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Differential effects of amphetamine and phencyclidine on the expression of growth-associated protein GAP-43
Neuroscience Research 40 (2001) 133– 140 www.elsevier.com/locate/neures
Differential effects of amphetamine and phencyclidine on the expression of growth-associated protein GAP-43 S. Vukosavic a, S. Ruzdijic a, R. Veskov a, Lj. Rakic b, S. Kanazir a,* a
Institute for Biological Research, 29 No6embar 142, Belgrade, Yugosla6ia b ICN Pharmaceuticals, Costa Mesa, CA, USA Received 10 October 2000; accepted 20 February 2001
Abstract The purpose of the present study was to test changes in the expression of growth-associated protein (GAP-43) after chronic treatment with two different psychotomimetic drugs: amphetamine and phencyclidine. Rats were treated chronically for 7 days (twice daily) with 5 mg/kg of amphetamine and phencyclidine and sacrificed after 2, 5 or 7 days of treatment, and following 7, 14 or 21 days of recovery after full treatment (7 days). Separate groups of rats were treated on the same regiment with haloperidol, and control group was treated with vehicle. To determine the effects of different psychotomimetic drugs on the expression of GAP-43 we have used Northern blotting and quantitative in situ hybridization. Treatment with amphetamine induced decrease of GAP-43 mRNA expression, that was detected also during recovery period, up to 14 days after the last day of 7 days treatments. On the contrary, PCP induced increase of GAP-43 mRNA expression, that was detectable from the first days of treatment until 21 days after the last day of treatment. Treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. It can be suggested that GAP-43 upregulation upon phencyclidine treatment occurs as a result of functional activation of pathways able to participate in remodeling, while amphetamine showed neurotoxic effect, decreasing expression of GAP-43 mRNA. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: GAP-43; Amphetamine; Phencyclidine; Haloperidol; In situ hybridization
1. Introduction The appearance of morphological changes related to the pathology of psychosis-like syndromes has been well described. However, the exact mechanisms causing these abnormalities remain unclear. In a search for molecular correlates of the alteration in brain structure and function observed in psychosis-like syndromes growth-associated protein GAP-43 (neuromodulin, B50, F1) (Benowitz and Routtenberg, 1987; Gispen et al., 1992) has been proposed as a useful marker. Several studies on post-mortem brain samples of schizophrenic patients clearly showed increased expression and increased phosphorylation of GAP-43 in selective regions such as associative cortices (Sower et al., 1995; PerroneBizzozero et al., 1996). GAP-43 protein is concentrated * Corresponding author. Tel.: + 381-11-764422; fax: +381-11761433. E-mail address: [email protected] (S. Kanazir).
in growth cones and axons (Skene, 1989) and it is substrate for protein kinase C. It also affects several intracellular messenger systems via binding to molecules such as calmodulin and GTP-binding protein Go. This neuronal membrane phosphoprotein is implicated in the initial establishment and reorganization of synaptic connections and it is found to be highly expressed in developing neurons during axonal growth and synaptogenesis (Kanazir et al., 1996). In the adult brain, selective brain regions such as limbic system and neocortex maintain high levels of GAP-43 throughout life (Benowitz et al., 1989; Kruger et al., 1993). Regionspecific upregulation of GAP-43 and its restricted localization to higher neocortical and limbic areas suggests that this protein marks neuronal circuits involved in the processing and storage of highest cortical functions such as memory (Neve et al., 1988; Perrone-Bizzozero et al., 1996). Since these processes are known to be altered in psychosis-related syndromes, it was proposed that GAP-43 levels might be altered in this group of
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disorders and several reports showed changes of GAP43 mRNA and protein expression in human postmortem schizophrenic brains (Perrone-Bizzozero et al., 1996; Blennow et al., 1999). Similarly, in another neurodegenerative disease, amyotrophic lateral sclerosis, surviving anterior horn motor neurons selectively upregulate GAP-43, implying the occurrence of compensatory sprouting (Parhad et al., 1992). The widespread aberrant neuritic growth accompanied by impaired synaptic plasticity in schizophrenia (Sower et al., 1995) and Alzheimer’s disease (Colleman et al., 1992; Callahan et al., 1994) suggests that severely compromised GAP-43 gene expression may contribute to the cascade of neurodegeneration. In an attempt to better understand and investigate psychiatric diseases two major animal models of psychosis-like syndromes were developed, based on the chronic, repetitive treatment of animals with two different psychotomimetic drugs such as amphetamine (Lillrank et al., 1991) or phencyclidine (Duncan et al., 1999; Olney et al., 1999). Knowing the importance of GAP43 gene expression in processes underlying brain plasticity and synaptic reorganization, and its possible role as a morphological correlate of psychosis-like syndromes, in this paper we have investigated changes in GAP-43 mRNA expression in selective regions of rat brain upon chronic treatment with these two different psychotomimetic drugs: amphetamine, as a direct dopaminergic agonist and phencyclidine, as noncompetitive NMDA antagonist. We have also involved chronic treatment with haloperidol, widely used antipsychotic drug, in an attempt to compare the effects of haloperidol applied on healthy brain with psychotomimetictreated brains. 2. Materials and methods
2.1. Animals and treatment Adult male Mill– Hill hooded rats were separated in experimental groups (three to five per group) and intraperitonealy injected for seven days in 12-h intervals with 5 mg/kg D-amphetamine (Sigma), 5 mg/kg phencyclidine (PCP, ICN-Galenika) or 5 mg/kg haloperidol (Sigma). The doses for each drug were chosen according to the literature: for amphetamine (Robinson and Becker, 1986; Lillrank et al., 1991; Axt et al., 1994), for phencyclidine (Ellison, 1995; Steinpreis, 1996) and for haloperidol (Burt et al., 1977). Randomly selected rats were allowed to recover up to 3 weeks after 7 days treatment. At 2, 5 and 7 days of treatment (24 h after the last injection) and after 7, 14 and 21 days of recovery period, randomly selected rats were killed by decapitation, brains were removed and immediately frozen in isopentane, cooled on dry ice, and stored at −80°C.
