The Journal of Neuroscience, August 1994, 14(B): 4794-4905

Multiple Types of /Ca*+ Release Channels Are Differentially Expressed in Rabbit

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-calledhousekeeping functions. 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 triggeredby 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; Fleischerand 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+], transientswithin a , 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 showdependence 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 , 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 cascade, for example, the successive activation of a plasma membrane receptor, G protein, and then 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 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, -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 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 isdepicted schematically at the top. AO. anteriorolfactory are basedon dominant tissuedistribution (sRyR and cRyR) or cortex;PO, primaryolfactory cortex: Tu, olfactorytubercle; LTO, lateral the origin of molecular cloning (bRyR). sRyR and cRyR are olfactory tract; CX, cerebral cortex; CP, caudate and putamen; Hl, hip- pocampus; Th, thalamus; ZI, zona incerta; Al, anterior cortical and involved in excitation+ontraction (E-C) coupling in skeletal medial amygdaloid nuclei, and amygdalohippocampal area; OT, nucleus and , respectively. These muscle RyRs have been of the optic tract; APT, anterror pretectal area; SuG, superficial gray immunohistochemically localized in chicken (Ellisman et al., layer of the superior colliculus; IC, inferior colliculus; Cb, cerebellum; 1990; Ouyang et al., 199 I; Walton et al., 199 1) and fish (Zupanc Mo5, motor trigeminal nucleus; 7, facial nucleus. et al., 1992) brain using the sRyR probe, and in rodent brain (Kuwashima ct al., 1992; Lai et al., 1992; Nakanishi et al., 1992; dominant type in brain (McPherson et al., 1991; Kuwashima Sharp et al.. 1993) using the sRyR and cRyR probes and the et al., 1992; Lai et al., 1992; Nakanishi et al., 1992).Widespread nonspecific probe. sRyR was exclusively localized in cerebellar distribution of RyR was shown by Western blotting (Kuwash- Purkinje cells (Ellisman et al., 1990; Walton et al., 1991; Ku- ima et al., 1992) and immunohistochemical (Nakanishi et al., washima et al., 1992), while cRyR was shown to be the pre- 1992) studies mainly using the probe nonspecific for all RyR 4796 Furuichi et al. - Neuronal Ryanodine Receptor Family

sRyR cRyR bRyR

Figure 2. Heterogeneous distribution of mRNAs of the neuronal RyR family in rabbit forebrain: film autoradiograms of coronal sections hybridized with the ?S-labeled antisense riboprobes for sRyR (A, D), cRyR (B, E), and bRyR (C, F). Films were treated as described for Figure 1. Anatomy is depicted schematically at the right. PCg, posterior cingulate cortex; FrPuM, frontoparietal cortex motor area; FrPuSS, frontoparietal cortex somatosensory area; CA3, CA3 pyramidal cell layer of the hippocampus; CP, caudate and putamen; PO, primary olfactory cortex; Hi, hippocampus; LHb, lateral habenular nucleus; T/z, thalamus (posterior thalamic nuclear group, paracentral, central medial, centrolateral, mediodorsal, paraven- tricular, ventrolateral, ventromedial, and ventroposterior thalamic nuclei); Rt, reticular thalamic nucleus; ZI, zona incerta; LM, dorsomedial hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; Arc, arcuate hypothalamic nucleus; A2, central, lateral, basolateral, medial, anterior cortical, and posterolateral cortical amygdaloid nuclei, and amygdalohippocampal area. types. However, now we know that none of the RyR probes particular neuron. Yoshida et al. (1992) reported that more than used in the previous immunological studies is capable of dis- 85% of 3H-ryanodine binding sites from rat brain were im- tinguishing the bRyR type specifically from either sRyR or cRyR munoprecipitated by the anti-cRyR monoclonal , and because of the cross-reactivity of the probes used. Therefore, it therefore claimed no cross-reactivity ofthe antibody with sRyR. remained unclear which type of RyR family was localized in a Smith and Nahorski (1993), on the other hand, claimed that

Figure 3. Differential localization of neuronal RyR mRNAs in the rabbit olfactory bulb: dark-field observations of coronal sections of olfactory bulbs hybridized with ?Mabeled sRyR (A), cRyR (B), and bRyR (C) probes and dipped into NTB2 emulsion followed by exposure for 12 d (B) or for 38 d (A and C). D, Bright-field observation of serial adjacent sections stained with hematoxylin and eosin. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GRL, granule cell layer. Scale bar, 100 pm. The Journal of Neuroscience, August 1994, 14(E) 4797

