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Concentration- and cell type-specific effects of calbindin D28k on vulnerability of hippocampal neurons to seizure-induced injury
Molecular Brain Research 75 Ž2000. 89–95 www.elsevier.comrlocaterbres
Research report
Concentration- and cell type-specific effects of calbindin D28k on vulnerability of hippocampal neurons to seizure-induced injury Devin S. Gary a , Karen Sooy b, Sic L. Chan a , Sylvia Christakos b, Mark P. Mattson
a,c,)
a
Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, UniÕersity of Kentucky, 211 Sanders-Brown Building, 800 South Limestone Street, Lexington, KY 40536, USA b Departments of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical and Graduate School of Biomedical Sciences, 185 South Orange AÕenue, Newark, NJ 07103, USA c Laboratory of Neurosciences, National Institute on Aging, 5600 Nathan Shock DriÕe, Baltimore, MD 21224, USA Accepted 5 October 1999
Abstract The calcium-binding protein calbindin D28k ŽCB. is expressed in limited subpopulations of neurons in the brain. In the hippocampus, CB is expressed in all dentate granule cells and a subpopulation of CA1 pyramidal neurons, but is absent from CA3 neurons. This pattern of CB expression is inversely correlated with neuronal vulnerability to seizure-induced damage suggesting the possibility that expression of CB confers resistance to excitotoxicity. While data from cell culture studies support an excitoprotective role for calbindin, it is not known whether CB is a key determinant of neuronal vulnerability in vivo. We therefore examined the pattern of damage to hippocampal neurons following intrahippocampal injection of the seizure-inducing excitotoxin kainate in CB homozygous ŽCB y ry . and CB heterozygous ŽCB q ry . knockout mice in comparison with wild-type mice ŽCB q rq .. Whereas the extent of damage to CA1 neurons was similar in CB y ry and CB q rq mice, damage to CA1 neurons was significantly reduced in CB q ry mice. Dentate granule neurons were not damaged following kainate-induced seizures in CB q rq, CB q ry or CB y ry mice. These findings suggest that CB can modify vulnerability of hippocampal CA1 neurons to seizure-induced injury, and that either CB is not a critical determinant of resistance of dentate granule neurons, or compensatory changes occur and lack of CB is not the only difference between CB y ry and CB q rq mice. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Calcium; Epilepsy; Excitotoxicity; Kainic acid; Microtubule associated protein; Spectrin
1. Introduction A major issue in the field of neurodegenerative disorder research concerns the selectivity of neuronal cell death. In both acute and chronic neurodegenerative conditions, only specific regions of the brain exhibit neuronal degeneration, and within those regions only certain neurons degenerate. For example, CA1 hippocampal neurons are vulnerable in stroke w28x and Alzheimer’s disease w11x, hippocampal CA3 and CA1 neurons are vulnerable to severe epileptic seizures w32x, striatal medium spiny neurons degenerate in Huntington’s disease w2x, and lower motor neurons in the spinal cord are vulnerable in amyotrophic lateral sclerosis w8x. The cellular and molecular mechanisms responsible for ) Corresponding author. [email protected]
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promoting or preventing neuronal death in such disorders are largely unknown. Much evidence supports a role for excessive elevation of intracellular calcium levels, resulting from overactivation of glutamate receptors under conditions of metabolic compromise and oxidative stress, in the pathogenesis of neuronal injury and death in each of the abovementioned disorders Žsee Refs. w22,33x for review.. It is therefore important to understand the extent to which differential expression of specific calcium-regulating proteins contributes the selective vulnerability of particular populations of neurons in neurodegenerative disorders. Administration of the excitotoxin kainic acid ŽKA. to laboratory rodents is a model of epilepsy that induces seizure activity and results in death of pyramidal neurons in regions CA3 and CA1 of the hippocampus w4,27x. This death is due largely to calcium influx resulting from the
0169-328Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 2 9 9 - 5
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activation of glutamate receptors. There is an interesting association between expression of the calcium-binding protein calbindin D28k ŽCB. and selective vulnerability to seizure-induced injury in that resistant dentate granule neurons contain high levels of calbindin, whereas vulnerable CA3 and CA1 neurons contain little or no calbindin w36x. Such correlations between expression of calcium-binding proteins and apparent resistance to neuronal degeneration in vivo have prompted cell culture studies that have directly addressed the issue of whether these proteins are in fact neuroprotective. Cultured hippocampal neurons expressing CB are relatively resistant to excitotoxic and metabolic insults and amyloid b-peptide toxicity w6,9,19,25x, and PC12 cells overexpressing CB exhibit increased resistance to apoptosis w41x, suggesting a neuroprotective role for CB. However, many factors in addition to expression of CB are likely to contribute to neuronal vulnerability in vivo. Examples include: differential inputs from excitatory and inhibitory neurons w19x; relative levels of expression of different subtypes of glutamate receptors w7x; levels of neurotrophic support w24x; and complement of stress proteins w17,43x. Moreover, it was recently reported that the extent of damage to cortical and striatal neurons following focal cerebral ischemia was decreased in mice lacking CB w13x, suggesting that CB may increase neuronal vulnerability under certain conditions. In order to directly address the role of CB in the selective vulnerability of hippocampal neurons to seizure-induced damage, we performed studies in mice expressing normal levels of CB, a reduced level of CB, or no CB w1x. Our data suggest that CB exerts a concentration-dependent effect on vulnerability of CA1 neurons to seizure-induced injury, and is either not responsible for resistance of dentate granule neurons or compensatory proteins are induced in the CB y ry mice.
