Introduction

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The pyrethroids constitute a major class of highly active synthetic derivatives of natural pyrethrins, toxins present in the flowers of Chrysanthemum cinerariaefolium. Pyrethroid insecticides are classified into two major groups on the basis of chemical structures (Vijverberg and van den Berken, 1982; Miyamoto et al., 1995). In rats or mice, type I pyrethroids, so-called noncyano compounds, cause the T (tremor) syndrome, which is characterized by an increased sensitivity to external stimuli and fine tremors progressing to whole body tremors and prostration (Verschoyle and Aldridge, 1980; Vijverberg and van den Berken, 1990). The type II compounds, containing an -cyano substituent on the alcohol moiety of the pyrethroid molecule, cause the CS (choreoathetosis and salivation) syndrome, which is characterized by pawing and burrowing, profuse salivation, and coarse tremor progressing to choreoathetosis and clonic seizure (Ray and Cremer, 1979; Vershoyle and Aldridge, 1980; Vijverberg and van den Berken, 1990). Although pyrethroids are reported to be rapidly metabolized in mammals and, hence, have a low toxicity, it is still unknown whether pyrethroids absorbed by newborn animals through the milk (Kavlock et al., 1979) or food affect the postnatal development of the nervous system.

Pyrethroids prolong the opening of the voltage-gated sodium channel, as a target in both insects and mammals, giving rise to a prolonged sodium tail current in mammalian as well as invertebrate neurons (Vijverberg et al., 1982; Narahashi, 1985; Vijverberg and van den Berken, 1990). Besides this excitatory effect on neurons, pyrethroid actions on the ion channels associated with several types of neurotransmitter receptors (Abbassy et al., 1983; Lawrence and Casida, 1983) and the neurotransmitter release from presynaptic terminals (Eells and Dubocovich, 1988) have been proposed. Over the last decade, on the other hand, evidence has been accumulating that the expression of a certain group of genes such as c-fos and brain-derived neurotrophic factor (BDNF) gene is controlled by a neural activity accompanying Ca²⁺ influx into neurons (Zafra et al., 1990; Bading et al., 1993; Sano et al., 1996; Tsuda, 1996). BDNF is a member of the neurotrophin family and plays a key role in the survival, differentiation, and synaptic plasticity of neurons (Thoenen, 1995). BDNF also plays an important role in the postnatal development of the mammalian central nervous system (Schwartz et al., 1997). Therefore, it is possible that the disturbance of neural activity, which can be caused by pyrethroids, influences the activity-dependent gene expression in neurons, resulting in an abnormal development of mammalian nervous systems. From this point of view, it is important to determine whether pyrethroids affect the expression of the c-fos and BDNF genes in neurons. Using primary cultures of mouse cerebellar granule cells, we clearly demonstrated that pyrethroid insecticides, type I and II pyrethroids, decreased the expression of c-fos and BDNF genes induced by neural activity.

	Materials and Methods

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Chemicals. cis- or trans-Permethrin, 3-phenoxybenzyl-(1R,S)-cis or trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate; cypermethrin, (S)--cyano-3-phenoxy-benzyl(1R)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane carboxylate; deltamethrin, (S)--cyano-3-phenoxybenzyl-cis-(1R,3R)-2,2-dimethyl-3-(2,2-dibromovinyl) cyclopropane carboxylate; and fenvalerate, (R,S)--cyano-3-phenoxybenzyl(R,S)-2-(4-chlorophenyl)-3-methyl butylate were purchased from Wako Pure Chemical Ind. (Osaka, Japan). Veratridine, (3,4,16)-4,9-epoxycevane-3,4,12,14,16,17,20-heptol 3-(3,4-dimethoxybenzoate), was purchased from Sigma (St. Louis, MO). Stock solutions of pyrethroids and veratridine were prepared with dimethyl sulfoxide. The dimethyl sulfoxide concentration in the medium was less than 0.2% (v/v).

