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Calbindin-D28k fails to protect hippocampal neurons against ischemia in spite of its cytoplasmic calcium buffering properties: evidence from calbindin-D28k knockout mice
Pergamon
PII:
Neuroscience Vol. 85, No. 2, pp. 361–373, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00632-5
CALBINDIN-D28k FAILS TO PROTECT HIPPOCAMPAL NEURONS AGAINST ISCHEMIA IN SPITE OF ITS CYTOPLASMIC CALCIUM BUFFERING PROPERTIES: EVIDENCE FROM CALBINDIN-D28k KNOCKOUT MICE G. J. KLAPSTEIN,* S. VIETLA,* D. N. LIEBERMAN,* P. A. GRAY,* M. S. AIRAKSINEN,† H. THOENEN,† M. MEYER† and I. MODY*‡ *Departments of Neurology and Physiology, UCLA School of Medicine RNRC 3-131, 710 Westwood Plaza, Los Angeles, CA 90095-1769, U.S.A. †Department of Neurochemistry, Max-Planck Institute for Psychiatry, Am Klopferspitz 18A, D-82152 Martinsried, Germany Abstract––Cytoplasmic calcium-binding proteins are thought to shield neurons against damage induced by excessive Ca2+ elevations. Yet, in theory, a mobile cellular Ca2+ buffer could just as well promote neuronal injury by facilitating the rapid dispersion of Ca2+ throughout the cytoplasm. In sharp contrast to controls, in mice lacking the gene for calbindin-D28k, synaptic responses of hippocampal CA1 pyramidal neurons which are normally extremely vulnerable to ischemia, recovered significantly faster and more completely after a transient oxygen-glucose deprivation in vitro, and sustained less cellular damage following a 12 min carotid artery occlusion in vivo. Other cellular and synaptic properties such as the altered adaptation of action potential firing, and altered paired-pulse and frequency potentiation at affected synapses in calbindin-D28k-deficient mice were consistent with a missing intraneuronal Ca2+ buffer. Our findings provide direct experimental evidence against a neuroprotective role for calbindin-D28k. 1998 IBRO. Published by Elsevier Science Ltd. Key words: calcium buffering, ischemia/hypoxia, neuronal vulnerability, oxygen-glucose deprivation, TUNEL, apoptosis.
Calcium plays a complex and often malign role in nerve cell function. Physiological Ca2+ entry into neurons regulates normal neuronal development, metabolism, ageing, and is involved in the control of synaptic transmission and its long-term modulation.16 Yet, an impaired cellular handling of Ca2+ yielding pathologically-elevated neuronal Ca2+ levels may constitute the final common pathway leading to neuronal degeneration.11,16,21 Accordingly, neurons are equipped with a sophisticated intracellular machinery which allows the continuous modulation of neuronal excitability but precludes excessive and sometimes fatal Ca2+ elevations. Increases in intraneuronal free Ca2+ ([Ca2+]i) are normally transient because several regulatory systems ‡To whom correspondence should be addressed. Abbreviations: ACSF, artificial cerebrospinal fluid; AHP, afterhyperpolarization; CV, Cresyl Violet; DAB, diaminobenzidine; EDTA, ethylenediaminetetra-acetate; EPSP, excitatory postsynaptic potential; HEPES, N-2hydroxyethylpiperazine-N -2-ethanesulphonic acid; ISI, inter-stimulus interval; NMDA, N-methyl--aspartate; OGD, oxygen/glucose deprivation; PB, phospate buffer; PFA, paraformaldehyde; RT, room temperature; TdT, terminal deoxyribonucleotidyl transferase; TTC, 2,3,5triphenyltetrazolium chloride; TUNEL, TdT-mediated biotin-14-dUTP nick-end labelling.
(mitochondria, endoplasmic reticulum, cytosolic Ca2+-binding proteins, and Ca2+ pumps) act in concert to buffer, sequester, or expel the bulk of Ca2+ entry.35 Many intracellular EF-hand Ca2+-binding proteins such as calbindin-D28k, parvalbumin, and calretinin are thought to function solely as cytoplasmic Ca2+-buffers.4,7,35 Of these, calbindin-D28k is confined to neurons in the mammalian CNS, but was originally discovered in the gut where it facilitates cellular Ca2+ transport and absorption.39 The currently favoured hypothesis for its role in nerve cells posits that calbindin-D28k conveys specific resistance against excessive Ca2+-dependent cellular vulnerability;10,22,28,31,46 however, see 14,36. Considering the effectiveness of exogenous cell permeant Ca2+-chelators against early excitotoxic and ischemic neuronal injury,51 we decided to examine ischemic/hypoxic neuronal damage in geneticallyengineered animals42 made null mutant for the presumed endogenous intracellular Ca2+-chelator calbindin-D28k.
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EXPERIMENTAL PROCEDURES
Calbindin-D28k null mutant mice As described in detail elsewhere,1 calbindin-D28k null mutant mice (calbindin-D28k/) were obtained by
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isolating calbindin-D28k genomic clones from a 129/Sv library. Using a 10.3 kb HindIII-EcoRI fragment as the targeting vector, a 1.3 kb ClaI-Eco47III fragment of the calbindin-D28k gene (containing part of the promoter and the first coding exon) was replaced with a 1.6 kb fragment containing the neomycin-resistance (neo) gene driven by the PGK promoter and bovine growth hormone polyadenylation signal. The chimeras were crossed to C57BL/6 wildtype mice. The calbindin-D28k knockouts develop normally, have no neuroanatomical deficits, and are behaviourally indistinguishable from their littermates in their normal cage environment. The only neurological deficit in the calbindinD28k/ mutants can be observed upon placing them on a runway where they display signs of cerebellar ataxia most likely resulting from the loss of calbindin-D28k from cerebellar Purkinje cells. The levels of other Ca2+-binding proteins (parvalbumin, calretinin, calbindin-D9k, calmodulin, and S100â) are all unaffected in calbindin-D28k/. As expected, in calbindin-D28k+/ animals calbindin-D28k levels determined by western blots are roughly half of those measured in calbindin-D28k+/+. All animals used in this study were obtained from heterozygous matings. All experiments were performed on littermates by investigators blinded with regard to the genotype of the animals. In vivo ischemia Mice were injected intraperitoneally with Napentobarbital (Abbott Laboratories, U.S.A., 85–100 mg/kg) under light halothane anaesthesia. A lateral incision was made in the neck, and superficial muscles were reflected to expose the trachea and carotid arteries. Transient brain ischemia was induced by ligating both common carotid arteries with 4-0 silk sutures threaded under each artery and pulled, with a half twist, ensuring a complete occlusion of blood flow. At the end of the hypoxic period (12 min), the ligatures were removed, and blood flow through the arteries was re-established. Body temperature was constantly monitored with a rectal probe and maintained with a heating pad and/or heat lamp both during surgery and following the hypoxic period until recovery from anaesthesia. Evaluation of ischemic damage Ischemic damage was assessed using several different methods. First, we used 2,3,5-triphenyltetrazolium chloride staining (TTC)20 to detect the ischemic infarct, with procedures slightly modified from those used by Isayama et al.23 Mice used ranged in age from 189 to 205 days. During surgery, body temperature was kept at 35.40.5C. Mice were allowed to recover for three days, then their brains were removed under halothane anaesthesia and placed into ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 4 CaCl2, 4 MgCl2, 26 NaHCO3, and 10 glucose. 500 µm coronal slices were made through the dorsal hippocampal region with a Vibratome. Slices were immersed in a thin layer of 2% TTC (w/v) in 0.2 M phosphate buffer (PB; 0.04 M NaH2PO4, 0.16 M Na2HPO4) in a Petri dish, and covered with a glass coverslip. The Petri dish was then wrapped with aluminum foil and set in a water bath at 37C for 10 min. The TTC solution was then replaced with fixative (10% paraformaldehyde (PFA) in 0.1 M PB) and slices were photographed for evaluation. Second, Cresyl Violet (CV) staining was performed on slices from a different set of animals. Briefly, mice used ranged in age from 117 to 151 days. Body temperature was maintained at 35.60.5C during surgery and then at 36.40.5C until recovery from anaesthetic. Mice were allowed to recover for 28 h following surgery. Brains were removed under halothane anaesthesia, fixed for 24–48 h in 4% PFA in 0.1 M PB, and cryopreserved overnight in 20% sucrose in 0.1 M PB. Coronal cryostat sections (30 µm) through the dorsal/septal hippocampus were mounted onto slides, desiccated, and frozen at 80C until needed. Frozen
sections were thawed overnight at 4C, then warmed to room temperature (RT). CV staining was performed using standard procedures. Thirdly, on adjacent sections (30 µm) to those used for CV staining, DNA fragmentation was assessed quantitatively in situ using the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-14-dUTP nick-end labelling procedure (TUNEL).15 Endogenous peroxidase activity was inactivated by immersion in 1% H2O2 in methanol for 10 min at RT. Sections were rehydrated through an ethanol series (100%, 95%, 70%), postfixed in 4% PFA in PB, and washed in 0.1 M Tris buffer (0.03 M Trizma base, 0.07 M Trizma HCl, pH 8). Nuclear permeabilization was achieved by proteinase K digestion (20 µg/ml in 0.1 M Tris, 0.05 M EDTA, pH 8) for 15 min at 37C, followed by a wash in ddH2O. Sections were then incubated with the labelling solution for 90 min at 37C (containing 0.625 units/µl TdT, 200 mM potassium cacodylate, 25 mM Tris–HCl, 0.25 mg/ ml bovine serum albumin, and 2.5 mM CoCl2, from terminal transferase kit, Boehringer Mannheim, U.S.A.; 10 µM biotin-14-dUTP from Gibco BRL, U.S.A.), washed in 0.1 M PB, and incubated in avidin–biotin–peroxidase solution in 0.1 M PB, (Vectastain Elite reagent kit, Vector Laboratories, U.S.A., mixed according to package instructions). Sections were then washed in PB and incubated in a diaminobenzidine (DAB)–Ni chromogen solution (8.9 mM DAB, 74.8 mM NH4Cl, 2.5 mM Ni(NH4)2(SO4)2 in 0.1 M PB), washed in PB, and counterstained with 0.05% OsO4 before being dehydrated through an ethanol-xylene series and coverslipped. Photographs were taken with a microscope-mounted camera using an interference-contrast filter. Cell counts were made by counting all positivelylabelled cells within the principal cell body layer contained