2. Materials and methods 2.1. Mice and KA administration The generation of the CB y ry mice was accomplished by a targeting approach described in detail previously w1x; mice were maintained on a C57BLr6= 129 background. Experiments were performed in young adult Ž3–4 months old. male homozygous ŽCB y ry . and heterozygous ŽCB q ry . CB knockout mice and wild-type littermate mice ŽCB q rq .. The CB y ry mice exhibit no overt phenotype, but do show deficits in motor coordination, and alterations in calcium regulation in cerebellar Purkinje cells w1x. We have observed no differences in hippocampal structure, or numbers of neuronsrsection in any of the subpopulations of hippocampal neurons ŽCA1, CA3 and dentate granule cells.. KA was injected stereotaxically into the dorsal aspect of the right hippocampus using methods described previously
w4,10x. Briefly, mice were anesthetized with Avertin, placed in a stereotaxic head holder, and the skull exposed along the midline. A convulsant dose of kainate Ž0.3 mgr0.5 ml sterile saline, pH 7.2. was injected unilaterally into dorsal hippocampus Žstereotaxic coordinates: AP s 2.0 mm posterior to bregma, ML s 2.4 mm lateral to bregma, DV s 1.8 mm below the surface of the skull.. Control mice were injected with 0.5 ml saline. Twenty-four hours following kainate administration, mice were anesthetized with sodium pentobarbital and perfused transcardially with saline followed by cold phosphate-buffered 4% paraformaldehyde. 2.2. Histological and Western blot analyses Coronal brain sections Ž30 mM. were cut on a freezing microtome and stained with Cresyl violet. Neuronal damage was quantified by methods similar to those used in our previous studies w4,10x. Briefly, Cresyl violet-positive undamaged neurons were counted in three 45 = fields in each region of interest ŽCA1, CA3 and upper limb of the dentate gyrus. in both the left and right hippocampi of each mouse. Neurons in which the cell body was crenated and the nucleus not readily visible were considered damaged, whereas neurons with a round to oval cell body and a visible nucleus were considered undamaged. Counts were made in three coronal brain sections from the middle third of the hippocampus in each mouse Žsections were chosen by unbiased sampling. and the mean number of undamaged neuronsrfieldrbrain was determined. Determinations were made in 11–13 micergroup. Comparisons of numbers of undamaged neurons in hippocampal regions among treatment groups were made using ANOVA followed by Fischer’s t-test for pairwise comparisons. Immunohistochemistry was performed in free-floating sections using methods described previously w4x. Briefly, sections were incubated for 2 h at room temperature in a solution containing 0.2% Triton X-100 and 0.015% blocking serum Žnormal goat serum for the spectrin and CB antibodies and normal horse serum for the MAP-2 antibody. in PBS. Primary antibody was then added to a final dilution of either 1:10,000 for the spectrin antibody or 1:500 for the MAP-2 antibody, and sections were incubated overnight at 48C. Sections were then washed with PBS, incubated for 2 h at room temperature in the presence of biotinylated secondary antibody, followed by washing in PBS. Sections were then incubated for 1 h in ABC reagent ŽVector Laboratories., washed in PBS, and incubated for 5 min in DAB solution ŽVector Laboratories.. Western blot analysis was performed as described previously Ž12% polyacrylamide gel. using CB antibody at a dilution of 1:1500 w6,20x. The primary antibodies were rabbit polyclonal antibodies specific for CB w20x, a calpain cleavage product of spectrin Žgenerous gift from R. Siman at Cephalon., and a mouse monoclonal antibody against MAP-2 Žclone AP20; Sigma..
