Differential expression of immediate-early genes, c-fos and zif268, in the visual cortex of young rats: effects of a noradrenergic neurotoxin on their expression

Pergamon PII:

Neuroscience Vol. 92, No. 2, pp. 473–484, 1999 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(99)00003-2

DIFFERENTIAL EXPRESSION OF IMMEDIATE-EARLY GENES, c-fos AND zif268, IN THE VISUAL CORTEX OF YOUNG RATS: EFFECTS OF A NORADRENERGIC NEUROTOXIN ON THEIR EXPRESSION Y. YAMADA,*† Y. HADA,*† K. IMAMURA,†‡ N. MATAGA,†§ Y. WATANABE† and M. YAMAMOTO* *Department of Ophthalmology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe-shi, Hyogo 650-0017, Japan †Department of Neuroscience, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita-shi, Osaka 565-0874, Japan

Abstract—We investigated the expression pattern of two immediate-early genes, zif268 and c-fos, under various visual conditions using immunohistochemical and northern blot analysis in the visual cortex of young rats. The basal expression of c-fos was low and was further reduced by dark rearing that lasted for one week. A marked and transient increase was induced upon visual stimulation applied immediately after dark rearing. Zif268 showed a relatively high basal level. Its expression was reduced by dark rearing of the animals, but returned rapidly to the basal expression level following the introduction of light. Administration of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a selective noradrenergic neurotoxin, suppressed the basal expression of c-fos messenger RNA. The response of c-fos to photo-stimulation was also significantly lower in the visual cortex of N-(2-chloroethyl)-N-ethyl-2-bromobenzylaminetreated young rats. In contrast, no significant change in zif268 expression was detected between normal and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine-treated animals. These findings suggest that differential expression of these immediate-early genes is involved in the activity-dependent regulation of cortical function. One possibility is that the noradrenergic system controls cortical function, including plasticity, by modifying the expression of c-fos. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: visual cortex, activity-dependent regulation, ocular dominance plasticity, c-fos, zif268, noradrenaline.

Synaptic plasticity and, in particular, the use-dependent change in synaptic efficacy are important features of the CNS. One of the most widely studied experimental models of this kind of synaptic plasticity is the ocular dominance modifiability of the visual cortex of young animals. 56 When a young kitten is deprived of vision in one eye for as briefly as a few days, many cells in the visual cortex lose their functional connections with the sutured closed eye, the majority thus becoming monocular (ocular dominance plasticity). 57 Previous studies have collectively indicated that ocular dominance plasticity ‡To whom correspondence should be addressed. §Present address: Laboratory for Neuronal Circuit Development, Brain Science Institute (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0106, Japan. Abbreviations: ABC, avidin–biotin complex; D, dark-reared animal group; DBH, dopamine-b-hydroxylase; DDL, animals exposed to ambient light after dark rearing; DDPS, animals that received flash stimulation after dark rearing; DSP4, N-(2chloroethyl)-N-ethyl-2-bromobenzylamine; HRP, horseradish peroxidase; IEG, immediate-early gene; LDPS, animals that received flash stimulation without preceding dark rearing; N, naive animal group; NA, noradrenaline; PBS, phosphate-buffered saline; PBS-T, PBS containing Triton X-100; SSC, standard saline citrate. 473

is regulated by neuronal activity in the visual cortex, and thus experimental manipulation of cortical excitability during monocular deprivation results in the suppression of this plasticity. 47,48 Studies have also demonstrated that the visual signal originating from the retina is transmitted to the visual cortex by the glutamatergic system. 2,21,53 Transcription factors of immediate-early genes (IEGs) are rapidly and transiently activated after cell stimulation without the requirement for de novo protein synthesis. 51 Among these, c-fos and zif268 (also termed egr-1, Krox 24 and NGFI-A) are considered to play important roles in activitydependent plasticity during development. 27 In fact, the expression of these genes in the primary visual cortex is modulated by intraocular injection of tetrodotoxin or by intraperitoneal injection of glutamate receptor ligands, such as kainate (agonist) or dizocilpine maleate (antagonist). 58,59 Furthermore, previous studies, using mammalian visual cortex of monkey, cat and rat, have demonstrated differences in the expression pattern between c-fos and zif268 genes or their proteins, both at the constitutive level and after visual deprivation or stimulation. 8,30,59 The constitutive expression of zif268 mRNA and protein

