Brain Research, 555 (1991) 169-172 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124757X
169
BRES 24757
A study of cerebellar inositol 1,4,5-trisphosphate receptor following climbing and parallel fibre deafferentation Peter P. Li 1, Marty A. Green I and Jerry J. W a r s h 1'2'3 1Section of Biochemical Psychiatry, Clarke Institute of Psychiatry, 2Department of Pharmacology and 3Institute of Medical Sciences, University of Toronto, Toronto, Ont. (Canada) (Accepted 23 April 1991)
Key words: Inositol 1,4,5-trisphosphate receptor; Cerebellum; Purkinje cell; Inferior olive-climbing fiber; Granule cell-parallel fiber
We have examined the influence of climbing fibre and parallel fibre afferent inputs on the inositol 1,4,5-trisphosphate (InsP3) receptor in rat cerebellum. Lesions of the inferior olive-climbing fibre projections to Purkinje cells by 3-acetylpyridine (3-AP) significanlty reduced the [3H]InsP3 binding density (-20%) with no apparent changes in the binding affinity 21 days after treatment. No further reduction in binding density was found in rats given a second dose 7 days after the initial injection. A significant reduction in the binding density was evident as early as 7 days post-lesion. However, 3-AP (0.5 mM) failed to inhibit [3H]InsP3 binding in vitro. Cerebellar granule cells were lesioned by two consecutive injections of methylazoxymethanol acetate at birth. In these 60-day-old granuloprival rats, the density and affinity of [3H]InsPa binding sites in the cerebellum remained comparable to the controls. Since lesions of the climbing fibres increase Purkinje cell activity, we suggest that changes in InsP3 receptor density may reflect an adaptative response to the heightened Purkinje cell activity. In addition, the results also indicate that expression of the InsP3 receptor in the cerebellum is largely independent of the presence of granule cell-parallel fibre synaptic innervation onto the Purkinje cells.
The pivotal role of inositol 1,4,5-trisphosphate (InsP3) as a calcium-mobilizing second messenger for many hormonal, neurotransmitter and growth factor signals h a s been well documented in a variety of cells 4. InsP 3 releases calcium from non-mitochondrial intracellular stores 4,29 through binding to specific receptors which have been identified in a variety of tissues 13,31,33. The InsP 3 receptor has been purified in rat 28 and mouse 15A6 cerebellum and more recently, the complementary D N A s encoding the InsP 3 receptor have been cloned x2,1s. Reconstitution and functional expression studies have revealed that the receptor protein behaves as an ionophoric receptor containing both recognition sites for InsP 3 and the calcium channel 11,19. In brain InsP 3 receptors are heterogeneously distributed with highest concentrations found in the cerebellum, where they appear to be selectively localized to Purkinje cell dendrites in the molecular layer 16,23,31,32. The cerebellar Purkinje cells are innervated by two major excitatory afferent systems: the climbing fibres which originate from the medullary inferior olivary nucleus, and the parallel fibres which derive from cerebellar granule cells 1°. Glutamate and aspartate appear to be the putative neurotransmitters released by the parallel and
the climbing fibres, respectively 24,3°. These excitatory amino acids are known to enhance phosphoinositide turnover with increased InsP 3 formation 21,26. The present work was aimed at determining whether the climbing or parallel fibre innervation of Purkinje cells transsynaptically modulate cerebellar InsP a receptors. To produce climbing fibre lesions, male Wistar rats (250-300 g; Charles River, Montreal) were injected intraperitoneally with 3-acetylpyridine (3-AP; 60 mg/kg, Sigma), a neurotoxin which produces selective destruction of the inferior olive-climbing fibre system s . Control animals received isotonic saline injections. Surviving animals were ataxic and showed difficulty in coordinating their movements. Animals were killed 21 days after the drug administration, and the cerebella quickly removed, frozen and stored at -70 °C. The extent of lesion was determined by examining Cresyl violet-stained sections of the inferior olive for surviving cells. To determine the effect of eliminating parallel fibre inputs on [3H]InsP3 binding, newborn pups at postpartum days 1 and 2 received subcutaneous injections of methylazoxymethanol acetate ( M A M , an antimitotic agent) at 20 mg/kg and 15 mg/kg, respectively 14. Controls were littermates that received isotonic saline. All animals
Correspondence: P.P. Li, Section of Biochemical Psychiatry, Clarke Institute of Psychiatry, 250 College Street, Toronto, Ont., Canada, M5T 1R8. Fax: (1) (416) 979-7871.
