Molecular Psychiatry (2001) 6, 285–292  2001 Nature Publishing Group All rights reserved 1359-4184/01 $15.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Developmental and stress-related changes of neurotrophic factor gene expression in an animal model of schizophrenia R Molteni1, BK Lipska2, DR Weinberger2, G Racagni1,3 and MA Riva1

1Center for Neuropharmacology, Department of Pharmacological Sciences, University of Milan, Milan, Italy; 2Clinical Disorders Branch, IRP, NIMH, NIH, Bethesda MD, USA; 3IRCCS San Giovanni di Dio-Fatebenefratelli, Brescia, Italy

The neonatal (PND 7) lesion of the ventral hippocampus (VH) with ibotenic acid represents a well-established experimental paradigm that recapitulates many schizophrenia-like phenom- ena. In order to investigate molecular changes that could contribute to long lasting conse- quences on brain function, we have investigated the effects of the VH lesion on the expression for the trophic factors FGF-2 and BDNF. We used RNase protection assay to measure their mRNA levels in cortical regions of prepubertal (PND 35) and young adult (PND 56) animals, both under basal condition as well as in response to an acute restraint stress. The expression of BDNF was not altered by the VH lesion in prefrontal (PFC) and frontal cortex (FC) of PND 35 or PND 56 rats. An acute restraint stress at PND 35 produced a significant increase of the expression in PFC of sham as well as lesioned animals. However in young adult animals a significant elevation of BDNF expression was observed only in sham rats. We also found that the VH lesion produced a significant reduction of basal BDNF mRNA levels in the cingulate cortex of young adult, but not prepubertal rats. This effect was not accompanied by changes in the acute modulation of the neurotrophin, which was up-regulated by stress in both experimental groups. Conversely the expression of FGF-2 at PND 35 and PND 56 was not altered by early postnatal VH lesion, and there were no major differences between sham and lesioned animals in response to the acute stress. The changes in trophic factor expression may be relevant for the long-term effects of VH lesion on synaptic plasticity and may determine an increased vulnerability of the brain under challenging situations. Molecular Psychiatry (2001) 6, 285–292. Keywords: brain derived neurotrophic factor; fibroblast -2; brain plasticity; ibotenic acid; hippocampus; stress

