Ž .Molecular Brain Research 47 1997 183–194

Research report

Early induction of mRNA for calbindin-D and BDNF but not NT-3 in28k

rat hippocampus after kainic acid treatment

S. Lee a, J. Williamson b, E.W. Lothman b, F.G. Szele c, M.F. Chesselet c,1, S. Von Hagen d,R.M. Sapolsky e, M.P. Mattson f, S. Christakos a,)

a Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USAb Department of Neurology, UniÕersity of Virginia, Health Science Center, CharlottesÕille, VA 22908, USA

c Department of Pharmacology, UniÕersity of PennsylÕania, Philadelphia, PA 19104, USAd Department of Pharmacology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA

e Department of Biological Sciences, Stanford UniÕersity, Stanford, CA 94305, USAf Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, UniÕersity of Kentucky, Lexington, KY 40536, USA

Accepted 17 December 1996

Abstract

Ž .The influence of kainic acid KA , which induces acute seizures, on expression of mRNA for the calcium-binding protein,Ž . Ž . w Ž .calbindin-D , brain-derived neurotrophic factor BDNF , neurotrophin-3 NT-3 and early-response genes c-fos, zif268 NGFI-A ,28k

Ž .x Ž .nur77 NGFI-B was examined in rat hippocampus by Northern blot analysis. A significant increase 3.2-fold in BDNF mRNA wasŽ . Ž .observed 1 h after KA injection 12 mgrkg i.p. and peak expression 9.4-fold occurred 3 h after KA. The induction of BDNF mRNA

Ž . Žwas preceded by the induction of c-fos, mRNA 30 min after KA and was followed by the induction of calbindin-D mRNA 3.5-fold28k.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-D and BDNF mRNAs were induced, a 3.4–3.8-fold decrease in NT-3 mRNA was observed by Northern analysis28k

3–24 h after KA treatment. Calbindin-D gene expression was also examined in rats with a chronic epileptic state characterized by28kŽ .recurrent seizures established with an episode of electrical stimulation-induced status epilepticus SE . When these animals were

examined 30 days post-SE, no changes in hippocampal calbindin-D mRNA were observed. Our findings suggest that the induction of28kŽ .calbindin-D mRNA which may be interrelated to the induction of BDNF mRNA is an early response which may not be related to28k

enhanced neuronal activity or seizures per se, but rather to maintaining neuronal viability.

1. Introduction

Ž .Kainic acid KA , an analog of the excitatory aminow xacid glutamate 58 , induces acute seizures in rats which

w xreproduce sequelae of human temporal lobe epilepsy 3,56 .Systemic injection of KA results in excitatory damage inselective brain regions. Affected areas include amygdaloid

Žcomplex, hippocampus the most extensively damaged area.is the CA3 region and related parts of the thalamus and

) Corresponding author. UMDNJ-New Jersey Medial School, 185 SouthŽ .Orange Avenue, Newark, NJ 07103-2714, USA. Fax: q1 201 982-5594.

1 Present address: Department of Neurology, University of Californiaat Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

w xneocortex 65 . It has been suggested that this neurotoxic-ity is caused by massive influx of extracellular calcium byactivation of KA-preferring glutamate receptors.

Neuronal cells are believed to have protective mecha-nisms against glutamate-induced calcium-mediated cyto-toxicity which involve, in part, the release of neu-

Ž .rotrophins. The nerve growth factor NGF family ofneurtrophins includes, in addition to NGF, brain-derived

Ž . Ž .neurotrophic factor BDNF , neurotrophin-3 NT-3 andŽ . w xneurotrophin-4r5 NT-4r5 60 . The neurotrophins bind

and activate members of the trk family of tyrosine kinaseŽreceptors NGF binds trkA, BDNF and NT-4r5 bind trkB

. w xand NT-3 is associated mainly with trk C 37 . Theneurotrophins are present in selected neuronal populationsin the peripheral and central nervous systems where theyplay important roles in supporting differentiation androrsurvival of subpopulations of neurons during development

0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0169-328X 97 00043-0

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194184

w x41 in addition to their reported role in protecting againstw xglutamate-induced cytotoxicity 10,11,43 . NGF and BDNF

mRNA and protein have been reported to be induced byw xseizure activity induced by KA 2,23,24,78 as well as by

w x w xdentate hilar lesion 38,40,63 , kindling 4,26 and injectionw x w xof bicuculline 77 or pentylenetetrazol 33 . Changes in

neurotrophin expression are not unique to epilepsy buthave been reported after brain injury induced by ischemiaw x w x42,72 and hypoglycemia 42 which also results in gluta-mate-induced increases in intracellular calcium which cancause cell death. It has recently been reported that onemechanism whereby NGF, BDNF and NT-3 can protectneurons against metabolicrexcitotoxic insults is by stabi-

