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Early induction of mRNA for calbindin-D28k and BDNF but not NT-3 in rat hippocampus after kainic acid treatment
Molecular Brain Research 47 Ž1997. 183–194
Research report
Early induction of mRNA for calbindin-D 28k and BDNF but not NT-3 in rat hippocampus after kainic acid treatment S. Lee a , J. Williamson b, E.W. Lothman b, F.G. Szele c , M.F. Chesselet R.M. Sapolsky e, M.P. Mattson f , S. Christakos a,)
c,1
, S. Von Hagen d ,
a
f
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA b Department of Neurology, UniÕersity of Virginia, Health Science Center, CharlottesÕille, VA 22908, USA c Department of Pharmacology, UniÕersity of PennsylÕania, Philadelphia, PA 19104, USA d Department of Pharmacology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA e Department of Biological Sciences, Stanford UniÕersity, Stanford, CA 94305, USA Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, UniÕersity of Kentucky, Lexington, KY 40536, USA Accepted 17 December 1996
Abstract The influence of kainic acid ŽKA., which induces acute seizures, on expression of mRNA for the calcium-binding protein, calbindin-D28k , brain-derived neurotrophic factor ŽBDNF., neurotrophin-3 ŽNT-3. and early-response genes wc-fos, zif268 ŽNGFI-A., nur77 ŽNGFI-B.x was examined in rat hippocampus by Northern blot analysis. A significant increase Ž3.2-fold. in BDNF mRNA was observed 1 h after KA injection Ž12 mgrkg i.p.. and peak expression Ž9.4-fold. occurred 3 h after KA. The induction of BDNF mRNA was preceded by the induction of c-fos, mRNA Ž30 min after KA. and was followed by the induction of calbindin-D28k mRNA Ž3.5-fold 3 h after KA; a maximal response was at 3–6 h after KA.. Region-specific changes, analyzed by immunocytochemistry and in situ hybridization, indicated that the most dramatic increases in calbindin protein and mRNA after KA treatment were in the dentate gyrus. Although calbindin-D28k and BDNF mRNAs were induced, a 3.4–3.8-fold decrease in NT-3 mRNA was observed by Northern analysis 3–24 h after KA treatment. Calbindin-D28k gene expression was also examined in rats with a chronic epileptic state characterized by recurrent seizures established with an episode of electrical stimulation-induced status epilepticus ŽSE.. When these animals were examined 30 days post-SE, no changes in hippocampal calbindin-D28k mRNA were observed. Our findings suggest that the induction of calbindin-D28k mRNA Žwhich may be interrelated to the induction of BDNF mRNA. is an early response which may not be related to enhanced neuronal activity or seizures per se, but rather to maintaining neuronal viability.
1. Introduction Kainic acid ŽKA., an analog of the excitatory amino acid glutamate w58x, induces acute seizures in rats which reproduce sequelae of human temporal lobe epilepsy w3,56x. Systemic injection of KA results in excitatory damage in selective brain regions. Affected areas include amygdaloid complex, hippocampus Žthe most extensively damaged area is the CA3 region. and related parts of the thalamus and
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Corresponding author. UMDNJ-New Jersey Medial School, 185 South Orange Avenue, Newark, NJ 07103-2714, USA. Fax: q1 Ž201. 982-5594. 1 Present address: Department of Neurology, University of California at Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.
neocortex w65x. It has been suggested that this neurotoxicity is caused by massive influx of extracellular calcium by activation of KA-preferring glutamate receptors. Neuronal cells are believed to have protective mechanisms against glutamate-induced calcium-mediated cytotoxicity which involve, in part, the release of neurotrophins. The nerve growth factor ŽNGF. family of neurtrophins includes, in addition to NGF, brain-derived neurotrophic factor ŽBDNF., neurotrophin-3 ŽNT-3. and neurotrophin-4r5 ŽNT-4r5. w60x. The neurotrophins bind and activate members of the trk family of tyrosine kinase receptors ŽNGF binds trkA, BDNF and NT-4r5 bind trkB and NT-3 is associated mainly with trk C. w37x. The neurotrophins are present in selected neuronal populations in the peripheral and central nervous systems where they play important roles in supporting differentiation andror survival of subpopulations of neurons during development
0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 0 4 3 - 0
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S. Lee et al.r Molecular Brain Research 47 (1997) 183–194
w41x in addition to their reported role in protecting against glutamate-induced cytotoxicity w10,11,43x. NGF and BDNF mRNA and protein have been reported to be induced by seizure activity induced by KA w2,23,24,78x as well as by dentate hilar lesion w38,40,63x, kindling w4,26x and injection of bicuculline w77x or pentylenetetrazol w33x. Changes in neurotrophin expression are not unique to epilepsy but have been reported after brain injury induced by ischemia w42,72x and hypoglycemia w42x which also results in glutamate-induced increases in intracellular calcium which can cause cell death. It has recently been reported that one mechanism whereby NGF, BDNF and NT-3 can protect neurons against metabolicrexcitotoxic insults is by stabilizing intracellular calcium w10,11x. It has been suggested that the stabilization of intracellular calcium may be mediated in part by the calcium-binding protein calbindin-D28k w11,53x which is induced in hippocampus by BDNF and NT-3 w19x. Calbindin-D28k , a major calcium-binding protein in brain Žit constitutes 0.1–1.5% of the total soluble protein in brain w14,15,69x., has a widespread but restricted distribution throughout the central nervous system w6x. In hippocampus, calbindin is localized in the dentate granule cells which are relatively resistant to seizure-induced insults w65x as well as ischemic injury w31x and in CA1 pyramidal neurons which are also relatively resistant to seizure damage w65x. Studies of hippocampal cells in culture have indicated that calbindin-positive neurons are protected against damage induced by glutamate and calcium ionophore and are better able to reduce free intracellular calcium than calbindin-negative neurons w53x. These findings suggest that calbindin, by buffering intracellular calcium, can prevent calcium-mediated neuronal death that could result from excitotoxic insult. More recently, direct evidence of the calcium buffering capacity of calbindin was demonstrated by Chard et al. w8x who reported that introduction of exogenous calbindin into dorsal root ganglia neurons resulted in reduction in the depolarization-induced rate of rise of intracellular calcium, indicating directly that calbindin can effectively regulate calcium-dependent aspects of neuronal function. Thus, the function of neurotrophic factors in regulating calbindin expression and thereby possibly preventing excitotoxic neuronal damage in neuropathies, such as epilepsy, has important therapeutic implications. In order to obtain an increased understanding of the possible interrelationship between calbindin, neurotrophins and seizure-induced excitotoxic insult, region-specific and time-dependent changes in hippocampal calbindin mRNA and protein as well as time-dependent changes in mRNA for two neurotrophins in hippocampus known to induce calbindin, BDNF and NT-3, were examined under conditions of acute status epilepticus ŽSE. induced by KA. To determine if the acutely observed changes were maintained in a chronic epileptic state, calbindin-D28k gene expression was also examined in the hippocampus of rats with recurrent spon-
taneous seizures induced with an episode of electrical stimulation-induced SE w44x.
2. Materials and methods 2.1. Materials w 32 PxdCTP Ž3000 Cirmmol, 10 mCirml. was purchased from New England Nuclear Products ŽBoston, MA.. Oligo ŽdT. cellulose and all restriction enzymes were purchased from Boehringer Mannheim ŽIndianapolis, IN.. Biotrans nylon membranes were obtained from ICN Biochemicals ŽCosta Mesa, CA. and formamide was from Sigma ŽSt. Louis, MO.. Guanidinium isothiocyanate and phenol Žultra pure, molecular biology grade. were from Fischer Scientific ŽSpringfield, NJ.. The Rad prime DNAlabeling system and agarose Želectrophoresis grade. were purchased from Gibco-BRL Life Technologies ŽGaithersburg, MD.. 2.2. Preparation of animals In all studies, adult male Sprague–Dawley rats Ž250– 300 g. were used and maintained with food and water ad libitum in a 12-h lightr12-h dark cycle. For studies involving systemic KA administration, 90 rats were used. Rats received either KA ŽSigma. Ž12 mgrkg body weight prepared in 10 mM phosphate-buffered saline; PBS pH 7.4. or saline by i.p. injection. Seizure activity was observed within 30–40 min following kainate treatment. Rats not exhibiting seizure activity after kainate injection were not used in the study. For studies using KA microinfusion in brain, 50 rats were anesthetized with Metofane ŽPitman-Moore, NJ. and KA Ž0.07 m g in 1 m l volume. was injected stereotaxically into Ammon’s horn of the dorsal hippocampus with a Hamilton syringe over 1 min followed by a 1 min wait for the KA to diffuse Žcoordinates: anteroposterior ŽAP., q4.1 mm; mediolateral ŽML., 2.1; dorsoventral ŽDV., 3.0 Žheight of l suture set equal to bregma. w25x. Control Žzero point. received 1.0 m l saline without KA. For studies in rats with a chronic epileptic state, rats were stereotaxically implanted with bipolar electrodes in the left ventral hippocampus ŽAP y3.6, ML 4.9, DV y5.0 to dura w59x.. Control rats were implanted with electrodes but not stimulated. One week after surgery, rats received a period of continuous hippocampal stimulation ŽCHS. Ž10-s, 50-Hz trains of 1-ms biphasic pulses set at 400 m A given once every 12 s for 90 min.. CHS established a condition of self-sustaining limbic SE ŽSSLSE. w44x. Rats were killed by decapitation 30 days after CHSinduced SSLSE. Chronic recurrent spontaneous hippocampal seizures begin in rats within 30 days post-CHS-induced SSLSE w44x.
