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Enhanced Calcium Transients in Glial Cells in Neonatal Cerebellar Cultures Derived from S100B Null Mice
Experimental Cell Research 257, 281–289 (2000) doi:10.1006/excr.2000.4902, available online at http://www.idealibrary.com on
Enhanced Calcium Transients in Glial Cells in Neonatal Cerebellar Cultures Derived from S100B Null Mice Zhi-gang Xiong,* ,1 David O’Hanlon,† Laurence E. Becker,‡ John Roder,§ John F. MacDonald,* and Alexander Marks† ,2 *Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8; †Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5G 1L6; ‡Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and §Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5
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S100B is the major low-affinity Ca -binding protein in astrocytes. In order to study the role of S100B in the maintenance of Ca 2ⴙ homeostasis, we generated S100B null mice by a targeted inactivation of the S100B gene. Absence of S100B expression was demonstrated by Northern and Western blotting for S100B mRNA and protein, respectively, and immunoperoxidase staining of sections of various brain regions. S100B null mice were viable, fertile, and exhibited no overt behavioral abnormalities up to 12 months of age. On the basis of light microscopy and immunohistochemical staining, there were no discernable alterations in the distribution and morphology of astrocytes or neurons in sections of adult brains of these mice. Astrocytes in cerebellar cultures derived from 6-day-old S100B null mice exhibited enhanced Ca 2ⴙ transients in response to treatment with KCl or caffeine. On the other hand, granule neurons, in the same cultures, exhibited normal Ca 2ⴙ transients in response to treatment with KCl, caffeine, or N-methyl-D-aspartate. These results demonstrate a specific decrease in Ca 2ⴙ-handling capacity in astrocytes derived from S100B null mice and suggest that S100B plays a role in the maintenance of Ca 2ⴙ homeostasis in astrocytes. © 2000 Academic Press Key Words: S100B; astrocytes; Ca 2ⴙ handling; knockout mice; cerebellar cultures.
INTRODUCTION
The dynamic equilibrium of Ca 2⫹ in astrocytes involves a complex array of Ca 2⫹ entry channels and intracellular buffering systems [1]. Calcium transients in astrocytes can cross cell borders via gap junctions resulting in intracellular Ca 2⫹ waves traveling from 1 Present address: Neurobiology Research, Legacy Clinical Research and Technology Center, Portland, OR 97208. 2 To whom correspondence and reprint requests should be addressed at Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, ON, M5G 1L6, Canada. Fax: ⫹1-416-978-8528. E-mail: [email protected].
one astrocyte to the next [2, 3]. Such Ca 2⫹ waves can be the basis of signaling between astrocytes to integrate extracellular signals and possibly a means to influence synaptic transmission between neurons [4, 5]. S100B is a small (⬃10 kDa) EF-hand Ca 2⫹-binding protein which constitutes 0.5% of cytoplasmic proteins in brain astrocytes [6 – 8]. The protein exists intracellularly as a non-covalent dimer [9] and binds Ca 2⫹ in vitro with a low binding affinity of 0.01–1.0 mM dependent on the presence of K ⫹, which lowers the affinity, or Zn 2⫹, which increases the affinity, of S100B for Ca 2⫹ [10]. The Ca 2⫹-binding ability of S100B and its high concentration in astrocytes suggest that the protein may play a role in maintaining Ca 2⫹ homeostasis in these cells, by analogy with other members of the S100 family of small EF-hand Ca 2⫹-binding proteins that have been implicated in the regulation of Ca 2⫹ homeostasis in other cell types [11]. S100B is widely distributed and conserved in the nervous system of vertebrates [12, 13] and its accumulation in the brain of mammals, including man, coincides with periods of central nervous system (CNS) maturation [14 –18]. The localization of the human S100B gene to the 21q22.3 region of chromosome 21 [19, 20] suggested the possibility that overexpression of the protein in trisomy 21 could promote the development of the Down syndrome (DS) phenotype [21]. However, subsequent analysis of human subjects with partial trisomy of chromosome 21, excluding the distal 21q22.3 region where the S100B locus is located, indicated that these subjects exhibited all the abnormal features of DS, including mental retardation, even in the absence of duplication of the S100B gene [22]. Furthermore, S100B transgenic mice, carrying multiple copies of the human S100B gene and overexpressing the protein in a gene dose-dependent manner [23], were found to be only slightly impaired in spatial learning [24], raising the question whether overexpression of S100B does in fact contribute to the severity of mental retardation in DS. In the present study, we used a complementary ge-
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0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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netic approach to study the potential function of S100B in the CNS, by creating S100B null mice in which the expression of S100B was abolished by introducing a targeted mutation in the S100B gene. We report that glial cells in cerebellar cultures derived from S100B null mice exhibit enhanced Ca 2⫹ transients in response to treatment with pharmacological agents that promote Ca 2⫹ entry or release. These results suggest that S100B plays a role in regulating Ca 2⫹ homeostasis in astrocytes. MATERIALS AND METHODS Targeting vector. Overlapping clones of the S100B gene were obtained by screening a mouse SvJ129 genomic library with a 264-bp EcoRI rat cDNA S100B fragment [25]. A clone carrying an 8.5-kb EcoRI fragment containing the three exons of the S100B gene and some 5⬘ upstream sequence was selected for derivation of a targeting vector and designated pMS 3.6. Exon 2 (which includes the ATG translation initiation codon) [26] and flanking 5⬘ and 3⬘ intron sequences were excised from pMS 3.6 with NdeI and replaced with a neomycin resistance (neo) selection cassette. The modified fragment was inserted into the EcoRI site of pGEM7-Tk (Stratagene, La Jolla, CA) in front of a thymidine kinase (tk) selection cassette to create the targeting vector pMS16. Derivation of S100B null mice. The targeting vector was linearized with NotI and introduced by electroporation into the embryonic stem (ES) cell line RI [27]. Doubly resistant colonies were isolated following selection in G418 and gancyclovir [28] and expanded. Genomic DNA of the selected colonies was screened for homologous recombination by Southern blotting [23] with an S100B genomic probe flanking the targeting construct at the 5⬘ end. Recombinant ES clones displaying an 8.4-kb EcoRV fragment (in which a novel EcoRV site is provided by the neo cassette) in addition to the wild-type 9.5 EcoRV fragment (Fig. 1) were injected into CD1 mouse blastocysts to generate aggregation chimeras. Following implantation into pseudopregnant females, male chimeras were selected by virtue of their fur color and high degree chimeras were mated with CD1 females. Germline transmission was identified on the basis of eye color of the offspring and confirmed by Southern blotting of tail DNA. S100B expression. S100B expression was assayed by Northern blotting with a rat antisense S100B riboprobe [25] and Western blotting and immunoperoxidase staining with anti-S100B antibodies, performed as previously described [18, 23]. Cerebellar cultures. Mouse cerebellar cultures were prepared from 6-day-old wild-type CD1 mice, S100B null mice, and S100B transgenic mice carrying 10 copies of the human S100B gene [23]. The mice were sacrificed by decapitation and the brains were removed. The cerebella were dissected out, freed of meninges, and cut into small pieces in ice-cold Hanks’ balanced salt solution. The tissue was digested with 0.05% trypsin–EDTA (Gibco-BRL, Grand Island, NY) for 20 min at 37°C and resuspended in growth medium, minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 10% heat-inactivated horse serum, and insulin (10 g/ml). The digested tissue was triturated using a fire-polished pasteur pipette and the cell suspension was plated onto 24-mm-diameter glass coverslips precoated with poly-D-lysine at a density of 3 ⫻ 10 5/cm 2. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO 2, 95% air. After 4 days, the medium was changed to growth medium without FBS and the cultures were maintained with twice weekly changes of medium. The cultures were used for Ca 2⫹ imaging studies 2–3 weeks after plating. Ca 2⫹ imaging. Ca 2⫹ imaging was performed as previously described [29]. The cells were perfused with a buffered physiological
