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Expression of c-Fos, Fos B, Jun B, and Zif268 transcription factor proteins in rat barrel cortex following apomorphine-evoked whisking behavior
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Neuroscience Vol. 106, No. 4, pp. 679^688, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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EXPRESSION OF c-Fos, Fos B, Jun B, AND Zif268 TRANSCRIPTION FACTOR PROTEINS IN RAT BARREL CORTEX FOLLOWING APOMORPHINE-EVOKED WHISKING BEHAVIOR R. K. FILIPKOWSKI,a;b * M. RYDZa;c and L. KACZMAREKa a
Department of Molecular and Cellular Neurobiology, Nencki Institute, Pasteura 3, 02-093 Warsaw, Poland b c
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
Division of Geriatric Medicine, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden
AbstractöApomorphine-evoked expression of transcription factor proteins: c-Fos, Fos B, Jun B, and Zif268 (also named Krox-24, NGFI-A, Egr-1), was investigated in rat somatosensory (barrel) cortex. The e¡ect of the N-methyl-D-aspartate receptor antagonist MK-801 on their expression was also analyzed. Apomorphine is a dopamine receptor agonist, eliciting motor activity, including enhanced whisking leading to the activation of vibrissae representation in the barrel cortex. Rats had their whiskers clipped on one side of the snout. The Zif268 levels were markedly reduced by this procedure alone. In contrast, apomorphine (5.0 mg/kg) evoked marked c-Fos elevation, less pronounced changes in Jun B and Zif268 and no change in Fos B. The greatest apomorphine-evoked c-Fos accumulation was observed in layers IV and V/VI of non-deprived barrel cortex and was not signi¢cantly in£uenced by MK-801 injection at 0.1 mg/kg. A higher dose of MK-801 (1.0 mg/kg) produced abnormalities in locomotor behavior and diminished c-Fos levels on the non-deprived side to the ones observed in the sensory stimulus-deprived cortex. We conclude that the response of the somatosensory cortex is selective with respect to both the gene activated and its cortical layer localization. Furthermore, sensory stimulation provides a major but not the only component to apomorphine-evoked barrel cortex gene activation. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: vibrissae, NMDA receptor, immediate early genes, AP-1, MK-801.
(PMBSF) (Woolsey and Van der Loos, 1970), also known as the MV area (Chapin and Lin, 1984). Because of its simplicity, laterality as well as ethological and behavioral importance, the rodent somatosensory cortex, representing mystacial vibrissae (large whiskers) of the snout, o¡ers a very convenient system to study the e¡ects of sensory stimulation upon cortical function. Sensory stimulation plays an important role in neuronal plasticity (Buonomano and Merzenich, 1998) and recent studies indicate that gene expression may be of pivotal importance for this phenomenon (Kaczmarek, 1993; Kaczmarek and Chaudhuri, 1997). The barrel cortex has already been a subject of investigation of stimulation-evoked gene expression (see review by Filipkowski, 2000). In particular, Steiner and Gerfen (1994) as well as LaHoste et al. (1996) reported that the treatment of rats with dopamine agonists, such as apomorphine, known to enhance motor (including whisking) activity (Beck et al., 1986; Berke et al., 1998; Szechtman et al., 1982; Young et al., 1991), resulted in the accumulation of immediate early genes, c-fos and zif268 mRNA as well as c-Fos protein in various brain regions including somatosensory cortex. This e¡ect was fully input-speci¢c, since clipping of the whiskers prevented the mRNA increase in the corresponding PMBSF (Steiner and Gerfen, 1994). Thus, it can be inferred that sensory input is critical for regulating c-fos and zif268 expression in the barrel cortex. Fur-
In the rodent vibrissae-barrel cortex system, sensory input produced by de£ection of the whiskers is conveyed via the brainstem and thalamus to cortical layer IV, where each cortical column, extending through all layers, contains a barrel, a discrete cluster of closely packed cells. There is one-to-one correspondence between each barrel and each vibrissa. The whisker-to-barrel system is contralateral, so the somatosensory cortex on one side represents vibrissae on the other side of the snout (Kossut, 1992). The part of the barrel ¢eld containing the large barrels and representing the vibrissae of the mystacial pad is called the postero-medial barrel sub¢eld
*Correspondence to: R.K. Filipkowski, Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA. Tel.: +1-516-367-8886; fax: +1-516-367-8880. E-mail address: ¢[email protected] (R. K. Filipkowski). Abbreviations : AP-1, activator protein-1 transcription factor; apo, mice treated with apomorphine; apoMK, mice treated with apomorphine and MK-801 (0.1 mg/kg); apoMK1, mice treated with apomorphine and MK-801 (1.0 mg/kg); CREB, cAMP-response element-binding protein transcription factor; DAB, 3,3P-diaminobenzidine; FL/HL, forelimb/hindlimb, a part of the cortex receiving sensory information from the limbs; MK, mice treated with MK-801; MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine, dizocilpine ; NMDA, N-methyl-D-aspartate; PBS, phosphate-bu¡ered saline ; PMBSF, postero-medial barrel sub¢eld. 679
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thermore, recently, Steiner and Kitai (2000) reported that selective intrastriatal infusion of the dopamine D1 receptor antagonist SCH-23390 attenuated apomorphineevoked gene expression in the cortex, thus suggesting that basal ganglia mediate this e¡ect, most probably via their role in the control of motor behavior. To further our knowledge on genomic response in the cortex, we decided to extend the aforementioned observations to protein levels of c-Fos and Zif268 as well as to investigate apomorphine- and sensory input-driven expression of two other transcription factor proteins, Jun B and Fos B. Fos and Jun proteins combine to produce the activator protein-1 (AP-1) transcription factor. Both AP-1 and Zif268 (also known as Krox-24, Egr1, NGFI-A) appear to be the major inducible gene expression regulators in the CNS (Herdegen and Leah, 1998; Morgan and Curran, 1991). Despite these apparent similarities, the aforementioned transcription factor proteins may play di¡erential roles in regulating neuronal gene expression. c-Fos is readily induced by many, even subtle, stimuli including, e.g., exposure to a novel environment (Anokhin et al., 1991; Filipkowski et al., 2000; Montero, 1997; Staiger et al., 2000). Inducibility of Jun B in the brain was observed in striking parallelism to c-Fos (Gass et al., 1993b; Kaminska et al., 1994, 1996; Kinouchi et al., 1994; Konopka et al., 1998; Luckman et al., 1996; Moratalla et al., 1993; Staiger et al., 2000). Hence, it could be inferred that Jun B and cFos form a major inducible AP-1 complex after stimula-
tion. In contrast, although Fos B levels were shown to increase in the brain in several experimental paradigms, expression of this protein was found not to change in many other physiological situations (Herdegen and Leah, 1998). Finally, Zif268 has been reported to be expressed at high protein levels in sensory cortex under `naive' conditions and was found to be down-regulated by sensory deprivation and up-regulated by stimulation (Kaczmarek and Chaudhuri, 1997). We focused on protein levels because an excellent spatial resolution provided by the immunocytochemical approach allowed us to study laminar distribution of the immunoreactivities in the somatosensory cortex. Furthermore, since the N-methyl-D-aspartate (NMDA) receptor is known to be critical for synaptic plasticity and has also been implicated in activation of c-fos, zif268 and the expression of some other immediate early genes, especially in vitro (Bading et al., 1993, 1995; Condorelli et al., 1994; Morgan and Linnoila, 1991; Vaccarino et al., 1992), we also analyzed its role in our preparation using the NMDA receptor antagonist MK-801. We employed the experimental model described before (Steiner and Gerfen, 1994). After clipping rats' mystacial vibrissae on one side of the snout and then applying apomorphine and MK-801, the expression of the immediate early gene proteins c-Fos, Fos B, Jun B, and Zif268 was evaluated in barrel cortex.
