bdnf mrna expression in rat hippocampus and prefrontal cortex: effects of neonatal ventral...

BDNF mRNA expression in rat hippocampus andprefrontal cortex: effects of neonatal ventral hippocampaldamage and antipsychotic drugs

Barbara K. Lipska, Zin Z. Khaing, Cynthia Shannon Weickert and Daniel R. WeinbergerClinical Brain Disorders Branch, Intramural Research Program, National Institute of Mental Health, Bethesda, MD,

20892-1385 USA

Keywords: animal model, clozapine, haloperidol, neurotrophin, schizophrenia

Abstract

Brain-derived neurotrophic factor (BDNF) plays an important role in development, synapse remodelling and responses to stress

and injury. Its abnormal expression has been implicated in schizophrenia, a neuropsychiatric disorder in which abnormal neural

development of the hippocampus and prefrontal cortex has been postulated. To clarify the effects of antipsychotic drugs used inthe therapy of schizophrenia on BDNF mRNA, we studied its expression in rats treated with clozapine and haloperidol and in rats

with neonatal lesions of the ventral hippocampus, used as an animal model of schizophrenia. Both antipsychotic drugs reduced

BDNF expression in the hippocampus of control rats, but did not signi®cantly lower its expression in the prefrontal cortex. The

neonatal hippocampal lesion itself suppressed BDNF mRNA expression in the dentate gyrus and tended to reduce its expressionin the prefrontal cortex. These results indicate that, unlike antidepressants, antipsychotics down-regulate BDNF mRNA, and

suggest that their therapeutic properties are not mediated by stimulation of this neurotrophin. To the extent that the lesioned rat

models some pathophysiological aspects of schizophrenia, our data suggest that a neurodevelopmental insult might suppressexpression of the neurotrophin in certain brain regions.

Introduction

Brain-derived neurotrophic factor (BDNF) promotes a variety of

neuromodulatory processes in the brain including neuronal survival,

neurite outgrowth and synapse formation (Altar & DiStefano, 1998).

Consistent with these roles, BDNF mRNA is abundantly expressed

throughout life in highly plastic regions of the rat and human brain,

e.g. the neocortex, dentate gyrus and CA1±4 regions of the

hippocampus (Phillips et al., 1990). It is involved in promoting

neuronal function during development, in response to injury and

stress, in supporting functional activity of dopaminergic, cholinergic,

glutaminergic and serotonergic neurons (Altar, 1999), and in

regulating activity of mature neurons in the hippocampus (Zafra

et al., 1990; Ballarin et al., 1991; Kang & Schuman, 1995; Levine

et al., 1995) and the neocortex (Desai et al., 1999; Marty et al., 1997).

As abnormal development of these brain regions has been frequently

implicated in the pathogenesis of schizophrenia (Weinberger, 1999;

Lewis & Gonzalez-Burgos, 2000), it is possible that BDNF is

abnormally regulated in schizophrenia (Weickert & Weinberger,

1998). Indeed, recent postmortem studies have shown that BDNF

mRNA is reduced in the hippocampus and prefrontal cortex of

patients with schizophrenia (Brouha et al., 1996; Kittel et al., 1997).

However, another recent study reported elevated levels of BDNF

protein in the anterior cingulate cortex and hippocampus of schizo-

phrenic patients (Takahashi et al., 2000). The potential role of BDNF

in schizophrenia is further complicated by the fact that the impact of

antipsychotic drugs on BDNF mRNA expression or BDNF protein

levels is unclear.