S. Lee et al.r Molecular Brain Research 47 (1997) 183–194
For all studies, brains were rapidly removed, brain areas were quickly dissected and immediately frozen on dry ice and stored at y808C.
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removed and quickly frozen on powered dry ice, cryostatcut into sections Ž10 m m. which were thaw-mounted onto gelatin-coated slides and stored at y708C. Radiolabeled
2.3. RNA isolation and Northern blot hybridization analysis Total RNA was isolated from pooled samples Žusually, from 3 animals. by the guanidinium thiocyanaterphenol chloroform procedure of Chomczynski and Sacchi w13x. PolyŽA.q RNA was obtained after two cycles of oligo ŽdT. cellulose chromatography. PolyŽA.q RNA was fractionated under denaturing conditions on a 1.2% formaldehyde agarose gel and transferred to a nylon membrane ŽBiotrans, ICN. using standard procedures as previously described w68,73x. Hybridization probes were labeled according to the random priming method of Feinberg and Vogelstein w27x using the Rad prime DNA labeling system ŽGibco-BRL Life Technologies.. The filters were hybridized at 428C overnight, washed and autoradiographed as previously described. For reprobing, hybridized probes were removed by washing in 50% formamide–10 mM phosphate buffer ŽpH 6.5. for 1 h at 658C. b-Actin cDNA andror 18S rRNA cDNA were used as control probes. Autoradiograms of varying exposures of Northern blots were analyzed using dual-wave length flying-spot scanner ŽShimadzu Scientific Instrument, Princeton, NJ.. The relative optical density of each Northern probed with calbindin, neurotrophin or early-response gene cDNA was divided by the relative optical density obtained after probing with b-actin or 18S rRNA cDNA to normalize for sample variation. 2.4. Preparation of DNA probes A 1.2-kb mouse calbindin-D28k cDNA insert from the EcoRI site of pIBI76 w76x, a 1.1-kb rat BDNF cDNA from the EcoRI site of pSK-rBC1 w50,51x, a 0.8-kb rat NT-3 cDNA from the XhoI site of pSK-rNT3 w50,51x ŽBDNF and NT-3 cDNA probes were kindly supplied by George D. Yancopoulos, Regeneron Pharmaceuticals, Tarrytown, NY., a 2.1-kb rat c-fos cDNA from the EcoRI site of pSp65 w22x, a 0.3-kb zif268 ŽNGFI-A. insert from the BamHI site of pB700-3.6 w71x Žobtained from Vikas Sukhatme, Beth Israel Hospital, Boston, MA. and a 2.5-kb rat nur77 ŽNGFI-B. insert from the EcoRI site of pJDM3 w54x were obtained by the restriction enzyme digestion of the respective plasmid preparations. A 2.1-kb chicken bactin cDNA was obtained from the HindIII site of pBR322 w17x. The 18S rRNA cDNA was from Ramareddy Guntaka ŽUniversity of Missouri at Columbia.. 2.5. In situ hybridization For the in situ hybridization experiments, rats were killed 6 h after KA injection. The brains were rapidly
Fig. 1. Northern blot analysis of calbindin-D28k mRNA in rat hippocampus after KA treatment. A: polyŽA.q RNA Ž8 m grlane., isolated from hippocampus from 3–4 adult male rats for each time point after systemic KA injection Ž12 mgrkg body weight. was used for Northern analysis Župper panel.. Results from the Northern blots using hippocampi from three separate groups of rats were used for densitometric analysis Žmean "S.E.M.; lower panel.. After normalization based on results obtained after re-hybridization with 18S rRNA or b-actin cDNAs, densities were expressed as percentage maximal response. B: polyŽA.q RNA Ž8 m grlane. was isolated from the ipsilateral Žinfused side. or contralateral hippocampus at various times after intrahippocampal infusion. Hippocampi from 5 rats were pooled for preparation of RNA at each time point. Densitometric scanning of autoradiograms and normalization with b-actin indicated that calbindin mRNA was elevated 1.6- and 1.9-fold on the contralateral and ipsilateral sides, respectively, at 1 h after KA injection. 3 h after KA injection, calbindin mRNA was induced 1.9- and 2.6-fold on the contralateral and ispsilateral sides, respectively Žresults are the mean of two independent measurements, using separate groups of rats..
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antisense and sense cRNAs were transcribed from cDNAs as previously described w12x using appropriate polymerases, 2.5 m M w 35 SxUTP Ž1000 Cirmmol, New England Nuclear., 10 m M cold UTP, with ATP, CTP and GTP in excess. The template cDNA Ž320 bp. was the same as used for Northern analysis. Both sense and antisense RNA probes were synthesized. In situ hybridization was carried out as previously described w12x. Briefly, tissue sections were brought to room temperature and dried rapidly under a stream of cold air, post-fixed in 3% paraformaldehyde, acetylated and dehydrated. Sections were incubated with 3 ng of probe Žf 400 000 dpmrng. in humid chambers at 508C for 3.5 h. After hybridization, non-specific binding was reduced by washing in 50% formamide in 2 = SSC Ž0.3 M NaClr 0.03 M sodium citrate. at 528C and treatment with RNase A Ž100 m grml. for 30 min at room temperature. After overnight incubation in 2 = SSCr0.05% Triton X-100, slides were dehydrated in graded ethanol, defatted in xylene and coated with Kodak NTB 3 emulsion diluted 1 : 1 with 300 mM ammonium acetate. After exposure at 48C for 29 days, the sections were developed in Kodak D-19 developer at full strength, lightly counterstained with hematoxylin eosin and mounted with Eukitt ŽCalibrated Instruments.. The sections were examined under bright-field and dark-field microscopy on a Leitz aristoplan microscope. 2.6. Calbindin-D2 8 k immunocytochemistry Rats were killed at 6, 12 and 24 h after systemic KA injection. Three ratsrtime point were studied. At the different times after KA, rats were anesthetized with an i.p. injection of 20% urethane ŽSigma. and then perfused intracardially with 0.1 M sodium phosphate-buffered saline ŽPBS. with 0.5% heparin ŽpH 7.4; LyphoMed, Melrose Park, IL. followed by 4% paraformaldehyde ŽSigma. in 0.1 M sodium PBS ŽpH 7.4.. The brains were removed, fixed in paraformaldehyde for 1 h, rinsed in PBS ŽpH 7.4. and then permeated with a cryoprotected sucrose solution Ž12.5 mM sodium phosphate monobasic, 38.5 mM sodium phosphate dibasic and 877 mM sucrose.. The brains were stored at y208C. After transfer to 10 mM PBS, 50-m m coronal sections were prepared using a vibratome. Sections Žfree-floating. were first incubated in PBS containing 0.2% Triton X-100 and 0.015% non-immune horse serum fol-
lowed by overnight incubation in PBSrTritonrnon-immune horse serum containing a 1 : 1500 dilution of a rabbit polyclonal rat calbindin antiserum w16x. Sections were immunostained using the biotin-avidin-peroxidase method described previously w25x. Control brains were similarly processed but without the primary antibody. Sections were photographed using an inverted Nikon Diaphot microscope with phase-contrast and bright-field optics. 2.7. Statistical analysis Results are expressed as the mean " S.E. and significance was determined by Dunnet’s multiple comparison t-statistic or by analysis of variance.
3. Results Northern blot analysis of hippocampal polyŽA.q RNA indicated that after systemic injection of KA Ž12 mgrkg bw., steady-state levels of calbindin-D28k mRNA were rapidly induced. The first significant increase was observed at 3 h after injection Ž P - 0.05; 3.5-fold induction.. Levels of calbindin mRNA returned to control levels at 12 h after KA treatment ŽFig. 1A.. Unlike the changes observed in hippocampus, no changes in calbindin-D28k mRNA were observed in cerebellum at any time after KA treatment Ž0.5–24 h; not shown.. Calbindin-D28k mRNA levels were also examined in a different experimental model in which rats received a unilateral injection of KA directly into Ammon’s horn of the dorsal hippocampus. This model allows the discrimination between a neurotoxic effect of KA which is observed in the ipsilateral side and a neuroexcitatory effect on the contralateral side which is most likely caused by hyperactivity. Calbindin-D28k mRNA was clearly increased at 1 and 3 h after KA infusion in both the ipsilateral and contralateral sides ŽFig. 1B.. At 6 and 16 h after KA infusion, levels of calbindin-D28k mRNA remained elevated in the ipsilateral but not in the contralateral side. Thus, calbindin-D28k mRNA can be induced not only by a direct action of KA on its receptor Žipsilateral side. but also as a result of hyperactivity in afferent excitatory pathways. The remainder of the studies involving KA were done using i.p. injection which results in selectivity of vulunerable neurons Žthose with high
Fig. 2. In situ hybridization histochemistry for calbindin-D28k . A,B: dark-field photomicrographs of sections of cerebellum of control ŽA. and KA-treated ŽB. rats were processed for in situ hybridization histochemistry with a 35 S-radiolabeled antisense RNA probe for calbindin-D28k . Arrows point to dense accumulations of silver grains over Purkinje cells. Scale bar shown in B: 70 m m for A and B. C–F: in situ hybridization histochemistry for calbindin-D28k in rat hippocampus. Sections were hybridzed with antisense ŽC,E. or sense ŽF. 35 S-radiolabeled RNA probes for calbindin mRNA. C,E: dark-field photomicrographs of sections of the same region of the hippocampus in KA-injected ŽC. and control ŽE. rats. Arrow in C points to labeling in the dentate gyrus. D: low-power bright-field photomicrograph of section illustrated in C Žcounterstaining: hematoxyllin eosin.. The arrow points to dentate gyrus. Scale bar shown in F s 100 m m Žin C,E.; s 500 m m Žin D.; s 80 m m Žin F.. Similar results were observed in the brains of two other control and KA-treated rats assayed at the same time using the same in situ hybridization conditions and exposure times. It should be noted, as has been previously reported w68x, that with longer autoradiographic exposure time calbindin-D28k can be observed using in situ hybridization, in the hippocampus of control rats.
