astrocytic basic fibroblast growth factor expression in dopaminergic regions after perinatal anoxia

Astrocytic Basic Fibroblast Growth Factor Expressionin Dopaminergic Regions after Perinatal Anoxia

Cecilia Flores, Jane Stewart, Natalina Salmaso, Ying Zhang, and Patricia Boksa

Background: Perinatal anoxia leads to persistent behav-ioral and neurochemical alterations suggestive of sensi-tized dopaminergic function. Because astrocytic basicfibroblast growth factor (bFGF) activity in the midbraindopaminergic cell body region is required for the devel-opment of enduring changes in dopaminergic functioninduced by stimulant drugs, we investigated the effects ofintrauterine anoxia on astrocytic bFGF expression indopaminergic regions at 2 weeks of age and after a stressmanipulation in adults.

Methods: We examined bFGF immunoreactivity in dopa-minergic regions of young and adult rats born by cesareansection, cesarean section � 15 min of intrauterine anoxia,or vaginally. bFGF immunoreactivity was also assessedbefore and after tail-pinch stress in adult animals exposedto the same perinatal interventions.

Results: Perinatal anoxia produced persistent decreasesin basal bFGF immunoreactivity in the ventral tegmentalarea (VTA), but enhanced the effect of stress on VTAbFGF immunoreactivity.

Conclusions: Perinatal anoxia has enduring effects onVTA bFGF immunoreactivity and influences adult neuro-adaptations to stress. The mechanisms whereby perinatalanoxia alters dopaminergic function may be similar tothose responsible for the development of sensitization tostimulant drugs and may involve bFGF. Biol Psychiatry2002;52:362–370 © 2002 Society of BiologicalPsychiatry

Key Words: Anoxia, basic fibroblast growth factor, do-pamine, FGF-2, sensitization, stress

Introduction

Sensitized functioning of the mesolimbic dopaminergicsystem appears to play an important role in the

psychotic symptoms of schizophrenia. In schizophrenic

patients, acute exposure to amphetamine, a stimulant drugthat causes excessive release of dopamine in striatal andcortical regions, evokes or exacerbates positive symptomsat doses that do not induce psychosis in healthy subjects(Lieberman et al 1987, 1997; Yui et al 1999). Interest-ingly, recent imaging studies demonstrate that a significantnumber of nonmedicated schizophrenic subjects show amarked elevation of amphetamine-induced striatal dopa-mine release in comparison to healthy volunteers (Abi-Dargham et al 1998; Laruelle 2000; Laruelle et al 1999).These findings, combined with the evidence that blockadeof D2 dopamine receptors is highly correlated with theability of neuroleptic drugs to alleviate positive symptoms(Kapur and Seeman 2001; Sanyal and Van Tol 1997;Seeman and Kapur 2000), suggest that in schizophreniathere may exist a sensitization or dysregulation of me-solimbic dopaminergic system functioning.

It is well known that chronic use of stimulant drugs suchas amphetamine leads to sensitization of its stimulanteffects in rats and monkeys (Castner et al 2000; Castnerand Goldman-Rakic 1999; Kalivas and Stewart 1991;Vanderschuren and Kalivas 2000). In otherwise healthyhumans, chronic use of stimulant drugs such as amphet-amine has been reported to provoke paranoid behaviorsthat can culminate in psychosis in some individuals (Bradyet al 1991; Ellinwood 1967; Satel et al 1991; Yui et al1999). Although psychotic-like symptoms generally dissi-pate after discontinuation of drug use, chronic abusers whoat one time experienced psychosis remain vulnerable tothese effects when challenged with drug or even stressorsseveral years later (Ellinwood 1967; Kramer et al 1967;Sato et al 1983). Questions that arise from such observa-tions are whether one can identify factors responsible forindividual differences in these responses to amphetamine-like stimulants and stress and whether the exaggeratedresponses to amphetamine seen in schizophrenic patientsreflect some underlying vulnerability within the mesolim-bic dopaminergic system.