To determine whether treatment with typical and atypical

antipsychotics in¯uences BDNF mRNA in brain regions implicated

in schizophrenia, we studied the effects of acute (a single intra-

peritoneal injection) and chronic (28 days) treatment with haloperidol

(0.5 and 1 mg/kg) and clozapine (10 mg/kg) on BDNF mRNA

expression in the rat prefrontal cortex and the hippocampus using an

in situ hybridization method that, with some modi®cations, has been

employed in our laboratory to measure BDNF mRNA in human tissue

(Brouha et al., 1996; Kittel et al., 1997). We have also assessed

BDNF mRNA expression in drug-free and neuroleptic-treated rats

with a neonatal excitotoxic lesion of the ventral hippocampus. We

have shown previously that the neonatally lesioned animals display in

adulthood a variety of abnormalities reminiscent of schizophrenia and

are used as an animal model of this disorder (Lipska et al., 1993,

1995a; Sams-Dodd et al., 1997; for review see Lipska & Weinberger,

2000). We hypothesized that, consistent with cognitive impairments

(Moghaddam et al., 1999) and molecular and morphological changes

in the prefrontal cortex of the neonatally lesioned animals (Bertolino

et al., 1999; Lipska & Weinberger, 2000), the neonatal ventral

hippocampal insult might impair BDNF mRNA expression in the

prefrontal cortex of adult animals. We have also tested whether the

lesion altered BDNF mRNA expression in other parts of the

hippocampal formation which are relatively spared by the lesion

(dentate gyrus and CA3 area) and which normally show high

expression of BDNF mRNA. Based on some of the previous reports

of increased BDNF mRNA after clozapine (Chlan-Fourney et al.,

1998), we also hypothesized that compromised BDNF mRNA

Correspondence: Dr B.K. Lipska, as above.E-mail: [email protected]

Received 15 January 2001, revised 3 April 2001, accepted 3 May 2001

European Journal of Neuroscience, Vol. 14, pp. 135±144, 2001 ã Federation of European Neuroscience Societies

136 B. K. Lipska et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 135±144

expression in the lesioned animals may be normalized by chronic

treatment with clozapine, an atypical antipsychotic drug.

Materials and methods

Surgery

All procedures were performed in accordance with the National

Institutes of Health guidelines for use and care of laboratory animals.

Animals were on a 12-h light : 12-h dark cycle (lights on at 06.00 h)

in a temperature-controlled environment and with ad lib. access to

food and water. Female Sprague-Dawley rats were purchased

pregnant at 14 days of gestation (Taconic, Germantown, NY, USA)

and housed individually in breeding cages. Litters of four to eight

male pups were formed (total n = 80 pups). Pups were randomly

assigned to lesion (n = 42) or sham (n = 38) status. Surgery was

performed as previously described (Lipska et al., 1993). Pups (7 days

of age, weight 15±18 g) were anaesthetized by hypothermia (placed

on wet ice for 10±20 min) and then immobilized by taping onto a

styrofoam platform ®xed to a stereotaxic Kopf instrument (David

Kopf Instruments, Tujunga, CA, USA). An incision was made in the

skin overlying the skull and 0.3 mL (3 mg) of ibotenic acid (in the

lesioned rats; Sigma Chemical Co., St Louis, MO, USA) or arti®cial

cerebrospinal ¯uid (in the sham-operated rats) was injected bilater-

ally, using an infusion pump through a Hamilton needle, into the

ventral hippocampus at a rate of 0.15 mL/min (AP, 3.0 mm relative to

bregma; ML, 63.5 mm; DV, 5.0 mm). The needle was left in place

for 4 min, to prevent diffusion of neurotoxin along the needle track,

and then withdrawn. The skin incision was closed with staples. The

pups were placed under a heating lamp and then returned to their

mothers. At postnatal day 24, the rats were weaned, separated by

lesion status, and housed two to three per cage.

Drug preparation

Ibotenic acid (Sigma) was dissolved in arti®cial cerebrospinal ¯uid

(5 mg in 500 mL) with the addition of 2 mL of 10 M NaOH, and

further neutralized to pH 7.4 by adding » 6 mL of 1 M NaOH.

Aliquots of 50 mL were stored at ±80 °C for up to several months. A

stock solution of haloperidol (Research Biochemicals Inc., Natick,

MA, USA) 20 mg/mL was prepared in 1% lactic acid by heating up

200 mg of haloperidol in 10 mL of 1% lactic acid until dissolved. To

obtain solutions of 0.5 and 1.0 mg/mL, a stock solution was diluted

with distilled water to a ®nal volume of 20 mL. Approximately

30 mL of 1 M NaOH was added to adjust the ®nal solutions to pH 5.3.

Clozapine (a generous gift from Sandoz Research Institute Berne Ltd,

Berne, Switzerland) was prepared as folows: 100 mg was dissolved in

a small amount of 0.1 M HCl (400 mL), diluted with distilled water to

40 mL and neutralized with 1 M NaOH to pH 5.2. Vehicle was

prepared by adding 2 mL of 1% lactic acid to 100 mL distilled water.

Treatment with antipsychotic drugs

Drug treatment started 2 months after the lesion (at postnatal day 56).

