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 from intracellular stores. We have mapped comparing their distribution with that of PKC and adenylate the distribution of 3H- 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- contains the highest level of inositol 1,4,5tris- togeny of IP, receptor binding in brain and performed lesion 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 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, 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), (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 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 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. Autoradiographic localization of 3H-IP, receptor binding sites in rat brain. Coronal sections of adult rat brain at 8 levels were processed for )H-IP, autoradiography (left half of each composite) or standard histology with toluidine blue (right half of each composite). A, Heavy labeling with ‘H-IP, is apparent in the anterior olfactory nucleus (a) but not in the olfactory bulb (0). B, Superficial layers of the neocortex are more intensely labeled than deep layers. The caudate-putamen (cp) is also labeled, while the corpus callosum (cc) and anterior commissure (UC) are devoid of staining. C, Caudate-putamen (cp) and olfactory tubercle (ot) are more heavily labeled than the globus pallidus (g) or the preoptic area (p). D and E, The CA1 region of the hippocampus (ca) shows marked selective labeling, with lower levels in molecular layer of the dentate (m). 3H-IP, labeling of the thalamus is low but detectable, while labeling of the hypothalamus (h) and midbrain (mb) are negligible. F, Although most of the brain stem, including the central gray (c) is devoid of labeling, significant binding is apparent in the substantia nigra (n). G and H, The cerebellum contains intense labeling restricted to the molecular layer and deep cerebellar nuclei, while the brain stem has none. 342 Worley et al. l IP, Receptor Autoradiography in Rat Brain

Figure 3. Effect of striatal lesion on )H-IP, binding. Quinolinic acid (200 nmol) wasinjected in the striatum unilaterally 4 d prior to sacrifice. In a horizontal section through the striatum (3) and substantia nigra (arrowheads), processedfor ‘H-IP, binding, ‘H-IP, binding sites are decreasedin the injected striatum (asterisk)and the ipsilateral nigra.

IP3 receptor distribution in mutant mice and in lesioned rats IP, binding is present in both the caudate and the zona reticulata of the substantia nigra (Fig. 3). To ascertain if IP, receptors, like forskolin binding sites and PKC, are contained in the descending striatonigral pathway, we evaluated the effect of unilateral quin- olinic acid lesions of the caudate on 3H-IP3 binding. Animals were sacrificed 4 d after injection to minimize possible trans- Figure 4. Effect of hippocampal lesion on )H-IP, binding sites. A, synaptic effects on binding. These caudate lesions induce a loss Toluidine blue stain of coronal section of rat brain lesioned in the left of IP, receptor-associated silver grains in the ipsilateral nigra, hippocampuswith 30 nmol of quinolinic acid. Pyramidal and granule indicating that IP, receptors are contained in the descending cellswhich stain darkly in unlesionedside are clearly damagedin treated hippocampus(arrows). B, 3H-IP, autoradiogramof an adjacentsection striatonigral pathway. demonstratingstriking loss of ‘H-IP, binding in lesioned hippocampus Previously, we showed that quinolinic acid lesions of the hip- (asterisk).C, Enhancedacetylcholinesterase staining of an adjacentsec- pocampus, which destroy intrinsic neuronal elements, deplete tion on the lesioned side indicates that the lesion sparedafferent cho- a major portion of 3H-PDBu binding (Worley et al., 1986a). linergic terminals. Similar lesions, evaluated 4 d postinjection, elicit a total deple- tion of IP, binding in the hippocampus (Fig. 4). The selectivity of this quinolinic acid lesion is ensured by the preservation of not in the normal location. The altered localization of IP, re- acetylcholinesterase staining in these sections, as acetylcholin- ceptors fits with the displaced pattern of Purkinje cells in reeler esterase is contained in the terminals of the septohippocampal mice. afferent projection (Lewis et al., 1967). Accordingly, in the hip- pocampus, IP, receptors appear to be localized to intrinsic neu- Ontogeny of IP3 receptors, protein kinase C, and ‘H-forskolin rons. binding To establish the localization ofIP, receptors in the cerebellum, We compared the autoradiographic localization of IP, receptors, we utilized strains of mutant mice. The lurcher mouse (lc/+) 3H-PDBu and 3H-forskolin binding in newborn and 5- and 16- displays a total loss of Purkinje cells with no other major neu- d-old rats (Fig. 6, Table 1). The principal localization of IP, ronal cell loss at the age studied (Rosenberg, 1986). IP, receptors receptors is in the cerebellum. The molecular layer of the new- are totally absent in cerebellar sections from Zurcher mice, while born cerebellum possessesapproximately 5% of the ‘H-IP, bind- a normal pattern is apparent in unaffected littermates (+I+) ing present in the adult molecular layer, while 16-d-old animals (Fig. 5). By contrast, in further mice, 3H-forskolin binding is display about 90% of adult levels. The width of the molecular retained in the cerebellum, although at reduced levels. The lower layer increases throughout this period, further accentuating de- levels of 3H-forskolin binding presumably are related to the velopmental differences in total cerebellar ‘H-IP, binding. The much smaller size of the molecular layer in lurcher mice com- increase in grain density with age parallels the development of pared with littermate control animals. The reeler mouse (rl/rl) dendrites in the cerebellum (Robain et al., 198 1). cerebellum is characterized by a greatly reduced granule cell In a previous study we demonstrated 3H-PDBu binding in population and by abnormal migration of the various elements fetal rat brain with substantial densities in the area of the cer- during ontogeny (Mullen and Herrup, 1979). In reeler mice, 3H- ebellum in 1g-d-old fetuses but negligible levels in higher centers IP,-associated silver grains in cerebellum are as dense as those (Murphy et al., 1983). In the present study we observe very little of the phenotypically normal littermate (+/+ or rl/+) but are 3H-PDBu binding in the newborn rat cerebellum, with grain The Journal of Neuroscience, January 1989, 9(l) 343

