Brain Research 956 (2002) 126–135www.elsevier.com/ locate/brainres

Research report

B rain-derived neurotrophic factor (BDNF) mRNA in rats withneonatal ibotenic acid lesions of the ventral hippocampus

,1*Paula C. Ashe , Jennifer Chlan-Fourney, Augusto V. Juorio, Xin-Min LiNeuropsychiatry Research Unit, Department of Psychiatry, University of Saskatchewan, 103 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N

5E4

Accepted 29 April 2002

Abstract

Increasing evidence suggests that schizophrenia is a neurodevelopmental disorder with a progressive course characterized by worseningof symptoms and morphological alterations within the brain. This suggests that a neurodegenerative component may exist inschizophrenia. The role of brain-derived neurotrophic factor (BDNF) in neurodevelopment, cell viability and synaptic plasticity led to theinvestigation of BDNF as a potential candidate molecule in the pathophysiology of schizophrenia. BDNF mRNA was examined by in situhybridization in the prefrontal cortex and hippocampus of animals with neonatal ibotenic acid lesions of the ventral hippocampus, aputative neurodevelopmental animal model of schizophrenia. Results demonstrate that animals with neonatal ibotenic acid lesions of theventral hippocampus have reduced basal levels of BDNF mRNA. It is possible that alterations in this trophic factor render animals moresusceptible to neurodegenerative insults. 2002 Published by Elsevier Science B.V.

Theme: Disorders of the nervous system

Topic: Neuropsychiatric disorders

Keywords: Schizophrenia; Prefrontal cortex; In situ hybridization; Stress

1 . Introduction also been demonstrated to modulate neurotransmitter syn-thesis, metabolism and release, postsynaptic ion channel

Brain-derived neurotrophic factor (BDNF), a member of fluxes, neuronal activity and long term potentiationthe neurotrophin family, was first isolated from pig brain [3,7,16,37]. The association of BDNF with neurodevelop-in 1982 [4] and was subsequently cloned and characterized ment, cell viability and synaptic strength make it anin the rat and the human [20,26]. In the hippocampus, attractive candidate for involvement in schizophrenia. Thiscortex and in vitro, BDNF has been demonstrated to claim is based on the evidence supporting abnormalregulate the survival, differentiation, morphology and neurodevelopment as a predisposing factor in the develop-synaptic remodeling of neurons [2,13,15,18,27,37]. It has ment of schizophrenia [41] in combination with growing

evidence that a neurodegenerative component is alsopresent in schizophrenia [9]. Support for a role for BDNFin schizophrenia also stems from the demonstration that itAbbreviations: BDNF, Brain-derived neurotrophic factor; PND, Post-is decreased by factors correlated with first episode onsetnatal day; ACSF, Artificial cerebrospinal fluid

*Corresponding author. Institute for Biological Sciences, National such as stress [35,36] and estrogen withdrawal [34]. InResearch Council, Montreal Road Campus, Ottawa, ON, Canada. Tel.: addition, stress induced decreases in BDNF are blocked by11-613-990-0849; fax:11-613-941-4475. 5-HT receptor antagonists, a receptor binding property of2E-mail address: paula.ashe@nrc.ca(P.C. Ashe).1 many neuroleptics [39]. Electroconvulsive treatmentPresent address: Section on Pharmacology, Laboratory of Clinical

(ECT), effective in treatment-resistant schizophrenia inScience, National Institute of Mental Health, Bldg 10, Rm 2D-57, 10Center Drive, Bethesda, MD 20892, USA. combination with neuroleptics [32], also upregulates the

0006-8993/02/$ – see front matter 2002 Published by Elsevier Science B.V.PI I : S0006-8993( 02 )03176-1