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in the brain is high and is reduced by visual deprivation. In contrast, basal levels of c-fos are relatively low and are transiently increased by stimulation. These findings suggested that expression of the c-fos and zif268 genes is regulated through distinct pathways to affect activity-dependent synaptic plasticity. In addition to the activity-dependent mechanism, non-retinal signals to the visual cortex have been implicated as a plausible neurochemical basis of ocular dominance plasticity. These modulatory systems include the noradrenergic, 3234,35 cholinergic 16,22 and serotonergic 17 systems. A number of studies have shown that the level of ocular dominance plasticity is reduced by the depletion and/or blockade of respective receptors for noradrenaline (NA), acetylcholine and serotonin in the visual cortex. However, little is known about the molecular mechanism by which such neuromodulatory systems control the activity-dependent mechanism. In the present study, we examined in detail the expression pattern of two IEGs, c-fos and zif268, in the visual cortex of young rats under various visual conditions using immunohistochemical and northern blot analysis. Also, to study the interaction between glutamate and NA (as a representative modulatory system) on the expression of activity-dependent genes in the visual cortex, we investigated whether the level of c-fos or zif268 expression induced by photic stimulation was affected by treatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), a selective noradrenergic neurotoxin. 25,26 EXPERIMENTAL PROCEDURES

All experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and approved by the animal research committee of the Osaka Bioscience Institute. All efforts were made to minimize animal suffering and to reduce the number of animals used in the present study.

N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine tration

adminis-

Male Wistar rats (four weeks old, n ˆ 22) were used for immunohistochemistry and northern blot analysis. DSP4 (Sigma) in 0.9% saline, prepared immediately before use, was administered intraperitoneally in a dose of 50 mg/kg. The same volume of 0.9% saline was injected into control animals. Five days after a single DSP4 treatment, the rats were placed under the following experimental conditions: (i) naive; (ii) 6 h of dark rearing starting at 9.00 a.m.; and (iii) exposure to flash stimulation for 30 min after the 6 h of darkness. Northern blot analysis Northern blot analysis was based on the method of Mataga et al. 38 Rats were killed by decapitation, and a 5mm-wide slice of the dorsal posterior cortex, which contains the primary visual cortex, was rapidly dissected from the caudal region of the corpus callosum on ice. The dorsomedial cortex, including the motor cortex, and the dorsoanterior cortex, including the somatosensory cortex, were also dissected out. Total RNA was extracted by the acid guanidine isothiocyanate–phenol–chloroform method. Total RNA (25–30 mg) was electrophoresed on 1% agarose/ formaldehyde (0.44 M) gels and transferred to nylon filters (Hybond N w, Amersham, Canada) by electroblotting. The filters were prehybridized overnight at room temperature in a buffer containing 50% formamide, 5 × standard saline citrate (SSC), 50 mg/ml sheared and denatured salmon sperm DNA and 5 × Denhardt’s solution. Complementary DNA probes for rat c-fos ( , 1.0 kb, Pst I fragment) 12 and zif268 ( , 1.6 kb, Bgl II fragment) 10 were radiolabeled with [a- 32P]dCTP (Amersham, Canada) by the random priming method (Pharmacia LKB, U.S.A.). Hybridization was carried out at 428C in prehybridization buffer containing radiolabeled probes (c-fos: 2 × 10 6 c.p.m./ml; zif268: 1 × 10 6 c.p.m./ml). Filters were washed three times at room temperature for 5 min in 1 × SSC, containing 0.1% sodium dodecyl sulfate, followed by a wash at 608C for 20 min in 0.2 × SSC/0.1% sodium dodecyl sulfate. The filters were exposed to X-ray films (RX w Fuji Film Co., Japan) at 2 808C using intensifying screens for two to three days. The autoradiographic signals corresponding to c-fos and zif268 mRNAs were analysed using NIH image (version 1.57) software for Macintosh and normalized by comparison with the value of glyceraldehyde 3-phosphate dehydrogenase gene mRNA.