Figure 5. 3H-IP, receptor binding in cerebellar sections of mutant mice. Coronal sections at the level of the cerebellum were taken from lurcher or reeler mice and processed for ‘H-IP, (4 and C) and 3H-forskolin (B) autoradiography. Littermate control (top) and mutant mouse cerebella were mounted in the same tissue block to ensure identical section thickness and autoradiographic conditions. A, Sections from an unaffected littermate (top) yield strong labeling with ‘H-IP, in the molecular layer and deep cerebellar nuclei as observed in rat. However, in the lurcher cerebellum (bottom, asterisk), no binding is observed. B, Adjacent sections to those used in a were processed with 3H-forskolin. Control littermate section shows typical pattern (top). In section from affected lurcher (bottom), labeling is present in the cerebellar molecular layer (arrows), which is thin due to degeneration of Purkinje cells. C, Section through cerebellum of affected reeler mouse (bottom) and control littermate (top) demonstrating preserved 3H-IP, binding despite loss of granule cells in this mutant. densities appearing in 5 and 16-d-old animals in association reaching adult levels by day 16. In the newborn rat striatum, with Purkinje cell ontogeny. Accordingly, the development of ‘H-forskolin binding is approximately 15% that of adult stria- PKC in the cerebellum parallels that of IP, receptors. The on- turn and displays a patchy appearance. Similar patchiness is togenetic development of 3H-PDBu binding in the hippocam- seen in newborn striatal 3H-PDBu binding. The patchiness ap- pus, caudate, and substantia nigra is fairly similar to that of the parent for 3H-forskolin and 3H-PDBu is also present at day 5 cerebellum. These autoradiographic findings fit well with bio- but is diminished at day 16 and absent in the adult striatum. chemical measures of PKC activity during development (Turner DARPP-32, a protein substrate for the dopamine-sensitive ad- et al., 1984). enylate cyclase shows a similar pattern of distribution in the Within the cerebellum, granule cells develop at a later stage striatum, with patchiness at birth that diminishes in the course than Purkinje cells (Altman, 1982). This accords with our ob- of ontogeny (Ouimet, 1984; Foster et al., 1987). servation that 3H-forskolin binding in the cerebellum develops somewhat later than 3H-PDBu or IP, receptor binding (Table Discussion l), as 3H-forskolin binding is localized to granule cell processes In the present study we have characterized the discrete local- (Worley et al., 1986b). Striatal 3H-forskolin binding also con- izations of IP, receptors throughout the brain. In most areas of tinues to increase between day 16 and adulthood. In contrast the brain, IP, receptor localization resembles that of PKC, though to the relatively late development of 3H-forskolin binding in the there are a number of differences. For instance, IP, receptor cerebellum, 3H-forskolin binding to the neocortex and CA 1 re- density is vastly higher in the cerebellum than any other brain gion of the hippocampus parallels that of 3H-PDBu and 3H-IP3, region, whereas PKC is more uniformly distributed. Certain

Table 1. Postnatal development of 3H-IP,, ‘H-PDBu, and 3H-forskolin binding in brain