Rats were given intraperitoneal injections of haloperidol (0.5 and

1 mg/kg) or clozapine (10 mg/kg) either acutely (a drug administered

on the last day of a 27-day regimen of vehicle injections) or

chronically (once daily for 28 days). A control group for both acute

and chronic treatment was injected with vehicle for 28 days. All rats

were killed 7 h after the last injection. Brains were frozen and

sectioned into 20-mm slices which were collected onto gelatin-coated

slides (two slices per slide) and stored at ±80 °C. Every 10th slide

was Nissl-stained and used for anatomical matching. The hippo-

campal slices were used for veri®cation of the lesion. Matched slides

through the prefrontal cortex and the hippocampus (four consecutive

slides per region per rat corresponding to ®gs 9 and 38 for prefrontal

cortex and hippocampus, respectively, of the atlas of Paxinos &

Watson, 1986) were used in the in situ hybridization study.

In situ hybridization histochemistry

An 35S-labelled riboprobe was synthesized using a clone containing

1095 base pairs of the rat BDNF cDNA sequence (Maisonpierre et al.,

1991). To produce an antisense ribonucleotide probe, the Bluescript

SK-vector was linearized with BamH1 restriction enzyme (New

England BioLabs, Beverly, MA, USA); 200 ng (1 mL) of linearized

plasmid was labelled using 150 mCi of [35S]UTP (NEN, Boston, MA,

USA) and 1 mL (5 units) T7 RNA polymerase (Stratagene, CA,

USA). For a sense probe, HindIII enzyme and T3 polymerase

(Promega, Madison, WI, USA) were used. The total volume of this

reaction was 10 mL: 1 mL each of 10 mM rATP, rCTP, rGTP

(Promega), 2 mL of 53 transcription buffer, 1 mL of 100 mM

dithiothreitol, 1 mL of RNasin and 1 mL diethyl pyrocarbonate

(DEPC)-treated water. After incubation for 15 min at 37 °C, labelled

RNA was digested with DNase (3 mL, 10 U/mL) and precipitated

with ethanol in the presence of yeast tRNA and ammonium acetate.

After precipitation, the probe was washed with 70% ethanol (speci®c

activity 2.2 3 109 dpm/mg), resuspended in DEPC water and added

to the hybridization cocktail containing 5 mL of hybridization buffer

(see below), 5 mL formamide, 20 mL 50% sodium thiosulphate,

200 mL 5 M dithiothreitol and 100 mL 10% sodium dodecyl sulphate

(Research Genetics). The hybridization buffer consisted of 1200 nM

NaCl, 20 mM Tris-HCl, 0.04% Ficoll, 0.04% bovine serum albumin

(Pierce, Rockford, IL, USA), 0.04% polyvinylpyrrolidone (PVP),

2 mM EDTA (Research Genetics, Huntsville, AL, USA), pH = 8,

0.02% salmon sperm DNA, 0.1% total yeast RNA, 0.01% yeast tRNA

(Invitrogen, Rockville, MD, USA) and 20% Dextran SO4. This

yielded a ®nal concentration of 5 ng/mL of the riboprobe, corres-

ponding to » 0.5 3 106 dpm per section (per 25 mL of hybridization

cocktail). The sections were ®xed in 4% formaldehyde, acetylated

using acetic anhydride, dehydrated in ethanol and defatted in

chloroform. In situ hybridization was carried out overnight at 55 °C

in humidi®ed chambers. After removing the coverslips in 23 sodium

chloride±sodium citrate (SSC), the sections were treated with RNase

A (20 mg/L for 30 min at 37 °C), washed in RNase A-free buffer for

30 min at 37 °C, washed in 23 SSC for 15 min at room temperature,

twice in 23 SSC for 1 h at 50 °C, once in 0.23 SSC for 1 h at 55 °C,

once in 0.23 SSC for 30 min at 60 °C and rinsed with 0.23 SSC at

room temperature. The sections were then dehydrated with 50±100%

ethanol solutions with ammonium acetate (300 mM), air-dried and

FIG. 1. A neonatal excitotoxic lesion of the ventral hippocampus shown in (A) a photomicrograph of a Nissl-stained brain section through the hippocampusof an adult rat and (B) in a diagram adapted from the atlas of Paxinos & Watson, 1986. BDNF mRNA expression is shown in coronal sections through (C)the hippocampus and (D) the frontal cortex. (A) An asterisk indicates a lesion site, with neuronal loss and gliosis. (B) A black area indicates the boundariesof a small lesion and a grey area depicts a large lesion. (C) Arrowheads delineate the borders of CA1/2 area, large straight arrows indicate the dentate gyrusand small straight arrows show CA3, the areas from which the measurements were taken. (D) An elongated oval delineates the area of the prefrontalcortex from which measurements were taken. Boxes in C and D are drawn over ®elds from which photomicrographs were taken. Scale bar, 800 mm (A),600 mm (B).