Numerous epidemiologic studies have concluded thatschizophrenic patients have an increased history of obstet-ric complications, particularly those related to labor anddelivery or to the neonatal period (Dalman et al 1999;Geddes et al 1999; Jones et al 1998; McNeil et al 2000;

From the Center for Studies in Behavioral Neurobiology Department of Psychology(CF, JS, NS), Concordia University, Montreal, Quebec; and Departments ofPsychiatry and of Neurology and Neurosurgery (YZ, PB), McGill University,Douglas Hospital Research Centre, Verdun, Quebec, Canada.

Address reprint requests to Dr. Cecilia Flores, Department of Neurology andNeurosurgery, McGill University, Centre for Neuronal Survival Room F116,Montreal Neurological Institute, 3801 University Street, Montreal Quebec,H3A 2B4, Canada.

Received October 26, 2001; revised January 23, 2002; accepted February 1, 2002.

© 2002 Society of Biological Psychiatry 0006-3223/02/$22.00PII S0006-3223(02)01363-X

Rosso et al 2000; Zornberg et al 2000). One commondenominator of these complications is perinatal hypoxia.To study the consequences of oxygen deprivation on thedeveloping dopaminergic system, animal models of peri-natal hypoxia have been used. Studies of rat models ofcesarean section (C-section) birth and of intrauterineanoxia have found changes in dopaminergic functionconsistent with increased responsiveness of the mesolim-bic dopaminergic system to stress and stimulant drugs.Specifically, compared with vaginally born rats, adult ratsborn by C-section, either with or without addition of aperiod of global anoxia, show elevated dopamine releasein the nucleus accumbens in response to repeated stress(Brake et al 1997b) and augmented locomotor activityfollowing amphetamine challenge (El-Khodor and Boksa1998). Furthermore, in adult rats born by C-section withadded intrauterine anoxia, repeated exposure to stressleads to sensitized behavioral responses to both stress andamphetamine (Brake et al 1997a; El-Khodor and Boksa2000). In addition, region-specific changes in numbers oftyrosine hydroxylase-immunoreactive neurons and levelsof tyrosine hydroxylase mRNA have been observed inanimals subjected to perinatal anoxia (Bjelke et al 1991;Gross et al 2000). It remains to be determined howperinatal oxygen deprivation results in altered functioningof the mesolimbic dopaminergic system.

As mentioned above, robust and enduring, perhapspermanent, changes in mesolimbic dopaminergic functionoccur after repeated exposure to stimulant drugs, includingamphetamine. When laboratory animals are repeatedlyexposed to amphetamine, they develop a progressiveincrease in sensitivity to the behavioral-activating effectsof this drug. This phenomenon, known as behavioralsensitization, is accompanied by increased responsivenessof the mesolimbic dopaminergic system, manifested asenhanced dopaminergic overflow in striatal terminal re-gions in response to subsequent drug challenge (Kalivasand Stewart 1991; Robinson et al 1988; Vanderschurenand Kalivas 2000). Moreover, behavioral cross-sensitiza-tion between stress and stimulant drugs has been demon-strated in rats and is associated with an increase inextracellular dopamine in the nucleus accumbens (Kalivasand Stewart 1991). Thus, there are clear similaritiesbetween the augmented dopaminergic responsiveness ac-companying behavioral sensitization and that observed atadulthood in animals born with birth anoxia. It is possible,therefore, that similar mechanisms could mediate thedevelopment of increased mesolimbic dopaminergic func-tion in models of behavioral sensitization and perinatalanoxia and perhaps, by analogy, in schizophrenia.

We have previously shown that repeated intermittentinjections of amphetamine result in persistent increases inastrocytic expression of the neuroprotective and neurotro-

phic substance, basic fibroblast growth factor (bFGF, alsoknown as FGF-2). These changes are observed in theventral tegmental area (VTA) and substantia nigra parscompacta (SNc), the cell body region of midbrain dopa-minergic cells; and are blocked by glutamatergic NMDAreceptor antagonists (Flores et al 1998). Most important,studies employing an anti-bFGF antibody to block bFGFactions indicate that endogenous bFGF in the VTA isrequired for the development of sensitization to the effectsof amphetamine (Flores et al 2000).