EXPERIMENTAL PROCEDURES
Treatment of animals All experiments described were done with adult male Wistar rats obtained from Nencki Institute Animal House. The animals were kept under the natural light^dark cycle with water and food provided ad libitum. The experiments were conducted during the light phase of the cycle. All e¡orts were made to minimize the number of animals used and their su¡ering. The rules established by the Ethical Committee on Animal Research of Nencki Institute and based on the disposition of the President of Polish Republic were followed strictly in all experiments. Experiment 1 On each of 20 days before the experiment, the rats were gently held in the same way as during the clipping procedure. On each of 10 days before the experiment, the rats received a s.c. saline injection in the neck, 4 h after the handling. On the experimental day, all rats had their mystacial vibrissae clipped with scissors on one side of the snout (both sides were used in separate animals), which took 1^3 min (see Fig. 1). Then the rats were divided into two groups: control (three rats) and apo (four rats). All rats received one s.c. injection 4 h after the clipping: the control group received one saline injection, the apo group received one apomorphine (Sigma, St. Louis, MO, USA; 5.0 mg/kg in 0.02% ascorbic acid) injection. One and a half hour after the injection the rats were treated with an overdose of chloral hydrate and processed for c-Fos immunocytochemistry as well as Nissl and cytochrome oxidase staining. Fig. 1. Experimental model used in all experiments. After vibrissae clipping, the deprived and non-deprived sides of PMBSF can be distinguished. Bottom, schematic diagram of a coronal section with the areas (dark gray) where immunopositive cells were counted. FL/HL, medial region of somatosensory cortex which includes forelimb and hindlimb areas. The ¢ducial mark was used to recognize sides of brains after the reaction.
Experiment 2 On each of 20 days before the experiment, the rats were gently held in the same way as during the clipping procedure. On each of 14 days before the experiment, the rats received a saline injection, i.p., 3.5 h after the handling. On each of the 7 last
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Fig. 2. Design of experiment 2 in which rats had their vibrissae trimmed at one side of the snout, and received one i.p. injection (at 3.5 h) and one s.c. injection (at 4 h). The table shows the di¡erent experimental groups and substances injected. Then, 1.5 h later, the animals were perfused for immunocytochemistry.
days of habituation before the experiment, all rats received one s.c. injection 4 h after the handling. On the experimental day, all rats had their mystacial vibrissae clipped on one side (see above). The rats were divided into four groups, three to six rats in each. All received two injections (at time points as during habituation). The saline group (three rats) received one saline (i.p.) and one ascorbic acid (0.02%, s.c.) injection. The second group (MK, four rats) received one MK-801 injection (0.1 mg/ kg in saline, i.p.) and one ascorbic acid injection (0.02%, s.c.). The third group (apo, six rats) received one saline (i.p.) and one apomorphine injection (5.0 mg/kg in 0.02% ascorbic acid, s.c.). The fourth group (apoMK, six rats) received MK-801 (0.1 mg/ kg in saline, i.p.) and apomorphine (5.0 mg/kg in 0.02% ascorbic acid, s.c.), see also Fig. 2. One and a half hours after the second injection the rats were treated with an overdose of chloral hydrate and were processed for c-Fos, Fos B, Jun B, and Zif268 immunocytochemistry as well as Nissl and cytochrome oxidase staining. Experiment 3 The same as above (experiment 2) except that only three groups were investigated: saline (four rats), apo (¢ve rats) and apoMK1 (four rats). The MK-801 concentration was changed to 1.0 mg/kg. After the experiment, the rats were treated with an overdose of chloral hydrate and were processed for c-Fos immunocytochemistry as well as Nissl and cytochrome oxidase staining. Immunocytochemistry One and a half hour after s.c. injection all rats were anesthetized with an overdose of chloral hydrate and immediately perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate bu¡er, pH 7.4. The brains were removed and stored for 24 h in the same ¢xative at 4³C and then in 30% sucrose with 0.02% sodium azide at 4³C until needed. Then the appropriate parts of the brain were slowly and gradually frozen in a heptane/ dry ice bath. The expression of c-Fos and other proteins was assessed as described before (Filipkowski et al., 2000; Kaminska et al., 1996). In brief, coronal brain cryostat sections were cut at 320³C, 45 Wm thick, 2.3^3.3 mm caudal to bregma (Paxinos
and Watson, 1986). The sections were washed in phosphatebu¡ered saline (PBS), incubated in 0.3% H2 O2 , incubated with one of the polyclonal antibodies (anti-c-Fos, 1:1000, Santa Cruz #sc-52, Santa Cruz, CA, USA; anti-Fos B, 1:2000, Santa Cruz #sc-48; anti-Jun B, 1:1000; anti-Zif268, 1:5000, the latter two from R. Bravo, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, USA) for 48 h at 4³C in PBS with azide (0.01%) and normal goat serum (3%). After that the sections were washed in PBS with Triton X-100 (0.3%, Sigma), incubated with goat anti-rabbit biotinylated secondary antibody (1:1000, Vector Laboratories, Burlingame, CA, USA) in PBS/Triton and normal goat serum (3%) for 2 h, washed in PBS/Triton, incubated with avidin^biotin complex (1:1000, 1:1000, in PBS/Triton, Vector) for 1 h and washed in PBS. The immunostaining reaction was developed using the glucose oxidase^3,3P-diaminobenzidine (DAB)^nickel method (Shu et al., 1988). The sections were mounted on gelatin-covered slides, air-dried, dehydrated in ethanol solutions and xylene, and embedded in Entellan (Merck KGaA, Darmstadt, Germany). Nissl staining The sections were mounted on slides and air-dried (2^4 days), washed in PBS followed by 70%, 100%, 70% ethanol, water, 0.5% Cresyl Violet solution with acetic acid (0.35 M) and sodium acetate (0.06 M), and ¢nally dehydrated in ethanol (70% and 100%) and xylene and embedded in Entellan (Merck). Cytochrome oxidase staining The sections were washed twice in PBS and incubated in staining solution (1% sucrose; 0.05% nickel sulfate; 0.025% DAB; 0.015% cytochrome c; 0.01% catalase; 2.5 WM imidazole in 0.05 M phosphate bu¡er, pH 7.4; all reagents from Sigma) for several hours at 37³C. The stained sections were again washed in PBS, mounted on slides and air-dried (2^4 days). The slides were then washed in PBS, dehydrated and mounted as above. Data gathering and analysis In experiments 1 and 3, c-Fos-stained nuclei were counted using the image analysis system (MCID, IMAGING Research,
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St. Catherines, ON, Canada) in two regions of the cortex (Fig. 1), PMBSF (the region known to receive input from the mystacial vibrissae) and FL/HL (the medial adjacent region which includes forelimb and hindlimb areas), in both hemispheres. Middle parts of these regions, 1 cm wide, were taken for the analysis, as described before (Filipkowski et al., 2000; Steiner and Gerfen, 1994). In experiment 2, the number of c-Fos-, Fos B-, Jun B-, and Zif268-stained nuclei in the PMBSF area was evaluated in cortical layers II/III, IV, V/VI (without the subplate), and VIb (also known as layer VII, subplate, deep layer VI bordering the white matter). The position of the regions and layers was con¢rmed by Nissl and cytochrome oxidase staining. The nuclei were counted with the researcher being blind to the treatment. Averages from two to four sections per animal were used for statistical analysis. In all experiments, separated one-way analyses of variance (ANOVAs) were applied. Values in the ¢gures are expressed as means+S.E.M.
RESULTS
animals had very little c-Fos immunoreactivity in both deprived and non-deprived PMBSF areas, where no signi¢cant di¡erence in c-Fos expression between the sides was detected (1.2-fold di¡erence, F1;4 = 0.13, P = 0.7). Similarly low c-Fos levels were detected in the FL/HL area with no di¡erence between sides either (0.9-fold difference, F1;4 = 0.04, P = 0.9). Apomorphine treatment resulted in an increase in c-Fos immunoreactivity in FL/LH (non-deprived side: 3.6-fold di¡erence, F1;5 = 9.25, P 6 0.03; deprived side: 2.6-fold, F1;5 = 14.22, P 6 0.02) and PMBSF (non-deprived side: 5.1-fold difference, F1;5 = 29.74, P 6 0.003; deprived side: 3.0-fold, F1;5 = 14.26, P 6 0.02). Thus, after apomorphine injection no di¡erence in c-Fos expression between the contraand ipsilateral sides was observed in the FL/HL area (1.3-fold, F1;6 = 0.97, P = 0.4) and only in PMBSF was the concentration of the protein two-fold greater on the non-deprived side (F1;6 = 15.71, P 6 0.01).