Brain-derived neurotrophic factor and antipsychotics 137


apposed with the 14C standards to BioMax Kodak ®lm for 11 days.

DEPC, sodium thiosulphate, salmon sperm DNA, ammonium acetate,

diethyl pyrocarbonate and total yeast RNA were purchased from

Sigma.

Analysis of autoradiographic images was performed using NIH

Image software. The regions analysed (medial prefrontal cortex,

CA1/2 and CA3 areas of the hippocampus, dentate gyrus) are

depicted in Fig. 1. In the lesioned rats, only those parts of the CA1/2

and CA3 areas of the hippocampus and dentate gyrus depicted in

Fig. 1C that were visibly spared by the lesion (i.e. contained intact

neurons, no gliosis as shown in Fig. 1A) were measured (the

hippocampal regions damaged by the lesion showed no expression of

BDNF and no neurons in Nissl-stained sections). Optical density was

converted to dpm/mm2 using 14C standards (Miller, 1991). Four

measurements were obtained per region per rat from four slides

containing two sections each, and averaged. The sense riboprobe did

not yield any signi®cant hybridization signal (not shown).

Statistical analysis

The densitometric data were analysed using a two-way analysis of

variance (ANOVA) with Drug and Lesion as independent factors.

Acute and chronic groups were analysed separately. A Fisher PLSD

test was used for post hoc comparisons.

Results

Lesion veri®cation

Analysis of Nissl-stained 20-mm brain sections through the

hippocampus of the lesioned rats showed that damage was restricted

to the ventral hippocampus (Fig. 1A and B). Consistent with our

previous reports (Lipska et al., 1993, 1995a; Lipska & Weinberger,

1995), neuronal loss, gliosis and some cavitation was mostly evident

in cytoarchitectural subdivisions CA1/2 of the hippocampal forma-

tion and in the ventral parts of the subiculum, whereas only minimal

damage was detected in the ventral dentate gyrus and the ventral CA3

sub®eld.

Hippocampal formation

BDNF mRNA expression was found in all cytoarchitectural divisions

of the hippocampus. The hybridization silver grain signal was located

over pyramidal (CA1±3) and granule (dentate gyrus) cell layers but

was not evenly distributed (Fig. 1C). The strongest hybridization

signal was observed in the dentate dyrus and the CA3 area, whereas

the BDNF mRNA expression over CA1/2 sub®elds was modest. As

previously noted (Hofer et al., 1990), the signal was not seen in all

cells of the hippocampal subregions; whilst most cells were strongly

labelled, occasional cells were often almost completely devoid of

silver grains (Fig. 3).

Both antipsychotic drugs markedly reduced BDNF mRNA expres-

sion in the CA1/2 sub®eld of the hippocampus and in the dentate

gyrus of control rats (Fig. 2A and B). Moreover, lesioned vehicle-

treated animals displayed profoundly attenuated BDNF mRNA

expression in parts of CA1/2 and in the dentate gyrus apparently

spared by the lesion as compared with sham-operated animals. In the

CA1/2 area after acute treatment (Fig. 2A), an ANOVA revealed a

signi®cant effect of Lesion (F1,35 = 6.91, P < 0.05) and Drug

(F2,35 = 7.37, P < 0.01, but no signi®cant Lesion±Drug interaction

(F2,35 = 2.15, P > 0.1). Post hoc tests showed that acute injections of

haloperidol and clozapine signi®cantly reduced BDNF levels in CA1/

2 of control rats (by 34 and 57%, respectively; P < 0.01) but had no

signi®cant effect in the lesioned rats whose basal (after vehicle

injections) BDNF mRNA expression was signi®cantly attenuated as

compared with sham-operated controls (by 41%; P < 0.01; Fig. 3A±

D).

Chronic treatment with antispychotics produced effects similar to

acute treatment in CA1/2 (Fig. 2A). An ANOVA revealed a signi®cant

effect of Lesion (F1,47 = 22.6, P < 0.0001) and Drug (F3,47 = 9.01,

P < 0.001), and a signi®cant Lesion±Drug interaction (F3,47 = 6.50,

P < 0.01). Both doses of haloperidol (0.5 and 1.0 mg/kg) and

clozapine attenuated BDNF mRNA expression in control rats (by 25,

31 and 55%, respectively; P < 0.01). In the lesioned animals, whose

basal levels of BDNF mRNA expression were signi®cantly attenuated

as compared to sham controls (by 41%; P < 0.001; Fig. 3), only a

high dose of haloperidol further reduced BDNF mRNA expression in

CA1/2 (by 54% as compared with the lesion vehicle group; P < 0.01;

Fig. 3E±H).

