Developmental changes in distribution patterns of phencyclidine-induced c-Fos in rat forebrain

Neuroscience Letters 239 (1997) 21–24

Developmental changes in distribution patterns of phencyclidineinduced c-Fos in rat forebrain Daisuke Sato a , b, Asami Umino a, Koyuki Kaneda a, Morikuni Takigawa b, Toru Nishikawa a ,* a

Department of Mental Disorder Research, National Institute of Neuroscience, NCNP, 4-1-1, Ogawa-Higashi, Kodaira-shi, Tokyo 187, Japan Department of Neuropsychiatry, Kagoshima University School of Medicine, 8-35-1, Sakuragaoka, Kagoshima-shi, Kagoshima-ken 890, Japan

b

Received 27 October 1997; received in revised form 17 November 1997; accepted 17 November 1997

Abstract In the forebrain of 56-day-old rats, histochemical studies revealed that the subcutaneous injection of a psychotomimetic phencyclidine (PCP; 1 and 10 mg/kg) induced a dose-related and dense nuclear c-Fos-like immunoreactivity in the pyriform cortex, layers IV–VI of the neocortex and septum, but a sparse c-Fos immunostaining in the olfactory tubercle and mid-lateral striatum. Infant rats at postnatal day 8 expressed much fewer and more confined c-Fos-positive cells in the neocortex than young adult rats following PCP injection. However, a similar expression pattern of PCP-induced c-Fos was observed in the pyriform cortex, mid-lateral striatum, olfactory tubercle and septum between the infant and adult periods. These developmental changes in the regional distribution of a neuronal activity marker, c-Fos, suggest that neuronal populations involved in PCP-induced abnormal behavior are influenced by postnatal development, at least, in the neocortex.  1997 Elsevier Science Ireland Ltd.

Keywords: c-Fos; Immunohistochemistry; Development; Phencyclidine; Neocortex

Phencyclidine (PCP) has long been considered to provide a more inclusive model of schizophrenia than amphetamines and cocaine, because not only schizophrenia-like positive but negative symptoms occur after its abuse and the single administration of subanesthetic doses to normal volunteers [6,16]. A group of schizophrenic patients suffer exacerbation of their psychotic symptoms by a challenge dose of PCP [6]. These observations indicate that PCP should affect the activity of certain neuron circuits that might be involved in the pathophysiology of schizophrenia. Because the onset of schizophrenic symptoms is clearly age-dependent and usually seen after puberty, it can be postulated that the PCP-induced disturbance of neuronal information processing may also change during development. In support of this hypothesis, marked changes have been observed in PCP-induced abnormal behavior as a model of schizophrenic symptoms during postnatal development [14]. Moreover, behavioral sensitization to PCP is shown to be a late-onset phenomenon [15]. In the cerebel* Corresponding author. Tel.: +81 423 461714; fax: +81 423 461744.

lum of the rat treated with acute ketamine, a PCP-like psychotomimetic arylcyclohexylamine [16], Na¨kki et al. [10] have demonstrated using a histochemical technique that there is a postnatal development of expression pattern of a stimulus-inducible molecule, heat shock protein 70 expression, in the rat. To get an insight into the possible postnatal alterations in neuronal responses to PCP, we have studied by immunocytochemistry the effects of the systemic administration of PCP on the expression of a neuron activity marker, c-Fos [9], in the brains of developing rats. The present animal experiments were performed in strict accordance with the guidelines of the National Institute of Neuroscience, National Center of Neurology and Psychiatry, and were approved by the Animal Investigation Committee of the institute. Male Wistar rats (Japan Clea Laboratories, Japan) at ages ranging from postnatal day 8 to 90 were used. The rats were kept at 25.5 ± 0.5°C in a humidity controlled room under a 12:12 h light-dark cycle (lights on at 0800 h) with free access to food and water. PCP HCl was a gift from Yamanouchi Pharmaceutical (Tsukuba, Japan). Other drugs and reagents used were ultrapure quality and commercially available. PCP HCl was dis-