salt solution containing 140 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl 2, 33 mM glucose, 1 M tetrodotoxin, 25 mM Hepes, pH 7.4, with an osmolarity of 320 –335 mosm. Ratio imaging of intracellular free Ca 2⫹ was performed fluorometrically using a fura-2-based technique. For loading, fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) (dissolved in dimethyl sulfoxide) was added to a final concentration of 5 M and the cells were incubated for 45 min at room temperature (22°C), followed by a further incubation in the physiological salt solution alone for an additional 60 min to ensure the deesterification of the loading reagent. Coverslips with fura-2-loaded cells were transferred to a perfusion chamber on an inverted microscope (Diophot 300, Nikon), illuminated with a 75-W xenon lamp, and viewed using a 40X UV fluor oil-immersion objective. For fura-2 excitation, the shutter and filter wheel were controlled by Axon Imaging Workbench (AIW) software (AIW 2.1, Axon Instruments, Foster City, CA) to provide sequential illumination at two alternating wavelengths, 340 and 380 nm. Fluorescence of fura-2 was detected at an emission wavelength of 510 nm. Video images were acquired using an intensified CCD camera (PTI IC-110). Digitized images were obtained by averaging four frames at video rates using an image processing board (Axon Image Lightening), also controlled by AIW 2.1 software. Fluorescence emission ratios following excitation at 340 and 380 nm were calculated by dividing averaged pixel values in circumscribed regions of individual responding cells in the field of view. The values were corrected for background fluorescence obtained by imaging a field with no cells. Pharmacological treatments were performed at room temperature. The agents KCl (Sigma, St. Louis, MO), caffeine (Sigma), NMDA (Tocris Neuramin, Buckhurst Hill, UK), and glycine (Sigma) were added to the final concentrations indicated in the figure legends. A multibarrel fast perfusion system was employed to achieve a rapid exchange of solutions. To minimize variations among batches of cells due to culture conditions, cerebellar cultures from the different groups to be compared (e.g., wild-type and null) were initiated at the same time and matched numbers of individual cells from each respective culture were analyzed on the same day. Variance analysis was used to determine differences between various groups, with significance defined as P ⬍ 0.05.
RESULTS
Derivation of S100B Null Mice A clone designated pMS 3.6 carrying an 8.5-kb EcoRI fragment containing the three exons of the S100B gene and some 5⬘ upstream sequence was selected from a mouse SvJ129 genomic library. The location of this clone in the mouse S100B gene locus and a partial restriction map are shown in Fig. 1. The targeting construct was derived by deleting exon 2 and part of its 5⬘ and 3⬘ flanking regions from pMS3.6 and replacing the excized DNA with a neo expression cassette. The modified fragment was inserted in front of a tk selection cassette to create the targeting vector pMS 16 (Fig. 1). Upon homologous recombination into ES cells at the S100B gene locus, the tk cassette is deleted and an 8.4-kb EcoRV fragment is created by a novel EcoRV site contributed by the neo cassette. Chimeric mice were obtained by injecting recombinant ES cells into CD1 blastocytes. Crossing of germline-targeted heterozygotes generated S100B homozygous null (⫺/⫺) mice with a frequency of approximately 25%, indicating no gestational bias against the null mice. When
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FIG. 1. Targeted disruption of the S100B gene. (A) Structure of the mouse wild-type S100B gene, the targeting construct, and the targeted gene. The S100B locus (top line) comprises three exons which are indicated as boxes and numbered, with coding regions indicated by solid boxes and untranslated regions by open boxes. The targeting vector (middle line) contains a neomycin resistance gene (NEO), which replaces exon 2, and a thymidine kinase gene (TK) to allow counterselection. Correct genomic targeting (bottom line) replaces exon 2 with the neo cassette and eliminates the tk cassette. The position of the 5⬘ probe used for Southern analysis is indicated. Restriction sites are N (NdeI), RI (EcoRI), RV (EcoRV), and S (SphI). (B) Southern blot analysis of genomic DNA (10 g) from S100B null (⫺/⫺), wild-type (⫹/⫹), and heterozygous (⫹/⫺) mice. Targeted inactivation of the S100B gene introduces an additional RV site in NEO. When hybridized to the 5⬘ probe, RV fragments are 9.5 kb in the wild-type and 8.4 kb in the mutant allele. (C) Northern blot analysis of adult brain RNA (10 g) showing the absence of S100B mRNA from S100B null mice. (D) Western blot analysis of total adult brain protein (10 g) showing the absence of S100B protein from S100B null mice.
compared with wild-type littermates, the null offspring had no obvious abnormalities, and, upon autopsy, no gross anatomical differences were seen in any organs, including brain. Southern blotting of tail DNA indicated the presence of a 9.5- or 8.4-kb fragment in wild-type (⫹/⫹) or null (⫺/⫺) mice, respectively, and of both fragments in heterozygous (⫹/⫺) mice, as expected for the respective genotypes (Fig. 1). The absence of expression of S100B in null mice was demonstrated by Northern and Western blotting (Fig. 1). Absence of Expression of S100B in Different Brain Regions of S100B Null Mice S100B null mice were viable, fertile, and exhibited no obvious motor or behavioral abnormalities on visual inspection in their normal cage environments up to 12 months of age. Immunoperoxidase staining demonstrated the absence of S100B protein expression in all brain regions of the null mice, whereas strong expression was seen in astrocytes in all brain regions, including Bergmann glia in the cerebellum in wild-type and
S100B transgenic mice (Fig. 2). There was an absence of S100B expression in Purkinje neurons and granule neurons in the cerebellum in wild-type and S100B transgenic mice (Fig. 2). The morphology and distribution of astrocytes and neurons were examined in the brains of adult mice by light microsopy following immunohistochemical and silver (Golgi) staining. Immunoperoxidase staining with anti-glial fibrillary acidic protein antibody indicated no apparent differences in the distribution and morphology of astrocytes between wild-type and null mice (data not shown). In addition, qualitative examination of neuronal populations in the cerebellum and hippocampus, using Golgi staining [30] and reactivity with anti-neurofilament antibody, revealed no abnormalities in the cell bodies, axons, dendrites, or dendritic spines in the S100B null mice (data not shown). Ca 2⫹ Imaging in Cerebellar Cultures Cerebellar cultures derived from brains of 6-day-old mice and cultured for 2–3 weeks consisted of fusiform
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FIG. 2. Immunoperoxidase staining of mouse brain regions for S100B protein; (A–C) cerebellum; (D and E) hippocampal formation (dentate gyrus); (F and H) cerebral cortex; (A, D, and F) S100B null mice; (B, E, and H) wild-type mice; (C) S100B transgenic mice. There is strong staining of Bergmann glia (arrows) and their process bracketing Purkinje neurons (arrowheads) in B and C and of astrocytes (arrows) in E and H. There is an absence of staining in A, D, and F. G denotes the granule cell layer.