Primary Culture of Mouse Cerebellar Granule Cells (CGCs). A primary culture of mouse CGCs was prepared from 1-week-old mice (ICR). The procedure for dissociating the cells was described previously (Ichikawa et al., 1998). The dissociated cells were suspended in DMEM containing 10% FCS and 25 mM KCl, and seeded in culture dishes (60 mm in diameter; Iwaki, Tokyo, Japan) that had been treated with PBS⁽⁾ containing 5 µg/ml poly(L-lysine) (Sigma). The cells were given fresh medium (+10% FCS) supplemented with 10 µM cytosine arabinoside (Sigma) 2 days later. All experiments were performed using CGCs at DIV 5. To avoid cytotoxic shock caused by medium replacement, we first incubated the fresh medium containing 10% FCS in a CO₂ incubator overnight to equilibrate the pH and temperature. After exposing the cells to 5 mM KCl for 24 h, we added concentrated KCl solution (2 M) to adjust the KCl concentration of the medium containing 5 to 25 mM KCl (high K⁺) and cultured the cells for the period indicated. For the control, we added corresponding buffer without KCl as a vehicle. We always prepared the medium containing 5 or 25 mM KCl by further adding KCl solution to the DMEM medium, which originally contains about 5.3 mM KCl, resulting in the final KCl concentrations 10.3 and 30.3 mM for 5 and 25 mM KCl, respectively.

Preparation of Total Cellular RNAs and Northern Blotting. Total cellular RNAs were extracted by the acid guanidine phenol-chloroform method. After removing the incubation solution, cells were scraped off using a rubber policeman in 500 µl of ISOGEN (Nippon Gene, Toyama, Japan), transferred into an Eppendorf tube and kept at room temperature (RT) for 5 min. Then, 100 µl of chloroform was added to the tube, and the mixture was vigorously vortexed for 20 s. After keeping it at RT for 3 min, centrifugation was performed at 14 krpm for 15 min and the supernatant was transferred into an Eppendorf tube. Then, 300 µl of isopropanol was added and the mixture vortex-mixed. After keeping it at RT for 10 min, centrifugation was done at 14 krpm for 10 min and the precipitates were stored at 20°C after a rinse with 70% ethanol. Precipitated RNAs were dissolved in 100 µl of diethylpyrocarbonate-treated water, and quantified by Beckman spectorophotometer. An aliquot of RNA solution was kept in ethanol at 20°C until use. Northern blot hybridization was performed according to Tabuchi et al. (1996). To compare the kinetics of c-fos, BDNF, and -actin mRNA expressions, we performed the hybridization using the same hybridization membrane filters after reprobing. For reprobing, the radiolabeled probes were removed from the filters by shaking them with boiled 0.1% SDS three times. After the radioactivities of c-fos, BDNF, and -actin mRNA bands had been measured by an Imaging scanner (BAS 2000; Fuji, Tokyo, Japan), the values of c-fos and BDNF mRNA bands were normalized to those of -actin ones and the relative ratio of c-fos or BDNF mRNA expression of each sample was calculated with the control as 100%. cDNA probes for hybridization were derived from mouse for c-fos (nucleotide position 36-1341) and -actin (nucleotide position 81-1208), and from rat for BDNF (nucleotide position 1-1892).

Assay of Lactate Dehydrogenase (LDH) Release. Cytotoxicity was evaluated by measuring LDH release into the extracellular fluid (LDH assay). The enzymatic activity of LDH was measured as described by Murphy et al. (1988). After stimulation with the drug, the incubation solution was transferred into fresh tubes and stored at 4°C until use. The LDH activities in the incubation solution and in the cell lysates were spectrophotometrically measured at 340 nm. The percentage of LDH release was defined as the LDH activity in the incubation solution divided by the additive values included in both the incubation solution and the cell lysates.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. The method of MTT assay, in which the conversion of MTT to colored formazan is measured, was performed according to the method of Hansen et al. (1989). After stimulation with the drug, 600 µl of 5 mg/ml MTT solution was added to 3 ml of the incubation medium and the cells were incubated in a CO₂ incubator for 20 min. Then, 2 ml of the incubation solution was discarded, and 1 ml of extraction buffer (20% SDS and 50% dimethylformamide) was added and the cells were further incubated overnight at 37°C. Finally, optical density was measured at 570 nm. The optical density value of unstimulated or time 0 sample was regarded as 100% survival.

Measurement of ⁴⁵Ca²⁺ Uptake. The Ca²⁺ influx into the CGCs was measured by the procedure of Lazarewicz et al. (1990). Ten minutes before the cells were stimulated, 10 µM permethrin was added to the culturing medium. After stimulation of the cells with high K⁺ or 10 µM veratridine in the presence of 1 µCi of ⁴⁵CaCl₂ (NEN, Boston, MA; >0.37 GBq/mg of calcium), incubation was carried out for 5 min, and the reaction was terminated by rapid aspiration of the medium and three washes with PBS⁽⁾ containing 2 mM EGTA. Cells were solubilized in 0.5 M NaOH and the radioactivity per dish was measured by liquid scintillation counter. The amounts of protein recovered from each dish were almost the same (approximately 2 mg/dish; data not shown).