Experimental manipulation of visual input Male Wistar rats (four weeks of age, n ˆ 67; SLC, Shizuoka, Japan) at the peak of the sensitive period for ocular dominance plasticity 13 were used for immunohistochemistry and northern blot analysis. Rats were grouped as follows: (i) usual 12-h/12-h light/dark cycle (N; n ˆ 13); (ii) one week of dark rearing to determine the effect of reduced visual activity (D; n ˆ 12); (iii) exposure to flash stimulation (0.5 Hz, 2 J) for 20, 30, 45 or 90 min after one week of dark rearing (DDPS; n ˆ 6–12 for each time); (iv) exposure to ambient light for 45 min after one week of dark rearing (DDL; n ˆ 6); and (v) exposure to flash stimulation for 45 min without the preceding dark rearing (LDPS; n ˆ 6). All experimental groups had ad libitum access to food and water. All animals were four weeks old at the beginning of dark rearing. All rats were killed at the same time of day to avoid possible circadian effects. Dark-reared animals were decapitated within 1 min of removal from the rearing room under a photographic darkroom lamp (,1 lx on the surface of the preparation table). For comparison, an additional 18 adult rats (11 weeks of age) were examined for the response pattern of IEGs to photic stimulation.

c-Fos and Zif268 immunohistochemistry Rats were deeply anesthetized with an overdose (100 mg/ kg) of sodium pentobarbital (Nembutal w; Abbot) and perfused transcardially with 0.9% saline and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. Each brain was removed and immersed in the same fixative at 48C overnight and cryoprotected with increasing concentrations of sucrose in 0.1 M phosphate buffer (15% sucrose for one day and 30% sucrose for several days at 48C until the brain sank). Using a sliding microtome, 50-mm frozen sections were cut and free-floating sections were placed in 10 mM phosphate-buffered saline (PBS), containing 0.1% hydrogen peroxide and 0.3% Triton X-100, for 2 h to block endogenous peroxidase activity. After washing with PBS, the sections were incubated with 0.3% Triton X-100, 2.5% bovine serum albumin, 2% normal goat serum and 10% blockase w (Yukijirushi, Japan) in 10 mM PBS overnight at 48C to block non-specific binding. The sections were then placed in a solution containing rabbit polyclonal antiserum selective for Zif268 [Santa Cruz Biotechnology, egr1 (C-19), 1:2000 for horseradish peroxidase (HRP), 1:50,000 for avidin–biotin complex (ABC)] or for c-Fos (Oncogene

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Science, Ab-5, 1:100,000) at 48C for 24–48 h. For detection of primary antibodies of Zif268, we used two different secondary antibodies as follows: (i) HRP-conjugated goat anti-rabbit immunoglobulin G at 1:500 in PBS at room temperature for 45 min, and (ii) biotinylated secondary antibody (goat anti-rabbit immunoglobulin G; Vector) at 1:250 in PBS for 30 min, followed by the ABC method for detection (Vectastain w Elite ABC Kit, Vector). After washing with PBS and 0.05 M Tris buffer, the reaction for visualization was performed in 0.05 M Tris buffer containing 0.02% diaminobenzidine, 0.0025% nickel chloride and 0.01% hydrogen peroxidase for 2–3 min. The sections adjacent to those used for immunostaining were always stained by Cresyl Violet for laminar analysis. Immunohistochemistry for dopamine-b -hydroxylase Tissue preparation for dopamine-b-hydroxylase (DBH) immunohistochemistry was the same as that for c-Fos and Zif268. Sections from control and DSP4-treated animals were incubated with PBS containing 0.3% Triton X-100 (PBS-T) at 48C for two days and then washed in distilled water (3 × 5 min). Sections were processed with 0.3% hydrogen peroxide in methanol for 10 min. After single washing in distilled water followed by PBS-T (2 × 5 min), non-specific binding was blocked by preincubation with 4% normal goat serum in PBS-T for 10 min. Sections were rinsed in PBS-T (3 × 5 min) and incubated in anti-DBH antibody (TE103, Eugene Tech International, U.S.A.) at 1:50 000 with PBS-T at 48C for 48–72 h. After incubation, the sections were washed in PBS-T (7 × 3 min) and placed in biotinylated secondary antibody (goat anti-rabbit immunoglobulin G; Vector) at 1:200 with PBS-T for 45 min. After washing in PBS-T, sections were incubated with peroxidase-conjugated streptavidin (DAKO, Denmark) at 1:600 in PBS-T for 45 min, then rinsed with PBS (2 × 5 min) and further washed with 0.05 M Tris buffer. The reaction products were visualized in 0.05 M Tris buffer, containing 0.01% diaminobenzidine, 0.06% nickel ammonium and 0.01% hydrogen peroxidase, for 2–3 min. The reaction was terminated by washing the sections in PBS-T (2 × 5 min) and PBS (1 × 5 min). All sections were mounted on gelatin/chrome–alumsubbed slides, dehydrated through graded concentrations of ethanol, cleared in xylene and then mounted with Permount w (Fisher Sci., U.S.A.). Semi-quantification of c-Fos and Zif268 immunoreactivity Three pairs of corresponding sections were selected from different groups of animals. c-Fos- and/or Zif268-immunoreactive cells in the visual cortex were semi-quantified, by a method modified from Simpson et al. 52 and Zhu et al. 60 Immunoreactive images were captured by a computerassisted microscope system (PROVIS AX-HDTV system, Olympus, Japan). Under bright-field microscopy, the c-Fos and Zif268 signals appeared as dark brown reaction products restricted to the nucleus of positively stained cells. Using image analysing software (MacASPECTw, Mitani, Japan), target criteria for area and optical density were set on the basis of visual identification of highly immunostained neurons. c-Fos- or Zif268-immunopositive cells in three sections from each brain were counted in a 100 × 200 pixel area (0.064 mm 2) in layers II/III, IV or V/ VI. The layer borders in the rat visual cortex were determined according to criteria used by Reid and Juraska. 46 Data analysis Statistical comparisons were made using either an unpaired t-test or a one-way ANOVA. The Bonferroni– Dunn post hoc test was used for comparison when significance was indicated by ANOVA.