or stellate glial cells and round granule cells. The distinct morphology of the two cell types permitted recording from individual glial cells and granule cells in the same culture. In addition, treatment with NMDA plus glycine provided further confirmation of the individual cell type, as only granule neurons, but not glial cells, display postsynaptic NMDA receptors that mediate Ca 2⫹ uptake in response to stimulation with NMDA. Ratio imaging of intracellular free Ca 2⫹ was performed by fura-2 microfluorimetry based on the measurement of the emitted fluorescence at 510 nm after excitation at 340 and 380 nm. Changes in free Ca 2⫹ concentrations were recorded in response to treatment with KCl for stimulation of Ca 2⫹ entry via voltage-gated channels, caffeine for release of Ca 2⫹ from intracellular stores, or NMDA and glycine for activation of NMDA receptor-mediated postsynaptic Ca 2⫹ influx. The colorcoded changes in intracellular Ca 2⫹ in individual glial cells in cerebellar cultures derived from wild-type, S100B transgenic, or S100B null mice, in response to treatment with KCl or caffeine, are shown in Fig. 3. Increases in intracellular Ca 2⫹ in response to these treatments are seen in glial cells in all three cultures, but are more pronounced in cultures derived from S100B null mice. Tracings of intracellular Ca 2⫹ transients in individual glial cells in response to treatment with KCl, caffeine, or NMDA plus glycine are shown in Fig. 4. Treatment with KCl or caffeine, but not NMDA plus glycine, induced intracellular Ca 2⫹ elevation. The amplitude of the response was higher in glial cells in cultures derived from S100B null mice than those in cultures derived from wild-type or S100B transgenic mice. In order to test for the specificity of these responses in glial cells, we examined intracellular Ca 2⫹ transients in individual granule cells in the same cultures. Treatment with KCl, caffeine, or NMDA plus glycine induced intracellular Ca 2⫹ elevation in granule
cells, with no difference in the amplitude of the responses between cultures derived from wild-type mice and null mice (Fig. 5). The responses recorded in glial cells are summarized in Figs. 6 and 7, and those in granule cells, in Fig. 8. Elevations in intracellular Ca 2⫹ were significantly higher in glial cells in cultures derived from S100B null mice than those in cultures from wild-type mice, in the case of treatment with both KCl (P ⫽ 0.0007) and caffeine (P ⫽ 0.03) (Fig. 6). There were no differences in Ca 2⫹ elevation in response to KCl or caffeine treatment in glial cells in cultures derived from wild-type or S100B transgenic mice (P ⬎ 0.05) (Fig. 7). Similarly, since granule cells do not express S100B, there were no differences in intracellular Ca 2⫹ elevations in response to KCl or caffeine treatment in granule cells derived from wild-type or S100B null mice (P ⬎ 0.05) (Fig. 8). DISCUSSION
S100B has been detected extracellularly in the CNS [31] and in conditioned medium of rat glioma cultures [32], raising speculation that it may exert a paracrine function following its secretion from astrocytes [33]. Results of experiments based on the addition of purified S100B in vitro to primary or transformed cultures of glia or neurons suggest that a disulfide-linked dimer of the protein acts extracellularly as a glial mitogen and neurotrophic factor in brain and peripheral nerves [33–36]. However, disulfide bond formation in the native protein is difficult to demonstrate in vitro [37–39] and it is thought that the protein exists intracellularly as a non-covalent dimer [9, 40 – 42]. It is possible that correct disulfide pairing occurs efficiently only extracellularly following the secretion of the protein from astrocytes [37, 40, 41]. In the present study, the brains of S100B null mice did not differ morphologically or
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FIG. 3. Pseudocolor representation of intracellular Ca 2⫹ changes in glial cells in response to treatment with KCl or caffeine. Mouse cerebellar cultures, derived from wild-type, S100B null, or S100B transgenic mice, were treated with 50 mM KCl or 10 mM caffeine, as indicated, and ratio imaging of intracellular free Ca 2⫹ was performed by fura-2 microfluorimetry in individual glial cells. The color scale is a linear pseudocolor representation of intracellular free Ca 2⫹ expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Low intracellular Ca 2⫹ levels corresponding to low ratios are represented by blue and high intracellular Ca 2⫹ levels corresponding to high ratios are represented by red.
histologically from the brains of wild-type mice, although detailed quantitative morphometric methodology was not applied. Therefore, at our level of discrimination, we could not provide evidence for an essential role for S100B as a trophic factor during CNS development. Furthermore, three other lines of preliminary experimental observations on small numbers of adult animals also did not uncover differences that could be attributed to an absence of an S100B paracrine neurotrophic effect in S100B null mice (A. Marks, W. Thompson, D. van der Kooy, and M. Wojtowicz, unpublished results). First, although S100B expressing Schwann cells that overlay neuromuscular junctions are critical for regeneration of peripheral axons following denervation [43], no defects in axonal regeneration were observed in S100B null mice. Second, a residual proliferative neuronal population is present near the lateral ventricle in the adult mouse forebrain and can be readily labeled with bromodeoxyuridine (BrdU) [44].
This neuronal population is located in the subependymal layer that is tightly juxtaposed to an S100B-producing ependymal cell layer which lines the wall of the ventricle [45]. There were no differences in the densities of BrdU-labeled neurons in this location between S100B null and wild-type mice. Third, there were no differences in neuronal densities between S100B null and wild-type mice in the subgranular zone of the dentate gyrus, a brain region in which adult neurogenesis also persists [46]. In order to examine the intracellular physiological consequences of the absence of S100B in astrocytes, we focused on the cerebellum, where the content of S100B is approximately twice that of other brain regions in the mouse [16]. This high concentration of S100B is found in Bergmann glia and their processes (Fig. 2). Primary cerebellar cultures established from neonatal mice contain round granule neurons and fusiform or stellate astrocytes derived from Bergmann glia [47,
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FIG. 4. Intracellular Ca 2⫹ transients in glial cells in response to pharmacological treatments. Representative tracings, recorded from an individual glial cell in cerebellar cultures, derived from wild-type, S100B null, or S100B transgenic mice, are illustrated. The cultures were treated sequentially with 50 mM KCl, 10 mM caffeine, or 100 M NMDA plus 3 M glycine, for the duration indicated by the time bars, with an interval of 300 s between each addition. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm.