2.2. Northern blot analysis Total brain RNA was isolated by the guanidine isothiocyanate (GTC)/ cesium chloride (CsCl) centrifugation method (Sambrook et al., 1989). Rat brains were homogenized in 4 M GTC, 4% N-sarcosyle, 50 mM Na –acetate pH 5.5 and 1% 2-mercaptoethanol. Homogenates were mixed with 0.5 g/ml CsCl and loaded on a 3-ml layer of 5.7 M CsCl. RNA was pelleted by centrifugation at 160 000× g for 20 h at room temperature. Pellets were resuspended in 10 mM Tris HCl pH 7.5, 1 mM EDTA, 0.5% SDS, and RNA was extracted with butanol:chloroform (1:4) and precipitated in cold ethanol. RNA (10mg) was then denatured and separated by electrophoresis on 1.5% agarose–formaldehyde gel, stained with ethidium bromide for control of loading and transferred to Hybond N+ membrane (Amersham) using capillary transfer overnight in 20×SSC buffer (Sambrook et al., 1989). RNA was UV crosslinked to the membrane (FUNA-UV-Linker, Spectroline). The GAP-43 probe for Northern hybridization was labeled by random priming using random primed DNA labeling kit (Boehringer Mannheim) according to the manufacturer’s instructions. Plazmid containing rat cDNA clone for GAP-43 (kindly provided by Dr E.E. Baetge) was linearized with EcoRI and cDNA insert was isolated with HindIII and subsequently labeled with 50 mCi of a-32P dCTP (ICN) in the presence of DNA polymerase-Klenow fragment. Labeled probe was purified on Nick Spin column (Pharmacia) and added to the hybridization buffer (1× 106 cpm/ml). Prehybridization and hybridization were performed in rapid hybridization buffer (Amersham) at 65°C, for 15 min and 4 h, respectively. After hybridization membranes were washed in 2× SSC, 0.1% SDS and 0.2× SC, 0.1% SDS, according to manufacturers recommendations, and exposed to phosphorimager screen (Molecular Dynamics). Quantification of signals was performed using image analysis software Image Quant (Molecular Dynamics).
2.3. In situ hybridization histochemistry In situ hybridization was performed as previously described (Kanazir et al., 1997). In brief, rat brain coronal sections (12 mm) were fixed in 4% paraformaldehyde, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, and delipidated in ethanol series (70, 96, 100% 1 min each) and chloroform (5 min). Sections were then incubated overnight at 55°C in 100 ml of hybridization buffer (50% formamide, 10% dextran sulfate, 5× Denhardt’s solution, 250 mg/ ml salmon DNA, 150 mg/ml tRNA, 2 × SSC and 20 mM 2-mercaptoethanol) containing radiolabeled antisense or sense riboprobe (10 000 cpm/ml) for GAP-43. A
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GAP-43 riboprobes were generated from a pBluescript KS vector (Stratagene) that contained a 1087 bp insert of rat cDNA clone (kindly provided by Dr E.E. Baetge). Following hybridization sections were washed in 2 ×SSC, treated with RNase A and subsequently washed for 4×15 min in 2×SSC, and overnight in 0.1 ×SSC. Sections were dried and exposed to Kodak NBT2 emulsion for 3 weeks, developed, counterstained with cresyl violet and examined under light microscopy. Quantification of in situ hybridization was performed using image analysis software Analyst (provided by A. Jovanovic, Mathematical Faculty, University of Belgrade, www.gisss.com).