Preparation of Nuclear-Mini Extracts and Gel-Mobility Assay. The procedures for preparing nuclear-mini extracts and conditions of gel-mobility assay were described previously (Sakurai et al., 1992). A synthetic 20-base pair oligonucleotide containing TPA-responsive element (5'-GATTCGTGACTCAGCACAGG-3') was end-labeled with [-³²P]dCTP (NEN) and used as a DNA probe to detect AP-1 DNA-binding activity. DNA-protein complexes were separated on 4% polyacrylamide gel in 1× TAE (6.7 mM Tris-HCl, pH 7.5, 3.3 mM sodium acetate, 2.5% glycerol, and 0.1 mM EDTA). After an electrophoresis, the gel was dried and subjected to autoradiography. Radioactivities of the bands detected by gel electrophoresis were scanned and quantified with NIH Image version 1.52 (National Institutes of Health, Bethesda, MD).

Immunoblotting Analysis. Nuclear-mini extracts (30 µg each) were denatured in 1× sample buffer (10 mM Tris-HCl, pH 6.8, 1% SDS, 1% -mercaptoethanol, 20% glycerol) for 5 min at 95°C and then separated on 10% SDS-polyacrylamide gel at 30 mA. Sample proteins were transferred onto a polyvinylidene difluoride membrane filter (Bio-Rad, Hercules, CA), and the filter was treated with blocking solution (3% bovine serum albumin, 0.1% Tween in PBS). The filter was incubated with rabbit anti-c-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C and then with goat biotinated anti-rabbit IgG antibody (Oncogene Science, Cambridge, MA) for 2 h at 37°C. c-Fos was finally detected with the ABC method (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA). The intensities of immunostained bands were scanned and quantified with NIH Image version 1.52.

	Results

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Induction of c-fos and BDNF Genes by Veratridine. To examine the inducibility of the c-fos and BDNF genes through an activation of sodium channel (Na⁺-ch), we stimulated the cultured mouse CGCs with veratridine, a potent Na⁺-ch agonist that can cause membrane depolarization in neurons, and monitored the mRNA expression of c-fos and BDNF genes by Northern blotting. As shown in Fig. 1, the addition of 10 µM veratridine to the medium containing 5 mM KCl markedly induced the c-fos mRNA expression at 30 min with maximum expression at 90 min after starting the incubation. Maximum expression level induced by veratridine was higher than that obtained by elevating the potassium (K⁺) concentration from 5 to 25 mM (high K⁺). On the other hand, the expression of BDNF mRNA became obvious at 90 min and reached a maximum at 180 min later. The -actin mRNA expression, which was examined as a control, did not change upon the stimulation of CGCs with veratridine. The most effective concentrations of veratridine for the c-fos induction were between 5 and 50 µM, which were also effective to induce the BDNF mRNA expression (data not shown). The addition of 2 µM nicardipine, a potent antagonist for L-type voltage-dependent calcium channels (L-VDCCs), before the stimulation of CGCs with veratridine reduced the c-fos and BDNF mRNA expression to the control level (data not shown), indicating that the increase in c-fos and BDNF mRNA expression induced by veratridine is mediated by the Ca²⁺ influx into CGCs through L-VDCCs.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Time course of c-fos and BDNF mRNA expressions induced by veratridine. After the incubation of CGCs in DMEM containing 25 mM KCl for 4 days, the medium was replaced with a fresh batch containing 5 mM KCl and the cells cultured for another 24 h. To the cultures with 5 mM KCl, veratridine or vehicle was added at 10 µM and the cells further incubated for the indicated times before total cellular RNA was extracted for Northern blotting. As a control, 40 µl of 2 M KCl solution was added to increase the KCl concentration to 25 mM. A, experimental schedule and the results of autoradiography after Northern blotting analysis of c-fos, BDNF, and -actin mRNA expressions. B, relative ratios of mRNA expression to the control [time 90 min at 25 mM KCl as 100% for both c-fos (a) and BDNF (b) mRNA expressions]. The experiments were performed twice and the same tendency was observed.