48]. In our primary cultures originating from wild-type mice, depolarization with KCl or treatment with caffeine stimulated Ca 2⫹ uptake or release, respectively,
FIG. 5. Intracellular Ca 2⫹ transients in granule cells in response to pharmacological treatments. Representative tracings, recorded from an individual granule cell in cerebellar cultures derived from wild-type or S100B null mice, are illustrated. The cultures were treated sequentially with 50 mM KCl, 10 mM caffeine, or 100 M NMDA plus 3 M glycine, for the duration indicated by the time bars, with an interval of 300 s between each addition. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm.
FIG. 6. Elevation in the intracellular Ca 2⫹ responses in glial cells derived from S100B null mice. Basal intracellular Ca 2⫹ and intracellular Ca 2⫹ transients evoked in response to treatment with 50 mM KCl or 10 mM caffeine were recorded from the total number of glial cells indicated in cerebellar cultures derived from wild-type or S100B null mice. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Data were obtained from a summation of values recorded from four independent cultures derived from wild-type (WT) or S100B null mice, respectively. Bars represent means ⫾ SEM.
in the astrocytes (Fig. 4). On the other hand, treatment with NMDA and glycine did not stimulate Ca 2⫹ entry (Fig. 4). These observations agree with the reported presence of voltage-gated Ca 2⫹ channels but absence of NMDA effects on Ca 2⫹ permeability in cultured astro-
FIG. 7. Normal Ca 2⫹ responses in glial cells derived from S100B transgenic mice. Basal intracellular Ca 2⫹ transients, evoked in response to treatment with 50 mM KCl or 10 mM caffeine, were recorded from the total number of glial cells indicated in cerebellar cultures derived from wild-type or S100B transgenic mice. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Data were obtained from a summation of values recorded from two independent cultures derived from wild-type (WT) or S100B transgenic (Trans) mice, respectively. Bars represent means ⫾ SEM. NS, not significant.
Ca 2⫹ HANDLING IN S100B-DEFICIENT ASTROCYTES
FIG. 8. Normal Ca 2⫹ responses in granule cells in cultures derived from S100B null mice. Basal intracellular Ca 2⫹ and intracellular Ca 2⫹ transients evoked in response to treatment with 50 mM KCl or 10 mM caffeine were recorded from the total number of granule cells indicated in cerebellar cultures derived from wild-type or S100B null mice. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Data were obtained from a summation of values recorded from four independent cultures derived from wild-type (WT) or S100B null mice, respectively. Bars represent means ⫾ SEM. NS, not significant.
cytes [49]. Glial cells in cultures derived from S100B null mice showed enhanced responses to treatment with pharmacological agents that stimulated Ca 2⫹ entry or release in comparison with glial cells in cultures derived from wild-type mice (Fig. 6). This suggests that the absence of S100B in glial cells from S100B null mice limits their Ca 2⫹-handling capacity to control increases in intracellular Ca 2⫹ levels. The specificity of the response in the glial cells was confirmed by parallel recordings from granule neurons in the same culture. Treatment with KCl or NMDA plus glycine stimulated Ca 2⫹ uptake by granule neurons, showing that they contained both voltage-gated Ca 2⫹ channels and postsynaptic NMDA receptors capable of mediating Ca 2⫹ entry in response to stimulation (Fig. 5). In addition, as S100B is expressed in astrocytes but not neurons in the cerebellum, granule cells in cultures derived from S100B null mice displayed normal responses to treatment with pharmacological agents that stimulate Ca 2⫹ entry or release (Fig. 8). The maintenance of intracellular Ca 2⫹ homeostasis is a critical component of the integrated response of the CNS to chemical signals or electrical activity. While this is the first report of a targeted inactivation of a major Ca 2⫹-binding protein in astrocytes, mice deficient in neuronal Ca 2⫹-binding proteins have been described previously. These include null mice with targeted inactivating mutations in the calbindin D 28k and calretinin genes [50, 51] and transgenic mice with reduced levels of calbindin D 28k resulting from expressed
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antisense transcripts [52]. Unlike the normal responses observed in the granule neurons in our S100B null mice (Fig. 5), abnormal Ca 2⫹ transients were observed in neurons from engineered mice with absent or reduced calbindin D 28k expression, as follows. In calbindin D 28k null mice, the peak amplitudes of dendritic calcium transients of cerebellar Purkinje cells were enhanced twofold in response to postsynaptic stimulation [50]. In mice with reduced levels of calbindin D 28k, there was a prolonged elevation of intracellular Ca 2⫹ in response to NMDA or KCl treatment [53]. The high concentration of S100B in astrocytes is consistent with its function as an intracellular Ca 2⫹ buffer in these cells. However, since S100B has been shown to interact in vitro with several target proteins in a Ca 2⫹-dependent manner [11], S100B could also modulate Ca 2⫹ homeostasis through alternative mechanisms. In the complete absence of S100B, i.e., in S100B null mice, a decreased Ca 2⫹-handling capacity in cultured glial cells could be demonstrated experimentally (Fig. 6). This indicates that S100B is a functional component of the complex system of plasma membrane exchangers, cytoplasmic organelles, and Ca 2⫹-binding proteins that regulate intracellular Ca 2⫹ in astrocytes [1]. However, it is interesting that there were no discernable differences in Ca 2⫹ handling between glial cells from wild-type mice and those from transgenic mice carrying 10 copies of the human S100B gene (Fig. 7). Both the endogenous mouse S100B gene and the human S100B transgene in the transgenic mice are induced during early postnatal development [16, 54]. Yet, we observed that Ca 2⫹ transients in glial cells in response to stimulation did not differ between wild-type and transgenic mice. This suggests that the S100B-dependent Ca 2⫹-handling capacity is present in excess in wild-type mice and is brought into play in our experimental conditions only at Ca 2⫹ concentrations that exceed a certain threshold. This explanation is consistent with a high concentration of S100B in astrocytes and its low binding affinity for Ca 2⫹ in vitro. We have no present evidence for developmental or behavioral consequences of the absence of S100B. It is possible that other buffer systems or feedback mechanisms compensate for the lack of S100B in intact animals. Alternatively, any disturbances in astrocytic Ca 2⫹ waves resulting from the absence of S100B could have subtle influences on neuronal synaptic transmission and hence animal behavior. For example, while mice lacking the expression of calbindin D 28k or calretinin exhibited no abnormalities in their normal cage environments, evidence of cerebellar incoordination with age could be brought out in these strains by experimental tests challenging their motor skills [50, 55]. A similar analysis of motor and cognitive functions in S100B null mice may provide evidence for subtle behavioral disturbances in these animals.
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This work was supported by grants from MRC to A.M. and J.F.M. and from NSERC to J.R. Z.-G.X. was an MRC Centennial Fellow. We thank Wanda Abramow-Newerley and Ela Czerwinska for expert technical assistance.