w xlizing intracellular calcium 10,11 . It has been suggestedthat the stabilization of intracellular calcium may be medi-ated in part by the calcium-binding protein calbindin-D28kw x11,53 which is induced in hippocampus by BDNF and

w xNT-3 19 .Calbindin-D , a major calcium-binding protein in28k

Žbrain it constitutes 0.1–1.5% of the total soluble proteinw x.in brain 14,15,69 , has a widespread but restricted distri-

w xbution throughout the central nervous system 6 . In hip-pocampus, calbindin is localized in the dentate granulecells which are relatively resistant to seizure-induced in-

w x w xsults 65 as well as ischemic injury 31 and in CA1pyramidal neurons which are also relatively resistant to

w xseizure damage 65 . Studies of hippocampal cells in cul-ture have indicated that calbindin-positive neurons areprotected against damage induced by glutamate and cal-cium ionophore and are better able to reduce free intra-

w xcellular calcium than calbindin-negative neurons 53 .These findings suggest that calbindin, by buffering intra-cellular calcium, can prevent calcium-mediated neuronaldeath that could result from excitotoxic insult. More re-cently, direct evidence of the calcium buffering capacity of

w xcalbindin was demonstrated by Chard et al. 8 who re-ported that introduction of exogenous calbindin into dorsalroot ganglia neurons resulted in reduction in the depolar-ization-induced rate of rise of intracellular calcium, indi-cating directly that calbindin can effectively regulate cal-cium-dependent aspects of neuronal function. Thus, thefunction of neurotrophic factors in regulating calbindinexpression and thereby possibly preventing excitotoxicneuronal damage in neuropathies, such as epilepsy, hasimportant therapeutic implications. In order to obtain anincreased understanding of the possible interrelationshipbetween calbindin, neurotrophins and seizure-induced exci-totoxic insult, region-specific and time-dependent changesin hippocampal calbindin mRNA and protein as well astime-dependent changes in mRNA for two neurotrophinsin hippocampus known to induce calbindin, BDNF andNT-3, were examined under conditions of acute status

Ž .epilepticus SE induced by KA. To determine if theacutely observed changes were maintained in a chronicepileptic state, calbindin-D gene expression was also28k

examined in the hippocampus of rats with recurrent spon-

taneous seizures induced with an episode of electricalw xstimulation-induced SE 44 .

2. Materials and methods

2.1. Materials

w32 x Ž .P dCTP 3000 Cirmmol, 10 mCirml was pur-Ž .chased from New England Nuclear Products Boston, MA .

Ž .Oligo dT cellulose and all restriction enzymes wereŽ .purchased from Boehringer Mannheim Indianapolis, IN .

Biotrans nylon membranes were obtained from ICN Bio-Ž .chemicals Costa Mesa, CA and formamide was from

Ž .Sigma St. Louis, MO . Guanidinium isothiocyanate andŽ .phenol ultra pure, molecular biology grade were from

Ž .Fischer Scientific Springfield, NJ . The Rad prime DNA-Ž .labeling system and agarose electrophoresis grade were

Žpurchased from Gibco-BRL Life Technologies Gaithers-.burg, MD .

2.2. Preparation of animals

ŽIn all studies, adult male Sprague–Dawley rats 250–.300 g were used and maintained with food and water ad

libitum in a 12-h lightr12-h dark cycle. For studies involv-ing systemic KA administration, 90 rats were used. Rats

Ž . Žreceived either KA Sigma 12 mgrkg body weight pre-.pared in 10 mM phosphate-buffered saline; PBS pH 7.4 or

saline by i.p. injection. Seizure activity was observedwithin 30–40 min following kainate treatment. Rats notexhibiting seizure activity after kainate injection were notused in the study.

For studies using KA microinfusion in brain, 50 ratsŽ .were anesthetized with Metofane Pitman-Moore, NJ and

Ž .KA 0.07 mg in 1 ml volume was injected stereotaxicallyinto Ammon’s horn of the dorsal hippocampus with aHamilton syringe over 1 min followed by a 1 min wait for

Ž Ž .the KA to diffuse coordinates: anteroposterior AP , q4.1Ž . Ž . Žmm; mediolateral ML , 2.1; dorsoventral DV , 3.0 height

. w x Ž .of l suture set equal to bregma 25 . Control zero pointreceived 1.0 ml saline without KA.