475 RESULTS

Effects of different light conditions on c-fos and zif268 messenger RNA expression We first studied c-fos and zif268 mRNA expression in the visual cortex of naive rats using northern blot analysis. The sizes of c-fos and zif268 transcripts were in agreement with previous studies. 10,12 In order to compare results obtained from different autoradiographic films, we normalized these values against those of glyceraldehyde 3-phosphate dehydrogenase expression, which was confirmed not to be affected by the one week of dark rearing. Expression patterns of c-fos and zif268 genes differed markedly, but both were higher in younger animals than in adults. The basal expression of c-fos mRNA in the visual cortex of four-week-old rats was five times higher than that of 11-week-old rats (unpaired t-test, P , 0.001). Robust expression of zif268 mRNA was found in the visual cortex of four-week-old rats and its expression level was 1.6 times higher than that of 11-week-old rats (unpaired t-test, P , 0.01). To evaluate whether mRNA expression of these IEGs in the visual cortex was regulated by visual input, rats were reared under total darkness. After one week of dark rearing, the expression of c-fos mRNA in the visual cortex of young rats was found to be significantly reduced to half of that found in normally reared animals (Fig. 1A; unpaired t-test, P , 0.01). Expression markedly and transiently increased upon photic stimulation after dark rearing. The expression of c-fos mRNA reached a peak, which was six times higher than the value of dark-reared animals, approximately 30 min after photic stimulation. The c-fos mRNA expression induced by visual stimulation was prominent when the photic stimulation was given after dark rearing. Photic stimulation without preceding dark rearing induced lower c-fos mRNA expression levels, although the difference was not statistically significant (Fig. 1A; LDPS vs DDPS). The expression of zif268 mRNA was also significantly reduced by half (unpaired t-test, P , 0.001) after one week of dark rearing (Fig. 1B). The expression of zif268 mRNA was much higher than that of c-fos in dark-reared animals. The expression of zif268 mRNA returned to the basal level within 20 min of photo-stimulation applied after one week of dark rearing. Messenger RNA expression at 20 min had reached saturation, as evidenced by the fact that photo-stimulation for 90 min induced almost the same amount of zif268 mRNA. The preceding dark rearing did not affect the induction of zif268 mRNA by visual stimulation (Fig. 1B, LDPS vs DDPS). Similar analyses were performed using samples obtained from the frontal and parietal cortical regions (Fig. 2). In these regions, expressions of c-fos and zif268 mRNAs were not affected by one