2.4. Statistical analysis Statistical analysis was performed using ANOVA and Tukey t-test for a posteriori pairwise comparison of means using SAS software (SAS Institute, 1990. SAS User guide: statistics. Vers. 6.0. SAS Institute, Cary, NC). Significant differences were considered to be these with PB 0.05.
3. Results In this study we have investigated the expression of growth-associated protein (GAP-43) in rats treated with amphetamine, PCP or haloperidol during the treatment (at 2, 5 and 7 days) and recovery period (7, 14 and 21 days after 7 days of treatment). Northern blot analysis revealed changes in GAP-43 mRNA expression on the level of total brain RNA (Fig. 1). Chronic treatment with amphetamine (5 mg/ kg/day) had diverse effect on GAP-43 mRNA expression. In the first days of treatment, amphetamine induced increase of GAP-43 mRNA expression for 19% after 5 days of treatment (Fig. 1D). However, after 7 days of treatment expression of GAP-43 mRNA was for 14% bellow control, and this trend of decreased expression of GAP-43 gene was continued during the period of recovery, declining from 22% after 14 days, to 27% bellow the control level after 21 days of recovery (Fig. 1D). Contrary to the effects of amphetamine, chronic treatment with phencyclidine (5 mg/kg/day) resulted in gradual increase of GAP-43 mRNA expression that was detectable from the beginning of treatment (41–75% over control after 2– 7 days of treatment) and it was up to 58– 98% above control during the recovery period (Fig. 1E). Treatment with haloperidol did not resulted in significant changes of GAP-43 mRNA expression, except for the small decrease (9%) of expression after 2 days of treatment (Fig. 1F). In order to examine regional changes of GAP-43 mRNA expression induced by chronic treatment with
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amphetamine, PCP or haloperidol, we have employed in situ hybridization. Using riboprobe specific for GAP43 mRNA we have analyzed changes of GAP-43 expression after chronic treatment with amphetamine, PCP or haloperidol in three brain regions: CA3 hippocampal region, the place of intense GAP-43 expression in adult brains; caudate-putamen, as a region that was primarily affected by the action of drugs used in the study; and fronto-parietal cortex, as another place of high GAP-43 mRNA expression, that is anatomically closely related and intensively interconnected with caudate-putamen. Representative photomicrographs of selected region (CA3) hybridized with GAP-43 riboprobe were shown on Fig. 2. Signals obtained by in situ hybridization were quantified using image analysis system Analyst and shown on Fig. 3. Hippocampal CA3 region is the site of increased expression of GAP-43 in adult rat brain (Fig. 2 A). However, after chronic treatment with amphetamine the expression of GAP-43 mRNA in this region was considerably changed. In situ hybridization confirmed the initial increase of GAP-43 mRNA as it was shown by Northern blotting expression after amphetamine treatment. In the CA3 region under action of this drug expression of GAP-43 was higher for approximately 40% after 2, 5 and 7 days of treatment (Figs. 2C and 3A). On the contrary, during the recovery period expression of GAP-43 mRNA was decreased for 11–28% bellow the control (Figs. 2D and 3A). In the same region, treatment with PCP resulted in increase of GAP-43 mRNA expression (Fig. 2E, F) for 36% after 2, 25% after 5, 37% after 7 days of treatment and for 88–99% after first and second week of recovery period (Fig. 3B). Haloperidol induced only 11% decrease of GAP-43 mRNA expression in CA3 region after 2 day of treatment, but during following time points of treatment and recovery period this expression was unchanged (Fig. 3C). In the cortical area (fronto-parietal cortex) effects of amphetamine treatment were similar to those observed in CA3 hippocampal region. After 2 days of treatment GAP-43 mRNA expression was increased (44% over control) and this increase was reduced to 35% at 5, and 21% over the control at 7 days of treatment (Fig. 3A). The trend of decline of GAP-43 mRNA expression was detected during recovery period as well. Thus, after 7 days of recovery expression of GAP-43 was reduced for 28%, after 14 days for 25% and after 21 days of recovery for 28% (Fig. 3A). The effects of PCP on GAP-43 mRNA expression in the cortical region were comparable to those detected in CA3 hippocampal region. Hence, in the period after 2–5 days of treatment GAP-43 mRNA expression was higher for 22%, and after 7 days for 34%, compared with control. Also, in the time of recovery, expression of GAP-43 mRNA showed tendency of persistent increase, from 64% after
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first week, reaching maximal increase of 95% over the control after 2 weeks of recovery (Fig. 3B). In the same region, haloperidol had effects on the expression of GAP-43 mRNA that were detectable after 5 and 7 days of treatment, and were observable as a slight increase of 24 and 16%, respectively (Fig. 3C). Caudate-putamen region is a major site of action of many psychostimulant drugs. Regardless of the fact that it is not a place of intense GAP-43 mRNA expression in adult brain, we have shown significant changes of this expression after chronic treatment with amphetamine (Fig. 3A). From the beginning of the treatment, expression of GAP-43 in this region was
decreased, precisely for 12–25% bellow control during the treatment, and furthermore it was decreased during the entire period of recovery (26, 24 and 13% after 7, 14 and 21 days of recovery, respectively). Contrary, the treatment with PCP revealed increased expression of GAP-43 in caudate-putamen region, for 37% after 2, 42% after 5 and 48% after 7 days of treatment (Fig. 3B). During the recovery period GAP-43 mRNA expression was consistently increased, from 52% after first, to 65% over the control level after second week of recovery. The effects of haloperidol on GAP-43 mRNA expression in caudate-putamen region were non-detectable (Fig. 3C).