The effects of antipsychotic drugs were slightly less pronounced in

the dentate gyrus than in CA1/2, but the neonatal lesion had an even

more profound effect in reducing BDNF mRNA in the dentate gyrus

than in CA1/2 (Fig. 2B). For acute treatment, an ANOVA showed a

signi®cant effect of Lesion (F1,35 = 163.83, P < 0.0001) and Drug

(F2,35 = 11.93, P < 0.001) and a signi®cant Lesion±Drug interaction

(F2,35 = 12.65, P < 0.0001). Post hoc comparisons showed that

haloperidol and clozapine signi®cantly reduced BDNF mRNA

expression in the dentate gyrus (by 21 and 55%, respectively;

P < 0.0.01) in control rats. Neither drug had an effect in the lesioned

animals whose basal (after vehicle injections) BDNF mRNA levels

were signi®cantly attenuated as compared to the sham-operated rats

(by 83%; P < 0.0001; Fig. 3I±L). For chronic treatment, an ANOVA

revealed a signi®cant lesion effect (F1,47 = 179.28, P < 0.05), no

signi®cant Drug effect (F3,47 = 2.40, P = 0.08), and a signi®cant

Lesion±Drug interaction (F3,47 = 3.3, P = 0.03). Post hoc compari-

sons showed that both doses of haloperidol and clozapine signi®-

cantly reduced BDNF mRNA expression in the dentate gyrus of

sham-operated rats (by 19, 33 and 30%, respectively). The effects of

antipsychotic drugs were not detected in the lesioned rats whose basal

(after vehicle) levels of BDNF mRNA were very low as compared

with vehicle- or antipsychotic drug-treated rats (P < 0.001 for all

sham vs. lesion comparisons; Fig. 2B).

The effects of antipsychotic drugs and the lesion differed in the

CA3 area of the hippocampus from those in the dentate gyrus and the

CA1/2 region (Fig. 2C). For acute treatment, ANOVA showed a main

effect of Lesion (F1,35 = 5.0, P < 0.05), but no effect of Drug

(F2,35 = 2.1, P = 0.13) and no signi®cant Lesion±Drug interaction

(F2,35 = 1.8, P = 0.17). In contrast to other hippocampal sub®elds,

there was no signi®cant difference in BDNF mRNA expression

between sham and lesioned vehicle-treated animals in the CA3 area.

In chronically treated rats, ANOVA showed a signi®cant effect of Drug

(F3,47 = 3.7, P < 0.02) but the effect of Lesion (F1,47 = 0.68,

P = 0.4) and the Drug±Lesion interaction (F3,47 = 0.27, P = 0.8)

were not signi®cant. Post hoc comparisons revealed that both doses

of chronic haloperidol signi®cantly attenuated BDNF mRNA expres-

sion in control and lesioned rats (P < 0.05), whereas clozapine had no

effect in either group of animals (Fig. 2C).

Prefrontal cortex

The area comprising the medial prefrontal cortex is depicted in

Fig. 1D. The hybridization signal was present throughout all layers of

the prefrontal cortex. This ®nding is in agreement with the study of

Yan et al. (1997) who reported the presence of BDNF protein in

layers I±III and V±VI of the neocortex. Clusters of silver grains were

overlying lightly Nissl-stained large cells, putatively pyramidal

neurons (not shown). Neither acute nor chronic treatment with



haloperidol or clozapine signi®cantly altered expression of BDNF

mRNA expression in the medial prefrontal cortex of the sham or

lesioned animals (Fig. 2D). ANOVAs for acutely or chronically treated

animals revealed no signi®cant effects of Lesion (F1,35 = 0.23,

P > 0.5 and F1,47 = 1.1, P > 0.3, respectively) or Drug (F2,35 = 1.40,

P > 0.2 and F3,47 = 1.0, P > 0.3, respectively). Lesion±Drug inter-

FIG. 2. Expression of BDNF mRNA (mean 6 SD) in (A) the hippocampal CA1/2 area, (B) the dentate gyrus, (C) the hippocampal CA3 area and (D) theprefrontal cortex of sham-operated rats (Sham) and rats with the neonatal lesion of the ventral hippocampus (Lesion). Rats were acutely (Acute) orchronically (Chronic; 28 days) treated with vehicle (Veh), haloperidol (Hal; 0.5 or 1.0 mg/kg) and clozapine (Cloz; 10 mg/kg). *P < 0.05 vs. a vehicle-treatedgroup of the same lesion status, #P < 0.05 vs. vehicle-treated sham animals.