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00879- 3

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D. Sato et al. / Neuroscience Letters 239 (1997) 21–24

solved in physiological saline and injected subcutaneously (s.c). Control animals received the same volume of saline instead. For immunohistochemical studies, animals received an acute injection of PCP (1 and 10 mg/kg, s.c.) or saline and were transcardially perfused 3 h later under pentobarbital anesthesia (40 mg/kg, i.p.) with physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Brains were post-fixed, and cut coronally at 40 mm after cryoprotection and freezing. Immunoctochemistry was achieved on free-floating coronal sections as previously described [20]. Following two 10 min rinses in 0.01 M phosphate buffered saline (PBS) and a 1 h incubation in normal goat serum, brain slices were incubated free-floating for 48 h at 4°C in 0.01 M PBS containing 0.2% Triton X-100 (PBST) and affinity-purified rabbit IgG polyclonal antibody against the peptide corresponding to residues 4–17 of human c-Fos protein (antibody Ab-2 [3]; Oncogene Science, Manhasset, NY, USA; 1:500 dilution). After two 10 min washes in 0.01 M PBST, brain sections were incubated for 1 h in biotinylated goat antirabbit IgG (secondary antibody; Vector Labs) and then rinsed twice in 0.1 M PBS. The reaction product was visualized by transferring the sections to a 50 mM Tris–HCl buffer (pH 7.6) containing 0.05% diaminobenzidine, 0.6% nickel ammonium sulfate and 0.01% H2O2. Nickel ion was added to the buffer to enhance the coloring reaction. Following two 10 min rinses in 0.01 M PBS and a subsequent wash in 0.01 M PBS, the stained sections were mounted on to subbed slides. The c-Fos-like immunostaining was found to be nuclear

and absent in the control sections incubated in the presence of the Fos peptide sequences against which the rabbit anti-cFos was generated (data not shown). The specificity of the immunostaining was further verified by incubation of the brain sections with normal rabbit serum, which produced no staining. Because preliminary studies indicated that a peak increase in the numbers of immunoreactive cells and the intensity of staining occurred from 2 to 4 h postinjection, all brain samples were taken 3 h after the injection of PCP or saline in this series of experiments. For quantitative analysis, coronal sections at the level of the rostral striatum (1.2 mm anterior to bregma according to the atlas of Paxinos and Watson [12]) were selected from each rat. The density of nuclei stained by anti-c-Fos antiserum in the pyriform cortex and neocortex was quantified by counting the number of immunoreactive nuclei per unit area indicated in Fig. 2. For comparison between the two groups, statistical evaluations were made by either unpaired two-tailed Student’s t-test or Mann–Whitney Utest. In adult rats (postnatal day 56 and 90), systemic injection of the lower dose of PCP (1 mg/kg, s.c.) resulted in a slight locomotor stimulation and discontinuous sniffing without apparent ataxia whereas the higher dose (10 mg/kg) induced a remarkable hyperactivity, stereotypy including head weaving, backpeddling and turning, and ataxic movements, as previously reported [19]. The infant rats displayed robust hyperlocomotion, stereotyped head movement and ataxia at 1 mg/kg PCP, but laid on their back with severe ataxia and repetitive movement of the limbs at 10 mg/kg. The inverse relationship between the locomotor stimulation effects and

Fig. 1. Photomicrographs showing the effects of saline (a,c,e,g,i,k) or PCP (10 mg/kg, s.c. 3 h before rat perfusion; b,d,f,h,j,l) on c-Fos-like immunoreactivity in the anterior portion of the neocortex (a–d), the striatum and septum (e–h) and the pyriform cortex (i–l) of the infant (a,b,e,f,i,j) and young adult (c,d,g,h,k,l) rats. Nuclear c-Fos-like immunoreactivity is observed as black dots in the brain slices. Abbreviations: Cg, cingulate cortex; LS, lateral septum; LV, lateral ventricle; Py, pyriform cortex; St, Striatum. Scale bar, 200 mm. Solid squares are the areas for counting the c–Fos–positive cell nuclei (see the legend for Fig. 2).