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Enhanced Calcium Transients in Glial Cells in Neonatal Cerebellar Cultures Derived from S100B Null Mice Zhi-gang Xiong,* ,1 David O’Hanlon,† Laurence E. Becker,‡ John Roder,§ John F. MacDonald,* and Alexander Marks† ,2 *Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8; †Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5G 1L6; ‡Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and §Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5
2ⴙ
S100B is the major low-affinity Ca -binding protein in astrocytes. In order to study the role of S100B in the maintenance of Ca 2ⴙ homeostasis, we generated S100B null mice by a targeted inactivation of the S100B gene. Absence of S100B expression was demonstrated by Northern and Western blotting for S100B mRNA and protein, respectively, and immunoperoxidase staining of sections of various brain regions. S100B null mice were viable, fertile, and exhibited no overt behavioral abnormalities up to 12 months of age. On the basis of light microscopy and immunohistochemical staining, there were no discernable alterations in the distribution and morphology of astrocytes or neurons in sections of adult brains of these mice. Astrocytes in cerebellar cultures derived from 6-day-old S100B null mice exhibited enhanced Ca 2ⴙ transients in response to treatment with KCl or caffeine. On the other hand, granule neurons, in the same cultures, exhibited normal Ca 2ⴙ transients in response to treatment with KCl, caffeine, or N-methyl-D-aspartate. These results demonstrate a specific decrease in Ca 2ⴙ-handling capacity in astrocytes derived from S100B null mice and suggest that S100B plays a role in the maintenance of Ca 2ⴙ homeostasis in astrocytes. © 2000 Academic Press Key Words: S100B; astrocytes; Ca 2ⴙ handling; knockout mice; cerebellar cultures.
INTRODUCTION
The dynamic equilibrium of Ca 2⫹ in astrocytes involves a complex array of Ca 2⫹ entry channels and intracellular buffering systems [1]. Calcium transients in astrocytes can cross cell borders via gap junctions resulting in intracellular Ca 2⫹ waves traveling from 1 Present address: Neurobiology Research, Legacy Clinical Research and Technology Center, Portland, OR 97208. 2 To whom correspondence and reprint requests should be addressed at Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, ON, M5G 1L6, Canada. Fax: ⫹1-416-978-8528. E-mail: [email protected].
one astrocyte to the next [2, 3]. Such Ca 2⫹ waves can be the basis of signaling between astrocytes to integrate extracellular signals and possibly a means to influence synaptic transmission between neurons [4, 5]. S100B is a small (⬃10 kDa) EF-hand Ca 2⫹-binding protein which constitutes 0.5% of cytoplasmic proteins in brain astrocytes [6 – 8]. The protein exists intracellularly as a non-covalent dimer [9] and binds Ca 2⫹ in vitro with a low binding affinity of 0.01–1.0 mM dependent on the presence of K ⫹, which lowers the affinity, or Zn 2⫹, which increases the affinity, of S100B for Ca 2⫹ [10]. The Ca 2⫹-binding ability of S100B and its high concentration in astrocytes suggest that the protein may play a role in maintaining Ca 2⫹ homeostasis in these cells, by analogy with other members of the S100 family of small EF-hand Ca 2⫹-binding proteins that have been implicated in the regulation of Ca 2⫹ homeostasis in other cell types [11]. S100B is widely distributed and conserved in the nervous system of vertebrates [12, 13] and its accumulation in the brain of mammals, including man, coincides with periods of central nervous system (CNS) maturation [14 –18]. The localization of the human S100B gene to the 21q22.3 region of chromosome 21 [19, 20] suggested the possibility that overexpression of the protein in trisomy 21 could promote the development of the Down syndrome (DS) phenotype [21]. However, subsequent analysis of human subjects with partial trisomy of chromosome 21, excluding the distal 21q22.3 region where the S100B locus is located, indicated that these subjects exhibited all the abnormal features of DS, including mental retardation, even in the absence of duplication of the S100B gene [22]. Furthermore, S100B transgenic mice, carrying multiple copies of the human S100B gene and overexpressing the protein in a gene dose-dependent manner [23], were found to be only slightly impaired in spatial learning [24], raising the question whether overexpression of S100B does in fact contribute to the severity of mental retardation in DS. In the present study, we used a complementary ge-
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netic approach to study the potential function of S100B in the CNS, by creating S100B null mice in which the expression of S100B was abolished by introducing a targeted mutation in the S100B gene. We report that glial cells in cerebellar cultures derived from S100B null mice exhibit enhanced Ca 2⫹ transients in response to treatment with pharmacological agents that promote Ca 2⫹ entry or release. These results suggest that S100B plays a role in regulating Ca 2⫹ homeostasis in astrocytes. MATERIALS AND METHODS Targeting vector. Overlapping clones of the S100B gene were obtained by screening a mouse SvJ129 genomic library with a 264-bp EcoRI rat cDNA S100B fragment [25]. A clone carrying an 8.5-kb EcoRI fragment containing the three exons of the S100B gene and some 5⬘ upstream sequence was selected for derivation of a targeting vector and designated pMS 3.6. Exon 2 (which includes the ATG translation initiation codon) [26] and flanking 5⬘ and 3⬘ intron sequences were excised from pMS 3.6 with NdeI and replaced with a neomycin resistance (neo) selection cassette. The modified fragment was inserted into the EcoRI site of pGEM7-Tk (Stratagene, La Jolla, CA) in front of a thymidine kinase (tk) selection cassette to create the targeting vector pMS16. Derivation of S100B null mice. The targeting vector was linearized with NotI and introduced by electroporation into the embryonic stem (ES) cell line RI [27]. Doubly resistant colonies were isolated following selection in G418 and gancyclovir [28] and expanded. Genomic DNA of the selected colonies was screened for homologous recombination by Southern blotting [23] with an S100B genomic probe flanking the targeting construct at the 5⬘ end. Recombinant ES clones displaying an 8.4-kb EcoRV fragment (in which a novel EcoRV site is provided by the neo cassette) in addition to the wild-type 9.5 EcoRV fragment (Fig. 1) were injected into CD1 mouse blastocysts to generate aggregation chimeras. Following implantation into pseudopregnant females, male chimeras were selected by virtue of their fur color and high degree chimeras were mated with CD1 females. Germline transmission was identified on the basis of eye color of the offspring and confirmed by Southern blotting of tail DNA. S100B expression. S100B expression was assayed by Northern blotting with a rat antisense S100B riboprobe [25] and Western blotting and immunoperoxidase staining with anti-S100B antibodies, performed as previously described [18, 23]. Cerebellar cultures. Mouse cerebellar cultures were prepared from 6-day-old wild-type CD1 mice, S100B null mice, and S100B transgenic mice carrying 10 copies of the human S100B gene [23]. The mice were sacrificed by decapitation and the brains were removed. The cerebella were dissected out, freed of meninges, and cut into small pieces in ice-cold Hanks’ balanced salt solution. The tissue was digested with 0.05% trypsin–EDTA (Gibco-BRL, Grand Island, NY) for 20 min at 37°C and resuspended in growth medium, minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 10% heat-inactivated horse serum, and insulin (10 g/ml). The digested tissue was triturated using a fire-polished pasteur pipette and the cell suspension was plated onto 24-mm-diameter glass coverslips precoated with poly-D-lysine at a density of 3 ⫻ 10 5/cm 2. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO 2, 95% air. After 4 days, the medium was changed to growth medium without FBS and the cultures were maintained with twice weekly changes of medium. The cultures were used for Ca 2⫹ imaging studies 2–3 weeks after plating. Ca 2⫹ imaging. Ca 2⫹ imaging was performed as previously described [29]. The cells were perfused with a buffered physiological