170 were weaned at 21 days of age and were killed at 60 days of age. Cerebella were removed and frozen for assay as described above. Specific [3H]InsP3 binding was determined as previously described 33 with minor modifications. Thawed cerebella were homogenized in 40 vols (w/v) of ice cold Tris-EDTA buffer (50 mM Tris-HC1, 1 mM EDTA, pH 8.3). The homogenates were then centrifuged at 35,000 g for 20 min at 4 °C, after which the supernatants were decanted and the tissue pellets were resuspended in buffer and used immediately in binding assays. The incubation mixtures contained approximately 0.3-0.5 mg membrane protein, 0.2 nM [3H]InsP3 (47 Ci/mmol; Amersham), InsP 3 (0-1000 nM; Sigma) and 1 mg/ml bovine serum albumin in a total volume of 1 ml of Tris-EDTA buffer. Triplicate samples were incubated at 4 °C for 10 min followed by centrifugation at 12,000 g for 10 min. The pellets were washed with 1 ml ice-cold buffer and subsequently solubilized with 0.5% sodium dodecyl sulphate overnight. The radioactivity in the pellets was quantified by liquid scintillation spectrometry (counting efficiency 50%). Non-specific binding was defined as the amount of radioactivity bound in the presence of 1/~M InsP 3. The protein content was determined using bovine serum albumin as the standard 25. Analyses of competition data and estimates of receptor binding parameters were performed using the E B D A computer program 17. Data were expressed as mean ___ S.E.M. and analyzed by two-tailed Student's t-tests or analysis of variance using the SYSTAT software program (SYSTAT, Evanston, IL). Mean differences with Pvalues < 0.05 were considered statistically significant. Histological examination of cresyl-violet stained medullary sections in rats at 3 weeks post-3-AP injection revealed selective and total destruction of the neurons in the inferior olive complex (data not shown). The density of [3H]InsP3 binding sites in cerebellum was significantly decreased by 20% with no apparent change in the binding affinity at 21 days after 3-AP injection (Table I). A second injection of 3-AP on day 7 failed to produce a greater decrease in the density of [3H]InsP3 binding sites in the cerebellum (Bmax: control, 24.2 + 2.4 pmol/mg protein; 3-AP, 19.3 + 1.3 pmol/mg protein, P < 0.01; Kd: control, 15.3 + 1.4 nM; 3-AP, 14.0 + 1.3 nM, P > 0.05; n = 7 per group). Neither the density nor the affinity of specific [3H]InsP3 binding was affected when cerebellar membrane of control rats was incubated with 0.5 mM 3-AP (60 mg/kg of 3-AP corresponds to 0.5 mM on the assumption of homogeneous drug distribution) in vitro (data not shown). The reduction in [3H]InsP3 binding density in the cerebellar membranes following 3-AP treatment is timedependent. The [3H]InsP3 binding was not different from
controls at 1 or 4 days post-injection. However, at 7 days post-3-AP injection, there was a statistically significant (P < 0.05) decrease (15%) in the cerebellar [3H]InsP3 binding density, which remained reduced thereafter up to 21 days post-injection (data not shown). In contrast to the results obtained for 3-AP, MAM did not produce statistically significant (P > 0.1) changes in the binding affinity or density of [3H]InsP3 binding sites between treated (K d = 8.1 + 0.6 nM; Bma x = 15.3 + 1.2 pmol/mg protein; n = 6) and control (K d = 8.9 + 0.7 nM; Bma x = 14.6 + 1.2 pmol/mg protein; n = 6) rat cerebella. The results indicate that InsP 3 receptor density, but not binding affinity, is decreased in the cerebellum following 3-AP lesion. The observations that the binding parameters of InsP 3 receptor were not altered by 3-AP in vitro, suggests that the in vivo effect of 3-AP is not related to a direct action on the InsP 3 receptors. The possibility that the decrease in InsP 3 receptor density could simply be a reflection of some