Figure 4. Differentiallocalization ofneuronal RyR mRNAsin rabbitcerebral cortex: dark-field observations ofcoronal sections ofthe frontoparietal cortex hybridized with ‘5S-labeledsRyR (A), cRyR (B), and bRyR (C) probes.D, Bright-fieldobservation of serialadjacent sections stained with hematoxylinand eosin.Scale bar, 500 pm. the >H-ryanodine binding properties of rat cortical membranes 443 1 nucleotide positions; Takeshima et al., 1989), the EcoRI-Sac1 were more similar to those of sRyR than cRyR. Neither group fragment of pHRR 12 (11,527-l 3,623; Nakai et al., 1990), and the EcoRI- EcoRI fragment of pBRR71 (11,834-13,353; Hakamata et al., 1992), consideredthe existence of bRyR in the brain. Therefore, the respectively. For the IP,RI probe, the EcoRI-NueI cDNA fragment RyR types and the predominant type expressedin brain should (7986-8569 nucleotide positions) cloned in pBluescript plasmid (Fu- be defined. ruichi et al., 1989, 1993) was used. Antisense and sense RNAs from In this study, we localized each member of the RyR family these cDNA fragments were synthesized by in vitro transcription using in the rabbit brain in detail by in situ hybridization using RyR T3 and T7 RNA polymerase (Promega) and (U-Y%UTP (Amersham). In situ hybridization. In situ hybridization was performed as described type-specific probes. The rabbit neuronal RyR family was com- previously (Furuichi et al., 1993). After hybridization, the samples were posedof all of theseRyRs. The present study has more clearly exposed to Hyper fl-max film (Amersham) for 5 d for the cRyR probe, demonstrated the heterogeneousdistribution of each neuronal 14 d for the bRyR probe, and 18 d for the sRyR probe. Some of the type, and is the first to localize bRyR mRNA in particular samples were dipped in NTB-2 emulsion (Kodak), exposed for 12 d for the cRyR probe and 38 d for the sRyR and bRyR probes at 4”C, and neurons.Upon examining the expressionlevels of all RyR-type then developed. The developed samples were lightly stained with 0.1% mRNAs, we confirmed that cRyR overwhelmingly predomi- cresyl violet. natesthroughout most of the rabbit brain. In comparisonwith sRyR and cRyR, the expressionofbRyR mRNA is highly char- Results acteristic and restricted, with the highest level in hippocampal mRNAs of the RyR-channel family showeddifferential distri- CA 1 pyramidal cells followed by the caudate, putamen, accum- butions in the rabbit brain, although the hybridization inten- bens nucleus, and dorsal thalamus. The heterogeneousdistri- sities with the antisenseprobes of these receptor types varied bution of functionally distinct RyR types may be involved in considerably (Figs. 1, 2; note the exposure times, seethe Fig. 1 the differential roles in neuronal Ca’+ signaling. caption). cRyR was the most common type of neuronal RyR in almost all regions (Fig. 1B). The distribution and approximate Materials and Methods expression levels are summarized in Table 1. No significant Cryosection preparutlon. Rabbits(std: JW/CSK, 13 weekold) wereob- hybridization signalswere observed with the senseprobes (for tainedfrom Nippon SLCCo. (Shizuoka,Japan). After beinganesthe- examples, seeFig. 1D). tized with sodiumpentobarbital(O.5 mg/kg of body weight),the rabbits wereintracardially perfused with phosphate-bufferedsaline (PBS) and Olfactory region. The mRNAs of the RyR family showed thenwith 4%paraformaldehyde in 0.1 M phosphatebuffer, pH 7.2 (PFA heterogeneousdistribution in the olfactory bulb (Fig. 3). cRyR fixer). After perfusion, were dissected, postfixed in PFA fixer for mRNA (Fig. 3B) was found at a high density in the olfactory 1.5 hr on ice, and immersedin 25%sucrose in PBSfor 16hr at 4°Cfor nerve layer, which consists of the axons of olfactory receptor cryoprotection.The brainswere frozen and sectionedat 12 pm. Rlboprobe preparation. pBluescriptplasmids harboring the cDNA neurons, olfactory nerve Schwann cells which ensheaththe ax- fragmentsof sRyR,cRyR, and bRyR, whichdiverged adequately from ons, and astrocyte subtypeswhich are morphologically divergent eachother, were used: the EcoRI-EcoRI fragment of pRR229 (1443- from others and may be associatedwith the axons (Bailey and 4798 Furuichi et al. - Neuronal Ryanodine Receptor Family