Fig. 1. Northern blot analysis of GAP-43 mRNA expression after 2, 5 and 7 days of treatment and 7, 14 and 21 day of recovery after treatment with amphetamine (A), phencyclidine (B) and haloperidol (C). A%, B%, C%: ethidium bromide staining showing quality of RNA samples loading. D, E, F: Histograms representing GAP-43 mRNA/ethidium bromide ratio, after three repeated experiments, expressed as a percent of control (Tukey t-test, *P B0.05).
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Fig. 2. Representative photomicrographs of in situ hybridization with GAP-43 riboprobe in CA3 hippocampal region of rats treated with saline (A), amphetamine for 7 days (C) and after 2 weeks of recovery (D), and phencyclidine after 7 days of treatment (E) and 2 weeks of recovery (F). Hybridization with sense riboprobe resulted in absence of hybridization signal (B). Scale bar = 15 mm.
4. Discussion Both amphetamine and phencyclidine are used in developing animal models of psychosis. These different psychotomimetic drugs can mimic different symptoms of psychosis-like syndromes possibly acting through different neurotransmitter systems. Knowing that several recent publications reported alterations in GAP-43 gene and protein expression in postmortem human samples from patients suffered from different types of psychosis (mostly schizophrenia), in this study we wanted to compare possible differential regulation of GAP-43 gene expression in rat brains after treatment with either amphetamine or phencyclidine. Chronic
treatment with haloperidol was used to investigate changes on the level of GAP-43 gene expression in rats treated in a similar regiment like psychotic-suffering patients. Using this approach we wanted to compare changes induced by psychotomimetic drugs with morphological changes induced by most commonly used anti-psychotic treatment. Quantitative in situ hybridization was used in order to examine levels of GAP-43 mRNA in selective brain regions after chronic treatment with amphetamine or phencyclidine. The effects of chronic application of these psychotomimetic drugs on the expression of GAP-43 was different: amphetamine induced bi-phasic response of GAP-43 mRNA expression, with initial
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increase (up to 5 days of treatments) and subsequent decrease, lasting up to 3 weeks after treatment. Conversely, phencyclidine induced continual upregulation of GAP-43 mRNA expression. Chronic treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. Previous studies on postmortem samples of schizophrenic patients showed increased levels of GAP43 protein in selected brain areas and it was pointed out at GAP-43 as a potential marker for morphological changes specific for schizophrenia and resembling psychiatric syndromes (Sower et al., 1995; Perrone-Bizzozero et al., 1996). It was also shown for other neuronal proteins that their alterations participate in the morphological substrates of schizophrenia. Thompson et al. (1998) reported altered levels of another neuronal protein, synaptosomal associated protein
SNAP-25, in postmortem samples from schizophrenic patients. Interestingly, Karson et al. (1999) reported decreased levels of SNAP-25 and synaptophysine in prefrontal cortex of schizophrenic patients, but the levels of their mRNAs were not decreased. Following this line, it was of interest for us to compare the effects of two different psychotomimetic drugs, amphetamine and phencyclidine, that are widely abused in drug-addicts, on the expression of major neurite-outgrowth protein, GAP-43. To confirm specificity of detected changes, we have used another drug, haloperidol that is known to have opposite effects to those obtained by use of psychostimulants. Results obtained by Northern blot analysis showed different response of GAP-43 mRNA expression on amphetamine treatment (Fig. 1A, D). Using in situ hybridization we have shown that during 7 days of
Fig. 3. Histograms representing quantitative in situ hybridization after chronic treatment with amphetamine (A), phencyclidine (B) and haloperidol (C). Expression of GAP-43 is represented as a number of silver grains per cell in the CA3 region (CA3; white bars), fronto-parietal cortex (Cx; grey bars) and caudate-putamen (CPu; black bars). Tukey t-test, *PB 0.05.
