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Proc. Nat!. Acad. Sci. USA Vol. 88, pp. 7076-7080, August 1991 Neurobiology

Localization of P-type calcium channels in the central (ion-channel blocker/Purkiqje /cerebeflum/finnel-web spider toxin) D. HILLMAN*, S. CHEN, T. T. AUNG, B. CHERKSEY, M. SUGIMORI, AND R. R. LLINAS Department of Physiology and Biophysics, New York University Medical Center, New York, NY 10016 Contributed by R. R. Llinds, May 29, 1991

ABSTRACT The distribution of the P-type calcium chan- polyclonal antibody was generated from this protein (17). In a nel in the mammalian has been dem- more recent study, a channel with similar characteristics was onstrated immunohistochemically by using a polydonal spe- cloned and sequenced (18). cific antibody. This antibody was generated after P-channel The present study describes the distribution of P channel isolation via a fraction from funnel-web spider toxin (FIX) that labeling in the and other regions of the central blocks the voltage-gated P channels in cerebellar Purklije cells. nervous system (CNS) by using the polyclonal antibody. In the cerebellar cortex, immunolabeling to the antibody appeared throughout the molecular layer, while all the other MATERIALS AND METHODS regions were negative. Intensely labeled patches of reactivity Tissue Prepartion. The of adult albino rats and the were seen on Purkije cell , especially at bifurcatlons; cerebella of mutant weaver mice (wv/wv) were studied im- much weaker reactivity was present in the and stem munohistochemically by using a polyclonal antibody against segment. Electron microscopic loItion revealed labeled the P-type voltage-gated (17). The animals patches of plasma membrane on the soma, min_ dendrites, were sacrificed under sodium pentobarbital anesthesia. Each spiny branchlets, and spines; portions of the smooth endoplas- animal was perfused through the vascular system via the mic reticulum were also labeled. Strong labeling was present in aorta with 4% paraformaldehyde and 0.2% glutaraldehyde in the periglomerular cells of the olfactory bulb and scattered 0.09 M phosphate buffer (19). The fixation was followed by in the deep layer of the entorhinal and pyriform Vibratome serial sectioning at 25-50 j.m for immunohisto- cortices. Neurons in the brainstem, habenula, nucleus of the logical analysis with a 300-pm sample taken at 600-ttm trapezoid body and inferior olive and along the floor Of the intervals. The 25- to 50-pm-thick sections were treated for 1 fourth ventricle were also labeled intensely. Medium-intensity hr in 1% sodium borohydride to remove unbound aldehyde reactions were observed in layer H pyramidal cells of the and then were rinsed thoroughly in phosphate-buffered saline frontal cortex, the CAl cells of the hippocampus, the lateral before the immune reaction. nucleus of the substantia nigra, lateral reticular nucleus,, and Immunologleal Techniques. A polyclonal antibody was pro- spinal fifth nucleus. Light labeling was seen in the neocortex, duced in rabbit from a bovine brain cerebellar protein isolated , and in some brainstem neurons. using two stages ofbatch-process affinity purification (20) with sFTX linked to Sepharose 4B (17). The purified protein Knowing the specific localization of voltage-gated ion channels solution was concentrated to a small volume. Rabbits were on the soma-dendritic membrane of neurons is fundamental to inoculated at multiple sites with a 1:1 mixture of the protein understanding their intrinsic and integrative functions. The ques- and Freund's incomplete adjuvant at 2-week intervals. After 3 tion of voltage-gated channel localiation has been of particular months, rabbits were periodically bled (through the ear vein) interest since the first report of dendritic action potentials in 10 days after inoculation. The serum fraction was processed to Purkinje cells over 2 decades ago (1, 2). Indeed, the existence of obtain the immunoglobulin (IgG) fraction by ammonium sul- was not in- fate precipitation followed by chromatography on a DEAE- such electroresponsiveness readily accepted until cellulose column (21). This IgG fraction was dialyzed against tradendritic recordings were made from