in a 40 field of view, at six different positions within the hippocampal formation, including three equally spaced areas from CA1, an area of CA3 taken just lateral to the ends of the dentate gyrus granule cell body layers, and areas from the central portions of both the upper and lower blades of the dentate gyrus granule cell body layers. Results are expressed as meansS.E.M. of counts from both hemispheres at three different mid-septal levels per animal per area. Statistical comparisons were made by one-way ANOVA and Duncan’s post hoc procedure using the SPSS statistical software package. Statistical significance is reported for Pc0.05. CV staining was performed on 30 µm sections adjacent to those used for TUNEL staining. Electrophysiological recordings in hippocampal slices Extracellular recordings. For the oxygen/glucose deprivation (OGD) experiments, coronal brain slices (350 µmthick) were prepared from halothane-anaesthetized mice using a Vibratome (Lancer Series 1000) as described previously,47 and were incubated, submerged at 35C in a recording chamber and perfused constantly with ACSF containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 4 CaCl2, 4 MgCl2, 26 NaHCO3, 10 glucose, and 0.05 picrotoxin in an atmosphere of warm, moist carbogen (95% O2/5% CO2). Recording electrodes (3–4 MÙ) were filled with extracellular fluid, and extracellular field excitatory postsynaptic potentials (EPSPs) were recorded from CA1 pyramidal cell apical dendrites. Constant current stimuli (20 µs duration) were delivered every 15 or 30 s via a bipolar stimulating electrode placed in stratum radiatum of area CA1. The OGD was induced for 6 or 8 min by switching the atmosphere of the slices to 95% N2/5% CO2 instead of carbogen, and perfusing the slice with ACSF which had an equimolar substitution of sucrose for glucose, and which was also equilibrated with 95% N2/5% CO2. For the assessment of synaptic paired-pulse and frequency facilitation using extracellular recordings, the slices were cut in the horizontal plane and were incubated as described above except for the following: ACSF consisted of
Neuronal vulnerability in calbindin null-mutants (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 4 MgCl2, 26 NaHCO3, 10 glucose, 0.05 picrotoxin, and 0.05 -2-amino-5-phosphonovalerate. Extracellular recording electrodes were placed in the apical dendritic fields of CA1 or CA3 pyramidal cells. Stimulating electrodes were placed either in stratum radiatum of area CA1 or onto the mossy fibres in the dentate gyrus. Whole-cell patch-clamp recordings. Coronal-cut brain slices (350 µm-thick) were incubated as described above, and perfused constantly with ACSF containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 4 MgCl2, 26 NaHCO3, 10 glucose, 0.05 picrotoxin, 2 kynurenic acid. Whole-cell patch current-clamp recordings were done with an Axoclamp 2A amplifier as previously described in detail.47 Borosilicate glass capillaries with an inner filament (KG-33, 1.5 mm o.d., 1.12 mm i.d., Garner Glass, U.S.A.) pulled to 2–2.5 µm outer tip diameters (0.5–0.8 µm diameter of the lumen) using a two-stage vertical Narishige PP-83 puller were filled with 130 mM K-methylsulphate, 5 mM KCl, 10 mM HEPES (pH 7.2, osmolarity 268–272 mosm). The solution contained no exogenous Ca2+-chelators to avoid possible interference with the endogenous Ca2+buffering capacity of neurons.25,47 Cell-attached recordings of N-methyl--aspartate channel openings. Dentate gyrus granule cells were acutely dissociated from CB+/+ and CB/ mouse hippocampal slices according to the method previously published for rat brain slices,24,25 with the exception that the time for incubation in pronase was reduced to 25 min. Fire polished, Sylgardcoated, thick-walled borosilicate (Garner Glass) glass pipettes (10–25 MÙ) were filled with the recording/ extracellular solution containing (in mM): 110 Na2SO4, 5 Cs2SO4, 1.8 CaCl2, 10 HEPES, 10 -glucose, 1 pyruvic acid, and 0.001 tetrodotoxin (Calbiochem, U.S.A.; final pH=7.25, osmolarity=285–305 mOsm). Cell-attached recordings were obtained at 22–25C using pipettes filled with 200 nM -aspartic acid and 10 µM glycine dissolved in the extracellular solution.24,27 Details of the single channel detection using the PAT program (courtesy of Dr J. Dempster) with a 50% threshold crossing algorithm, and event analysis including the determination of burst, cluster and supercluster durations, using an in-house program are provided elsewhere.24
RESULTS
In vitro oxygen/glucose deprivation We directly tested the relationship between Ca2+dependent neuronal vulnerability and calbindin-D28k content in both an in vitro and an in vivo model of ischemia. Pyramidal neurons of the hippocampal CA1 region are among the most vulnerable cells to ischemia,45 and synaptic responses to the activation of Schaffer collateral/commissural input to these cells can be used as a measure of their synaptic excitability during impaired metabolic conditions.30 We have investigated how transiently exposing hippocampal slices to various periods of OGD affects the rate of rise (dV/dt) of field EPSPs recorded in the stratum radiatum of the CA1 region of hippocampal slices prepared from three different types of mice genetically engineered for the lack of calbindinD28k.1 The three preparations consisted of null mutant homozygous calbindin-D28k knockout mice
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(calbindin-D28k/, n=10), and their heterozygous (calbindin-D28k+/, n=10), and wild-type littermates (calbindin-D28k+/+, n=10). A transient, 3 min anoxia alone (not shown), or a 6 min period of OGD resulted in no significant differences in the recovery rates of the EPSPs in the three preparations (Fig. 1A, Table 1). A slightly longer metabolic challenge, a transient 8 min OGD, caused a marked and prolonged depression (>2 h) of the synaptic responses in slices obtained from calbindin-D28k+/, and calbindin-D28k+/+ animals (Fig. 1B, Table 1). In contrast, the synaptic responses fully abolished during OGD (Table 1) in calbindin-D28k deficient pyramidal neurons recovered to 72% of control, with a five-fold faster rate of recovery than that observed in calbindin-D28k+/+ preparations (Fig. 1B, Table 1). In vivo ischemia To generate ischemia in vivo, mice were subjected to a bilateral carotid artery ligation for 12 min under pentobarbital anaesthesia while their body temperatures were maintained constant (see Experimental Procedures). Three days after the transient ischemia, slices were prepared and stained with the vital dye Tetrazolium Red (TTC),20,23 a colourless reagent which is metabolized by functional mitochondrial enzymes into a red product, and which is frequently used to assess ischemic brain damage. Considerable damage was observed in the subiculum, area CA1, and the hilus of the dentate gyrus of calbindinD28k+/+ animals, while only small TTC-negative areas could be observed in the subiculum and hilus of calbindin-D28k+/ animals. No detectable damage was found in any of the CB/ animals (n=6, data not shown). To assess the damage quantitatively at the cellular level, standard Cresyl Violet staining and TUNEL staining, an immunohistochemical procedure detecting DNA fragmentation, were performed on adjacent 30 µm coronal hippocampal sections from a different set of animals, fixed 28 h following surgery (n=3 calbindin-D28k/, n=2 calbindin-D28k+/, n=3 calbindin-D28k+/+). In the CV-stained sections, ischemic damage was considerably more evident in calbindin-D28k+/+ than in calbindin-D28k/ animals (Figs 2, 3). Throughout the calbindin-D28k+/+ hippocampus, but especially in areas CA1 and the dentate gyrus, many cells appeared shrunken and nuclei appeared condensed. TUNEL-positive cells were frequently found clustered in the principal cell body layers and hilus of the dentate gyrus, with occasional presumable interneurons also found in the dendritic field regions throughout the hippocampus (Fig. 4). Cell counts of TUNEL-positive cells in the principal cell body layers of CA1, CA3 and the dentate gyrus were higher in calbindin-D28k+/+ than in
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Fig. 1. Effects of oxygen/glucose deprivation (OGD) on evoked dendritic field EPSPs in hippocampal CA1 of calbindin-D28k/, calbindin-D28k+/, and calbindin-D28k+/+ mice. For the period of time indicated by the bar (A: 6 min, B: 8 min) an equimolar substitution of sucrose for glucose was made in the perfusion solution equilibrated with 95% N2/5% CO2 which was also substituted for carbogen in the atmosphere. Symbols represent the average of the slopes of dendritic field EPSPs, normalized such that control levels=1.0 and maximum effect=0.0. Error bars represent S.E.M. Single exponential curves were fitted using a non-linear least squares Simplex method to the recovery phase of the averaged data. The following recovery time constants were obtained (all in min). For 6 min OGD: calbindin-D28k/, 4.45; calbindin-D28k+/, 6.08; calbindin-D28k+/+, 9.26; for 8 min OGD: calbindin-D28k/, 6.17; calbindinD28k+/, 5.71; calbindin-D28k+/+, 29.60. Below each graph are representative traces of CA1 dendritic field EPSPs, elicited under control conditions (Control), during the maximum effect OGD (Anoxia) and following return to control conditions (Recovery), for each genotype (, calbindin-D28k/; , calbindin-D28k+/; , calbindin-D28k+/+). All experiments and analyses of EPSP slopes were conducted and analysed blindly with respect to the genotype of the animals. Table 1. Maximum inhibition of excitatory postsynaptic potential slope during oxygen/glucose deprivation, and its recovery following return to control conditions Maximum inhibition (%)
Recovery at 30 min (%)
Genotype
6 min OGD
8 min OGD
6 min OGD
8 min OGD
CB/ CB+/ CB+/+
917 (8) 952 (15) 1000 (10)
934 (11) 952 (16) 934 (11)
8513 (7) 6512 (15) 9311 (10)
7210 (11)* 269 (15) 2119 (10)
Data for maximum % inhibition show meansS.E.M. during the period of OGD, normalized to control. Data for recovery are meansS.E.M. obtained at 30 min following the start of OGD, as depicted in Fig. 1. Numbers in parentheses indicate the number of experiments. The asterisk indicates a significant difference from both other groups in pairwise comparisons (two-tailed t-test, P<0.05). CB, calbindin-D28k.
