Enhancement of AP-1 DNA-binding activity during amphetamine- and phencyclidine-mediated behaviour in rats

Neuropharmacology 50 (2006) 924e933 www.elsevier.com/locate/neuropharm

Enhancement of AP-1 DNA-binding activity during amphetamine- and phencyclidine-mediated behaviour in rats Desanka Milanovic a,1, Vesna Pesic a,1, Ljubisav Rakic b, Selma Kanazir a, Sabera Ruzdijic a,* a

Institute for Biological Research, Department of Neurobiology and Immunology, Laboratory of Molecular Neurobiology, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia and Montenegro b Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11000 Belgrade, Serbia and Montenegro Received 22 September 2005; received in revised form 22 December 2005; accepted 4 January 2006

Abstract Amphetamine (AMPH) and phencyclidine (PCP) induce a variety of behavioural and synaptic changes in the brain, many of which are believed to involve the regulation of gene expression. In this study, we examined the effects of AMPH (5 mg/kg), PCP (5 mg/kg) and their combination (5 mg/kg each) on rat motor activity as well as on the activation of the AP-1 transcription factor in rat brains. AMPH administration, followed by PCP, led to a statistically significant elevation of locomotor activity. It was found that the behavioural response of rats was more pronounced when the two drugs were administered together. The electrophoretic mobility shift assay (EMSA) revealed a significant increase in AP-1-binding activity after treatments with AMPH, PCP or their combination. Super shift/shift inhibition analysis demonstrated the presence of c-Fos and c-Jun protein families in the transcriptional complex bound to AP-1 sequences. Further, our results suggest that the enhanced behavioural changes after AMPH and PCP administration were associated with increased expression of AP-1 proteins (Fos and Jun) in the cortex, striatum and hippocampus and that their binding to AP-1 sites on the DNA contributes to long-term changes in rat brain. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Amphetamine; Phencyclidine; Locomotor activity; AP-1 binding activity; c-Fos; c-Jun

1. Introduction Increasing evidence suggests that exposure to drugs, such as amphetamine (AMPH) and phencyclidine (PCP), leads to dramatic behavioural changes. In humans, exposure to drugs of abuse leads to short- and long-term adaptive alterations related to the pathology of a schizophrenia-like state (Robinson and Berridge, 1993). AMPH and PCP are similar in that they can induce psychosis in non-psychotic individuals. They also have a tendency to reactivate and precipitate psychotic episodes in schizophrenic patients in remission (Steinpreis, 1996). PCP-induced psychosis mimics the productive and * Corresponding author. Tel.: þ381 11 2078 336; fax: þ381 11 761 433. E-mail address: [email protected] (S. Ruzdijic). 1 Equal contribution. 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.01.011

deficit symptoms of schizophrenia, whereas AMPH produces only productive symptoms. AMPH activates the forebrain dopaminergic (DA-ergic) system. It enhances DA release from presynaptic terminals by impulse-independent mechanisms (Seiden et al., 1993). PCP is a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist. It induces increased DA release in the mesocorticolimbic terminal regions by Caþþ- and impulsedependent mechanisms known as vesicular release (Raiteri et al., 1979). Nevertheless, the exact role of NMDA receptors in the behavioural and neurochemical PCP-induced effects is still unclear due to the interaction of PCP with DA transport and other receptors such as sigma receptors (Steinpreis, 1996). Both PCP and AMPH induce a hyperdopaminergic state in the brain and produce psychomimetic symptoms in humans (Robinson and Berridge, 1993).