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3. Results and discussion Relative levels of CB protein in hippocampi of CB q rq, CB q ry and CB y ry mice were determined by Western blot analysis. Hippocampi from CB y ry mice contained no detectable CB, and the CB level in hippocampi from CB q ry mice was approximately 50% lower than the level in hippocampi from CB q rq mice ŽFig. 1A.. Levels of CB immunoreactivity were high in the cell bodies, dendrites and axons Žmossy fibers. of essentially all granule cells ŽFig. 1B.. CB immunoreactivity was present at relatively low levels in CA1 pyramidal neurons and was absent from CA3 neurons. In order to elucidate the possible modulatory roles for CB on seizure-induced injury to hippocampal neurons, we injected KA into the dorsal hippocampus of CB q rq, CB q ry and CB y ry mice. KA-induced damage to CA1 neurons in the ipsilateral hippocampus was significantly less in CB q ry mice compared to CB q rq and CB y ry mice ŽFigs. 2 and 3.. There was a trend towards reduced damage to CA3 neurons in CB q ry mice, although it fell short of statistical significance Ž p - 0.10.. There was no evidence of damage to dentate granule neurons in mice of any of the three genotypes Ždata not shown.. Mice from all the groups showed moderate damage to CA1 and CA3 neurons in the contralateral hippocampus, and no significant differences were seen among the three groups ŽFig. 3.. In order to relate the data on neuronal damage to alterations in neuronal calcium homeostasis, we also performed immunohistochemical analyses using antibodies against calpaincleaved spectrin and MAP-2, two markers of calciummediated proteolysis w12,29,34,35x. KA-induced a large increase in levels of calpain-cleaved spectrin immunoreactivity in regions CA1 and CA3 of CB q rq and CB y ry mice, and the level of the increase was lower in region CA1 the CB q ry mice ŽFig. 4.. Levels of MAP-2 immunoreactivity were markedly reduced in regions CA1 and CA3 of KA-injected CB q rq and CB y ry mice ŽFig. 5.. The extent of decrease in MAP-2 immunoreactivity in region CA1 was less in the CB q ry mice compared to mice of the other two genotypes. The present findings suggest that the level of CB may modify neuronal vulnerability to seizure-induced injury, but that its effects can be concentration-dependent and cell type-specific. When calbindin levels were artificially lowered to approximately half the normal level, CA1 neurons were more resistant to seizure-induced injury. However, complete lack of calbindin did not further increase resistance of CA1 neurons, but rather increased vulnerability. Although the mechanism whereby intermediate levels of CB allow for optimal survival of CA1 neurons is unknown, previous findings suggest several possibilities. Based upon their observations that ischemic neuronal injury is decreased in CB y ry mice and electrophysiological analyses, Klapstein et al. w13x proposed that lack of CB results in increased activation of calcium-sensitive potas-
Fig. 1. Characterization of calbindin expression in wild-type mice and CB knockout mice. ŽA. Western blot analysis of CB in hippocampal homogenates Žeach lane was loaded with 50 mg protein. from CBqrq, CBqry and CByry mice. ŽB. Hippocampal sections from a CBq rq mouse Župper. and a CByry mouse Žlower. immunostained with CB antibody. Note high levels of CB immunoreactivity in dentate granule cells Žincluding their mossy fiber terminals in region CA3., moderate levels of immunoreactivity in CA1 neurons, and lack of immunoreactivity in CA3 neurons.
sium channels. Perhaps the moderate decrease in CB levels in CB q ry mice might allow for activation of such potassium channels as well as for calcium buffering. Studies of developing neurons in culture have shown that there is a narrow range of intracellular free calcium levels that is optimal for promoting cell survival and neurite outgrowth w18,21x. It may therefore be the case that an intermediate levels of CB results in an intracellular calcium concentration that is within the optimum range. A study of the relationship between cellular expression of CB and neuronal vulnerability to transient global forebrain ischemia in gerbils revealed a strong inverse correlation in immature animals ŽCA3 neurons and neurons at the base of the granule cell layer that lacked CB were vulnerable., but less of a correlation in adult animals Žonly CA1 neurons being vulnerable. w38x. The latter findings suggest that as hip-
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Fig. 2. Micrographs showing Cresyl violet staining of region CA1 of hippocampus in a CB q rq control mouse 24 h following administration of saline Župper left., and CB q rq, CB q ry and CB y ry mice 24 h following administration of KA Žsee Section 2; photographs were taken using a 40 = objective.. Note increased damage to CA1 neurons in CB q rq and CB y ry mice compared to the CB q ry mouse.
pocampal circuitry matures, factors in addition to CB expression come into play in determining selective vulnerability. Dentate granule neurons were not damaged by KA in mice of any of the three genotypes, indicating that the resistance of the granule neurons to seizure-induced injury is not the result of their high content of CB or that compensatory changes occur and the lack of calbindin is not the only difference between CB y ry and CB q rq mice. Multiple factors that confer resistance of dentate granule neurons may be involved. One possibility is that the position of the granule neurons in the circuitry involved in KA-induced seizures is such that they are subjected to less severe excitation. However, studies of levels of excitability and gene expression in dentate granule neurons following seizure induction w37,40x suggest that granule neurons are subjected to high levels of glutamate receptor activation. A second possibility is that dentate neurons express, or are exposed to, multiple proteins that make them resistant to injury. For example, levels of antioxidant enzymes have been shown to be related to neuronal vulnerability to excitotoxic and metabolic injury in cell culture studies w23x, and differences in the cellular localization of such enzymes have been demonstrated in hippocampus of adult rodents w14,39x. The expression of several neurotrophic factors including BDNF, NGF and bFGF is rapidly increased in dentate gyrus following seizure induction w30x, and these factors have been shown to protect hippocampal neurons against excitotoxic insults
Žsee Ref. w24x for review.. In addition, certain stress-responsive proteins are greatly increased in dentate granule neurons following seizures w15,16x.
Fig. 3. Quantification of KA-induced damage to hippocampal neurons. Mice were administered KA or saline, via unilateral injection into the dorsal hippocampus. Twenty-four hours later, mice were euthanized and coronal brain sections stained with Cresyl violet. Numbers of undamaged neurons in regions CA1 and CA3 of ipsilateral Žip. and contralateral Žcon. hippocampus were quantified Žsee Section 2.. Values are the mean and S.E.M. Ž ns11s13 mice.. U p- 0.01 compared to corresponding values for CBqrq and CByry mice.