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treatment amphetamine induced increase of GAP-43 mRNA expression in both CA3 and cortical regions, while only in the caudate-putamen region it was significantly decreased (Fig. 3A). The ability of repeated amphetamine treatment to alter patterns of synaptic connectivity in prefrontal cortex, increasing the lenght of dendrites of layer III pyramidal neurons, was previously shown by Robinson and Kolb (1997, 1999). Thus, the observed increased expression of GAP-43 mRNA in the cortical and CA3 regions during the repeated amphetamine treatment could be related to the similar morphological changes. However, during the recovery period, expression of GAP-43 was diminished after amphetamine treatment in all examined regions. Considering that caudate-putamen is the main location where amphetamine excerts its dopamine-stimulatory action in the brain, it can be suggested that this region was the first to show downregulation of GAP-43. This downregulation could be explained as a consequence of reorganization of neural connections in order to avoid and survive strong, amphetamine-induced, dopamine stimulation. On the other hand, downregulation of GAP-43 mRNA observed in recovery could be due to the fact that amphetamine was shown to be neurotoxic in higher doses or after longer treatment, as in our experiment, inducing axonolisis of dopaminergic projections (Gibb et al., 1997). Chronic exposure to D-amphetamine results in significant neurotoxicity in rat neocortical neurons in vitro and apoptotic pathways are involved in amphetamine-induced neurotoxicity (Stumm et al., 1999). Likewise, repeated treatment with amphetamine leads to alterations in phosphorylation/ dephosphorylation activities that can be detected in the incubated synaptosomes (Iwata et al., 1997). However, downregulation of GAP-43 mRNA expression detected after amphetamine treatment in our experiments could be also a result of neuronal loss. Phencyclidine is a psychotomimetic drug that acts as an NMDA antagonist, thus inhibiting glutamatergic transmission (Ellison, 1995; Steinpreis, 1996). Several studies indicated protective effects of PCP in rat brain ischemia (Barone et al., 1994), cortical lesion (Pohl et al., 1999) and other forms of brain injury (French, 1986). Several decades of research attempting to explain schizophrenia in terms of the dopamine hyperactivity hypothesis have produced disappointing results. A new hypothesis focusing on hypofunction of the NMDA glutamate transmitter system is emerging as a potentially more promising concept. Thus, in this study, we have analyzed changes of GAP-43 mRNA expression after chronic PCP treatment, with the idea to induce hypofunction of glutamatergic system in rat brain. It is known that repeated PCP administration to healthy subjects can induce psychotic symptoms. NMDA antagonists induce positive, negative, and cognitive schizophrenic-like symptoms in healthy volunteers and
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precipitate thought disorder and delusions in schizophrenia patients (Duncan et al., 1999). Thus, PCP was used in an attempt to develop an animal model of psychosis (Javitt and Zukin, 1991). It was also shown that PCP has properties of both indirect and direct dopaminergic agonist, increasing dopamine release and blocking the excitatory glutamatergic input on dopaminergic cells in striatal region (French, 1986). In this study we have shown that chronic application of PCP induced strong and continual upregulation of GAP-43 mRNA expression, detectable even after 3 weeks of recovery period. It can be suggested that aside from its NMDA antagonistic and dopamine-agonistic actions, PCP could possess some additional properties, resulting in increased sprouting and neurite outgrowth. Moreover, we have shown that given in doses of 5 mg/kg/day during 7 days, haloperidol does not significantly affect basic morphology that can be followed by the expression of GAP-43 gene, except for the small increase of GAP-43 mRNA expression observed in the cortical region, and small decrease in the CPu region. The significance of that finding is dual: first, it was shown that psychotomimetic drugs amphetamine and phencyclidine has opposite effects on GAP-43 mRNA expression, while haloperidol, the drug with different mode of action, did not induced long-lasting changes in GAP-43 mRNA expression. Second, using in this regiment, haloperidol has no toxic effects in rats. Given the relationship of GAP-43 expression to the establishment and remodeling of synaptic connections, here presented results support the hypothesis that chronic treatment with psychostimulant drugs, suggested to be used as a tool for development of animal model of schizophrenia, is associated with perturbations of synaptic connections in limbic and neocortical areas of the rat brain. Furthermore, our finding that GAP-43 levels were not affected in brains of saline and haloperidol treated rats suggest that these alterations are specific for the effect of amphetamine and phencyclidine.
Acknowledgements This work was supported by grant no. 03E16 from the Ministry of Science and Technology, Republic of Serbia, Yugoslavia. We are grateful to Dr E.E. Baetge for providing GAP-43 cDNA clone.