Experiment 1
Experiment 2
In this experiment, animals with unilateral removal of vibrissae were treated with apomorphine and c-Fos levels were analyzed on both sides of the cerebral cortex in the areas representing the vibrissae (PMBSF) as well as adjacent representations of the forelimb/hindlimb (FL/HL). After the injections, the control animals remained inactive. In contrast, apomorphine treatment produced behavioral changes including sni¤ng, whisking and continuous snout contact with surfaces, all lasting until death. The brains of all the rats were processed for c-Fos immunocytochemistry, which was scored for throughout the entire cortical thickness. In the barrel cortex of experimental animals, apomorphine-driven c-Fos up-regulation was clearly observed (Fig. 3). This increase was greatly diminished after removal of the vibrissae. Control
The design of experiment 2 was similar to the previous one, however, MK-801 (0.1 mg/kg), an NMDA receptor antagonist, was used along with apomorphine. At this dose, MK-801 was previously found to block long-term but not short-term memory formation and is known to block more than 50% of brain NMDA receptors (Sierocinska et al., 1991). The behavior of control and apo animals was as described for experiment 1. No behavioral e¡ect of MK-801 (0.1 mg/kg) could be observed in MK and apoMK animals. In this experiment, we focused on the PMBSF area, where we investigated c-Fos, Fos B, Jun B, and Zif268 immunoreactivity after unilateral vibrissae clipping and apomorphine injection, with emphasis on the laminar distribution of the investigated proteins, and the role of the NMDA receptor in their expression. Figs. 4 and 5 show the results of this experiment. The clipping itself (Fig. 4, upper part, and saline group in Fig. 5) resulted in no change in expression of c-Fos, Fos B, and Jun B. In contrast, Zif268 immunoreactivity was reduced on the deprived side (e.g., ¢ve-fold in layer II/III, F1;4 = 9.56, P 6 0.05).
Fig. 3. Apomorphine injection caused a marked and signi¢cant increase in c-Fos expression in FL/HL and PMBSF areas (experiment 1). This increase was much bigger in the barrel cortex not deprived of sensory input. The results of immunocytochemistry were quanti¢ed in the deprived (solid bars) and non-deprived (hatched bars) sides of the cortex (see Experimental Procedures, also Fig. 1). Data are means and S.E.M. Statistically signi¢cant di¡erences are marked with *P 6 0.05 and **P 6 0.01.
c-Fos. As in experiment 1, apomorphine treatment resulted in a signi¢cant c-Fos induction in both the deprived and the non-deprived sides, in all layers. On the deprived side, the di¡erence was most signi¢cant in layer II/III (2.2-fold increase, F1;7 = 110.24, P 6 0.00002). In the non-deprived side, it was markedly signi¢cant in all investigated layers, namely in layer II/III (3.5-fold increase, F1;7 = 11.25, P 6 0.02), layer IV (4.6-fold increase, F1;7 = 15.46, P 6 0.006), layer V/VI (5.6-fold, F1;7 = 8.86, P 6 0.03), and layer VIb (12.3-fold, F1;7 = 13.14, P 6 0.009). In the apo group, the c-Fos levels on the deprived side were signi¢cantly lower in all layers (two-fold in layer II/III, F1;10 = 12.73, P 6 0.006; six-fold in layer IV, F1;10 = 39.04, P 6 0.0001; two-fold in layer V/VI, F1;10 = 7.63, P 6 0.03), except for layer VIb, where no di¡erence between sides was detected (F1;10 = 1.09, P = 0.3). Pre-injection of MK-801 (0.1 mg/
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Fig. 4. Experiment 2; immunocytochemistry showing c-Fos, Fos B, Jun B, and Zif268 expression in PMBSF following vibrissae clipping (saline group, vibrissae clipped from one side of the snout; upper panel) and apomorphine injection (apo group, vibrissae clipped, apomorphine injected, lower panel). Neighboring sections of one rat from each group are shown. Pictures of Nissl and cytochrome oxidase (Cyt Ox) staining of neighboring sections are included. Calibration bar = 0.5 mm.
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Fig. 5. Experiment 2; the numbers of nuclei containing c-Fos, Fos B, Jun B, and Zif268 in all layers (II/III, IV, V/VI, and VIb), sides and experimental groups (saline, MK, apo, apoMK) in the PMBSF area. Data are means and S.E.M. The results of immunocytochemistry were quanti¢ed in all cortical layers in the deprived (solid bars) and non-deprived (hatched bars) sides of the cortex (see Experimental Procedures). Statistically signi¢cant di¡erences are marked with *P 6 0.05, **P 6 0.01 and ***P 6 0.001. An asterisk above a column shows a signi¢cant di¡erence between the value represented and the control (apo vs. saline; apoMK vs. MK) ; an asterisk above a pair of columns marks a di¡erence between the deprived and nondeprived sides of the cortex.
kg) did not signi¢cantly a¡ect apomorphine-induced cFos expression. Fos B. No signi¢cant e¡ect on Fos B expression of either deprivation or apomorphine or MK-801 injections in any of the layers examined was found (Figs. 4 and 5). Jun B. Apomorphine administration resulted in an induction of Jun B immunoreactivity in layer VIb on both sides. In the apo group, the increase was 4.3-fold for the deprived side (F1;6 = 14.06, P 6 0.01) and 6.4-fold for the non-deprived side (F1;6 = 7.38, P 6 0.04). Signi¢cant increases in Jun B accumulation after apomorphine administration could also be observed in cortical layers IV and VI in MK-801-treated rats. In all layers except VIb, sensory deprivation revealed stronger Jun B expression on the non-deprived side of the barrel cortex. Zif268. Apomorphine treatment elicited only small and non-signi¢cant increases in Zif268 immunoreactivity in the PMBSF area, except for layer VIb, where there was a statistically signi¢cant Zif268 elevation following the treatment (e.g., 7.5-fold for the non-deprived side in
the apo group, F1;7 = 6.17, P 6 0.04). The basic e¡ect of sensory deprivation was observed in all groups (not only saline, but also MK, apo, and apoMK) in layers II/III, IV, and V/VI. No e¡ect of MK-801 pre-injection was noted. Experiment 3 In this experiment, the e¡ects of a higher (1.0 mg/kg) dose of MK-801 were analyzed on c-Fos expression. This dose was chosen because this and even higher doses have been used in a number of previous reports on cortical gene expression (Jones et al., 1999; Kaczmarek and Chaudhuri, 1997; Kinouchi et al., 1994; Sullivan et al., 1996; Watanabe et al., 1998). The behavior of control and apo animals was as described for experiment 1, whereas MK-801 (1.0 mg/kg) caused stereotypical behavior, namely partial immobility (ataxia), swaying, weaving and crawling of injected rats. Figure 6 shows the results of experiment 3. As in the two previous ones, apomorphine injection caused an increase in c-Fos immunocytochemistry on both the deprived (4.4-fold, F1;7 = 9.41, P 6 0.02) and the non-
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Fig. 6. Experiment 3; the NMDA receptor antagonist MK-801 (1.0 mg/kg) decreases apomorphine-evoked c-Fos expression in barrel cortex not deprived of sensory input. The results of immunocytochemistry were quanti¢ed in the deprived (solid bars) and non-deprived (hatched bars) sides of the cortex of control (saline), apo as well as apoMK1 animals (see Experimental Procedures, also Fig. 1). Data are means and S.E.M. Statistically signi¢cant di¡erences are marked with *P 6 0.05 and ***P 6 0.001.
deprived (8.8-fold, F1;7 = 37.11, P 6 0.0005) side and there was a signi¢cant di¡erence between the sides (two-fold, F1;8 = 10.32, P 6 0.02). Co-injection of MK801 (1.0 mg/kg) resulted in c-Fos expression levels diminishing to those evoked by apomorphine alone (for the non-deprived side: 4.8-fold, F1;6 = 6.01, P 6 0.05) with no di¡erence between the sides (1.3-fold, F1;6 = 0.35, P = 0.6).