FIG. 3. Light (A, C, E, G, I, K) and dark(B, D, F, H, J, L) ®eld autoradiographicimages of BDNF mRNA expression in (A±H)the hippocampus CA1/2 area and (I±L) thedentate gyrus. (A and B) CA1 area of acontrol rat treated with vehicle. (C and D)Corresponding region of a vehicle-treated ratwith a neonatal lesion in the ventralhippocampus. The lesioned rats showedsigni®cantly less BDNF mRNA expression inthe CA1/2 area of the hippocampus than didcontrols. (E and F) CA1 area of a control rattreated chronically with haloperidol (1.0 mg/kgi.p.). (G and H) Corresponding region of a ratwith a neonatal lesion in the ventralhippocampus, treated chronically withhaloperidol 1.0 mg/kg i.p.The haloperidol-treated lesioned rats showedsigni®cantly less BDNF mRNA expression inthe CA1/2 area of the hippocampus than didhaloperidol-treated controls. (I and J) Dentategyrus of a control rat treated with vehicle. (Kand L) Corresponding region of a rat with aneonatal lesion in the ventral hippocampus,treated with vehicle. Expression of BDNFmRNA was signi®cantly reduced in the dentategyrus of the lesioned rats. Scale bar, 25 mm.



actions were also not signi®cant (F2,35 = 0.42, P > 0.5 and

F3,47 = 0.21, P > 0.5, for acute and chronic treatment, respectively).

The results suggest, however, that BDNF mRNA expression levels

tended to be lower in the medial prefrontal cortex of control rats

treated chronically with clozapine as compared with chronic vehicle

injections (by 34%; Fig. 2D). BDNF mRNA expression was also

lower in the lesioned vehicle-treated rats as compared with sham-

operated vehicle-treated animals although the difference did not reach

statistical signi®cance (by 18%; Fig. 2D).

Discussion

Effects of antipsychotic drugs

The results of the current study revealed that antipsychotic drugs

attenuated the expression of BDNF mRNA in the normal rat

hippocampus but did not signi®cantly lower its expression in the

prefrontal cortex. The extent of the change depended on the speci®c

subregion, type of drug, dosage and length of administration. In

particular, both haloperidol and clozapine after acute and chronic

administration markedly down-regulated expression of BDNF mRNA

in the dentate gyrus and CA1/2 sub®elds of the rat hippocampus. In

the CA3 sub®eld, only chronic treatment with haloperidol (but not

clozapine) signi®cantly reduced BDNF mRNA expression in control

rats. In contrast, haloperidol had no signi®cant effect in the medial

prefrontal cortex, whereas chronic treatment with clozapine tended to

lower BDNF mRNA expression in this region.

The ®nding of reduced BDNF mRNA in the hippocampus of

haloperidol- and clozapine-treated rats contrasts markedly with the

action of antidepressants that elevate BDNF mRNA in this brain

region (Nibuya et al., 1995; Russo-Neustadt et al., 1999). The

antidepressant-induced BDNF elevations have been attributed to

increased synaptic levels of the neurotransmitters serotonin and

noradrenaline in the forebrain (Celada et al., 1996; for review see

Altar, 1999) and enhanced growth of serotonergic and noradrenergic

®bers (Mamounas et al., 2000). These effects may be mediated

through 5-hydroxytryptamine (5-HT)2A and b-adrenoreceptor sub-

types (Vaidya et al., 1997). Although the exact mechanisms of

boosting the BDNF levels by antidepressants are not well understood,

stimulation of intracellular pathways involving cyclic adenosine

monophosphate (cAMP) (Fujimaki et al., 2000) and cAMP response

element binding protein (CREB) (Duman et al., 1997) have been

implicated.