S. Lee et al.r Molecular Brain Research 47 (1997) 183–194
concentrations of KA type glutamate receptors.. Regionspecific changes in calbindin-D28k mRNA and protein after i.p. KA injection was examined by in situ hybridiza-
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tion and immunocytochemistry, respectively. In Fig. 2A,B, a dense accumulation of silver grains was observed over Purkinje cells in the cerebellum of both control ŽA. and
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KA-injected ŽB. rats in sections processed for in situ hybridization with 35 S-radiolabeled antisense probe for calbindin. Sections hybridized under the same conditions with the sense probe did not show specific labeling in any brain area examined. Despite the presence of a strong cerebellar autoradiographic signal in the same sections, no specific labeling was detected in the hippocampus of control rats in our experimental conditions ŽFig. 2E.. In contrast, labeling was clearly present in the dentate gyrus 6 h after KA administration ŽFig. 2C,D.. No increases in labeling were observed in other hippocampal regions or in caudate-putamen Žstriatum. of the same rats Žnot shown.. KA treatment was also found to induce a marked increase in calbindin immunoreactivity at 6 and 12 h which, similar to results of in situ hybridization, was also most obvious in the dentate gyrus ŽFig. 3.. By 24 h, calbindin immunoreactivity was reduced towards control levels ŽFig. 3.. Since BDNF and NT-3 have been reported to enhance the expression of calbindin in the hippocampus w19x, the influence of KA on the expression of mRNAs for these neurotrophins was examined by Northern analysis. A significant increase in BDNF mRNA was observed 1 h after KA injection Ž P - 0.05; 3.2-fold induction. and peak ex-
pression Ž9.4-fold. occurred 3 h after KA ŽFig. 4A.. In contrast, KA treatment resulted in a marked 3.4–3.8-fold decrease in the levels of NT-3 mRNA in the hippocampus ŽFig. 4B.. Since it has been suggested that immediate-early genes may play a role in regulating genes which respond later to trans-synaptic activation, changes in the expression of c-fos, zif268 and nur77 mRNAs were examined after KA treatment. The induction of both BDNF and calbindin-D28k mRNAs was preceded by induction of c-fos mRNA. Levels of c-fos mRNA were undetectable in control rats Žzero time. even after prolonged radiographic exposure but were significantly induced 30 min after KA ŽFig. 5.. Although 1.5- and 1.9-fold inductions in zif268 and nur77 mRNAs, respectively, were observed at 30 min, Northern analysis indicated that the first significant induction Ž P - 0.05. in these early-response genes was at 1 h with peak expression Ž6.3- and 7.4-fold for zif268 and nur77 mRNAs, respectively. occurring at 3 h after KA ŽFig. 5.. A direct comparison of the time relationship of the response to KA of the immediate-early genes, BDNF, NT-3 and calbindin mRNAs is shown in Fig. 6. Calbindin-D28k gene expression was also examined in
Fig. 3. KA induces a transient increase in hippocampal calbindin immunoreactivity. Rats were injected with saline Žcontrol. or KA Ž12 mgrkg body weight.. KA-treated rats were killed at 6, 12 and 24 h following injection. KA induced a marked increase in calbindin immunoreactivity at 6 and 12 h which was most obvious in the molecular layer of the dentate gyrus. Increased calbindin immunoreactivity was also observed in region CA1. By 24 h, calbindin immunoreactivity was reduced towards control levels.
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Fig. 4. Northern analysis of BDNF and NT-3 mRNAs in rat hippocampus after KA treatment. Left panel: representative Northern blot analysis. PolyŽA.q RNA was isolated from the hippocampus at various times after systemic administration of KA and fractionated on a 1.2% formaldehyde-agarose gel. The mRNA was transferred to a nylon membrane and hybridized with calbindin ŽFig. 1A., BDNF and NT-3 cDNAs. Basal levels of BDNF mRNA were observed at 0 and 0.5 h after longer radiographic exposure. ŽFor details concerning the experiment, see legend to Fig. 1A.. Right panel: graphical representation of the results of Northern analyses obtained in three separate experiments.
rats with a chronic epileptic state characterized by recurrent seizure established with an episode of electrical stimulation-induced SE. When these animals were examined 30 days post-SE, no changes in hippocampal calbindin-D28k mRNA were observed ŽFig. 7.. Calbindin-D28k mRNA was also not altered in cerebellum ŽFig. 7., amygdala Žnot shown. or frontoparietal cortex Žnot shown. under these conditions.
4. Discussion In this study, we report that calbindin mRNA and protein rapidly increase after KA treatment and the most pronounced increases are observed in the dentate gyrus. Although a previous study indicated an induction of calbindin mRNA Žas well as 72-kDa heat-shock protein and 78-kDa glucose-regulated protein mRNAs. in rat hippocampus after KA administration w45x, region-specific changes and changes in protein had not as yet been examined. In our study, we found that the induction of calbindin mRNA is preceded at 1 h and coincident at 3 and 6 h after KA with the induction of BDNF mRNA. Previously reported region-specific changes in BDNF mRNA after KA treatment indicate, similar to changes in calbindin mRNA, that the greatest, earliest increase occurs in the
dentate granule cell layer Žwhich is most resistant to seizure-induced injury., followed by an increase in CA1 w24x. The extent of elevation in the CA1 region was approximately half that observed in the dentate granule cell layer. Increases in BDNF mRNA in the CA3 region, similar to increases in the CA1 region, have also been reported w24x. Due to the correlation of the increases in calbindin mRNA and BDNF mRNA in the dentate gyrus as well as the observation that the induction of BDNF mRNA precedes the induction of calbindin mRNA, it is possible that the induction of calbindin after KA may be mediated, in part, by BDNF. Besides the hippocampus, induction of BDNF mRNA by KA was also previously noted in the cortex Žlayer II and layers IV and VI w24x.. Although KA did not induce calbindin mRNA in cerebellum, it will be of interest in future studies to determine whether calbindin, which is present in layers II and IV w6x, and its mRNA can be induced in these areas of the cortex similar to BDNF mRNA. Until recently, unlike studies related to calbindin in peripheral tissues w14,15,30,32x, very little has been known about the signalling mechanisms involved in regulating neuronal calbindin. However, correlative evidence between decreases in neuronal calbindin and neurodegeneration in studies of ischemic injury w5,62x, seizure activity w1,68x and chronic neurodegenerative disorders ŽAlzheimer, Hunting-
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Fig. 5. Changes in immediate-early genes Žc-fos, zifr268 and nur 77. in rat hippocampus after KA treatment assessed by Northern blot hybridization. The same Northern blots probed for calbindin-D28k andror BDNF and NT-3 were also hybrized with c-fos, zif268 and nur77 cDNAs. After normalization based on results obtained upon re-hybridization with 18S rRNA Žshown in bottom panel inset., densities were expressed as percent maximal response Žmean"S.E.M... Data was obtained from three separate Northern analyses except for the 24-h time point which represents the mean of duplicate determinations.