For studies in rats with a chronic epileptic state, ratswere stereotaxically implanted with bipolar electrodes in

Žthe left ventral hippocampus AP y3.6, ML 4.9, DVw x.y5.0 to dura 59 . Control rats were implanted with

electrodes but not stimulated. One week after surgery, ratsreceived a period of continuous hippocampal stimulationŽ . ŽCHS 10-s, 50-Hz trains of 1-ms biphasic pulses set at

.400 mA given once every 12 s for 90 min . CHS estab-Ž .lished a condition of self-sustaining limbic SE SSLSE

w x44 . Rats were killed by decapitation 30 days after CHS-induced SSLSE. Chronic recurrent spontaneous hippocam-pal seizures begin in rats within 30 days post-CHS-induced

w xSSLSE 44 .

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194 185

For all studies, brains were rapidly removed, brain areaswere quickly dissected and immediately frozen on dry iceand stored at y808C.

2.3. RNA isolation and Northern blot hybridization analy-sis

ŽTotal RNA was isolated from pooled samples usually,.from 3 animals by the guanidinium thiocyanaterphenol

w xchloroform procedure of Chomczynski and Sacchi 13 .Ž .qPoly A RNA was obtained after two cycles of oligo

Ž . Ž .qdT cellulose chromatography. Poly A RNA was frac-tionated under denaturing conditions on a 1.2% formal-dehyde agarose gel and transferred to a nylon membraneŽ .Biotrans, ICN using standard procedures as previously

w xdescribed 68,73 . Hybridization probes were labeled ac-cording to the random priming method of Feinberg and

w xVogelstein 27 using the Rad prime DNA labeling systemŽ .Gibco-BRL Life Technologies . The filters were hy-bridized at 428C overnight, washed and autoradiographedas previously described. For reprobing, hybridized probeswere removed by washing in 50% formamide–10 mM

Ž .phosphate buffer pH 6.5 for 1 h at 658C.b-Actin cDNA andror 18S rRNA cDNA were used as

control probes. Autoradiograms of varying exposures ofNorthern blots were analyzed using dual-wave length fly-

Žing-spot scanner Shimadzu Scientific Instrument, Prince-.ton, NJ . The relative optical density of each Northern

probed with calbindin, neurotrophin or early-response genecDNA was divided by the relative optical density obtainedafter probing with b-actin or 18S rRNA cDNA to normal-ize for sample variation.

2.4. Preparation of DNA probes

A 1.2-kb mouse calbindin-D cDNA insert from the28kw xEcoRI site of pIBI76 76 , a 1.1-kb rat BDNF cDNA from

w xthe EcoRI site of pSK-rBC1 50,51 , a 0.8-kb rat NT-3w x ŽcDNA from the XhoI site of pSK-rNT3 50,51 BDNF

and NT-3 cDNA probes were kindly supplied by GeorgeD. Yancopoulos, Regeneron Pharmaceuticals, Tarrytown,

.NY , a 2.1-kb rat c-fos cDNA from the EcoRI site ofw x Ž .pSp65 22 , a 0.3-kb zif268 NGFI-A insert from the

w x ŽBamHI site of pB700-3.6 71 obtained from Vikas.Sukhatme, Beth Israel Hospital, Boston, MA and a 2.5-kb

Ž .rat nur77 NGFI-B insert from the EcoRI site of pJDM3w x54 were obtained by the restriction enzyme digestion ofthe respective plasmid preparations. A 2.1-kb chicken b-actin cDNA was obtained from the HindIII site of pBR322w x17 . The 18S rRNA cDNA was from Ramareddy GuntakaŽ .University of Missouri at Columbia .

2.5. In situ hybridization

For the in situ hybridization experiments, rats werekilled 6 h after KA injection. The brains were rapidly

removed and quickly frozen on powered dry ice, cryostat-Ž .cut into sections 10 mm which were thaw-mounted onto

gelatin-coated slides and stored at y708C. Radiolabeled

Fig. 1. Northern blot analysis of calbindin-D mRNA in rat hippocam-28kŽ .q Ž .pus after KA treatment. A: poly A RNA 8 mgrlane , isolated from

hippocampus from 3–4 adult male rats for each time point after systemicŽ .KA injection 12 mgrkg body weight was used for Northern analysis

Ž .upper panel . Results from the Northern blots using hippocampi fromŽthree separate groups of rats were used for densitometric analysis mean

."S.E.M.; lower panel . After normalization based on results obtainedafter re-hybridization with 18S rRNA or b-actin cDNAs, densities were

Ž .q Žexpressed as percentage maximal response. B: poly A RNA 8. Ž .mgrlane was isolated from the ipsilateral infused side or contralateral

hippocampus at various times after intrahippocampal infusion. Hip-pocampi from 5 rats were pooled for preparation of RNA at each timepoint. Densitometric scanning of autoradiograms and normalization withb-actin indicated that calbindin mRNA was elevated 1.6- and 1.9-fold onthe contralateral and ipsilateral sides, respectively, at 1 h after KAinjection. 3 h after KA injection, calbindin mRNA was induced 1.9- and

Ž2.6-fold on the contralateral and ispsilateral sides, respectively results arethe mean of two independent measurements, using separate groups of

.rats .