DISCUSSION
The major ¢ndings of this study can be summarized as follows. Sensory deprivation/stimulation procedures had di¡erential e¡ects on various transcription factor proteins in the barrel cortex and these e¡ects were cortical layer-speci¢c. Decreasing the sensory input down-regulated only Zif268 but none of the other proteins tested. The down-regulatory e¡ect of sensory deprivation on Zif268 was observed in all cortical layers except VIb. In contrast, apomorphine treatment resulted in a striking up-regulation of c-Fos protein levels throughout the cortical thickness. There was a similar, but lesser e¡ect on Jun B levels. Removal of the sensory input diminished this response in all layers, except VIb. In contrast, expression of Fos B was not a¡ected by any of the manipulations employed. The NMDA receptor antagonist MK-801, at 0.1 mg/kg, did not produce any signi¢cant e¡ects on either behavior or expression of the proteins under study. However, treatment with this compound at the higher dose of 1.0 mg/kg resulted in gross behavioral abnormalities along with decreased c-Fos activation. Apomorphine is a dopamine receptor agonist that acts at both D1 and D2 receptors. Behaviorally, apomorphine
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treatment produces an increased motor activity including sni¤ng, whisking, licking and snout contact ¢xation, i.e., establishment of a continuous contact of the snout with surfaces (Beck et al., 1986; Szechtman et al., 1982). These responses, observed also in our experiments, obviously involve increased stimulation of the whiskers, and hence might be responsible for sensory input-driven cFos activation. However, dopamine receptor agonists such as apomorphine as well as cocaine and amphetamine induce by themselves c-fos, fos B, Zif268 and jun B expression in the striatum and cortex (Berke et al., 1998; Cole et al., 1992; Deutch and Duman, 1996; Dragunow et al., 1990; Gerfen et al., 1998; Graybiel et al., 1990; Jung and Bennett, 1996; Keefe and Gerfen, 1996; LaHoste et al., 1996; Moratalla et al., 1992; Nguyen et al., 1992; Robertson et al., 1989; Young et al., 1991). Thus, the e¡ects of apomorphine in the somatosensory cortex could also result from a direct stimulation of dopamine receptors. Our results suggest that indeed there are two components to apomorphine action ^ sensory, that is dominant, and non-sensory, possibly through direct stimulation of cortical dopamine receptors. This ¢nding is in contrast to data reported by Steiner and Gerfen (1994) regarding c-fos and zif268 mRNA expression following the same paradigm of apomorphine treatment. These authors reported that apomorphine-evoked accumulation of both mRNAs was fully abolished by sensory deprivation. In contrast, in our hands, the removal of the whiskers a¡ected apomorphine-evoked c-Fos protein expression in the deprived part of the cortex only partially. Steiner and Gerfen (1994) also described an apomorphine-dependent increase in zif268 mRNA expression level. This increase was even bigger than the decrease they observed after clipping of the vibrissae in the control group, without apomorphine injection. We did not con¢rm this observation at the protein level. It is not clear what di¡erences between experimental paradigms are responsible for this discrepancy, although it should be noted that di¡erential regulation of mRNA vs. protein expression has been observed in other situations (Hisanaga et al., 1992; Kiessling et al., 1993; Worley et al., 1993). However, similar to Steiner and Gerfen (1994) as well as others (Melzer and Steiner, 1997; Staiger et al., 2000; see also Kaczmarek and Chaudhuri, 1997) we recorded decreased expression of Zif268 following sensory deprivation. Jun B expression appeared increased in barrel cortex after apomorphine stimulation with higher levels reached on the non-deprived side. The most striking, and sensory input-independent increase was observed in layer VIb. However, the changes observed in other layers were not as marked as those of c-Fos. Inducibility of Jun B in the brain was observed by others and a striking parallelism to c-Fos was repeatedly noted (Gass et al., 1993b; Kaminska et al., 1994, 1996; Kinouchi et al., 1994; Konopka et al., 1998; Luckman et al., 1996; Moratalla et al., 1993) also in barrel cortex (Staiger et al., 2000). Hence, our results go along with the prevalent notion that Jun B and c-Fos form a major inducible AP1 complex in the brain after stimulation (Kaminska et al., 2000).
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Fos B expression in rat barrel cortex was not altered by any manipulations employed in this study. Although the levels of this protein have been shown to increase in the brain in several experimental paradigms, in a number of other conditions, Fos B expression, contrary to that of, e.g., c-Fos, was found not to change (Herdegen and Leah, 1998). It is important to further investigate their di¡erential expression patterns and biological functions with focus on possible di¡erent target genes for various Fos proteins in the brain. Recently another AP-1 protein, c-Jun, was also found not to change after stimulation of whiskers (Staiger et al., 2000). Thus both proteins seem to be involved (together with Jun D, see, e.g., Kaminska et al., 1995, 1996) in the basal AP-1 complex in the sensory cortex. We also wanted to study the role of NMDA receptors in apomorphine-induced activation of the transcription factor proteins. There are multiple examples that NMDA receptor activation mediates c-fos and zif268 expression (Platenik et al., 2000). In our experiments, we applied ^ together with apomorphine ^ the NMDA receptor antagonist MK-801. This added to the complexity of the system as we simultaneously: (i) activated dopamine receptors through apomorphine injection, (ii) possibly activated NMDA receptors through apomorphineevoked whisking behavior, and (iii) attempted to block NMDA receptors with MK-801 injection. Two doses of MK-801 were selected (see Results). MK-801 at 0.1 mg/ kg did not a¡ect overt behavior of the rats and did not reduce the sensory input-driven response to apomorphine of any of the investigated proteins. Treatment with the higher dose of MK-801, 1.0 mg/kg, resulted in a signi¢cant decline of c-Fos immunoreactivity on the non-deprived side of the cortex, but nevertheless, the levels of c-Fos throughout the cortical thickness were still well above those observed in animals not treated with apomorphine. Importantly, at the same time, MK801 (1.0 mg/kg) e¤ciently disabled locomotor behavior of the animals. It can thus be suggested that the lack of a proper motor response, including alteration in sni¤ng and whisking, was responsible for the observed phenomenon. Notably, at this dose, MK-801 is known to induce cortical c-fos expression on its own (Gao et al., 1998; Gass et al., 1993a; Hughes et al., 1993; Sharp et al., 1991; Wedzony and Czyrak, 1996; Zhang et al., 1999). Thus the important conclusion resulting from these considerations is to put into question the usefulness of applying such a high dose of MK-801 to investigate the role of NMDA receptors in regulation of cortical gene expression. A special comment should be given to the distinct pattern of gene expression observed in layer VIb. In this layer, in contrast to the other layers, no e¡ect of vibrissae clipping (compare deprived vs. non-deprived side) was noted in the case of c-Fos, Jun B, and Zif268
expression, also the apomorphine-evoked induction was more pronounced in the case of Jun B and Zif268. This region is known for its speci¢c role during the early phases of cortical histogenesis. Cells in layer VIb are among the ¢rst to di¡erentiate. They show speci¢c patterns of gene expression during development and adulthood (Alcantara and Greenough, 1993; Gaspar et al., 1995; Gomez-Pinilla and Cotman, 1992; Valverde et al., 1989). We did not observe c-Fos induction following tactile stimulation in this sublayer (Filipkowski et al., 2000). It seems that the prevailing signals which lead to changes in expression of the investigated transcription factors in layer VIb are non-sensory. This signaling may be due to the direct action of apomorphine on dopamine receptors as D1 mRNA was found to be abundant in VIb cells of the posterior frontal cortex (Gaspar et al., 1995). The immediate early gene products we have studied are involved in the formation of transcription factors, and thus their only known biological function is to control gene expression. Because of this, they have been repeatedly hypothesized to play a pivotal role in orchestrating genomic responses underlying long-term neuronal plasticity (Kaczmarek, 2000). Sensory stimulation appears to be an important trigger for cortical plasticity which in adults seems to be restricted mostly to layers II/ III (Darian-Smith and Gilbert, 1994; Daw et al., 1992; Glazewski, 1998). These layers receive the major input from layer IV. Thus, it is interesting to note that the greatest di¡erences in c-Fos expression observed between the deprived and non-deprived sides (and hence possibly the greatest sensory component to gene activation) were noted in layer IV. Also, in this layer, another transcription factor, cAMP-response element-binding protein (CREB), was implicated in barrel cortex plasticity (Glazewski et al., 1999) and CREB-dependent expression was recently found to be induced only in layer IV of the barrel corresponding to the spared whisker after deprivation of surrounding vibrissae (Barth et al., 2000). As suggested by these authors, we may conclude that the molecular events initiating cortical plasticity ^ possibly involving basal ganglia activity ^ occur presynaptically, i.e., in neurons with cell bodies located in layer IV, and projecting to layers II/III. Furthermore, our results provide additional evidence for the hypothesis that increased c-Fos/AP-1 expression comprises one of the molecular events involved in this phenomenon. AcknowledgementsöWe wish to thank Rodrigo Bravo for a kind gift of anti-Jun B and anti-Zif268 antibodies and Krzysztof Wedzony for priceless methodological advice. This work was supported by State Committee for Scienti¢c Research (KBN, Poland) Grant 6 P04A 036 19. R.K.F. was the holder of a Young Scientist Award of the Foundation for Polish Science. M.R. was the holder of a KIRT scholarship between the Karolinska Institute and Warsaw Medical University.