Table 1. Heterogeneous distribution of the ryanodine receptor family

sRyR cRyR bRyR Olfactory regions Olfactory bulb Olfactory nerve layer -/+ +++ -/+ Glomerular layer -/+ ++ ++ External plexiform layer + + + Mitral cell layer ++ ++ ++ Granule cell layer + ++ + Anterior olfactory nu. + +++ + Primary olfactory cortex ++ ++++ ++ Olfactory tubercles + +++ + Lateral olfactory nu. + ++ + Cerebral cortex Frontal cortex +/++ +++ + Frontoparietal cortex, motor area +/++ +++ +/++ Frontoparietal cortex, somatosensory area +/++ +++ +/+ + Striate cortex + ++ + Anterior cingulate cortex ++ ++ ++ Posterior cingulate cortex ++ ++ ++ Agranular region frontopariental cortex, motor area ++ ++ ++ Retrosplenial cortex + ++ + Hippocampal formation Entorhinal region + + + Subiculum + + + CA1 pyramidal cell layer +++ +++ ++++ CA2 pyramidal cell layer ++ +++ ++ CA3 pyramidal cell layer ++ +++ + CA4 pyramidal cell layer + +++ + Dentate gyrus ++ ++++ + Basal ganglia Caudate + ++ +++ Putamen + ++ +++ Accumbens nu. -/+ ++ ++ Substantia nigra, reticular -/+ + -/+ Thalamus Lateral habenula nu. + ++ -/+ Reticular th. nu. -/+ ++ + Laterodorsal th. nu. -/+ + ++ Lateral posterior th. nu. -/+ + ++ Posterior th. nuclear group -/+ + ++ Paracentral th. nu. -/+ + ++ Central medial th. nu. -/+ + ++ Centrolateral th. nu. -/+ + ++ Mediodorsal th. nu. -/+ + ++ Paraventricular th. nu. -/+ + ++ Anteroventral th. nu. + + +++ Ventroposterior th. nu. + + +++ Ventroposterior th. nu. medial + + +++ Ventroposterior th. nu. lateral + + +++ Ventrolateral th. nu. + + +++ Ventromedial th. nu. + + +++ Medial geniculate nu. + + +++ Zona incerta -/+ ++ -/+ Anterior pretectal area + ++ + Hypothalamus + + + Amygdala + + + The Journal of Neuroscience, August 1994, 14(8) 4799

Table 1. Continued

SRYR CRYR bRyR

Cerebellum Molecular layer -/-I- + -/+ Purkinje cell layer +++t i-+/+-l-t -/+ Granule cell layer -/-I- +++ -/-I- Cerebellar nu. -/t ++ ++ Brainstem Nu. optic tract -/+ ++ + Superificial gray layer of superior colliculus + ++ + Inferior colliculus -/+ ++ + Dorsal nu. lateral lemniscus -/+ ++ + Ventral nu. lateral lemniscus -/-I- ++ + Dorsal cochlear nu. -/t ++ + External cuneate nu. -/+ ++ + Lateral superior olive -/t- ++ + Motor trigeminal nu. + +++ ++ Facial nu. + +++ ++ Lateral reticular nu. -/+ ++ + Nu. spinal tract trigeminal nerve + ++ ++ th., thalamic; nu., nucleus. Approximate expression levels of each RyR-type mRNA: + + + +, very high; + + +, high; + +, medium; +, low; -, not srgnificant. These expression levels were not equilibrated among RyR types.