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expression of BDNF [23,30]. These findings lead to the to lesion or sham status and anaesthetized by hypothermiainference that BDNF may be altered in schizophrenia. (15 min on wet ice). Xylocaine was applied to the headDirect investigations of BDNF in schizophrenia also and an incision was made to expose the skull. The pupssuggest it may be altered. Findings indicate that hippocam- were placed in a modified neonatal stereotaxic apparatuspal BDNF mRNA is reduced [5] as is serum BDNF in [8]. Bilateral injections of 1.5mg (6)-ibotenic acid (RBI,schizophrenic patients [38]. An allele variant of the BDNF Natick, MA, USA) in 0.3ml of artificial cerebrospinal fluidgene has also been identified in a population of schizophre- (ACSF; 124 mM NaCl, 5 mM KCl, 2 mM CaCl?2H O, 12 2

nic patients [40]. These factors suggest that disruptions of mM MgCl , 1 mM NaH PO , 24 mM NaHCO and 112 2 4 3

BDNF may play a role in the etiology of this disorder by mMD-glucose) (lesion) or ACSF alone (sham) werecompromising neuroplasticity or altering neurotrans- infused into the ventral hippocampus (AP23.0, ML 63.5,mission. VD25.0 relative to bregma) at a flow rate of 0.15ml /min.

The animal model established by Lipska et al. [25] was Rats were weaned at PND 25 and housed in groups of fourused to test the hypothesis that neurodevelopmental abnor- with two lesion and two sham per cage.malities modeling schizophrenia create a functionallycompromised system with alterations in factors necessary2 .3. Stress paradigmfor maintaining neuron viability and neuronal communica-tion. The overall result would be a system more susceptible At PND 75 lesion and sham animals, in groups of four,to neuronal atrophy and/or death caused by environmental were exposed to either a stress or a non-stress condition.factors such as stress. The stress condition was a forced swim test consisting of

15 min in 30 cm of 258C water. The control environment

2 . Materials and methods

2 .1. Animals

Timed pregnant Sprague–Dawley rats were obtainedfrom Charles River Canada (Montreal, Quebec, Canada) at14 days gestation. All animals were housed in clear plasticcages at a room temperature of 19–218C on a 12-hlight /dark cycle. Animals were allowed access to food andwater ad libitum. All procedures involving animals weredone in accordance with the guidelines of the CanadianCouncil on Animal Care and were approved by theUniversity of Saskatchewan Committee of Animal Careand Supply.

2 .2. Surgery

Post-natal day (PND) seven male rats were randomized

Fig. 2. BDNF mRNA in the prefrontal cortex. Two-way ANOVArevealed a significant effect of treatment (control environment vs. forcedswim) (F 588.43, P,0.001) and a significant interaction between(1,54)

treatment and lesion status (ACSF vs. ibotenic acid) (F 513.03,(1,54)

P,0.01). * significant with respect to ACSF-Control,P,0.05; **significant with respect to ACSF-Control,P,0.01; [ significant withFig. 1. Haematoxylin and eosin stained sections through the ventralrespect to Ibotenic Acid-Control,P,0.01; 1 significant with respect tohippocampus of ACSF and ibotenic acid treated animals. Scale bar, 500ACSF-Swim,P,0.05.mm.

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was identical to the swim environment with the exception Only animals meeting the inclusion criteria were includedthat bedding material was placed in the bottom of the cage in subsequent experiments.instead of water.

2 .6. In situ hybridization2 .4. Tissue processing

Prior to hybridization, sections were rehydrated in PBS,Twenty-four hours post-stress, animals were transcar- acetylated in 0.1 M triethanolamine containing 0.25%

dially perfused with PBS followed by 4% paraformal- acetic anhydride, rinsed in 23 SSC, dehydrated, delipi-35dehyde in PBS. Brains were removed, cut into two blocks dated and air dried. S-aCTP (12.5 mCi /ml; Dupont