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Res. 57, 719^729. (Accepted 26 June 2001)
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Neuroscience Vol. 106, No. 4, pp. 679^688, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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EXPRESSION OF c-Fos, Fos B, Jun B, AND Zif268 TRANSCRIPTION FACTOR PROTEINS IN RAT BARREL CORTEX FOLLOWING APOMORPHINE-EVOKED WHISKING BEHAVIOR R. K. FILIPKOWSKI,a;b * M. RYDZa;c and L. KACZMAREKa a
Department of Molecular and Cellular Neurobiology, Nencki Institute, Pasteura 3, 02-093 Warsaw, Poland b c
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
Division of Geriatric Medicine, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden
AbstractöApomorphine-evoked expression of transcription factor proteins: c-Fos, Fos B, Jun B, and Zif268 (also named Krox-24, NGFI-A, Egr-1), was investigated in rat somatosensory (barrel) cortex. The e¡ect of the N-methyl-D-aspartate receptor antagonist MK-801 on their expression was also analyzed. Apomorphine is a dopamine receptor agonist, eliciting motor activity, including enhanced whisking leading to the activation of vibrissae representation in the barrel cortex. Rats had their whiskers clipped on one side of the snout. The Zif268 levels were markedly reduced by this procedure alone. In contrast, apomorphine (5.0 mg/kg) evoked marked c-Fos elevation, less pronounced changes in Jun B and Zif268 and no change in Fos B. The greatest apomorphine-evoked c-Fos accumulation was observed in layers IV and V/VI of non-deprived barrel cortex and was not signi¢cantly in£uenced by MK-801 injection at 0.1 mg/kg. A higher dose of MK-801 (1.0 mg/kg) produced abnormalities in locomotor behavior and diminished c-Fos levels on the non-deprived side to the ones observed in the sensory stimulus-deprived cortex. We conclude that the response of the somatosensory cortex is selective with respect to both the gene activated and its cortical layer localization. Furthermore, sensory stimulation provides a major but not the only component to apomorphine-evoked barrel cortex gene activation. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: vibrissae, NMDA receptor, immediate early genes, AP-1, MK-801.
(PMBSF) (Woolsey and Van der Loos, 1970), also known as the MV area (Chapin and Lin, 1984). Because of its simplicity, laterality as well as ethological and behavioral importance, the rodent somatosensory cortex, representing mystacial vibrissae (large whiskers) of the snout, o¡ers a very convenient system to study the e¡ects of sensory stimulation upon cortical function. Sensory stimulation plays an important role in neuronal plasticity (Buonomano and Merzenich, 1998) and recent studies indicate that gene expression may be of pivotal importance for this phenomenon (Kaczmarek, 1993; Kaczmarek and Chaudhuri, 1997). The barrel cortex has already been a subject of investigation of stimulation-evoked gene expression (see review by Filipkowski, 2000). In particular, Steiner and Gerfen (1994) as well as LaHoste et al. (1996) reported that the treatment of rats with dopamine agonists, such as apomorphine, known to enhance motor (including whisking) activity (Beck et al., 1986; Berke et al., 1998; Szechtman et al., 1982; Young et al., 1991), resulted in the accumulation of immediate early genes, c-fos and zif268 mRNA as well as c-Fos protein in various brain regions including somatosensory cortex. This e¡ect was fully input-speci¢c, since clipping of the whiskers prevented the mRNA increase in the corresponding PMBSF (Steiner and Gerfen, 1994). Thus, it can be inferred that sensory input is critical for regulating c-fos and zif268 expression in the barrel cortex. Fur-
In the rodent vibrissae-barrel cortex system, sensory input produced by de£ection of the whiskers is conveyed via the brainstem and thalamus to cortical layer IV, where each cortical column, extending through all layers, contains a barrel, a discrete cluster of closely packed cells. There is one-to-one correspondence between each barrel and each vibrissa. The whisker-to-barrel system is contralateral, so the somatosensory cortex on one side represents vibrissae on the other side of the snout (Kossut, 1992). The part of the barrel ¢eld containing the large barrels and representing the vibrissae of the mystacial pad is called the postero-medial barrel sub¢eld
*Correspondence to: R.K. Filipkowski, Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA. Tel.: +1-516-367-8886; fax: +1-516-367-8880. E-mail address: ¢[email protected] (R. K. Filipkowski). Abbreviations : AP-1, activator protein-1 transcription factor; apo, mice treated with apomorphine; apoMK, mice treated with apomorphine and MK-801 (0.1 mg/kg); apoMK1, mice treated with apomorphine and MK-801 (1.0 mg/kg); CREB, cAMP-response element-binding protein transcription factor; DAB, 3,3P-diaminobenzidine; FL/HL, forelimb/hindlimb, a part of the cortex receiving sensory information from the limbs; MK, mice treated with MK-801; MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine, dizocilpine ; NMDA, N-methyl-D-aspartate; PBS, phosphate-bu¡ered saline ; PMBSF, postero-medial barrel sub¢eld. 679
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thermore, recently, Steiner and Kitai (2000) reported that selective intrastriatal infusion of the dopamine D1 receptor antagonist SCH-23390 attenuated apomorphineevoked gene expression in the cortex, thus suggesting that basal ganglia mediate this e¡ect, most probably via their role in the control of motor behavior. To further our knowledge on genomic response in the cortex, we decided to extend the aforementioned observations to protein levels of c-Fos and Zif268 as well as to investigate apomorphine- and sensory input-driven expression of two other transcription factor proteins, Jun B and Fos B. Fos and Jun proteins combine to produce the activator protein-1 (AP-1) transcription factor. Both AP-1 and Zif268 (also known as Krox-24, Egr1, NGFI-A) appear to be the major inducible gene expression regulators in the CNS (Herdegen and Leah, 1998; Morgan and Curran, 1991). Despite these apparent similarities, the aforementioned transcription factor proteins may play di¡erential roles in regulating neuronal gene expression. c-Fos is readily induced by many, even subtle, stimuli including, e.g., exposure to a novel environment (Anokhin et al., 1991; Filipkowski et al., 2000; Montero, 1997; Staiger et al., 2000). Inducibility of Jun B in the brain was observed in striking parallelism to c-Fos (Gass et al., 1993b; Kaminska et al., 1994, 1996; Kinouchi et al., 1994; Konopka et al., 1998; Luckman et al., 1996; Moratalla et al., 1993; Staiger et al., 2000). Hence, it could be inferred that Jun B and cFos form a major inducible AP-1 complex after stimula-
tion. In contrast, although Fos B levels were shown to increase in the brain in several experimental paradigms, expression of this protein was found not to change in many other physiological situations (Herdegen and Leah, 1998). Finally, Zif268 has been reported to be expressed at high protein levels in sensory cortex under `naive' conditions and was found to be down-regulated by sensory deprivation and up-regulated by stimulation (Kaczmarek and Chaudhuri, 1997). We focused on protein levels because an excellent spatial resolution provided by the immunocytochemical approach allowed us to study laminar distribution of the immunoreactivities in the somatosensory cortex. Furthermore, since the N-methyl-D-aspartate (NMDA) receptor is known to be critical for synaptic plasticity and has also been implicated in activation of c-fos, zif268 and the expression of some other immediate early genes, especially in vitro (Bading et al., 1993, 1995; Condorelli et al., 1994; Morgan and Linnoila, 1991; Vaccarino et al., 1992), we also analyzed its role in our preparation using the NMDA receptor antagonist MK-801. We employed the experimental model described before (Steiner and Gerfen, 1994). After clipping rats' mystacial vibrissae on one side of the snout and then applying apomorphine and MK-801, the expression of the immediate early gene proteins c-Fos, Fos B, Jun B, and Zif268 was evaluated in barrel cortex.