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194186

antisense and sense cRNAs were transcribed from cDNAsw xas previously described 12 using appropriate poly-

w35 x Žmerases, 2.5 mM S UTP 1000 Cirmmol, New England.Nuclear , 10 mM cold UTP, with ATP, CTP and GTP in

Ž .excess. The template cDNA 320 bp was the same as usedfor Northern analysis. Both sense and antisense RNAprobes were synthesized. In situ hybridization was carried

w xout as previously described 12 . Briefly, tissue sectionswere brought to room temperature and dried rapidly undera stream of cold air, post-fixed in 3% paraformaldehyde,acetylated and dehydrated. Sections were incubated with 3

Ž .ng of probe f400 000 dpmrng in humid chambers at508C for 3.5 h. After hybridization, non-specific bindingwas reduced by washing in 50% formamide in 2=SSCŽ .0.3 M NaClr 0.03 M sodium citrate at 528C and treat-

Ž .ment with RNase A 100 mgrml for 30 min at roomtemperature. After overnight incubation in 2=SSCr0.05%Triton X-100, slides were dehydrated in graded ethanol,defatted in xylene and coated with Kodak NTB emulsion3

diluted 1 : 1 with 300 mM ammonium acetate. After expo-sure at 48C for 29 days, the sections were developed inKodak D-19 developer at full strength, lightly counter-stained with hematoxylin eosin and mounted with EukittŽ .Calibrated Instruments . The sections were examined un-der bright-field and dark-field microscopy on a Leitzaristoplan microscope.

2.6. Calbindin-D immunocytochemistry28k

Rats were killed at 6, 12 and 24 h after systemic KAinjection. Three ratsrtime point were studied. At the dif-ferent times after KA, rats were anesthetized with an i.p.

Ž .injection of 20% urethane Sigma and then perfusedintracardially with 0.1 M sodium phosphate-buffered salineŽ . ŽPBS with 0.5% heparin pH 7.4; LyphoMed, Melrose

. Ž .Park, IL followed by 4% paraformaldehyde Sigma inŽ .0.1 M sodium PBS pH 7.4 . The brains were removed,

Ž .fixed in paraformaldehyde for 1 h, rinsed in PBS pH 7.4and then permeated with a cryoprotected sucrose solutionŽ12.5 mM sodium phosphate monobasic, 38.5 mM sodium

.phosphate dibasic and 877 mM sucrose . The brains werestored at y208C. After transfer to 10 mM PBS, 50-mmcoronal sections were prepared using a vibratome. SectionsŽ .free-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-im-mune horse serum containing a 1 : 1500 dilution of a rabbit

w xpolyclonal rat calbindin antiserum 16 . Sections wereimmunostained using the biotin-avidin-peroxidase method

w xdescribed previously 25 . Control brains were similarlyprocessed but without the primary antibody. Sections werephotographed using an inverted Nikon Diaphot microscopewith phase-contrast and bright-field optics.

2.7. Statistical analysis

Results are expressed as the mean"S.E. and signifi-cance was determined by Dunnet’s multiple comparisont-statistic or by analysis of variance.

3. Results

Ž .qNorthern blot analysis of hippocampal poly A RNAŽindicated that after systemic injection of KA 12 mgrkg

.bw , steady-state levels of calbindin-D mRNA were28k

rapidly induced. The first significant increase was ob-Ž .served at 3 h after injection P-0.05; 3.5-fold induction .

Levels of calbindin mRNA returned to control levels at 12Ž .h after KA treatment Fig. 1A . Unlike the changes ob-

served in hippocampus, no changes in calbindin-D28k

mRNA were observed in cerebellum at any time after KAŽ .treatment 0.5–24 h; not shown . Calbindin-D mRNA28k

levels were also examined in a different experimentalmodel in which rats received a unilateral injection of KAdirectly into Ammon’s horn of the dorsal hippocampus.This model allows the discrimination between a neurotoxiceffect of KA which is observed in the ipsilateral side and aneuroexcitatory effect on the contralateral side which ismost likely caused by hyperactivity. Calbindin-D mRNA28k

was clearly increased at 1 and 3 h after KA infusion inŽ .both the ipsilateral and contralateral sides Fig. 1B . At 6

and 16 h after KA infusion, levels of calbindin-D28k

mRNA remained elevated in the ipsilateral but not in thecontralateral side. Thus, calbindin-D mRNA can be28k

induced not only by a direct action of KA on its receptorŽ .ipsilateral side but also as a result of hyperactivity inafferent excitatory pathways. The remainder of the studiesinvolving KA were done using i.p. injection which results