ton and Parkinson diseases. w35x had been reported. Using rat hippocampal cultures, recent evidence has indicated that NT-3 w19,36x, BDNF w19,36x, fibroblast growth factor ŽFGF. w19x and tumor necrosis factors ŽTNFs. w9x can all induce calbindin. In addition, in vivo corticosterone administration has been reported to increase calbindin expression in rat hippocampus w34x, specifically in the CA1 region w39x. Retinoic acid has also been reported to induce calbindin protein and mRNA in medulloblastoma cells, which express a neuronal phenotype w74x, and the content of calbindin in cultured Purkinje cells can be increased by insulin-like growth factor I w57x. Thus, neuronal calbindin can be regulated by steroids as well as by factors that affect signal transduction pathways. Although a 20-fold induction of calbindin in response to NT-3 compared to about a 2-fold induction by BDNF has previously been reported in rat hippocampal cultures w19x, in this study
NT-3 mRNA was found to be decreased at times when calbindin mRNA was maximally induced after KA. In previous studies, NT-3 mRNA has been reported to be unchanged or markedly decreased after seizure induced by KA, hilus lesion or kindling w2,4,28x. Region-specific changes indicated that NT-3 mRNA was most dramatically reduced in the dentate granule cell layer w4,28x. Thus, it is likely that, within the same region of the hippocampus, calbindin and BDNF mRNAs are increased while NT-3 mRNA is decreased. Since NT-3 mRNA is down-regulated and it has been reported that changes in mRNA content for neurotrophins are reflected in protein production w28x, NT3, which may be involved in inducing calbindin during hippocampal neurogenesis w19x, may not play a role in the up-regulation of calbindin observed after KA-induced seizures. However, it is possible that trkC receptor mRNA up-regulation, which has been reported after kindling induced seizures coincident with NT-3 mRNA down-regulation w4x, may also occur after KA-induced seizures and may compensate, in part, for the decrease in NT-3 mRNA. Since the expression of mRNAs for FGF w28x and TNF w55x have also been reported to be induced in hippocampus early times after seizure, it is possible that these factors, which induce calbindin in hippocampal cultures, may also contribute to the up-regulation of calbindin observed after KA. FGF w10x and TNFs w9x as well as BDNF w11x have all been reported to protect hippocampal neurons against excitotoxic insults by stabilizing calcium homeostasis, further suggesting an interrelationship between these factors and calbindin. Although the induction of calbindin mRNA after KA treatment was preceded by the induction of BDNF mRNA, the most rapid genomic response was the increase in c-fos, mRNA. c-Fos is a member of a heterodimer transcription complex, which combines with members of the c-jun family, binds to AP1 consensus sequences and results in activation or repression of genes w21x. In the nervous system, few physiological targets for c-fos and c-jun have been definitively identified. However, one candidate has been reported to be the proenkephalin gene w70x. Early increases in c-fos protein after KA treatment w61x and in c-fos mRNA after electrical stimulation w67x are observed predominately in dentate gyrus, similar to changes in calbindin protein and mRNA and BDNF mRNA, with lower levels of c-fos induced in CA3 and CA1 pyramidal cell layers. Region-specific changes in the mRNA for zif268, a zinc finger containing protein, which binds to a G-rich DNA consensus sequence, and the mRNA for nur77, an orphan steroid receptor, have been reported after electrical stimulation or administration of convulsant drug to be similar to those reported for c-fos mRNA w18,64,67,75x. It has been suggested the induction of these putative transcription factor mRNAs in brain is part of a programmed response of neurons which can ultimately result in long-term plastic changes induced by seizure activity w18,29,64,67x. The molecular mechanism of tran-
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Fig. 6. Time relationship of the response to KA of the immediate-early genes, BDNF, NT-3 and calbindin mRNAs. Using all data obtained from 0 Žcontrol. –3 h, plots were fitted to a quadratic polynomial which fits the early part of the data. Each of the various curves, except the NT-3 curve, was adjusted vertically so that the zero point begins at the same level. Analysis of all the data, as shown above, indicates Žassuming the steeper slopes occur at an earlier point in the time sequence. that the time relationship of induction is: early, c-fos, nur77, zif268 mRNAs; middle, BDNF mRNA; and later, calbindin mRNA. NT-3 mRNA decreases at times when the immediate-early genes, BDNF and calbindin mRNA are induced.
scriptional up-regulation of these early-response genes has been reported to involve calcium influx into neurons w18,66x. Calcium response elements ŽCREs., having the
Fig. 7. Relative levels of calbindin-D28k mRNA in rat hippocampus and cerebellum examined 30 days after continuous hippocampal stimulationinduced SE. Left panel: representative Northern analysis of calbindin mRNA in hippocampus and cerebellum of C, control rats or rats sacrificed 30 days post-SE. For each lane polyŽA.q RNA Ž8 m g. was prepared from tissue pooled from 3–4 rats for hippocampus and from 2–3 rats for cerebellum. Right panel: graphic representation Žmean" S.E.M.. of densitometric quantitation of Northern blot analysis. After normalization based on results obtained upon hybridization with b-actin or 18S rRNA cDNA, the densities were expressed as relative signal intensity. Results were obtained from three Northern analyses using separate groups of rats.
consensous TGACGTCA, have been reported in the promoter regions of c-fos w66x, zif268 w7x and nur77 w77x. Increases in intracellular calcium have also been reported to induce later responsive genes, such as BDNF w78x and NGF w48x. Since the expression of calbindin in peripheral tissues is modulated by calcium w15,32x and since calbindin in hippocampus is induced both by perforant path stimulation w46x and KA administration, which result in elevations in intracellular calcium, it is possible that the induction of the early-response genes and the later responsive BDNF and calbindin mRNAs by KA could be initiated by a common calcium-dependent mechanism. Thus, the relationship suggested by the present findings is that in response to KA there is activation of KA-preferring glutamate receptors and a subsequent influx of calcium which triggers the increase in the immediate-early genes, in BDNF mRNA and in the number of neurons expressing calbindin which results in increased buffering of intracellular calcium and protection against brain injury. Although calcium may indeed be involved in the regulation of these genes in the same neurons in the hippocampus, further studies are needed to determine whether there is a direct involvement of IEGs in the regulation of BDNF and whether the subsequent increase in BDNF affects the transcription of the calbindin gene. Studies using the multiple promoters of the BDNF gene w28,43x and promoter region of the calbindin gene w30x, should enable us to obtain new insight concerning the multiple factors involved in the transcription response to neuronal activation.
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An association between neurotrophic factors and calbindin is suggested not only in these studies in which early times after KA were examined but also when the effect of KA is examined after several days. Histological evaluation after systemic administration of KA has indicated both acute neuropathological changes Žwithin 3–5 h after i.p. injection. as well as subsequent neuropathological changes Ž24 h–16 days. in brain areas unaffected at early times after KA. Using in vitro bioassays for growth factor activity, Lowenstein et al. w47x reported prolonged increases in neurotrophic activity in hippocampal extracts observed 4 and 7 days after kainate which were associated with mossy fiber reorganization. We previously reported that 6 days following systemic KA injection there is a striking appearance of calbindin in astrocytes w52x. Using cultured neocortical and hippocampal astrocytes, we found that TNF but not NGF or bFGF can induce calbindin in astrocytes. Thus, at longer times after KA administration, TNF and perhaps other factors not yet examined induce calbindin, promoting the survival of supporting astrocytes, a rich source of neurotrophic factors which may play a role in a later effect of KA related to synaptic reorganization. A question raised by the current investigation is whether the induction of calbindin expression at early times after KA is a response to seizure or to neuronal injury coincident with seizure. Although KA-induced seizures result in an induction in hippocampal calbindin expression, recurrent spontaneous seizures established with an episode of electrical stimulation-induced SE failed to result in a change in calbindin mRNA. As a result of SSLSE, the hippocampus becomes chronically epileptic w44x and similar to findings observed after injection of KA into the hippocampus w20x, there is a loss of inhibition in the CA1 area w44x. Also, hippocampal slices from rats sacrificed 30 days after continuous hippocampal stimulation-induced SSLSE were found to be hyperexcitable compared to those from control animals w44x. However, unlike the effect of KA, 30 days after SSLSE recurrent spontaneous seizures are less intense, non-convulsive and not specifically excitotoxic. When calbindin mRNA was examined by in situ hybridization as well as by Northern analysis after traditional commissure kindling Ždegenerative morphological changes are not observed in this model of epilepsy; w68x., no changes in calbindin mRNA in hippocampus and other brain areas were observed 30 min, 1, 6 or 24 h after the last kindled seizure w68x. Thus, we suggest that the induction of calbindin mRNA may not be related to enhanced neuronal activity but rather to maintaining neuronal viability in response to the toxic effects of KA. In our studies, we observed an increase in calbindin mRNA not only after systemic injection but also after intrahippocampal infusion, both in the ipsilateral and contralateral sides of the hippocampus. The induction of calbindin mRNA in the contralateral side, which may result from hyperactivity in the afferent excitatory pathways, may also be a response to neuronal vulnerability. Excitotoxic damage involving cal-
cium-dependent mechanisms and selectively vulnerable cell types has been reported at sites remote from the site of intracerebral injection of KA w49,65x. In addition to calbindin, whether the induced changes in the expression of the neurotrophins are a response to seizure has also been a matter of debate. Although increases in BDNF and NGF mRNAs after hilus lesion or after KA administration in adult rats were reported to be a response to seizure w78x, Dugich-Djordjevic et al. w23x found that on post-natal day 8, seizures were induced by KA but the seizures were not associated with elevations in BDNF mRNA. The authors suggested, similar to our suggestion for the induction of calbindin, that the rapid response of BDNF mRNA in the adult rat may be correlated with a neuropathological outcome of seizure and may not be primarily regulated by neuronal activity. The induction of calbindin and BDNF may represent two components of a chain of genomic events which result in calcium stabilization and neuroprotection after excitotoxic insults. Further studies are needed to identify other components of this chain of genomic events and to determine how the neuroprotective actions of the neurotrophins and calbindin may be used to develop new therapeutic strategies for protecting against brain injury after excitotoxic insults.