calbindin-D28k/ littermates (Fig. 5). In the subicular third of CA1 (area 1), TUNEL-positive cell counts in calbindin-D28k/ and calbindinD28k+/ animals were 15% and 64% of wild-type, respectively. In the central third of CA1 (area 2), the respective cell counts were 16% and 76% of wild-type. In the lateral third of CA1 (area 3), the respective cell counts were 16% and 97% of wild-type. In CA3 (area
4), the respective cell counts were 22% and 65% of wild-type. In the upper blade of the dentate gyrus (area 5), the respective cell counts were 2% and 157% of wild-type. In the lower blade of the dentate gyrus (area 6), the respective cell counts were 3% and 102% of wild-type. There were significantly fewer (P<0.05) TUNEL-positive cells in calbindin-D28k/ than in calbindin-D28k+/+ mice in every hippocampal region
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Fig. 2. Hippocampal damage induced by ischemia. Calbindin knockout mice (calbindin-D28k/, bottom), their heterozygote (calbindin-D28k+/, not shown) or wild-type (calbindin-D28k+/+, top) littermates were subjected to a 12 min bilateral carotid artery occlusion and allowed to recover for 28 h. Subsequent CV staining of 30 µm hippocampal sections shows severe damage in the subiculum, CA1, and dentate gyrus of calbindin-D28k+/+ compared with calbindin-D28k/ mice. Scale bar=400 µm.
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Fig. 3. Higher magnification views of sections shown in Fig. 2. CV-stained 30 µm sections from area CA1 (top row) and dentate gyrus (bottom row) of calbindin-D28k+/+ (left) and calbindin-D28k/ (right) mice which have undergone 12 min bilateral carotid artery occlusion with 28 h recovery. Scale bar=50 µm.
examined. TUNEL-positive cells in calbindinD28k+/ animals were significantly more numerous than in calbindin-D28k/ animals in areas 2 and 3 (CA1) and areas 5 and 6 (dentate gyrus), but were not significantly different from calbindin-D28k+/+ animals in any area. Cellular properties of hippocampal neurons devoid of calbindin-D28k We also wanted to examine whether other aspects of cellular and synaptic physiology normally reliant on intracellular Ca2+ buffering were altered when calbindin-D28k was eliminated from various hippocampal neurons and their axon terminals. In wholecell current-clamp recordings, we studied the adaptation of action potential firing and post-spike afterhyperpolarizations (AHP) in dentate gyrus granule cells from calbindin-D28k+/+ and calbindinD28k/ animals (n=4 each). These cellular parameters are governed by Ca2+-dependent K+ conductances (gKCa) which are dependent on the amount of intracellular Ca2+ buffering.48 Figure 6 shows examples from three different neurons. In a
total of 24 dentate gyrus granule cells (n=8 from each preparation), the number of action potentials elicited by a 200 ms long 0.5 nA depolarizing current pulse from the comparable resting membrane potential (calbindin-D28k/: 82.72.6 mV; calbindin-D28k+/+: 82.13.2 mV) was significantly (P<0.01, two-tailed t-test) larger (11.72.4) in calbindin-D28k+/+ than in calbindin-D28k/ animals (3.41.8). The input resistances (RN) of granule cells were comparable in the two preparations (calbindin-D28k+/+: 16812 MÙ; calbindin-D28k/: 15719 MÙ). In calbindinD28k/ granule cells (n=8), the amplitude and the area of the post-spike AHP measured after the first action potential elicited by the threshold current pulse were respectively 217% and 176% of those recorded in calbindin-D28k+/+ granule cells (n=8; Fig. 6). Effects of calbindin-D28k loss on synaptic function Considering the dependence of neurotransmitter release at central synapses on presynaptic Ca2+ and its buffering,41,49 we next compared frequency
Neuronal vulnerability in calbindin null-mutants
Fig. 4. TUNEL staining of hippocampal slices showing DNA fragmentation. Dark staining indicates cells undergoing apoptotic or necrotic cell death. Photographs were taken at 40 (top row, each set) or 100 (oil immersion, bottom row, each set) of the CA1 pyramidal cell body layer (top set) and dentate granule cell layer (bottom set) of calbindin-D28k+/+ (left) and calbindin-D28k/ (right) mice. Scale bar=25 µm for 40 and 10 µm for 100 photographs.
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at the mossy fibre synapses onto CA3 pyramidal cells, where the mossy fibre terminals are devoid of calbindin-D28k in the calbindin-D28k/ mutants, there was a significant (P<0.05) 1.8-times slower rate of potentiation and a 20% smaller steady-state value attained during frequency potentiation at 33 Hz compared to that found in calbindin-D28k+/+ slices (Fig. 7). A 30–33% decrease in paired-pulse facilitation was also found in the dendritic region of CA3 pyramidal cells. This decrease in facilitation was significant (P<0.05, two-tailed t-test; n=8 slices for calbindin-D28k/, and n=6 for calbindin-D28k+/+) for short (20, 30 and 50 ms) ISIs. Fig. 5. Cell counts of TUNEL-positive cells in six different hippocampal regions of calbindin-D28k+/+, calbindinD28k+/ and calbindin-D28k/ mice which have undergone 12 min bilateral carotid artery occlusion with 28 h recovery. The total number of TUNEL-positive cells were counted in the cell body layer within a field of view as seen with a 40 objective. These areas corresponded approximately to the size and position of the rectangles indicated in the inset. A camera lucida attached to the microscope served to identify and later count the TUNEL-positive cells. Error bars indicate S.E.M. Calbindin-D28k/ differed from calbindin-D28k+/+ in all areas and from Calbindin-D28k+/ in all areas except CA3 (area 4). Calbindin-D28k+/+ and calbindin-D28k+/ were not significantly different (n=8 hippocampal sections from each of three calbindinD28k+/+, six from each of two calbindin-D28k+/, and eight from each of three calbindin-D28k/ mice).