D. Sato et al. / Neuroscience Letters 239 (1997) 21–24

the doses of PCP has also been observed at postnatal day 12 [14]. At postnatal day 56, in accordance with our previous report [20], there was a sparse expression of the c-Fos-like immunoreactivity in the brain after saline injection at the level of the striatum (Fig. 1c,g,k). An acute administration of PCP (1 and 10 mg/kg, s.c.) caused a dense nuclear c-Foslike immunoreactivity (see Fig. 1d,h,l for 10 mg/kg) in the forebrain regions including the pyriform cortex (Fig. 1l), layers IV–VI of the neocortex (Fig. 1d), and the lateral septum (Fig. 1h). In the medial frontal and cinglate cortex, moderate to low expression of the proto-oncogene product was observed. Sporadic c-Fos immunostaining was seen in the olfactory tubercle, mid-lateral striatum (Fig. 1h), hippocampus and layers I–III of most of the neocortical areas although there was a moderate to low expression of c-Fos in layers II–III of the cinglate and medial frontal cortex (Fig. 1d) and in the periventricular portion of the striatum (Fig. 1h). While a similar overall pattern of immunostaining was observed after either dose of PCP, the c-Fos-positive brain cells were less prominent after injection of PCP at 1 mg/kg than at 10 mg/kg (data not shown). There was no apparent difference in the distribution pattern of PCPinduced c-Fos between postnatal day 56 and 90 (data not shown). In 8-day-old rats, an acute injection of PCP (1 and 10 mg/ kg, s.c.) and saline failed to cause c-Fos-like immunoreactivity in layers I–V of the neocortex (Fig. 1a,b) and the periventricular portion of the striatum (Fig. 1e,f) but induced the gene product in the deeper parts of layer VI of the neocortex (Fig. 1a,b). However, a similar pattern of c-Fos immunoreactivity was observed in the pyriform cortex (Fig. 1i–l), mid-lateral striatum (Fig. 1e–h) and olfactory tubercle of the adult and neonatal rats following PCP and saline administration. The two doses of PCP induced no apparent difference in the overall distribution of c-Fos. These observations agreed with the quantitative analysis of the c-Fos expression. As shown in Fig. 2, saline admin-

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istration induced the c-Fos expression at low density in the pyriform cortex and in layer VI of the neocortex of adult and infant rats, and in the layer V of the adult neocortex. An acute injection of PCP (10 mg/kg) caused a similar increase in the number of c-Fos-like immunorective cell nuclei per mm2 in the pyriform cortex at postnatal day 8 (278% of saline control) and 56 (286%) whereas adult animals displayed a much higher rate of augmentation in density (5429% of saline control) than pups (217%) in layer VI of the neocortex (Fig. 2). There was no c-Fos-positive nuclei in V layer of the pups following saline and PCP (10 mg/kg) application (Fig. 2). The present study demonstrates the differential effects of postnatal development on the expression patterns of PCPinduced c-Fos in the forebrain regions. There was a marked difference in the distribution of PCP-induced c-Fos in the neocortex between infant and young adult rats. In contrast, the c-Fos expression pattern remained rather constant in the pyriform cortex and striatum. The overall distribution patterns of c-Fos induced by PCP in the adult rat forebrains seem to be consistent with those of previously reported c-fos mRNA (PCP; 8.6 mg/kg) [18]. These patterns are distinct from those following injection of other schizophrenomimetic dopamine agonists such as amphetamines and cocaine [5,20], but resemble those after acute injection of dizocilpine (MK-801), a selective noncompetitive antagonist for the N-methyl-D-aspartate (NMDA) type glutamate receptor ([4] and our unpublished observations). Because PCP has been shown to be a potent non-competitive NMDA antagonist [7], the above similarity suggests that the PCP-induction of c-Fos may be chiefly due to the reduced neurotransmission via the NMDA receptor. However, the regional variation in the c-Fos positive cell density after PCP injection does not parallel that of the cerebral NMDA receptor [1,21]. For instance, a very sparse c-Fos expression was observed in the striatum which is concentrated with the NMDA receptor. This discrepancy may be explained by the possibility that the proto-oncogene