D. Milanovic et al. / Neuropharmacology 50 (2006) 924e933

Most studies focusing on neuronal processes that induce drug-provoked psychosis have been carried out on animal models. All drugs of abuse, including AMPH and PCP, produce an acute response that is often characterized by enhanced arousal or euphoria. In studies on rodents, enhanced arousal was observed predominantly as increased locomotor activity and stereotypic behaviour (Sturgeon et al., 1979; Steinpreis and Salamone, 1992; Vasilev et al., 2003). Biochemical adaptations induced, at least in part, by long-term changes in neuronal gene expression are believed to mediate long-lasting changes in locomotor activity (Herdegen and Leah, 1998). In addition, many studies demonstrate the importance of the striatum and its ventral extension, the nucleus accumbens, in mediating these common responses. These brain regions also exhibit persistent changes in specific signaling proteins after long-term exposure to AMPH, PCP and other drugs of abuse (Hughes and Dragunow, 1995). Numerous studies have shown that addictive drugs, such as AMPH or PCP, induce the effect of immediate-early genes (IEGs) in areas of the brain involved in motor activity and reward (Graybiel et al., 1990; Morgan and Curran, 1991; Turgeon et al., 1997; Herdegen and Leah, 1998). This effect can play an important role in both acute activities of psychostimulant drugs and their ability to contribute to the development of behavioural sensitization, tolerance, and addiction (Vukosavic et al., 2001; Milanovic et al., 2003; Self, 2004). The IEGs constitute a class of transcription factors where the Fos protein, via heterodimeric association with Jun, another IEG protein product, forms the (AP-1) complex that binds to specific DNA binding sites involved in the promotion or repression of the transcription of specific genes (Nye et al., 1995; Turgeon et al., 1997; Karin et al., 1997). AP-1 exists either as a homo- or hetero-dimer composed of Fos (c-Fos and Fos B), Fos-related antigens (Fra 1 and Fra 2) or Jun (c-Jun, Jun B and Jun D) protein families in different combinations (e.g., c-Fos/c-Jun, Jun B/Jun D, Fra/c-Jun, etc.). The AP-1 complex was originally considered a transcriptional activator, which by various manipulations, such as drugs of abuse, lesions, seizures and behavioural training, can apparently influence target gene expression (Morgan and Curran, 1991; Sharp et al., 1993). In the present study, we investigated the differential effects of AMPH, PCP and their combination on the motor activity in rats and the effects of drug-induced behaviour on the modulation of DNA binding activity of the AP-1 complex. In an attempt to investigate the composition of AP-1 complex after different drug treatments, the members of the Fos and Jun protein families were also evaluated. The observed cross-sensitization to AMPH and PCP suggests that the drugs share a common pathway of sensitization (Balla et al., 2003). We hope that the effectiveness of the two agents can be assessed through a better understanding of the molecular mechanisms underlying psychomotor stimulation at the behavioural level after the co-administration of AMPH and PCP. This field has not been adequately examined, and many questions remain unanswered, including the effects of dosage, the time between two injections, and the sequence of injections (Pesic et al., 2003).

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2. Materials and methods 2.1. Animals and drugs Adult Wistar male rats (250e300 g) were used. The animals were housed in groups of 4e5 animals per cage under standard conditions (23
2  C, 60e 70% relative humidity, 12 h/12 light/dark cycle, food and water ad libitum) in the Institute for Biological Research, Belgrade. The animals were maintained in accordance with the principles enunciated in the Guide for Care and Use of Laboratory Animals, NIH publication No 85-23. D-Amphetamine sulphate (AMPH) and 1-[1-phenylcyclohexyl] piperidine hydrochloride (PCP), obtained from Sigma-ALDRICH Chemie, Germany, were dissolved in saline (each 5 mg in 1.0 ml 0.9% NaCl).

2.2. Motor activity measurement system for behavioural tests Motor activity was monitored in an open field by an automatic device, the Auto-Track System (Version 3.0 A, Columbus Institute, OH, USA). Each monitoring instrument (Opto-Varimex) consisted of a Plexiglas cage (44.2  43.2  20 cm) connected to the Auto-Track interface and inter-crossed by horizontal and vertical infrared beams. The interruption of the beam generated an electrical impulse, which was processed and sent to a computer linked to the Auto-Track interface. The Auto-Track system detected 11 behavioural parameters, including locomotor and stereotypic activities. The type of activity, as characterized by the animal’s movements, was determined using a user-defined box size (set to five beams in the present study). To eliminate environmental influences on the animals during the experimental sessions, the OptoVarimexes were placed in light- and sound-attenuated chambers with artificially regulated illumination (100 Lx) and ventilation.

2.3. Behavioural tests Each rat was naive and tested only once. The experiments were performed between 09:00 and 15:00. The rats, divided into 4 groups (n ¼ 5 per group), received two i.p. injections each. The rats were first habituated to the experimental cages for 15 min and then injected twice at 5 min intervals with the following: saline (1.0 ml/kg; controls), AMPH (5.0 mg/kg) þ saline, saline þ PCP (5.0 mg/kg) and AMPH þ PCP (each 5 mg/kg). Motor activity was recorded 120 min after the second injection. AMPH and PCP doses were the same as those applied by previously (Graybiel et al., 1990; Turgeon et al., 1997; Jentsch et al., 1998; Vukosavic et al., 2001).

2.4. Preparation of brain nuclear extracts The rats were killed by rapid decapitation 30 min, 1 h, 2 h, 4 h, and 6 h after the treatments (n ¼ 5 for each time point). The brains with the cerebellum and brain stem removed, were quickly placed on ice and immediately used for nuclear extract preparation (Milanovic et al., 2003). Brain tissue was homogenized with a Dounce homogenizer in 4 vol. (w/v) of buffer containing 0.25-M sucrose, 15-mM TriseHCl pH 7.9, 60-mM KCl, 15mM NaCl, 5-mM EDTA, 1.0-mM EGTA, 0.15-mM spermine, 0.15-mM spermidine, 1.0-mM DTT and the following protease inhibitors: 0.1-mM PMSF, 2-mg/ml leupeptin and 5 mg/ml aprotinin. The cells were collected by centrifugation (2000  g, 10 min, 4  C), resuspended in 4 vol of a solution containing 10-mM HEPES pH 7.9, 1.5-mM MgCl2, 10-mM KCl and protease inhibitors. The nuclei were pelleted by centrifugation (4000  g, 10 min, 4  C). The pellets were resuspended in a buffer containing 10-mM HEPES pH 7.9, 0.75-mM MgCl2, 0.5-mM EDTA, 0.5-M KCl, 12.5% glycerol, plus the protease inhibitors, and then incubated on a rotary wheel for 30 min. After 30 min of cell extraction, nuclei were collected by centrifugation (14 000  g, 30 min, 4  C) and the supernatant was dialyzed against a solution containing 10-mM TriseHCl pH 7.9, 1.0-mM EDTA, 5-mM MgCl2, 10-mM KCl, 10% glycerol and protease inhibitors. The extracts of different samples were stored in aliquots at 80  C before their analyses.