Figure 5. Differential localization of neuronal RyR mRNAs in rabbit hippocampus: bright-field observations ofcoronal sections of the hippocampus hybridized with 35S-labeled sRyR (A, D), cRyR (B, E), and bRyR (C, F) probes. A-C are CA1 pyramidal cell layers, and D-F are dentate granular cell layers. Silver grains are seen in and around cell bodies counterstained with cresyl violet. Note: for an easy detection of grains located in dentate granular cells, the sections hybridized with the cRyR probe (B, E) are more lightly stained with cresyl violet than the others. Scale bar, 50 pm. 4800 Furuichi et al. - Neuronal Ryanodine Receptor Family

QT< APT /‘.. ’ MLV Th AY .I p ..,. Rt ‘: ..-

Figure 6. Differential localization of cRyR and bRyR mRNAs in rabbit thalamus: film autoradiograms of parasagittal sections hybridized with ‘5S-labeled cRyR (A) and bRyR (B) probes (higher magnification of the region around the thalamus in Fig. 1). Anatomy is depicted schematically at the right. CAI, hippocampal CA I pyramidal cell layer; DC, dentate gyrus; LS, lateral septal nucleus; LV, lateral ventricle; CP, caudate and putamen; Tu. olfactory tubercle; A V, anteroventral thalamic nucleus; Th, thalamus (laterodorsal and lateral posterior thalamic nuclei, posterior thalamic nuclear. ventronosterior. ventromedial, and ventrolateral thalamic nuclei); Rt, reticular thalamic nucleus; ZI, zona incerta; APT, anterior pretectal area; F~PaM, frontoparietal cortex motor area.

Shipley, 1993). In the glomerular layer consistingof periglomer- (Figs. 1B; 2&E). The highest labeling intensity was seenover ular cells, cRyR mRNA (Fig. 3B) was predominantly seenin the dentate granule cell layer (Figs. 5E, 6A). sRyR mRNA was the superficial part, while bRyR mRNA (Fig. 3C) was observed present almost uniformly in the CA l-CA4 pyramidal cells and in the deeper half. RyR immunoreactivity was also shown in dentate granule cells (Figs. IA; 2A,C, 5A,D), whereas bRyR the glomerular layer of the fish olfactory bulb (Zupanc et al., mRNA expression was much higher in CA1 pyramidal cells 1992).cRyR and bRyR mRNAs was found in the external plexi- than in the other parts of the hippocampus (Figs. 2C,F, 5C,F, form layer consistingoftufted cells. In the mitral cell and granule 6B). This CA1 pyramidal cell layer was the predominant site cell layers, the mRNAs ofall three receptor types were observed. for the labeling of bRyR mRNA (Fig. 1C). In other parts of the olfactory system, the anterior olfactory Basal ganglia. As shown in Figures 1, 2, and 6, both cRyR nucleus, piriform cortex, and olfactory tubercle expressed all and bRyR mRNA were detected in the caudate, putamen, and three receptor types, but cRyR expressionwas predominant. accumbensnucleus, in which medium and large cells lightly Cerebral c0rte.y. The labeling density of cRyR mRNA was counterstained with cresyl violet were labeled (Fig. 7A,B). In obviously greater than that of the other RyR mRNAs (Figs. 1C, contrast, sRyR mRNA expression was not so marked. In the 2&E), and showed a laminar distribution (Fig. 4B), especially substantia nigra, low labeling levels of cRyR mRNA were ob- in the frontoparietal cortex, a very high level in layer VI, a served (Fig. 1B). moderate level in layers II-III, and a low level in layers I, IV, Thalamus. Very low levels of sRyR mRNA were seenin the and V. Low-level expressionof sRyR (Figs. 1A; 2A,D; 4A) and thalamus (Figs. IA, 20). Either cRyR or bRyR was highly ex- bRyR mRNAs (Figs. 1C, 2C,F, 4C) was observed with no sig- pressedin particular subdivisions of the thalamus (Figs. 1, 2). nificant laminar dominance. Intense labeling of cRyR mRNA was seenmainly in the epi- Hippocampal formation. Although the mRNAs of all three thalamus (e.g., lateral habenular nucleus) and the ventral thal- receptor types were expresseddifferentially in the CAl-CA4 amus (e.g., reticular thalamic nucleus and zona incerta) (Figs, pyramidal cell layers of Ammon’s horn, the dentate gyrus, the 2E, 6A). In marked contrast to cRyR mRNA, bRyR mRNA polymorphic region, the subiculum, and the entorhinal cortex was found in the dorsal thalamus (Figs. 2F, 6B), at fairly high (Figs. i,2), cRyR predominance wasstriking in all ofthese areas densities in the ventral nuclei (medium and large cells lightly The Journal of Neuroscience, August 1994, 14(E) 4801