Introduction during early stages of gestation, that will affect the function of prefrontal-intracortical connectivity.6–9 Schizophrenia is a complex disorder characterized by Although several factors can contribute to cortical hallucinations, delusions, cognitive deficits and dra- maldevelopment, one possibility is the involvement of matic functional impairment. The neurodevelopmental neurotrophic factors.10 These are important hypothesis of schizophrenia provides a starting point regulators of brain development and neuronal shaping to understand structural and functional abnormalities and play a role in synaptic plasticity as well as in the which have been observed in the brain of schizo- maintenance of cellular homeostasis.11–15 There is a phrenic patients, particularly at the level of prefrontal characteristic temporal and spatial pattern of cortex and hippocampal formation.1–3 expression for these molecules during brain develop- In vivo imaging and postmortem studies on schizo- ment.16,17 Abnormalities in their expression profiles phrenic subjects have demonstrated the presence of during development may affect the functional con- neuropathological abnormalities in the hippocampus nectivity of specific neuronal phenotypes that become that may correlate with the deficits in episodic mem- evident after the brain has reached full maturation. For ory, a form of explicit memory linked to hippocampal example the lack of FGF-2 signaling in FGF-2-deficient function.4,5 Such abnormalities may be the result of mice affects migration of a fraction of neuronal precur- alterations in brain development, probably occurring sor during cerebral cortex development18 and reduces the number of cortical neurons and glial cells at maturity.19 Correspondence: MA Riva, Center for Neuropharmacology, Via Because of the importance of growth factors for the Balzaretti 9, 20133 Milan, Italy. E-mail: marivaȰmailserver. unimi.it cortical abnormalities implicated in schizophrenia, we Received 21 September 2000; revised and accepted 27 Nov- studied the expression profile of brain-derived neuro- ember 2000 trophic factor (BDNF) and fibroblast growth factor-2 Neonatal VH lesion alters cortical trophic factor expression R Molteni et al 286 (FGF-2) in a developmental animal model of schizo- Surgery phrenia. Rat pups were lesioned as described previously.48 BDNF, a member of the neurotrophin family, has a Briefly, pregnant Sprague–Dawley rats obtained at 12– wide distribution in the CNS, where it is synthesized 14 days of gestation (Harlan, Indianapolis, IN, USA) predominantly by neurons and is highly expressed in were housed individually in breeding cages with a 12- hippocampus, cerebral cortex, limbic areas and cere- h light/dark cycle and fed ad libitum. Litters of four to bellum.20,21 This neurotrophin plays an important role eight male pups were formed. On the 7th day of age in regulating the survival and differentiation of selec- (PND 7) pups (weight 15–18 g), within each litter were tive populations of neurons during development22,23 randomized to Sham or Lesion status and anaesthet- and is an important mediator of neuronal adaptive ized by hypothermia (placed on ice for 15–20 min). An responses.12–14 The expression of BDNF is regulated by incision was made in the skin overlying the skull and neuronal activity, shows marked and transient changes 0.3 ␮lof10␮g ␮l−1 of ibotenic acid solution (in the in response to a number of neuronal insults, including lesioned rats) or artificial cerebrospinal fluid (in sham- ischemia, hypoglycemia, epileptic activity, immobiliz- operated rats) was infused bilaterally, through a Hamil- 17,24–28 ation stress and trauma and its modulation ton needle, using an infusion pump into the ventral involves a complex interplay between different neuro- hippocampal formation (AP −3.0 mm, ML ± 3.5 mm, 29,30 transmitter systems. VD −5.0 mm, relative to Bregma) at the rate of 0.15 ␮l FGF-2 is a trophic molecule that has a critical role min−1. The pups were warmed and then returned to in the maturation and survival of a variety of neuronal their mothers. On the 25th day of age rats were weaned, phenotypes.15,31 Its expression is regulated by neuronal 32,33 34–36 separated according to the lesion status and grouped activity, neurotransmitters and hormones as two to a cage. well as by learning and physical .37,38 It is well established that FGF-2 has neuroprotective activity over a wide range of neurons39–41 and it may render the 42,43 Experimental procedure cells more resistant to potential lethal damage. The effect of the neonatal VH ibotenic acid lesion on The animal model employed in our study is obtained neurotrophic gene expression as well as the effect of by bilateral infusion of the excitotoxin ibotenic acid in acute stress in sham and lesion animals was assessed the ventral hippocampus (VH) of rats on the 7th day at PND 35 (before puberty) and PND 56 (after puberty). of age44 and reproduces some fundamental features of Rats were randomly assigned to four groups (Sham/No schizophrenia, including dopaminergic overactivity Stress; Sham/Stress; Lesion/No Stress; Lesion/Stress). and emergence of behavioral abnormalities in early The animals undergoing restraint stress were placed for adulthood. Adult rats with the neonatal VH lesion 2 h in transparent plexiglas containers whereas those express exaggerated locomotor response to ampheta- in the control group were left undisturbed in their mine and to acute stress,44 augmented apomorphine- induced stereotypy,45 and altered prepulse inhibition home cages. Rats were killed by decapitation 1 h after of the acoustic startle response.46 Moreover, the the end of immobilization, their rapidly expression of behavioral phenotypes is strain-depen- removed and the following cortical regions were dis- dent.47 Neonatal damage of the hippocampus also dis- sected from 2-mm thick slices: prefrontal cortex PFC rupts complex behaviors such as working memory and (defined as Cg1, Cg3 and IL subregions corresponding social interaction.48 Some of these behavioral abnor- to the plates 6–9 according to the atlas of Paxinos and 51 malities are reversed by treatment with typical and Watson, frontal cortex FC (comprising regions Fr1–3, atypical antipsychotics.49 Using this paradigm, we Par1 and AI from the same slice as PFC) and cingulate have analyzed the expression profile of BDNF and FGF- cortex CC (comprising Cg1 and Cg3 areas from the slice 2 in prefrontal, frontal and cingulate cortex in prepub- corresponding to plates 10–13 in the atlas of Paxinos 51 ertal and mature animals. Since an impairment in and Watson. We have investigated PFC and CC, as coping with specific challenging situations may rep- compared to FC, because they are connected with the resent a long-lasting effect observed after the lesion as hippocampal formation (that is lesioned in our well as other developmental perturbations,50 we have paradigm) and because functional1 and anatomical52 also investigated whether the stress-dependent regulat- changes have been detected at this level in schizo- ory mechanism of neurotrophic factor expression27,28,35 phrenic subjects. All the specimens were frozen on dry could be altered as a consequence of the neonatal hip- ice and stored at −80°C for further analysis. pocampal lesion.