EXPERIMENTAL PROCEDURES
Treatment of animals All experiments described were done with adult male Wistar rats obtained from Nencki Institute Animal House. The animals were kept under the natural light^dark cycle with water and food provided ad libitum. The experiments were conducted during the light phase of the cycle. All e¡orts were made to minimize the number of animals used and their su¡ering. The rules established by the Ethical Committee on Animal Research of Nencki Institute and based on the disposition of the President of Polish Republic were followed strictly in all experiments. Experiment 1 On each of 20 days before the experiment, the rats were gently held in the same way as during the clipping procedure. On each of 10 days before the experiment, the rats received a s.c. saline injection in the neck, 4 h after the handling. On the experimental day, all rats had their mystacial vibrissae clipped with scissors on one side of the snout (both sides were used in separate animals), which took 1^3 min (see Fig. 1). Then the rats were divided into two groups: control (three rats) and apo (four rats). All rats received one s.c. injection 4 h after the clipping: the control group received one saline injection, the apo group received one apomorphine (Sigma, St. Louis, MO, USA; 5.0 mg/kg in 0.02% ascorbic acid) injection. One and a half hour after the injection the rats were treated with an overdose of chloral hydrate and processed for c-Fos immunocytochemistry as well as Nissl and cytochrome oxidase staining. Fig. 1. Experimental model used in all experiments. After vibrissae clipping, the deprived and non-deprived sides of PMBSF can be distinguished. Bottom, schematic diagram of a coronal section with the areas (dark gray) where immunopositive cells were counted. FL/HL, medial region of somatosensory cortex which includes forelimb and hindlimb areas. The ¢ducial mark was used to recognize sides of brains after the reaction.
Experiment 2 On each of 20 days before the experiment, the rats were gently held in the same way as during the clipping procedure. On each of 14 days before the experiment, the rats received a saline injection, i.p., 3.5 h after the handling. On each of the 7 last
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Fig. 2. Design of experiment 2 in which rats had their vibrissae trimmed at one side of the snout, and received one i.p. injection (at 3.5 h) and one s.c. injection (at 4 h). The table shows the di¡erent experimental groups and substances injected. Then, 1.5 h later, the animals were perfused for immunocytochemistry.
days of habituation before the experiment, all rats received one s.c. injection 4 h after the handling. On the experimental day, all rats had their mystacial vibrissae clipped on one side (see above). The rats were divided into four groups, three to six rats in each. All received two injections (at time points as during habituation). The saline group (three rats) received one saline (i.p.) and one ascorbic acid (0.02%, s.c.) injection. The second group (MK, four rats) received one MK-801 injection (0.1 mg/ kg in saline, i.p.) and one ascorbic acid injection (0.02%, s.c.). The third group (apo, six rats) received one saline (i.p.) and one apomorphine injection (5.0 mg/kg in 0.02% ascorbic acid, s.c.). The fourth group (apoMK, six rats) received MK-801 (0.1 mg/ kg in saline, i.p.) and apomorphine (5.0 mg/kg in 0.02% ascorbic acid, s.c.), see also Fig. 2. One and a half hours after the second injection the rats were treated with an overdose of chloral hydrate and were processed for c-Fos, Fos B, Jun B, and Zif268 immunocytochemistry as well as Nissl and cytochrome oxidase staining. Experiment 3 The same as above (experiment 2) except that only three groups were investigated: saline (four rats), apo (¢ve rats) and apoMK1 (four rats). The MK-801 concentration was changed to 1.0 mg/kg. After the experiment, the rats were treated with an overdose of chloral hydrate and were processed for c-Fos immunocytochemistry as well as Nissl and cytochrome oxidase staining. Immunocytochemistry One and a half hour after s.c. injection all rats were anesthetized with an overdose of chloral hydrate and immediately perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate bu¡er, pH 7.4. The brains were removed and stored for 24 h in the same ¢xative at 4³C and then in 30% sucrose with 0.02% sodium azide at 4³C until needed. Then the appropriate parts of the brain were slowly and gradually frozen in a heptane/ dry ice bath. The expression of c-Fos and other proteins was assessed as described before (Filipkowski et al., 2000; Kaminska et al., 1996). In brief, coronal brain cryostat sections were cut at 320³C, 45 Wm thick, 2.3^3.3 mm caudal to bregma (Paxinos
and Watson, 1986). The sections were washed in phosphatebu¡ered saline (PBS), incubated in 0.3% H2 O2 , incubated with one of the polyclonal antibodies (anti-c-Fos, 1:1000, Santa Cruz #sc-52, Santa Cruz, CA, USA; anti-Fos B, 1:2000, Santa Cruz #sc-48; anti-Jun B, 1:1000; anti-Zif268, 1:5000, the latter two from R. Bravo, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, USA) for 48 h at 4³C in PBS with azide (0.01%) and normal goat serum (3%). After that the sections were washed in PBS with Triton X-100 (0.3%, Sigma), incubated with goat anti-rabbit biotinylated secondary antibody (1:1000, Vector Laboratories, Burlingame, CA, USA) in PBS/Triton and normal goat serum (3%) for 2 h, washed in PBS/Triton, incubated with avidin^biotin complex (1:1000, 1:1000, in PBS/Triton, Vector) for 1 h and washed in PBS. The immunostaining reaction was developed using the glucose oxidase^3,3P-diaminobenzidine (DAB)^nickel method (Shu et al., 1988). The sections were mounted on gelatin-covered slides, air-dried, dehydrated in ethanol solutions and xylene, and embedded in Entellan (Merck KGaA, Darmstadt, Germany). Nissl staining The sections were mounted on slides and air-dried (2^4 days), washed in PBS followed by 70%, 100%, 70% ethanol, water, 0.5% Cresyl Violet solution with acetic acid (0.35 M) and sodium acetate (0.06 M), and ¢nally dehydrated in ethanol (70% and 100%) and xylene and embedded in Entellan (Merck). Cytochrome oxidase staining The sections were washed twice in PBS and incubated in staining solution (1% sucrose; 0.05% nickel sulfate; 0.025% DAB; 0.015% cytochrome c; 0.01% catalase; 2.5 WM imidazole in 0.05 M phosphate bu¡er, pH 7.4; all reagents from Sigma) for several hours at 37³C. The stained sections were again washed in PBS, mounted on slides and air-dried (2^4 days). The slides were then washed in PBS, dehydrated and mounted as above. Data gathering and analysis In experiments 1 and 3, c-Fos-stained nuclei were counted using the image analysis system (MCID, IMAGING Research,
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St. Catherines, ON, Canada) in two regions of the cortex (Fig. 1), PMBSF (the region known to receive input from the mystacial vibrissae) and FL/HL (the medial adjacent region which includes forelimb and hindlimb areas), in both hemispheres. Middle parts of these regions, 1 cm wide, were taken for the analysis, as described before (Filipkowski et al., 2000; Steiner and Gerfen, 1994). In experiment 2, the number of c-Fos-, Fos B-, Jun B-, and Zif268-stained nuclei in the PMBSF area was evaluated in cortical layers II/III, IV, V/VI (without the subplate), and VIb (also known as layer VII, subplate, deep layer VI bordering the white matter). The position of the regions and layers was con¢rmed by Nissl and cytochrome oxidase staining. The nuclei were counted with the researcher being blind to the treatment. Averages from two to four sections per animal were used for statistical analysis. In all experiments, separated one-way analyses of variance (ANOVAs) were applied. Values in the ¢gures are expressed as means+S.E.M.
RESULTS
animals had very little c-Fos immunoreactivity in both deprived and non-deprived PMBSF areas, where no signi¢cant di¡erence in c-Fos expression between the sides was detected (1.2-fold di¡erence, F1;4 = 0.13, P = 0.7). Similarly low c-Fos levels were detected in the FL/HL area with no di¡erence between sides either (0.9-fold difference, F1;4 = 0.04, P = 0.9). Apomorphine treatment resulted in an increase in c-Fos immunoreactivity in FL/LH (non-deprived side: 3.6-fold di¡erence, F1;5 = 9.25, P 6 0.03; deprived side: 2.6-fold, F1;5 = 14.22, P 6 0.02) and PMBSF (non-deprived side: 5.1-fold difference, F1;5 = 29.74, P 6 0.003; deprived side: 3.0-fold, F1;5 = 14.26, P 6 0.02). Thus, after apomorphine injection no di¡erence in c-Fos expression between the contraand ipsilateral sides was observed in the FL/HL area (1.3-fold, F1;6 = 0.97, P = 0.4) and only in PMBSF was the concentration of the protein two-fold greater on the non-deprived side (F1;6 = 15.71, P 6 0.01).