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Fig. 1. Effects of visual stimulation on c-fos (A) and zif268 (B) mRNA expression in the visual cortex of young rats. The results of northern analysis of rat visuocortical RNA are presented at the top of each graph. Lane N represents a naive animal reared under the normal dark/light cycle and killed during the light phase. Lane D represents an animal reared in a total darkness for one week and killed under a dark-room light. Other lanes (DDPS) represent animals that received strobe flash stimulation for the indicated time period following dark rearing. Columns in the lower graph represent mean and S.E.M. of quantified values from the densitometric analysis of blots. LDPS represents data from animals reared under a normal dark/light cycle and killed after flash stimulation for 45 min. DDL represents rats reared for one week in complete darkness and killed after 45 min exposure to ambient light. The number of rats used for each experiment was as follows: N, n ˆ 10; LDPS, n ˆ 6; D, n ˆ 9; DDL, n ˆ 6; DDPS, 20, 30 and 90 min, n ˆ 6; DDPS 45 min, n ˆ 9. Statistical analysis was performed using the Bonferroni–Dunn post hoc test. Asterisk indicates a comparison with the D value and # indicates the result of comparison with the N value. The number of each symbol indicates the level of significance (1, P , 0.05; 2, P , 0.01; 3, P , 0.001). N and D values were compared using unpaired t-test († in A and § in B). G3PDH, glyceraldehyde 3-phosphate dehydrogenase.

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Fig. 2. Effects of visual stimulation on c-fos (A, C) and zif268 (B, D) mRNA expression outside the visual cortex. The results of northern analysis are shown for the parietal (A, B) and frontal (C, D) cortices of young rats. The convention is the same as for Fig. 1. Three rats were used in each experiment.

week of dark rearing. However, expression of c-fos mRNA in both the parietal and frontal cortices was observed to increased 30–45 min after visual stimulation (Fig. 2A, C). The mRNA increase in the frontal cortex was found to be significant only when a statistical method of lower threshold was used (Fig. 2C; Fisher post hoc test, P , 0.01). In the frontal and parietal cortical regions, expression of zif268 was not affected by dark rearing or following visual stimulation. Effects of different light conditions on c-Fos and Zif268 immunoreactivity To visualize the spatial distribution of c-Fos and Zif268 proteins in rat visual cortex, we performed an immunohistochemical analysis. A relatively small number of c-Fos-immunopositive cells was found in the visual cortex of naive and young rats (four weeks old). The number of immunopositive cells was higher in layer IV (95.8 cells/counted area) than the other layers (layers II/III, 67.1; layers V/VI,

62.1 cells/counted area) in the visual cortex. In the visual cortex of adult rats (11 weeks old), a small population of cells showed immunoreactivity against c-Fos (data not shown). As shown in Fig. 3A–C, one week of dark rearing markedly reduced c-Fos immunoreactivity (Fig. 3A vs B) and photic stimulation for 45 min after the dark rearing dramatically increased it (Fig. 3A or B vs C). An increase in the number of c-Fos-immunopositive cells was observed throughout the cortical layers. Semi-quantitative analysis revealed that the increase in c-Fosimmunopositive cells induced by visual stimulation after dark adaptation was about 20-fold in layers II/ III, 11-fold in layer IV and seven-fold in layers V/ VI, respectively (Fig. 3D). No regional difference was observed in the increase between the occipital cortex, area 1, monocular part (Oc1M) and occipital cortex, area 1, binocular part (Oc1B). In contrast to c-Fos expression, a relatively large amount of Zif268-immunopositive cells was observed in all the cortical layers of naive and young rats (layers II/III, 165.9; layer IV, 225.8;

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Fig. 3. Effects of photic stimulation after one week of dark rearing on c-Fos and Zif268 immunoreactivity in visual cortex of young rats. (A–C) A visuocortical section immunostained with anti-c-Fos antibody. (A) Naive control. (B) Animal was killed immediately after dark rearing for one week. (C) The rat received photic stimulation for 45 min after one week of dark rearing. (E–G) Zif268 immunoreactivity found in the visual cortex of a naive control animal (E), a dark-reared animal (F) and after photic stimulation (G). Scale bar ˆ 300 mm. (D, H) Number of immunopositive cells per counted area (200 × 100 pixels) is summarized for the three different cortical sublayers. Each column shows the mean and S.E.M. calculated from the averaged value of three animals (three sections/animals). Statistical comparisons (one-way ANOVA) were made. Asterisks indicates a comparison between D (dark-reared) and PS (photo-stimulated), # indicates comparison between N (Naive) and PS. The number of each symbol indicates the level of significance (1, P , 0.05; 2, P , 0.01; 3, P , 0.001).