Purkinje cell degeneration induced indirectly following 3-AP lesion cannot be excluded, although it seems unlikely, as previous histological studies have indicated that 3-AP treatment of adult rats has no apparent neurotoxic effect on Purkinje cells in the cerebellum 3'6'9'27. The finding that there was a 20% reduction in the InsP 3 receptor density after ablation of the climbing fibres suggests that some of these receptors may be localized on the climbing fibre terminals. Such an interpretation, however, is difficult to reconcile with the apparent lack of [3H]InsP3 binding in the cerebellum of p c d 16 and lurcher 32 mutant mice, and the inability to detect InsP3 receptor protein in presynaptic nerve terminals in contact with Purkinje cell dendrites immunohistochemically 23. It has been shown that, following climbing fibre lesions, Purkinje cells simple spike activity increases markedly 7'2°. As the synaptic transmission between parallel fibre and Purkinje cell is thought to involve the quisqualate-selective glutamate receptor cou-
TABLE I Effect of 3-acetylpyridine on specific [3H]InsP3 binding in the rat cerebellum
Animals received an intraperitoneally injection of 3-acetylpyridine (60 mg/kg) and were killed 21 days later. Specific [3H]InsP3binding was determined in the cerebella as described in the text. Results are the mean + S.E.M. for 8 animals, each rat brain cerebellum being assayed in triplicate for the saturation isotherms. The K a and Bmax were determined by a non-linear curve-fitting program (EBDA).
Control 3-AP
K a (nM)
B,~a~ (pmol/mg protein)
15.1 + 1.6 13.8 + 0.8
23.4 + 1.5 19.8 + 1.5*
*P < 0.01 vs controls (2-tailed Student's t-test).
171 pied to phosphoinositide turnover and increased InsP3 production 5, a m o r e likely possibility is that the decrease in InsP 3 r e c e p t o r density might reflect an adaptive response to the heightened Purkinje cell single spike discharges following climbing fibre deafferentation. In rats t r e a t e d within 24 h of birth with the cytotoxic agent, M A M , there was a selective destruction of granule cells and deafferentation of Purkinje cells which retain n o r m a l dendritic spines in the molecular layer 14. However, d e p l e t i o n of granule cells following M A M treatment did not reduce InsP 3 r e c e p t o r density or affinity in the rat cerebellum. A previous study in reeler m u t a n t mouse which is characterized by a greatly reduced granule cell p o p u l a t i o n in the cerebellum also indicated that [3H]InsP3 binding sites are similar to those of the phenotypically n o r m a l littermate 32. Similarly, the expression of calbindin-D(28k), a Purkinje cell-specific protein, is also not affected by the elimination of granule cells 22. Taken together, the above results suggest that the granule cell-parallel fibre afferents do not regulate the expression
of InsP 3 r e c e p t o r in the P u r k i n j e cells. In addition it raises the possibility that the InsP 3 r e c e p t o r in the cerebellum is solely constitutive to P u r k i n j e cells and its expression is regulated by signals or mechanisms intrinsic to these cells. These findings are consistent with the p r o p o s a l that the d e v e l o p m e n t o f Purkinje cells might be i n d e p e n d e n t of synaptic inputs from the granule cells 1'2'6. In s u m m a r y , the p r e s e n t study shows decreases in cerebeUar InsP 3 r e c e p t o r density following climbing fibre, but not parallel fibre deafferentation, and in addition, suggests a relationship b e t w e e n InsP 3 r e c e p t o r density and r e p o r t e d Purkinje cell simple spike discharges 7'2°. Such changes might affect the p r o p e r t y of calcium oscillations within the Purkinje cells including the frequency and intensity of the oscillation in response to agonist stimulation.