The purpose of this study was to explore whether birthcomplications would lead to changes in expression ofbFGF, a molecule known to mediate lasting changes indopaminergic function. Hence, we assessed bFGF immu-noreactivity in brains of rats born by C-section, byC-section with 15 min of added intrauterine anoxia, orvaginally, at the ages of 2 weeks and 3.5 months. Weanalyzed bFGF expression in the VTA and SNc becausesensitization to the behavioral and dopaminergic effects ofstimulant drugs is known to be initiated in the cell bodyregion of midbrain dopaminergic neurons (Kalivas andWeber 1988; Vezina 1993, 1996; Vezina and Stewart1990); because repeated amphetamine treatment results inincreased bFGF expression in these two regions (Floresand Stewart 2000); and finally because bFGF in the VTAis necessary for the development of sensitization to am-phetamine (Flores et al 2000).

In addition, because repeated stress given to adult ratsborn by C-section with global anoxia results in sensitizedbehavioral responses to subsequent tail-pinch stress and toamphetamine (Brake et al 1997a; El-Khodor and Boksa2000), we examined whether birth condition would havelong-lasting effects on bFGF expression in cell body andterminal regions of midbrain dopaminergic neurons;whether changes in bFGF expression in dopaminergicregions would be seen in response to repeated stress atadulthood; and whether any effect of repeated stress onbFGF expression would be dependent on birth condition.

Methods and Materials

Animals and Intrauterine AnoxiaAll procedures were performed in accordance with the guidelinesestablished by the Canadian Council on Animal Care and wereapproved by the Concordia University and the McGill UniversityAnimal Care Committees. Efforts were made to minimize animalsuffering and to reduce the number of animals used.

Timed pregnant Sprague–Dawley rat dams (Charles River,Quebec, Canada) at 22 days of gestation were used to generatepups born under three birth conditions: C-section with 15 min ofintrauterine anoxia, C-section alone, and vaginal birth. To avoidpossible prematurity, C-sections were only begun after one of agroup of timed pregnant dams, mated at the same time, had givenbirth vaginally and only pups from litters with all members

Perinatal Anoxia and bFGF Expression 363BIOL PSYCHIATRY2002;52:362–370

weighing � 5 g at delivery were used. Only male pups wereretained for the study.

Intrauterine anoxia was induced in rats delivered by C-sectionaccording to a modified version of the methods described byBjelke et al (1991). Detailed descriptions of the procedure havebeen given previously (El-Khodor and Boksa 1997, 1998, 2000,2001). Briefly, at 22 days of gestation, dams were decapitated (toavoid the confound of anesthetic use) and hysterectomized. Theuterus was quickly isolated and immersed into a 37°C bath for 15min. The pups were then delivered from the uterus and weregently tapped to stimulate breathing (30–40 sec). Pups deliveredby this procedure were designated as the C-section � 15 minanoxia group (C-sec � 15� group). Survival was 97% following15 min of birth anoxia. (In general, the immature central nervoussystems (CNS) of any species can sustain longer periods ofhypoxia than does the adult. The rat is able to sustain longerperiods of hypoxia at birth than can the human neonate. Ratssubjected to 15–20 min of anoxia during C-section beginbreathing at birth without artificial resuscitation other thanpalpation. Thus, 15 min of hypoxia may be considered a“moderate” hypoxic episode in the neonatal rat. Quantification ofbrain lactate, a marker of CNS hypoxia, and brain adenosinetriphosphate indicate that this model produces consistent andreproducible CNS hypoxia in offspring (Berger et al 2000;El-Khodor and Boksa 1997).

A second group of pups was delivered via C-section without aperiod of added anoxia (C-sec group). Pups in this group wereimmediately delivered, and breathing was induced by gentletapping for a few seconds. The time between decapitation of thedam and delivery of the last pup in a litter was less than 1.5 min,and survival was 100% in the C-sec group. After delivery, pupsfrom C-sec � 15� and C-sec groups had their umbilical cordsligated and were placed on a heating pad for 1–2 hours untilgiven to surrogate dams.