RNA preparation Materials and methods The tissue was homogenized in 4 M guanidinium iso- Materials thiocyanate (containing 25 mM sodium citrate pH 7.5, General reagents were purchased from Sigma (Milan, 0.5% sarcosyl and 0.1% 2-mercaptoethanol) and total Italy), whereas molecular biology reagents were RNA was isolated by phenol-chloroform extraction.53 obtained from Ambion (Austin, TX, USA), New Quantification was carried out by spectrophotometric England Biolabs (Beverly, MA, USA) and Promega analysis and RNA aliquots were re-precipitated in etha- (Milan, Italy). nol for RNase protection assay.

Molecular Psychiatry Neonatal VH lesion alters cortical trophic factor expression R Molteni et al 287 RNA probes synthesis for RNase protection assay single gel, optical densities from all within-age samples A transcription (MAXI script, Ambion) was used were analyzed separately. We analyzed by two-way to generate cRNA probes and 32P-CTP was used as a analysis of variance (ANOVA) the data, with status radiolabelled nucleotide. The following plasmids were (sham and lesion) and treatment (no stress and stress) employed in the RNase protection assay: plasmid pSK- as independent factors and BDNF or FGF-2 levels as rB(C1), containing rat BDNF coding region, was lin- dependent variables. When appropriate, further differ- earized with Xma I and used to generate a 32P-labeled ences were analyzed by Single Contrast post-hoc test 800-bases antisense cRNA probe, which included 740 (SCPHT). Optical densities from experimental groups of BDNF sequence and 60 bases of the polylinker were then expressed and presented as mean percent of region; plasmid RobFGF103, containing rat FGF-2 control values for graphic clarity. The mean percent coding region, was linearized with Nco I and used to represents the ratio between neurotrophic factor and generate a 32P-labeled 524-bases antisense cRNA probe, GAPDH levels expressed as % of control (sham which included 477 of FGF-2 sequence and 47 bases animals). The mean value of the control group within of the polylinker region. A cRNA probe to glyceral- a single experiment was set to 100 and all other values dehyde 3-phosphate-dehydrogenase (GAPDH) was were expressed as ‘percent’ of control animals. used as internal standard. By in vitro transcription with a T3 polymerase a 376-bases probe was obtained from Results the plasmid pTRI-GAPDH-Rat (Ambion) which, fol- lowing RNase protection assay, yielded a protected In order to investigate if changes in neurotrophic factor fragment of 316 bases. levels may represent a molecular consequence of the neonatal ibotenic acid lesion, the expression of BDNF RNase protection assay and FGF-2 was assessed by RNase protection assay in The RNase protection assay was performed on a 10-␮g prefrontal, frontal and cingulate cortex before and sample of total RNA as described previously.36 Briefly, after puberty. after ethanol-precipitation total RNA, obtained from Basal BDNF mRNA levels were not altered by the different tissues, was dissolved in 20 ␮l of hybridiz- neonatal VH lesion in prefrontal cortex (PFC) both at ation solution (80% formamide, 40 mM PIPES pH 6.4, 35 and 56 PND (Figure 1). An acute restraint stress (2 h) 400 mM sodium acetate pH 6.4 and 1 mM EDTA) con- produced a significant increase of BDNF expression = = taining 150 000 cpm of BDNF and FGF-2 and before puberty (F1,29 18.664, P 0.002, two-way 50 000 cpm of GAPDH 32P-labeled cRNA probes ANOVA). Although this increase was more pro- (specific activity Ͼ108 cpm ␮g−1). After being heated at nounced in lesioned animals, single contrast post-hoc 85°C for 10 min, the cRNA probes were allowed to test (SCPHT) showed a significant effect of stress in ° = = hybridize the endogenous RNAs at 45 C overnight. sham (F1,29 6.017, P 0.042) as well as in lesion rats = = At the end of the hybridization, the solution was (F1,29 12.858, P 0.002). Conversely, we observed a diluted with 200 ␮l of RNase digestion buffer (300 mM significant effect of the lesion on stress-induced modi- NaCl, 10 mM Tris HCl pH 7.4, 5 mM EDTA pH 7.4) fication of BDNF mRNA levels in young adult animals. containing a 1/200 dilution of a RNase cocktail (1 mg Although, at PND 56, there was a small increase in ml−1 RNase A and 20 U ml−1 RNase T1) and incubated basal BDNF levels in PFC of lesioned rats, this was not ° ␮ ␮ = for 30 min at 30 C. Proteinase K (10 g) and SDS (10 l significant with respect to sham animals (F1,28 0.226, of 20% stock solution) were then added to the sample P = 0.639). However the induction of BDNF and mRNA and the mixture was incubated at 37°C for an expression after immobilization was significant in = = additional 15 min. At the end of the incubation the sham (F1,28 8.356, P 0.018; SCPHT) but not in = = sample was extracted with phenol/chloroform and lesioned animals (F1,28 1.209, P 0.284; SCPHT) ethanol precipitated. The pellet, containing the (Figure 1), suggesting that at this age there is a reduced RNA:RNA hybrids, was dried and resuspended in load- function of stress-dependent regulation of BDNF as ing buffer (80% formamide, 0.1% xylene cyanol, 0.1% consequence of the neonatal lesion. bromophenol blue, 2 mM EDTA), boiled at 95°C for In FC the lesion did not affect the basal expression 5 min and separated on a 5% polyacrylamide gel under of the neurotrophin at PND 35 or 56. The acute stress denaturing conditions (7 M urea). The protected frag- produced a reduction of BDNF mRNA levels at PND = = ments were visualized by autoradiography. 35 (F1,26 14.332, P 0.0009; two-way ANOVA) which = was significant only in lesioned rats (F1,26 11.597, P RNA calculation and statistical analysis = 0.004; SCPHT), without changing its expression in The levels of mRNA for FGF-2, BDNF or GAPDH were young adult animals (Table 1). calculated by measuring the peak densitometric area of Despite no significant effects in PFC and FC, the VH the autoradiography analyzed with a LKB laser densi- lesion produced a significant reduction of BDNF = tometer. In order to ensure that the autoradiographic expression in CC of young adult animals (F1,28 9.144, = bands were in the linear range of intensity, different P 0.006; two-way ANOVA) but not at PND 35 (F1,28 exposure times were used. GAPDH was employed as = 1.933, P = 0.181; two-way ANOVA) (Figures 2 and internal standard for RNase protection assay: its 3). We observed a slight, although not significant, expression was not altered by lesion or by stress. reduction of BDNF after restraint stress in lesioned rats Because all within-age samples were processed on a at PND 35, resembling the effect in FC. Conversely, as