1 Altman, J., Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer, J. Comp. Neurol., 145 (1972) 399-464. 2 Altman, J., Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer, J. Comp. Neurol., 145 (1972) 465-514. 3 Baetens, D., Garcia-Segura, L.M. and Perrelet, A., Effects of CF destruction on large dendrite spines of Purkinje cells, Exp. Brain Res., 48 (1982) 256-262. 4 Berridge, M.J., Inositol trisphosphate and diacylglycerol: two interacting second messengers, Ann. Rev. Biochem., 56 (1987) 159-193. 5 Blackstone, C.D., Supattapone, S. and Synder, S.H., Inositolphospholipid-linked glutamate receptors mediate cerebellar parallel-fibre-Purkinje-cellsynaptic transmission, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 4316-4320. 6 Bradley, P. and Berry, M., Quantitative effects of climbing fibre deafferentation on the adult Purkinje cell dendrite tree, Brain Research, 112 (1976) 133-140. 7 Colin, E, Manil, J. and Desclin, J.C., The olivocerebellar system. I. Delayed and slow inhibitory effects: an overlooked salient feature of cerebellar climbing fibers, Brain Research, 53 (1980) 3-27. 8 Desclin, J.C. and Escubi, J., Effects of 3-acetylpyridine on the central nervous system of the rat as demonstrated by silver methods, Brain Research, 77 (1974) 341-364. 9 Desclin, J.C. and Colin, E, The olivocerebellar system. II. some ultrastructural correlates of inferior olive destruction in the rat, Brain Research, 187 (1980) 29-46. 10 Eccles, J.C., Ito, M. and Szentagothai, J., The Cerebellum as a Neuronal Machine, Springer, New York, 1967. 11 Ferris, C.D., Huganir, R.L., Supattapone, S. and Snyder, S.H., Purified inositol 1,4,5-trisphosphate receptor mediates calcium flux in reconstituted lipid vesicles, Nature, 342 (1989) 87-89. 12 Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. and Mikoshiha, K., Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P,,oo, Nature, 342 (1989) 32-38. 13 Guillemette, G., BaUa, T., Baukal, A.J. and Catt, K.J., Inositol 1,4,5-trisphosphate binds to a specific receptor and releases microsomal calcium in the anterior pituitary gland, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8195-8199.