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Fig. 4. Photomicrographs of DBH immunohistochemistry in coronal sections of the rat visual cortex. (A) Control animal. (B) DSP4-treated animal. The top is the dorsal surface of the cortex. Scale bar ˆ 300 mm.

layers V/VI, 167.4 cells/counted area). As shown in Fig. 3F, a reduction in the number of Zif268-immunopositive cells was found in layers II/III of the visual cortex of dark-reared animals. Light exposure for 45 min after dark rearing induced a small increase in the immunopositive cells throughout the layers (Fig. 3F, G). The increase was 40% in layers II/III, 40% in layer IV and 31% in layers V/ VI; however, no statistically significant differences were detected. No apparent difference in the change induced by visual stimulation was found between the Oc1M and Oc1B. The effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine on c-Fos and Zif268 immunoreactivity To examine the effects of DSP4 injection into the visual cortex of young rats (four weeks old), we performed immunohistochemical staining for DBH. As shown in Fig. 4A and B, DSP4 administration resulted in a dramatic reduction in DBH-immunoreactive fibers. Many small rounded vesicles, which might indicate regeneration of noradrenergic fibers, were observed throughout the cortical layers. By staining adjacent sections with anti-c-Fos or Zif268 antibody, we examined the effects of DSP4 on the basal expression of immunoreactivity in the visual cortex of young rats. As shown in Fig. 5A and B, DSP4 administration markedly reduced the

number of c-Fos-immunopositive cells in all cortical layers (layers II/III, 17.8%; layer IV, 14.1%; layers V/VI, 24.9% of control), whereas only a small decrease was found in the number of Zif268-immunopositive cells in the visual cortex of DSP4-treated animals (layers II/III, 88.5%; layer IV, 87.4%; layers V/VI, 95.0% of control). These results suggest that the central noradrenergic system plays an important role in the induction of c-Fos in the visual cortex of young rats. Effect of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine on c-fos and zif268 messenger RNA expression in the visual cortex To examine the effect of DSP4 on the expression of c-fos or zif268 mRNA, northern blot analysis was performed using cortical samples obtained from DSP4-treated animals (four weeks old) that received 30 min of visual stimulation after the preceding dark rearing for 6 h. DSP4 treatment significantly reduced the visually evoked increase in c-fos mRNA to 75.7% of the control level (P , 0.01, unpaired t-test; Fig. 6A, control-PS vs DSP4-PS). In addition, DSP4 reduced the expression of c-fos mRNA in the visual cortex of naive animals up to 62.9% of the control level (P , 0.05, unpaired t-test; Fig. 6A, control-N vs DSP4-N), supporting our previous analysis of c-Fos protein.

Fig. 5. The effects of DSP4 on c-Fos and Zif268 immunoreactivity in rat visual cortex. (A, C) Sections from control animals. (B, D) Sections from DSP4-treated animals. (A, B) Immunostained for c-Fos. (C, D) Immunostained for Zif268. (E, F) Number of immunopositive cells per counted area (200 × 100 pixels) for c-Fos (E) and Zif268 (F) summarized for the three different cortical sublayers. Each column shows the mean and S.E.M. of values calculated from four sections from two animals (cont., control rats; DSP4, toxin-treated rats). Scale bar in D ˆ 300 mm (applies to A–D).

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analysis, we investigated the expression pattern of two IEGs, c-fos and zif268, under various visual conditions in the visual cortex of young rats. The expression of c-fos mRNA and c-Fos protein in the visual cortex of naive animals was relatively low and was further reduced by one week of dark rearing. A marked and transient increase in the expression of c-fos mRNA and c-Fos protein was induced by visual stimulation after dark rearing. In contrast to c-fos, the basal expression of zif268 mRNA and Zif268 protein was relatively high in the visual cortex of young rats. Its expression was reduced by dark rearing and increased to the basal level upon the introduction of light after dark rearing. DSP4, a selective noradrenergic neurotoxin, suppressed the basal expression of c-fos mRNA and c-Fos protein. The response of c-fos mRNA to photic stimulation was also reduced in the visual cortex of young rats by DSP4 treatment. In contrast, the expression of zif268 mRNA and Zif268 protein was less affected by DSP4 injection. Expression patterns of c-fos and zif268 in response to visual stimulation