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2.5. Total protein isolation from brain structures Sensomotor cortex, striatum and hippocampus were taken from the rats (n ¼ 4/group) that were treated with AMPH, PCP, and AMPH þ PCP (5 mg/kg each). Brain structures were pooled from 4 dissected brains. Whole protein extracts prepared from the brain structures were homogenized in RIPA buffer (300-mM NaCl, 20-mM HEPES pH 7.5, 0.2% SDS, 2% Nadeoxycholate, 2% Triton X-100). Before SDS-PAGE electrophoresis, the protein extracts were clarified by centrifugation at 10 000  g. Protein concentrations were determined following the method of Bradford (1976). Western analyses were performed using primary antibodies raised against Fos, Fra, and Jun (Santa Cruz Biotechnology Inc.; see Section 2.8 below).

2.6. Electrophoretic mobility shift assay (EMSA) The nuclear extracts were used in the EMSA (Milanovic et al., 2003). Ten micrograms of protein was incubated with radioactive oligonucleotide probes at ambient temperature for 20 min in a binding buffer containing 1.0-mg poly (dI-dC)-poly (dI-dC), 4% glycerol, 1.0-mM MgCl2, 0.5-mM EDTA, 0.5-mM DTT, 50-mM NaCl and 10-mM TriseHCl, pH 7.5. For competition experiments, an excess of radio inert oligoprobe or poly (dI-dC)-poly (dI-dC) was added before the radiolabelled probe. The samples were then loaded onto a non-denaturing 4% acrylamide/0.05% N,N0 -methylene-bis-acrylamide gel and subjected to electrophoresis at 200 V, for 3e4 h in 25-mM Tris-borate buffer containing 0.5-mM EDTA. After electrophoresis, the gels were dried and exposed to X-ray films. To quantify DNA binding activity, the gels were exposed on radiosensitive phosphorus screens and scanned by laser densitometry (Phosphor Imager SI, Molecular Dynamics, U.S.A). The radioactivity was quantified using Image Quant software (Molecular Dynamics). The activities were converted to percentages of DNA binding and compared with the values obtained in the protein extracts isolated from saline-treated rats (controls), which were taken as 100%.

2.7. Oligonucleotides and antibodies In the initial EMSA studies, 21-mer double-stranded oligonucleotides containing the consensus AP-1 site (TGAC/GTCA) with the following sequences: 50 -CGCTTGATGAGTCAGCCGGAA-30 and reverse complement, 30 -GCGAAC TACTCAGTCGGCCTT-50 , were used. AP-1 was purchased from Promega, Madison, WI, U.S.A. The AP-1 sequences were radiolabelled with [alfa-32P] deoxy-CTP using Klenow fragment of DNA polymerase 1 (Boehringer, Mannheim, Germany) and purified by Quick Spin columns (Boehringer, Mannheim, Germany)following the manufacturer’s procedures. For the super shift assay, 1.0 mg of polyclonal antibodies against c-Fos, Fra-2, Jun D, JunB, c-jun/AP-1 (Santa Cruz Biotechnology, Inc.) were added to the mixture containing protein extracts without the labelled probe. The nuclear extracts were incubated with antibodies (4  C, 14e16 h) and then with radioactive probes at ambient temperature for 20 min before gel retardation electrophoresis on a 4% acrylamide gel. The procedure was repeated as stated above.

2.8. Immunoprecipitation and Western-blot analysis Nuclear extract proteins (60e70 mg) were diluted with RIPA buffer (300 mM NaCl, 20 mM HEPES pH 7.5, 0.2% SDS, 2% Na-deoxycholate, 2% Triton X-100) to a final volume of 375 ml and incubated with 1.0-mg Jun D [sc-74, rabbit polyclonal IgG, Santa Cruz Biotechnology, (Santa Cruz, CA, U.S.A.)] on a rotating wheel, for 30 min at 4  C. Protein A sepharose (Amersham Biosciences, UK) was added (25 ml per sample) and mixed by rotation for 60 min. A control experiment without the Jun D primary antibody was performed to confirm the IgG band. The bound protein was collected by centrifugation and the pellet washed five times with RIPA buffer. Finally, the immunoadsorbed Jun D complex was solubilized and denatured in 50-mM TriseHCl (pH 7.4) containing 10-mM EDTA, 2% SDS, 1% 2-mercaptoethanol and 0.001% bromphenol blue. The samples were boiled for 5 min, clarified by centrifugation, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Aliquots containing 20 g of proteins from the cortex, striatum, and hippocampus were separated by electrophoresis on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes. The blots were incubated in 5% non-fat dry milk in TBST (50-mM TriseHCl, pH 7.4, 150-mM NaCl, 0.05% Tween 20) for 60 min. The blots were then incubated overnight on a shaker at þ4  C in a dilution of different antibodies (c-Fos, Fra-1, Fra-2, c-Jun) in a blocking buffer. After three washes with the incubation buffer, the membrane was incubated for 60 min at ambient temperature with the secondary antibody (anti-rabbit IgG, peroxidase-linked from Amersham Biosciences, UK) diluted 5000 times and incubated in TBST buffer (60 min, ambient temperature). The membrane was washed three times with incubation buffer. X-ray film was subsequently exposed to the membrane treated with the Enhanced Chemiluminescence System [(ECS) Amersham Biosciences, UK]. Semi-quantitative densitometric analyses of proteins were performed on the X-ray film using a Multi-Analyst/PC Software Image Analysis System (Bio-Rad Gel Doc 1000).