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Fig. 4. Micrographs showing hippocampi in brain sections stained with an antibody against calpain-cleaved spectrin. Mice were administered KA or saline, via unilateral injection into the dorsal hippocampus. Twenty-four hours later, mice were euthanized and coronal brain sections were immunostained with an antibody against calpain-cleaved spectrin. Note increased immunoreactivity in region CA1 of CB q rq and CB y ry mice compared to the CB q ry mouse.
Fig. 5. Micrographs showing hippocampi in brain sections stained with an antibody against MAP-2. Mice were administered KA or saline, via unilateral injection into the dorsal hippocampus. Twenty-four hours later, mice were euthanized and coronal brain sections were immunostained with an antibody against MAP-2. Note decreased immunoreactivity in region CA1 of CB q rq and CB y ry mice compared to the CB q ry mouse.
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The patterns of KA-induced proteolysis of spectrin and MAP-2 observed in the present study were similar to those observed in previous studies in which status epilepticus was induced in rats by KA or electrical stimulation w3,34,35,42x. The reduced levels of MAP-2 and spectrin proteolysis in region CA1 of CB q ry mice tightly paralleled the decreased vulnerability of CA1 neurons in the CB q ry mice. These findings are consistent with a role for cellular calcium overload in the neuronal damage, and suggest a pivotal role for CB in protecting CA1 neurons against seizure-induced injury. The lack of increased vulnerability of CA3 neurons in CB y ry mice, and the complete resistance of dentate granule neurons to seizureinduced injury in both CB q rq and CB y ry mice further suggests either a specific role for CB expression in resistance of CA1 neurons to excitotoxic injury, or the induction of compensatory proteins in certain brain regions of the CB y ry mice. In addition, since specific spatiotemporal patterns of neuronal damage w17x and calpain activation w3x have been reported after KA treatment, examination of changes in CB q rq, CB q ry and CB y ry mice at various times after KA administration may provide insight related to differential early andror longterm effects of calbindin in discrete brain regions. Because excitotoxicity may contribute to the degeneration of hippocampal CA1 neurons in disorders ranging from stroke w31x to Alzheimer’s disease w22x, our findings suggest a role for CB in suppressing degeneration of CA1 neurons in those disorders. Moreover, beyond its apparent influence on neuronal vulnerability to excitotoxicity, CB may play roles in modulating the various physiological processes that involve glutamate receptors and calcium influx. As evidence, cultured hippocampal neurons overexpressing calbindin show no alteration in evoked neurotransmitter release, but do exhibit a suppression of posttetanic potentiation w5x consistent with a postsynaptic localization of calbindin and decrease in the magnitude of calcium elevation in the postsynaptic region. Moreover, Molinari et al. w26x reported impaired hippocampal longterm potentiation and spatial memory deficits in mice with reduced levels of CB as the result of overexpression of CB antisense. Thus, physiological and pathophysiological roles for CB appear quite complex. Acknowledgements This work was supported by the National Institute on Aging. References w1x M.S. Airaksinen, J. Eilers, O. Garaschuk, H. Thoenen, A. Konnerth, M. Meyer, Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene, Proc. Natl. Acad. Sci. U.S.A. 94 Ž1997. 1488–1493.
w2x M.F. Beal, R.J. Ferrante, K.J. Swartz, N.W. Kowall, Chronic quinolinic acid lesions in rats closely resemble Huntington’s disease, J. Neurosci. 11 Ž1991. 1649–1659. w3x X. Bi, V. Chang, R. Siman, G. Tocco, M. Baudry, Regional distribution and time-course of calpain activation following kainateinduced seizure activity in adult rat brain, Brain Res. 726 Ž1996. 98–108. w4x A.J. Bruce, W. Boling, M.S. Kindy, J. Peschon, P.J. Kraemer, M.K. Carpenter, F.W. Holtsberg, M.P. Mattson, Altered neuronal and microglial responses to brain injury in mice lacking TNF receptors, Nat. Med. 2 Ž1996. 788–794. w5x P.S. Chard, J. Jordan, C.J. Marcuccilli, R.J. Miller, J.M. Leiden, R.P. Roos, G.D. Ghadge, Regulation of excitatory transmission at hippocampal synapses by calbindin D28k, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 5144–5148. w6x B. Cheng, S. Christakos, M.P. Mattson, Tumor necrosis factors protect neurons against excitotoxicrmetabolic insults and promote maintenance of calcium homeostasis, Neuron 12 Ž1994. 139–153. w7x D.W. Choi, Glutamate neurotoxicity and diseases of the nervous system, Neuron 1 Ž1988. 623–634. w8x S.M. Chou, H.S. Wang, K. Komai, Colocalization of NOS and SOD1 in neurofilament accumulation within motor neurons of amyotrophic lateral sclerosis: an immunohistochemical study, J. Chem. Neuroanat. 10 Ž1996. 249–258. w9x Q. Guo, S. Christakos, N. Robinson, M.P. Mattson, Calbindin blocks the pro-apoptotic actions of mutant presenilin-1: reduced oxidative stress and preserved mitochondrial function, Proc. Natl. Acad. Sci. U.S.A. 95 Ž1998. 3227–3232. w10x Q. Guo, W. Fu, B.L. Sopher, M.W. Miller, C.B. Ware, G.M. Martin, M.P. Mattson, Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice, Nat. Med. 5 Ž1999. 101–106. w11x B.T. Hyman, G.W. Van Hoesen, A.R. Damasio, C.L. Barnes, Alzheimer’s disease: cell-specific pathology isolates the hippocampal formation, Science 225 Ž1984. 1168–1170. w12x G.V. Johnson, J.M. Litersky, R.S. Jope, Degradation of microtubule-associated protein 2 and brain spectrin by calpain: a comparative study, J. Neurochem. 56 Ž1991. 