(one containing the prefrontal cortex and the other con- NEN, Boston, MA, USA) labeled antisense RNA probetaining the hippocampus), post-fixed then cryoprotected in was transcribed in vitro from BDNF cDNA in pBluescript30% sucrose in 0.1 M PBS at 48C. Following cryoprotec- SK-, designated pSK-rB(C1), obtained from Dr. Rontion, brains were flash frozen in isopentane/dry ice at Lindsay (Regeneron Pharmaceuticals, Tarrytown, NY,250 8C and stored at270 8C until sectioned. Tenmm USA). Prehybridized sections were incubated in hybridiza-sections through the prefrontal cortex and the hippocampus tion buffer (0.6 M NaCl, 0.08 M Tris, pH 7.5, 0.004 Mwere thaw mounted onto poly-L-lysine coated slides. EDTA, 0.1% w/v sodium pyrophosphate, 10% w/v dex-Sections through the hippocampus designated for histologi- tran sulfate, 0.2% w/v SDS, 0.02% w/v heparin sodiumcal analysis were collected at 100mm intervals and thaw salt, 50% v/v ultraformamide, 100 mM DTT) containing

35 6mounted onto poly-L-lysine coated slides. S-aCTP labeled BDNF antisense RNA (0.5310 cpm/section) overnight at 548C. Sections were washed in 23

2 .5. Histology SSC then treated with 5mg/ml RNase A in RNasedigestion buffer (0.5 M NaCl, 10 mM Tris (pH 8.0) and 1

Sections through the hippocampus were stained with mM EDTA) for 30 min. Sections were washed in RNasehaematoxylin and eosin (H&E) and evaluated for animal buffer for 30 min, 23 SSC for 4 min, 23 SSC/50%inclusion or exclusion based of the following exclusion formamide at 508C for 20 min then 0.13 SSC at 508C forcriteria: damage in ACSF treated animals, damage outside 15 min. Sections were dehydrated in ascending ethanolsthe hippocampus in ibotenic acid treated animals, unilater- containing 0.3 M ammonium acetate, air dried then dippedal damage in ibotenic acid treated animals or lack of in NTB2 photographic emulsion (Eastman Kodak Co.,damage in ibotenic acid treated animals. Animals were Rochester, NY, USA) diluted 1:1 with nanopure water andalso excluded if they demonstrated convulsive behavior. left to expose at 48C. When proper exposure time was

Fig. 3. In situ hybridization of BDNF mRNA in the prefrontal cortex of lesion and sham animals exposed to a control environment or swim stress. (A)ACSF, control environment; (B) ACSF, swim stress; (C) Ibotenic acid, control environment; (D) Ibotenic acid, swim stress. Scale bar, 100mm.

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confirmed through test slides, sample slides were de- and do lesion and sham animals respond differently toveloped in D-19 Kodak developer (Eastman Kodak Co., stress.Rochester, NY, USA) and fixed with Kodak general fixer(Eastman Kodak Co., Rochester, NY, USA). Slides werelightly counterstained with cresyl violet, dehydrated, 3 . Resultscleared and mounted with DPX Neutral Mounting Medium(Aldrich, Chemical Co. Inc., Milwaukee, WI, USA). Spe- 3 .1. Histological analysiscificity of the BDNF RNA probe was confirmed by theinclusion of sense RNA probes, competition assays with Sections through the ventral hippocampus of lesion andcold-labeled BDNF antisense probes and RNase digestion sham rats, stained with haematoxylin and eosin wereof test slides prior to hybridization (data not shown). assessed for animal inclusion or exclusion. Rats infused

with ACSF that were included in the study demonstratedno observable cell loss in or around the ventral hippocam-

2 .7. Quantificationpus (see Fig. 1). Of the total number of animals infusedwith ACSF, approximately 70% were included in further

BDNF mRNA was quantified using an Axioskop micro-experiments. Animals excluded generally showed small

scope (Carl Zeiss, Inc., Thornwood, NY, USA) interfacedrims of unilateral or bilateral cell loss in the CA3 pyrami-

to a PC computer using Northern Eclipse Image Analysisdal region of the ventral hippocampus; less than 300mm

Software (Empix Imaging Inc., Mississauga, Ontario,anterior–posteriorly and ventro–dorsally. Animals infused