14 HiUman, D.E., Chen, S. and Ackman, J., Perinatal methylazoxymethanol acetate uncouples coincidence of orientation of cerebellar folia and parallel fibres, Neuroscience, 24 (1988) 99-110. 15 Maeda, N., Niinobe, M., Nakahira, K. and Mikoshiba, K., Purification and characterization of P,~o protein, a glycoprotein characteristic of Purkinje cell, from mouse cerebellum, J. Neurochem., 51 (1988) 1724-1730. 16 Maeda, N., Niinobe, M. and Mikoshiba, K., A cerebellar Purkinje cell marker P4oo protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex, EMBO J., 9 (1990) 61-67. 17 McPherson, G.A., Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC, J. Pharmacol. Methods, 14 (1985) 213-218. 18 Mignery, G.A., Newton, C.L., Archer, B.T. and Sudhof, T.C., Structure and expression of the rat inositol 1,4,5-trisphosphate receptor, J. Biol. Chem., 265 (1990) 12679-12685. 19 Miyawaki, A., Furuichi, T., Maeda, N. and Mikoshiba, K., Expressed cerebellar-type inositol 1,4,5-trisphosphate receptor, P4oo, has calcium release activity in a fibroblast L cell line, Neuron, 5 (1990) 11-18. 20 Montarolo, P.G., Palestini, M. and Strata, P., The inhibitory effect of the olivocerebellar input on the cerebellar Purkinje cells in the rat, J. Physiol., 332 (1982) 187-202. 21 Nicoletti, E, Wroblewski, J.T., Novelli, A., Alho, A., Guidotti, A. and Costa, E., The activation of inositol phospholipid metabolism as a signal-transducing system for excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6 (1989) 1905-1911. 22 Parkes, C.O., Mariani, J. and Thomasset, M., 28K Cholecalcin (CaBP) levels in abnormal cerebella:Studies on mutant mice and harmaline- and 3-acetylpyridine-treated rats, Brain Research, 339 (1985) 265-269. 23 Ross, C.A., Meldolesi J., Milner, T.A., Satoh, T., Supattapone, S. and Snyder, S.H., Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons, Nature, 339 (1989) 468-470. 24 Sandoval, M.E. and Cotman, C.W., Evaluation of glutamate as a neurotransmitter of cerebeUar parallel fibers, Neuroscience, 3 (1978) 199-206. 25 Sedmak, J.J. and Grossherg, S.E., A rapid, sensitive, and
This work was supported in part by grants to EL. (Ontario Mental Health Foundation) and J.W. (Medical Research Council of Canada).
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26 27 28 29
versatile assay for protein using Coomassie brilliant blue G250, Anal. Biochem., 79 (1987) 544-552. Sladeczek, F., Pins, J.P., Recasens, M., Bockaert, J. and Weiss, S., Glutamate stimulates inositoi phosphate formation in striatal neurones, Nature, 317 (1985) 717-719. Sotelo, C., Hillman, D.E., Zamora, A.K. and Llinas, R., Climbing fibre deafferentation: its action on Purkinje cell dendritic spines, Brain Research, 98 (t975) 574-581. Supattapone, S., Worley, P.F., Baraban, J.M. and Snyder, S.H., Solubilization, purification, and characterization of an inositol trisphosphate receptor, J. Biol. Chem., 263 (1988) 1530-1534. Volpe, P., Krause, K-H., Hashimoto, S., Zorzato, E, Pozzan, T., Meldolesi, J. and Lew, D.P., 'Calciosome,' a cytoplasmic organeUe: the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of non-muscle cells?, Proc. Natl. Acad. Sci. U.S.A., 85 (1988)
1091-1095. 30 Wiklund, L., Toggenburger, G. and Cuenod, M., Aspartate: possible neurotransmitter in cerebellar climbing fibres, Science, 246 (1982) 78-80. 31 Worley, P.E, Baraban, J.M., Colvin, J.S. and Snyder, S.H., Inositol trisphosphate receptor localization in brain: variable stoichiometry with protein kinase C, Nature, 325 (1987) 159161. 32 Worley, P.E, Baraban, J.M. and Snyder, S.H., Inositol trisphosphate receptor binding: autoradiographic localization in rat brain, J. Neurosci., 9 (1989) 339-346. 33 Young, L.T., Li, P.E, Kish, S.J., Chiu, A.S. and Warsh, J.J., [3H]Inositol 1,4,5-trisphosphate binding in human cerebral cortex, Neurosci. Lett., 87 (1988) 283-287.