Fig. 6. Effects of DSP4 on c-fos and zif268 mRNA expression in the visual cortex of young rats. Quantified and normalized values of northern blot analysis are shown for c-fos (A) and zif268 (B). Each column indicates the mean and S.E.M. (n ˆ 3). DSP4, animals were injected with DSP4 dissolved in saline (50 mg/kg, i.p.); control, animals were injected only with saline. N, rats were reared under the usual light/dark cycle and killed during the light phase; D, rats were kept in complete darkness for 6 h and killed under a dark-room light; PS, rats were killed after flash stimulation for 30 min, which was given immediately after dark rearing. Statistical comparisons (unpaired t-tests) were performed between corresponding values of control and DSP4: *P , 0.05; **P , 0.01.

However, DSP4 administration induced no significant change in the expression of zif268 mRNA under any visual conditions examined in the present study. These results suggest that the function of the central noradrenergic system is important for cortical induction of c-fos mRNA evoked by visual activation, while the induction of zif268 mRNA is less affected by the noradrenergic system. DISCUSSION

Using northern blot and immunohistochemical

Our results are basically in agreement with those of previous reports on changes in the expression of transcription factor mRNA and protein induced by visual stimulation in the visual cortex of rat, 28,58,59 cat 3,30,43,49 and monkey. 8,9 First, there is a clear difference in the levels of constitutive expression between c-fos and zif268 in the visual cortex of young rats: the expression of c-fos mRNA is relatively low, while that of zif268 mRNA is high. 18,20,59 Previous studies have demonstrated that both c-fos and zif268 mRNA or protein are more highly expressed in the early postnatal period (e.g., five to 20 weeks in the cat) than in the adult. 18,29,40,59 We also found that the basal expression of c-fos mRNA in the visual cortex of fourweek-old rats was relatively low, although still significantly higher than that of adult rats, which was barely detectable by the techniques used in the present study. We can therefore detect a significant decrease in the expression of c-fos level following prolonged dark rearing (one week). The expression of zif268 mRNA was also higher in the visual cortex of young rats. Due to this high basal expression, a significant reduction in expression of zif268 mRNA was also detected following one week of dark rearing. We used young four-week-old rats in our experiments, since they were clearly in the sensitive period for ocular dominance plasticity, which has been defined precisely by a previous electrophysiological study. 13 Our comparison of adjacent sections stained with either anti-c-Fos or Zif268 antibody indicates that the number of immunopositive cells for Zif268 at baseline level was about 2.5-fold higher than that for c-Fos in the visual cortex of young rats.

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Second, there is a clear difference in the spatiotemporal pattern of c-fos and zif268 induction. The expression of c-fos mRNA in the visual cortex was increased markedly by photic stimulation after dark adaptation, and gradually decreased even in the presence of continuous visual stimulation. The expression of zif268 mRNA was also enhanced by visual stimulation, however, it was saturated at the level of basal expression without showing any decrease upon prolonged visual stimulation. The saturation was not due to the high sensitivity of the detection method for immunoreactivity analysis (ABC method), since the less sensitive HRPconjugated secondary antibody detection method yielded almost identical results. Finally, the pattern of expression outside the visual cortex was different between c-fos and zif268. We found that the increase in c-fos mRNA and c-Fos protein was induced in the frontal and parietal cortical regions by visual activation after dark rearing. In contrast, the expression of zif268 in these regions showed no change upon visual activation. The increase in c-fos mRNA in the frontal and parietal cortex upon visual stimulation after prolonged rearing in total darkness may be induced secondarily by uncontrollable stress 11 or the environmental effects of being exposed to novel objects. 60 Previous experiments have shown that c-Fos is induced in the visual cortex without preceding dark rearing, suggesting the importance of novelty associated with the stimulation. 4,44,45 Correspondingly, we found no quantitative difference in the induction of c-fos between animals that experienced ambient light stimulation and those that underwent strobe flash stimulation after dark rearing. The different expression patterns of c-fos and zif268 mRNAs and proteins suggest different roles for these transcription factors in the regulation of neuronal function. Regulation of c-fos and zif268 expression by the central noradrenergic system In the present study, we focused on the role of the central noradrenergic system in the regulation of activity-dependent expression of c-fos and zif268 in the visual cortex. It is unlikely that NA simply increases the excitability of cortical cells. Previously, enhanced release of NA either by activation of the locus coeruleus or by microiontophoresis of exogenous NA has been shown to modulate neuronal responses to afferent synaptic input in the auditory, somatosensory and visual cortices. 14,33,50,54,55 Previous studies have reported that the spatiotemporal tuning of visual cortical neurons may depend not only on local interactions between visual afferents and target neurons, but also on the activation state of the noradrenergic cerulcocortical projection system in cortical area 17 of the rat and cat. 36,41,54 Furthermore, many experimental