Canada). Percent area covered by silver grains in in-with ibotenic acid showed varying degrees of cell loss in

dividual cells in the prefrontal cortex was measured inthe ventral hippocampus. The dorsal hippocampus showed

minimum of 100 cells per animal over three sections peranimal per experiment. The prefrontal cortical area ana-lyzed corresponded to cingulate 1 and 3 as defined byPaxinos and Watson [31]. All prefrontal sections analyzedwere approximately12.2 mm relative to Bregma asdetermined by reference to Paxinos and Watson. Percentarea covered by silver grains in the dentate gyrus, CA3 andCA1 regions of the hippocampus were measured over theentire area in three sections per animal per experiment.Sections analyzed were dorsal to the area of the lesion andcorresponded to approximately24.16 mm relative toBregma as determined by reference to Paxinos and Wat-son. None of the sections analyzed demonstrated anyobvious signs of cell loss over the areas measured.Averaged percent area values were converted to percentcontrol values and averaged across two experiments fromeach set of animals processed. In total, four independentsurgery sets and subsequent analyses were performed. Allthe resulting data was pooled for the final statisticalanalysis. All statistical analyses were performed on valuesexpressed as a percentage of control.

2 .8. Statistics

Two-way analysis of variance (ANOVA) (SPSS forWindows, SPSS Inc., Chicago, IL, USA) was used for allstatistical analyses. Newman–Keuls post-hoc testing wasused to explore all significant interactions. Two-wayANOVA with status (ACSF versus ibotenic acid) andtreatment (control or swim) as independent variables andpercent control mRNA as a dependent variable was

Fig. 4. BDNF mRNA in the dentate gyrus of the hippocampus. Two-wayperformed to assess the effect of stress on BDNF mRNAANOVA revealed a significant effect of treatment (control environment

in lesion and sham animals. Specific questions addressedvs. forced swim) (F 533.27, P,0.001) and a significant interaction(1,28)were: does baseline expression differ between lesion andbetween treatment and status (ACSF vs. ibotenic acid) (F 54.30,(1,28)

sham animals, does stress affect the expression of BDNFP,0.05). * significant with respect to ACSF-Control (P,0.05).

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little to no cell loss in included animals, but excluded effect for lesion status (F 50.70,P50.41). Exploration(1,54)

animals treated with ibotenic acid often demonstrated of the interaction revealed a significant change in baselinedorsal hippocampal damage or ablation, often in conjunc- BDNF mRNA in lesion versus sham animals (P,0.05)tion with thalamic damage. This was evident in approxi- and a significantly different response to stress in lesionmately 25% of ibotenic acid treated animals and was often animals as compared to sham (P,0.01). Overall, BDNFassociated with convulsive behavior. Animals in the mRNA was reduced in ibotenic acid treated animals toibotenic acid treated group were also excluded on the basis 88% of control values (n511 and 19, respectively) andof unilateral damage only (|10%), limited extent of was increased in both lesion (139%) and sham (122%)damage not meeting the criteria (|15%), or behavioral animals in response to stress (n511 and 17, respectively)abnormalities (seizures) (|10%). Included ibotenic acid (see Figs. 2 and 3). Significantly different responses totreated animals showed significant cell loss in the CA3 and stress were seen in lesion versus sham animals such that aCA1 pyramidal layers of the ventral hippocampus. This relative 51% increase in BDNF mRNA was demonstratedregion of damage averaged a distance of approximately 1.5 in ibotenic acid treated animals, whereas only a 22%mm anterior–posterior through the ventral hippocampus relative increase was demonstrated in sham animals.(24.30 to 25.80 relative to Bregma with reference toPaxinos and Watson [31]) and was most dramatic in the

3 .3. BDNF mRNA in the dentate gyrus of theCA3 region. The ventricular region surrounding the hip-hippocampuspocampus was greatly enlarged in the majority of animals

(See Fig. 1). In total, approximately 45% of animalsTwo-way ANOVA revealed a significant main effect ofinfused with ibotenic acid were included in subsequent in

treatment (swim stress versus control environment)situ hybridization experiments.(F 533.27, P,0.001) and a significant interaction(1,28)

between lesion status (ACSF versus ibotenic acid) and3 .2. BDNF mRNA in the prefrontal cortextreatment (F 54.30, P,0.05). There was no main(1,28)