Žin selectivity of vulunerable neurons those with high

Ž .Fig. 2. In situ hybridization histochemistry for calbindin-D . A,B: dark-field photomicrographs of sections of cerebellum of control A and KA-treated28kŽ . 35B rats were processed for in situ hybridization histochemistry with a S-radiolabeled antisense RNA probe for calbindin-D . Arrows point to dense28k

accumulations of silver grains over Purkinje cells. Scale bar shown in B: 70 mm for A and B. C–F: in situ hybridization histochemistry for calbindin-D28kŽ . Ž . 35in rat hippocampus. Sections were hybridzed with antisense C,E or sense F S-radiolabeled RNA probes for calbindin mRNA. C,E: dark-field

Ž . Ž .photomicrographs of sections of the same region of the hippocampus in KA-injected C and control E rats. Arrow in C points to labeling in the dentateŽ .gyrus. D: low-power bright-field photomicrograph of section illustrated in C counterstaining: hematoxyllin eosin . The arrow points to dentate gyrus.

Ž . Ž . Ž .Scale bar shown in Fs100 mm in C,E ; s500 mm in D ; s80 mm in F . Similar results were observed in the brains of two other control andKA-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

w xreported 68 , that with longer autoradiographic exposure time calbindin-D can be observed using in situ hybridization, in the hippocampus of control28k

rats.

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194 187

.concentrations of KA type glutamate receptors . Region-specific changes in calbindin-D mRNA and protein28k

after i.p. KA injection was examined by in situ hybridiza-

tion and immunocytochemistry, respectively. In Fig. 2A,B,a dense accumulation of silver grains was observed over

Ž .Purkinje cells in the cerebellum of both control A and

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194188

Ž .KA-injected B rats in sections processed for in situhybridization with 35S-radiolabeled antisense probe forcalbindin. Sections hybridized under the same conditionswith the sense probe did not show specific labeling in anybrain area examined. Despite the presence of a strongcerebellar autoradiographic signal in the same sections, nospecific labeling was detected in the hippocampus of con-

Ž .trol rats in our experimental conditions Fig. 2E . Incontrast, labeling was clearly present in the dentate gyrus 6

Ž .h after KA administration Fig. 2C,D . No increases inlabeling were observed in other hippocampal regions or in

Ž . Ž .caudate-putamen striatum of the same rats not shown .KA treatment was also found to induce a marked increasein calbindin immunoreactivity at 6 and 12 h which, similarto results of in situ hybridization, was also most obvious in

Ž .the dentate gyrus Fig. 3 . By 24 h, calbindin immuno-Ž .reactivity was reduced towards control levels Fig. 3 .

Since BDNF and NT-3 have been reported to enhancew xthe expression of calbindin in the hippocampus 19 , the

influence of KA on the expression of mRNAs for theseneurotrophins was examined by Northern analysis. A sig-nificant increase in BDNF mRNA was observed 1 h after

Ž .KA injection P-0.05; 3.2-fold induction and peak ex-

Ž . Ž .pression 9.4-fold occurred 3 h after KA Fig. 4A . Incontrast, KA treatment resulted in a marked 3.4–3.8-folddecrease in the levels of NT-3 mRNA in the hippocampusŽ .Fig. 4B .

Since it has been suggested that immediate-early genesmay play a role in regulating genes which respond later totrans-synaptic activation, changes in the expression ofc-fos, zif268 and nur77 mRNAs were examined after KAtreatment. The induction of both BDNF and calbindin-D28k

mRNAs was preceded by induction of c-fos mRNA. Lev-Žels of c-fos mRNA were undetectable in control rats zero

.time even after prolonged radiographic exposure but wereŽ .significantly induced 30 min after KA Fig. 5 . Although

1.5- and 1.9-fold inductions in zif268 and nur77 mRNAs,respectively, were observed at 30 min, Northern analysis

Ž .indicated that the first significant induction P-0.05 inthese early-response genes was at 1 h with peak expressionŽ6.3- and 7.4-fold for zif268 and nur77 mRNAs, respec-

. Ž .tively occurring at 3 h after KA Fig. 5 . A direct compar-ison of the time relationship of the response to KA of theimmediate-early genes, BDNF, NT-3 and calbindin mR-NAs is shown in Fig. 6.

Calbindin-D gene expression was also examined in28k

Ž . ŽFig. 3. KA induces a transient increase in hippocampal calbindin immunoreactivity. Rats were injected with saline control or KA 12 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.