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Differential effects of amphetamine and phencyclidine on the expression of growth-associated protein GAP-43 S. Vukosavic a, S. Ruzdijic a, R. Veskov a, Lj. Rakic b, S. Kanazir a,* a
Institute for Biological Research, 29 No6embar 142, Belgrade, Yugosla6ia b ICN Pharmaceuticals, Costa Mesa, CA, USA Received 10 October 2000; accepted 20 February 2001
Abstract The purpose of the present study was to test changes in the expression of growth-associated protein (GAP-43) after chronic treatment with two different psychotomimetic drugs: amphetamine and phencyclidine. Rats were treated chronically for 7 days (twice daily) with 5 mg/kg of amphetamine and phencyclidine and sacrificed after 2, 5 or 7 days of treatment, and following 7, 14 or 21 days of recovery after full treatment (7 days). Separate groups of rats were treated on the same regiment with haloperidol, and control group was treated with vehicle. To determine the effects of different psychotomimetic drugs on the expression of GAP-43 we have used Northern blotting and quantitative in situ hybridization. Treatment with amphetamine induced decrease of GAP-43 mRNA expression, that was detected also during recovery period, up to 14 days after the last day of 7 days treatments. On the contrary, PCP induced increase of GAP-43 mRNA expression, that was detectable from the first days of treatment until 21 days after the last day of treatment. Treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. It can be suggested that GAP-43 upregulation upon phencyclidine treatment occurs as a result of functional activation of pathways able to participate in remodeling, while amphetamine showed neurotoxic effect, decreasing expression of GAP-43 mRNA. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: GAP-43; Amphetamine; Phencyclidine; Haloperidol; In situ hybridization
1. Introduction The appearance of morphological changes related to the pathology of psychosis-like syndromes has been well described. However, the exact mechanisms causing these abnormalities remain unclear. In a search for molecular correlates of the alteration in brain structure and function observed in psychosis-like syndromes growth-associated protein GAP-43 (neuromodulin, B50, F1) (Benowitz and Routtenberg, 1987; Gispen et al., 1992) has been proposed as a useful marker. Several studies on post-mortem brain samples of schizophrenic patients clearly showed increased expression and increased phosphorylation of GAP-43 in selective regions such as associative cortices (Sower et al., 1995; PerroneBizzozero et al., 1996). GAP-43 protein is concentrated * Corresponding author. Tel.: + 381-11-764422; fax: +381-11761433. E-mail address: [email protected] (S. Kanazir).
in growth cones and axons (Skene, 1989) and it is substrate for protein kinase C. It also affects several intracellular messenger systems via binding to molecules such as calmodulin and GTP-binding protein Go. This neuronal membrane phosphoprotein is implicated in the initial establishment and reorganization of synaptic connections and it is found to be highly expressed in developing neurons during axonal growth and synaptogenesis (Kanazir et al., 1996). In the adult brain, selective brain regions such as limbic system and neocortex maintain high levels of GAP-43 throughout life (Benowitz et al., 1989; Kruger et al., 1993). Regionspecific upregulation of GAP-43 and its restricted localization to higher neocortical and limbic areas suggests that this protein marks neuronal circuits involved in the processing and storage of highest cortical functions such as memory (Neve et al., 1988; Perrone-Bizzozero et al., 1996). Since these processes are known to be altered in psychosis-related syndromes, it was proposed that GAP-43 levels might be altered in this group of
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disorders and several reports showed changes of GAP43 mRNA and protein expression in human postmortem schizophrenic brains (Perrone-Bizzozero et al., 1996; Blennow et al., 1999). Similarly, in another neurodegenerative disease, amyotrophic lateral sclerosis, surviving anterior horn motor neurons selectively upregulate GAP-43, implying the occurrence of compensatory sprouting (Parhad et al., 1992). The widespread aberrant neuritic growth accompanied by impaired synaptic plasticity in schizophrenia (Sower et al., 1995) and Alzheimer’s disease (Colleman et al., 1992; Callahan et al., 1994) suggests that severely compromised GAP-43 gene expression may contribute to the cascade of neurodegeneration. In an attempt to better understand and investigate psychiatric diseases two major animal models of psychosis-like syndromes were developed, based on the chronic, repetitive treatment of animals with two different psychotomimetic drugs such as amphetamine (Lillrank et al., 1991) or phencyclidine (Duncan et al., 1999; Olney et al., 1999). Knowing the importance of GAP43 gene expression in processes underlying brain plasticity and synaptic reorganization, and its possible role as a morphological correlate of psychosis-like syndromes, in this paper we have investigated changes in GAP-43 mRNA expression in selective regions of rat brain upon chronic treatment with these two different psychotomimetic drugs: amphetamine, as a direct dopaminergic agonist and phencyclidine, as noncompetitive NMDA antagonist. We have also involved chronic treatment with haloperidol, widely used antipsychotic drug, in an attempt to compare the effects of haloperidol applied on healthy brain with psychotomimetictreated brains. 2. Materials and methods
2.1. Animals and treatment Adult male Mill– Hill hooded rats were separated in experimental groups (three to five per group) and intraperitonealy injected for seven days in 12-h intervals with 5 mg/kg D-amphetamine (Sigma), 5 mg/kg phencyclidine (PCP, ICN-Galenika) or 5 mg/kg haloperidol (Sigma). The doses for each drug were chosen according to the literature: for amphetamine (Robinson and Becker, 1986; Lillrank et al., 1991; Axt et al., 1994), for phencyclidine (Ellison, 1995; Steinpreis, 1996) and for haloperidol (Burt et al., 1977). Randomly selected rats were allowed to recover up to 3 weeks after 7 days treatment. At 2, 5 and 7 days of treatment (24 h after the last injection) and after 7, 14 and 21 days of recovery period, randomly selected rats were killed by decapitation, brains were removed and immediately frozen in isopentane, cooled on dry ice, and stored at −80°C.