Molecular Psychiatry Neonatal VH lesion alters cortical trophic factor expression R Molteni et al 288

Figure 1 Effect of acute restraint stress on BDNF mRNA levels in prefrontal (PFC) of sham and lesion rats assessed at PND 35 and PND 56. The results, expressed as % of sham rats, represent the mean ± SEM of seven to eight samples for each experimental group. *P Ͻ 0.05 and **P Ͻ 0.01 vs control (non-stressed) rats (two-way ANOVA with SCPHT).

Table 1 Effect of acute restraint stress on BDNF mRNA lev- els in frontal cortex (FC) of sham and lesion rats assessed at PND 35 and PND 56. The results are expressed as % of sham rats and represent the mean ± SEM of seven to eight samples for each experimental group

Group PND-35 PND-56 (% of sham levels)

Sham 100 ± 7 100 ± 7 Lesion 119 ± 15 94 ± 7 Sham + Stress 77 ± 486± 6 Lesion + Stress 74 ± 8* 89 ± 6

* P Ͻ 0.01 vs control (non-stressed) rats (two-way ANOVA with SCPHT).

depicted in Figure 3, BDNF mRNA levels were consist- ently up-regulated by immobilization in the CC at PND Figure 2 RNase protection analysis of BDNF and FGF-2 = = mRNA levels in the cingulate cortex of sham (S) or VH 56 both in sham (F1,28 23.068, P 0.00016; SCPHT) = = lesioned (L) young adult (PND-56) rats under basal conditions as well as lesioned rats (F1,28 15.003, P 0.0016; (S-C and L-C) or after 2 h immobilization stress (S-R and L- SCPHT). R). Arrows indicate the protected fragments for BDNF, FGF- The analysis of FGF-2 indicates that its basal 2 or GAPDH, used as internal standard. The lane marked as expression is not affected by the lesion before and after Probe represents an aliquot of the hybridization solution with puberty in any cortical areas considered (Figures 4, 5 the original cRNA probes. The autoradiographic film was and Table 2). The acute restraint stress elicited a sig- exposed at −70°C with an intensifying screen for 5 h (GAPDH) = nificant upregulation in PFC at PND 35 (F1,29 9.333, or 18 h (BDNF and FGF-2). = = P 0.005; two-way ANOVA) as well as 56 (F1,28 14.741, P = 0.0008; two-way ANOVA). The effect at = = Discussion PND 35 was significant in sham (F1,28 9.201, P = 0.012; SCPHT) but not in lesioned animals (F1,28 The results of this study indicate that neonatal VH 2.037, P = 0.34; SCPHT), whereas a similar increase at lesion, an animal model for schizophrenia-related dys- = = PND 56 was observed in sham (F1,28 7.006, P 0.028; function, reduces BDNF expression in adult CC and = = SCPHT) and lesioned rats (F1,28 7.748, P 0.022; alters its stress-related modulation in PFC. According SCPHT) at adult age. The immobilization stress did not to its neurodevelopmental hypothesis, schizophrenia affect FGF-2 expression in FC of sham or lesion rats may be caused by subtle defects occurring in brain before and after puberty (Table 2). Conversely an elev- development, which disrupt late-maturing, highly ation of FGF-2 expression was produced by stress in evolved neocortical functions.6–9 Behavioral and func- = = CC of young adult lesioned rats (F1,28 7.026, P 0.03; tional abnormalities become fully manifest in adult life = SCPHT), but not in sham operated animals (F1,28 1.32, as a consequence of disturbance in processes that may P = 0.263; SCPHT), as shown in Figure 5. involve neurotrophic factors. Hence the changes

Molecular Psychiatry Neonatal VH lesion alters cortical trophic factor expression R Molteni et al 289

Figure 3 Effects of acute restraint stress on BDNF mRNA levels in cingulate cortex (CC) of sham and lesion rats assessed at PND 35 and PND 56. The results, expressed as % of sham rats, represent the mean ± SEM of seven to eight samples for each experimental group. *P Ͻ 0.01 vs sham-operated animals and **P Ͻ 0.01 vs control (non-stressed) rats (two-way ANOVA with SCPHT). observed in the regulatory profile of BDNF and, to less ment with the delayed onset of behavioral abnormali- extent, FGF-2 may represent one of the molecular ties, displayed by neonatal lesioned rats only postpub- events contributing to long-term impairment in brain ertally, as well as with the evidence of prefrontal function and