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A study of cerebellar inositol 1,4,5-trisphosphate receptor following climbing and parallel fibre deafferentation Peter P. Li 1, Marty A. Green I and Jerry J. W a r s h 1'2'3 1Section of Biochemical Psychiatry, Clarke Institute of Psychiatry, 2Department of Pharmacology and 3Institute of Medical Sciences, University of Toronto, Toronto, Ont. (Canada) (Accepted 23 April 1991)
Key words: Inositol 1,4,5-trisphosphate receptor; Cerebellum; Purkinje cell; Inferior olive-climbing fiber; Granule cell-parallel fiber
We have examined the influence of climbing fibre and parallel fibre afferent inputs on the inositol 1,4,5-trisphosphate (InsP3) receptor in rat cerebellum. Lesions of the inferior olive-climbing fibre projections to Purkinje cells by 3-acetylpyridine (3-AP) significanlty reduced the [3H]InsP3 binding density (-20%) with no apparent changes in the binding affinity 21 days after treatment. No further reduction in binding density was found in rats given a second dose 7 days after the initial injection. A significant reduction in the binding density was evident as early as 7 days post-lesion. However, 3-AP (0.5 mM) failed to inhibit [3H]InsP3 binding in vitro. Cerebellar granule cells were lesioned by two consecutive injections of methylazoxymethanol acetate at birth. In these 60-day-old granuloprival rats, the density and affinity of [3H]InsPa binding sites in the cerebellum remained comparable to the controls. Since lesions of the climbing fibres increase Purkinje cell activity, we suggest that changes in InsP3 receptor density may reflect an adaptative response to the heightened Purkinje cell activity. In addition, the results also indicate that expression of the InsP3 receptor in the cerebellum is largely independent of the presence of granule cell-parallel fibre synaptic innervation onto the Purkinje cells.
The pivotal role of inositol 1,4,5-trisphosphate (InsP3) as a calcium-mobilizing second messenger for many hormonal, neurotransmitter and growth factor signals h a s been well documented in a variety of cells 4. InsP 3 releases calcium from non-mitochondrial intracellular stores 4,29 through binding to specific receptors which have been identified in a variety of tissues 13,31,33. The InsP 3 receptor has been purified in rat 28 and mouse 15A6 cerebellum and more recently, the complementary D N A s encoding the InsP 3 receptor have been cloned x2,1s. Reconstitution and functional expression studies have revealed that the receptor protein behaves as an ionophoric receptor containing both recognition sites for InsP 3 and the calcium channel 11,19. In brain InsP 3 receptors are heterogeneously distributed with highest concentrations found in the cerebellum, where they appear to be selectively localized to Purkinje cell dendrites in the molecular layer 16,23,31,32. The cerebellar Purkinje cells are innervated by two major excitatory afferent systems: the climbing fibres which originate from the medullary inferior olivary nucleus, and the parallel fibres which derive from cerebellar granule cells 1°. Glutamate and aspartate appear to be the putative neurotransmitters released by the parallel and
the climbing fibres, respectively 24,3°. These excitatory amino acids are known to enhance phosphoinositide turnover with increased InsP 3 formation 21,26. The present work was aimed at determining whether the climbing or parallel fibre innervation of Purkinje cells transsynaptically modulate cerebellar InsP a receptors. To produce climbing fibre lesions, male Wistar rats (250-300 g; Charles River, Montreal) were injected intraperitoneally with 3-acetylpyridine (3-AP; 60 mg/kg, Sigma), a neurotoxin which produces selective destruction of the inferior olive-climbing fibre system s . Control animals received isotonic saline injections. Surviving animals were ataxic and showed difficulty in coordinating their movements. Animals were killed 21 days after the drug administration, and the cerebella quickly removed, frozen and stored at -70 °C. The extent of lesion was determined by examining Cresyl violet-stained sections of the inferior olive for surviving cells. To determine the effect of eliminating parallel fibre inputs on [3H]InsP3 binding, newborn pups at postpartum days 1 and 2 received subcutaneous injections of methylazoxymethanol acetate ( M A M , an antimitotic agent) at 20 mg/kg and 15 mg/kg, respectively 14. Controls were littermates that received isotonic saline. All animals
Correspondence: P.P. Li, Section of Biochemical Psychiatry, Clarke Institute of Psychiatry, 250 College Street, Toronto, Ont., Canada, M5T 1R8. Fax: (1) (416) 979-7871.