potentiation and paired-pulse facilitation of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mediated component of EPSPs elicited by various inter-stimulus intervals at two different synapses deliberately chosen to have the calbindinD28k deficiency in calbindin-D28k/ mutants restricted solely to pre- or postsynaptic elements. The presynaptic buffering role of calbindin-D28k was assessed at the mossy fibre-CA3 pyramidal cell synapse where the mossy fibres have high levels of calbindin-D28k in calbindin-D28k+/+, while the CA3 pyramidal cells are normally devoid of the protein.4 At a second hippocampal synapse, the Schaffer collateral/commissural input onto CA1 pyramidal cells, the excitatory terminals lack calbindin-D28k even in calbindin-D28k+/+ animals.4 Therefore, the excitatory axon terminals at this synapse are similar in calbindin-D28k/ and calbindin-D28k+/+, and the postsynaptic cells are devoid of calbindin-D28k only in calbindin-D28k/. The time-course of EPSP potentiation during a train of stimuli can best be fitted by a single exponential.41,49 At the Schaffer collateral/commissural synapses stimulated at a frequency of 33 Hz, there were no significant differences between the calbindin-D28k/ and calbindinD28k+/+ preparations (Fig. 7). Furthermore, there was no statistically significant change, not even at short (25–50 ms) inter-stimulus intervals (ISIs), in the level of paired-pulse facilitation of CA1 field EPSPs evoked by Schaffer collateral/commissural stimulation in calbindin-D28k/ mice (Fig. 7). In contrast,
N-methyl--aspartate channel properties are unaltered in calbindin-D28k null mutants The openings of N-methyl--aspartate (NMDA) channels are sensitive to rises in intracellular Ca2+ which can affect the channel properties directly,32,33 through activation of the protein phosphatase calcineurin,27,50 or through the Ca2+-dependent modulator, calmodulin.13 If calbindin-D28k levels surrounding the inner mouth of the NMDA channel are sufficiently high, and the binding of Ca2+ to calbindin-D28k is considerably faster than the Ca2+binding proteins involved in the regulation of NMDA channel activity, the loss of calbindin-D28k may significantly alter the openings of NMDA channels. A change in the properties of NMDA channels in calbindin-D28k knockouts may also be responsible for the altered sensitivity to ischemia or OGD. In order to assess any possible alterations in NMDA channel properties, we recorded from six dentate gyrus granule cells each obtained from four calbindin-D28k+/+ and four calbindin-D28k/ mice. We chose to record from these neurons because of their homogeneity with regard to their calbindinD28k content under control conditions.3,4 The cell-attached recording mode was preferred to avoid any interference with intracellular Ca2+-buffering mechanisms or second messenger systems.24,27 As shown on Table 2, the properties of NMDA channels were similar regardless of the presence of calbindin-D28k in the cells. DISCUSSION
Our findings are consistent with a Ca2+-buffering role of calbindin-D28k in central neurons. But, in contrast to previous studies reporting correlations between calbindin-D28k content and neuronal survival after injury,10,18,22,28,31,46 hippocampal cells of calbindin-D28k/ mice were more resistant to the effect of a transient OGD and ischemia than their calbindin-D28k containing counterparts. Our main evidence for the role of calbindin-D28k as a Ca2+-buffer consists of the differences in the activation of gKCa between calbindin-D28k+/+ and calbindin-D28k/ neurons. The frequency of
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Fig. 6. Increased adaptation of action potential firing and post-spike AHP in calbindin-D28k/ mouse granule cells. The figure shows the responses of one calbindin-D28k+/+ and two calbindin-D28k/ neurons to hyperpolarizing and depolarizing current pulses (200 ms). In the case of neurons M1715 (calbindin-D28k+/+) and M1513 (calbindin-D28k/) the pulses were injected at the resting membrane potential of the cells (81 and 82 mV, respectively). In neuron M1703 (calbindin-D28k/) the cell was held depolarized at 64 mV (+20 mV from its actual resting potential of 84 mV). The same high degree of adaptation was observed at this depolarized membrane potential. The resting RN of the two neurons M1715 (calbindin-D28k+/+ ) and M1513 (calbindin-D28k/ ) are indicated on the top right panel. The numbers show the linear regression fits to the I–V plots with the meanS.E.M. of the slope. The traces on the bottom panel are superimposed action potentials showing the second spike in the trains from the three granule cells. The action potential recorded in the control neuron (M1715; calbindinD28k+/+) is shown with a thicker line. Note the more than doubling of the amplitudes of the rapid post-spike AHP in the two calbindin-D28k/ granule cells. Similar results were obtained in six other calbindin-D28k/ neurons.
action potential firing in central neurons is regulated by several voltage- and second-messenger gated ion channels including a large number of K+ conductances. Of these, a fast gKCa is partly responsible for the rapid repolarization of action potentials in various types of neuron,29,48 and can be blocked by fast intracellular Ca2+ chelators such as 1,2-bis(2aminophenoxy)-ethane-N,N,N ,N -tetraacetic acid (BAPTA) in both CA1 pyramidal48 and dentate gyrus granule cells.47 The enhancement of the postspike AHP in calbindin-D28k/ granule cells can thus be explained by the absence of an intracellular Ca2+-buffering system with fast kinetics, and together with the increased adaptation of firing frequency
observed in calbindin-D28k/ granule cells, illustrates the effectiveness of calbindin-D28k in fine tuning neuronal firing patterns.26 Based on experimental and theoretical predictions at the mossy fibre–CA3 pyramidal cell synapse41,49 and the attenuation of post-tetanic potentiation in cultured hippocampal neurons transfected with a calbindin-D28k gene containing adenovirus,8 it was surprising to see decreased frequency potentiation and paired pulse facilitation in the absence of presynaptic calbindin-D28k. However, if gKCa contributes to the control of neurotransmitter release at this synapse, just as it does at other synaptic junctions,6,43,44 an enhanced activation of gKCa in terminals without calbindin-D28k is
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Fig. 7. Frequency potentiation and paired-pulse facilitation of mossy fibre but not Schaffer collateral/ commissural fibre-evoked responses is decreased in calbindin-D28k/ mice. The frequency potentiation of field EPSPs (left panels), was recorded in the str. radiatum of the CA1 and CA3 regions, respectively, evoked by low intensity repetitive stimulation (33 Hz) of the Schaffer collateral/commissural fibres and mossy fibres. The facilitation ratio (PnP1)/P1, where Pn and P1 are the rates of rise (dV/dt) of the nth and first EPSPs during the paired-pulse stimulation, was plotted against time. The rate of frequency potentiation can be assessed by the indicated fits of the average values to single exponential functions (solid lines). The 95% confidence intervals are shown with thin lines. Error bars represent S.E.M. (n=8 slices for both calbindin-D28k/ and calbindin-D28k+/+). The two curves and their asymptotes are similar for the CA1 responses in calbindin-D28k+/+ and calbindin-D28k/ preparations, but are significantly different (P<0.05) for the CA3 EPSPs. Right panels: The reduction in paired-pulse facilitation in the CA3 region of calbindin-D28k/ mice is significant (P<0.05, two-tailed t-test) at the three shortest ISI. In contrast, there are no differences in the CA1 region. The facilitation ratio was calculated as above for n=2 at ISIs ranging between 20 and 1000 ms. Error bars represent S.E.M. (n=8 slices for calbindin-D28k/ and n=6 for calbindin-D28k+/+). Table 2. Similar N-methyl--aspartate channel properties in calbindin-D28k+/+ and calbindinD28k/ mice Genotype
NMDA channel properties (in ms) Open time Burst length Total open time/burst Cluster length Total open time/cluster Supercluster length
CB+/+
CB/
2.520.26(6) 6.491.96(6) 3.980.64(6) 16.843.66(6) 7.131.34(6) 124.842.7(6)
3.020.41(6) 6.962.05(6) 5.261.31(6) 16.354.00(6) 8.982.63(6) 119.222.9(6)
NMDA channel openings were recorded in the cell-attached mode in six dentate gyrus granule cells each acutely dissociated from calbindin-D28k+/+ (n=4) and calbindin-D28k/ mice (n=4). With 200 nM -aspartate and 10 mM glycine in the pipette, no significant differences were detected between the basal opening properties of NMDA channels in neurons with or without calbindin-D28k. CB, calbindin-D28k.
conceivable.40 An increase in gKCa similar to that seen at the somata of calbindin-D28k-deficient neurons, may override the effect of an elevated Ca2+ entry1 on transmitter release.
The early events in OGD including the lasting depression of synaptic transmission are harbingers of neuronal death.30 This is the first time the effect of OGD on synaptic transmission has been examined in
Neuronal vulnerability in calbindin null-mutants
mice, and it is clearly more severe than that of oxygen deprivation alone which does not lead to an irreversible loss of synaptic function in this species.52 The enhanced recovery of calbindin-D28k deficient CA1 pyramidal cell synaptic responses after a certain threshold level of OGD in vitro is consistent with a deleterious rather than a neuroprotective46 effect of calbindin-D28k in neurotoxicity.10,22,28,31 Likewise, 28 h after transient ischemia in vivo, hippocampi of calbindin-D28k+/+ animals had a greater number of abnormally shrunken principal cells with condensed, pyknotic-looking nuclei than did their calbindinD28k/ littermates. These results are mirrored by the number of cells labelled by TUNEL stain, which detects DNA fragmentation and is the method of choice for detecting imminent apoptotic or necrotic ischemic cell death.9 That wild-type mice expressing normal levels of calbindin suffer more TUNELpositive neurons following transient ischemia than their calbindin-D28k/ littermates is irreconcilable with a generally neuroprotective role for this protein. The use of littermates avoided possible complications resulting from the variability in ischemic neuronal damage seen in various mouse strains.5,54 The dentate granule cell damage found by us in the wild-type mice has been observed in this species before,38 although it is contrary to the more common observation that granule cells are relatively resistant to ischemic damage in adult rats.45 Other studies17,19 also indicate that the resistance of the granule cells may be age or species dependent. As NMDA channel properties are similar in calbindin-D28k-containing and calbindin-D28kdeficient cells, these excitatory amino acid channels are probably not responsible for the differences in neurotoxicity observed between the two prepara-
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tions. The absence of calbindin-D28k may protect neurons by enhancing the Ca2+-dependent inactivation of voltage-gated Ca2+ entry25 thereby reducing the net Ca2+ influx during prolonged depolarizations. In contrast, the presence of a mobile cytoplasmic Ca2+ buffer such as calbindin-D28k could enhance the dispersion of Ca2+ throughout the cytoplasm,53,55 thus delivering the Ca2+ that enters the cells,12 or that is being released from intracellular stores,37 much faster and more effectively to the various cellular compartments where it may exert its malignant effects on neuronal survival. CONCLUSION
Clearly, Ca2+-binding proteins have a complex role in neurons, but should not be necessarily regarded as effective protectors against Ca2+-dependent neuronal damage. Consistent with this notion is the recent observation in midbrain dopaminergic neurons describing a lack of neuroprotective role of calbindin-D28k against 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) toxicity.2 As pointed out in a recent review,12 the entire Ca2+-overload hypothesis of ischemic-hypoxic neuronal damage may have to be refined depending on the nature of the cellular killing mechanism activated, and this will ultimately lead to novel cytoprotective therapies for stroke.34 Acknowledgements—We want to thank B. Oyama for expert technical assistance. This research was supported by NINDS grants to I.M., an AHFMR Fellowship to G.J.K., an APA MFP Fellowship to P.A.G., a Howard Hughes Predoctoral Fellowship to D.N.L., and by grants from the Academy of Finland and Regeneron Pharmaceuticals (Tarrytown) to M.S.A.