plasticity. cortical cellular changes emerging in early adult- The gene expression for BDNF was not altered by the hood.54,55 These effects might contribute to long-term VH lesion in prepubertal rats in cortical regions, but alterations in brain plasticity. The lack of BDNF up- its expression was decreased in the CC of young adult regulation in this region may render the cells of PFC rats. Postmortem studies provide some evidence for a more vulnerable to the action of acute or prolonged decreased population of neuronal and glial cells, stressors, which are thought to be important in exacer- smaller clusters and increased numbers of inhibitory bating psychiatric symptoms. Indeed, stress-reduction interneurons in the anterior cingulate cortex of schizo- strategies have significant impact in reducing relapse- phrenics.52 It is known that the regulation of BDNF rate of schizophrenics.56 Such observations suggest that mRNA involves a complex interplay between different schizophrenia may develop when a threshold of stress neurotransmitter systems,30 GABA being inhibitory on tolerance is exceeded in a vulnerable individual.57 neurotrophin expression.29,30 We could speculate that Moreover, some schizophrenic patients are susceptible the selective reduction of BDNF levels observed in CC to relapse due to psychological stressors, even when might be caused by an increased inhibitory tone, as a maintained on antipsychotic medication.58 consequence of the neonatal VH lesion, becoming fully The difference in the modulation of BDNF manifest only in adulthood. expression following stress might depend upon several Another consideration emerging from our studies is mechanisms. It is known that stress not only increases that the machinery controlling acute modulation of the levels of circulating corticosteroids but also neurotrophic factors may be altered as a consequence induces the release of several neurotransmitters. Glu- of the neonatal lesion. It is known that the production cocorticoid hormones are probably not responsible for of FGF-2 and BDNF in the adult CNS can be modulated BDNF modulation at this level, as they decrease rather under different circumstances.24–26,32–36 It is generally than increase the production of the neurotrophin.27 believed that these events represent adaptive mech- Dopamine might be a likely candidate, as it is well anisms that, within the CNS, are important to cope established that dopamine projections to the PFC are with challenging situations. Functional changes of sys- activated by stressful stimuli.59 Previous studies have tems involved in the regulation of neurotrophic factor shown that, despite the fact that stress-induced loco- biosynthesis may increase CNS vulnerability to the motor activity is increased in VH lesioned rats,44 these potential damage brought about by different types of animals display reduced dopamine release.60 Accord- insults. It is known that stress affects neurotrophin ingly the lack of BDNF up-regulation in PFC after gene expression with a precise anatomical profile. For restraint stress may be related to a reduced dopami- example, 2 h of immobilization reduced BDNF mRNA nergic response. levels in hippocampus27 while increasing its Glutamate is another neurotransmitter that is expression in hypothalamus and pituitary gland.28 Our released in the PFC in response to stress61 and may data demonstrate that acute restraint stress increases contribute to the up-regulation of BDNF. Glutamate, BDNF mRNA levels in PFC of sham and lesion rats through its NMDA and non-NMDA receptors, stimu- before puberty. However in young adult animals such lates BDNF transcription in cortex and hippocampus.29 effect is observed only in sham operated rats, suggest- Accordingly, the lack of stress-induced modulation of ing that such regulatory mechanism is impaired as a BDNF levels in PFC may be the result of glutamatergic consequence of the neonatal lesion. The temporal hypofunctionality, a situation that has been hypothes- specificity of BDNF modulation by stress is in agree- ized in schizophrenia.62,63 It has been recently