salt solution containing 140 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl 2, 33 mM glucose, 1 M tetrodotoxin, 25 mM Hepes, pH 7.4, with an osmolarity of 320 –335 mosm. Ratio imaging of intracellular free Ca 2⫹ was performed fluorometrically using a fura-2-based technique. For loading, fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) (dissolved in dimethyl sulfoxide) was added to a final concentration of 5 M and the cells were incubated for 45 min at room temperature (22°C), followed by a further incubation in the physiological salt solution alone for an additional 60 min to ensure the deesterification of the loading reagent. Coverslips with fura-2-loaded cells were transferred to a perfusion chamber on an inverted microscope (Diophot 300, Nikon), illuminated with a 75-W xenon lamp, and viewed using a 40X UV fluor oil-immersion objective. For fura-2 excitation, the shutter and filter wheel were controlled by Axon Imaging Workbench (AIW) software (AIW 2.1, Axon Instruments, Foster City, CA) to provide sequential illumination at two alternating wavelengths, 340 and 380 nm. Fluorescence of fura-2 was detected at an emission wavelength of 510 nm. Video images were acquired using an intensified CCD camera (PTI IC-110). Digitized images were obtained by averaging four frames at video rates using an image processing board (Axon Image Lightening), also controlled by AIW 2.1 software. Fluorescence emission ratios following excitation at 340 and 380 nm were calculated by dividing averaged pixel values in circumscribed regions of individual responding cells in the field of view. The values were corrected for background fluorescence obtained by imaging a field with no cells. Pharmacological treatments were performed at room temperature. The agents KCl (Sigma, St. Louis, MO), caffeine (Sigma), NMDA (Tocris Neuramin, Buckhurst Hill, UK), and glycine (Sigma) were added to the final concentrations indicated in the figure legends. A multibarrel fast perfusion system was employed to achieve a rapid exchange of solutions. To minimize variations among batches of cells due to culture conditions, cerebellar cultures from the different groups to be compared (e.g., wild-type and null) were initiated at the same time and matched numbers of individual cells from each respective culture were analyzed on the same day. Variance analysis was used to determine differences between various groups, with significance defined as P ⬍ 0.05.
RESULTS
Derivation of S100B Null Mice A clone designated pMS 3.6 carrying an 8.5-kb EcoRI fragment containing the three exons of the S100B gene and some 5⬘ upstream sequence was selected from a mouse SvJ129 genomic library. The location of this clone in the mouse S100B gene locus and a partial restriction map are shown in Fig. 1. The targeting construct was derived by deleting exon 2 and part of its 5⬘ and 3⬘ flanking regions from pMS3.6 and replacing the excized DNA with a neo expression cassette. The modified fragment was inserted in front of a tk selection cassette to create the targeting vector pMS 16 (Fig. 1). Upon homologous recombination into ES cells at the S100B gene locus, the tk cassette is deleted and an 8.4-kb EcoRV fragment is created by a novel EcoRV site contributed by the neo cassette. Chimeric mice were obtained by injecting recombinant ES cells into CD1 blastocytes. Crossing of germline-targeted heterozygotes generated S100B homozygous null (⫺/⫺) mice with a frequency of approximately 25%, indicating no gestational bias against the null mice. When
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FIG. 1. Targeted disruption of the S100B gene. (A) Structure of the mouse wild-type S100B gene, the targeting construct, and the targeted gene. The S100B locus (top line) comprises three exons which are indicated as boxes and numbered, with coding regions indicated by solid boxes and untranslated regions by open boxes. The targeting vector (middle line) contains a neomycin resistance gene (NEO), which replaces exon 2, and a thymidine kinase gene (TK) to allow counterselection. Correct genomic targeting (bottom line) replaces exon 2 with the neo cassette and eliminates the tk cassette. The position of the 5⬘ probe used for Southern analysis is indicated. Restriction sites are N (NdeI), RI (EcoRI), RV (EcoRV), and S (SphI). (B) Southern blot analysis of genomic DNA (10 g) from S100B null (⫺/⫺), wild-type (⫹/⫹), and heterozygous (⫹/⫺) mice. Targeted inactivation of the S100B gene introduces an additional RV site in NEO. When hybridized to the 5⬘ probe, RV fragments are 9.5 kb in the wild-type and 8.4 kb in the mutant allele. (C) Northern blot analysis of adult brain RNA (10 g) showing the absence of S100B mRNA from S100B null mice. (D) Western blot analysis of total adult brain protein (10 g) showing the absence of S100B protein from S100B null mice.
compared with wild-type littermates, the null offspring had no obvious abnormalities, and, upon autopsy, no gross anatomical differences were seen in any organs, including brain. Southern blotting of tail DNA indicated the presence of a 9.5- or 8.4-kb fragment in wild-type (⫹/⫹) or null (⫺/⫺) mice, respectively, and of both fragments in heterozygous (⫹/⫺) mice, as expected for the respective genotypes (Fig. 1). The absence of expression of S100B in null mice was demonstrated by Northern and Western blotting (Fig. 1). Absence of Expression of S100B in Different Brain Regions of S100B Null Mice S100B null mice were viable, fertile, and exhibited no obvious motor or behavioral abnormalities on visual inspection in their normal cage environments up to 12 months of age. Immunoperoxidase staining demonstrated the absence of S100B protein expression in all brain regions of the null mice, whereas strong expression was seen in astrocytes in all brain regions, including Bergmann glia in the cerebellum in wild-type and
S100B transgenic mice (Fig. 2). There was an absence of S100B expression in Purkinje neurons and granule neurons in the cerebellum in wild-type and S100B transgenic mice (Fig. 2). The morphology and distribution of astrocytes and neurons were examined in the brains of adult mice by light microsopy following immunohistochemical and silver (Golgi) staining. Immunoperoxidase staining with anti-glial fibrillary acidic protein antibody indicated no apparent differences in the distribution and morphology of astrocytes between wild-type and null mice (data not shown). In addition, qualitative examination of neuronal populations in the cerebellum and hippocampus, using Golgi staining [30] and reactivity with anti-neurofilament antibody, revealed no abnormalities in the cell bodies, axons, dendrites, or dendritic spines in the S100B null mice (data not shown). Ca 2⫹ Imaging in Cerebellar Cultures Cerebellar cultures derived from brains of 6-day-old mice and cultured for 2–3 weeks consisted of fusiform
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FIG. 2. Immunoperoxidase staining of mouse brain regions for S100B protein; (A–C) cerebellum; (D and E) hippocampal formation (dentate gyrus); (F and H) cerebral cortex; (A, D, and F) S100B null mice; (B, E, and H) wild-type mice; (C) S100B transgenic mice. There is strong staining of Bergmann glia (arrows) and their process bracketing Purkinje neurons (arrowheads) in B and C and of astrocytes (arrows) in E and H. There is an absence of staining in A, D, and F. G denotes the granule cell layer.