1630–1638. w13x G.J. Klapstein, S. Vietla, D.N. Lieberman, P.A. Gray, M.S. Airaksinen, H. Thoenen, M. Meyer, I. Mody, Calbindin D28k fails to protect hippocampal neurons against ischemia in spite of its cytoplasmic calcium buffering properties: evidence from calbindin D28k knockout mice, Neuroscience 85 Ž1998. 361–373. w14x S. Knollema, H.W. Hom, H. Schirmer, J. Korf, G.J. Ter Horst, Immunolocalization of glutathione reductase in the murine brain, J. Comp. Neurol. 373 Ž1996. 157–172. w15x E. Little, G. Tocco, M. Baudry, A.S. Lee, S.S. Schreiber, Induction of glucose-regulated protein Žglucose-regulated protein 78rBiP and glucose-regulated protein 94. and heat shock protein 70 transcripts in the immature rat brain following status epilepticus, Neuroscience 75 Ž1996. 209–219. w16x D.H. Lowenstein, The stress protein response and its potential relationship to prolonged seizure activity, Clin. Neuropharmacol. 18 Ž1995. 148–158. w17x Z. Magloczky, T.F. Freund, Selective neuronal death in the contralateral hippocampus following unilateral kainate injection into the CA3 subfield, Neuroscience 56 Ž1993. 317–336. w18x M.P. Mattson, S.B. Kater, Calcium regulation of neurite elongation and growth cone motility, J. Neurosci. 7 Ž1987. 4034–4043. w19x M.P. Mattson, S.B. Kater, Excitatory and inhibitory neurotransmitters in the generation and degeneration of hippocampal neuroarchitecture, Brain Res. 478 Ž1989. 337–348. w20x M.P. Mattson, B. Rychlik, C. Chu, S. Christakos, Evidence of calcium-reducing and excitoprotective roles for the calcium-binding protein calbindin D28k in cultured hippocampal neurons, Neuron 6 Ž1991. 41–51.
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Research report
Concentration- and cell type-specific effects of calbindin D28k on vulnerability of hippocampal neurons to seizure-induced injury Devin S. Gary a , Karen Sooy b, Sic L. Chan a , Sylvia Christakos b, Mark P. Mattson
a,c,)
a
Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, UniÕersity of Kentucky, 211 Sanders-Brown Building, 800 South Limestone Street, Lexington, KY 40536, USA b Departments of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical and Graduate School of Biomedical Sciences, 185 South Orange AÕenue, Newark, NJ 07103, USA c Laboratory of Neurosciences, National Institute on Aging, 5600 Nathan Shock DriÕe, Baltimore, MD 21224, USA Accepted 5 October 1999
Abstract The calcium-binding protein calbindin D28k ŽCB. is expressed in limited subpopulations of neurons in the brain. In the hippocampus, CB is expressed in all dentate granule cells and a subpopulation of CA1 pyramidal neurons, but is absent from CA3 neurons. This pattern of CB expression is inversely correlated with neuronal vulnerability to seizure-induced damage suggesting the possibility that expression of CB confers resistance to excitotoxicity. While data from cell culture studies support an excitoprotective role for calbindin, it is not known whether CB is a key determinant of neuronal vulnerability in vivo. We therefore examined the pattern of damage to hippocampal neurons following intrahippocampal injection of the seizure-inducing excitotoxin kainate in CB homozygous ŽCB y ry . and CB heterozygous ŽCB q ry . knockout mice in comparison with wild-type mice ŽCB q rq .. Whereas the extent of damage to CA1 neurons was similar in CB y ry and CB q rq mice, damage to CA1 neurons was significantly reduced in CB q ry mice. Dentate granule neurons were not damaged following kainate-induced seizures in CB q rq, CB q ry or CB y ry mice. These findings suggest that CB can modify vulnerability of hippocampal CA1 neurons to seizure-induced injury, and that either CB is not a critical determinant of resistance of dentate granule neurons, or compensatory changes occur and lack of CB is not the only difference between CB y ry and CB q rq mice. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Calcium; Epilepsy; Excitotoxicity; Kainic acid; Microtubule associated protein; Spectrin
1. Introduction A major issue in the field of neurodegenerative disorder research concerns the selectivity of neuronal cell death. In both acute and chronic neurodegenerative conditions, only specific regions of the brain exhibit neuronal degeneration, and within those regions only certain neurons degenerate. For example, CA1 hippocampal neurons are vulnerable in stroke w28x and Alzheimer’s disease w11x, hippocampal CA3 and CA1 neurons are vulnerable to severe epileptic seizures w32x, striatal medium spiny neurons degenerate in Huntington’s disease w2x, and lower motor neurons in the spinal cord are vulnerable in amyotrophic lateral sclerosis w8x. The cellular and molecular mechanisms responsible for ) Corresponding author. [email protected]
Fax:
q 1-410-558-8465;
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promoting or preventing neuronal death in such disorders are largely unknown. Much evidence supports a role for excessive elevation of intracellular calcium levels, resulting from overactivation of glutamate receptors under conditions of metabolic compromise and oxidative stress, in the pathogenesis of neuronal injury and death in each of the abovementioned disorders Žsee Refs. w22,33x for review.. It is therefore important to understand the extent to which differential expression of specific calcium-regulating proteins contributes the selective vulnerability of particular populations of neurons in neurodegenerative disorders. Administration of the excitotoxin kainic acid ŽKA. to laboratory rodents is a model of epilepsy that induces seizure activity and results in death of pyramidal neurons in regions CA3 and CA1 of the hippocampus w4,27x. This death is due largely to calcium influx resulting from the