Repressive Effect of Permethrin on the Veratridine- or High K⁺-Induced c-fos and BDNF mRNA Expression. To examine the effect of permethrin on the induction of c-fos and BDNF genes, we added cis- or trans-permethrin at given concentrations before the stimulation of CGCs with veratridine. As shown in Fig. 2, both the cis- and trans-permethrins dose dependently repressed the c-fos induction, with the trans-permethrin more effectively than cis-permethrin. The same inhibitory effect of permethrin was obtained on the induction of BDNF gene (Fig. 2).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effect of permethrin on the c-fos and BDNF mRNA expressions induced by veratridine. After the incubation of CGCs at 5 mM KCl for 24 h, trans- or cis-permethrin was added at the indicated concentrations 10 min before the addition of 10 µM veratridine or vehicle and the cells were incubated for another 90 min before total cellular RNAs was prepared for Northern blotting analysis. A, experimental schedule and the results of autoradiography. B, relative ratios of mRNA expression to the control (samples treated with veratridine only as 100%). The experiments were performed twice and the same tendency was observed.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effect of permethrin on the c-fos and BDNF mRNA expressions induced by high K+. After the incubation of CGCs at 5 mM KCl for 24 h, trans- or cis-permethrin was added at the indicated concentrations 10 min before the addition of 40 µl of 2 M KCl solution (25 mM KCl) or vehicle (5 mM KCl) and the cells were incubated for another 90 min before total cellular RNA was prepared for Northern blotting analysis. A, experimental schedule and the results of autoradiography. B, relative ratios of mRNA expression to the control (samples treated with 25 mM KCl only as 100%). The experiments were performed twice and the same tendency was observed.

Effect of Permethrin on Ca²⁺ Influx into CGCs. Because the Ca²⁺ influx into CGCs through L-VDCCs induces the expression of c-fos and BDNF genes, we examined the effect of permethrin on the Ca²⁺ influx induced by veratridine or high K⁺. As shown in Fig. 4, the ⁴⁵Ca²⁺ uptake by CGCs was markedly stimulated, with 10 µM veratridine more effective than high K⁺. Addition of 10 µM permethrin 10 min before the stimulation reduced the ⁴⁵Ca²⁺ uptake induced by veratridine or high K⁺ to about 40 to 60% of the control. The repressive effect was stronger with trans-permethrin than with cis-permethrin. The treatment of CGCs with permethrin at 5 mM KCl also decreased the ⁴⁵Ca²⁺ uptake by CGCs.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of permethrin on the 45Ca2+ uptake by CGCs treated with veratridine or high K+. After the incubation of CGCs at 5 mM KCl for 24 h, 10 µM trans- or cis-permethrin was added 10 min before stimulating the CGCs with 10 µM veratridine or high K+. 45Ca2+ (1 µCi) was added just before the stimulation and the incorporated radioactivities were measured 5 min after the stimulation. The columns represent the mean ± S.D. from three separate experiments, each of which was performed in duplicate. Two groups were assessed using Student's t test followed by the F test. * or , P < .05 versus the control, which was obtained by the stimulation with veratridine (*) or high K+ () without permethrin.

Noncytotoxicity of Permethrin for the CGCs under High K⁺. To examine the cytotoxic effect of permethrin on CGCs, we measured the changes in LDH release from CGCs into the medium and in MTT-reducing activity of CGCs 24 h after incubating the cells with permethrin under high K⁺. The high K⁺ condition reduced the extent of LDH release (Fig. 5A) and increased the level of MTT-reducing activity (Fig. 5B), whereas the low K⁺ condition had the reverse effect (Fig. 5, A and B), consistent with the previous report (Ichikawa et al., 1998). When cis-permethrin was added with high K⁺ at concentrations that inhibit the c-fos and BDNF mRNA expressions, no obvious change in LDH release or in MTT-reducing activity was observed below 50 µM although the MTT-reducing activity tended to decrease at concentrations above 100 µM (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of permethrin on LDH-release and MTT-reducing activity. After the incubation of CGCs at 5 mM KCl for 24 h, cis-permethrin was added at the indicated concentrations 10 min before stimulating the cells with high K+ and the incubation was performed for another 24 h to measure the LDH-release from the CGCs (A) and the MTT-reducing activity in CGCs (B). The columns represent the mean ± S.D. from three separate experiments. Two groups were assessed using Student's t test followed by the F test. *P < .05 versus the control obtained at 5 mM KCl. NS, nonsignificant versus the sample obtained at 25 mM KCl in the absence of permethrin.