Acknowledgements Grants from the NIH to S.C., E.W.L., M.F.C., R.M.S. and M.P.M. supported this study.
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Research report
Early induction of mRNA for calbindin-D 28k and BDNF but not NT-3 in rat hippocampus after kainic acid treatment S. Lee a , J. Williamson b, E.W. Lothman b, F.G. Szele c , M.F. Chesselet R.M. Sapolsky e, M.P. Mattson f , S. Christakos a,)
c,1
, S. Von Hagen d ,
a
f
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA b Department of Neurology, UniÕersity of Virginia, Health Science Center, CharlottesÕille, VA 22908, USA c Department of Pharmacology, UniÕersity of PennsylÕania, Philadelphia, PA 19104, USA d Department of Pharmacology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA e Department of Biological Sciences, Stanford UniÕersity, Stanford, CA 94305, USA Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, UniÕersity of Kentucky, Lexington, KY 40536, USA Accepted 17 December 1996
Abstract The influence of kainic acid ŽKA., which induces acute seizures, on expression of mRNA for the calcium-binding protein, calbindin-D28k , brain-derived neurotrophic factor ŽBDNF., neurotrophin-3 ŽNT-3. and early-response genes wc-fos, zif268 ŽNGFI-A., nur77 ŽNGFI-B.x was examined in rat hippocampus by Northern blot analysis. A significant increase Ž3.2-fold. in BDNF mRNA was observed 1 h after KA injection Ž12 mgrkg i.p.. and peak expression Ž9.4-fold. occurred 3 h after KA. The induction of BDNF mRNA was preceded by the induction of c-fos, mRNA Ž30 min after KA. and was followed by the induction of calbindin-D28k mRNA Ž3.5-fold 3 h after KA; a maximal response was at 3–6 h after KA.. Region-specific changes, analyzed by immunocytochemistry and in situ hybridization, indicated that the most dramatic increases in calbindin protein and mRNA after KA treatment were in the dentate gyrus. Although calbindin-D28k and BDNF mRNAs were induced, a 3.4–3.8-fold decrease in NT-3 mRNA was observed by Northern analysis 3–24 h after KA treatment. Calbindin-D28k gene expression was also examined in rats with a chronic epileptic state characterized by recurrent seizures established with an episode of electrical stimulation-induced status epilepticus ŽSE.. When these animals were examined 30 days post-SE, no changes in hippocampal calbindin-D28k mRNA were observed. Our findings suggest that the induction of calbindin-D28k mRNA Žwhich may be interrelated to the induction of BDNF mRNA. is an early response which may not be related to enhanced neuronal activity or seizures per se, but rather to maintaining neuronal viability.
1. Introduction Kainic acid ŽKA., an analog of the excitatory amino acid glutamate w58x, induces acute seizures in rats which reproduce sequelae of human temporal lobe epilepsy w3,56x. Systemic injection of KA results in excitatory damage in selective brain regions. Affected areas include amygdaloid complex, hippocampus Žthe most extensively damaged area is the CA3 region. and related parts of the thalamus and
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Corresponding author. UMDNJ-New Jersey Medial School, 185 South Orange Avenue, Newark, NJ 07103-2714, USA. Fax: q1 Ž201. 982-5594. 1 Present address: Department of Neurology, University of California at Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.
neocortex w65x. It has been suggested that this neurotoxicity is caused by massive influx of extracellular calcium by activation of KA-preferring glutamate receptors. Neuronal cells are believed to have protective mechanisms against glutamate-induced calcium-mediated cytotoxicity which involve, in part, the release of neurotrophins. The nerve growth factor ŽNGF. family of neurtrophins includes, in addition to NGF, brain-derived neurotrophic factor ŽBDNF., neurotrophin-3 ŽNT-3. and neurotrophin-4r5 ŽNT-4r5. w60x. The neurotrophins bind and activate members of the trk family of tyrosine kinase receptors ŽNGF binds trkA, BDNF and NT-4r5 bind trkB and NT-3 is associated mainly with trk C. w37x. The neurotrophins are present in selected neuronal populations in the peripheral and central nervous systems where they play important roles in supporting differentiation andror survival of subpopulations of neurons during development
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w41x in addition to their reported role in protecting against glutamate-induced cytotoxicity w10,11,43x. NGF and BDNF mRNA and protein have been reported to be induced by seizure activity induced by KA w2,23,24,78x as well as by dentate hilar lesion w38,40,63x, kindling w4,26x and injection of bicuculline w77x or pentylenetetrazol w33x. Changes in neurotrophin expression are not unique to epilepsy but have been reported after brain injury induced by ischemia w42,72x and hypoglycemia w42x which also results in glutamate-induced increases in intracellular calcium which can cause cell death. It has recently been reported that one mechanism whereby NGF, BDNF and NT-3 can protect neurons against metabolicrexcitotoxic insults is by stabilizing intracellular calcium w10,11x. It has been suggested that the stabilization of intracellular calcium may be mediated in part by the calcium-binding protein calbindin-D28k w11,53x which is induced in hippocampus by BDNF and NT-3 w19x. Calbindin-D28k , a major calcium-binding protein in brain Žit constitutes 0.1–1.5% of the total soluble protein in brain w14,15,69x., has a widespread but restricted distribution throughout the central nervous system w6x. In hippocampus, calbindin is localized in the dentate granule cells which are relatively resistant to seizure-induced insults w65x as well as ischemic injury w31x and in CA1 pyramidal neurons which are also relatively resistant to seizure damage w65x. Studies of hippocampal cells in culture have indicated that calbindin-positive neurons are protected against damage induced by glutamate and calcium ionophore and are better able to reduce free intracellular calcium than calbindin-negative neurons w53x. These findings suggest that calbindin, by buffering intracellular calcium, can prevent calcium-mediated neuronal death that could result from excitotoxic insult. More recently, direct evidence of the calcium buffering capacity of calbindin was demonstrated by Chard et al. w8x who reported that introduction of exogenous calbindin into dorsal root ganglia neurons resulted in reduction in the depolarization-induced rate of rise of intracellular calcium, indicating directly that calbindin can effectively regulate calcium-dependent aspects of neuronal function. Thus, the function of neurotrophic factors in regulating calbindin expression and thereby possibly preventing excitotoxic neuronal damage in neuropathies, such as epilepsy, has important therapeutic implications. In order to obtain an increased understanding of the possible interrelationship between calbindin, neurotrophins and seizure-induced excitotoxic insult, region-specific and time-dependent changes in hippocampal calbindin mRNA and protein as well as time-dependent changes in mRNA for two neurotrophins in hippocampus known to induce calbindin, BDNF and NT-3, were examined under conditions of acute status epilepticus ŽSE. induced by KA. To determine if the acutely observed changes were maintained in a chronic epileptic state, calbindin-D28k gene expression was also examined in the hippocampus of rats with recurrent spon-
taneous seizures induced with an episode of electrical stimulation-induced SE w44x.
2. Materials and methods 2.1. Materials w 32 PxdCTP Ž3000 Cirmmol, 10 mCirml. was purchased from New England Nuclear Products ŽBoston, MA.. Oligo ŽdT. cellulose and all restriction enzymes were purchased from Boehringer Mannheim ŽIndianapolis, IN.. Biotrans nylon membranes were obtained from ICN Biochemicals ŽCosta Mesa, CA. and formamide was from Sigma ŽSt. Louis, MO.. Guanidinium isothiocyanate and phenol Žultra pure, molecular biology grade. were from Fischer Scientific ŽSpringfield, NJ.. The Rad prime DNAlabeling system and agarose Želectrophoresis grade. were purchased from Gibco-BRL Life Technologies ŽGaithersburg, MD.. 2.2. Preparation of animals In all studies, adult male Sprague–Dawley rats Ž250– 300 g. were used and maintained with food and water ad libitum in a 12-h lightr12-h dark cycle. For studies involving systemic KA administration, 90 rats were used. Rats received either KA ŽSigma. Ž12 mgrkg body weight prepared in 10 mM phosphate-buffered saline; PBS pH 7.4. or saline by i.p. injection. Seizure activity was observed within 30–40 min following kainate treatment. Rats not exhibiting seizure activity after kainate injection were not used in the study. For studies using KA microinfusion in brain, 50 rats were anesthetized with Metofane ŽPitman-Moore, NJ. and KA Ž0.07 m g in 1 m l volume. was injected stereotaxically into Ammon’s horn of the dorsal hippocampus with a Hamilton syringe over 1 min followed by a 1 min wait for the KA to diffuse Žcoordinates: anteroposterior ŽAP., q4.1 mm; mediolateral ŽML., 2.1; dorsoventral ŽDV., 3.0 Žheight of l suture set equal to bregma. w25x. Control Žzero point. received 1.0 m l saline without KA. For studies in rats with a chronic epileptic state, rats were stereotaxically implanted with bipolar electrodes in the left ventral hippocampus ŽAP y3.6, ML 4.9, DV y5.0 to dura w59x.. Control rats were implanted with electrodes but not stimulated. One week after surgery, rats received a period of continuous hippocampal stimulation ŽCHS. Ž10-s, 50-Hz trains of 1-ms biphasic pulses set at 400 m A given once every 12 s for 90 min.. CHS established a condition of self-sustaining limbic SE ŽSSLSE. w44x. Rats were killed by decapitation 30 days after CHSinduced SSLSE. Chronic recurrent spontaneous hippocampal seizures begin in rats within 30 days post-CHS-induced SSLSE w44x.