or stellate glial cells and round granule cells. The distinct morphology of the two cell types permitted recording from individual glial cells and granule cells in the same culture. In addition, treatment with NMDA plus glycine provided further confirmation of the individual cell type, as only granule neurons, but not glial cells, display postsynaptic NMDA receptors that mediate Ca 2⫹ uptake in response to stimulation with NMDA. Ratio imaging of intracellular free Ca 2⫹ was performed by fura-2 microfluorimetry based on the measurement of the emitted fluorescence at 510 nm after excitation at 340 and 380 nm. Changes in free Ca 2⫹ concentrations were recorded in response to treatment with KCl for stimulation of Ca 2⫹ entry via voltage-gated channels, caffeine for release of Ca 2⫹ from intracellular stores, or NMDA and glycine for activation of NMDA receptor-mediated postsynaptic Ca 2⫹ influx. The colorcoded changes in intracellular Ca 2⫹ in individual glial cells in cerebellar cultures derived from wild-type, S100B transgenic, or S100B null mice, in response to treatment with KCl or caffeine, are shown in Fig. 3. Increases in intracellular Ca 2⫹ in response to these treatments are seen in glial cells in all three cultures, but are more pronounced in cultures derived from S100B null mice. Tracings of intracellular Ca 2⫹ transients in individual glial cells in response to treatment with KCl, caffeine, or NMDA plus glycine are shown in Fig. 4. Treatment with KCl or caffeine, but not NMDA plus glycine, induced intracellular Ca 2⫹ elevation. The amplitude of the response was higher in glial cells in cultures derived from S100B null mice than those in cultures derived from wild-type or S100B transgenic mice. In order to test for the specificity of these responses in glial cells, we examined intracellular Ca 2⫹ transients in individual granule cells in the same cultures. Treatment with KCl, caffeine, or NMDA plus glycine induced intracellular Ca 2⫹ elevation in granule
cells, with no difference in the amplitude of the responses between cultures derived from wild-type mice and null mice (Fig. 5). The responses recorded in glial cells are summarized in Figs. 6 and 7, and those in granule cells, in Fig. 8. Elevations in intracellular Ca 2⫹ were significantly higher in glial cells in cultures derived from S100B null mice than those in cultures from wild-type mice, in the case of treatment with both KCl (P ⫽ 0.0007) and caffeine (P ⫽ 0.03) (Fig. 6). There were no differences in Ca 2⫹ elevation in response to KCl or caffeine treatment in glial cells in cultures derived from wild-type or S100B transgenic mice (P ⬎ 0.05) (Fig. 7). Similarly, since granule cells do not express S100B, there were no differences in intracellular Ca 2⫹ elevations in response to KCl or caffeine treatment in granule cells derived from wild-type or S100B null mice (P ⬎ 0.05) (Fig. 8). DISCUSSION
S100B has been detected extracellularly in the CNS [31] and in conditioned medium of rat glioma cultures [32], raising speculation that it may exert a paracrine function following its secretion from astrocytes [33]. Results of experiments based on the addition of purified S100B in vitro to primary or transformed cultures of glia or neurons suggest that a disulfide-linked dimer of the protein acts extracellularly as a glial mitogen and neurotrophic factor in brain and peripheral nerves [33–36]. However, disulfide bond formation in the native protein is difficult to demonstrate in vitro [37–39] and it is thought that the protein exists intracellularly as a non-covalent dimer [9, 40 – 42]. It is possible that correct disulfide pairing occurs efficiently only extracellularly following the secretion of the protein from astrocytes [37, 40, 41]. In the present study, the brains of S100B null mice did not differ morphologically or
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FIG. 3. Pseudocolor representation of intracellular Ca 2⫹ changes in glial cells in response to treatment with KCl or caffeine. Mouse cerebellar cultures, derived from wild-type, S100B null, or S100B transgenic mice, were treated with 50 mM KCl or 10 mM caffeine, as indicated, and ratio imaging of intracellular free Ca 2⫹ was performed by fura-2 microfluorimetry in individual glial cells. The color scale is a linear pseudocolor representation of intracellular free Ca 2⫹ expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Low intracellular Ca 2⫹ levels corresponding to low ratios are represented by blue and high intracellular Ca 2⫹ levels corresponding to high ratios are represented by red.
histologically from the brains of wild-type mice, although detailed quantitative morphometric methodology was not applied. Therefore, at our level of discrimination, we could not provide evidence for an essential role for S100B as a trophic factor during CNS development. Furthermore, three other lines of preliminary experimental observations on small numbers of adult animals also did not uncover differences that could be attributed to an absence of an S100B paracrine neurotrophic effect in S100B null mice (A. Marks, W. Thompson, D. van der Kooy, and M. Wojtowicz, unpublished results). First, although S100B expressing Schwann cells that overlay neuromuscular junctions are critical for regeneration of peripheral axons following denervation [43], no defects in axonal regeneration were observed in S100B null mice. Second, a residual proliferative neuronal population is present near the lateral ventricle in the adult mouse forebrain and can be readily labeled with bromodeoxyuridine (BrdU) [44].
This neuronal population is located in the subependymal layer that is tightly juxtaposed to an S100B-producing ependymal cell layer which lines the wall of the ventricle [45]. There were no differences in the densities of BrdU-labeled neurons in this location between S100B null and wild-type mice. Third, there were no differences in neuronal densities between S100B null and wild-type mice in the subgranular zone of the dentate gyrus, a brain region in which adult neurogenesis also persists [46]. In order to examine the intracellular physiological consequences of the absence of S100B in astrocytes, we focused on the cerebellum, where the content of S100B is approximately twice that of other brain regions in the mouse [16]. This high concentration of S100B is found in Bergmann glia and their processes (Fig. 2). Primary cerebellar cultures established from neonatal mice contain round granule neurons and fusiform or stellate astrocytes derived from Bergmann glia [47,
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FIG. 4. Intracellular Ca 2⫹ transients in glial cells in response to pharmacological treatments. Representative tracings, recorded from an individual glial cell in cerebellar cultures, derived from wild-type, S100B null, or S100B transgenic mice, are illustrated. The cultures were treated sequentially with 50 mM KCl, 10 mM caffeine, or 100 M NMDA plus 3 M glycine, for the duration indicated by the time bars, with an interval of 300 s between each addition. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm.