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194 189

Ž .qFig. 4. Northern analysis of BDNF and NT-3 mRNAs in rat hippocampus after KA treatment. Left panel: representative Northern blot analysis. Poly ARNA was isolated from the hippocampus at various times after systemic administration of KA and fractionated on a 1.2% formaldehyde-agarose gel. The

Ž .mRNA was transferred to a nylon membrane and hybridized with calbindin Fig. 1A , BDNF and NT-3 cDNAs. Basal levels of BDNF mRNA wereŽ .observed at 0 and 0.5 h after longer radiographic exposure. For 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 recur-rent seizure established with an episode of electrical stimu-lation-induced SE. When these animals were examined 30days post-SE, no changes in hippocampal calbindin-D28k

Ž .mRNA were observed Fig. 7 . Calbindin-D mRNA28kŽ . Žwas also not altered in cerebellum Fig. 7 , amygdala not

. Ž .shown or frontoparietal cortex not shown under theseconditions.

4. Discussion

In this study, we report that calbindin mRNA andprotein rapidly increase after KA treatment and the mostpronounced increases are observed in the dentate gyrus.Although a previous study indicated an induction of cal-

Žbindin mRNA as well as 72-kDa heat-shock protein and.78-kDa glucose-regulated protein mRNAs in rat hip-

w xpocampus after KA administration 45 , region-specificchanges and changes in protein had not as yet beenexamined. In our study, we found that the induction ofcalbindin mRNA is preceded at 1 h and coincident at 3 and6 h after KA with the induction of BDNF mRNA. Previ-ously reported region-specific changes in BDNF mRNAafter KA treatment indicate, similar to changes in calbindinmRNA, that the greatest, earliest increase occurs in the

Ždentate granule cell layer which is most resistant to.seizure-induced injury , followed by an increase in CA1

w x24 . The extent of elevation in the CA1 region wasapproximately half that observed in the dentate granulecell layer. Increases in BDNF mRNA in the CA3 region,similar to increases in the CA1 region, have also been

w xreported 24 . Due to the correlation of the increases incalbindin mRNA and BDNF mRNA in the dentate gyrusas well as the observation that the induction of BDNFmRNA precedes the induction of calbindin mRNA, it ispossible that the induction of calbindin after KA may bemediated, in part, by BDNF. Besides the hippocampus,induction of BDNF mRNA by KA was also previously

Ž w x.noted in the cortex layer II and layers IV and VI 24 .Although KA did not induce calbindin mRNA in cerebel-lum, it will be of interest in future studies to determine

w xwhether calbindin, which is present in layers II and IV 6 ,and its mRNA can be induced in these areas of the cortexsimilar to BDNF mRNA.

Until recently, unlike studies related to calbindin inw xperipheral tissues 14,15,30,32 , very little has been known

about the signalling mechanisms involved in regulatingneuronal calbindin. However, correlative evidence betweendecreases in neuronal calbindin and neurodegeneration in

w x w xstudies of ischemic injury 5,62 , seizure activity 1,68 andŽchronic neurodegenerative disorders Alzheimer, Hunting-

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194190

Ž .Fig. 5. Changes in immediate-early genes c-fos, zifr268 and nur 77 inrat hippocampus after KA treatment assessed by Northern blot hybridiza-tion. The same Northern blots probed for calbindin-D andror BDNF28k

and NT-3 were also hybrized with c-fos, zif268 and nur77 cDNAs. Afternormalization based on results obtained upon re-hybridization with 18S

Ž .rRNA shown in bottom panel inset , densities were expressed as percentŽ .maximal response mean"S.E.M. . Data was obtained from three sepa-

rate Northern analyses except for the 24-h time point which represents themean of duplicate determinations.

. w xton and Parkinson diseases 35 had been reported. Usingrat hippocampal cultures, recent evidence has indicated

w x w xthat NT-3 19,36 , BDNF 19,36 , fibroblast growth factorŽ . w x Ž . w xFGF 19 and tumor necrosis factors TNFs 9 can allinduce calbindin. In addition, in vivo corticosterone admin-istration has been reported to increase calbindin expression

w xin rat hippocampus 34 , specifically in the CA1 regionw x39 . Retinoic acid has also been reported to induce cal-bindin protein and mRNA in medulloblastoma cells, which

w xexpress a neuronal phenotype 74 , and the content ofcalbindin in cultured Purkinje cells can be increased by

w xinsulin-like growth factor I 57 . Thus, neuronal calbindincan be regulated by steroids as well as by factors thataffect signal transduction pathways. Although a 20-foldinduction of calbindin in response to NT-3 compared toabout a 2-fold induction by BDNF has previously been