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Neuroscience Vol. 85, No. 2, pp. 361–373, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00632-5
CALBINDIN-D28k FAILS TO PROTECT HIPPOCAMPAL NEURONS AGAINST ISCHEMIA IN SPITE OF ITS CYTOPLASMIC CALCIUM BUFFERING PROPERTIES: EVIDENCE FROM CALBINDIN-D28k KNOCKOUT MICE G. J. KLAPSTEIN,* S. VIETLA,* D. N. LIEBERMAN,* P. A. GRAY,* M. S. AIRAKSINEN,† H. THOENEN,† M. MEYER† and I. MODY*‡ *Departments of Neurology and Physiology, UCLA School of Medicine RNRC 3-131, 710 Westwood Plaza, Los Angeles, CA 90095-1769, U.S.A. †Department of Neurochemistry, Max-Planck Institute for Psychiatry, Am Klopferspitz 18A, D-82152 Martinsried, Germany Abstract––Cytoplasmic calcium-binding proteins are thought to shield neurons against damage induced by excessive Ca2+ elevations. Yet, in theory, a mobile cellular Ca2+ buffer could just as well promote neuronal injury by facilitating the rapid dispersion of Ca2+ throughout the cytoplasm. In sharp contrast to controls, in mice lacking the gene for calbindin-D28k, synaptic responses of hippocampal CA1 pyramidal neurons which are normally extremely vulnerable to ischemia, recovered significantly faster and more completely after a transient oxygen-glucose deprivation in vitro, and sustained less cellular damage following a 12 min carotid artery occlusion in vivo. Other cellular and synaptic properties such as the altered adaptation of action potential firing, and altered paired-pulse and frequency potentiation at affected synapses in calbindin-D28k-deficient mice were consistent with a missing intraneuronal Ca2+ buffer. Our findings provide direct experimental evidence against a neuroprotective role for calbindin-D28k. 1998 IBRO. Published by Elsevier Science Ltd. Key words: calcium buffering, ischemia/hypoxia, neuronal vulnerability, oxygen-glucose deprivation, TUNEL, apoptosis.
Calcium plays a complex and often malign role in nerve cell function. Physiological Ca2+ entry into neurons regulates normal neuronal development, metabolism, ageing, and is involved in the control of synaptic transmission and its long-term modulation.16 Yet, an impaired cellular handling of Ca2+ yielding pathologically-elevated neuronal Ca2+ levels may constitute the final common pathway leading to neuronal degeneration.11,16,21 Accordingly, neurons are equipped with a sophisticated intracellular machinery which allows the continuous modulation of neuronal excitability but precludes excessive and sometimes fatal Ca2+ elevations. Increases in intraneuronal free Ca2+ ([Ca2+]i) are normally transient because several regulatory systems ‡To whom correspondence should be addressed. Abbreviations: ACSF, artificial cerebrospinal fluid; AHP, afterhyperpolarization; CV, Cresyl Violet; DAB, diaminobenzidine; EDTA, ethylenediaminetetra-acetate; EPSP, excitatory postsynaptic potential; HEPES, N-2hydroxyethylpiperazine-N -2-ethanesulphonic acid; ISI, inter-stimulus interval; NMDA, N-methyl--aspartate; OGD, oxygen/glucose deprivation; PB, phospate buffer; PFA, paraformaldehyde; RT, room temperature; TdT, terminal deoxyribonucleotidyl transferase; TTC, 2,3,5triphenyltetrazolium chloride; TUNEL, TdT-mediated biotin-14-dUTP nick-end labelling.
(mitochondria, endoplasmic reticulum, cytosolic Ca2+-binding proteins, and Ca2+ pumps) act in concert to buffer, sequester, or expel the bulk of Ca2+ entry.35 Many intracellular EF-hand Ca2+-binding proteins such as calbindin-D28k, parvalbumin, and calretinin are thought to function solely as cytoplasmic Ca2+-buffers.4,7,35 Of these, calbindin-D28k is confined to neurons in the mammalian CNS, but was originally discovered in the gut where it facilitates cellular Ca2+ transport and absorption.39 The currently favoured hypothesis for its role in nerve cells posits that calbindin-D28k conveys specific resistance against excessive Ca2+-dependent cellular vulnerability;10,22,28,31,46 however, see 14,36. Considering the effectiveness of exogenous cell permeant Ca2+-chelators against early excitotoxic and ischemic neuronal injury,51 we decided to examine ischemic/hypoxic neuronal damage in geneticallyengineered animals42 made null mutant for the presumed endogenous intracellular Ca2+-chelator calbindin-D28k.
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EXPERIMENTAL PROCEDURES
Calbindin-D28k null mutant mice As described in detail elsewhere,1 calbindin-D28k null mutant mice (calbindin-D28k/) were obtained by
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isolating calbindin-D28k genomic clones from a 129/Sv library. Using a 10.3 kb HindIII-EcoRI fragment as the targeting vector, a 1.3 kb ClaI-Eco47III fragment of the calbindin-D28k gene (containing part of the promoter and the first coding exon) was replaced with a 1.6 kb fragment containing the neomycin-resistance (neo) gene driven by the PGK promoter and bovine growth hormone polyadenylation signal. The chimeras were crossed to C57BL/6 wildtype mice. The calbindin-D28k knockouts develop normally, have no neuroanatomical deficits, and are behaviourally indistinguishable from their littermates in their normal cage environment. The only neurological deficit in the calbindinD28k/ mutants can be observed upon placing them on a runway where they display signs of cerebellar ataxia most likely resulting from the loss of calbindin-D28k from cerebellar Purkinje cells. The levels of other Ca2+-binding proteins (parvalbumin, calretinin, calbindin-D9k, calmodulin, and S100â) are all unaffected in calbindin-D28k/. As expected, in calbindin-D28k+/ animals calbindin-D28k levels determined by western blots are roughly half of those measured in calbindin-D28k+/+. All animals used in this study were obtained from heterozygous matings. All experiments were performed on littermates by investigators blinded with regard to the genotype of the animals. In vivo ischemia Mice were injected intraperitoneally with Napentobarbital (Abbott Laboratories, U.S.A., 85–100 mg/kg) under light halothane anaesthesia. A lateral incision was made in the neck, and superficial muscles were reflected to expose the trachea and carotid arteries. Transient brain ischemia was induced by ligating both common carotid arteries with 4-0 silk sutures threaded under each artery and pulled, with a half twist, ensuring a complete occlusion of blood flow. At the end of the hypoxic period (12 min), the ligatures were removed, and blood flow through the arteries was re-established. Body temperature was constantly monitored with a rectal probe and maintained with a heating pad and/or heat lamp both during surgery and following the hypoxic period until recovery from anaesthesia. Evaluation of ischemic damage Ischemic damage was assessed using several different methods. First, we used 2,3,5-triphenyltetrazolium chloride staining (TTC)20 to detect the ischemic infarct, with procedures slightly modified from those used by Isayama et al.23 Mice used ranged in age from 189 to 205 days. During surgery, body temperature was kept at 35.40.5C. Mice were allowed to recover for three days, then their brains were removed under halothane anaesthesia and placed into ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 4 CaCl2, 4 MgCl2, 26 NaHCO3, and 10 glucose. 500 µm coronal slices were made through the dorsal hippocampal region with a Vibratome. Slices were immersed in a thin layer of 2% TTC (w/v) in 0.2 M phosphate buffer (PB; 0.04 M NaH2PO4, 0.16 M Na2HPO4) in a Petri dish, and covered with a glass coverslip. The Petri dish was then wrapped with aluminum foil and set in a water bath at 37C for 10 min. The TTC solution was then replaced with fixative (10% paraformaldehyde (PFA) in 0.1 M PB) and slices were photographed for evaluation. Second, Cresyl Violet (CV) staining was performed on slices from a different set of animals. Briefly, mice used ranged in age from 117 to 151 days. Body temperature was maintained at 35.60.5C during surgery and then at 36.40.5C until recovery from anaesthetic. Mice were allowed to recover for 28 h following surgery. Brains were removed under halothane anaesthesia, fixed for 24–48 h in 4% PFA in 0.1 M PB, and cryopreserved overnight in 20% sucrose in 0.1 M PB. Coronal cryostat sections (30 µm) through the dorsal/septal hippocampus were mounted onto slides, desiccated, and frozen at 80C until needed. Frozen
sections were thawed overnight at 4C, then warmed to room temperature (RT). CV staining was performed using standard procedures. Thirdly, on adjacent sections (30 µm) to those used for CV staining, DNA fragmentation was assessed quantitatively in situ using the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-14-dUTP nick-end labelling procedure (TUNEL).15 Endogenous peroxidase activity was inactivated by immersion in 1% H2O2 in methanol for 10 min at RT. Sections were rehydrated through an ethanol series (100%, 95%, 70%), postfixed in 4% PFA in PB, and washed in 0.1 M Tris buffer (0.03 M Trizma base, 0.07 M Trizma HCl, pH 8). Nuclear permeabilization was achieved by proteinase K digestion (20 µg/ml in 0.1 M Tris, 0.05 M EDTA, pH 8) for 15 min at 37C, followed by a wash in ddH2O. Sections were then incubated with the labelling solution for 90 min at 37C (containing 0.625 units/µl TdT, 200 mM potassium cacodylate, 25 mM Tris–HCl, 0.25 mg/ ml bovine serum albumin, and 2.5 mM CoCl2, from terminal transferase kit, Boehringer Mannheim, U.S.A.; 10 µM biotin-14-dUTP from Gibco BRL, U.S.A.), washed in 0.1 M PB, and incubated in avidin–biotin–peroxidase solution in 0.1 M PB, (Vectastain Elite reagent kit, Vector Laboratories, U.S.A., mixed according to package instructions). Sections were then washed in PB and incubated in a diaminobenzidine (DAB)–Ni chromogen solution (8.9 mM DAB, 74.8 mM NH4Cl, 2.5 mM Ni(NH4)2(SO4)2 in 0.1 M PB), washed in PB, and counterstained with 0.05% OsO4 before being dehydrated through an ethanol-xylene series and coverslipped. Photographs were taken with a microscope-mounted camera using an interference-contrast filter. Cell counts were made by counting all positivelylabelled cells within the principal cell body layer contained in a 40 field of view, at six different positions within the hippocampal formation, including