Our ®ndings of reduced BDNF mRNA expression in the hippo-

campal formation by typical and atypical antipsychotics are in

agreement with one recently published study that assessed BDNF

protein levels after a 29-day treatment with haloperidol and

risperidone (Angelucci et al., 2000), which, however, also found a

signi®cant reduction of BDNF in the frontal cortex after haloperidol,

and with a report by Chlan-Fourney et al. (1998) who found reduced

BDNF mRNA. Our ®ndings additionally indicate that single doses of

haloperidol and clozapine are suf®cient to produce a signi®cant

suppression of BDNF mRNA in the hippocampal areas CA1/2 and in

the dentate gyrus, suggesting that these drugs can alter BDNF mRNA

synthesis by short-term changes in synaptic transmission in addition

to possible long-term adaptive mechanisms involving synaptic

plasticity and rearrangement of connections. It is unclear, however,

which neurotransmitter systems might be involved.

Haloperidol and clozapine were shown to down-regulate D1 and

D4 dopamine receptor mRNA in the hippocampus (D'Souza et al.,

1997; Ritter & Meador-Woodruff, 1997), suggesting that blockade of

these dopamine receptor subtypes might interfere with BDNF gene

expression. Clozapine is also a potent 5-HT2A inhibitor and was

shown to in¯uence the serotonergic system by decreasing the

expression of 5-HT6 receptors in all hippocampal sub®elds

(Frederick & Meador-Woodruff, 1999) and increasing the densities

of 5-HT transporters in the ventral hippocampus (Ase et al., 1999).

Moreover, both haloperidol and clozapine reduce serotonin concen-

trations in the hippocampus (Burnet et al., 1996). Because enhanced

serotonergic transmission has been associated with the up-regulation

of BDNF mRNA in the hippocampus (Nibuya et al., 1995), it is

conceivable that the suppression of 5-HT function by antipsychotics

might lead to the reduction of BDNF mRNA. The glutamatergic

system may also play a role in mediating the effects of antipsychotics

because both drugs, although in complex and differential fashions,

alter the expression of certain a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid (AMPA)- and kainate-receptor subunits in

the hippocampus (Eastwood et al., 1996; Meador-Woodruff et al.,

1996), and glutamate potently stimulates expression of BDNF mRNA

in the hippocampus (Zafra et al., 1990; Wetmore et al., 1994). In the

CA3 region, however, only chronic treatment with haloperidol had a

signi®cant effect on BDNF down-regulation, and clozapine had no

effect at all.

In contrast to the hippocampus, neither drug had a signi®cant effect

in the prefrontal cortex, although chronic clozapine showed a trend

for a reduction BDNF mRNA levels. It has been suggested that the

activation of 5-HT2A receptors in the neocortex results in enhanced

presynaptic release of glutamate, stimulation of AMPA receptors and

a subsequent increase of BDNF mRNA synthesis (Vaidya et al.,

1997). Because clozapine antagonizes 5-HT2A receptors, which are

abundant in the prefrontal cortex (Pompeiano et al., 1994; Jakab &

Goldman-Rakic, 1998), it is conceivable that it could reduce BDNF

mRNA via this mechanism. Moreover, clozapine and other atypical

drugs, but not haloperidol, have been shown to induce internalization

of 5-HT2A receptors after chronic treatment and their subsequent loss

from apical dendrites of pyramidal neurons in the medial prefrontal

cortex (Willins et al., 1999).

BDNF is important in long-term potentiation of hippocampal

neurons and in learning and memory (Jankowsky & Patterson, 1999;

Hall et al., 2000; Liu et al., 2000), and its loss has been implicated in

the failure of cognitive functions in dementia and Alzheimer's

disease (Altar & DiStefano, 1998; Phillips et al., 1991). Whatever the

mechanisms responsible for the down-regulation of BDNF mRNA

following antipsychotic treatment, our ®ndings suggest that anti-

psychotic drugs, unlike antidepressants, would not improve cognition

by stimulating BDNF production. This notion is partially supported

by the observations that chronic haloperidol reduces spine density in

the prefrontal cortex and the striatum (Benes et al., 1985; Kelley et al.,

1997), although no effect on spine density was observed in the

hippocampus (Uranova et al., 1991). Thus, to the extent that BDNF

and synaptic density play a role in cognition (Elston, 2000), we would

not expect these drugs to bene®t cognitive abilities via these

mechanisms (Weinberger & Gallhofer, 1997).

Effects of the neonatal hippocampal lesion

We also found that the neonatal lesion of the ventral hippocampus

profoundly attenuated expression of BDNF mRNA in areas of the

hippocampus outside the lesion site, particularly in the dentate gyrus.

The lesion had no signi®cant effect in the prefrontal cortex although

some reduction in the BDNF mRNA expression was also seen there.