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For all studies, brains were rapidly removed, brain areas were quickly dissected and immediately frozen on dry ice and stored at y808C.
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removed and quickly frozen on powered dry ice, cryostatcut into sections Ž10 m m. which were thaw-mounted onto gelatin-coated slides and stored at y708C. Radiolabeled
2.3. RNA isolation and Northern blot hybridization analysis Total RNA was isolated from pooled samples Žusually, from 3 animals. by the guanidinium thiocyanaterphenol chloroform procedure of Chomczynski and Sacchi w13x. PolyŽA.q RNA was obtained after two cycles of oligo ŽdT. cellulose chromatography. PolyŽA.q RNA was fractionated under denaturing conditions on a 1.2% formaldehyde agarose gel and transferred to a nylon membrane ŽBiotrans, ICN. using standard procedures as previously described w68,73x. Hybridization probes were labeled according to the random priming method of Feinberg and Vogelstein w27x using the Rad prime DNA labeling system ŽGibco-BRL Life Technologies.. The filters were hybridized at 428C overnight, washed and autoradiographed as previously described. For reprobing, hybridized probes were removed by washing in 50% formamide–10 mM phosphate buffer ŽpH 6.5. for 1 h at 658C. b-Actin cDNA andror 18S rRNA cDNA were used as control probes. Autoradiograms of varying exposures of Northern blots were analyzed using dual-wave length flying-spot scanner ŽShimadzu Scientific Instrument, Princeton, NJ.. The relative optical density of each Northern probed with calbindin, neurotrophin or early-response gene cDNA was divided by the relative optical density obtained after probing with b-actin or 18S rRNA cDNA to normalize for sample variation. 2.4. Preparation of DNA probes A 1.2-kb mouse calbindin-D28k cDNA insert from the EcoRI site of pIBI76 w76x, a 1.1-kb rat BDNF cDNA from the EcoRI site of pSK-rBC1 w50,51x, a 0.8-kb rat NT-3 cDNA from the XhoI site of pSK-rNT3 w50,51x ŽBDNF and NT-3 cDNA probes were kindly supplied by George D. Yancopoulos, Regeneron Pharmaceuticals, Tarrytown, NY., a 2.1-kb rat c-fos cDNA from the EcoRI site of pSp65 w22x, a 0.3-kb zif268 ŽNGFI-A. insert from the BamHI site of pB700-3.6 w71x Žobtained from Vikas Sukhatme, Beth Israel Hospital, Boston, MA. and a 2.5-kb rat nur77 ŽNGFI-B. insert from the EcoRI site of pJDM3 w54x were obtained by the restriction enzyme digestion of the respective plasmid preparations. A 2.1-kb chicken bactin cDNA was obtained from the HindIII site of pBR322 w17x. The 18S rRNA cDNA was from Ramareddy Guntaka ŽUniversity of Missouri at Columbia.. 2.5. In situ hybridization For the in situ hybridization experiments, rats were killed 6 h after KA injection. The brains were rapidly
Fig. 1. Northern blot analysis of calbindin-D28k mRNA in rat hippocampus after KA treatment. A: polyŽA.q RNA Ž8 m grlane., isolated from hippocampus from 3–4 adult male rats for each time point after systemic KA injection Ž12 mgrkg body weight. was used for Northern analysis Župper panel.. Results from the Northern blots using hippocampi from three separate groups of rats were used for densitometric analysis Žmean "S.E.M.; lower panel.. After normalization based on results obtained after re-hybridization with 18S rRNA or b-actin cDNAs, densities were expressed as percentage maximal response. B: polyŽA.q RNA Ž8 m grlane. was isolated from the ipsilateral Žinfused side. or contralateral hippocampus at various times after intrahippocampal infusion. Hippocampi from 5 rats were pooled for preparation of RNA at each time point. Densitometric scanning of autoradiograms and normalization with b-actin indicated that calbindin mRNA was elevated 1.6- and 1.9-fold on the contralateral and ipsilateral sides, respectively, at 1 h after KA injection. 3 h after KA injection, calbindin mRNA was induced 1.9- and 2.6-fold on the contralateral and ispsilateral sides, respectively Žresults are the mean of two independent measurements, using separate groups of rats..
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antisense and sense cRNAs were transcribed from cDNAs as previously described w12x using appropriate polymerases, 2.5 m M w 35 SxUTP Ž1000 Cirmmol, New England Nuclear., 10 m M cold UTP, with ATP, CTP and GTP in excess. The template cDNA Ž320 bp. was the same as used for Northern analysis. Both sense and antisense RNA probes were synthesized. In situ hybridization was carried out as previously described w12x. Briefly, tissue sections were brought to room temperature and dried rapidly under a stream of cold air, post-fixed in 3% paraformaldehyde, acetylated and dehydrated. Sections were incubated with 3 ng of probe Žf 400 000 dpmrng. in humid chambers at 508C for 3.5 h. After hybridization, non-specific binding was reduced by washing in 50% formamide in 2 = SSC Ž0.3 M NaClr 0.03 M sodium citrate. at 528C and treatment with RNase A Ž100 m grml. for 30 min at room temperature. After overnight incubation in 2 = SSCr0.05% Triton X-100, slides were dehydrated in graded ethanol, defatted in xylene and coated with Kodak NTB 3 emulsion diluted 1 : 1 with 300 mM ammonium acetate. After exposure at 48C for 29 days, the sections were developed in Kodak D-19 developer at full strength, lightly counterstained with hematoxylin eosin and mounted with Eukitt ŽCalibrated Instruments.. The sections were examined under bright-field and dark-field microscopy on a Leitz aristoplan microscope. 2.6. Calbindin-D2 8 k immunocytochemistry Rats were killed at 6, 12 and 24 h after systemic KA injection. Three ratsrtime point were studied. At the different times after KA, rats were anesthetized with an i.p. injection of 20% urethane ŽSigma. and then perfused intracardially with 0.1 M sodium phosphate-buffered saline ŽPBS. with 0.5% heparin ŽpH 7.4; LyphoMed, Melrose Park, IL. followed by 4% paraformaldehyde ŽSigma. in 0.1 M sodium PBS ŽpH 7.4.. The brains were removed, fixed in paraformaldehyde for 1 h, rinsed in PBS ŽpH 7.4. and then permeated with a cryoprotected sucrose solution Ž12.5 mM sodium phosphate monobasic, 38.5 mM sodium phosphate dibasic and 877 mM sucrose.. The brains were stored at y208C. After transfer to 10 mM PBS, 50-m m coronal sections were prepared using a vibratome. Sections Žfree-floating. were first incubated in PBS containing 0.2% Triton X-100 and 0.015% non-immune horse serum fol-
lowed by overnight incubation in PBSrTritonrnon-immune horse serum containing a 1 : 1500 dilution of a rabbit polyclonal rat calbindin antiserum w16x. Sections were immunostained using the biotin-avidin-peroxidase method described previously w25x. Control brains were similarly processed but without the primary antibody. Sections were photographed using an inverted Nikon Diaphot microscope with phase-contrast and bright-field optics. 2.7. Statistical analysis Results are expressed as the mean " S.E. and significance was determined by Dunnet’s multiple comparison t-statistic or by analysis of variance.
3. Results Northern blot analysis of hippocampal polyŽA.q RNA indicated that after systemic injection of KA Ž12 mgrkg bw., steady-state levels of calbindin-D28k mRNA were rapidly induced. The first significant increase was observed at 3 h after injection Ž P - 0.05; 3.5-fold induction.. Levels of calbindin mRNA returned to control levels at 12 h after KA treatment ŽFig. 1A.. Unlike the changes observed in hippocampus, no changes in calbindin-D28k mRNA were observed in cerebellum at any time after KA treatment Ž0.5–24 h; not shown.. Calbindin-D28k mRNA levels were also examined in a different experimental model in which rats received a unilateral injection of KA directly into Ammon’s horn of the dorsal hippocampus. This model allows the discrimination between a neurotoxic effect of KA which is observed in the ipsilateral side and a neuroexcitatory effect on the contralateral side which is most likely caused by hyperactivity. Calbindin-D28k mRNA was clearly increased at 1 and 3 h after KA infusion in both the ipsilateral and contralateral sides ŽFig. 1B.. At 6 and 16 h after KA infusion, levels of calbindin-D28k mRNA remained elevated in the ipsilateral but not in the contralateral side. Thus, calbindin-D28k mRNA can be induced not only by a direct action of KA on its receptor Žipsilateral side. but also as a result of hyperactivity in afferent excitatory pathways. The remainder of the studies involving KA were done using i.p. injection which results in selectivity of vulunerable neurons Žthose with high
Fig. 2. In situ hybridization histochemistry for calbindin-D28k . A,B: dark-field photomicrographs of sections of cerebellum of control ŽA. and KA-treated ŽB. rats were processed for in situ hybridization histochemistry with a 35 S-radiolabeled antisense RNA probe for calbindin-D28k . Arrows point to dense accumulations of silver grains over Purkinje cells. Scale bar shown in B: 70 m m for A and B. C–F: in situ hybridization histochemistry for calbindin-D28k in rat hippocampus. Sections were hybridzed with antisense ŽC,E. or sense ŽF. 35 S-radiolabeled RNA probes for calbindin mRNA. C,E: dark-field photomicrographs of sections of the same region of the hippocampus in KA-injected ŽC. and control ŽE. rats. Arrow in C points to labeling in the dentate gyrus. D: low-power bright-field photomicrograph of section illustrated in C Žcounterstaining: hematoxyllin eosin.. The arrow points to dentate gyrus. Scale bar shown in F s 100 m m Žin C,E.; s 500 m m Žin D.; s 80 m m Žin F.. Similar results were observed in the brains of two other control and KA-treated rats assayed at the same time using the same in situ hybridization conditions and exposure times. It should be noted, as has been previously reported w68x, that with longer autoradiographic exposure time calbindin-D28k can be observed using in situ hybridization, in the hippocampus of control rats.
