The Journal of Neuroscience, August 1994, 14(B): 4794-4905 Multiple Types of Ryanodine Receptor/Ca*+ Release Channels Are Differentially Expressed in Rabbit Brain Teiichi Furuichi,’ Daisuke Furutama, I,2 Yasuhiro Hakamata,3 Junichi Nakai,3 Hiroshi Takeshima,4 and Katsuhiko Mikoshiba115 ‘Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-l Shirokanedai, Minato-ku, Tokyo 108, 2First Department of Internal Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569, 3Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-01, 41nternational Institute for Advanced Studies, Shimadzu Corporation N-80-3F, 1 Nishinokyo-Kuwabara-cho, Nakagyo-ku, Kyoto 604, and 5Department of Molecular Neurobiology, Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, 3-l-l Koyadai, Tsukuba 30.5, Japan The neuronal Ca2+ signal is induced by a rise in the intra- [Key words: ryanodine receptor, Ca2+-induced Ca2+ re- cellular free Ca*+ concentration ([Ca*+],), and is thought to lease, intracellular free CaZ+, neuronal Ca*+ signal, inositol be important for higher brain function. Dynamic changes in 1,4,5-trisphosphate receptor, in situ hybridization] [Ca2+li are affected by the spatial distributions of various Ca*+-increasing molecules (channels and receptors). The Dynamic changesin the intracellular free Cal+ concentration ryanodine receptor (RyR) is an intracellular channel through ([Caz+],) play a crucial role in numerous neuronal functions, which Ca*+ is released from intracellular stores. To define including the movement of the growth cone, releaseof neuro- the contribution of neuronal Ca2+ signaling via the RyR chan- transmitters, initiation and maintenanceof long-lastingchanges nel, we examined RyR type-specific gene expression in rab- in synaptic transmissionefficacy such as long-term potentiation bit brain by in situ hybridization histochemistry. The neuronal (LTP) in the hippocampus and long-term depression(LTD) in RyR was composed of three distinct types, two types dom- the cerebellum, the transcription of immediate-early genes,and inant in skeletal (sRyR) and cardiac (cRyR) muscle, respec- neurotoxicity (Kennedy, 1989) in addition to the ubiquitous tively, and a novel brain type (bRyR). sRyR was distinguished so-called housekeepingfunctions. Neuronal [Cal+], is well reg- by its high level of expression in cerebellar Purkinje cells. ulated by homeostatic mechanisms(Ca’+ transporting and buf- cRyR was predominantly expressed throughout nearly the fering systems)that maintain its resting cytosolic concentration entire brain, and was characterized by its markedly high level (-lo-’ M) (Blaustein, 1988). In order to act as signals,[Ca*+], of expression in the olfactory nerve layer, layer VI of the must increaseat least severalfold. With neuronal activity, a rise cerebral cortex, the dentate gyrus, cerebellar granule cells, in the [Ca*+], is triggered by two pathways: (I) Ca*+ influx across the motor trigeminal nucleus, and the facial nucleus. bRyR the plasmamembrane by voltage-operated Ca2+channels (Tsien expression was the least widely distributed throughout the et al., 1989)and Ca*+-permeableligand-gated ion channels(Bar- brain, and was high in the hippocampal CA1 pyramidal layer, nard, 1992) and (2) mobilization of Ca*+ from internal stores caudate, putamen, and dorsal thalamus. This investigation by ryanodine receptors(RyRs) (Endo, 1977; Fleischer and Inui, demonstrates that the heterogeneous distribution of neu- 1989) and inositol I ,4,Strisphosphate receptors (IP,Rs) (Ber- ronal RyRs may be implicated in distinct Ca2+-associated ridge and Irvine, 1989; Furuichi et al., 1992). These different brain functions. Moreover, it should be noted that cRyR, a Ca2+-increasingsystems show the distinct spatiotemporal dy- typical CICR channel, is distributed widely throughout the namics of [Ca2+], transients within a cell, which are thought to brain, suggesting that in a variety of cell types, the ampli- be important for the functional diversity of Cal+ signaling.The fication of neuronal Ca*+ signals is functionally accompanied spatial dynamics of Ca*+ signalingare crucially affected by the by a rise in [Ca2+la such as Ca*+ influx stimulated by neuronal heterogeneousspatial distribution of a variety ofplasmalemmal activity. This widespread distribution of the neuronal RyR and organellar channel types within the cell. This local [Ca2+], family indicates that Ca2+ signals via the intracellular stores elevation is thought to be a possiblemechanism underlying the should be considered in studies of neuronal Ca2+ dynamics. functional specialization of neuronal Ca2+signals, and appears to decide a range of target proteins, activated or inactivated, many of which generally have their