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activation of glutamate receptors. There is an interesting association between expression of the calcium-binding protein calbindin D28k ŽCB. and selective vulnerability to seizure-induced injury in that resistant dentate granule neurons contain high levels of calbindin, whereas vulnerable CA3 and CA1 neurons contain little or no calbindin w36x. Such correlations between expression of calcium-binding proteins and apparent resistance to neuronal degeneration in vivo have prompted cell culture studies that have directly addressed the issue of whether these proteins are in fact neuroprotective. Cultured hippocampal neurons expressing CB are relatively resistant to excitotoxic and metabolic insults and amyloid b-peptide toxicity w6,9,19,25x, and PC12 cells overexpressing CB exhibit increased resistance to apoptosis w41x, suggesting a neuroprotective role for CB. However, many factors in addition to expression of CB are likely to contribute to neuronal vulnerability in vivo. Examples include: differential inputs from excitatory and inhibitory neurons w19x; relative levels of expression of different subtypes of glutamate receptors w7x; levels of neurotrophic support w24x; and complement of stress proteins w17,43x. Moreover, it was recently reported that the extent of damage to cortical and striatal neurons following focal cerebral ischemia was decreased in mice lacking CB w13x, suggesting that CB may increase neuronal vulnerability under certain conditions. In order to directly address the role of CB in the selective vulnerability of hippocampal neurons to seizure-induced damage, we performed studies in mice expressing normal levels of CB, a reduced level of CB, or no CB w1x. Our data suggest that CB exerts a concentration-dependent effect on vulnerability of CA1 neurons to seizure-induced injury, and is either not responsible for resistance of dentate granule neurons or compensatory proteins are induced in the CB y ry mice.
2. Materials and methods 2.1. Mice and KA administration The generation of the CB y ry mice was accomplished by a targeting approach described in detail previously w1x; mice were maintained on a C57BLr6= 129 background. Experiments were performed in young adult Ž3–4 months old. male homozygous ŽCB y ry . and heterozygous ŽCB q ry . CB knockout mice and wild-type littermate mice ŽCB q rq .. The CB y ry mice exhibit no overt phenotype, but do show deficits in motor coordination, and alterations in calcium regulation in cerebellar Purkinje cells w1x. We have observed no differences in hippocampal structure, or numbers of neuronsrsection in any of the subpopulations of hippocampal neurons ŽCA1, CA3 and dentate granule cells.. KA was injected stereotaxically into the dorsal aspect of the right hippocampus using methods described previously
w4,10x. Briefly, mice were anesthetized with Avertin, placed in a stereotaxic head holder, and the skull exposed along the midline. A convulsant dose of kainate Ž0.3 mgr0.5 ml sterile saline, pH 7.2. was injected unilaterally into dorsal hippocampus Žstereotaxic coordinates: AP s 2.0 mm posterior to bregma, ML s 2.4 mm lateral to bregma, DV s 1.8 mm below the surface of the skull.. Control mice were injected with 0.5 ml saline. Twenty-four hours following kainate administration, mice were anesthetized with sodium pentobarbital and perfused transcardially with saline followed by cold phosphate-buffered 4% paraformaldehyde. 2.2. Histological and Western blot analyses Coronal brain sections Ž30 mM. were cut on a freezing microtome and stained with Cresyl violet. Neuronal damage was quantified by methods similar to those used in our previous studies w4,10x. Briefly, Cresyl violet-positive undamaged neurons were counted in three 45 = fields in each region of interest ŽCA1, CA3 and upper limb of the dentate gyrus. in both the left and right hippocampi of each mouse. Neurons in which the cell body was crenated and the nucleus not readily visible were considered damaged, whereas neurons with a round to oval cell body and a visible nucleus were considered undamaged. Counts were made in three coronal brain sections from the middle third of the hippocampus in each mouse Žsections were chosen by unbiased sampling. and the mean number of undamaged neuronsrfieldrbrain was determined. Determinations were made in 11–13 micergroup. Comparisons of numbers of undamaged neurons in hippocampal regions among treatment groups were made using ANOVA followed by Fischer’s t-test for pairwise comparisons. Immunohistochemistry was performed in free-floating sections using methods described previously w4x. Briefly, sections were incubated for 2 h at room temperature in a solution containing 0.2% Triton X-100 and 0.015% blocking serum Žnormal goat serum for the spectrin and CB antibodies and normal horse serum for the MAP-2 antibody. in PBS. Primary antibody was then added to a final dilution of either 1:10,000 for the spectrin antibody or 1:500 for the MAP-2 antibody, and sections were incubated overnight at 48C. Sections were then washed with PBS, incubated for 2 h at room temperature in the presence of biotinylated secondary antibody, followed by washing in PBS. Sections were then incubated for 1 h in ABC reagent ŽVector Laboratories., washed in PBS, and incubated for 5 min in DAB solution ŽVector Laboratories.. Western blot analysis was performed as described previously Ž12% polyacrylamide gel. using CB antibody at a dilution of 1:1500 w6,20x. The primary antibodies were rabbit polyclonal antibodies specific for CB w20x, a calpain cleavage product of spectrin Žgenerous gift from R. Siman at Cephalon., and a mouse monoclonal antibody against MAP-2 Žclone AP20; Sigma..