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concentrations of KA type glutamate receptors.. Regionspecific changes in calbindin-D28k mRNA and protein after i.p. KA injection was examined by in situ hybridiza-
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tion and immunocytochemistry, respectively. In Fig. 2A,B, a dense accumulation of silver grains was observed over Purkinje cells in the cerebellum of both control ŽA. and
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KA-injected ŽB. rats in sections processed for in situ hybridization with 35 S-radiolabeled antisense probe for calbindin. Sections hybridized under the same conditions with the sense probe did not show specific labeling in any brain area examined. Despite the presence of a strong cerebellar autoradiographic signal in the same sections, no specific labeling was detected in the hippocampus of control rats in our experimental conditions ŽFig. 2E.. In contrast, labeling was clearly present in the dentate gyrus 6 h after KA administration ŽFig. 2C,D.. No increases in labeling were observed in other hippocampal regions or in caudate-putamen Žstriatum. of the same rats Žnot shown.. KA treatment was also found to induce a marked increase in calbindin immunoreactivity at 6 and 12 h which, similar to results of in situ hybridization, was also most obvious in the dentate gyrus ŽFig. 3.. By 24 h, calbindin immunoreactivity was reduced towards control levels ŽFig. 3.. Since BDNF and NT-3 have been reported to enhance the expression of calbindin in the hippocampus w19x, the influence of KA on the expression of mRNAs for these neurotrophins was examined by Northern analysis. A significant increase in BDNF mRNA was observed 1 h after KA injection Ž P - 0.05; 3.2-fold induction. and peak ex-
pression Ž9.4-fold. occurred 3 h after KA ŽFig. 4A.. In contrast, KA treatment resulted in a marked 3.4–3.8-fold decrease in the levels of NT-3 mRNA in the hippocampus ŽFig. 4B.. Since it has been suggested that immediate-early genes may play a role in regulating genes which respond later to trans-synaptic activation, changes in the expression of c-fos, zif268 and nur77 mRNAs were examined after KA treatment. The induction of both BDNF and calbindin-D28k mRNAs was preceded by induction of c-fos mRNA. Levels of c-fos mRNA were undetectable in control rats Žzero time. even after prolonged radiographic exposure but were significantly induced 30 min after KA ŽFig. 5.. Although 1.5- and 1.9-fold inductions in zif268 and nur77 mRNAs, respectively, were observed at 30 min, Northern analysis indicated that the first significant induction Ž P - 0.05. in these early-response genes was at 1 h with peak expression Ž6.3- and 7.4-fold for zif268 and nur77 mRNAs, respectively. occurring at 3 h after KA ŽFig. 5.. A direct comparison of the time relationship of the response to KA of the immediate-early genes, BDNF, NT-3 and calbindin mRNAs is shown in Fig. 6. Calbindin-D28k gene expression was also examined in
Fig. 3. KA induces a transient increase in hippocampal calbindin immunoreactivity. Rats were injected with saline Žcontrol. or KA Ž12 mgrkg body weight.. KA-treated rats were killed at 6, 12 and 24 h following injection. KA induced a marked increase in calbindin immunoreactivity at 6 and 12 h which was most obvious in the molecular layer of the dentate gyrus. Increased calbindin immunoreactivity was also observed in region CA1. By 24 h, calbindin immunoreactivity was reduced towards control levels.
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Fig. 4. Northern analysis of BDNF and NT-3 mRNAs in rat hippocampus after KA treatment. Left panel: representative Northern blot analysis. PolyŽA.q RNA was isolated from the hippocampus at various times after systemic administration of KA and fractionated on a 1.2% formaldehyde-agarose gel. The mRNA was transferred to a nylon membrane and hybridized with calbindin ŽFig. 1A., BDNF and NT-3 cDNAs. Basal levels of BDNF mRNA were observed at 0 and 0.5 h after longer radiographic exposure. ŽFor details concerning the experiment, see legend to Fig. 1A.. Right panel: graphical representation of the results of Northern analyses obtained in three separate experiments.
rats with a chronic epileptic state characterized by recurrent seizure established with an episode of electrical stimulation-induced SE. When these animals were examined 30 days post-SE, no changes in hippocampal calbindin-D28k mRNA were observed ŽFig. 7.. Calbindin-D28k mRNA was also not altered in cerebellum ŽFig. 7., amygdala Žnot shown. or frontoparietal cortex Žnot shown. under these conditions.
4. Discussion In this study, we report that calbindin mRNA and protein rapidly increase after KA treatment and the most pronounced increases are observed in the dentate gyrus. Although a previous study indicated an induction of calbindin mRNA Žas well as 72-kDa heat-shock protein and 78-kDa glucose-regulated protein mRNAs. in rat hippocampus after KA administration w45x, region-specific changes and changes in protein had not as yet been examined. In our study, we found that the induction of calbindin mRNA is preceded at 1 h and coincident at 3 and 6 h after KA with the induction of BDNF mRNA. Previously reported region-specific changes in BDNF mRNA after KA treatment indicate, similar to changes in calbindin mRNA, that the greatest, earliest increase occurs in the
dentate granule cell layer Žwhich is most resistant to seizure-induced injury., followed by an increase in CA1 w24x. The extent of elevation in the CA1 region was approximately half that observed in the dentate granule cell layer. Increases in BDNF mRNA in the CA3 region, similar to increases in the CA1 region, have also been reported w24x. Due to the correlation of the increases in calbindin mRNA and BDNF mRNA in the dentate gyrus as well as the observation that the induction of BDNF mRNA precedes the induction of calbindin mRNA, it is possible that the induction of calbindin after KA may be mediated, in part, by BDNF. Besides the hippocampus, induction of BDNF mRNA by KA was also previously noted in the cortex Žlayer II and layers IV and VI w24x.. Although KA did not induce calbindin mRNA in cerebellum, it will be of interest in future studies to determine whether calbindin, which is present in layers II and IV w6x, and its mRNA can be induced in these areas of the cortex similar to BDNF mRNA. Until recently, unlike studies related to calbindin in peripheral tissues w14,15,30,32x, very little has been known about the signalling mechanisms involved in regulating neuronal calbindin. However, correlative evidence between decreases in neuronal calbindin and neurodegeneration in studies of ischemic injury w5,62x, seizure activity w1,68x and chronic neurodegenerative disorders ŽAlzheimer, Hunting-
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Fig. 5. Changes in immediate-early genes Žc-fos, zifr268 and nur 77. in rat hippocampus after KA treatment assessed by Northern blot hybridization. The same Northern blots probed for calbindin-D28k andror BDNF and NT-3 were also hybrized with c-fos, zif268 and nur77 cDNAs. After normalization based on results obtained upon re-hybridization with 18S rRNA Žshown in bottom panel inset., densities were expressed as percent maximal response Žmean"S.E.M... Data was obtained from three separate Northern analyses except for the 24-h time point which represents the mean of duplicate determinations.