three equally spaced areas from CA1, an area of CA3 taken just lateral to the ends of the dentate gyrus granule cell body layers, and areas from the central portions of both the upper and lower blades of the dentate gyrus granule cell body layers. Results are expressed as meansS.E.M. of counts from both hemispheres at three different mid-septal levels per animal per area. Statistical comparisons were made by one-way ANOVA and Duncan’s post hoc procedure using the SPSS statistical software package. Statistical significance is reported for Pc0.05. CV staining was performed on 30 µm sections adjacent to those used for TUNEL staining. Electrophysiological recordings in hippocampal slices Extracellular recordings. For the oxygen/glucose deprivation (OGD) experiments, coronal brain slices (350 µmthick) were prepared from halothane-anaesthetized mice using a Vibratome (Lancer Series 1000) as described previously,47 and were incubated, submerged at 35C in a recording chamber and perfused constantly with ACSF containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 4 CaCl2, 4 MgCl2, 26 NaHCO3, 10 glucose, and 0.05 picrotoxin in an atmosphere of warm, moist carbogen (95% O2/5% CO2). Recording electrodes (3–4 MÙ) were filled with extracellular fluid, and extracellular field excitatory postsynaptic potentials (EPSPs) were recorded from CA1 pyramidal cell apical dendrites. Constant current stimuli (20 µs duration) were delivered every 15 or 30 s via a bipolar stimulating electrode placed in stratum radiatum of area CA1. The OGD was induced for 6 or 8 min by switching the atmosphere of the slices to 95% N2/5% CO2 instead of carbogen, and perfusing the slice with ACSF which had an equimolar substitution of sucrose for glucose, and which was also equilibrated with 95% N2/5% CO2. For the assessment of synaptic paired-pulse and frequency facilitation using extracellular recordings, the slices were cut in the horizontal plane and were incubated as described above except for the following: ACSF consisted of
Neuronal vulnerability in calbindin null-mutants (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 4 MgCl2, 26 NaHCO3, 10 glucose, 0.05 picrotoxin, and 0.05 -2-amino-5-phosphonovalerate. Extracellular recording electrodes were placed in the apical dendritic fields of CA1 or CA3 pyramidal cells. Stimulating electrodes were placed either in stratum radiatum of area CA1 or onto the mossy fibres in the dentate gyrus. Whole-cell patch-clamp recordings. Coronal-cut brain slices (350 µm-thick) were incubated as described above, and perfused constantly with ACSF containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 4 MgCl2, 26 NaHCO3, 10 glucose, 0.05 picrotoxin, 2 kynurenic acid. Whole-cell patch current-clamp recordings were done with an Axoclamp 2A amplifier as previously described in detail.47 Borosilicate glass capillaries with an inner filament (KG-33, 1.5 mm o.d., 1.12 mm i.d., Garner Glass, U.S.A.) pulled to 2–2.5 µm outer tip diameters (0.5–0.8 µm diameter of the lumen) using a two-stage vertical Narishige PP-83 puller were filled with 130 mM K-methylsulphate, 5 mM KCl, 10 mM HEPES (pH 7.2, osmolarity 268–272 mosm). The solution contained no exogenous Ca2+-chelators to avoid possible interference with the endogenous Ca2+buffering capacity of neurons.25,47 Cell-attached recordings of N-methyl--aspartate channel openings. Dentate gyrus granule cells were acutely dissociated from CB+/+ and CB/ mouse hippocampal slices according to the method previously published for rat brain slices,24,25 with the exception that the time for incubation in pronase was reduced to 25 min. Fire polished, Sylgardcoated, thick-walled borosilicate (Garner Glass) glass pipettes (10–25 MÙ) were filled with the recording/ extracellular solution containing (in mM): 110 Na2SO4, 5 Cs2SO4, 1.8 CaCl2, 10 HEPES, 10 -glucose, 1 pyruvic acid, and 0.001 tetrodotoxin (Calbiochem, U.S.A.; final pH=7.25, osmolarity=285–305 mOsm). Cell-attached recordings were obtained at 22–25C using pipettes filled with 200 nM -aspartic acid and 10 µM glycine dissolved in the extracellular solution.24,27 Details of the single channel detection using the PAT program (courtesy of Dr J. Dempster) with a 50% threshold crossing algorithm, and event analysis including the determination of burst, cluster and supercluster durations, using an in-house program are provided elsewhere.24
RESULTS
In vitro oxygen/glucose deprivation We directly tested the relationship between Ca2+dependent neuronal vulnerability and calbindin-D28k content in both an in vitro and an in vivo model of ischemia. Pyramidal neurons of the hippocampal CA1 region are among the most vulnerable cells to ischemia,45 and synaptic responses to the activation of Schaffer collateral/commissural input to these cells can be used as a measure of their synaptic excitability during impaired metabolic conditions.30 We have investigated how transiently exposing hippocampal slices to various periods of OGD affects the rate of rise (dV/dt) of field EPSPs recorded in the stratum radiatum of the CA1 region of hippocampal slices prepared from three different types of mice genetically engineered for the lack of calbindinD28k.1 The three preparations consisted of null mutant homozygous calbindin-D28k knockout mice
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(calbindin-D28k/, n=10), and their heterozygous (calbindin-D28k+/, n=10), and wild-type littermates (calbindin-D28k+/+, n=10). A transient, 3 min anoxia alone (not shown), or a 6 min period of OGD resulted in no significant differences in the recovery rates of the EPSPs in the three preparations (Fig. 1A, Table 1). A slightly longer metabolic challenge, a transient 8 min OGD, caused a marked and prolonged depression (>2 h) of the synaptic responses in slices obtained from calbindin-D28k+/, and calbindin-D28k+/+ animals (Fig. 1B, Table 1). In contrast, the synaptic responses fully abolished during OGD (Table 1) in calbindin-D28k deficient pyramidal neurons recovered to 72% of control, with a five-fold faster rate of recovery than that observed in calbindin-D28k+/+ preparations (Fig. 1B, Table 1). In vivo ischemia To generate ischemia in vivo, mice were subjected to a bilateral carotid artery ligation for 12 min under pentobarbital anaesthesia while their body temperatures were maintained constant (see Experimental Procedures). Three days after the transient ischemia, slices were prepared and stained with the vital dye Tetrazolium Red (TTC),20,23 a colourless reagent which is metabolized by functional mitochondrial enzymes into a red product, and which is frequently used to assess ischemic brain damage. Considerable damage was observed in the subiculum, area CA1, and the hilus of the dentate gyrus of calbindinD28k+/+ animals, while only small TTC-negative areas could be observed in the subiculum and hilus of calbindin-D28k+/ animals. No detectable damage was found in any of the CB/ animals (n=6, data not shown). To assess the damage quantitatively at the cellular level, standard Cresyl Violet staining and TUNEL staining, an immunohistochemical procedure detecting DNA fragmentation, were performed on adjacent 30 µm coronal hippocampal sections from a different set of animals, fixed 28 h following surgery (n=3 calbindin-D28k/, n=2 calbindin-D28k+/, n=3 calbindin-D28k+/+). In the CV-stained sections, ischemic damage was considerably more evident in calbindin-D28k+/+ than in calbindin-D28k/ animals (Figs 2, 3). Throughout the calbindin-D28k+/+ hippocampus, but especially in areas CA1 and the dentate gyrus, many cells appeared shrunken and nuclei appeared condensed. TUNEL-positive cells were frequently found clustered in the principal cell body layers and hilus of the dentate gyrus, with occasional presumable interneurons also found in the dendritic field regions throughout the hippocampus (Fig. 4). Cell counts of TUNEL-positive cells in the principal cell body layers of CA1, CA3 and the dentate gyrus were higher in calbindin-D28k+/+ than in
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Fig. 1. Effects of oxygen/glucose deprivation (OGD) on evoked dendritic field EPSPs in hippocampal CA1 of calbindin-D28k/, calbindin-D28k+/, and calbindin-D28k+/+ mice. For the period of time indicated by the bar (A: 6 min, B: 8 min) an equimolar substitution of sucrose for glucose was made in the perfusion solution equilibrated with 95% N2/5% CO2 which was also substituted for carbogen in the atmosphere. Symbols represent the average of the slopes of dendritic field EPSPs, normalized such that control levels=1.0 and maximum effect=0.0. Error bars represent S.E.M. Single exponential curves were fitted using a non-linear least squares Simplex method to the recovery phase of the averaged data. The following recovery time constants were obtained (all in min). For 6 min OGD: calbindin-D28k/, 4.45; calbindin-D28k+/, 6.08; calbindin-D28k+/+, 9.26; for 8 min OGD: calbindin-D28k/, 6.17; calbindinD28k+/, 5.71; calbindin-D28k+/+, 29.60. Below each graph are representative traces of CA1 dendritic field EPSPs, elicited under control conditions (Control), during the maximum effect OGD (Anoxia) and following return to control conditions (Recovery), for each genotype (, calbindin-D28k/; , calbindin-D28k+/; , calbindin-D28k+/+). All experiments and analyses of EPSP slopes were conducted and analysed blindly with respect to the genotype of the animals. Table 1. Maximum inhibition of excitatory postsynaptic potential slope during oxygen/glucose deprivation, and its recovery following return to control conditions Maximum inhibition (%)
Recovery at 30 min (%)
Genotype
6 min OGD
8 min OGD
6 min OGD
8 min OGD
CB/ CB+/ CB+/+
917 (8) 952 (15) 1000 (10)
934 (11) 952 (16) 934 (11)
8513 (7) 6512 (15) 9311 (10)
7210 (11)* 269 (15) 2119 (10)
Data for maximum % inhibition show meansS.E.M. during the period of OGD, normalized to control. Data for recovery are meansS.E.M. obtained at 30 min following the start of OGD, as depicted in Fig. 1. Numbers in parentheses indicate the number of experiments. The asterisk indicates a significant difference from both other groups in pairwise comparisons (two-tailed t-test, P<0.05). CB, calbindin-D28k.