results indicate that the central noradrenergic system plays important roles in the regulation of ocular dominance plasticity in the cat visual cortex. 23,24,31,32,34,35 The release of NA in the cerebral cortex is thought to be regulated by N-methyl-d-aspartate receptordependent and nitric oxide-mediated processes. 42 In addition, visual stimulation enhances the release of NA. 37 In the present study, we showed that both basal and visually evoked expression of c-fos was significantly reduced in the visual cortex in which endogenous noradrenergic fibers had been degenerated by DSP4 treatment. It is therefore possible that NA inversely regulates the activity-dependent process in the visual cortex via N-methyl-d-aspartate- and/or nitric oxide-mediated processes. We employed a shorter duration of dark rearing in this series of experiments. If one week of dark rearing had been imposed as in the former series of experiments, the values of dark-reared animals would be expected to show an even greater decrease and those of dark-reared/photo-stimulated animals an even greater increase. However, it is possible that visual activation following one week of dark rearing is sufficiently large to mask the differences in response between control and DSP4-treated animals. It is therefore necessary to elucidate in a future study what level of dark adaptation is the best for detecting the effect of DSP4 on the expression of these IEGs. Our findings in the visual cortex were in accordance with previous findings that NA plays an important role in the expression of c-fos in the rat CNS. 6,7,19 We were unable to detect any changes in zif268 expression in the visual cortex of DSP4treated animals. One explanation is that expression of zif268 is regulated by modulatory systems other than the noradrenergic system. Another possibility is that we could not completely eliminate the noradrenergic afferents to the visual cortex. Based on previous studies, we estimate that about 20–30% of NA remains in the visual cortex of DSP4-treated animals. 15,39 It is thus possible that more severe and specific degeneration of NA fibers induced a significant reduction in the expression of zif268 mRNA level. Actually, Bhat and Baraban 5 have shown using DSP4 that expression of zif268 does depend upon activation of noradrenergic neurons. They reported an intriguing regional difference in the reduction of zif268 mRNA following DSP4 administration. Messenger RNA in the cortex and striatum was markedly suppressed, but that in the hippocampus and piriform cortex was not affected. This may be due to the differential effect of DSP4 on the central noradrenergic system and/or to regional differences in compensatory mechanisms after suppression of the central noradrenergic system. Bhat and Baraban pretreated animals with a selective serotonin uptake inhibitor 30 min prior to DSP4 injection, in order to prevent any effect of DSP4 on serotonin projection. 5

Noradrenergic regulation of IEGs in the visual cortex

We omitted this process in our study so that DSP4 would be able to affect not only the central NA, but also the serotonin system, and to reduce the effect of DSP4 on the expression of zif268. Currently, there is no evidence that expression of c-fos is critical for ocular dominance plasticity, which is known to be accompanied by anatomical changes in terminal arborization of thalamic fibers. 1 However, it seems likely that c-fos and the subsequent late gene expression are involved in the activity-dependent modification of the neuronal network required for de novo protein synthesis. CONCLUSIONS

We have reported a differential expression pattern

483

of c-fos and zif268 in the rat visual cortex following dark rearing and visual activation. These experiments using a specific noradrenergic neurotoxin, DSP4, showed that the noradrenergic system in the visual cortex of young rats plays an important role in the regulation of c-fos expression. It is suggested that the central noradrenergic system affects activity-dependent plasticity by modulating the signal transduction mediated by c-fos.

Acknowledgements—We are grateful to Mr Tsuyoshi Shiomitsu for assistance and excellent technical support throughout the course of this study. We also thank Dr Shoji Nakamura for his helpful advice on DbH immunohistochemistry.

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