characteristic subcellular Received June 4, 1993; revised Nov. IS, 1993; accepted Feb. 8, 1994. localizations and someof which are known to show dependence We thank Drs. K. Imoto. K. Kouda. T. Inoue. and M. Yuzaki for their heloful on a narrow [Ca2+], range. Thus, to understand the Ca*+ sig- discussion. This work was supported dy grants from the Human Frontier Scidnce naling function in a particular neuron, it is important to know Program, the Toray Scientific Research Foundation, the Semi Life Science Foun- dation, and the Yamanouchi Foundation for Research on Metabolic Disorders, what kinds of Ca*+-increasingmolecules are expressedin the and by the Ministry of Education, Science and Culture of Japan. cell. Correspondence should be addressed to Teiichi Furuichi, Department of Mo- lecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-l The organellar Ca’ + releasechannels IP,R and RyR are re- Shirokanedai, Minato-Ku, Tokyo 108, Japan. sponsible for distinct Ca’+ mobilization systemsfrom internal Copyright 0 1994 Society for Neuroscience 0270-6474/94/144794-12$05.00/O stores, but the physiological roles of Cal+ signaling via these The Journal of Neuroscience, August 1994, 74(8) 4795 channels in the brain are still unclear, in contrast to the plas- malemmal Ca’+ channels. The IP,R channel releases CaL+ in response to binding of the second messenger IP, produced by the phosphoinositide signal transduction cascade, for example, the successive activation of a plasma membrane receptor, G protein, and then phospholipase C (Berridge and Irvine, 1989). Although phosphoinositide turnover is considered to be in- volved in crucial parts of higher brain function, such as LTP in the hippocampus (Murphy and Miller, 1988; Lester and Jahr, 1990) and LTD in the cerebellum (Ito and Karachot, 1990; Linden et al., 199 l), there is no strong evidence with respect to the substantial role of IP,-induced Ca’+ release (IICR) in brain function. The functional role of RyR-mediated Ca’+ release in the dynamic changes in neuronal [Ca>+], is even more unclear. Release of Ca’+ from the intracellular stores of neurons by caf- feine, analogous to muscle RyR function, has been reported in peripheral (Kuba and Nishi, 1976; Kuba, 1980; Smith et al., 1983; Neering and McBurney, 1984; Thayer et al., 1988a,b) and central (Martinez-Serrano and Satrustegui, 1989; Murphy and Miller, 1989; Glaum et al., 1990) neurons. Ca*+ waves and oscillations in frog sympathetic neurons (Kuba and Takeshita, 198 1; Smith et al., 1983; Lipscombe et al., 1988) appear to be due partly to Ca”-induced Ca’+ release (CICR), which would be compatible with cardiac RyR function. Thus, caffeine-sen- sitive a’nd Ca’+ -induced CaZ+ release in neurons may be attrib- utable to neuronal RyR function. The existence of Ca’+ stores in the dendritic spines of the dentate molecular layer was dem- onstrated using a pyroantimonate precipitation technique (Fif- kova et al., 1983). Caffeine abolishes posttetanic potentiation in rat hippocampal synapses (Lee et al., 1987), and dantrolene and thapsigargin, drugs that inhibit the CICR activity of RyR and deplete intracellular Ca’+ pools, respectively, can inhibit the induction of LTP in the CA1 region of rat hippocampal slices (Obenaus et al., 1989; Harvey and Collingridge, 1992). Recently, upon stimulation of associative-commissural inputs, the sustained [Ca?+], elevation in the spine heads of hippocam- pal CA3 pyramidal cell dendrites was recorded (Miiller and Cormor, 199 1; see also Regehr and Tank, 1992), which has been considered to be due to CICR (regenerative [Caz+], transients). These results suggest that these neuronal RyRs are probably important for higher brain function (Miller, 1992; Bliss and Collingridge, 1993). In cerebellum, it was also shown that cul- tured cerebellar neurons definitely have caffeine-induced Ca’+ release activity (Brorson et al., 1991; Yuzaki and Mikoshiba, 1992). Through molecular cloning, we now know that mammals Flgure 1. Heterogeneous distribution of mRNAs of the neuronal RyR have at least three distinct types of RyR: two muscle RyRs, the family in rabbit brain: film autoradiograms of parasagittal sections hy- bridized with the ‘3-labeled antisense riboprobes for sRyR (A), cRyR skeletal type (sRyR) (Takeshima et al., 1989) and the cardiac (B), bRyR (C), and sense riboprobe for bRyR (conf, D). Films were type (cRyR) (Nakai et al., 1990; Otsu et al., 1990); and a novel exposed for 18 d (sRyR, A). 5 d (cRyR, B), or 14 d (bRyR, C, control, brain type RyR (bRyR) (Hakamata et al., 1992). The type names 0. Anatomy is depictedschematically at the top. AO. anteriorolfactory are basedon dominant tissuedistribution (sRyR and cRyR) or cortex;PO, primaryolfactory