs (Kenny et al., 2003
). However, these differential results do not clarify whether or not MPEP is acting on the pre-synaptic dopamine neuron or the post-synaptic neurons of the motive circuitry.
The numerous glutamate receptors and acceptor sites that affect their function allow for many different pharmacological interactions. The development of drugs that target specific metabotropic receptors may allow for drugs with significant therapeutic benefit without the strong effects of most antagonists of the NMDA and AMPA receptors (Spooren et al., 2001). It is apparent that diminished glutamatergic activity at the receptors linked to increased intracellular calcium can decrease the consumption of ethanol. As MPEP interferes with the self-administration of other drugs of abuse, it is possible that either this drug or another mGlu5 receptor antagonist may be of benefit as an adjunct to the psychotherapy of alcohol and substance abuse.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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