Decrease in c-Fos Synthesis and AP-1 DNA-Binding Activity in CGCs Treated with Permethrin. To know whether the level of c-Fos or BDNF protein synthesis is also reduced by permethrin in concert with the repression of mRNA expression, we examined the effect of permethrin on the c-Fos synthesis using an immunoblotting analysis or a gel-mobility assay. As shown in Fig. 6, A and B, the c-Fos synthesis increased upon stimulation of the CGCs with high K⁺ and the addition of cis-permethrin inhibited the increase in a dose-dependent manner. The gel-mobility assay also showed that the DNA-binding activity of AP-1 decreased as the concentrations of permethrin increased (Fig. 6, A and C), indicating a decrease in AP-1 molecules included in the nuclear extracts prepared from the CGCs treated with cis-permethrin.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Decrease in c-Fos synthesis and AP-1 DNA-binding activity induced by cis-permethrin. A, after the incubation of CGCs at 5 mM KCl for 24 h, cis-permethrin was added at the indicated concentrations 10 min before stimulating the cells with high K+ and the nuclear mini-extracts were prepared for immunoblotting and gel-mobility analyses 6 h after the incubation. The relative ratio (%) of c-Fos proteins () or AP-1 DNA-binding activity () to the control (samples treated with 25 mM KCl only as 100%) is shown. The results obtained by immunoblotting (B) or gel-mobility assay (C) are also shown. The experiments were performed twice for each experiment and the same tendency was observed.

Effects of Other Pyrethroids on c-fos mRNA Expression. Although cypermethrin, a typical type II pyrethroid, inhibited the increase in c-fos mRNA expression induced by veratridine or high K⁺ (data not shown), the extent of the inhibition was less than that with either cis- or trans-permethrin, as shown by the greater IC₅₀ value of cypermethrin than permethrin (Table 1). Deltamethrin and fenvalerate, both type II pyrethroids, also repressed the c-fos induction caused by veratridine or high K⁺, but to a lesser extent than permethrin (Table 1). The tendency of repression caused by pyrethroids for the high K⁺-induced c-fos induction was almost the same as that for the veratridine-induced one.

	Discussion

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In the present study, we clearly demonstrated that the exposure of cultured CGCs to permethrin repressed the induction of c-fos and BDNF genes caused by a direct activation of Na⁺-ch evoked via the administration of veratridine or by a membrane depolarization evoked via elevation of the KCl concentration in the medium from 5 to 25 mM (high K⁺). Because veratridine also evokes membrane depolarization through an activation of Na⁺-ch, the activation of c-fos and BDNF genes induced by veratridine should also be mediated by membrane depolarization. In addition, the observation that the induction of these genes was blocked by nicardipine (data not shown; Ichikawa et al., 1998) indicates that the Ca²⁺ influx into CGCs through L-VDCC, which can be caused by membrane depolarization (Ichikawa et al., 1998), accounts for the induction. As shown in Fig. 4, however, the increase in ⁴⁵Ca²⁺ uptake caused by veratridine or high K⁺ was reduced by the treatment of CGCs with cis- or trans-permethrin. Therefore, it is very likely that the permethrin-induced repression of c-fos and BDNF gene induction is mediated by a reduction of Ca²⁺ influx into CGCs through L-VDCC. Within the range of concentrations effective for repressing the c-fos and BDNF gene induction, permethrin did not induce the cell death 24 h after the incubation (Fig. 5), indicating that the cytotoxic effects of permethrin, which are obvious at concentrations above 100 µM (data not shown), do not account for the repression of gene induction. The treatment of CGCs with permethrin alone (10 µM) could induce neither the c-fos mRNA expression (Fig. 3) nor the Ca²⁺ influx (Fig. 4).

It is well established that pyrethroids act on Na⁺-ch and prolong sodium currents, leading to repetitive bursts of action potentials (Vijverberg et al., 1982; Narahashi, 1985; Vijverberg and van den Berken, 1990). Recently, radioligand binding and electrophysiological experiments have revealed that the pyrethroid binding site is intrinsic to the sodium channel -subunit (Smith and Soderlund, 1998). This direct effect of pyrethroids on Na⁺-ch molecules on the cell membrane would have a strong excitatory action on neurons, which could give rise to multiple toxic syndromes in mammals. In the present study, however, permethrin repressed the gene expression and Ca²⁺ influx, which was enhanced by a stimulus such as activation of Na⁺-ch or high K⁺. The effective range of permethrin concentrations for repressing the c-fos and BDNF gene induction was almost the same as that (10 to 20 µM) for inducing sodium tail currents (Vijverberg et al., 1982). However, the time courses of responses of Na⁺-ch and gene expression to veratridine or high K⁺ are different because the initial response of Na⁺-ch is observed within a minute, which can be detected by an electrophysiological method (Vijverberg et al., 1982), whereas that of gene expression is after 15 min with the c-fos induction (data not shown). Because the incubation of CGCs for at least 15 min after stimulating the CGCs in culture is needed to cause the changes in gene expression, the long exposure of CGCs to permethrin seems to affect not only Na⁺-ch but also other molecules controlling the Ca²⁺ signalings leading to gene expression. In support of this, it has been reported that some ion channels and neurotransmitter receptors could be targeted by pyrethroids (Abbassy et al., 1983; Lawrence and Casida, 1983; Eells and Dubocovich, 1988), and, furthermore, calcineurin, a neural calcium/calmodulin-dependent protein phosphatase, could be specifically inhibited by cypermethrin (Enan and Matsumura, 1992). Thus, it is possible that not only the Na⁺-ch but also some membrane or intracellular molecules are targeted by pyrethroids, resulting in the blockade of Ca²⁺ influx or intracellular Ca²⁺ signalings required for the c-fos and BDNF gene induction.