Two-way ANOVA revealed a significant main effect of effect of status (F 52.96,P50.10). Exploration of the(1,28)

treatment (swim stress versus control environment) interaction revealed a significant change in baseline BDNF(F 588.43, P,0.001) and a significant interaction mRNA in lesion versus sham animals (P,0.05) (n56 and(1,54)

between lesion status (ACSF versus ibotenic acid) and 12, respectively). Overall, BDNF mRNA in ibotenic acidtreatment (F 513.03, P,0.01). There was no main treated animals was reduced to 75% of control values (see(1,54)

Fig. 5. In situ hybridization of BDNF mRNA in the dentate gyrus of the hippocampus of lesion and sham animals exposed to a control environment orswim stress. (A) ACSF, control environment; (B) ACSF, swim stress; (C) Ibotenic acid, control environment; (D) Ibotenic acid, swim stress. Scale bar, 100mm.

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Figs. 4 and 5) and was increased to similar overall levels 4 . Discussionfollowing stress in lesion (127%) and sham (124%)animals (n57 and 7, respectively) as compared to control 4 .1. BDNF mRNA in the prefrontal cortexlevels. Relative increases in BDNF mRNA followingexposure to stress, however, were not equivalent as a 52% The results of the present study demonstrate that basalincrease in ibotenic acid treated animals was demonstrated BDNF mRNA levels are reduced in the prefrontal cortexwhereas only a 24% increase in sham animals was seen. of rats with neonatal ibotenic acid lesions of the ventralFollowing stress, the induction of BDNF mRNA appears hippocampus. Reductions in baseline BDNF mRNA ex-to be distributed throughout the granular region of the pression could result in alterations in neuronal morpholo-dentate gyrus rather than an induction within a specific gy, specifically dendritic atrophy [27], as well as reducedsubset of cells. synaptic plasticity [37] and reduced neuronal survival [13].

The inter-relationship between BDNF and the neurotrans-3 .4. BDNF mRNA in the CA3 region of the mitters GABA and glutamate also suggests that reductionshippocampus in basal BDNF mRNA expression could relate to altera-

tions in inhibitory and excitatory neurotransmission in theTwo-way ANOVA revealed a significant main effect of prefrontal cortex. Specifically, it has been demonstrated

lesion status (ACSF versus ibotenic acid) (F 511.46, that BDNF expression is positively correlated with gluta-(1,25)

P,0.01) and a significant main effect of treatment (swim matergic neurotransmission and negatively correlated withstress versus control environment) (F 544.91, P, GABAergic neurotransmission [42,43]. A bi-directional(1,25)

0.001). There was no lesion status by treatment interaction regulatory relationship exists, however, between these(F 50.70, P50.41). Overall, neonatal ibotenic acid systems. This implies that altered BDNF expression can(1,25)

lesions resulted in reduced levels of BDNF mRNA in the directly influence glutamatergic neurotransmission just asCA3 region of the hippocampus as compared to shamanimals (n57 and 9, respectively) (see Figs. 6 and 7).There was a reduction of BDNF mRNA to 70% of controllevels under baseline conditions. Further contributing tothe main effect of lesion status, BDNF mRNA was inducedin lesioned animals to 123% of control following exposureto stress, whereas, sham animals showed an induction to141% of control (n56 and 7, respectively). Despite theoverall lower levels of BDNF mRNA in lesioned animalsas compared to controls, however, similar relative in-creases in BDNF mRNA following stress were seen; a53% increase from baseline in lesioned animals and a 41%increase in sham animals.