Experiment 1
Experiment 2
In this experiment, animals with unilateral removal of vibrissae were treated with apomorphine and c-Fos levels were analyzed on both sides of the cerebral cortex in the areas representing the vibrissae (PMBSF) as well as adjacent representations of the forelimb/hindlimb (FL/HL). After the injections, the control animals remained inactive. In contrast, apomorphine treatment produced behavioral changes including sni¤ng, whisking and continuous snout contact with surfaces, all lasting until death. The brains of all the rats were processed for c-Fos immunocytochemistry, which was scored for throughout the entire cortical thickness. In the barrel cortex of experimental animals, apomorphine-driven c-Fos up-regulation was clearly observed (Fig. 3). This increase was greatly diminished after removal of the vibrissae. Control
The design of experiment 2 was similar to the previous one, however, MK-801 (0.1 mg/kg), an NMDA receptor antagonist, was used along with apomorphine. At this dose, MK-801 was previously found to block long-term but not short-term memory formation and is known to block more than 50% of brain NMDA receptors (Sierocinska et al., 1991). The behavior of control and apo animals was as described for experiment 1. No behavioral e¡ect of MK-801 (0.1 mg/kg) could be observed in MK and apoMK animals. In this experiment, we focused on the PMBSF area, where we investigated c-Fos, Fos B, Jun B, and Zif268 immunoreactivity after unilateral vibrissae clipping and apomorphine injection, with emphasis on the laminar distribution of the investigated proteins, and the role of the NMDA receptor in their expression. Figs. 4 and 5 show the results of this experiment. The clipping itself (Fig. 4, upper part, and saline group in Fig. 5) resulted in no change in expression of c-Fos, Fos B, and Jun B. In contrast, Zif268 immunoreactivity was reduced on the deprived side (e.g., ¢ve-fold in layer II/III, F1;4 = 9.56, P 6 0.05).
Fig. 3. Apomorphine injection caused a marked and signi¢cant increase in c-Fos expression in FL/HL and PMBSF areas (experiment 1). This increase was much bigger in the barrel cortex not deprived of sensory input. The results of immunocytochemistry were quanti¢ed in the deprived (solid bars) and non-deprived (hatched bars) sides of the cortex (see Experimental Procedures, also Fig. 1). Data are means and S.E.M. Statistically signi¢cant di¡erences are marked with *P 6 0.05 and **P 6 0.01.
c-Fos. As in experiment 1, apomorphine treatment resulted in a signi¢cant c-Fos induction in both the deprived and the non-deprived sides, in all layers. On the deprived side, the di¡erence was most signi¢cant in layer II/III (2.2-fold increase, F1;7 = 110.24, P 6 0.00002). In the non-deprived side, it was markedly signi¢cant in all investigated layers, namely in layer II/III (3.5-fold increase, F1;7 = 11.25, P 6 0.02), layer IV (4.6-fold increase, F1;7 = 15.46, P 6 0.006), layer V/VI (5.6-fold, F1;7 = 8.86, P 6 0.03), and layer VIb (12.3-fold, F1;7 = 13.14, P 6 0.009). In the apo group, the c-Fos levels on the deprived side were signi¢cantly lower in all layers (two-fold in layer II/III, F1;10 = 12.73, P 6 0.006; six-fold in layer IV, F1;10 = 39.04, P 6 0.0001; two-fold in layer V/VI, F1;10 = 7.63, P 6 0.03), except for layer VIb, where no di¡erence between sides was detected (F1;10 = 1.09, P = 0.3). Pre-injection of MK-801 (0.1 mg/
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Fig. 4. Experiment 2; immunocytochemistry showing c-Fos, Fos B, Jun B, and Zif268 expression in PMBSF following vibrissae clipping (saline group, vibrissae clipped from one side of the snout; upper panel) and apomorphine injection (apo group, vibrissae clipped, apomorphine injected, lower panel). Neighboring sections of one rat from each group are shown. Pictures of Nissl and cytochrome oxidase (Cyt Ox) staining of neighboring sections are included. Calibration bar = 0.5 mm.
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Fig. 5. Experiment 2; the numbers of nuclei containing c-Fos, Fos B, Jun B, and Zif268 in all layers (II/III, IV, V/VI, and VIb), sides and experimental groups (saline, MK, apo, apoMK) in the PMBSF area. Data are means and S.E.M. The results of immunocytochemistry were quanti¢ed in all cortical layers in the deprived (solid bars) and non-deprived (hatched bars) sides of the cortex (see Experimental Procedures). Statistically signi¢cant di¡erences are marked with *P 6 0.05, **P 6 0.01 and ***P 6 0.001. An asterisk above a column shows a signi¢cant di¡erence between the value represented and the control (apo vs. saline; apoMK vs. MK) ; an asterisk above a pair of columns marks a di¡erence between the deprived and nondeprived sides of the cortex.
kg) did not signi¢cantly a¡ect apomorphine-induced cFos expression. Fos B. No signi¢cant e¡ect on Fos B expression of either deprivation or apomorphine or MK-801 injections in any of the layers examined was found (Figs. 4 and 5). Jun B. Apomorphine administration resulted in an induction of Jun B immunoreactivity in layer VIb on both sides. In the apo group, the increase was 4.3-fold for the deprived side (F1;6 = 14.06, P 6 0.01) and 6.4-fold for the non-deprived side (F1;6 = 7.38, P 6 0.04). Signi¢cant increases in Jun B accumulation after apomorphine administration could also be observed in cortical layers IV and VI in MK-801-treated rats. In all layers except VIb, sensory deprivation revealed stronger Jun B expression on the non-deprived side of the barrel cortex. Zif268. Apomorphine treatment elicited only small and non-signi¢cant increases in Zif268 immunoreactivity in the PMBSF area, except for layer VIb, where there was a statistically signi¢cant Zif268 elevation following the treatment (e.g., 7.5-fold for the non-deprived side in
the apo group, F1;7 = 6.17, P 6 0.04). The basic e¡ect of sensory deprivation was observed in all groups (not only saline, but also MK, apo, and apoMK) in layers II/III, IV, and V/VI. No e¡ect of MK-801 pre-injection was noted. Experiment 3 In this experiment, the e¡ects of a higher (1.0 mg/kg) dose of MK-801 were analyzed on c-Fos expression. This dose was chosen because this and even higher doses have been used in a number of previous reports on cortical gene expression (Jones et al., 1999; Kaczmarek and Chaudhuri, 1997; Kinouchi et al., 1994; Sullivan et al., 1996; Watanabe et al., 1998). The behavior of control and apo animals was as described for experiment 1, whereas MK-801 (1.0 mg/kg) caused stereotypical behavior, namely partial immobility (ataxia), swaying, weaving and crawling of injected rats. Figure 6 shows the results of experiment 3. As in the two previous ones, apomorphine injection caused an increase in c-Fos immunocytochemistry on both the deprived (4.4-fold, F1;7 = 9.41, P 6 0.02) and the non-
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Fig. 6. Experiment 3; the NMDA receptor antagonist MK-801 (1.0 mg/kg) decreases apomorphine-evoked c-Fos expression in barrel cortex not deprived of sensory input. The results of immunocytochemistry were quanti¢ed in the deprived (solid bars) and non-deprived (hatched bars) sides of the cortex of control (saline), apo as well as apoMK1 animals (see Experimental Procedures, also Fig. 1). Data are means and S.E.M. Statistically signi¢cant di¡erences are marked with *P 6 0.05 and ***P 6 0.001.
deprived (8.8-fold, F1;7 = 37.11, P 6 0.0005) side and there was a signi¢cant di¡erence between the sides (two-fold, F1;8 = 10.32, P 6 0.02). Co-injection of MK801 (1.0 mg/kg) resulted in c-Fos expression levels diminishing to those evoked by apomorphine alone (for the non-deprived side: 4.8-fold, F1;6 = 6.01, P 6 0.05) with no di¡erence between the sides (1.3-fold, F1;6 = 0.35, P = 0.6).
