Lnositol 1,4,5=Trisphosphate Receptor Binding: Autoradiographic Localization in Rat Brain

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Lnositol 1,4,5=Trisphosphate Receptor Binding: Autoradiographic Localization in Rat Brain The Journal of Neuroscience, January 1989, 9(l): 339-348 lnositol 1,4,5=Trisphosphate Receptor Binding: Autoradiographic Localization in Rat Brain Paul F. Worley,1s2 Jay M. Baraban,lv3 and Solomon H. Snyderi.3,4 Departments of ‘Neuroscience, *Neurology, 3Psychiatry and Behavioral Sciences, and 4Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 lnositol 1,4,5trisphosphate is a second messenger gener- al., 1987a). In the present study, we have employed autora- ated by stimulation of the phosphoinositide cycle, thought diography to localize IP, receptors in brain in greater detail, to release calcium from intracellular stores. We have mapped comparing their distribution with that of PKC and adenylate the distribution of 3H-inositol 1,4,5trisphosphate receptor cyclase, labeled by 3H-phorbol 12,13-dibutyrate (3H-PDBu) and binding sites in rat brain by autoradiographic techniques. The 3H-forskolin, respectively. We have also characterized the on- cerebellum contains the highest level of inositol 1,4,5tris- togeny of IP, receptor binding in brain and performed lesion phosphate binding sites in brain, which appear to be selec- studies to identify neuronal elements enriched in IP, receptors. tively localized to Purkinje cells. Moderate levels of binding sites are present in the hippocampus, cerebral cortex, cau- Materials and Methods date, and substantia nigra. Lesion studies indicate that bind- *H-IP, autoradiography.3H-IP, autoradiography was carried out as de- ing in the hippocampus is restricted to intrinsic neuronal scribed (Worley et al., 1987a). Male Sprague-Dawley rats were anes- thetized with pentobarbital and perfused via the left cardiac ventricle elements and in the nigra is found on terminals of the stria- with buffer containing 50 mM sodium phosphate, pH 7.5, and 100 mM tonigral projection. Overall, the autoradiographic distribution NaCl, followed by 50 mM sodium phosphate, pH 7.5, in 0.3 M sucrose. of inositol 1,4,5-trisphosphate receptors resembles the dis- Brains were removed and frozen in brain paste on microtome chucks. tribution of phorbol ester binding sites associated with pro- Cryostat sections (10 PM) were thaw-mounted on subbed slides and stored at -20°C until use. IP, binding sites were labeled by incubating tein kinase C. However, the inositol 1,4,5-trisphosphate re- brain sections for 10 min at 4°C in Buffer A (20 mM NaCl, 20 mM Tris- ceptor has a more restricted distribution since it is not HCl, pH 7.7, 100 mM KCl, 1 mM EDTA, 1 mg/ml BSA) containing 50 detectable in the spinal cord or olfactory bulb, regions with nM ‘H-IP, (NEN/DuPont, Boston; 3.6 Ci/mmol), then washed twice for substantial levels of protein kinase C. 2 min in the same buffer. Preliminary experiments verified that specific IP, binding reaches a plateau under these incubation conditions. Non- specific labeling was determined in the presence of 5 PM unlabeled IP, The phosphoinositide (PI) cycle appears to be a major second- and represents less than 5% of total ‘H-IP, binding in all areas. Auto- messenger system for a wide range of neurotransmitters, hor- radiograms were prepared by apposing labeled sections to Ultrofilm for mones, and growth factors. In the PI cycle, phosphatidylinositol 4-8 weeks. Autoradiograms were analyzed using a computerized mi- 4,5bisphosphate is hydrolyzed in response to receptor stimu- crodensitometer (Loats Associates, Westminster, MD), and optical den- sities were converted to equivalent disintegrations/min (DPM)/mg of lation to generate inositol 1,4$trisphosphate (IP,) and diacyl- methacrylate (autoradiographic 3H-microscales, Amersham, Chicago), glycerol (Berridge and Irvine, 1984). Diacylglycerol stimulates as described by Unnerstall et al. (1982). 3H-PDBu and ‘H-forskolin protein kinase C (PKC), while IP, acts at membrane sites within autoradiography were performed as described (Worley et al., 1986a, b). the cell to release calcium from intracellular stores (Williamson Lesion studies.Quinolinic acid (Sigma, St. Louis) was used to lesion et al., 1985; Nishizuka, 1986). Putative IP, receptors have been the hippocampal formation and caudate nucleus (Schwartz et al., 1983) as described (Worley et al., 1986a). The lesions were characterized for identified by the binding of radiolabeled IP, to membrane prep- extent and selectivity by histologic examination of the brain sections arations of peripheral tissues (Baukal et al., 1985; Spat et al., following toluidine blue or acetylcholinesterase stains. 