Fig. 2. The density of brain cell nuclei expressing c-Fos-like immunoreactivity in layers V and VI of the neocortex and the pyriform cortex of the rats at postnatal day 8 (PD8) and 56 (PD56) after systemic administration of saline and PCP. Rats received a s.c. injection of PCP (10 mg/kg) or saline and were perfused 3 h thereafter. The immunostained nuclei were counted in each area surrounded by the solid square that is indicated in Fig. 1 (a,c,i,k). Their density is expressed as the number of c-Fos-positive cell nuclei per mm2. Data are the mean ± SEM of data obtained from four to five animals. *P , 0.05, **P , 0.01 as compared to the respective saline-treated controls.

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responses could be accounted for not only by direct NMDA receptor blocking but also transsynaptically generated change in neuronal activity following the primary action of PCP. From the NMDA antagonist property of PCP, the developmental changes in the PCP-induced c-Fos expression can also be assumed to be associated with those of the NMDA receptor ion channel complex. As the distribution and function of mRNAs and proteins of the NMDA receptor have indeed been shown to be changed during postnatal development [1,17], further investigation is needed to clarify the possible relationship. The regional variation in the development of the c-Fos induction is likely to be related to that of certain functionspecific neuron circuits involved in the information processing in response to PCP. In fact, brain area-selective postnatal alterations in the c-Fos expression pattern have been reported in the rats treated with kainate [13], pentylenetetrazole[13], methamphetamine [11] and cocaine [8]. The neocortex represented the most prominent changes in the early gene expression patterns after PCP application between postnatal days 8 and 56. The lack of PCP-induced c-Fos-like immunoreactivity in the I–V layers of the infant neocortex is likely to be connected to immaturity of the neocortical circuits, because (1) nerve cell migration in the neocortex has been shown to be largely finished before birth while the neuronal network formation persists during neonatal periods [2], and (2) c-fos gene expression may reflect changes in neuronal activity [9]. The fact that the PCP-induced c-Fos expression pattern and abnormal behavior in the 56-day-old rats are similar to those of rats at postnatal day 90, but not 8, indicates that neuronal responses to the drug may mature between postnatal day 8 and 56. The present findings, therefore, suggest that the maturation of a subset of the neocortical networks might be needed for the adult type behavioral response to PCP that has been considered to be an animal model of a subgroup of schizophrenic symptoms. The authors thank Mrs. M. Asakawa and M. Kurita for preparing this manuscript. This work was partly supported by the Research Grant for Nervous and Mental Disorders from the Ministry of Health and Welfare (Japan), a Grantin-Aid for Scientific Research (C) from the Ministry of Education, Science and Culture (Japan) and a grant from the Social Insurance Agency Contract Fund commissioned to Japan Health Sciences Foundation. [1] Akazawa, T., Shigemoto, R., Bessho, Y., Nakanishi, S. and Mizuno, N., Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats, J. Comp. Neurol., 347 (1994) 150–160. [2] Bayer, S.A. and Altman, J., Neocortical Development, Raven Press, New York, 1991, 255 pp. [3] Detogni, P., Niman, H., Raymond, V., Sawchenko, P. and Verma, I.M., Detection of fos protein during osteogenesis by monoclonal antibodies, Mol. Cell. Biol., 8 (1988) 2251–2256.

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