2.9. Statistical analyses Significant difference between data sets was determined using STATISTICA 6.0 software. The results of the behavioural tests are presented graphically, either as mean values
SEM of the locomotor activity at 10-min intervals, or as the total locomotor activity during the first and second hours. The total locomotor activity values were transformed (sqrt) and analyzed by two-way analysis of variance (ANOVA) with treatment and time (repeated measure) as factors. Subsequent comparisons were made with Tukey HSD test. The protein and AP-1 binding levels were quantified by densitometry. Data for Figs. 2 and 4 were analyzed by two-way ANOVA with treatment and time as factors, followed by Tukey HSD test. Analysis of the data for Fig. 2 was performed after its transformation (ln). Results were presented graphically as percentages (mean
SEM, N ¼ 5) of the densities obtained from control animals assumed to be 100%. The data for Fig. 5A were analyzed by oneway ANOVA with treatment as factor. Comparisons with control values were made using t-test. The data were presented as percentages (mean
SEM, N ¼ 4) of the densities obtained for the respective structures of control animals. The differences were considered significant at P < 0.05.

3. Results 3.1. The effects of AMPH, PCP and AMPHþPCP on the locomotor activity of rats The time-profile of the locomotor activity during 120 min is presented in Fig. 1A. Injection of AMPH (5 mg/kg), PCP (5 mg/kg) or their combination (AMPH þ PCP, 5 mg/kg each) induced a significant increase in locomotor activity in comparison with the controls at almost all the time points examined. This increase was much higher with the injection of AMPH þ PCP than with the injection of AMPH or PCP alone. Locomotor activity counts during the first and second hours of recording are presented in Fig. 1B. Analysis of the total locomotor activities by ANOVA revealed significant effect of the treatment, F(2,12) ¼ 21.886, P < 0.01, time, F(1,12) ¼ 585.739, P < 0.01 and significant interaction F(2,12) ¼ 4.993, P < 0.01. Post hoc comparisons revealed significantly higher locomotion in AMPH þ PCP-treated rats than in AMPH- or PCP-treated rats during the first and second hours of recording (#P < 0.01 and &P < 0.01, respectively). Also, in all the three drug-treated groups, locomotor activity was higher in the first hour than in the second hour (P < 0.05).

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of a single dose of AMPH or PCP. Increase in AP-1 binding by about 50%, 130%, and 240% after an injection of AMPH þ PCP was observed at the 2-, 4-, and 6-h time points, respectively. Two-way ANOVA revealed a nearly significant effect of the treatment (F(2, 60) ¼ 2.716, P ¼ 0.07), but showed a significant effect of the time (F(4, 60) ¼ 41.803, P < 0.05) and a significant interaction (F(8, 60) ¼ 3.789, P < 0.05). Subsequent analysis showed that at the 6-h time point, the intensity of AP-1 binding was significantly higher in the AMPH þ PCP-treated rats than in AMPH- or PCP-treated rats (#P < 0.05, &P < 0.05, respectively). The intensity of AP-1 binding in all the drug-treated groups at the ½-h time point was not significantly different from that of the controls, whereas at the 1-h time point it was significantly higher in both AMPH- and AMPHþPCP-treated rats (Fig. 2E, *P < 0.05). At the 2-h, 4-h and 6-h time points, the intensity of AP-1 binding was significantly higher in all the drug-treated groups (*P < 0.05) than that of the controls.

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Fig. 1. The effects of AMPH (5 mg/kg, i.p.), PCP (5 mg/kg, i.p.) and their combination (5 mg/kg each) on the locomotor activity of rats during a 120min registration period. A represents mean
SEM activity counts at 10 min-intervals. B shows the locomotor activity counts during the first and second hours of registration. **P < 0.01 compared to control; #P < 0.01 compared to AMPH treated rats; &P < 0.01 compared to PCP treated rats.

Comparison with the activity of controls showed that all the drug-treatments induced significant elevations in locomotor activity during the first and second hours of motor activity monitoring (**P < 0.01).