Pups born vaginally served as control animals (vaginal group).These pups were removed from their dams at 0–12 hours afterbirth and were placed on a heating pad for 1–2 hours beforebeing placed with surrogate dams. To minimize effects ofdifferential rearing, pups from all groups were cross-fosteredwith surrogate dams in mixed litters of 12 pups per dam. Toidentify animals from different birth groups, a small quantity ofindelible India ink was injected into one of the paws of each pup.Animals in each birth group were born from at least 4 dams in theexperiment with 2-week-old offspring (experiment 1 below) andfrom at least 7 to 8 dams in the experiment with adult offspring(experiment 2 below).

Tissue Preparation and ImmunohistochemistryAnimals received an overdose of pentobarbital (120 mg/kg) andwere perfused transcardially with 200 mL of ice-cold phosphate-buffered saline followed by 100 mL of an ice-cold solution of 4%paraformaldehyde (w/v) and 15% picric acid (vol/vol) in 0.1mol/L phosphate buffer (PB, pH 6.9). Brains were stored overnight in fixative solution. Coronal 50-�m sections were cut on aVibratome, collected in ice-cold PB, and stored overnight at 4°C.Brains sections were processed for bFGF immunohistochemistryaccording to the avidin-biotin method (Hsu et al 1981), as we

have previously described (Flores et al 1998, 1999). Free-floating sections were incubated for 24 hours at 4°C with amouse monoclonal antibody (Upstate Technology, Lake Placid,NY) that recognizes biologically active bFGF (Matsuzaki et al1989). The bFGF antibody was diluted 1:500 with 0.3% TritonX-100 (Sigma) in PB and 1% normal horse serum (Vector,Burlingame, CA). Sections were then rinsed (3 � 5 min) in coldPB and incubated for 1 hour at room temperature in a solution ofrat adsorbed biotinylated antimouse antibody (Vector) diluted1:200 with PB and 1% normal horse serum. After washings (3 �5 min) in cold PB, sections were incubated in an avidin-horseradish peroxidase complex (Vectastain Elite ABC Kit,Vector) for 30 min at room temperature and then rinsed again(3 � 5 min) in cold PB. Sections were then incubated for 10 min,at room temperature and under constant agitation, in a solution of0.05% 3,3�-diaminobenzidine (Sigma) in PB. Then, withoutwashing, sections were incubated in a 3,3�-diaminobenzidine/PBsolution (pH 7.8) with 0.01% H2O2 and 8% NiCl2. Thisincubation lasted 8 min.

Sections were mounted on gelatin-coated slides, dried for atleast 1 day, gradually dehydrated in ethanol, cleared in Hemo-De, and coverslipped with Permount. To reveal anatomicallandmarks, midbrain sections were lightly counterstained forNissl substance by using 0.1% cresyl violet.

Image AnalysisbFGF immunostained sections were observed under a Leicamicroscope (Leitz DMRB). Quantitative analysis was conductedusing an image analysis system (NIH Image 1.6) on digitizedimages of sampling areas of VTA and SNc, nucleus accumbens(NAcc; including both the shell and core), dorsal striatum (STR),and medial prefrontal cortex (PFC) layer V and VI of thepregenual cingulate cortex area 2. Boundaries of cortical andsubcortical structures were defined using Zilles (1985) andPaxinos and Watson (1997) as guides, respectively. Samplingareas of the VTA and SNc were taken from sections correspond-ing to plates 38 and 39; of the NAcc and STR from sectionscorresponding to plates 11, 12, and 13; and of the PFC fromsections corresponding to plate 11 (Paxinos and Watson 1997).Images for each structure were taken from three sections fromeach brain. All analysis was conducted blind to experimentalcondition.

Experimental DesignsEXPERIMENT 1: EARLY EFFECTS OF PERINATAL AN-

OXIA ON bFGF EXPRESSION. This experiment (see Figure 1).was conducted to assess early effects of birth condition on bFGFexpression in the VTA and SNc, the cell body region of midbraindopaminergic neurons. Animals born by C-sec, C-sec � 15�, orvaginally were removed from their litters at the age of 2 weeks,perfused transcardially, and their brains were processed for bFGFimmunoreactivity.