Molecular Psychiatry Neonatal VH lesion alters cortical trophic factor expression R Molteni et al 290

Figure 4 Effect of acute restraint stress on FGF-2 mRNA levels in prefrontal (PFC) of sham and lesion rats assessed at PND 35 and PND 56. The results, expressed as % of sham rats, represent the mean ± SEM of seven to eight samples for each experimental group. *P Ͻ 0.05 and **P Ͻ 0.01 vs control (non-stressed) rats (two-way ANOVA with SCPHT).

Table 2 Effect of acute restraint stress on FGF-2 mRNA lev- in young adult CC, we did not observe any significant els in frontal cortex (FC) of sham and lesion rats assessed at difference between sham and VH lesioned rats, sug- PND 35 and PND 56. The results, expressed as % of sham gesting that in this region the neonatal lesion reduces rats, represent the mean ± SEM of seven to eight samples for the basal expression of the neurotrophin without alter- each experimental group ing its modulation by stress. The regulation of FGF-2 expression is somewhat dif- Group PND-35 PND-56 ferent from BDNF. Our data indicate that the mRNA (% of sham levels) levels for FGF-2 in cortical regions are not altered fol- lowing VH lesion. Furthermore its expression is up- ± ± Sham 100 5 100 9 regulated in adult PFC in sham as well as in lesioned Lesion 105 ± 11 114 ± 5 animals, suggesting that different mechanisms might Sham + Stress 118 ± 8 108 ± 11 Lesion + Stress 114 ± 13 101 ± 7 be involved in the changes of BDNF and FGF-2 expression in response to stress. We may hypothesize that stress regulates FGF-2 expression through the elev- ation of glucocorticoid hormones as we have pre- observed that glutamate neurotransmission regulates viously demonstrated that , a glucocort- stimulus-induced increase of dopamine release in pre- icoid analogue, increases FGF-2 mRNA levels in the frontal cortex. In particular, the activation of both rat brain.35 NMDA and AMPA receptor subtypes in the ventral teg- In summary, we have shown that the VH lesion in mental area as well as AMPA receptors in the PFC, con- developing rats may alter the basal expression of BDNF tributes to dopamine release in the PFC in response in young adult animals. Furthermore it appears that the to stress.64 Hence it is possible to hypothesize that the stress-related mechanisms involved in the acute modu- abnormal regulation of BDNF expression in PFC after lation of BDNF in PFC in adulthood might be altered acute stress may be the result of a complex dysregul- as a consequence of the neonatal lesion. These data ation involving dopaminergic as well as glutama- may help in understanding how stress or other chal- tergic neurotransmission. lenging situations affect brain function in the context Although stress up-regulates BDNF expression also of neurodevelopmental injury. According to our results

Figure 5 Effect of acute restraint stress on FGF-2 mRNA levels in cingulate cortex (CC) of sham and lesion rats assessed at PND 35 and PND 56. The results, expressed as % of sham rats, represent the mean ± SEM of seven to eight samples for each experimental group. *P Ͻ 0.05 vs control (not stressed) rats (two-way ANOVA with SCPHT).

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