3.2. Time-course of AP-1 binding activity following AMPH, PCP and AMPH and PCP application The AP-1 binding activity in the initial EMSA experiments was measured using brain nuclear extracts of rats treated with AMPH, PCP or AMPH þ PCP at different time points after treatment (from 30 min to 6 h). The AP-1 oligonucleotides formed a single shifted protein-DNA complex with the nuclear extract (Fig. 2, panels A, B, and C). To confirm the binding specificity to AP-1, competition studies were performed and the results, presented in Fig. 2D, show a gradual reduction of the specific AP-1 complex after incubation with radio inert AP-1 probes at concentrations 10e100 times higher than that of the labelled probes. The results were quantified by densitometry and the data were presented graphically as percentages of the densities obtained from control animals (Fig. 2E). Quantitative analysis of the autoradiographs revealed a significant increase in AP-1 binding activity 1 h after administration of the drugs and until the 6-h time point. AP-1 binding increased after administration

Experiments were undertaken to determine the protein components of the AP-1 transcription complex. The EMSAsupershift/shift inhibition assay enabled us to further retard the AP-1 probe-protein complex with an antibody that specifically recognized the bound protein (Fig. 3). Therefore, in our experiments we analyzed a set of antibodies to identify the potential proteins that bind to the specific AP-1 complex involved in the regulation of transcription after treatment with AMPH, PCP or their combination. Fig. 3 shows that the anti-Jun D antibody was very effective in inducing an upward shift of the probe/protein complex on gels, thus revealing the presence of Jun D in the AP-1 complex after all the three treatments. After incubation with anti-c-Jun and anti-Jun B, antibodies diminishment of the specific AP-1 complex was observed, pointing to the participation of c-Jun and Jun B proteins in AP-1 hetero- or homo-dimmers after AMPH, PCP and AMPH þ PCP stimulation (Fig. 3, panels A, B and C). After incubation with anti c-Fos, antibody shifts in the inhibition (40%e45%) of the band were observed in the AMPH-, PCPand AMPH þ PCP-treated samples (Fig. 3AeC). The addition of anti-Fra antibody also resulted in a reduction of the AP-1 bands in the AMPH and PCP-treated rats (Fig. 3A,B), whereas in the AMPH þ PCP-treated rats, the shift in the inhibition of the bands was similar to that observed after incubation with anti c-Fos antibody (Fig. 3C). 3.4. Detection of Jun D Since the incubation of brain nuclear extracts with the antiJun D antibody resulted in the formation of a supershift band in the gel mobility assay that indicated that the AP-1-DNA complex only super shifted in the presence of the Jun D protein, we examined the time course of changes of the level of Jun D protein expression in the same brain nuclear extracts. Two immune-specific bands (having molecular masses of 45

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Time (h) Fig. 2. The time course of AP-1 DNA-binding activity in rat brains after acute AMPH, PCP, and AMPH/PCP treatments. Ten micrograms of nuclear extract proteins were incubated with 32P-labelled AP-1 oligonucleotides. DNA protein complexes were separated by gel electrophoresis as described in the Experimental Procedures. Amphetamine (A); phencyclidine (B); AMPH þ PCP (C); DNA binding specificity of AP-1 sequences (D). Ten mg of nuclear extract protein were incubated with 32P-labelled AP-1 oligonucleotides in the presence of an excess of radio inert AP-1 oligonucleotides (10e100). Arrows indicate the position of specific bands. C e control; P e 32P-labelled AP-1 probe. The AP-1 binding profile was quantified by densitometry of EMSA autoradiograms. The AP-1 DNA-binding activities are expressed as the percentage changes (mean
SEM) compared to the control (E). The dotted line represents the control value (100%). *P < 0.05, compared to controls; #P < 0.05, compared to AMPH; &P < 0.05, compared to PCP, n ¼ 5, N ¼ 5.

and 32 kDa) were detected in the presence of the anti-Jun D antibody during the immune precipitation procedure (Fig. 4). In the control experiment without the Jun D antibody, one of the co-precipitated bands (45 kDa) was detected. Thus, the band with a higher molecular mass was identified as IgG and the other with lower molecular mass band (32 kDa) as Jun D. The autoradiograms were analyzed by densitometry. The values corresponding to Jun D were normalized using the values for IgG and the data were presented graphically as percentages of the values obtained from control animals (Fig. 4D). Two-way ANOVA revealed significant effects of treatment (F[2, 48] ¼ 215.466, P < 0.05), time (F[3, 48] ¼ 29.780, P < 0.05) and interaction (F(6, 48) ¼ 5.756, P < 0.05). Subsequent multiple comparisons showed that at all the examined time points, the expression of Jun D protein was significantly higher in the AMPH þ PCP-treated rats than the one in AMPH- or PCPtreated rats (#P < 0.05, &P < 0.05, respectively). A comparison with the control values revealed that in AMPH- and AMPHþPCP-treated rats, the expression of Jun D was significantly high at all time points (*P < 0.05),