1986a, b; Guillemette et al., 1987) and the brain (Willcocks et Mice were obtained from Jackson Laboratories (Bar Harbor, ME). al., 1987; Worley et al., 1987a, b; Supattapone et al., 1988a). The mutants used were lurcher and reeler (Mullen and Herrup, 1979; Rosenberg, 1986). Cerebellar sections were processed for autoradiog- In initial studies, we showed biochemically and autoradiograph- raphy as described above. ically that IP, receptor binding sites are distributed heteroge- neously in the brain, closely resembling the localization of PKC Results in some areas but with differences in other regions (Worley et Regional distribution of IP3 binding sites The techniques employed in this study for autoradiographic Received Mar. 28, 1988; revised May 23, 1988; accepted May 26, 1988. analysis of 3H-IP, binding are the same as those used in our This work was supported by USPHS Grants MH-18501 and DA-00266, Phy- sician Scientist Award AG-00256 (to P.F.W.), Research Scientist Award DA- earlier report (Worley et al., 1987a). To ensure that )H-IP, binds 00074 (to S.H.S.). J.M.B. is a Lucille P. Markey and A.P. Sloan Fellow, and this selectively to IP, receptors, we showed that the relative potencies work was supported in part by a grant from the Lucille P. Markey Charitable of various inositol phosphates in competing for binding sites in Trust and the Blades Center for Research in Alcoholism. We wish to thank V.S. Wilson for excellent technical assistance, Nancy Bruce and Dawn C. Dodsonfor brain sections is the same as in homogenate binding studies. excellent secretarial assistance, and M.L. Oster-Granite for helpful discussions. Moreover, we conducted studies to rule out the possibility that Correspondence should be addressed to Solomon H. Snyder, M.D., Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 N. Wolfe 3H-IP, was binding to inositol phosphatases or IP, kinase (Wor- Street, Baltimore, MD 21205. ley et al., 1987b). Copyright 0 1989 Society for Neuroscience 0270-6474/89/010339-08$02.00/O In confirmation of our earlier findings, IP,-associated grains 340 Worley et al. l IP, Receptor Autoradiography in Rat Brain A B Figure 1. Autoradiographic localiza- tions of (A) 3H-IP,, (B) )H-PDBu, and (C) 3H-forskolin in adult rat brain. Ad- jacent sagittal brain sections from adult rat were processed for autoradiography to illustrate differential localization of these ligands as discussed in text. Al- though the density of staining in the C cerebellar molecular layer appears sim- ilar for all 3 ligands, the number of binding sites for 3H-forskolin is ap- proximately IO-fold lower than for either 3H-IP, or )H-PDBu, which are comparable (Worley et al., 1986b, 1987a). Structures are labeled as fol- lows: d, deep cerebellar nuclei; n, sub- stantia nigra; s, striatum; c, CA1 region of the hippocampus; m, mossy fiber layer; *, molecular layer of cerebellum. are most dense in the cerebellar molecular layer (Figs. 1 and 2). 3H-forskolin binding differs strikingly from that of IP, in the This localization is in striking contrast to the underlying brain hippocampus, with highest densities in the molecular layer of stem, which is devoid of autoradiographic grains. Moderate the dentate gyrus and the mossy fiber projection to CA3. levels of grain density are apparent in the deep nuclei of the Levels of IP, receptors in the caudate are somewhat lower cerebellum and certain white matter regions extending into the than those in the hippocampus, with still lower, barely detect- cerebellar folia. able levels in the adjacent globus pallidus. These relative dis- PKC, labeled with a tritiated phorbol ester, 3H-PDBu, is also tributions are similar to those of 3H-PDBu. However, levels of enriched in the molecular layer of the cerebellum and the deep 3H-PDBu binding in the caudate are similar to those in the nuclei. By contrast, 3H-forskolin, which labels the G,-adenylate cerebellum, while IP, receptor binding is much greater in the cyclase complex (Seamon et al., 1984), shows pronounced den- cerebellum than the caudate. Relative to the cerebellum, 3H- sity in the molecular layer of the cerebellum without labeling forskolin densities in the caudate are the highest of the 3 ligands the deep nuclei (Worley et al., 1986b). examined. For 3H-forskolin and 3H-PDBu, grain densities are The second highest density of IP, receptors occurs in the readily visualized in the descending striatonigral pathway and hippocampus, resembling levels in the cerebellar deep nuclei. the substantia nigra. Within the hippocampus, binding is most concentrated in the In the cerebral cortex, IP, binding is most concentrated in the CA 1 region; much less binding occurs in CA3 and CA4, though superficial layers. In the more anterior cortex, discrete densities a moderate level of binding is apparent in the dentate gyrus. In of IP, binding sites are apparent in the anterior olfactory nucleus. contrast to the closely similar localization of IP, receptors and IP, receptors are not detected in the adjacent olfactory bulb, PKC in the cerebellum, they differ in their localizations in the contrasting with the high density of PKC in the external plex- hippocampus, as PKC is present in high levels in all regions of iform layer of the olfactory bulb reported previously (Worley the hippocampus. As observed previously (Worley et al., 1986b), et al., 1986a). The Journal of Neuroscience, January 1989, 9(l) 341 Figure 2.
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