3 .5. BDNF mRNA in the CA1 region of thehippocampus

Two-way ANOVA revealed a significant main effect oflesion status (ACSF versus ibotenic acid) (F 55.67,(1,22)

P,0.05) and a significant main effect of treatment (swimstress versus control environment) (F 521.87, P,(1,22)

0.001). There was no lesion status by treatment interaction(F 50.12, P50.73). Overall, neonatal ibotenic acid(1,22)

lesions appeared to decrease levels of BDNF mRNA in theCA1 region of the hippocampus as compared to shamanimals (n58 and 5, respectively) (see Figs. 8 and 9).Baseline BDNF mRNA levels in ibotenic acid treatedanimals was reduced to 74% of control levels. Furthercontributing to the main effect of lesion status was thelesser induction of BDNF mRNA in lesion (122%) versus

Fig. 6. BDNF mRNA in the CA3 region of the hippocampus. Two-waysham (141%) animals following exposure to stress (n56ANOVA revealed a significant effect of lesion status (ACSF vs. Ibotenicand 7, respectively). There was, however, no significant Acid) (F 511.46, P,0.01) and a significant effect of treatment(1,25)difference in the relative degree of the upregulation in (control environment vs. swim stress) (F 544.91, P,0.001). There(1,25)

lesion (48%) and sham (41%) animals. was no interaction between lesion status and treatment.

P.C. Ashe et al. / Brain Research 956 (2002) 126–135132

glutamate can directly regulate BDNF expression following stress compared to the previous reports of its[16,19,21]. As will be discussed later, this inter-relation- reduction following stress.ship and the reduction in baseline BDNF levels can have The demonstration that lesioned animals exhibit sig-significant consequences with respect to brain function, nificantly increased BDNF mRNA expression compared tosynaptic connectivity, neurotransmission, and neuronal sham animals following exposure to stress suggests thatviability and/or vulnerability. these animals are hyperresponsive to the effects of stress.

Previous investigations using restraint stress demonstrate In addition to this, one can conclude that the ability tothat stress results in a decrease in BDNF expression regulate BDNF expression is not impaired. Three possible[35,36], however, the present study demonstrates a signifi- explanations may account for the relative overexpressioncant upregulation of BDNF mRNA following swim stress. of BDNF mRNA; a relative hyper-release of glutamateIn addition, animals with neonatal ibotenic acid lesions resulting in a proportional increase in BDNF mRNA, ademonstrate significantly greater increases in BDNF compensatory mechanism in response to increased de-mRNA expression than do sham animals exposed to stress. mands on an already compromised system or, a dysregula-This finding is contrary to what was expected according to tion of BDNF expression. The glutamatergic system isprevious reports and the hypothesis tested in this study. It suggested to be hypofunctional in the animal model ofwas hypothesized that animals with these neonatal lesions schizophrenia used in the present study [22]. Al-Amin etwould show reductions in baseline BDNF, as was demon- al. [1] demonstrated that rats with neonatal ibotenic acidstrated, and also show significantly reduced BDNF mRNA lesions are hypersensitive to NMDA antagonists, sug-levels as compared to sham animals exposed to swim gesting a possible dysregulation of the glutamatergicstress. It is well documented that stress increases glutamate system. It was concluded that destruction of glutamatergicrelease in the prefrontal cortex [28] and that glutamate can projection fibers results in reduced glutamate release in thedirectly increase BDNF expression [42,43]. It is possible prefrontal cortex. Investigation of stimulus-induced aminothat the increased glutamate release following stress has a acid release from prefrontal cortical and hippocampaldirect effect of increasing BDNF mRNA, although the slices prepared from adult rats lesioned as neonates revealsresults by Smith et al. [35,36] are not in accordance with a reduction in release [33], thereby supporting this conclu-this conclusion. Recently, a corroborating report of in- sion. These findings suggest that increased glutamatecreased BDNF mRNA in the prefrontal cortex following release is not responsible for the increase in BDNFrestraint stress was published [29], however no comment expression, however, stimulus-induced alterations in gluta-was made regarding the demonstration of increased BDNF mate have not been investigated in vivo. In addition,

Fig. 7. In situ hybridization of BDNF mRNA in the CA3 region of the hippocampus of lesion and sham animals exposed to a control environment or swimstress. (A) ACSF, control environment; (B) ACSF, swim stress; (C) Ibotenic acid, control environment; (D) Ibotenic acid, swim stress. Scale bar, 100mm.