w xreported in rat hippocampal cultures 19 , in this study

NT-3 mRNA was found to be decreased at times whencalbindin mRNA was maximally induced after KA. Inprevious studies, NT-3 mRNA has been reported to beunchanged or markedly decreased after seizure induced by

w xKA, hilus lesion or kindling 2,4,28 . Region-specificchanges indicated that NT-3 mRNA was most dramatically

w xreduced in the dentate granule cell layer 4,28 . Thus, it islikely that, within the same region of the hippocampus,calbindin and BDNF mRNAs are increased while NT-3mRNA is decreased. Since NT-3 mRNA is down-regulatedand it has been reported that changes in mRNA content for

w xneurotrophins are reflected in protein production 28 , NT-3, which may be involved in inducing calbindin during

w xhippocampal neurogenesis 19 , may not play a role in theup-regulation of calbindin observed after KA-inducedseizures. However, it is possible that trkC receptor mRNAup-regulation, which has been reported after kindling in-duced seizures coincident with NT-3 mRNA down-regu-

w xlation 4 , may also occur after KA-induced seizures andmay compensate, in part, for the decrease in NT-3 mRNA.

w xSince the expression of mRNAs for FGF 28 and TNFw x55 have also been reported to be induced in hippocampusearly times after seizure, it is possible that these factors,which induce calbindin in hippocampal cultures, may alsocontribute to the up-regulation of calbindin observed after

w x w x w xKA. FGF 10 and TNFs 9 as well as BDNF 11 have allbeen reported to protect hippocampal neurons against exci-totoxic insults by stabilizing calcium homeostasis, furthersuggesting an interrelationship between these factors andcalbindin.

Although the induction of calbindin mRNA after KAtreatment 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 transcriptioncomplex, which combines with members of the c-junfamily, binds to AP1 consensus sequences and results in

w xactivation or repression of genes 21 . In the nervoussystem, few physiological targets for c-fos and c-jun havebeen definitively identified. However, one candidate has

w xbeen reported to be the proenkephalin gene 70 . Earlyw xincreases in c-fos protein after KA treatment 61 and in

w xc-fos mRNA after electrical stimulation 67 are observedpredominately in dentate gyrus, similar to changes incalbindin protein and mRNA and BDNF mRNA, withlower levels of c-fos induced in CA3 and CA1 pyramidalcell layers. Region-specific changes in the mRNA forzif268, a zinc finger containing protein, which binds to aG-rich DNA consensus sequence, and the mRNA fornur77, an orphan steroid receptor, have been reported afterelectrical stimulation or administration of convulsant drugto be similar to those reported for c-fos mRNAw x18,64,67,75 . It has been suggested the induction of theseputative transcription factor mRNAs in brain is part of aprogrammed response of neurons which can ultimatelyresult in long-term plastic changes induced by seizure

w xactivity 18,29,64,67 . The molecular mechanism of tran-

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194 191

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 0Ž .control –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

Žadjusted vertically so that the zero point begins at the same level. Analysis of all the data, as shown above, indicates assuming 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 hasbeen reported to involve calcium influx into neuronsw x Ž .18,66 . Calcium response elements CREs , having the

Fig. 7. Relative levels of calbindin-D mRNA in rat hippocampus and28k

cerebellum examined 30 days after continuous hippocampal stimulation-induced SE. Left panel: representative Northern analysis of calbindinmRNA in hippocampus and cerebellum of C, control rats or rats sacri-

Ž .q Ž .ficed 30 days post-SE. For each lane poly A RNA 8 mg wasprepared from tissue pooled from 3–4 rats for hippocampus and from

Ž2–3 rats for cerebellum. Right panel: graphic representation mean"

.S.E.M. of densitometric quantitation of Northern blot analysis. Afternormalization based on results obtained upon hybridization with b-actinor 18S rRNA cDNA, the densities were expressed as relative signalintensity. Results were obtained from three Northern analyses usingseparate groups of rats.

consensous TGACGTCA, have been reported in the pro-w x w x w xmoter regions of c-fos 66 , zif268 7 and nur77 77 .

Increases in intracellular calcium have also been reportedw xto induce later responsive genes, such as BDNF 78 and

w xNGF 48 . Since the expression of calbindin in peripheralw xtissues is modulated by calcium 15,32 and since calbindin

in hippocampus is induced both by perforant path stimula-w xtion 46 and KA administration, which result in elevations

in intracellular calcium, it is possible that the induction ofthe early-response genes and the later responsive BDNFand calbindin mRNAs by KA could be initiated by acommon calcium-dependent mechanism. Thus, the rela-tionship suggested by the present findings is that in re-sponse to KA there is activation of KA-preferring gluta-mate receptors and a subsequent influx of calcium whichtriggers the increase in the immediate-early genes, in BDNFmRNA and in the number of neurons expressing calbindinwhich results in increased buffering of intracellular cal-cium and protection against brain injury. Although calciummay indeed be involved in the regulation of these genes inthe same neurons in the hippocampus, further studies areneeded to determine whether there is a direct involvementof IEGs in the regulation of BDNF and whether thesubsequent increase in BDNF affects the transcription ofthe calbindin gene. Studies using the multiple promoters of

w xthe BDNF gene 28,43 and promoter region of the cal-w xbindin gene 30 , should enable us to obtain new insight

concerning the multiple factors involved in the transcrip-tion response to neuronal activation.

