The distribution and regulation of mu opioid receptor mRNA in rat basal ganglia

79 THE DISTRIBUTION AND REGULATION OF MU OPIOID RECEPTOR mRNA IN RAT BASAL GANGLIA Delfs JM, Yu L, Reisine T, Chesselet M-F, Dept. of Pharmacology, Univ. of Pennsylvania, Philadelphia, PA 19104 and Dept. of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202 Receptor autoradiographic studies have shown that mu (p.) opioid receptors, are present in discrete locations throughout the brain (1). These receptors are abundant in the basal ganglia, most notably the striosomes, a distinct anatomical compartment of the striatum (2). In the present study, the distribution of ~t opioid receptor mRNA was examined in the basal ganglia with in situ hybridization histochemistry at the single cell level (3). Furthermore, the regulation of the ~t opioid receptor in the globus pallidus was examined after short and long-term haloperidol treatments, which alter striatal enkephalin levels (4). For the distribution study and short-term haloperidol treatments, adult male SpragueDawley rats (250-300g) were used. For the short-term haloperidol experiments, rats were injected daily with haloperidol (1 m g / k g , s.c.) or vehicle (Tween 80/d2H20) and were sacrificed by decapitation 24 hours after the final drug injection. For chronic studies, 12 female Sprague-Dawley rats were administered hatoperidol decanoate or vehicle (sesame oil) via intramuscular injection (21 mg/kg; average dose 1 m g / k g / d a y ) once every 3 weeks for 7 months and in drinking water (average intake 1 m g / k g / d a y ) for the final 3 weeks. Animals were killed by decapitation 1 week after the final drug administration. In situ hybridization histochemistry was performed as previously described on cryostatcut 10 ~tm thick fresh-frozen sections (5, 6). Radiolabelled antisense and sense cRNA probes were transcribed from cDNAs using RNA polymerases, 2.5 ~tM 35S-UTP (DuPont), 10 ~tM cold UTP and ATP, GTP and CTP in excess. The ~t opioid receptor cDNA consisted of a 1.4 kilobase sequence from the 3'-untranslated region of the clone, which was isolated from a rat brain cDNA library (7). The cDNA for preproenkephalin, a 970 base pair sequence isolated from a rat striatal library, was kindly donated by Dr. S. L. Sabol (NIMH). For double labelling experiments, a digoxigenin-labelled cRNA probe for tyrosine hydroxylase was synthesized with digoxigenin-labelled UTP (Boehringer-Mannheim) and sections were hybridized with both the radiolabelled probe for the ~t opioid receptor mRNA and the digoxigenin-labelled probe followed by an overnight incubation with the anti-digoxigenin antibody at 4°C. An alkaline phosphatase-catalyzed chromagen reaction was used to detect the digoxigenin-labelled hybrids (6). Slides were exposed to 3HUltrofilm (2-4 weeks) or dipped in Ilford photographic emulsion (12-24 days). Autoradiographic films were analyzed using the Macintosh based IMAGE analysis program (NIH). On emulsion coated sections, the level of labelling over individual neurons in the globus pallidus was measured with the MORPHON image analysis system (8). Only slides processed concurrently in the same experiment were compared with ANOVA. The Dunnett's post-hoc test was used to compare treated with control rats with p<0.05 considered statistically significant. In the striatum, the majority of labelled neurons were found in clusters which corresponded to ~t opioid receptor binding patches in serially adjacent sections processed for 3H-naloxone binding. In target areas of striatal efferent neurons, dense labelling was found in the globus pallidus (external pallidum), which receives dense enkephalinergic projections from the striatum. In contrast, no labelled neurons were found in the entopeduncular nucleus (internal pallidum) and few in the substantia nigra pars reticulata, areas which do not receive a significant enkephalinergic projection in the rat.

80 Double-labelling experiments performed with a radiolabelled probe for receptor mRNA and a digoxigenin-labelled probe for tyrosine hydroxylase dopaminergic neurons of the substantia nigra pars compacta also express receptor mRNA. The subthalamic nucleus, a region functionally related ganglia, contained a subpopulation of labelled neurons. These data indicate Ix opioid receptor m R N A in discrete neuronal populations in the basal suggest that enkephalin may be an endogenous ligand at Ix opioid receptors pallidus.

the Ix opioid revealed that the Ix opioid to the basal expression of ganglia and in the globus

In order to determine if alterations in striatopallidal enkephalin levels can regulate IX opioid receptor mRNA in the globus pallidus, levels of mRNA for enkephalin and the Ix receptor were measured with in situ hybridization in rats treated with haloperidol for 3 or 7 days (1 m g / k g , s.c.) or 8 months (1 mg/kg, depot). As seen in figure 1, enkephalin and Ix opioid receptor mRNA levels were unchanged after 3 days of haloperidol treatment. In contrast, enkephalin mRNA was increased in the striatum and Ix opioid receptor mRNA levels were decreased in the GP after both 7 days and 8 months of haloperidol treatment (Fig. 1). The results provide the first evidence of regulation of ~t opioid receptor mRNA in vivo and suggest that Ix receptor expression may be under tonic enkephalin regulation in the globus pallidus. Supported by U.S. Public Health Service grants MH44894, MH17168, DA08951 and NS29230 200 Enk-Strlatum 150

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Fig. 1. Time course of changes in enkephalin mRNA in the striaturn and IXopioid receptor (IxOR) mRNA in the globus pallidus (GP) after short and long-term haloperidol treatments. Data are means of the level of labelling in 6-8 rats expressed as percent of control. * p<0.05, ANOVAwith Dunnett's post hoc test. 1. A. Mansour, H. Khachaturian, M.E. Lewis, H. Akil and S. Watson (1988) Trends Neurosci. 11, 308-314 2. M. Herkenham and C.B. Pert (1982) J. Neurosci. 2, 1129-1149 3. J.M. Delfs, H. Kong, A. Mestek, Y. Chen, L. Yu, T. Reisine and M.-F. Chesselet (1994) J. Comp. Neurol. 345, 46-68 4. J.M. Delfs, L. Yu, G.D. Ellison, T. Reisine and M.-F. Chesselet (1994) J. Neurochem. 63, 777-780 5. M.-F. Chesselet, L. Weiss, C. Wuenschell, A.J. Tobin and H.U. Affolter (1987) J. Comp. Neurol. 262, 125-140 6. M.-F. Chesselet (1990) In Situ Hybridization Histochemistry. Boca Raton: CRC Press 7. Y. Chen, A. Mestek, J. Liu, J.A. Hurley and L. Yu (1993) Mol. Pharmacol. 44, 8-12 8. M.-F. Chesselet and L. Weiss-Wunder (1994) In Situ Hybridization in Neurobiology: Advances in Methodology. New York: Oxford Press, pp. 114-123