DISCUSSION
The major ¢ndings of this study can be summarized as follows. Sensory deprivation/stimulation procedures had di¡erential e¡ects on various transcription factor proteins in the barrel cortex and these e¡ects were cortical layer-speci¢c. Decreasing the sensory input down-regulated only Zif268 but none of the other proteins tested. The down-regulatory e¡ect of sensory deprivation on Zif268 was observed in all cortical layers except VIb. In contrast, apomorphine treatment resulted in a striking up-regulation of c-Fos protein levels throughout the cortical thickness. There was a similar, but lesser e¡ect on Jun B levels. Removal of the sensory input diminished this response in all layers, except VIb. In contrast, expression of Fos B was not a¡ected by any of the manipulations employed. The NMDA receptor antagonist MK-801, at 0.1 mg/kg, did not produce any signi¢cant e¡ects on either behavior or expression of the proteins under study. However, treatment with this compound at the higher dose of 1.0 mg/kg resulted in gross behavioral abnormalities along with decreased c-Fos activation. Apomorphine is a dopamine receptor agonist that acts at both D1 and D2 receptors. Behaviorally, apomorphine
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treatment produces an increased motor activity including sni¤ng, whisking, licking and snout contact ¢xation, i.e., establishment of a continuous contact of the snout with surfaces (Beck et al., 1986; Szechtman et al., 1982). These responses, observed also in our experiments, obviously involve increased stimulation of the whiskers, and hence might be responsible for sensory input-driven cFos activation. However, dopamine receptor agonists such as apomorphine as well as cocaine and amphetamine induce by themselves c-fos, fos B, Zif268 and jun B expression in the striatum and cortex (Berke et al., 1998; Cole et al., 1992; Deutch and Duman, 1996; Dragunow et al., 1990; Gerfen et al., 1998; Graybiel et al., 1990; Jung and Bennett, 1996; Keefe and Gerfen, 1996; LaHoste et al., 1996; Moratalla et al., 1992; Nguyen et al., 1992; Robertson et al., 1989; Young et al., 1991). Thus, the e¡ects of apomorphine in the somatosensory cortex could also result from a direct stimulation of dopamine receptors. Our results suggest that indeed there are two components to apomorphine action ^ sensory, that is dominant, and non-sensory, possibly through direct stimulation of cortical dopamine receptors. This ¢nding is in contrast to data reported by Steiner and Gerfen (1994) regarding c-fos and zif268 mRNA expression following the same paradigm of apomorphine treatment. These authors reported that apomorphine-evoked accumulation of both mRNAs was fully abolished by sensory deprivation. In contrast, in our hands, the removal of the whiskers a¡ected apomorphine-evoked c-Fos protein expression in the deprived part of the cortex only partially. Steiner and Gerfen (1994) also described an apomorphine-dependent increase in zif268 mRNA expression level. This increase was even bigger than the decrease they observed after clipping of the vibrissae in the control group, without apomorphine injection. We did not con¢rm this observation at the protein level. It is not clear what di¡erences between experimental paradigms are responsible for this discrepancy, although it should be noted that di¡erential regulation of mRNA vs. protein expression has been observed in other situations (Hisanaga et al., 1992; Kiessling et al., 1993; Worley et al., 1993). However, similar to Steiner and Gerfen (1994) as well as others (Melzer and Steiner, 1997; Staiger et al., 2000; see also Kaczmarek and Chaudhuri, 1997) we recorded decreased expression of Zif268 following sensory deprivation. Jun B expression appeared increased in barrel cortex after apomorphine stimulation with higher levels reached on the non-deprived side. The most striking, and sensory input-independent increase was observed in layer VIb. However, the changes observed in other layers were not as marked as those of c-Fos. Inducibility of Jun B in the brain was observed by others and a striking parallelism to c-Fos was repeatedly noted (Gass et al., 1993b; Kaminska et al., 1994, 1996; Kinouchi et al., 1994; Konopka et al., 1998; Luckman et al., 1996; Moratalla et al., 1993) also in barrel cortex (Staiger et al., 2000). Hence, our results go along with the prevalent notion that Jun B and c-Fos form a major inducible AP1 complex in the brain after stimulation (Kaminska et al., 2000).
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Fos B expression in rat barrel cortex was not altered by any manipulations employed in this study. Although the levels of this protein have been shown to increase in the brain in several experimental paradigms, in a number of other conditions, Fos B expression, contrary to that of, e.g., c-Fos, was found not to change (Herdegen and Leah, 1998). It is important to further investigate their di¡erential expression patterns and biological functions with focus on possible di¡erent target genes for various Fos proteins in the brain. Recently another AP-1 protein, c-Jun, was also found not to change after stimulation of whiskers (Staiger et al., 2000). Thus both proteins seem to be involved (together with Jun D, see, e.g., Kaminska et al., 1995, 1996) in the basal AP-1 complex in the sensory cortex. We also wanted to study the role of NMDA receptors in apomorphine-induced activation of the transcription factor proteins. There are multiple examples that NMDA receptor activation mediates c-fos and zif268 expression (Platenik et al., 2000). In our experiments, we applied ^ together with apomorphine ^ the NMDA receptor antagonist MK-801. This added to the complexity of the system as we simultaneously: (i) activated dopamine receptors through apomorphine injection, (ii) possibly activated NMDA receptors through apomorphineevoked whisking behavior, and (iii) attempted to block NMDA receptors with MK-801 injection. Two doses of MK-801 were selected (see Results). MK-801 at 0.1 mg/ kg did not a¡ect overt behavior of the rats and did not reduce the sensory input-driven response to apomorphine of any of the investigated proteins. Treatment with the higher dose of MK-801, 1.0 mg/kg, resulted in a signi¢cant decline of c-Fos immunoreactivity on the non-deprived side of the cortex, but nevertheless, the levels of c-Fos throughout the cortical thickness were still well above those observed in animals not treated with apomorphine. Importantly, at the same time, MK801 (1.0 mg/kg) e¤ciently disabled locomotor behavior of the animals. It can thus be suggested that the lack of a proper motor response, including alteration in sni¤ng and whisking, was responsible for the observed phenomenon. Notably, at this dose, MK-801 is known to induce cortical c-fos expression on its own (Gao et al., 1998; Gass et al., 1993a; Hughes et al., 1993; Sharp et al., 1991; Wedzony and Czyrak, 1996; Zhang et al., 1999). Thus the important conclusion resulting from these considerations is to put into question the usefulness of applying such a high dose of MK-801 to investigate the role of NMDA receptors in regulation of cortical gene expression. A special comment should be given to the distinct pattern of gene expression observed in layer VIb. In this layer, in contrast to the other layers, no e¡ect of vibrissae clipping (compare deprived vs. non-deprived side) was noted in the case of c-Fos, Jun B, and Zif268
expression, also the apomorphine-evoked induction was more pronounced in the case of Jun B and Zif268. This region is known for its speci¢c role during the early phases of cortical histogenesis. Cells in layer VIb are among the ¢rst to di¡erentiate. They show speci¢c patterns of gene expression during development and adulthood (Alcantara and Greenough, 1993; Gaspar et al., 1995; Gomez-Pinilla and Cotman, 1992; Valverde et al., 1989). We did not observe c-Fos induction following tactile stimulation in this sublayer (Filipkowski et al., 2000). It seems that the prevailing signals which lead to changes in expression of the investigated transcription factors in layer VIb are non-sensory. This signaling may be due to the direct action of apomorphine on dopamine receptors as D1 mRNA was found to be abundant in VIb cells of the posterior frontal cortex (Gaspar et al., 1995). The immediate early gene products we have studied are involved in the formation of transcription factors, and thus their only known biological function is to control gene expression. Because of this, they have been repeatedly hypothesized to play a pivotal role in orchestrating genomic responses underlying long-term neuronal plasticity (Kaczmarek, 2000). Sensory stimulation appears to be an important trigger for cortical plasticity which in adults seems to be restricted mostly to layers II/ III (Darian-Smith and Gilbert, 1994; Daw et al., 1992; Glazewski, 1998). These layers receive the major input from layer IV. Thus, it is interesting to note that the greatest di¡erences in c-Fos expression observed between the deprived and non-deprived sides (and hence possibly the greatest sensory component to gene activation) were noted in layer IV. Also, in this layer, another transcription factor, cAMP-response element-binding protein (CREB), was implicated in barrel cortex plasticity (Glazewski et al., 1999) and CREB-dependent expression was recently found to be induced only in layer IV of the barrel corresponding to the spared whisker after deprivation of surrounding vibrissae (Barth et al., 2000). As suggested by these authors, we may conclude that the molecular events initiating cortical plasticity ^ possibly involving basal ganglia activity ^ occur presynaptically, i.e., in neurons with cell bodies located in layer IV, and projecting to layers II/III. Furthermore, our results provide additional evidence for the hypothesis that increased c-Fos/AP-1 expression comprises one of the molecular events involved in this phenomenon. AcknowledgementsöWe wish to thank Rodrigo Bravo for a kind gift of anti-Jun B and anti-Zif268 antibodies and Krzysztof Wedzony for priceless methodological advice. This work was supported by State Committee for Scienti¢c Research (KBN, Poland) Grant 6 P04A 036 19. R.K.F. was the holder of a Young Scientist Award of the Foundation for Polish Science. M.R. was the holder of a KIRT scholarship between the Karolinska Institute and Warsaw Medical University.
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Res. 57, 719^729. (Accepted 26 June 2001)
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