This raises the possibility that the hippocampal subregions may also

be directly damaged by the neurotoxin, although the assessment of

Nissl-stained sections through these regions does not reveal

discernable losses of neurons or gliosis. Our recent data suggest,



however, that hippocampal sub®elds outside the ventral hippocampus

suffer DNA damage in response to neonatal insult (Khaing et al.,

2000). It is also possible, however, as the hippocampus and prefrontal

cortex develop extensively after birth, that the restricted lesion in the

ventral aspects of the hippocampal areas CA1/2 had dramatic effects

on synaptogenesis and neuronal development in other parts of the

hippocampal formation and, although in a much more subtle way,

affected development of the prefrontal cortex to which the ventral

hippocampus projects (Jay & Witter, 1991; Carr & Sesack, 1996).

Reduced levels of N-acetylaspartate, a marker of neuronal metabolic

activity, in the prefrontal cortex of adult rats with the neonatal

hippocampal lesions may support this notion (Bertolino et al., 1999).

The neonatal ventral hippocampal lesion might have disturbed

function of interconnected brain regions by producing loss of neural

targets for some neurons (as in the case of the dentate gyrus neurons

that send projections to areas CA3, CA1/2) or loss of neuronal inputs

in other areas (as in the case of the prefrontal cortex that would lose

glutamatergic projections from the ventral hippocampus and may also

suffer reduced glutamatergic excitation from the thalamus, which is

deafferented by the hippocampal lesion). It is noteworthy in this

regard that unilateral electrolytic lesions of the hippocampus in

neonatal (postnatal day 1) rats, which produce severe spatial learning

disabilities, attenuate BDNF mRNA expression in the contralateral

hippocampus for up to 20 weeks postlesion without producing other

neurochemical changes, such as in choline-acetyltransferase or

GABA decarboxylase activity (van Praag et al., 1998). Thus,

unilateral damage of the hippocampus has long-lasting adverse

effects on neurotrophin synthesis on the seemingly healthy side that

can possibly have detrimental effects on development of the

contralateral hippocampus, and possibly on learning and memory.

Another possibility for reduced BDNF mRNA in rats with the

neonatal ventral hippocampal lesion may be their increased vulner-

ability to stress. We have previously shown that behavioural

responses to stressful stimuli are exaggerated (Lipska et al., 1993),

that stress-induced dopamine release is attenuated (Lipska et al.,

1995b; Lillrank et al., 1999) and that BDNF mRNA response to acute

restraint stress is blunted in the prefrontal cortex of the lesioned rats

(Molteni et al., 2001). Repeated stress decreases BDNF mRNA in the

hippocampus and the dentate gyrus (Smith et al., 1995) and up-

regulates TrkB receptors in all hippocampal sub®elds (Nibuya et al.,

1999). Thus, it is conceivable that stress experienced chronically by

the neonatally lesioned rats may contribute to the loss of BDNF

mRNA in vulnerable brain regions. However, we did not observe

diminished BDNF mRNA in the CA3 sub®eld, the region whose

pyramidal neurons have been reported to be most sensitive to stress

and to atrophy following repeated stressful events (McEwen, 1999),

and which show elevations in TrkB mRNA following chronic stress

(Nibuya et al., 1999).

Finally, it should be noted that, similarly to the normal animals, we

did not ®nd evidence for a bene®cial effect of antipsychotic drugs on

the BDNF mRNA expression in the neonatally lesioned rats.

Antipsychotics had either no effect or further lowered BDNF

mRNA levels in the brains of the lesioned rats.

In summary, our results indicate that antipsychotic drugs exert

suppressive effects on BDNF gene expression in the brain regions

studied. Thus, the results raise the possibility that reduced BDNF

mRNA levels observed in brain tissue obtained from patients with

schizophrenia might re¯ect the consequences of long-term treatment

with antipsychotic drugs and may not be necessarily related to the

pathogenesis of the disease. However, to the extent that rats with the

neonatal lesion of the hippocampus model some pathophysiological

aspects of schizophrenia, our data also suggest that a neurodevelop-

mental insult affecting the ventral portion of the hippocampus might

suppress BDNF production in distant sites.

Acknowledgements

We would like to thank Mr Michael Valentine for his technical assistance.

Abbreviations

AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF,brain-derived neurotrophic factor; DEPC, diethyl pyrocarbonate; 5-HT,5-hydroxytryptamine (serotonin); SSC, sodium chloride±sodium citrate.

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