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3. Results and discussion Relative levels of CB protein in hippocampi of CB q rq, CB q ry and CB y ry mice were determined by Western blot analysis. Hippocampi from CB y ry mice contained no detectable CB, and the CB level in hippocampi from CB q ry mice was approximately 50% lower than the level in hippocampi from CB q rq mice ŽFig. 1A.. Levels of CB immunoreactivity were high in the cell bodies, dendrites and axons Žmossy fibers. of essentially all granule cells ŽFig. 1B.. CB immunoreactivity was present at relatively low levels in CA1 pyramidal neurons and was absent from CA3 neurons. In order to elucidate the possible modulatory roles for CB on seizure-induced injury to hippocampal neurons, we injected KA into the dorsal hippocampus of CB q rq, CB q ry and CB y ry mice. KA-induced damage to CA1 neurons in the ipsilateral hippocampus was significantly less in CB q ry mice compared to CB q rq and CB y ry mice ŽFigs. 2 and 3.. There was a trend towards reduced damage to CA3 neurons in CB q ry mice, although it fell short of statistical significance Ž p - 0.10.. There was no evidence of damage to dentate granule neurons in mice of any of the three genotypes Ždata not shown.. Mice from all the groups showed moderate damage to CA1 and CA3 neurons in the contralateral hippocampus, and no significant differences were seen among the three groups ŽFig. 3.. In order to relate the data on neuronal damage to alterations in neuronal calcium homeostasis, we also performed immunohistochemical analyses using antibodies against calpaincleaved spectrin and MAP-2, two markers of calciummediated proteolysis w12,29,34,35x. KA-induced a large increase in levels of calpain-cleaved spectrin immunoreactivity in regions CA1 and CA3 of CB q rq and CB y ry mice, and the level of the increase was lower in region CA1 the CB q ry mice ŽFig. 4.. Levels of MAP-2 immunoreactivity were markedly reduced in regions CA1 and CA3 of KA-injected CB q rq and CB y ry mice ŽFig. 5.. The extent of decrease in MAP-2 immunoreactivity in region CA1 was less in the CB q ry mice compared to mice of the other two genotypes. The present findings suggest that the level of CB may modify neuronal vulnerability to seizure-induced injury, but that its effects can be concentration-dependent and cell type-specific. When calbindin levels were artificially lowered to approximately half the normal level, CA1 neurons were more resistant to seizure-induced injury. However, complete lack of calbindin did not further increase resistance of CA1 neurons, but rather increased vulnerability. Although the mechanism whereby intermediate levels of CB allow for optimal survival of CA1 neurons is unknown, previous findings suggest several possibilities. Based upon their observations that ischemic neuronal injury is decreased in CB y ry mice and electrophysiological analyses, Klapstein et al. w13x proposed that lack of CB results in increased activation of calcium-sensitive potas-
Fig. 1. Characterization of calbindin expression in wild-type mice and CB knockout mice. ŽA. Western blot analysis of CB in hippocampal homogenates Žeach lane was loaded with 50 mg protein. from CBqrq, CBqry and CByry mice. ŽB. Hippocampal sections from a CBq rq mouse Župper. and a CByry mouse Žlower. immunostained with CB antibody. Note high levels of CB immunoreactivity in dentate granule cells Žincluding their mossy fiber terminals in region CA3., moderate levels of immunoreactivity in CA1 neurons, and lack of immunoreactivity in CA3 neurons.
sium channels. Perhaps the moderate decrease in CB levels in CB q ry mice might allow for activation of such potassium channels as well as for calcium buffering. Studies of developing neurons in culture have shown that there is a narrow range of intracellular free calcium levels that is optimal for promoting cell survival and neurite outgrowth w18,21x. It may therefore be the case that an intermediate levels of CB results in an intracellular calcium concentration that is within the optimum range. A study of the relationship between cellular expression of CB and neuronal vulnerability to transient global forebrain ischemia in gerbils revealed a strong inverse correlation in immature animals ŽCA3 neurons and neurons at the base of the granule cell layer that lacked CB were vulnerable., but less of a correlation in adult animals Žonly CA1 neurons being vulnerable. w38x. The latter findings suggest that as hip-
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Fig. 2. Micrographs showing Cresyl violet staining of region CA1 of hippocampus in a CB q rq control mouse 24 h following administration of saline Župper left., and CB q rq, CB q ry and CB y ry mice 24 h following administration of KA Žsee Section 2; photographs were taken using a 40 = objective.. Note increased damage to CA1 neurons in CB q rq and CB y ry mice compared to the CB q ry mouse.