different points in the phosphate buffer and the volume was concentrated. The IgG dendritic tree of Purkinje cells (3, 4). fraction was further purified by absorption with rat liver The calcium-dependent nature of these potentials was ini- powder. Samples were frozen until used. tially shown in avians (5) and later in mammals (4,6). The recent Immunohbistological Techniques. Nonspecific immune availability of specific calcium-channel blockers has allowed a blocking with normal goat serum for 30 mini was followed by more precise identification of these conductances and the 1-2 days ofincubation (at 40C) with primary antibody diluted different types of calcium channels involved. Specifically, cal- 1:2000, 1:4000, 1:8000, and 1:16,000 and containing 0.5% cium-channel blockers such as the dihydropyridines (7, 8) and normal goat serum plus 0.1% bovine serum albumin. After a a-conotoxin (9, 10) were ineffective in Purkinje cells, while thorough washing in phosphate-buffered saline, the sections these responses were blocked by a funnel-web spider toxin underwent reaction with the anti-rabbit Vectastain Elite ABC (FTX) (11). The results obtained from the venom study were kit (Vector Laboratories). Before reaction with the diamino- confirmed at both macroscopic current and single-channel benzidine chromagen, the sections were washed thoroughly levels for the calcium channels induced by rat brain mRNA in Tris buffer. The sections were mounted and dried on injection into Xenopus oocytes (12, 13). The toxin (FTX) charged glass slides (Fisher Scientific). Three types ofcontrol specifically responsible for this calcium-channel block was then preparations were made: (i) complete reaction mixtures isolated from the venom and a synthetic analog (sFTX) was without antibody; (it) 12-hr prereaction absorption of antigen made (14, 15). The calcium channel, called the P channel, was then isolated from bovine cerebella by using sFTX (16), and a Abbreviations: CNS, central nervous system; FTX, funnel-web spider toxin; sFrX, synthetic FTX. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Physiology and Biophysics, New York University Medical Center, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 550 First Avenue, New York, NY 10016. 7076 Neurobiology: Hillman et al. Proc. Natl. Acad. Sci. USA 88 (1991) 7077

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fair~~~~~~~ _ EX. FIG. 1. P channel immunoreactions in the cerebellum. (A, B, and E) Light micrographs of P channel immunoreactions on Vibratome slices ofrat cerebellum. (A) Low-power image showing the distribution oflabeling in the molecular layer and nonreactive granule cells. Note the intense reaction through the secondary and tertiary bifurcation zones. Reactivity in the Purkinje cell somata and primary dendritic segments is weak. (B) Medium-power image shows general cytoplasmic reactions in dendritic segments with intense reactivity at bifurcation zones. Note the larger dendrites leading to spiny branches and the dots (arrowhead) representing reactivity in spine heads. (C and D) Electron micrographs of P channel antibody reactions in the cerebellar molecular layer. The reactive patches (arrowheads) on spine heads (C) and on the main (D) are located on the plasma membrane. (E) The reactive axonal boutons outline Deiters' neurons (S) in the vestibular nucleus. to antibody at ratios of 10:1, 30:1, and 100:1; (iii) absorption Control preparations did not show any immune reaction after of antigen with calbindin antibody (M. R. Celio, Freibourg, absorption ofantigen to antibody at 100:1. A series ofantigen Switzerland). absorption concentrations showed that the limit of the ratio For ultrastructural observation sections were washed with was slightly >30:1, where a weak labeling remained. Calbin- 0.1 M phosphate buffer and postfixed in 2% glutaraldehyde din antibody absorbed with the sFTX-selective antigen frac- followed by 2% aqueous OS04. Sections were dehydrated in tion did not show a reduced staining intensity. ethyl alcohol and flat embedded in Durcupan (Fluka). The results obtained at the light microscopic level revealed intensely reacting Purkinje cell dendrites near the secondary RESULTS and tertiary bifurcations (Fig. 1A). The interbifurcation seg- Purkinje Cell Light Microscopic Studies. Light microscopic ments were generally less intense. By contrast, the soma and localization of the reaction product in Purkinje cells was proximal part of the primary segment were only lightly to obtained with a 1:4000 dilution of the primary antibody. moderately reactive (Fig. 1A). 7078 Neurobiology: Hillman et al. Po.Ni.Aa.SiProc. Natl. Acad Sci. USAS 888(91(1991)