Fgure 7. Localization of bRyR mRNA in rabbit caudate, putamen, and thalamus: dark-field (A and C) and bright-field (B and D) observations of coronal sections of the caudate and putamen (A and B) and the thalamus (C and D) hybridized with %S-labeled bRyR probe. Open triangles and solid arrowheads represent positive and negative cells, respectively. Scale bar, 100 pm. stainedwith cresyl violet were positive; Fig. 7C, D), and at slight densitiesin the dorsal nuclei. Cerebellum. In the cerebellar cortex, differencesin hybridiza- tion patterns with the various probes of the RyR family were very conspicuous(Fig. U-C), in strong contrast to resultswith the IP,Rl probe (Fig. 80). IP,Rl mRNA was abundantly lo- calized in both the dendrites and somata of Purkinje cells (Fu- ruichi et al., 1989, 1993). sRyR mRNA showeda characteristic high expressionlevel in Purkinje cells (Fig. 9A,D), as had pre- viously been demonstrated by immunohistochemical analyses (Ellisman et al., 1990; Walton et al., 1991). A high density of cRyR mRNA was seen in the granule cell layer (Fig. 9&E). cRyR mRNA coexisted with sRyR mRNA in Purkinje cells, and was also present in interneurons of the molecular layer. Labeling for bRyR mRNA was not significant in the cells of the cerebellarcortex (Fig. 9C,F), but was slightly above background in the film autoradiogram (Fig. 1CD). Although not in all cells, somebRyR mRNA signalswere observed in the granular layer, and a few signalswere sometimesseen in Purkinje cells. In the cerebellar nuclei, bRyR mRNA (Fig. 10) coexisted with cRyR mRNA (data not shown). Other brain regions. As shown in Figures 1 and 2 and Table 1, hybridization signalsof the three receptor mRNAs were ob- Figure 8. Differential localization of mRNAs of the RyR family and served in cells in the hypothalamus, amygdala, superior and IP,R I in rabbit cerebellar lobes: dark-field observations of coronal sec- inferior colliculus, dorsal cochlearnucleus, external cuneate nu- tions of cerebellar lobes hybridized with YYabeled probes of the RyR cleus,and so on. Most intriguing was the high cRyR expression family (A, sRyR; B, cRyR; C, bRyR) and IP,Rl (D). Scale bar, 500 Km. 4802 Furuichl et al. * Neuronal Ryanodlne Receptor Family

Figure 9. Differentiallocalization of neuronalRyR mRNAsin the rabbitcerebellar cortex (highermagnifications of Fig. 8): dark-field(,4-C) and bright-field(D-F) observationsof coronalsections of the cerebellarcortex hybridizedwith YGlabeledsRyR (A and D), cRyR (Band E), and bRyR (C and F) probes.Open tnangle.~ representPurkinje cell bodies.Sobd arrowheads point to positiveneurons in the molecularlayer. ML, molecular layer; PCL, Purkinjecell layer: CCL, granulecell layer. Scalebar, 50 pm. seenin the motor trigeminal nucleus(Fig. 1 lA,D), facial nucleus plexes may be made up of more than one subunit type arranged (Fig. 1l&E), and lateral reticular nucleus (Fig. 1lC,F). bRyR in a tetrameric structure. Receptor/channel kinetics of heter- mRNA also coexisted in these nuclei (for examples, see Fig. omeric RyR complexes could vary depending upon thesedif- 1C). Intermediate labeling intensity of cRyR mRNA was ob- ferent subunit combinations, thereby modulatingneuronal RyRs served in the nucleus of the spinal tract trigeminal nerve (Fig. involved in a variety of brain functions. On the other hand, 12). A similar labeling pattern of sRyR and bRyR mRNAs was theseorganellar channels could be located in spatially different seenat low-level densities(data not shown). Ca’+ stores involved in differential Cal+ signalingwithin indi- vidual cells (i.e., dendritic, axonal, and/or somatic Ca’+ storage Discussion organelles),which is inferred from the observation that in cer- The rabbit neuronal RyR family is composedof three distinct ebellar Purkinje cells, the localization of immunoreactivity of types: bRyR, which was recently isolated from the brain (Hak- chicken sRyR (Walton et al., 199I) and rat cRyR (Sharp et al., amata et al., 1992), and sRyR and cRyR, which were originally 1993) appearsto be spatially different from that of the other well characterized as muscle types. These neuronal RyRs are classof the organellar channel family, IP,R 1. differentially expressedin various brain areas, and are distin- Recently, the channel properties of caffeine-sensitiveand ry- guished by their own peculiar distribution in particular brain anodine-sensitiveCal+ releasechannels in the brain have been areas,such as the olfactory bulb, cerebral cortex, hippocampus, examined by various systems(Ashley, 1989; Bezprozvanny et thalamus,and cerebellarcortex. This heterogeneousdistribution al., 1991; McPherson et al., 1991; Lai et al., 1992). Theseresults of multiple neuronal RyRs throughout the brain suggeststheir suggestedthat neuronal RyR is almost indistinguishablephar- differential roles in brain function. In addition, thesethree dis- macologically(sensitivity to caffeine, ryanodine, Ca’+, and ATP) tinct types of RyRs coexist within individual neurons, such as from the RyRs in a variety of muscle tissues, but is slightly hippocampal CA1 pyramidal cells. Both sRyR and cRyR are different in electrophysiological properties (e.g., channel con- known to function as homotetrameric receptor/channel com- ductance). It is now apparent, however, that in thesestudies the plexes (Lai et al., 1988; Anderson et al., 1989). bRyR seemsto authors probably recorded channel activities of a complex of form an analogoustetramer. Therefore, the coexpressionof these neuronal RyR channels and that most of this activity was at- distinct RyR types in a cell suggeststhat neuronal RyR com- tributable to cRyR channels, which are dominant throughout The Journal of Neuroscience, August 1994, M(8) 4803