170 were weaned at 21 days of age and were killed at 60 days of age. Cerebella were removed and frozen for assay as described above. Specific [3H]InsP3 binding was determined as previously described 33 with minor modifications. Thawed cerebella were homogenized in 40 vols (w/v) of ice cold Tris-EDTA buffer (50 mM Tris-HC1, 1 mM EDTA, pH 8.3). The homogenates were then centrifuged at 35,000 g for 20 min at 4 °C, after which the supernatants were decanted and the tissue pellets were resuspended in buffer and used immediately in binding assays. The incubation mixtures contained approximately 0.3-0.5 mg membrane protein, 0.2 nM [3H]InsP3 (47 Ci/mmol; Amersham), InsP 3 (0-1000 nM; Sigma) and 1 mg/ml bovine serum albumin in a total volume of 1 ml of Tris-EDTA buffer. Triplicate samples were incubated at 4 °C for 10 min followed by centrifugation at 12,000 g for 10 min. The pellets were washed with 1 ml ice-cold buffer and subsequently solubilized with 0.5% sodium dodecyl sulphate overnight. The radioactivity in the pellets was quantified by liquid scintillation spectrometry (counting efficiency 50%). Non-specific binding was defined as the amount of radioactivity bound in the presence of 1/~M InsP 3. The protein content was determined using bovine serum albumin as the standard 25. Analyses of competition data and estimates of receptor binding parameters were performed using the E B D A computer program 17. Data were expressed as mean ___ S.E.M. and analyzed by two-tailed Student's t-tests or analysis of variance using the SYSTAT software program (SYSTAT, Evanston, IL). Mean differences with Pvalues < 0.05 were considered statistically significant. Histological examination of cresyl-violet stained medullary sections in rats at 3 weeks post-3-AP injection revealed selective and total destruction of the neurons in the inferior olive complex (data not shown). The density of [3H]InsP3 binding sites in cerebellum was significantly decreased by 20% with no apparent change in the binding affinity at 21 days after 3-AP injection (Table I). A second injection of 3-AP on day 7 failed to produce a greater decrease in the density of [3H]InsP3 binding sites in the cerebellum (Bmax: control, 24.2 + 2.4 pmol/mg protein; 3-AP, 19.3 + 1.3 pmol/mg protein, P < 0.01; Kd: control, 15.3 + 1.4 nM; 3-AP, 14.0 + 1.3 nM, P > 0.05; n = 7 per group). Neither the density nor the affinity of specific [3H]InsP3 binding was affected when cerebellar membrane of control rats was incubated with 0.5 mM 3-AP (60 mg/kg of 3-AP corresponds to 0.5 mM on the assumption of homogeneous drug distribution) in vitro (data not shown). The reduction in [3H]InsP3 binding density in the cerebellar membranes following 3-AP treatment is timedependent. The [3H]InsP3 binding was not different from
controls at 1 or 4 days post-injection. However, at 7 days post-3-AP injection, there was a statistically significant (P < 0.05) decrease (15%) in the cerebellar [3H]InsP3 binding density, which remained reduced thereafter up to 21 days post-injection (data not shown). In contrast to the results obtained for 3-AP, MAM did not produce statistically significant (P > 0.1) changes in the binding affinity or density of [3H]InsP3 binding sites between treated (K d = 8.1 + 0.6 nM; Bma x = 15.3 + 1.2 pmol/mg protein; n = 6) and control (K d = 8.9 + 0.7 nM; Bma x = 14.6 + 1.2 pmol/mg protein; n = 6) rat cerebella. The results indicate that InsP 3 receptor density, but not binding affinity, is decreased in the cerebellum following 3-AP lesion. The observations that the binding parameters of InsP 3 receptor were not altered by 3-AP in vitro, suggests that the in vivo effect of 3-AP is not related to a direct action on the InsP 3 receptors. The possibility that the decrease in InsP 3 receptor density could simply be a reflection of some Purkinje cell degeneration induced indirectly following 3-AP lesion cannot be excluded, although it seems unlikely, as