48]. In our primary cultures originating from wild-type mice, depolarization with KCl or treatment with caffeine stimulated Ca 2⫹ uptake or release, respectively,
FIG. 5. Intracellular Ca 2⫹ transients in granule cells in response to pharmacological treatments. Representative tracings, recorded from an individual granule cell in cerebellar cultures derived from wild-type or S100B null mice, are illustrated. The cultures were treated sequentially with 50 mM KCl, 10 mM caffeine, or 100 M NMDA plus 3 M glycine, for the duration indicated by the time bars, with an interval of 300 s between each addition. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm.
FIG. 6. Elevation in the intracellular Ca 2⫹ responses in glial cells derived from S100B null mice. Basal intracellular Ca 2⫹ and intracellular Ca 2⫹ transients evoked in response to treatment with 50 mM KCl or 10 mM caffeine were recorded from the total number of glial cells indicated in cerebellar cultures derived from wild-type or S100B null mice. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Data were obtained from a summation of values recorded from four independent cultures derived from wild-type (WT) or S100B null mice, respectively. Bars represent means ⫾ SEM.
in the astrocytes (Fig. 4). On the other hand, treatment with NMDA and glycine did not stimulate Ca 2⫹ entry (Fig. 4). These observations agree with the reported presence of voltage-gated Ca 2⫹ channels but absence of NMDA effects on Ca 2⫹ permeability in cultured astro-
FIG. 7. Normal Ca 2⫹ responses in glial cells derived from S100B transgenic mice. Basal intracellular Ca 2⫹ transients, evoked in response to treatment with 50 mM KCl or 10 mM caffeine, were recorded from the total number of glial cells indicated in cerebellar cultures derived from wild-type or S100B transgenic mice. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Data were obtained from a summation of values recorded from two independent cultures derived from wild-type (WT) or S100B transgenic (Trans) mice, respectively. Bars represent means ⫾ SEM. NS, not significant.
Ca 2⫹ HANDLING IN S100B-DEFICIENT ASTROCYTES
FIG. 8. Normal Ca 2⫹ responses in granule cells in cultures derived from S100B null mice. Basal intracellular Ca 2⫹ and intracellular Ca 2⫹ transients evoked in response to treatment with 50 mM KCl or 10 mM caffeine were recorded from the total number of granule cells indicated in cerebellar cultures derived from wild-type or S100B null mice. Intracellular free Ca 2⫹ is expressed as the ratio of fura-2 emission at 510 nm following sequential excitation at 340 and 380 nm. Data were obtained from a summation of values recorded from four independent cultures derived from wild-type (WT) or S100B null mice, respectively. Bars represent means ⫾ SEM. NS, not significant.
cytes [49]. Glial cells in cultures derived from S100B null mice showed enhanced responses to treatment with pharmacological agents that stimulated Ca 2⫹ entry or release in comparison with glial cells in cultures derived from wild-type mice (Fig. 6). This suggests that the absence of S100B in glial cells from S100B null mice limits their Ca 2⫹-handling capacity to control increases in intracellular Ca 2⫹ levels. The specificity of the response in the glial cells was confirmed by parallel recordings from granule neurons in the same culture. Treatment with KCl or NMDA plus glycine stimulated Ca 2⫹ uptake by granule neurons, showing that they contained both voltage-gated Ca 2⫹ channels and postsynaptic NMDA receptors capable of mediating Ca 2⫹ entry in response to stimulation (Fig. 5). In addition, as S100B is expressed in astrocytes but not neurons in the cerebellum, granule cells in cultures derived from S100B null mice displayed normal responses to treatment with pharmacological agents that stimulate Ca 2⫹ entry or release (Fig. 8). The maintenance of intracellular Ca 2⫹ homeostasis is a critical component of the integrated response of the CNS to chemical signals or electrical activity. While this is the first report of a targeted inactivation of a major Ca 2⫹-binding protein in astrocytes, mice deficient in neuronal Ca 2⫹-binding proteins have been described previously. These include null mice with targeted inactivating mutations in the calbindin D 28k and calretinin genes [50, 51] and transgenic mice with reduced levels of calbindin D 28k resulting from expressed
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antisense transcripts [52]. Unlike the normal responses observed in the granule neurons in our S100B null mice (Fig. 5), abnormal Ca 2⫹ transients were observed in neurons from engineered mice with absent or reduced calbindin D 28k expression, as follows. In calbindin D 28k null mice, the peak amplitudes of dendritic calcium transients of cerebellar Purkinje cells were enhanced twofold in response to postsynaptic stimulation [50]. In mice with reduced levels of calbindin D 28k, there was a prolonged elevation of intracellular Ca 2⫹ in response to NMDA or KCl treatment [53]. The high concentration of S100B in astrocytes is consistent with its function as an intracellular Ca 2⫹ buffer in these cells. However, since S100B has been shown to interact in vitro with several target proteins in a Ca 2⫹-dependent manner [11], S100B could also modulate Ca 2⫹ homeostasis through alternative mechanisms. In the complete absence of S100B, i.e., in S100B null mice, a decreased Ca 2⫹-handling capacity in cultured glial cells could be demonstrated experimentally (Fig. 6). This indicates that S100B is a functional component of the complex system of plasma membrane exchangers, cytoplasmic organelles, and Ca 2⫹-binding proteins that regulate intracellular Ca 2⫹ in astrocytes [1]. However, it is interesting that there were no discernable differences in Ca 2⫹ handling between glial cells from wild-type mice and those from transgenic mice carrying 10 copies of the human S100B gene (Fig. 7). Both the endogenous mouse S100B gene and the human S100B transgene in the transgenic mice are induced during early postnatal development [16, 54]. Yet, we observed that Ca 2⫹ transients in glial cells in response to stimulation did not differ between wild-type and transgenic mice. This suggests that the S100B-dependent Ca 2⫹-handling capacity is present in excess in wild-type mice and is brought into play in our experimental conditions only at Ca 2⫹ concentrations that exceed a certain threshold. This explanation is consistent with a high concentration of S100B in astrocytes and its low binding affinity for Ca 2⫹ in vitro. We have no present evidence for developmental or behavioral consequences of the absence of S100B. It is possible that other buffer systems or feedback mechanisms compensate for the lack of S100B in intact animals. Alternatively, any disturbances in astrocytic Ca 2⫹ waves resulting from the absence of S100B could have subtle influences on neuronal synaptic transmission and hence animal behavior. For example, while mice lacking the expression of calbindin D 28k or calretinin exhibited no abnormalities in their normal cage environments, evidence of cerebellar incoordination with age could be brought out in these strains by experimental tests challenging their motor skills [50, 55]. A similar analysis of motor and cognitive functions in S100B null mice may provide evidence for subtle behavioral disturbances in these animals.
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This work was supported by grants from MRC to A.M. and J.F.M. and from NSERC to J.R. Z.-G.X. was an MRC Centennial Fellow. We thank Wanda Abramow-Newerley and Ela Czerwinska for expert technical assistance.
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