EXPERIMENT 2: EFFECTS OF PERINATAL ANOXIA ON

bFGF EXPRESSION IN ADULT RATS: BASAL LEVELS AND

RESPONSE TO REPEATED STRESS. Pups born by C-sec,C-sec � 15�, or vaginally were weaned at 21 days of age, and

364 C. Flores et alBIOL PSYCHIATRY2002;52:362–370

randomly assigned to mixed groups of 3 animals per cage (seeFigure 1). All animals were maintained on a 12-hour light/dark cycle with food and water provided ad libitum. At 3months of age, half of the animals in each birth group wereadministered repeated tail-pinch stress for 8 consecutive days.On each test day, animals were removed from their homecages, placed individually in holding cages, and left un-touched for 10 min. Following this, a plastic clothes pin wasplaced 1 cm from the base of the tail for 15 min. Animals werethen returned to their home cages. The stress procedure wasalways applied in the same room and at the same time (from9 –12 AM). Nonstressed animals were left undisturbedthroughout the experiment. One week after the last day oftail-pinch stress, all animals (stressed and nonstressed groups)were perfused transcardially, and their brains were processedfor bFGF immunohistochemistry.

Statistical Analysis

Analyses were done on the number of bFGF immunoreactivecells per square millimeter. Data were analyzed using one- ortwo-way analyses of variance (ANOVAs). When appropriate,post hoc analyses of significant effects were carried out usingFisher’s Protected Least Significant Difference (PLSD) test.

Results

bFGF Expression in Dopaminergic Regions

Consistent with previous observations, the bFGF antibodyused in our study detected bFGF immunoreactivity inastrocytes in all areas examined (Flores et al 1998, 1999;Szele et al 1995; Woodward et al 1992).

Effects of Perinatal Anoxia on Astrocytic bFGFExpression at 2 Weeks of Age

The ANOVA for the data on bFGF expression in the VTAand SNc at 2 weeks revealed that the effects of birthcondition did not reach statistical significance (Figure 2;VTA p � .11, SNc p � .15). When the C-sec � 15� groupwas compared with the C-sec and vaginal groups com-bined, it was found to have significantly fewer bFGF-immunoreactive cells in both regions (VTA p � .05, SNcp � .03).

Figure 1. Diagram outlining the experimental manipulationsconducted in experiments 1 and 2.

Figure 2. Basic fibroblast growth factor (bFGF) immunoreactiv-ity in the ventral tegmental area (VTA) and substantia nigracompacta (SNc) of 2-week-old rat pups that had been born undervarious conditions: cesarean section (C-sec), C-sec � 15� anoxia,or vaginally. The data are expressed as the mean � SEM numberof bFGF-immunoreactive cells per square millimeter. *Signifi-cant differences between the C-sec � 15� group and the othertwo groups combined [VTA, F(1,13) � 4.6, p � .05; SNc,F(1,13) � 5.6, p � .03). n per group � 5.


Effects of Perinatal Anoxia on Astrocytic bFGFExpression in Adult Rats

The decreased basal expression of bFGF in the VTAobserved in 2-week-old pups born by C-sec � 15� wassimilar and more pronounced in adult animals. As shownin Figure 3, there were significantly fewer bFGF-immu-noreactive cells in the VTA of adult (3.5-month) animalsborn by C-sec � 15� than in adult animals born vaginally(p � .01). A similar, but nonsignificant trend was alsoseen for the difference between C-sec � 15� and C-secgroups (p � .07).

In addition to the effect on basal VTA bFGF immuno-reactivity, birth condition modulated the effects of therepeated stress manipulation on VTA bFGF expression. Itcan be seen in Figure 3 that repeated tail-pinch stress (oncea day for 8 consecutive days) produced a large andsignificant (p � .03) increase in bFGF immunoreactivityin animals born by C-sec � 15�, whereas the manipulationhad no significant effect on bFGF immunoreactivity in theother two groups. Thus, perinatal anoxia had long-lastingconsequences on basal expression of bFGF in the VTAand altered the effects of adult exposure to repeatedtail-pinch stress on expression of bFGF protein.