( )S. Lee et al.rMolecular Brain Research 47 1997 183–194192

An association between neurotrophic factors and cal-bindin is suggested not only in these studies in which earlytimes after KA were examined but also when the effect ofKA is examined after several days. Histological evaluationafter systemic administration of KA has indicated both

Žacute neuropathological changes within 3–5 h after i.p..injection as well as subsequent neuropathological changes

Ž .24 h–16 days in brain areas unaffected at early timesafter KA. Using in vitro bioassays for growth factor activ-

w xity, Lowenstein et al. 47 reported prolonged increases inneurotrophic activity in hippocampal extracts observed 4and 7 days after kainate which were associated with mossyfiber reorganization. We previously reported that 6 daysfollowing systemic KA injection there is a striking appear-

w xance of calbindin in astrocytes 52 . Using cultured neocor-tical and hippocampal astrocytes, we found that TNF butnot NGF or bFGF can induce calbindin in astrocytes.Thus, at longer times after KA administration, TNF andperhaps other factors not yet examined induce calbindin,promoting the survival of supporting astrocytes, a richsource of neurotrophic factors which may play a role in alater effect of KA related to synaptic reorganization.

A question raised by the current investigation is whetherthe induction of calbindin expression at early times afterKA is a response to seizure or to neuronal injury coinci-dent with seizure. Although KA-induced seizures result inan induction in hippocampal calbindin expression, recur-rent spontaneous seizures established with an episode ofelectrical stimulation-induced SE failed to result in a changein calbindin mRNA. As a result of SSLSE, the hippocam-

w xpus becomes chronically epileptic 44 and similar to find-ings observed after injection of KA into the hippocampusw x w x20 , there is a loss of inhibition in the CA1 area 44 .Also, hippocampal slices from rats sacrificed 30 days aftercontinuous hippocampal stimulation-induced SSLSE werefound to be hyperexcitable compared to those from control

w xanimals 44 . However, unlike the effect of KA, 30 daysafter SSLSE recurrent spontaneous seizures are less in-tense, non-convulsive and not specifically excitotoxic.When calbindin mRNA was examined by in situ hybridiza-tion as well as by Northern analysis after traditional com-

Žmissure kindling degenerative morphological changes arew x.not observed in this model of epilepsy; 68 , no changes in

calbindin mRNA in hippocampus and other brain areaswere observed 30 min, 1, 6 or 24 h after the last kindled

w xseizure 68 . Thus, we suggest that the induction of cal-bindin mRNA may not be related to enhanced neuronalactivity but rather to maintaining neuronal viability inresponse to the toxic effects of KA. In our studies, weobserved an increase in calbindin mRNA not only aftersystemic injection but also after intrahippocampal infusion,both in the ipsilateral and contralateral sides of the hip-pocampus. The induction of calbindin mRNA in the con-tralateral side, which may result from hyperactivity in theafferent excitatory pathways, may also be a response toneuronal vulnerability. Excitotoxic damage involving cal-

cium-dependent mechanisms and selectively vulnerable celltypes has been reported at sites remote from the site of

w xintracerebral injection of KA 49,65 . In addition to cal-bindin, whether the induced changes in the expression ofthe neurotrophins are a response to seizure has also been amatter of debate. Although increases in BDNF and NGFmRNAs after hilus lesion or after KA administration in

w xadult rats were reported to be a response to seizure 78 ,w xDugich-Djordjevic et al. 23 found that on post-natal day

8, seizures were induced by KA but the seizures were notassociated with elevations in BDNF mRNA. The authorssuggested, similar to our suggestion for the induction ofcalbindin, that the rapid response of BDNF mRNA in theadult rat may be correlated with a neuropathological out-come of seizure and may not be primarily regulated byneuronal activity. The induction of calbindin and BDNFmay represent two components of a chain of genomicevents which result in calcium stabilization and neuropro-tection after excitotoxic insults. Further studies are neededto identify other components of this chain of genomicevents and to determine how the neuroprotective actions ofthe neurotrophins and calbindin may be used to developnew therapeutic strategies for protecting against brain in-jury 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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