Morphine withdrawal in A2A adenosine receptor knockout mice A.Bailey, H. M. B. Lesscher, M. D. W. Kelly, L. Davis, C. Leden1, S. M. O. Hourani & I. Kitchen. School of Biomedical and Life Sciences, University of Surrey, Guildford, GU2 7XH. 1 Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire, Universite Libre de Bruxelles, Belgium.

Print abstract Search PubMed for: Bailey A Lesscher HMB Kelly MDW Davis C Ledent C Hourani SMO Kitchen I

Much evidence supports the hypothesis that A_2A receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might be involved (Zarrindast et al., 1999). We have evaluated morphine withdrawal signs in wild-type and A_2A receptor knockout mice generated by Ledent's group (Ledent et al., 1997). To investigate further if µ opioid receptor expression and G-protein activation are altered in the absence of the A_2A receptor gene under conditions of morphine withdrawal, quantitative autoradiography of µ opioid receptors and µ opioid receptor-stimulated guanylyl 5'-[-[³⁵S]thio]-triphosphate ([³⁵S]GTPS) autoradiography were carried out in brain sections of morphine-withdrawn wild-type and A_2A receptor knockout mice. Moreover, to investigate whether D₂ dopamine receptor expression is altered in withdrawn A_2A receptor knockout mice, quantitative autoradiography of D₂ dopamine receptors was carried out in brain sections of morphine-withdrawn wild-type and mutant mice.

Wild type and A_2A receptor knockout mice (8-10 week old) were treated for 7 days with morphine sulphate (24 mg/kg/day), administered subcutaneously via osmotic minipumps. Seven days after the pumps were implanted, naloxone HCl (10 mg/kg, i.p) was injected in order to precipitate opioid withdrawal. Both objective (weight loss, urine and faecal matter, number of jumps, number of wet dog shakes, number of face washes, number of paw tremors, number of writhes) and subjective measures (diarrhoea score, ptosis score) of withdrawal behaviour were scored for a period of 30 minutes according to a measurement protocol described by Kitchen et al. (1993).

Following behavioural assessment, mice were killed and adjacent coronal brain sections were cut from morphine-withdrawn wild-type and A_2A receptor knockout mice for the determination of total and non-specific binding of [³H]D-Ala²-MePhe⁴-Gly-ol⁵ enkephalin (DAMGO) (4 nM) and [3H]raclopride (4 nM) to µ opioid and D₂ dopamine receptors respectively. DAMGO (3 µM) stimulated [³⁵S]GTPS (0.04 nM) autoradiography was also carried out in brain sections of withdrawn wild-type and mutant mice. The autoradiographic procedures were performed as detailed previously (Kitchen et al., 1997; Kirschke et al., 2002).

A significant enhancement in the number of jumps (+/+: 3.3±1.9; -/- 45.3 ±19.0, P<0.01, n=9-12), paw tremors (+/+: 95.3 ±15.7; -/-: 327.7 ±101.6, P<0.01, n=9-12) and writhes (+/+: 19.9 ±7.5; -/-: 46.7 ±13.1, P<0.05, n=9-12) was observed in morphine-withdrawn A2A receptor knockout mice compared to wild-type. No significant changes in D₂ and µ receptor binding (P>0.05) were observed in any of the brain regions analysed. However, a significant increase in the % level of µ receptor-stimulated [³⁵S]GTPS binding (+/+: 27.7 ± 8.0%; -/-: 71.5 ± 11.9%, P<0.05, n=4) was observed in the nucleus accumbens of withdrawn knockout mice.

The data indicate that adenosine, via its action on A_2A receptors, can control the expression of opioid withdrawal and that inhibition of µ receptor G-protein activation is involved in the adenosinergic control of morphine withdrawal.

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