whereas in the PCP-treated rats, it was high only at the 2and 4-h time points (*P < 0.05). It should be emphasized that for AMPH- and AMPH þ PCP-treated rats, the highest level of Jun D expression was obtained at the 2-h time point (P < 0.05, comparison with the respective treatments at ½-h, 4-h and 6-h time points). 3.5. Analysis of Fos- and Jun-related protein expression in specific regions of the brain To examine whether AMPH and PCP induced any changes in Jun- and Fos-related proteins in different regions of the brain, we investigated the expressions of c-Jun, Jun B, Jun D (35 kDa), c-Fos (55 kDa), Fos B (62 kDa), and Fra 2 (35 kDa) proteins by Western blot analysis using extracts from sensomotor cortex, striatum, and hippocampus 2 h after treatments with AMPH, PCP and their combination. As expected, quantitative and qualitative changes in Fos- and Junrelated protein expression were observed in different brain structures (Fig. 5).

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One-way ANOVA demonstrated that the effect of treatment was not significant. A significant increase in c-Fos protein levels in comparison to appropriate controls (Table 1, *P < 0.05) was detected in all the examined structures after the treatments. Surprisingly, a difference in the intensity of the Fos B (62 kDa) band corresponded to the prominent increase of the Fos B protein level in the striatum with slight quantitative difference between control and treated rats. Fos B protein was hardly detected in the cortex and hippocampus. Western blots with Fra-2 antibody demonstrated a consistent increase in proteins exclusively in the 33e40 kDa range, and a prominent Fra-2 (35 kDa) expression in the control, as well as after different drug treatments (Fig. 5). Semi-quantitative analysis of the change of relative concentration of Fra-2 protein revealed non-significant differences between the control and drug-treated rats in all the examined brain structures. Additional experiments, which included Western analysis with Fra-1 antibody after the above treatments, were performed to assess the cross-reactivity of the used Fra antiserum. The Fra-1 antibody recognized a 41 kDa protein and proteins in the 33e40 kDa range (data not shown). Incubation of the protein blots with anti-c-Jun antibody (that recognizes not only c-Jun, but also Jun B and Jun D), revealed the presence of three bands at approximately 35e40 kDa. As can be seen from the blots, all three proteins were induced in the cortex, striatum, and hippocampus 2 h after the treatments.

4. Discussion

Fig. 3. Supershift/shift inhibition analysis of AP-1 complexes with antibodies against different transcription factors 2 h after AMPH (A), PCP (B) and AMPH/PCP administration (C). Prior to gel-shift assay, the protein extracts were incubated with 1 mg of the indicated antibodies (for 14e16 h at 4  C). In the control samples, the antibody was omitted from the assay mixture and the lane designated P was loaded with radiolabelled AP-1 probe alone. The arrows indicate the supershifted bands; arrowheads indicate the positions of the specific bands.

Increases in c-Fos protein levels were observed in all the examined structures. The data obtained by densitometry are presented as percentage changes of the densities as compared to appropriate structures in the control animals (Table 1).

The results obtained in this study show that a single dose of AMPH, PCP or their combination induces in adult rats both AP-1 DNA-binding activity and behavioural changes. This is in line with the earlier findings on locomotion produced by AMPH or PCP (Steinpreis and Salamone, 1992; Jaber et al., 1995). However, only a few studies provide information on the effects of the combination of AMPH and PCP (Turgeon and Case, 2001; Balla et al., 2003; Pesic et al., 2003). The differences in the applied doses and in the order and interval of applications make comparison of our results with the data of other authors somewhat difficult. We found that the administration of AMPH, followed by PCP, resulted in the elevation of rat locomotor activity. This suggests that the behavioural response was stronger when the two drugs were given together rather than separately. The curves that represent the locomotor activity of rats treated with AMPH/PCP and AMPH have similar time-dependent profiles, differing only in the extent of change. The pronounced excitatory behavioural effects of AMPH are generally believed to be closely associated with increased concentration of DA in the brain. Administration of AMPH causes a direct stimulation of DA release from the presynaptic mesoaccumbal and nigrostriatal terminals (Sharp et al., 1987; Seiden et al., 1993). As a positive correlation of AMPH-induced locomotor activity with DA release has been proven, it can be assumed that the locomotor activity described in the present study

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Fig. 4. The time course of Jun D protein expression in rat brains following AMPH (A), PCP (B), and AMPH/PCP (C) administration, as analyzed by Western blotting. After immune-purification of nuclear extracts with rabbit anti-Jun D antibody, two immune-specific bands (45 and 32 kDa) were detected by Western blot analysis. Jun D was detected in control nuclear extracts (lane 1), 30 min (lane 2), 2 h (lane 3), 4 h (lane 4) and 6 h (lane 5) after AMPH, PCP, and AMPH/PCP administration. Densitometric data corresponding to Jun D were normalized using the values for IgG and presented as the percentages (mean
SEM) relative to the control values (D). The dotted line represents the control value (100%). *P < 0.05, compared to the controls; #P < 0.05, compared to AMPH; & P < 0.05, compared to PCP, n ¼ 5, N ¼ 5.

also resulted from an elevated concentration of DA in the striatum. However, the influence of other neurotransmitter systems cannot be ruled out. AMPH also induces pronounced and rapid dose- and time-dependent increases of norepinephrine, acetylcholine, and serotonin (Steketee, 2003).