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FIG. 2. P channel and calbindin reactions in the cerebellum of a mutant weaver mouse (wv/wv). (A) The P channel reaction is very light in Purkinje cell somata and dendrites. (B) A corresponding region of the mutant after reaction with the calbindin antibody shows the extent of Purkinje cell dendrites (arrowheads) that do not react with the P channel antibody in A. Elongated, densely stained patches were frequently observed dendritic segments; these segments extended into a narrow along the axis of main dendrites (Fig. 1 A and B). The spiny molecular layer. Spiny branches were not reactive in mutant branchlets, while strongly labeled, were less noticeable at low weaver mice. Magnification. because of their smaller volume (Fig. lA). Spine Brainsteu and Cerebral Cortex. Neurons demonstrating an heads were visible throughout the molecular layer as small dots, immune reaction on somata and dendrites were distributed while spine necks were not readily visible because oftheir small throughout the CNS. The level of the reaction was generally diameter. At high concentrations ofantibody, the reaction prod- uniform for neurons of the same type. Qualitatively, three uct appeared to fill the dendritic cytoplasm and soma. levels ofreaction were identified. Cells in level 1 were highly Purkiuje Cell Ultrastructural Studies. Ultrastructural in- reactive, as seen in cerebellar Purkinje cells. Level 2 cells vestigations of preembedded immune reactions revealed la- showed medium reactivity and were clearly distinguished bel on somata, dendrites, and spine profiles. With the ex- from the background, while level 3 cells were just above the of cell no other of the background level. ception Purkinje , components Level 1 labeling was in a few neuronal dis- cerebellum were reactive. The label penetrated only 1-2 A~m found types into the dendritic that were tributed along the neuraxis. Prominently labeled periglomer- segments opened by sectioning. ular cells were seen at the periphery of the olfactory bulb The distribution of label occurred as dense thickenings ofthe (Fig. 3A), while the remainder of the olfactory bulb was not plasma membrane and endoplasmic reticulum that extended labeled. In the archicortex, intense label was found in neu- into the cytoplasm (Fig. 1 C and D). Spine heads consistently rons ofthe entorhinal (Fig. 3B) and pyriform. areas. Dendritic showed a plasma membrane reaction, which was character- and possibly axonic projections were seen in the neuropile ized by patches of reactivity opposite, or adjacent to, the surrounding these neurons. Along the ventricular axis, reac- synaptic site (Fig. 1C). In the soma, some rough endoplasmic tive cells were seen in the habenula, (Fig. 3C), in the ventral reticulum with prominent -ribosomes, showed a reaction but periaqueduct gray, and along the floor of the fourth ventricle the Golgi apparatus was negative. (Fig. 3D). A similar specific reaction was found in the bulk of Other Cerebellar Cell Types. Other neurons in the cerebel- cells of the trapezoid body nucleus (Fig. 3E) and in the lum, including granule cells, lacked discernable label. Pur- (Fig. 3F). kinje cell axons and their collaterals were lightly labeled, as Cells showing level 2 reactivity were found throughout the were the projection courses ofthese axons into the vestibular neural axis with layer II ofthe frontal cortex being prominent. and . The contained In the diencephalon, the nucleus of the lateral substantia positive stained axons ofPurkinje cells, which had a regularly nigra was more prominent than any other cells in the region. spaced distribution within the and appeared as The CAl cells and dendrites were more reactive than any bundled