Density of ligand binding (% of adult levels) at indicated age (d) Cerebellar molecular layer Neocortex Hippocampus CA 1 Striatum Region 0 5 16 0 5 16 0 5 16 0 5 16 Ligand )H-IP, 5 36 89 cl5 45 115

od 5d 16d

Figure 6. Ontogeny of second-messenger systems. Parasagittal sections through rat brains were taken at days 0, 5, and 16 and processed for Nissl stain with toluidine blue (A) or autoradiography with ‘H-IP, (B), ‘H-PDBu (C’), and 3H-forskolin (0). A, Toluidine blue micrographs demonstrate the morphologic development of the caudate-putamen (CD),substantia nigra (n), hippocampal regions CA1 (ca), dentate hilus (h), neocortex, and cerebellum (cb), with the deep cerebellar nucleus (d) and molecular layer (ml). B, )H-IP, binding is first detected at day 0 in a fine ribbon of cerebellar molecular layer (arrows) and becomes more prominent at day 5 in both the deep cerebellar nucleus (d) and the molecular layer (arrows). By day 16, binding is enhanced in the cerebellar molecular layer and deep cerebellar nuclei and extends into the folia. Less intensely labeled forebrain structures that demonstrate binding by day 16 include the CA 1 region of the hippocampus (ca) and the caudate-putamen (cp). C, 3H-PDBu labels the caudate-putamen at day 0 with a patchy pattern, and by day 5 the substantia nigra (n) is well demarcated. Binding to the cerebellar molecular layer and deep cerebellar nucleus is similar to the pattern seen with 3H-IP, binding. By day 16, ‘H-PDBu binding is most enriched in the hippocampal CA1 region (ca). Labeling of the neocortex, which at days 0 and 5 is less than the caudate-putamen, becomes comparable to the caudate-putamen at day 16. D, ‘H-forskolin binding at day 0 is enriched in the caudate-putamen (cp), substantia nigra (n), and brain stem. Labeling of the caudate- putamen is patchy at day 0 and day 5 but homogeneous by day 16. The cerebellar molecular layer, which is labeled at day 5 by 3H-IP, and )H- PDBu, is distinctly low in 3H-forskolin binding at day 5 (arrows). By contrast, at day 16, the cerebellar molecular layer is labeled. Similarly, the dentate hilus (h), is intensely labeled in the 16-d-old animal (and adult) but not in the 5-d-old animal. The Journal of Neuroscience, January 1989. 9(l) 345