Figure 12. Localization ofcRyR mRNA in the spinal tract ofthe rabbit brain: dark-held (A) and bright-field (B) observations of sagittal sections of spinal tracts hybridized with Y+labeled cRyR probe. Scale bar, 500 0.

most of the brain. cRyR functions as a Ca’+-induced Ca2+re- lease(CICR) channel in cardiac muscle (Fabiato and Fabiato, 1973; Fabiato, 1985). It is believed that in central neuronsCa’+ entry through voltage-activated Ca’+ channels or Ca’+-per- meable ligand-gated ion channels is the primary signal for ini- Figure IO. Localization of bRyR mRNA in rabbit cerebellar nuclei: tiating the [Ca”‘], transient, and that CICR probably amplifies dark-held (A) and bright-field (B) observations of sagittal sections of the effects of Ca” influx (Bliss and Collingridge, 1993). This cerebellar nuclei hybridized with Y34abeled bRyR probe. The urrow- regenerative [Caz+], rise by CICR is thought to be involved in he&s in B are pointing to large cells with many positive grains. Scale bar, 50 pm.

Figure II. Localization of cRyR mRNA in the facial, motor trigeminal, and lateral reticular nuclei of the rabbit brain: dark-field (,4-C) and bright-field (D-F) observations of sagittal sections hybridized with ‘Wabeled cRyR probe. A and D, facial nucleus; B and E, motor trigeminal nucleus; C and F, lateral reticular nucleus. Scale bar, 50 pm. 4804 Furuichi et al. - Neuronal Ryanodine Receptor Family oscillatory [Caz+], elevation in a cell in some cases. We showed PD, Airey JA, Sutko JL (1990) Identification and localization of the widespread localization of cRyR in rabbit brain, suggesting ryanodine binding proteins in the avian central nervous system. Neu- ron 5: 135-146. that upon the primary [Caz+], elevation, the CICR events could Endo M (1977) release from the . occur throughout the brain. The present results also indicate Physiol Rev 57:71-108. that sRyR and bRyR are only a fraction of neuronal RyR and Fabiato A (1985) Time and calcium dependence of activation and have the expressionpattern distinct from that ofcRyR. Purkinje inactivation of calcium-induced release of calcium from the sarco- cells, which highly expresssRyR mRNA, are known to possess plasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol 85:247-289. subsurfacecistemae just beneath the plasma membrane, like Fabiato A, Fabiato F (1973) Activation of skinned cardiac cells. Sub- the sarcoplasmicreticulum of (Henkart et al., cellular affects of cardioactive drugs. Eur J Cardiol l/2: 143-155. 1976), and some fractions of sRyR proteins in Purkinje cells F&ova E, Markham JA, Delay RJ (1983) Calcium in the spine ap- exist in these subsurfacemembrane cisternae (Walton et al., paratus of dendritic spines in the dentate molecular layer. Brain Res 266:163-168. 1991). Thus, the neuronal sRyR in Purkinje cells may form a Fleischer S, Inui M (1989) Biochemistry and biophysics of excitation- “foot”-like structure, thereby opened through somemechanical contraction coupling. Annu Rev Biophys Biophys Chem 18:333-364. interaction with high-voltage-activated Cal+ channels,just like Furuichi T, Yoshikawa S, Miyawaki A, Wada K, Maeda N, Mikoshiba the model in skeletal muscle (Schneider and Chandler, 1973; K (1989) Primary structure and functional expression of the inositol Catterall, 1991). It has been well documented that the opening 