2.2. Northern blot analysis Total brain RNA was isolated by the guanidine isothiocyanate (GTC)/ cesium chloride (CsCl) centrifugation method (Sambrook et al., 1989). Rat brains were homogenized in 4 M GTC, 4% N-sarcosyle, 50 mM Na –acetate pH 5.5 and 1% 2-mercaptoethanol. Homogenates were mixed with 0.5 g/ml CsCl and loaded on a 3-ml layer of 5.7 M CsCl. RNA was pelleted by centrifugation at 160 000× g for 20 h at room temperature. Pellets were resuspended in 10 mM Tris HCl pH 7.5, 1 mM EDTA, 0.5% SDS, and RNA was extracted with butanol:chloroform (1:4) and precipitated in cold ethanol. RNA (10mg) was then denatured and separated by electrophoresis on 1.5% agarose–formaldehyde gel, stained with ethidium bromide for control of loading and transferred to Hybond N+ membrane (Amersham) using capillary transfer overnight in 20×SSC buffer (Sambrook et al., 1989). RNA was UV crosslinked to the membrane (FUNA-UV-Linker, Spectroline). The GAP-43 probe for Northern hybridization was labeled by random priming using random primed DNA labeling kit (Boehringer Mannheim) according to the manufacturer’s instructions. Plazmid containing rat cDNA clone for GAP-43 (kindly provided by Dr E.E. Baetge) was linearized with EcoRI and cDNA insert was isolated with HindIII and subsequently labeled with 50 mCi of a-32P dCTP (ICN) in the presence of DNA polymerase-Klenow fragment. Labeled probe was purified on Nick Spin column (Pharmacia) and added to the hybridization buffer (1× 106 cpm/ml). Prehybridization and hybridization were performed in rapid hybridization buffer (Amersham) at 65°C, for 15 min and 4 h, respectively. After hybridization membranes were washed in 2× SSC, 0.1% SDS and 0.2× SC, 0.1% SDS, according to manufacturers recommendations, and exposed to phosphorimager screen (Molecular Dynamics). Quantification of signals was performed using image analysis software Image Quant (Molecular Dynamics).
2.3. In situ hybridization histochemistry In situ hybridization was performed as previously described (Kanazir et al., 1997). In brief, rat brain coronal sections (12 mm) were fixed in 4% paraformaldehyde, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, and delipidated in ethanol series (70, 96, 100% 1 min each) and chloroform (5 min). Sections were then incubated overnight at 55°C in 100 ml of hybridization buffer (50% formamide, 10% dextran sulfate, 5× Denhardt’s solution, 250 mg/ ml salmon DNA, 150 mg/ml tRNA, 2 × SSC and 20 mM 2-mercaptoethanol) containing radiolabeled antisense or sense riboprobe (10 000 cpm/ml) for GAP-43. A
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GAP-43 riboprobes were generated from a pBluescript KS vector (Stratagene) that contained a 1087 bp insert of rat cDNA clone (kindly provided by Dr E.E. Baetge). Following hybridization sections were washed in 2 ×SSC, treated with RNase A and subsequently washed for 4×15 min in 2×SSC, and overnight in 0.1 ×SSC. Sections were dried and exposed to Kodak NBT2 emulsion for 3 weeks, developed, counterstained with cresyl violet and examined under light microscopy. Quantification of in situ hybridization was performed using image analysis software Analyst (provided by A. Jovanovic, Mathematical Faculty, University of Belgrade, www.gisss.com).
2.4. Statistical analysis Statistical analysis was performed using ANOVA and Tukey t-test for a posteriori pairwise comparison of means using SAS software (SAS Institute, 1990. SAS User guide: statistics. Vers. 6.0. SAS Institute, Cary, NC). Significant differences were considered to be these with PB 0.05.