areas of the CNS possessabundant PKC with no detectable IP, sensitive to calcium ions, which inhibit binding 50% at 300 nM receptor binding, as exemplified by the external plexiform layer (Worley et al., 1987b). Conceivably, this reflects a feedback of the olfactory bulb and the substantia gelatinosa of the spinal mechanism whereby calcium released by IP, prevents further cord (Worley et al., 1987a). This discrepancy may be related to interactions of IP, with its receptor. Heparin, which binds to diacylglycerol production from sources other than PIP, in these the IP, receptor (Supattapone et al., 1988a) and potently dis- areas. It would be of interest to compare the localization of IP, places 3H-IP, binding (Worley et al., 1987b), also potently blocks receptors to the specific C that generates IP,. A IP,-induced calcium release (Hill et al., 1987; Ghosh et al., recent immunohistochemical study localized 2 distinct phos- 1988). These observations suggest that the IP, receptor binding phoinositide-specific enzymes in rat brain sites that we have characterized, and which are enriched in the (Gerfen et al., 1988). One of these closely mimics the localiza- cerebellum, are involved in calcium release similar to peripheral tions we have observed of phorbol ester binding associated with IP, receptors. The regional distribution of3H- protein kinase C (Worley et al., 1986a) and of the G, protein, generation from 3H-inositol added to brain slices differs from which may be linked to the phosphinositide cycle (Worley et that ofthe IP, receptor, with the cerebellum displaying low levels al., 1986~). (Rooney and Nahorski, 1986). However, such a comparison is Based on studies of mutant mice, cerebellar IP, receptors meaningful only if phosphoinositide turnover in each region is appear to be selectively localized to Purkinje cells. Multiple stimulated by ligands that physiologically regulate such turnover types of PKC have been identified (Huang et al., 1986; Kikkawa in the various brain areas. et al., 1987; Ohno et al., 1987). Type I appears to be brain It is noteworthy that the distribution of a cytoplasmic, 28 kDa specific (Shearman et al., 1987) and selectively concentrated in calcium binding protein () resembles that of 3H-IP3 Purkinje cells of the cerebellum (Brandt et al., 1987; Kitano et binding sites (Bairnbridge and Miller, 1982; Bairnbridge et al., al., 1987), closely resembling the distribution of PKC monitored 1982; Miller and Baimbridge, 1983). For instance, in the cer- by autoradiography with 3H-PDBu (Worley et al., 1986a) and ebellum this calcium binding protein is also selectively localized of 3H-IP3 receptor binding. Other studies have demonstrated to Purkinje cells and in the hippocampus is concentrated in CA, the presence of type II PKC in granule cells of the cerebellum and the molecular layer of the dentate gyrus. Also, levels of (Brandt et al., 1987), which appear to be devoid of 3H-IP, bind- calbindin in cerebellum are approximately 1O-fold higher than ing. Thus, the localization of IP, receptor binding in cerebellum in hippocampus, similar to the IP, receptor. The similar local- parallels that of type I PKC. izations of calbindin and the IP, receptor suggest mutual roles IP, receptors are not restricted to one part of neurons. For in modulating neuronal . instance, in the hippocampus, IP, receptors are localized to all layers of CA 1, suggesting that it is present in cell bodies and References dendrites of intrinsic neurons. In the cerebellum, IP, receptors are present throughout the molecular layer, which contains Pur- Altman, J. (1982) Morphological development of the rat cerebellum and some of its mechanisms. In The Cerebellum-New Vistas.S. L. kinje cell dendrites. In the striatonigral pathway, IP, receptors Palay and V. Chan-Palay, eds., pp. 849, Springer-Verlag, Berlin. appear to be contained in axons and terminals. In the cerebellum Amar, S., P. Kitabgi, and J.-P. Vincent (1987) Stimulation of inositol it is probable, though not definitely established, that IP, recep- phosphate production by neurotensin in neuroblastoma NIE115 cells: tors are present throughout Purkinje cell axons and extend into Implication of GTP-binding proteins and relationship with the cyclic terminals in the deep cerebellar nuclei. These conclusions, based GMP response. J. Neurochem. 49: 999-1006. Baimbridge, K. G., and J. J. Miller (1982) Immunohistochemical on lesion studies, cannot totally exclude transsynaptic effects. localization of calcium-binding protein in the cerebellum, hippocam- However, we examined animals 4 d after lesions, a period that pal formation and olfactory bulb of the rat. Brain Res. 245: 223-229. is probably not long enough to permit major transsynaptic al- Bairnbridge. K. G.. J. J. Miller. and C. 0. Parkes (1982) Calcium- terations. bindingprotein distribution in the rat brain. Brain Res: 239: 519- 525. Recently, we solubilized IP, receptors from the cerebellum Baukal, A. J., G. Guillemette, R. Rubin, A. Spat, and K. J. Catt (1985) and purified the receptor protein to homogeneity (Supattapone Binding sites for in the bovine adrenal cortex. et al., 1988a). It is a glycoprotein with a subunit size of 270 kDa Biochem. Biophys. Res. Commun. 133: 532-538. that is phosphorylated by cyclic AMP-dependent protein kinase Berridge, M. J., and R. F. Irvine (1984) Inositol trisphosphate, a novel second messenger in cellular . Nature 312: 315- but not by PKC or calcium- kinase (Supattapone et -... 5LI. al., 1988b). The IP, receptor appears to be identical to a pre- Brandt, S. J., J. E. Niedel, R. M. Bell, and W. S. Young, III (1987) viously isolated phosphoprotein of the cerebellum referred to Distinct patterns of expression of different protein kinase C mRNAs as PCPP-260 (Walaas et al., 1987). Interestingly, PCPP-260 was in rat tissues.Cell 49: 57-63. shown to be localized to Purkinje cells in the cerebellum by DeCamilli, P., P. E. Miller, P. Levitt, U. Walter, and P. Greengard (1984) Anatomy of cerebellar Purkinie cells in the rat determined experiments utilizing mutant mice similar to those in the present by a specific immunohistochemical marker. Neuroscience I I: 76 l- study. The identity of the IP, receptor with PCPP-260 also fits 817. well with evidence that PCPP-260 is phosphorylated by cyclic Foster, G. A., M. Schultzberg, T. Hiikfelt, M. Goldstein, H. C. Hem- GMP-dependent protein kinase (Weeks et al., 1988), as cyclic mings, Jr., C. C. Ouimet, S. I. Walaas, and P. Greengard (1987) GMP-dependent protein kinase is present in extremely high Development of a dopamine and cyclic adenosine 3’:5’-monophos- phate-regulated phosphoprotein (DARPP-32) in the prenatal rat cen- levels in the Purkinje cells of the cerebellum (DeCamilli et al., tral nervous sytem, and its relationship to the arrival of presumptive 1984). A physiological link of the IP, receptor and cyclic GMP dopaminergic-innervation. J. Neurosci. 7: 1994-2018. - - is suggested by the ability of IP, to elevate cyclic GMP levels Gerfen. C. R.. W. C. Choi. P. G. Suh. and S. G. Rhee (1988) Phos- in permeabilized neuroblastoma cells (Amar et al., 1987). pholipase d I and II brain :‘Immunohistochemical localiza- tion in neuronal systems in rat brain. Proc. Natl. Acad. Sci. USA 85: A presumed major function of IP, is to release intracellular 3208-3212. calcium after binding to its receptors (Benidge and Irvine, 1984; Ghosh, T. K., P. S. Eis, J. M. Mullaney, C. L. Ebert, and D. L. Gill Williamson et al., 1985). IP, receptor binding in brain is highly (1988) Competitive, reversible, and potent antagonism of inositol 346 Worley et al. - IP, Receptor Autoradiography in Rat Brain

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