As depicted in Figure 4, basal bFGF immunoreactivity

in the SNc of adult rats appeared to be lower in the C-secand C-sec � 15�groups than in vaginal control rats, but theeffect of birth condition was not statistically significant. Incontrast to the effects observed in the VTA, exposure torepeated tail-pinch stress did not alter bFGF expression inthe SNc in any of the birth groups.

The effects of birth condition on astrocytic bFGFimmunoreactivity during basal conditions and in responseto repeated tail-pinch stress in dopaminergic terminalregions in the NAcc, PFC, and STR were analyzed inbrains of 3.5-month-old rats 1 week after the last stressexposure (see Figure 5). Under basal conditions, thenumber of bFGF-immunoreactive cells was significantlyincreased in the NAcc of the C-sec � 15� group comparedwith the C-sec (p � .05) and vaginal (p � .02) groups. Nosignificant effects of birth condition were found in theother two brain regions (PFC and STR). Exposure to stressdid not significantly alter bFGF expression in terminalregions in any of the birth groups.

Discussion

Adult rats subjected to birth complications that involveperinatal hypoxia exhibit behavioral and neurochemical

Figure 3. Basic fibroblast growth factor (bFGF) immunoreactiv-ity in the ventral tegmental area (VTA) of adult rats that had beenborn under various conditions: cesarean section (C-sec), C-sec �15� anoxia, or vaginally. The figure shows bFGF immunoreac-tivity during basal conditions in animals at 3.5 months of age(control, open bars) and after repeated stress at adulthood (stress,dark bars). The data are expressed as the mean � SEM numberof bFGF-immunoreactive cells per square millimeter. A two-wayanalysis of variance for birth condition by stress manipulationrevealed a significant interaction [F(2,23) � 3.58, p � .04].*different from vaginal control, p � .01; †different from C-seccontrol, p � .07 (Fisher PLSD). §different from C-sec � 15�control, p � .03 (Student’s t test for independent samples). n pervaginal stress, C-sec-stress, and C-sec � 15� control groups � 5;n per vaginal control, C-sec � 15�-stress groups � 4; n per C-seccontrol group � 6. PLSD, protected least significant difference.

Figure 4. Basic fibroblast growth factor (bFGF) immunoreactiv-ity in the substantia nigra compacta (SNc) of adult rats that hadbeen born under various conditions: cesarean section (C-sec),C-sec � 15� anoxia, or vaginally. The figure shows bFGFimmunoreactivity during basal conditions in animals at 3.5months of age (control, open bars) and after repeated stress atadulthood (stress, dark bars). The data are expressed as themean � SEM number of bFGF-immunoreactive cells per squaremillimeter. Two-way analysis of variance for birth condition bystress manipulation revealed no significant main effects [birthcondition: F(2,23) � 1.86, p � .17; stress manipulation:F(1,23) � .09, p � .76] and no significant interaction [F(2,23) �.1, p � .9]. n per vaginal-stress, C-sec-stress, and C-sec � 15�control groups � 5; n per vaginal control, C-sec � 15�-stressgroups � 4; n per C-sec control group � 6.


responses suggestive of sensitized functioning within themesolimbic dopaminergic system (Brake et al 1997a,1997b; El-Khodor and Boksa 1998, 2000). The experi-ments reported here were carried out to examine whetherthese effects of perinatal complications might be mediatedby mechanisms similar to those shown to be involved inthe development of sensitization to amphetamine. Becauseastrocytic bFGF expression is increased in the cell bodyregions of midbrain dopaminergic neurons in response torepeated amphetamine treatment (Flores et al 1998; Floresand Stewart 2000) and because blockade of bFGF activityin this region during drug treatment prevents the develop-ment of sensitization to the effects of amphetamine (Floreset al 2000), we examined the effects of birth complicationsand of an adult stress manipulation on bFGF immunore-activity in dopaminergic regions.