In contrast to AMPH, administration of PCP increased locomotion during the first 40 min. PCP is a non-competitive NMDA receptor antagonist. L-Glutamate, by primarily reacting with NMDA receptors, stimulates GABA-ergic outflow from the basal ganglia so that the inhibition of NMDA

Fig. 5. The protein levels of c-Fos, Fos B, Fra2, c-Jun, Jun B and Jun D in the prefrontal cortex, striatum, and hippocampus determined by Western blot assay. Two hours after the drugs were injected, the brain structures were dissected and the protein extracts isolated in RIPA buffer. Aliquots of 20 mg of proteins were subjected to SDS-PAGE and immunoblot analysis using corresponding antibodies. Line 1 e control sample; line 2 e AMPH; line 3 e PCP; line 4 e AMPH þ PCP. The arrows indicate the approximate molecular masses of the Fos and Jun-related proteins.

D. Milanovic et al. / Neuropharmacology 50 (2006) 924e933 Table 1 C-Fos protein level in cortex, striatum and hippocampus 2 h after injection of AMPH (5 mg/kg), PCP (5 mg/kg) or AMPH þ PCP (5 mg/kg both)

AMPH PCP AMPH þ PCP

Cortex

Striatum

Hippocampus

135
13* 128
7* 143
9*

141
11* 149
12* 164
14*

154
16* 167
16* 155
20*

Data were expressed as a percentage (mean
SEM) of control values assumed to be 100% (n ¼ 4; N ¼ 4). *P < 0.05, compared to control.

receptors by PCP decreases the GABA-ergic outflow also, leading to behavioural disinhibition similar to that seen after AMPH administration (Carlsson et al., 1999). Also, PCP activates sigma receptors and influences other neurotransmitter systems, e.g. DA-ergic, serotonergic, and cholinergic (Kitaichi et al., 1996; Steinpreis and Salamone, 1992; Gleason and Shannon, 1997; Sarter and Bruno, 1999). PCP also inhibits DA reuptake, facilitates DA release and affects its synthesis. This explains the ability of PCP to induce amphetamine-like behaviour. In contrast to AMPH, which acts within the terminal regions of DA-ergic neurons alone (Seiden et al., 1993), PCP acts on the cell bodies of DA neurons. Mathe et al. (1999) reported that PCP and MK-801, in a dose-dependent manner, augmented neuronal activity (burst firing) in DA neurons localized in the ventral tegmental area (VTA), a region which projects largely towards subcortical sites, i.e. the nucleus accumbens and the prefrontal cortex. When PCP was given shortly after AMPH, the locomotor response increased. The curve retained the same profile but showed increased activation. Our study demonstrated that the combination of the two psychostimulants significantly increased the locomotor activity during the first and second hours. As elevation of extra cellular dopamine concentration in the nucleus accumbens is important for locomotor effects of both AMPH and PCP (Sharp et al., 1987; Adams and Moghaddam, 1998), it could be speculated that the difference in locomotion was due to higher accumbal dopamine concentrations in AMPH þ PCP-treated rats than those in AMPH-treated rats. It was recently demonstrated that increased dopamine levels in the prefrontal cortex also correlated with the time course of hyperactivity following PCP administration (Adams and Moghaddam, 1998). Jentsch et al. (1998) showed that lesions of the prefrontal cortex blocked the ability of PCP (but not of AMPH) to induce hyper locomotion and increase the dopamine utilization in the nucleus accumbens. Our results suggest that the additional locomotor activity observed in the AMPH/PCP-treated group, over that of the AMPH-treated group, depended on the effects of PCP on the prefrontal cortex. However, further studies are needed to determine the brain structures, sites and doses that are responsible for such motor effects (Balla et al., 2003; Pesic et al., 2003). AMPH- and PCP-induced alterations of behaviour are good examples of how stimulation of neurotransmitter receptors leads to a signalling cascade involving second messengers, protein kinases, activation of transcription factors, and longlasting changes in gene expression (Simpson et al., 1995;