axons in the central white matter and cerebellar other hippocampal structures. The dentate granule cells were peduncles. However, the site of Purkinje cell preterminal only slightly above background. Scattered brainstem cells axons and synaptic boutons in the deep cerebellar and had level 2 reactions; specifically stained groups were the Deiters' nucleus were indicated by an intense pattern of lateral reticular nucleus and the spinal fifth nucleus. Vestib- staining forming perineuronal plexi in the neuropile (Fig. 1E). ular and deep cerebellar nuclei showed axonal plexus and Cerebella of Mutant Weaver Mice. In homozygous mutant bouton staining that represent the distribution ofthe Purkinje weaver mice (wv/wv), the layer is incompletely cell axonic terminals (Fig. 1E). developed, generating regions of cerebellar cortex without Lightly stained cells were seen in most regions ofthe brain. granule cells; other regions have interspersed granule cells Somata were visible as distinct bodies, but the reaction was and a narrow molecular layer. In those areas where granule near background. Neuropile in general had a reaction that cells were lacking, the Purkinje cell somata and dendrites was distinct from the white matter. Finally, as in the case of were present (as indicated by calbindin labeling) but the the cerebellum, preabsorption of the antibody with antigen molecular layer lacked organization (Fig. 2). These Purkinje completely removed the nonspecific reaction, including all cell somata had a barely discernable field of small dendrites background staining except for occasional erythrocytes. and axons and reacted weakly to the P channel antibody. Most important perhaps is the fact that the bifurcation points DISCUSSION ofthe dendritic trees were not noticeable (Fig. 2A). In regions where a narrow molecular layer was evident, there was a light Studies in dorsal root cells revealed at least three to moderate reaction in the somata and the bifurcating main major types of voltage-gated calcium channels named L, T, Neurobiology: Hillman et al. Proc. Natl. Acad. Sci. USA 88 (1991) 7079

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FIG. 3. P channel antibody reactions in neurons in different regions of the brain. Intensely reacting groups of neurons (arrows) are found in the olfactory glomeruli (A), deep layers of the entorhinal cortex (B), the lateral habenular nuclei (C), the floor of the fourth ventricle (D), nucleus of the trapezoid body (E), and the inferior olivary nucleus (F). The reaction is primarily in the somata but dendritic and possibly axonal reactions are seen in the entorhinal cortex.

and N (22, 23). The present study directly demonstrates the Dihydropyridine-sensitive calcium channels (L-type) are localization ofa fourth type ofvoltage-gated calcium channel, found in both the peripheral nervous system and the CNS (7, the P channel, identified in previous investigations (11-16). In 8, 29). Autoradiographic labeling of dihydropyridine blocking the present work, using immunohistochemical techniques, agents revealed a wide distribution within the brain, with cell the P channel has been specifically localized to Purkinje cell and neuropile regions being primary targets (29). These in- dendrites and found to a lesser extent in their somata and clude the somata and proximal dendrites of the hippocampal axons, as well as in other neurons throughout the brain. dentate and CA regions, cerebellar Purkinje cells, olfactory Histochemical localization of the N-type voltage-gated bulb mitral cells, and subiculum layers (8, 29). calcium channel using 125I-labeled w-conotoxin has given a The distribution of FTX binding sites identified morpho- general picture of the distribution of that binding site in the logically agrees quite well with the electrophysiological find- CNS (24-27). A recent report of immunolabeling with an ings relating to the distribution of P-type calcium channels. antibody to w-conotoxin is in disagreement with these radi- Indeed, as proposed on electrophysiological grounds 2 dec- oligand binding analyses since the antibody reactivity was ades ago (3), the dense reaction in the main dendrites of seen in Purkinje cells as well