calbindin-D28k/ littermates (Fig. 5). In the subicular third of CA1 (area 1), TUNEL-positive cell counts in calbindin-D28k/ and calbindinD28k+/ animals were 15% and 64% of wild-type, respectively. In the central third of CA1 (area 2), the respective cell counts were 16% and 76% of wild-type. In the lateral third of CA1 (area 3), the respective cell counts were 16% and 97% of wild-type. In CA3 (area
4), the respective cell counts were 22% and 65% of wild-type. In the upper blade of the dentate gyrus (area 5), the respective cell counts were 2% and 157% of wild-type. In the lower blade of the dentate gyrus (area 6), the respective cell counts were 3% and 102% of wild-type. There were significantly fewer (P<0.05) TUNEL-positive cells in calbindin-D28k/ than in calbindin-D28k+/+ mice in every hippocampal region
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Fig. 2. Hippocampal damage induced by ischemia. Calbindin knockout mice (calbindin-D28k/, bottom), their heterozygote (calbindin-D28k+/, not shown) or wild-type (calbindin-D28k+/+, top) littermates were subjected to a 12 min bilateral carotid artery occlusion and allowed to recover for 28 h. Subsequent CV staining of 30 µm hippocampal sections shows severe damage in the subiculum, CA1, and dentate gyrus of calbindin-D28k+/+ compared with calbindin-D28k/ mice. Scale bar=400 µm.
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Fig. 3. Higher magnification views of sections shown in Fig. 2. CV-stained 30 µm sections from area CA1 (top row) and dentate gyrus (bottom row) of calbindin-D28k+/+ (left) and calbindin-D28k/ (right) mice which have undergone 12 min bilateral carotid artery occlusion with 28 h recovery. Scale bar=50 µm.
examined. TUNEL-positive cells in calbindinD28k+/ animals were significantly more numerous than in calbindin-D28k/ animals in areas 2 and 3 (CA1) and areas 5 and 6 (dentate gyrus), but were not significantly different from calbindin-D28k+/+ animals in any area. Cellular properties of hippocampal neurons devoid of calbindin-D28k We also wanted to examine whether other aspects of cellular and synaptic physiology normally reliant on intracellular Ca2+ buffering were altered when calbindin-D28k was eliminated from various hippocampal neurons and their axon terminals. In wholecell current-clamp recordings, we studied the adaptation of action potential firing and post-spike afterhyperpolarizations (AHP) in dentate gyrus granule cells from calbindin-D28k+/+ and calbindinD28k/ animals (n=4 each). These cellular parameters are governed by Ca2+-dependent K+ conductances (gKCa) which are dependent on the amount of intracellular Ca2+ buffering.48 Figure 6 shows examples from three different neurons. In a
total of 24 dentate gyrus granule cells (n=8 from each preparation), the number of action potentials elicited by a 200 ms long 0.5 nA depolarizing current pulse from the comparable resting membrane potential (calbindin-D28k/: 82.72.6 mV; calbindin-D28k+/+: 82.13.2 mV) was significantly (P<0.01, two-tailed t-test) larger (11.72.4) in calbindin-D28k+/+ than in calbindin-D28k/ animals (3.41.8). The input resistances (RN) of granule cells were comparable in the two preparations (calbindin-D28k+/+: 16812 MÙ; calbindin-D28k/: 15719 MÙ). In calbindinD28k/ granule cells (n=8), the amplitude and the area of the post-spike AHP measured after the first action potential elicited by the threshold current pulse were respectively 217% and 176% of those recorded in calbindin-D28k+/+ granule cells (n=8; Fig. 6). Effects of calbindin-D28k loss on synaptic function Considering the dependence of neurotransmitter release at central synapses on presynaptic Ca2+ and its buffering,41,49 we next compared frequency
Neuronal vulnerability in calbindin null-mutants
Fig. 4. TUNEL staining of hippocampal slices showing DNA fragmentation. Dark staining indicates cells undergoing apoptotic or necrotic cell death. Photographs were taken at 40 (top row, each set) or 100 (oil immersion, bottom row, each set) of the CA1 pyramidal cell body layer (top set) and dentate granule cell layer (bottom set) of calbindin-D28k+/+ (left) and calbindin-D28k/ (right) mice. Scale bar=25 µm for 40 and 10 µm for 100 photographs.
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at the mossy fibre synapses onto CA3 pyramidal cells, where the mossy fibre terminals are devoid of calbindin-D28k in the calbindin-D28k/ mutants, there was a significant (P<0.05) 1.8-times slower rate of potentiation and a 20% smaller steady-state value attained during frequency potentiation at 33 Hz compared to that found in calbindin-D28k+/+ slices (Fig. 7). A 30–33% decrease in paired-pulse facilitation was also found in the dendritic region of CA3 pyramidal cells. This decrease in facilitation was significant (P<0.05, two-tailed t-test; n=8 slices for calbindin-D28k/, and n=6 for calbindin-D28k+/+) for short (20, 30 and 50 ms) ISIs. Fig. 5. Cell counts of TUNEL-positive cells in six different hippocampal regions of calbindin-D28k+/+, calbindinD28k+/ and calbindin-D28k/ mice which have undergone 12 min bilateral carotid artery occlusion with 28 h recovery. The total number of TUNEL-positive cells were counted in the cell body layer within a field of view as seen with a 40 objective. These areas corresponded approximately to the size and position of the rectangles indicated in the inset. A camera lucida attached to the microscope served to identify and later count the TUNEL-positive cells. Error bars indicate S.E.M. Calbindin-D28k/ differed from calbindin-D28k+/+ in all areas and from Calbindin-D28k+/ in all areas except CA3 (area 4). Calbindin-D28k+/+ and calbindin-D28k+/ were not significantly different (n=8 hippocampal sections from each of three calbindinD28k+/+, six from each of two calbindin-D28k+/, and eight from each of three calbindin-D28k/ mice).