1,4,5-trisphosphate-binding protein P,,,,,.Nature 342:32-38. Furuichi T, Miyawaki A, Maeda N, Nakade S, Nakanishi S, Nakagawa of both sRyR and cRyR channels is stimulated by both ryano- T, Yoshikawa S, Mikoshiba K (1992) Structure and function of the dine and caffeine. In contrast, bRyR appearsto releaseCa’+ in inositol 1,4,5-trisphosphate receptor. In: Neuroreceptors, ion chan- responseto ryanodine but not to caffeine(Giannini et al., 1992). nels and the brain (Kawai N, Nakajima T, Barnard EA, eds), pp 103- Thus, bRyR appearsto have a receptor/channel property dis- 1 I I. Amsterdam: Elsevier. tinct from that of sRyR and cRyR. Finally, the resultsof recent Furuichi T, Simon-Chazottes D, Fujino I, Yamada N, Hasegawa M, Mivawaki A. Yoshikawa S. Guknet J-L. Mikoshiba K (19931\ , Wide- intriguing studies on intracellular Ca’+ releasehave suggested spread expression of inositbl 1,4,5-trisphosphate receptor type 1 gene that cyclic adenosinediphosphate ribose (cADPR) is a major (insp3rf) in the mouse central nervous system. Recept Channels 1:1 I- candidate(s)for the endogenousligand of RyRs (Galione, 1992, 24. 1993; Lee, 1993; Takasawa et al., 1993; White et al., 1993). Galione A (1992) Cal+ -induced Ca’+ release and its modulation by cyclic ADP-ribose. Trends Pharmacol Sci 13:304-306. Indeed, extracts from rabbit brain contain FADPR-synthesizing Galione A (1993) Cvclic ADP-ribose: a new wav to control calcium. enzymes(Rusinko and Lee, 1989). Therefore, someof neuronal Science 2g9:325-3i6. RyR-mediated [Ca”], increasesmay be triggered by this pu- Giannini G, Clementi E, Cele R, Marziali G, Sorrentino V (1992) tative intracellular messenger,cADPR. Expression of a ryanodine receptor-Ca’ + channel that is regulated by TGF-0. Science 257:9 l-94. In conclusion, the presentdata indicate that each functionally Glaum SR, Scholz WK, Miller RJ (1990) Acute- and long-term glu- divergent type of neuronal RyR family is differentially localized tamate-mediated regulation of [Ca’+ 1, in rat hippocampa~pyram~dal in the rabbit brain and appearsto be involved in the amplifi- neurons in vitro. J Pharmacol Exo Ther 253: 1293-l 302. cation of Ca’+ signals by CICR or may transduce electrical Hakamata Y, Nakai J, Takeshima I?, Imoto K (1992) Primary struc- information (membrane depolarization) or intracellular mes- ture and distribution of a novel ryanodine receptor/calcium-release channel from rabbit brain. FEBS Lett 3 12:229-235. senger(s),such as cADPR, into Ca’+ signals. Harvey J, Collingridge GL (1992) Thapsigargin blocks the induction of long-term potentiation in rat hippocampal slices. Neurosci Lett References 139: 197-200. Anderson K, Lai FA, Lin Q-Y, Rousseau E, Erickson H, Meissner G Henkart M, Landis DMD, Reese TS (1976) Similarity of junctions (1989) Structural and functional characterization of the purified car- between plasma membranes and in muscle diac ryanodine receptor-Ca’+ release channel complex. J Biol Chem and neurons. J Cell Biol 70:338-347. 