We found that 15 min of perinatal anoxia reduced basalastrocytic bFGF expression in the VTA and SNc 2 weeksafter birth. This effect persisted and was more pronouncedin the VTA in adult animals, so that by 3.5 months of age,animals born by C-section with 15 min of anoxia showedsignificantly reduced bFGF expression in the VTA, but notthe SNc, compared with either vaginally born control orC-sectioned animals. Interestingly, when the animals fromthe three treatment groups were repeatedly exposed to themild stressor, bFGF expression was enhanced in the VTAonly in adult animals exposed to anoxia at birth. Thesefindings indicate that an anoxic insult in early life haslong-lasting effects on astrocytic bFGF expression andinfluences adult neuroadaptations to stress.

bFGF is a major neurotrophic and neuroprotectivefactor of midbrain dopaminergic neurons. It promotes theirgrowth and survival in culture and in the adult brain(Bouvier and Mytilineou 1995; Hou et al 1997; Reuss andUnsicker 2000; Takayama et al 1995). bFGF expressionhas been shown to increase after injury to midbrain

dopaminergic cells (Chadi et al 1994) and to participate intheir survival and sprouting (Chadi et al 1993; Otto andUnsicker 1990). In our study, bFGF expression wasreduced as early as 2 weeks of age in the cell body regionsof midbrain dopaminergic cells in animals exposed toanoxia at birth. Although at present we have no directevidence bearing on the issue, it is interesting to speculatethat the decrease in bFGF expression might be associatedwith delayed maturation of dopaminergic neurons. Suchdelayed maturation might indirectly render these neuronsmore plastic in later life and contribute to the observedenhanced responsiveness of the midbrain dopaminergicsystem to stress and amphetamine following birth hypoxia(Brake et al 1997a, 1997b; El-Khodor and Boksa 1997,1998, 2000). This suggestion is supported by the findingthat when rats in our study were exposed to a relativelymild stress manipulation, the anoxia group appeared moresensitive to it, as measured by increases in VTA bFGFimmunoreactivity.

The developmental expression of bFGF in the rat brainsuggests that bFGF is involved in postnatal neuronalmaturation. bFGF mRNA and protein expression is quitelow in newborn rats and increases significantly over thenext few postnatal weeks, reaching a peak around postna-tal day 20 (Caday et al 1990; Eckenstein et al 1994; Kuziset al 1995; Riva and Mocchetti 1991). Thus, the persistentdecrease in bFGF expression observed in the dopaminer-gic cell body region of animals born by C-sec � 15�anoxia is consistent with the idea that perinatal anoxiaslows down or temporarily arrests developmental pro-cesses in this region. In some instances, the effects ofinsults close to the time of birth, including injury todopaminergic neurons (Neal-Beliveau and Joyce 1999),have been shown to be particularly disruptive (Kolb andCioe 2000; Kolb et al 2000a, 2000b). For instance,whereas cortical injury at 7 to 12 days of age leads to

Figure 5. Basic fibroblast growth factor (bFGF) immunoreactivity in the nucleus accumbens (NAcc, including both core and shell),dorsal striatum (STR), and medial prefrontal cortex (PFC) of adult rats that had been born under various conditions: cesarean section(C-sec), C-sec � 15� anoxia, or vaginally. The figure shows bFGF immunoreactivity during basal conditions in animals at 3.5 monthsof age (control, open bars) and after repeated stress at adulthood (stress, dark bars). The data are expressed as the mean � SEM numberof bFGF-immunoreactive cells per square millimeter. Two-way ANOVA for birth condition by stress manipulation revealed nosignificant main effects [NAcc, birth condition: F(2,22) � 1.19, p � .32; stress manipulation: F(1,22) � .08, p � .77; STR, birthcondition: F(2,21) � .35, p � .7; stress manipulation: F(1,21) � 2.02, p � .16; PFC, birth condition: F(2,22) � .18, p � .83; stressmanipulation: F(1,22) � .46, p � .5]; however, in the NAcc, one-way analysis of variance revealed a significant effect of birthcondition [F(2,12) � 3.6, p � .05]. *Post hoc tests show significant difference from vaginal control and C-sec control groups (ps �.05). n per group � 4 to 6.


relatively normal cortical morphology and significantfunctional recovery, cortical injury on the first few days oflife leads to abnormal morphology and severe behavioraldisruption in adult animals (Kolb and Cioe 2000). Simi-larly, newborn mice have been found to be more vulner-able than 1-week-old animals to neuronal cell deathfollowing nerve crush injury, and interestingly, this effectis attenuated in transgenic mice that overexpress bFGF(Kuzis et al 1999).