potentiation and paired-pulse facilitation of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mediated component of EPSPs elicited by various inter-stimulus intervals at two different synapses deliberately chosen to have the calbindinD28k deficiency in calbindin-D28k/ mutants restricted solely to pre- or postsynaptic elements. The presynaptic buffering role of calbindin-D28k was assessed at the mossy fibre-CA3 pyramidal cell synapse where the mossy fibres have high levels of calbindin-D28k in calbindin-D28k+/+, while the CA3 pyramidal cells are normally devoid of the protein.4 At a second hippocampal synapse, the Schaffer collateral/commissural input onto CA1 pyramidal cells, the excitatory terminals lack calbindin-D28k even in calbindin-D28k+/+ animals.4 Therefore, the excitatory axon terminals at this synapse are similar in calbindin-D28k/ and calbindin-D28k+/+, and the postsynaptic cells are devoid of calbindin-D28k only in calbindin-D28k/. The time-course of EPSP potentiation during a train of stimuli can best be fitted by a single exponential.41,49 At the Schaffer collateral/commissural synapses stimulated at a frequency of 33 Hz, there were no significant differences between the calbindin-D28k/ and calbindinD28k+/+ preparations (Fig. 7). Furthermore, there was no statistically significant change, not even at short (25–50 ms) inter-stimulus intervals (ISIs), in the level of paired-pulse facilitation of CA1 field EPSPs evoked by Schaffer collateral/commissural stimulation in calbindin-D28k/ mice (Fig. 7). In contrast,
N-methyl--aspartate channel properties are unaltered in calbindin-D28k null mutants The openings of N-methyl--aspartate (NMDA) channels are sensitive to rises in intracellular Ca2+ which can affect the channel properties directly,32,33 through activation of the protein phosphatase calcineurin,27,50 or through the Ca2+-dependent modulator, calmodulin.13 If calbindin-D28k levels surrounding the inner mouth of the NMDA channel are sufficiently high, and the binding of Ca2+ to calbindin-D28k is considerably faster than the Ca2+binding proteins involved in the regulation of NMDA channel activity, the loss of calbindin-D28k may significantly alter the openings of NMDA channels. A change in the properties of NMDA channels in calbindin-D28k knockouts may also be responsible for the altered sensitivity to ischemia or OGD. In order to assess any possible alterations in NMDA channel properties, we recorded from six dentate gyrus granule cells each obtained from four calbindin-D28k+/+ and four calbindin-D28k/ mice. We chose to record from these neurons because of their homogeneity with regard to their calbindinD28k content under control conditions.3,4 The cell-attached recording mode was preferred to avoid any interference with intracellular Ca2+-buffering mechanisms or second messenger systems.24,27 As shown on Table 2, the properties of NMDA channels were similar regardless of the presence of calbindin-D28k in the cells. DISCUSSION
Our findings are consistent with a Ca2+-buffering role of calbindin-D28k in central neurons. But, in contrast to previous studies reporting correlations between calbindin-D28k content and neuronal survival after injury,10,18,22,28,31,46 hippocampal cells of calbindin-D28k/ mice were more resistant to the effect of a transient OGD and ischemia than their calbindin-D28k containing counterparts. Our main evidence for the role of calbindin-D28k as a Ca2+-buffer consists of the differences in the activation of gKCa between calbindin-D28k+/+ and calbindin-D28k/ neurons. The frequency of
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Fig. 6. Increased adaptation of action potential firing and post-spike AHP in calbindin-D28k/ mouse granule cells. The figure shows the responses of one calbindin-D28k+/+ and two calbindin-D28k/ neurons to hyperpolarizing and depolarizing current pulses (200 ms). In the case of neurons M1715 (calbindin-D28k+/+) and M1513 (calbindin-D28k/) the pulses were injected at the resting membrane potential of the cells (81 and 82 mV, respectively). In neuron M1703 (calbindin-D28k/) the cell was held depolarized at 64 mV (+20 mV from its actual resting potential of 84 mV). The same high degree of adaptation was observed at this depolarized membrane potential. The resting RN of the two neurons M1715 (calbindin-D28k+/+ ) and M1513 (calbindin-D28k/ ) are indicated on the top right panel. The numbers show the linear regression fits to the I–V plots with the meanS.E.M. of the slope. The traces on the bottom panel are superimposed action potentials showing the second spike in the trains from the three granule cells. The action potential recorded in the control neuron (M1715; calbindinD28k+/+) is shown with a thicker line. Note the more than doubling of the amplitudes of the rapid post-spike AHP in the two calbindin-D28k/ granule cells. Similar results were obtained in six other calbindin-D28k/ neurons.
action potential firing in central neurons is regulated by several voltage- and second-messenger gated ion channels including a large number of K+ conductances. Of these, a fast gKCa is partly responsible for the rapid repolarization of action potentials in various types of neuron,29,48 and can be blocked by fast intracellular Ca2+ chelators such as 1,2-bis(2aminophenoxy)-ethane-N,N,N ,N -tetraacetic acid (BAPTA) in both CA1 pyramidal48 and dentate gyrus granule cells.47 The enhancement of the postspike AHP in calbindin-D28k/ granule cells can thus be explained by the absence of an intracellular Ca2+-buffering system with fast kinetics, and together with the increased adaptation of firing frequency
observed in calbindin-D28k/ granule cells, illustrates the effectiveness of calbindin-D28k in fine tuning neuronal firing patterns.26 Based on experimental and theoretical predictions at the mossy fibre–CA3 pyramidal cell synapse41,49 and the attenuation of post-tetanic potentiation in cultured hippocampal neurons transfected with a calbindin-D28k gene containing adenovirus,8 it was surprising to see decreased frequency potentiation and paired pulse facilitation in the absence of presynaptic calbindin-D28k. However, if gKCa contributes to the control of neurotransmitter release at this synapse, just as it does at other synaptic junctions,6,43,44 an enhanced activation of gKCa in terminals without calbindin-D28k is
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Fig. 7. Frequency potentiation and paired-pulse facilitation of mossy fibre but not Schaffer collateral/ commissural fibre-evoked responses is decreased in calbindin-D28k/ mice. The frequency potentiation of field EPSPs (left panels), was recorded in the str. radiatum of the CA1 and CA3 regions, respectively, evoked by low intensity repetitive stimulation (33 Hz) of the Schaffer collateral/commissural fibres and mossy fibres. The facilitation ratio (PnP1)/P1, where Pn and P1 are the rates of rise (dV/dt) of the nth and first EPSPs during the paired-pulse stimulation, was plotted against time. The rate of frequency potentiation can be assessed by the indicated fits of the average values to single exponential functions (solid lines). The 95% confidence intervals are shown with thin lines. Error bars represent S.E.M. (n=8 slices for both calbindin-D28k/ and calbindin-D28k+/+). The two curves and their asymptotes are similar for the CA1 responses in calbindin-D28k+/+ and calbindin-D28k/ preparations, but are significantly different (P<0.05) for the CA3 EPSPs. Right panels: The reduction in paired-pulse facilitation in the CA3 region of calbindin-D28k/ mice is significant (P<0.05, two-tailed t-test) at the three shortest ISI. In contrast, there are no differences in the CA1 region. The facilitation ratio was calculated as above for n=2 at ISIs ranging between 20 and 1000 ms. Error bars represent S.E.M. (n=8 slices for calbindin-D28k/ and n=6 for calbindin-D28k+/+). Table 2. Similar N-methyl--aspartate channel properties in calbindin-D28k+/+ and calbindinD28k/ mice Genotype
NMDA channel properties (in ms) Open time Burst length Total open time/burst Cluster length Total open time/cluster Supercluster length
CB+/+
CB/
2.520.26(6) 6.491.96(6) 3.980.64(6) 16.843.66(6) 7.131.34(6) 124.842.7(6)
3.020.41(6) 6.962.05(6) 5.261.31(6) 16.354.00(6) 8.982.63(6) 119.222.9(6)
NMDA channel openings were recorded in the cell-attached mode in six dentate gyrus granule cells each acutely dissociated from calbindin-D28k+/+ (n=4) and calbindin-D28k/ mice (n=4). With 200 nM -aspartate and 10 mM glycine in the pipette, no significant differences were detected between the basal opening properties of NMDA channels in neurons with or without calbindin-D28k. CB, calbindin-D28k.
conceivable.40 An increase in gKCa similar to that seen at the somata of calbindin-D28k-deficient neurons, may override the effect of an elevated Ca2+ entry1 on transmitter release.
The early events in OGD including the lasting depression of synaptic transmission are harbingers of neuronal death.30 This is the first time the effect of OGD on synaptic transmission has been examined in
Neuronal vulnerability in calbindin null-mutants
mice, and it is clearly more severe than that of oxygen deprivation alone which does not lead to an irreversible loss of synaptic function in this species.52 The enhanced recovery of calbindin-D28k deficient CA1 pyramidal cell synaptic responses after a certain threshold level of OGD in vitro is consistent with a deleterious rather than a neuroprotective46 effect of calbindin-D28k in neurotoxicity.10,22,28,31 Likewise, 28 h after transient ischemia in vivo, hippocampi of calbindin-D28k+/+ animals had a greater number of abnormally shrunken principal cells with condensed, pyknotic-looking nuclei than did their calbindinD28k/ littermates. These results are mirrored by the number of cells labelled by TUNEL stain, which detects DNA fragmentation and is the method of choice for detecting imminent apoptotic or necrotic ischemic cell death.9 That wild-type mice expressing normal levels of calbindin suffer more TUNELpositive neurons following transient ischemia than their calbindin-D28k/ littermates is irreconcilable with a generally neuroprotective role for this protein. The use of littermates avoided possible complications resulting from the variability in ischemic neuronal damage seen in various mouse strains.5,54 The dentate granule cell damage found by us in the wild-type mice has been observed in this species before,38 although it is contrary to the more common observation that granule cells are relatively resistant to ischemic damage in adult rats.45 Other studies17,19 also indicate that the resistance of the granule cells may be age or species dependent. As NMDA channel properties are similar in calbindin-D28k-containing and calbindin-D28kdeficient cells, these excitatory amino acid channels are probably not responsible for the differences in neurotoxicity observed between the two prepara-
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tions. The absence of calbindin-D28k may protect neurons by enhancing the Ca2+-dependent inactivation of voltage-gated Ca2+ entry25 thereby reducing the net Ca2+ influx during prolonged depolarizations. In contrast, the presence of a mobile cytoplasmic Ca2+ buffer such as calbindin-D28k could enhance the dispersion of Ca2+ throughout the cytoplasm,53,55 thus delivering the Ca2+ that enters the cells,12 or that is being released from intracellular stores,37 much faster and more effectively to the various cellular compartments where it may exert its malignant effects on neuronal survival. CONCLUSION
Clearly, Ca2+-binding proteins have a complex role in neurons, but should not be necessarily regarded as effective protectors against Ca2+-dependent neuronal damage. Consistent with this notion is the recent observation in midbrain dopaminergic neurons describing a lack of neuroprotective role of calbindin-D28k against 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) toxicity.2 As pointed out in a recent review,12 the entire Ca2+-overload hypothesis of ischemic-hypoxic neuronal damage may have to be refined depending on the nature of the cellular killing mechanism activated, and this will ultimately lead to novel cytoprotective therapies for stroke.34 Acknowledgements—We want to thank B. Oyama for expert technical assistance. This research was supported by NINDS grants to I.M., an AHFMR Fellowship to G.J.K., an APA MFP Fellowship to P.A.G., a Howard Hughes Predoctoral Fellowship to D.N.L., and by grants from the Academy of Finland and Regeneron Pharmaceuticals (Tarrytown) to M.S.A.
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