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Turgeon et al., 1997; Morgan and Curran, 1991; Milanovic et al., 2003). To investigate this aspect at the molecular level, we analyzed the time-dependence of the transcriptional regulation by AP-1 DNA binding after the administration of AMPH, PCP and AMPH þ PCP. Our findings revealed that the highest level of AP-1 binding activity was between 4 and 6 h after drug administration, with maximal binding at the 6-h time point. We also observed detectable levels of AP-1 binding in control rat tissues. Previously, it was reported that striatal extracts from the normal rat have substantial capacity to bind the AP-1 DNA consensus sequence (Huang and Walters, 1996). The increased AP-1 DNA binding that occurred immediately after administration of the drugs could have been induced by the stress of experimental handling that has previously been reported to cause a higher basal Fos-like immunoreactivity (Chapman and Zahm, 1996) and induce AP-1 binding (Ogita and Yoneda, 1994). The biphasic stimulation of AP-1 binding over a more protracted time period was demonstrated in both in vitro and in vivo experiments (Sonnenberg et al., 1989; Szekely et al., 1990) as the result of changes in composition of the AP-1 complex apparently suited to serve different functions. The components of the AP-1 complex at the 2-h time point were examined by adding specific antibodies to the gel shift reaction. It was found that in the samples incubated with anti-Jun D antibody that AP-1-specific band was clearly shifted, whereas after addition of anti-c-Jun and anti-Jun B the band was reduced. This indicated that the AP-1 complex, 2 h after administration of psychostimulants, contained predominantly Jun dimers, e.g. c-Jun-Jun D, Jun D-Jun B, etc., which were supported by the induction of high level of Jun proteins in different brain structures in our study. A characteristic of Jun D is its high basal expression in many cell types. Hence, Jun D was earlier considered non-inducible and hence was not classified as an immediate, early gene (Herdegen and Leah, 1998). However, according to Platenik et al. (2000), participation of Jun D in AP-1 complex depends on the time and experimental model. To see whether the composition of AP-1 protein correlates with the induction of Jun D proteins, Western blot analysis was performed with the same antibodies to Jun D as those in the supershift experiments. One to 2 h after administration of the psychostimulants, Jun D protein increased and it remained so up to the 4e6 h interval. This raises the possibility that Jun D levels contributed significantly to the formation of AP-1 complex at 2 h. The composition of novel AP-1 complexes at subsequent time points, when AP-1 binding activity markedly increases, remains to be established. In the present study, we demonstrated that acute treatments with psychostimulants induced various changes of Fos, Fra and Jun transcription factors in the sensomotor cortex, striatum and hippocampus. Different effects of acute and chronic AMPH or cocaine doses on the induction of Fos and Fra proteins and AP-1 binding activity in the rat striatum have been described (Graybiel et al., 1990; Simpson et al., 1995; Nye et al., 1995; Turgeon et al., 1997). The current study showed that acute treatment with AMPH, PCP or their combination led to a significant increase in relative concentration of c-Fos protein in all

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the examined brain structures. The expression of Fra-2 protein (35e37 kDa) was observed after 2 h in all the examined groups. Supershift and shift inhibition analyses of AP-1 complexes, induced by AMPH, PCP or AMPH þ PCP, showed that the components of the acute AP-1 complex were c-Fos and Fra-2, as well as c-Jun, Jun D and Jun B proteins. With the Jun D antibody, a single supershifted band was observed in the AP-1 complex. In contrast, the antisera for c-Fos, Fra-2, Jun B and c-Jun produced shift inhibition of the AP-1 complexes, suggesting the involvement of these proteins in the regulation of AP-1-binding activity after drug treatment. Composition-dependent AP-1 binding affinities were demonstrated previously for different Fos/Jun family heterodimers in vitro (Karin et al., 1997). Differences in dimer affinities could explain the apparent discrepancy between the presence of c-Fos and its detectability in the AP-1 complex. On the other hand, Fos protein undergoes extensive post-translational modifications, such as serine and threonine phosphorylation (Morgan and Curran, 1991). The phosphorylation state of AP-1 proteins, including c-Jun, has been shown to affect AP1 binding and transcriptional activity (Kallunki et al., 1996). It is important to bear in mind that the relationship between the presence and function of inducible transcription factors is not completely predictable. C-Fos can be induced and bound to the AP-1 binding site (Herdegen and Leah, 1998). However, induced AP-1 binding does not necessarily mean that the expression of AP-1-driven target genes has increased (Platenik et al., 2000). Hollen et al. (1997) described changes in composition of the AP-1 complex over time even though its binding activity was not induced. However, a direct correlation between c-Fos binding, AP-1 composition and behavioural changes was detected, suggesting that knowledge of the composition of AP-1 could provide further insight into the effects of this transition In conclusion, our results clearly demonstrated that 2 h after administration to adult rats of AMPH, PCP and their combination, the locomotor activity increased and AP-1 protein binding activity reached its highest level. This might be the result of the neurons receiving and generating electrophysiological signals and relaying them to the nucleus and transforming them into genetic changes with lasting functional consequences (Herdegen and Leah, 1998). In the course of regulation of target genes, they either activate or inhibit their transcription (Morgan and Curran, 1991; Platenik et al., 2000). The set of target genes that are regulated by AP-1 complex in the brain has not been identified. Thus, the potentiation of AP-1 binding affinity and the variations in its heterodimer composition are of major importance, because the transcriptional regulation of genes plays a key role in molecular mechanism underlying neuronal plasticity and diseases, such as schizophrenia. These genes could be of great interest as they may be involved in psychotic behaviour. Acknowledgement This study was supported by grant #143004 from the Ministry for Science and Environmental Protection, Republic of Serbia.

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