previous histological studies have indicated that 3-AP treatment of adult rats has no apparent neurotoxic effect on Purkinje cells in the cerebellum 3'6'9'27. The finding that there was a 20% reduction in the InsP 3 receptor density after ablation of the climbing fibres suggests that some of these receptors may be localized on the climbing fibre terminals. Such an interpretation, however, is difficult to reconcile with the apparent lack of [3H]InsP3 binding in the cerebellum of p c d 16 and lurcher 32 mutant mice, and the inability to detect InsP3 receptor protein in presynaptic nerve terminals in contact with Purkinje cell dendrites immunohistochemically 23. It has been shown that, following climbing fibre lesions, Purkinje cells simple spike activity increases markedly 7'2°. As the synaptic transmission between parallel fibre and Purkinje cell is thought to involve the quisqualate-selective glutamate receptor cou-
TABLE I Effect of 3-acetylpyridine on specific [3H]InsP3 binding in the rat cerebellum
Animals received an intraperitoneally injection of 3-acetylpyridine (60 mg/kg) and were killed 21 days later. Specific [3H]InsP3binding was determined in the cerebella as described in the text. Results are the mean + S.E.M. for 8 animals, each rat brain cerebellum being assayed in triplicate for the saturation isotherms. The K a and Bmax were determined by a non-linear curve-fitting program (EBDA).
Control 3-AP
K a (nM)
B,~a~ (pmol/mg protein)
15.1 + 1.6 13.8 + 0.8
23.4 + 1.5 19.8 + 1.5*
*P < 0.01 vs controls (2-tailed Student's t-test).
171 pied to phosphoinositide turnover and increased InsP3 production 5, a m o r e likely possibility is that the decrease in InsP 3 r e c e p t o r density might reflect an adaptive response to the heightened Purkinje cell single spike discharges following climbing fibre deafferentation. In rats t r e a t e d within 24 h of birth with the cytotoxic agent, M A M , there was a selective destruction of granule cells and deafferentation of Purkinje cells which retain n o r m a l dendritic spines in the molecular layer 14. However, d e p l e t i o n of granule cells following M A M treatment did not reduce InsP 3 r e c e p t o r density or affinity in the rat cerebellum. A previous study in reeler m u t a n t mouse which is characterized by a greatly reduced granule cell p o p u l a t i o n in the cerebellum also indicated that [3H]InsP3 binding sites are similar to those of the phenotypically n o r m a l littermate 32. Similarly, the expression of calbindin-D(28k), a Purkinje cell-specific protein, is also not affected by the elimination of granule cells 22. Taken together, the above results suggest that the granule cell-parallel fibre afferents do not regulate the expression
of InsP 3 r e c e p t o r in the P u r k i n j e cells. In addition it raises the possibility that the InsP 3 r e c e p t o r in the cerebellum is solely constitutive to P u r k i n j e cells and its expression is regulated by signals or mechanisms intrinsic to these cells. These findings are consistent with the p r o p o s a l that the d e v e l o p m e n t o f Purkinje cells might be i n d e p e n d e n t of synaptic inputs from the granule cells 1'2'6. In s u m m a r y , the p r e s e n t study shows decreases in cerebeUar InsP 3 r e c e p t o r density following climbing fibre, but not parallel fibre deafferentation, and in addition, suggests a relationship b e t w e e n InsP 3 r e c e p t o r density and r e p o r t e d Purkinje cell simple spike discharges 7'2°. Such changes might affect the p r o p e r t y of calcium oscillations within the Purkinje cells including the frequency and intensity of the oscillation in response to agonist stimulation.
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This work was supported in part by grants to EL. (Ontario Mental Health Foundation) and J.W. (Medical Research Council of Canada).
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