as other cerebellar neurons in Purkinje cells indicates a strong antigen localization distally cerebellar slices (28). on the dendritic arbor, especially at bifurcation points for the 7080 Neurobiology: Hillman et al. Proc. Nati. Acad. Sci. USA 88 (1991) main dendritic segments. The immunolabeling response in The results reported here directly demonstrate the distri- spiny branches was less obvious at the light microscopic level bution of P channel previously characterized by biophysical because of the smaller mass of these projections. The label and pharmacological approaches and strongly suggest that distribution also corresponded quite closely to that predicted the newly described sequence named B1 by Mori et al. (18) from Purkinje cell intradendritic recordings (4), as well as to represents, in fact, the P channel. In support ofthis view, the the fluorescence changes observed after intracellular injec- mutant weaver mouse strain, which has a reduced number of tion of the calcium-sensitive dyes arsenazo 11 (30) and fura 2 Purkinje cells and a low level of cDNA hybridizable mRNA (31-33). In addition, the distribution of P channels along the (18) demonstrated reduced P channel labeling. However, this spiny branchlets, the spines themselves, and to the bifurca- reduction, rather than being due to the fewer granule cells as tion points in the large dendrites agrees well with the calcium proposed by Mori et al., is due to a reduced expression ofthis dye imaging for both calcium-dependent plateau and action channel in the Purkinje cell. These findings suggest that potential generation (31, 33). P-type calcium-channel expression in the cerebellar cortex Indeed, this ultrastructural study revealed the labeling of may be enhanced by the formation of synaptic contacts Purkinje cell plasma and endoplasmic reticulum membranes. between Purkinje cell dendrites and parallel fibers. The membrane reaction occurred as patches on main den- drites, spiny branches, and spines. Dense patches observed This research was supported by National Institute of Neurological at branch points in light microscopy may represent massively Disorders and Stroke Grant NS13742 and National Institute on Aging labeled sites on membranes. These may be the inwardly Grant AG09480. thickened membrane reactions on spiny branches, spine 1. Llinds, R., Nicholson, C., Freeman, J. A. & Hillman, D. E. (1968) necks, and spine heads seen ultrastructurally. Immunola- Science 160, 1132-1135. beled dense projections penetrating the cytoplasm are com- 2. Fujita, Y. (1968) J. Neurophysiol. 31, 131-141. monly found for other transmembrane proteins such as the 3. Llinas, R. & Nicholson, C. (1971) J. Neurophysiol. 34, 532-551. 4. Llinds, R. & Sugimori, M. (1980) J. Physiol. (London) 305, 197-213. glycine receptor (34). In addition to the membrane reactivity, 5. Llinis, R. & Hess, R. (1976) Proc. Natl. Acad. Sci. USA 73, 2520-2523. a general reaction on the surface of microtubules and mito- 6. Llinds, R. & Sugimori, M. (1980) J. Physiol. (London) 305, 171-195. chondria was observed in the ultrastructural preparations. 7. Ahlijanian, M. K., Westenbroek, R. E. &Catterall, W. A. (1990) The weaker reactivity in the somata, primary dendrites, and 4, 819-832. 8. Turner, T. & Goldin, S. (1988) Ann. N. Y. Acad. Sci. 522, 278-283. spiny branchlets may be due to (i) the presence of some 9. Olivera, B. M., McIntosh, J. M., Cruz, L. J., Luque, F. A. & Gray, antigenicity throughout the Purkinje cell, (il) the reaction of W. R. (1984) Biochemistry 23, 5087-5090. a polyclonal antibody component with other nonmembrane 10. Abe, T., Saisu, H., Nishiuuchi, Y. & Sakakibara, S. (1986) Neurosci. antigens, or (iii) antigen redistribution. Nevertheless, some Lett. 71, 203-208. 11. Llinds, R. & Sugimori, M. (1987) Soc. Neurosci. Abstr. 13, 228. component of the P channel antibody contains a specific IgG 12. Lin, J.-W., Rudy, B., Cherksey, B., Sugimori, M. & Llinas, R. (1989) for the protein. For example, control preparations show Soc. Neurosci. Abstr. 15, 652. absorption of all components of the antibody preparation, 13. Lin, J.-W., Rudy, B. & LlinAs, R. (1990) Proc. Nat!. Acad. Sci. USA 87, and reductions in the antibody concentrations by 8000- and 4538-4542. 