264:1329-1335. Ito M, Karachot L (1990) Messengers mediating long-term desensi- Ashley RH (1989) Activation and conductance properties of ryano- tization in cerebellar Purkinje cells. Neuroreport 1: 129-l 32. dine-sensitive calcium channels from brain microsomal membranes Kennedy MB (1989) Regulation of neuronal function by calcium. incorporated into planar lipid bilayers. J Membr Biol 111: 179-l 89. Trends Neurosci 12:4 17420. Bailey MS, Shipley MT (1993) Astrocyte subtypes in the rat olfactory Kuba K (1980) Release of calcium ions linked to the activation of bulb: morphological heterogeneity and differential laminar distribu- potassium conductance in a caffeine-treated sympathetic neurone. J tion. J Comp Neural 328:501-526. Physiol (Lond) 298:251-269. Barnard EA (1992) Receptor classes and the transmitter-gated ion Kuba K, Nishi S (1976) Rhythmic hyperpolarizations and depolar- channels. Trends Biochem Sci 17:368-374. ization of sympathetic ganglion cells induced by caffeine. J Neuro- Berridge MJ, Irvine RF (1989) Inositol phosphate and cell signalling. physiol 391547-563. Nature 341:197-205. Kuba K, Takeshita S (1981) Stimulation of intracellular Ca’+ oscil- Bezprozvanny I, Watras J, Ehrlich B (1991) Bell-shaped calcium- lations in a sympathetic neurone. J Theor Biol 93: 1009-103 1. response curves of Ins( 1,4,5)P,- and calcium-gated channels from Kuwashima G, Futatsugi A, Niinobe M, Nakanishi S, Mikoshiba K endoplasmic reticulum of cerebellum. Nature 35 I:75 l-754. (1992) Two types of ryanodine receptors in mouse brain: skeletal Blaustein MP (1988) Calcium transport and buffering in neurons. muscle type exclusively in Purkinje cells and cardiac muscle type in Trends Neurosci 11:438-443. various neurons. Neuron 9: 1133-l 142. Bliss TVP, CoIlingridge GL (1993) A synaptic model of memory: long- Lai FA, Frickson HP, Rausseau E, Liu Q-Y, Meissner G (1988) Pu- term potentiati& h the hippocamp&. Nature 36 I:3 l-39. - rification and reconstitution of the calcium release channel from skel- Brorson JR. Bleakman D. Gibbons SJ. Miller RJ (199 1) The nronerties etal muscle. Nature 33 1:3 15-3 19. of intracellular calcium stores in cultured rat‘ceredellar neurons. J Lai FA, Dent M, Wickenden C, Xu L, Kumari G, Misra M, Lee HB, Neurosci 11:4024-4043. Sar M, Meissner G (1992) Expression of a cardiac Cal+ release Calabresi P, Maj R, Pisani A, Mercuri NB, Bemardi G (1992) Long- channel isoform in mammalian brain. Biochem J 288:553-564. term synaptic depression in the striatum: physiological and phar- Lee HC (1993) Potentiation of calcium- and caffeine-induced calcium macological characterization. J Neurosci 12:4224-4233. releaseby cyclic ADP-ribose.J Biol Chem268:293-299. Catterall WA (199 1) Excitation-contraction coupling in vertebrate Lee W-L, Anwyl R, Rowan M (1987) Caffeine inhibits post-tetanic skeletal muscle: a tale of two calcium channels. Cell 64:87 l-874. potentiation but does not alter long-term potentiation in the rat hip- Ellisman MH, Deerinck TJ, Ouyang Y, Beck CF, Tanksley SJ, Walton pocampal slice. Brain Res 462:250-256. The Journal of Neuroscience, August 1994, 14(g) 4805

Lester RA, Jaher CE (1990) Quisqualate receptor mediated depression of an enzyme catalyzing the conversion of NAD’ into cyclic metab- of calcium currents in hippocampal neurons. Neuron 4:741-749. elite with intracellular Ca’ + -mobilizing activity. J Biol Chem 264: Linden DJ, Dikinson MH, Smeyne M, Conner JA (I 99 1) A long-term 11725-11731. depression ofAMPA currents in cultured cerebellar Purkinje neurons. Schneider MF, Chandler WK (1973) Voltage dependent charge move- Neuron 7:8 l-89. ment in skeletal muscle: a possible step in excitation