pocampal circuitry matures, factors in addition to CB expression come into play in determining selective vulnerability. Dentate granule neurons were not damaged by KA in mice of any of the three genotypes, indicating that the resistance of the granule neurons to seizure-induced injury is not the result of their high content of CB or that compensatory changes occur and the lack of calbindin is not the only difference between CB y ry and CB q rq mice. Multiple factors that confer resistance of dentate granule neurons may be involved. One possibility is that the position of the granule neurons in the circuitry involved in KA-induced seizures is such that they are subjected to less severe excitation. However, studies of levels of excitability and gene expression in dentate granule neurons following seizure induction w37,40x suggest that granule neurons are subjected to high levels of glutamate receptor activation. A second possibility is that dentate neurons express, or are exposed to, multiple proteins that make them resistant to injury. For example, levels of antioxidant enzymes have been shown to be related to neuronal vulnerability to excitotoxic and metabolic injury in cell culture studies w23x, and differences in the cellular localization of such enzymes have been demonstrated in hippocampus of adult rodents w14,39x. The expression of several neurotrophic factors including BDNF, NGF and bFGF is rapidly increased in dentate gyrus following seizure induction w30x, and these factors have been shown to protect hippocampal neurons against excitotoxic insults
Žsee Ref. w24x for review.. In addition, certain stress-responsive proteins are greatly increased in dentate granule neurons following seizures w15,16x.
Fig. 3. Quantification of KA-induced damage to hippocampal neurons. Mice were administered KA or saline, via unilateral injection into the dorsal hippocampus. Twenty-four hours later, mice were euthanized and coronal brain sections stained with Cresyl violet. Numbers of undamaged neurons in regions CA1 and CA3 of ipsilateral Žip. and contralateral Žcon. hippocampus were quantified Žsee Section 2.. Values are the mean and S.E.M. Ž ns11s13 mice.. U p- 0.01 compared to corresponding values for CBqrq and CByry mice.
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Fig. 4. Micrographs showing hippocampi in brain sections stained with an antibody against calpain-cleaved spectrin. Mice were administered KA or saline, via unilateral injection into the dorsal hippocampus. Twenty-four hours later, mice were euthanized and coronal brain sections were immunostained with an antibody against calpain-cleaved spectrin. Note increased immunoreactivity in region CA1 of CB q rq and CB y ry mice compared to the CB q ry mouse.
Fig. 5. Micrographs showing hippocampi in brain sections stained with an antibody against MAP-2. Mice were administered KA or saline, via unilateral injection into the dorsal hippocampus. Twenty-four hours later, mice were euthanized and coronal brain sections were immunostained with an antibody against MAP-2. Note decreased immunoreactivity in region CA1 of CB q rq and CB y ry mice compared to the CB q ry mouse.
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The patterns of KA-induced proteolysis of spectrin and MAP-2 observed in the present study were similar to those observed in previous studies in which status epilepticus was induced in rats by KA or electrical stimulation w3,34,35,42x. The reduced levels of MAP-2 and spectrin proteolysis in region CA1 of CB q ry mice tightly paralleled the decreased vulnerability of CA1 neurons in the CB q ry mice. These findings are consistent with a role for cellular calcium overload in the neuronal damage, and suggest a pivotal role for CB in protecting CA1 neurons against seizure-induced injury. The lack of increased vulnerability of CA3 neurons in CB y ry mice, and the complete resistance of dentate granule neurons to seizureinduced injury in both CB q rq and CB y ry mice further suggests either a specific role for CB expression in resistance of CA1 neurons to excitotoxic injury, or the induction of compensatory proteins in certain brain regions of the CB y ry mice. In addition, since specific spatiotemporal patterns of neuronal damage w17x and calpain activation w3x have been reported after KA treatment, examination of changes in CB q rq, CB q ry and CB y ry mice at various times after KA administration may provide insight related to differential early andror longterm effects of calbindin in discrete brain regions. Because excitotoxicity may contribute to the degeneration of hippocampal CA1 neurons in disorders ranging from stroke w31x to Alzheimer’s disease w22x, our findings suggest a role for CB in suppressing degeneration of CA1 neurons in those disorders. Moreover, beyond its apparent influence on neuronal vulnerability to excitotoxicity, CB may play roles in modulating the various physiological processes that involve glutamate receptors and calcium influx. As evidence, cultured hippocampal neurons overexpressing calbindin show no alteration in evoked neurotransmitter release, but do exhibit a suppression of posttetanic potentiation w5x consistent with a postsynaptic localization of calbindin and decrease in the magnitude of calcium elevation in the postsynaptic region. Moreover, Molinari et al. w26x reported impaired hippocampal longterm potentiation and spatial memory deficits in mice with reduced levels of CB as the result of overexpression of CB antisense. Thus, physiological and pathophysiological roles for CB appear quite complex. Acknowledgements This work was supported by the National Institute on Aging. References w1x M.S. Airaksinen, J. Eilers, O. Garaschuk, H. Thoenen, A. Konnerth, M. Meyer, Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene, Proc. Natl. Acad. Sci. U.S.A. 94 Ž1997. 1488–1493.
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