Although it is still uncertain whether the repressive effect of permethrin on Ca²⁺ influx and gene expression is due to mechanisms including the direct action of permethrin on Na⁺-ch, it is clear that permethrin leads to a decrease in production of c-Fos along with a reduction in c-fos mRNA expression, as was shown in Fig. 6. It is well established that the c-fos and BDNF genes, both of which are termed immediate-early genes (Sano et al., 1996), can be markedly induced by kainic acid-induced seizure in neurons of the rodent brain (Smeyne et al., 1992; Timmusk et al., 1993). c-Fos consists of an AP-1 transcriptional factor that specifically binds to the TPA (12-O-tetradecanoylphorbol-13-acetate)-responsive element-binding motif on the promoters of genes and plays an important role in the transcriptional regulation of genes. Because the treatment of CGCs with permethrin reduced the AP-1 DNA-binding activities (Fig. 6C), the decrease in c-Fos production induced by permethrin should lead to a decrease in AP-1 DNA-binding activity in neurons, which might give rise to disturbances in the transcriptional regulation involved in the rapid response to synaptic transmission of neurons. On the other hand, BDNF is now recognized not only as a neurotrophic factor but also as a factor controlling synaptic plasticity (Thoenen, 1995; Kafitz et al., 1999). In addition, BDNF plays an important role in the postnatal development of rodent brain. In the rat cerebellum, the BDNF mRNA expression begins to be highly activated at two or three postnatal weeks (Neveu and Arenas, 1996), when the CGCs in the internal granular cell layer receive the glutamatergic afferents of mossy fibers. Mice with a targeted gene deletion of BDNF exhibit an abnormal cerebellar development (Schwartz et al., 1997). BDNF is also involved in the formation of ocular dominance columns in the visual cortex of mammals (Cabelli et al., 1995; Huang et al., 1999), which is known as a typical activity- or experience-dependent neural network formation in the postnatal development of the brain. Therefore, it seems likely that the exposure of newborn animals to pyrethroids induces a decrease in the Ca²⁺ influx into neurons and, consequently, a reduction in c-fos and BDNF mRNA expression and protein syntheses in the developing brain, resulting in the blockade of the activity-dependent neural network formation.

Pyrethroid insecticides have been classified into two groups, type I and type II pyrethroids, on the basis of differences in chemical structure and inducible syndromes (Vijverberg and van den Berken, 1982). When cypermethrin, a type II pyrethroid, was administered orally to neonatal and adult rats, it was found to be more toxic than permethrin (Cantalamessa, 1993). Although cypermethrin was effective in repressing the induction of the c-fos (Table 1) and BDNF genes (data not shown), the repressive effect was weaker than that of permethrin (Table 1). Other type II pyrethroids, deltamethrin and fenvalerate, also repressed the c-fos induction less extensively than permethrin (Table 1). In addition, trans-permethrin was more effective in repressing the c-fos induction than cis-permethrin (Fig. 2; Table 1), although cis-permethrin should be more toxic to animals than trans-permethrin (Vijverberg and Bercken, 1990). These differences of pyrethroid toxicity between animal and cell culture experiments seem to be mainly due to the fact that the hydrolysis rate of pyrethroids is an important factor determining the toxic levels of pyrethroids in vivo (Gaughan et al., 1976, 1978; Cantalamessa, 1993). In any case, it is possible that a long exposure to pyrethroids leads to a decrease in the activity-dependent cellular responses of neurons through a disturbance in, at least in part, Ca²⁺ influx and gene expression of neurons, which might affect the postnatal development of mammalian brain.