In contrast to our findings of decreased bFGF proteinexpression in adult rats following birth by C-section with15 min of anoxia, increases in bFGF mRNA levels inhomogenates of SN/VTA from 4-week-old rats born byC-section with 19 to 20 min of anoxia, compared withC-section control rats, have been reported (Andersson et al1995). Although we did not measure bFGF expression at4 weeks of age in the present experiments, the persistentdecrease in bFGF protein expression observed at 2 weeksand 3.5 months following birth anoxia suggests that bFGFprotein expression would also be low at 4 weeks. Althoughthis remains to be tested, it should be noted that differingtime periods of global anoxia (e.g., 15 min in our study vs.19–20 min in the Andersson et al study) have been shownto produce important differences in the magnitude anddirection of biochemical changes observed in the C-section/anoxia model (Bjelke et al 1991; Chen et al 1997;Dell’Anna et al 1995).

The finding that among all the terminal regions studiedin the adult animals, basal levels of bFGF expression werehigher only in the NAcc of the C-sec � anoxia group isinteresting. At this time, however, speculation about itsfunctional importance is premature. Nonetheless, it can benoted that the responsiveness of astrocytes to particulartreatments is regionally specific, as the functional proper-ties of these cells depends largely on their neuronalenvironment (Hosli and Hosli 1993, 2000).

As mentioned, adult animals born by C-sec � 15�exhibit enhanced amphetamine-induced behavioral re-sponses suggestive of increased dopamine release in theNAcc (El-Khodor and Boksa 1998). Moreover, adult ratssubjected to perinatal anoxia and repeatedly treated withsaline injections show sensitized behavioral responding toan amphetamine challenge (Brake et al 1997a). Indeed, invivo voltammetry studies indicate that birth by C-sectionwith 15 min of global anoxia results in elevated dopaminerelease in the NAcc in response to repeated stress exposurein adult rats (Brake et al 1997b). Here we show that thissame perinatal insult induces long-lasting suppression ofbasal expression of astrocytic bFGF in the VTA andincreased bFGF expression in response to repeated stress.Astrocytic bFGF expression in the VTA is altered inresponse to repeated amphetamine treatment and is re-quired for the development of sensitization to the effects

of amphetamine (Flores and Stewart 2000). We canspeculate, therefore, that the functioning of the mesolim-bic dopaminergic system of animals born by C-sec � 15�anoxia is enhanced in response to stress at adulthood, atleast in part, in a manner similar to what is seen in thedevelopment of sensitization to amphetamine.

In summary, the data from our study demonstrate thatperinatal anoxia leads to enduring changes in basal levelsof bFGF expression in the VTA, the cell body region ofdopaminergic neurons, and renders bFGF responses in thisregion more sensitive to the effects of stressors in later life.These results suggest that the effects of perinatal oxygendeprivation on the functioning of the mesolimbic dopami-nergic system may be mediated, at least in part, byastrocytic bFGF. These results also suggest that themechanisms underlying the development of long-lastingalterations in mesolimbic dopaminergic functioning ob-served in animals subjected to birth anoxia may be similar,in part, to those responsible for the development ofsensitization to the effects of stimulant drugs.

This work was supported by Grant Nos. MOP-6678 (to JS) andMT-13361 (to PB) from Canadian Institutes of Health Research (CIHR).CF was supported by a Schizophrenia Society of Canada/CIHR postdoc-toral fellowship.

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astrocytic basic fibroblast growth factor expression in dopaminergic regions after perinatal anoxia

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