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4 .2. BDNF mRNA in the hippocampus

The present study demonstrates a significant reduction inbaseline BDNF mRNA in the dentate gyrus of the hip-pocampus in animals with neonatal ibotenic acid lesions.An overall reduction in BDNF mRNA expression is alsoseen in both the CA3 and the CA1 fields of the hippocam-pus following the neonatal lesion. In response to stress,however, differences exist between the various hippocam-pal regions. Although stress consistently increases theexpression of BDNF mRNA in all regions, only thedentate gyrus demonstrates a hyperresponsivity. The rela-tive response to stress in the CA3 and CA1 regions arecomparable in lesion and sham animals, whereas thedentate gyrus of lesioned animals demonstrates a signifi-cant upregulation of BDNF mRNA following exposure tostress as compared to sham animals.

The upregulation of BDNF mRNA in the hippocampusfollowing stress can potentially be explained throughmechanisms similar to those described for the prefrontalcortex. In addition, however, the differences in BDNFregulation between the various hippocampal regions alsolead to the suggestion that differential input to the dentategyrus may account for its hyperresponsivity as comparedto the CA3 and CA1 fields. The predominant inputs to theCA3 and CA1 fields are glutamatergic whereas the dentatereceives both glutamatergic and GABAergic input [11,12].It is also known that BDNF expression is dependent on thebalance of glutamatergic and GABAergic input [42,43].Fig. 8. BDNF mRNA in the CA1 region of the hippocampus. Two-way

ANOVA revealed a significant effect of lesion status (ACSF vs. ibotenic The possibility exists, therefore, that alterations inacid) (F 55.67,P,0.05) and a significant effect of treatment (control(1,22) GABAergic neurotransmission following exposure toenvironment vs. swim stress) (F 521.87, P,0.001). There was no(1,22) stress in lesioned animals may account for the differentialinteraction between lesion status and treatment.

regulation of BDNF mRNA in the dentate gyrus.

different stimuli may result in different glutamate release,therefore, one cannot conclude definitively that glutamate 5 . Conclusionsrelease is reduced. The role that BDNF plays in neuronalprotection suggests that its expression may be upregulated The present study demonstrates a reduction in basalin response to a threat to the system as is the situation with BDNF mRNA levels in the prefrontal cortex and theischemia [24] and kainic acid toxicity [43]. The func- hippocampal formation of animals with neonatal ibotenictionally compromised system set up by the neonatal lesion acid lesions of the hippocampus. Reductions in basalmay demonstrate increased vulnerability to stress, there- BDNF levels could result in alterations in neuronal con-fore, protection from atrophy and/or death may require a nectivity by producing a situation of increased neuronalgreater upregulation of BDNF to overcome the threat. atrophy and fewer synapses. Alterations in synaptic densityAlternatively, dysregulation of BDNF expression may have not been investigated in the model, however, theresult from the neonatal lesion. This results in the possi- reduction in BDNF mRNA would suggest that this maybility that unnecessary overexpression of BDNF mRNA occur. As stated in the hypothesis, reductions in BDNFmay put further stress on the system. It has been reported may also increase the vulnerability of neurons to stress.that overexpression of BDNF can potentiate NMDA-in- Hyperresponsivity to stress is demonstrated by increases induced necrotic cell death, at least under cell culture BDNF mRNA in the prefrontal cortex and the dentateconditions [17]. It is questionable whether necrotic cell gyrus. This could be a direct attempt to compensate for thedeath would occur in this system, however, the conditions increased vulnerability of the system induced by theunder which this would occur could be present as there is a neonatal lesion. The present results support the hypothesispotential for increased glutamate release following stress that a neurodevelopmentally-compromised system canand a marked increase in BDNF mRNA was demonstrated result in significant consequences in adulthood, includingin the present study. alterations in neuronal viability and neuronal communica-

P.C. Ashe et al. / Brain Research 956 (2002) 126–135134

Fig. 9. In situ hybridization of BDNF mRNA in the CA1 region of the hippocampus of lesion and sham animals exposed to a control environment or swimstress. (A) ACSF, control environment; (B) ACSF, swim stress; (C) Ibotenic acid, control environment; (D) Ibotenic acid, swim stress. Scale bar, 100mm.

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