14. Cherksey, B., Sugimori, M., Lin, J.-W. & Llin.s, R. (1989) Biophys. J. 16,000-fold helped to define greater specificity for light and 55, 438a (abstr.). electron microscopy. 15. Llinas, R., Sugimori, M., Lin, J.-W. & Cherksey, B. (1989) Proc. Nat!. Three qualitative levels of reaction, indicating the amount Acad. Sci. USA 86, 1689-1693. of reactive substances in specific cell types, were identified. 16. Cherksey, B., Lin, J.-W., Sugimori, M. & Llinas, R. (1990) Biophys. J. 57, 305a (abstr.). The high intensity (level 1) immunolocalization of the FTX- 17. Cherksey, B., Sugimori, M. & Llinas, R. (1991) Ann. N. Y. Acad. Sci., in sensitive P channel protein in such diverse regions as Pur- press. kinje cell axons, periglomerular cells, and deep entorhinal 18. Mori, Y., Friedrich, T., Kim, M.-S., Mikami, A., Nakai, J., Ruth, P., and pyriform cells indicates that the P channel may be widely Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., Mikoshiba, K., Imoto, K., Tanabe, T. & Numa, S. (1991) Nature (London) 380,398-402. distributed in the brain. In other regions ofthe brain, the level 19. Hillman, D. E. & Chen, S. (1984) Brain Res. 295, 325-343. ofreactivity was specific and quite uniform for each neuronal 20. Cherksey, B., Sugimori, M., Rudy, B. & Llinas, R. (1988) Soc. Neurosci. type. The inferior olivary neurons (providing a major afferent Abstr. 14, 901. source to Purkinje cells) were moderately intense, although 21. Harlow, E. & Land, D. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). the paths were not detected. Level 2 or 3 reactivity 22. Nowycky, M. C., Fox, A. P. &-Tsien, R. W. (1985) Nature (London) throughout the nervous system may indicate a broad but 316, 440-443. lower density of P channels in specific neuronal groups. Just 23. Tsien, R., Lipscombe, D., Madison, K., Bley, R. & Fox, P. (1988) Trends above background reactivity (level 3) could represent a Neurosci. 11, 431-438. 24. Kerr, L. M., Filoux, F., Olivera, B. M., Jackson, H. & Wamsley, J. K. nonspecific reaction or the presence of P channels in most (1988) Eur. J. Pharmacol. 146, 181-183. CNS neurons. Absorption of the antibody with antigen at 25. Maeda, N., Wada, K., Yuzaki, M. & Mikoshiba, K. (1989) Brain Res. 1:100 completely removed all the reaction product including 489, 21-30. the background. 26. Takemura, M., Kiayama, H., Fukui, H., Toyama, M. & Wada, H. (1987) Biochem. Biophys. Res. Commun. 149, 982-988. The presence of high-threshold calcium channels has been 27. Takemura, M., Kiayama, H., Fukui, H., Toyama, M. & Wada, H. (1988) demonstrated in a number of neuronal types throughout the Brain Res. 451, 386-389. CNS (35). Those in Purkinje cells seem to be unusual in that 28. Fortier, L. P., Trtemblay, J. P., Rafrafi, J. & Hawkes, R. (1991) Mol. they are insensitive to both dihydropyridine and c-conotoxin Brain Res. 9, 209-215. 29. Cortes, R., Supavilia, P. & Palacios, J. M. (1984) J. Neurotrans. 60, (15, 36). Voltage-gated components of the calcium current 169-197. expressed from rat brain mRNA in Xenopus oocytes was 30. Ross, W. N. & Werman, R. (1987) J. Physiol. (London) 389, 319-336. insensitive to both w-conotoxin and dihydropyridine (37). 31. Tank, D. W., Sugimori, M., Connor, J. A. & Llinas, R. R. (1988) Science This current was similar in every respect to that generated by 242, 773-777. 32. Hockberg, P. E., Tseng, H. Y. & Connor, J. A. (1989) J. Neurosci. 9, P-type channel activation, including blockage by FTX (12, 2272-2284. 13). Similar results, reported after injection of a mRNA 33. Sugimori, M. & Llinas, R. (1990) Soc. Neurosci. Abstr. 16, 894. hybridized with a calcium-channel cDNA isolated from rab- 34. Wenthold, R. J., Altshuler, R. A. & Hampson, D. R. (1990) J. Electron bit cerebellum (18) confirm the biophysical and pharmaco- Microsc. Tech. 15, 81-96. logical properties previously reported for the P channel 35. Llinas, R. (1988) Science 242, 1654-1664. 36. Regan, L. J., Sah, D. W. Y. & Bean, B. P. (1991) Neuron 6, 269-280. incorporated into lipid bilayers (15) and expressed in Xeno- 37. Leonard, J. P., Nargeot, J., Snutch, T. P., Davidson, N. & Lester, H. A. pus oocytes (12, 13). (1987) J. Neurosci. 7, 875-881.