ton and Parkinson diseases. w35x had been reported. Using rat hippocampal cultures, recent evidence has indicated that NT-3 w19,36x, BDNF w19,36x, fibroblast growth factor ŽFGF. w19x and tumor necrosis factors ŽTNFs. w9x can all induce calbindin. In addition, in vivo corticosterone administration has been reported to increase calbindin expression in rat hippocampus w34x, specifically in the CA1 region w39x. Retinoic acid has also been reported to induce calbindin protein and mRNA in medulloblastoma cells, which express a neuronal phenotype w74x, and the content of calbindin in cultured Purkinje cells can be increased by insulin-like growth factor I w57x. Thus, neuronal calbindin can be regulated by steroids as well as by factors that affect signal transduction pathways. Although a 20-fold induction of calbindin in response to NT-3 compared to about a 2-fold induction by BDNF has previously been reported in rat hippocampal cultures w19x, in this study
NT-3 mRNA was found to be decreased at times when calbindin mRNA was maximally induced after KA. In previous studies, NT-3 mRNA has been reported to be unchanged or markedly decreased after seizure induced by KA, hilus lesion or kindling w2,4,28x. Region-specific changes indicated that NT-3 mRNA was most dramatically reduced in the dentate granule cell layer w4,28x. Thus, it is likely that, within the same region of the hippocampus, calbindin and BDNF mRNAs are increased while NT-3 mRNA is decreased. Since NT-3 mRNA is down-regulated and it has been reported that changes in mRNA content for neurotrophins are reflected in protein production w28x, NT3, which may be involved in inducing calbindin during hippocampal neurogenesis w19x, may not play a role in the up-regulation of calbindin observed after KA-induced seizures. However, it is possible that trkC receptor mRNA up-regulation, which has been reported after kindling induced seizures coincident with NT-3 mRNA down-regulation w4x, may also occur after KA-induced seizures and may compensate, in part, for the decrease in NT-3 mRNA. Since the expression of mRNAs for FGF w28x and TNF w55x have also been reported to be induced in hippocampus early times after seizure, it is possible that these factors, which induce calbindin in hippocampal cultures, may also contribute to the up-regulation of calbindin observed after KA. FGF w10x and TNFs w9x as well as BDNF w11x have all been reported to protect hippocampal neurons against excitotoxic insults by stabilizing calcium homeostasis, further suggesting an interrelationship between these factors and calbindin. Although the induction of calbindin mRNA after KA treatment was preceded by the induction of BDNF mRNA, the most rapid genomic response was the increase in c-fos, mRNA. c-Fos is a member of a heterodimer transcription complex, which combines with members of the c-jun family, binds to AP1 consensus sequences and results in activation or repression of genes w21x. In the nervous system, few physiological targets for c-fos and c-jun have been definitively identified. However, one candidate has been reported to be the proenkephalin gene w70x. Early increases in c-fos protein after KA treatment w61x and in c-fos mRNA after electrical stimulation w67x are observed predominately in dentate gyrus, similar to changes in calbindin protein and mRNA and BDNF mRNA, with lower levels of c-fos induced in CA3 and CA1 pyramidal cell layers. Region-specific changes in the mRNA for zif268, a zinc finger containing protein, which binds to a G-rich DNA consensus sequence, and the mRNA for nur77, an orphan steroid receptor, have been reported after electrical stimulation or administration of convulsant drug to be similar to those reported for c-fos mRNA w18,64,67,75x. It has been suggested the induction of these putative transcription factor mRNAs in brain is part of a programmed response of neurons which can ultimately result in long-term plastic changes induced by seizure activity w18,29,64,67x. The molecular mechanism of tran-
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Fig. 6. Time relationship of the response to KA of the immediate-early genes, BDNF, NT-3 and calbindin mRNAs. Using all data obtained from 0 Žcontrol. –3 h, plots were fitted to a quadratic polynomial which fits the early part of the data. Each of the various curves, except the NT-3 curve, was adjusted vertically so that the zero point begins at the same level. Analysis of all the data, as shown above, indicates Žassuming the steeper slopes occur at an earlier point in the time sequence. that the time relationship of induction is: early, c-fos, nur77, zif268 mRNAs; middle, BDNF mRNA; and later, calbindin mRNA. NT-3 mRNA decreases at times when the immediate-early genes, BDNF and calbindin mRNA are induced.
scriptional up-regulation of these early-response genes has been reported to involve calcium influx into neurons w18,66x. Calcium response elements ŽCREs., having the
Fig. 7. Relative levels of calbindin-D28k mRNA in rat hippocampus and cerebellum examined 30 days after continuous hippocampal stimulationinduced SE. Left panel: representative Northern analysis of calbindin mRNA in hippocampus and cerebellum of C, control rats or rats sacrificed 30 days post-SE. For each lane polyŽA.q RNA Ž8 m g. was prepared from tissue pooled from 3–4 rats for hippocampus and from 2–3 rats for cerebellum. Right panel: graphic representation Žmean" S.E.M.. of densitometric quantitation of Northern blot analysis. After normalization based on results obtained upon hybridization with b-actin or 18S rRNA cDNA, the densities were expressed as relative signal intensity. Results were obtained from three Northern analyses using separate groups of rats.
consensous TGACGTCA, have been reported in the promoter regions of c-fos w66x, zif268 w7x and nur77 w77x. Increases in intracellular calcium have also been reported to induce later responsive genes, such as BDNF w78x and NGF w48x. Since the expression of calbindin in peripheral tissues is modulated by calcium w15,32x and since calbindin in hippocampus is induced both by perforant path stimulation w46x and KA administration, which result in elevations in intracellular calcium, it is possible that the induction of the early-response genes and the later responsive BDNF and calbindin mRNAs by KA could be initiated by a common calcium-dependent mechanism. Thus, the relationship suggested by the present findings is that in response to KA there is activation of KA-preferring glutamate receptors and a subsequent influx of calcium which triggers the increase in the immediate-early genes, in BDNF mRNA and in the number of neurons expressing calbindin which results in increased buffering of intracellular calcium and protection against brain injury. Although calcium may indeed be involved in the regulation of these genes in the same neurons in the hippocampus, further studies are needed to determine whether there is a direct involvement of IEGs in the regulation of BDNF and whether the subsequent increase in BDNF affects the transcription of the calbindin gene. Studies using the multiple promoters of the BDNF gene w28,43x and promoter region of the calbindin gene w30x, should enable us to obtain new insight concerning the multiple factors involved in the transcription response to neuronal activation.
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An association between neurotrophic factors and calbindin is suggested not only in these studies in which early times after KA were examined but also when the effect of KA is examined after several days. Histological evaluation after systemic administration of KA has indicated both acute neuropathological changes Žwithin 3–5 h after i.p. injection. as well as subsequent neuropathological changes Ž24 h–16 days. in brain areas unaffected at early times after KA. Using in vitro bioassays for growth factor activity, Lowenstein et al. w47x reported prolonged increases in neurotrophic activity in hippocampal extracts observed 4 and 7 days after kainate which were associated with mossy fiber reorganization. We previously reported that 6 days following systemic KA injection there is a striking appearance of calbindin in astrocytes w52x. Using cultured neocortical and hippocampal astrocytes, we found that TNF but not NGF or bFGF can induce calbindin in astrocytes. Thus, at longer times after KA administration, TNF and perhaps other factors not yet examined induce calbindin, promoting the survival of supporting astrocytes, a rich source of neurotrophic factors which may play a role in a later effect of KA related to synaptic reorganization. A question raised by the current investigation is whether the induction of calbindin expression at early times after KA is a response to seizure or to neuronal injury coincident with seizure. Although KA-induced seizures result in an induction in hippocampal calbindin expression, recurrent spontaneous seizures established with an episode of electrical stimulation-induced SE failed to result in a change in calbindin mRNA. As a result of SSLSE, the hippocampus becomes chronically epileptic w44x and similar to findings observed after injection of KA into the hippocampus w20x, there is a loss of inhibition in the CA1 area w44x. Also, hippocampal slices from rats sacrificed 30 days after continuous hippocampal stimulation-induced SSLSE were found to be hyperexcitable compared to those from control animals w44x. However, unlike the effect of KA, 30 days after SSLSE recurrent spontaneous seizures are less intense, non-convulsive and not specifically excitotoxic. When calbindin mRNA was examined by in situ hybridization as well as by Northern analysis after traditional commissure kindling Ždegenerative morphological changes are not observed in this model of epilepsy; w68x., no changes in calbindin mRNA in hippocampus and other brain areas were observed 30 min, 1, 6 or 24 h after the last kindled seizure w68x. Thus, we suggest that the induction of calbindin mRNA may not be related to enhanced neuronal activity but rather to maintaining neuronal viability in response to the toxic effects of KA. In our studies, we observed an increase in calbindin mRNA not only after systemic injection but also after intrahippocampal infusion, both in the ipsilateral and contralateral sides of the hippocampus. The induction of calbindin mRNA in the contralateral side, which may result from hyperactivity in the afferent excitatory pathways, may also be a response to neuronal vulnerability. Excitotoxic damage involving cal-
cium-dependent mechanisms and selectively vulnerable cell types has been reported at sites remote from the site of intracerebral injection of KA w49,65x. In addition to calbindin, whether the induced changes in the expression of the neurotrophins are a response to seizure has also been a matter of debate. Although increases in BDNF and NGF mRNAs after hilus lesion or after KA administration in adult rats were reported to be a response to seizure w78x, Dugich-Djordjevic et al. w23x found that on post-natal day 8, seizures were induced by KA but the seizures were not associated with elevations in BDNF mRNA. The authors suggested, similar to our suggestion for the induction of calbindin, that the rapid response of BDNF mRNA in the adult rat may be correlated with a neuropathological outcome of seizure and may not be primarily regulated by neuronal activity. The induction of calbindin and BDNF may represent two components of a chain of genomic events which result in calcium stabilization and neuroprotection after excitotoxic insults. Further studies are needed to identify other components of this chain of genomic events and to determine how the neuroprotective actions of the neurotrophins and calbindin may be used to develop new therapeutic strategies for protecting against brain injury after excitotoxic insults.
Acknowledgements Grants from the NIH to S.C., E.W.L., M.F.C., R.M.S. and M.P.M. supported this study.
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