3. Results In this study we have investigated the expression of growth-associated protein (GAP-43) in rats treated with amphetamine, PCP or haloperidol during the treatment (at 2, 5 and 7 days) and recovery period (7, 14 and 21 days after 7 days of treatment). Northern blot analysis revealed changes in GAP-43 mRNA expression on the level of total brain RNA (Fig. 1). Chronic treatment with amphetamine (5 mg/ kg/day) had diverse effect on GAP-43 mRNA expression. In the first days of treatment, amphetamine induced increase of GAP-43 mRNA expression for 19% after 5 days of treatment (Fig. 1D). However, after 7 days of treatment expression of GAP-43 mRNA was for 14% bellow control, and this trend of decreased expression of GAP-43 gene was continued during the period of recovery, declining from 22% after 14 days, to 27% bellow the control level after 21 days of recovery (Fig. 1D). Contrary to the effects of amphetamine, chronic treatment with phencyclidine (5 mg/kg/day) resulted in gradual increase of GAP-43 mRNA expression that was detectable from the beginning of treatment (41–75% over control after 2– 7 days of treatment) and it was up to 58– 98% above control during the recovery period (Fig. 1E). Treatment with haloperidol did not resulted in significant changes of GAP-43 mRNA expression, except for the small decrease (9%) of expression after 2 days of treatment (Fig. 1F). In order to examine regional changes of GAP-43 mRNA expression induced by chronic treatment with
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amphetamine, PCP or haloperidol, we have employed in situ hybridization. Using riboprobe specific for GAP43 mRNA we have analyzed changes of GAP-43 expression after chronic treatment with amphetamine, PCP or haloperidol in three brain regions: CA3 hippocampal region, the place of intense GAP-43 expression in adult brains; caudate-putamen, as a region that was primarily affected by the action of drugs used in the study; and fronto-parietal cortex, as another place of high GAP-43 mRNA expression, that is anatomically closely related and intensively interconnected with caudate-putamen. Representative photomicrographs of selected region (CA3) hybridized with GAP-43 riboprobe were shown on Fig. 2. Signals obtained by in situ hybridization were quantified using image analysis system Analyst and shown on Fig. 3. Hippocampal CA3 region is the site of increased expression of GAP-43 in adult rat brain (Fig. 2 A). However, after chronic treatment with amphetamine the expression of GAP-43 mRNA in this region was considerably changed. In situ hybridization confirmed the initial increase of GAP-43 mRNA as it was shown by Northern blotting expression after amphetamine treatment. In the CA3 region under action of this drug expression of GAP-43 was higher for approximately 40% after 2, 5 and 7 days of treatment (Figs. 2C and 3A). On the contrary, during the recovery period expression of GAP-43 mRNA was decreased for 11–28% bellow the control (Figs. 2D and 3A). In the same region, treatment with PCP resulted in increase of GAP-43 mRNA expression (Fig. 2E, F) for 36% after 2, 25% after 5, 37% after 7 days of treatment and for 88–99% after first and second week of recovery period (Fig. 3B). Haloperidol induced only 11% decrease of GAP-43 mRNA expression in CA3 region after 2 day of treatment, but during following time points of treatment and recovery period this expression was unchanged (Fig. 3C). In the cortical area (fronto-parietal cortex) effects of amphetamine treatment were similar to those observed in CA3 hippocampal region. After 2 days of treatment GAP-43 mRNA expression was increased (44% over control) and this increase was reduced to 35% at 5, and 21% over the control at 7 days of treatment (Fig. 3A). The trend of decline of GAP-43 mRNA expression was detected during recovery period as well. Thus, after 7 days of recovery expression of GAP-43 was reduced for 28%, after 14 days for 25% and after 21 days of recovery for 28% (Fig. 3A). The effects of PCP on GAP-43 mRNA expression in the cortical region were comparable to those detected in CA3 hippocampal region. Hence, in the period after 2–5 days of treatment GAP-43 mRNA expression was higher for 22%, and after 7 days for 34%, compared with control. Also, in the time of recovery, expression of GAP-43 mRNA showed tendency of persistent increase, from 64% after
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first week, reaching maximal increase of 95% over the control after 2 weeks of recovery (Fig. 3B). In the same region, haloperidol had effects on the expression of GAP-43 mRNA that were detectable after 5 and 7 days of treatment, and were observable as a slight increase of 24 and 16%, respectively (Fig. 3C). Caudate-putamen region is a major site of action of many psychostimulant drugs. Regardless of the fact that it is not a place of intense GAP-43 mRNA expression in adult brain, we have shown significant changes of this expression after chronic treatment with amphetamine (Fig. 3A). From the beginning of the treatment, expression of GAP-43 in this region was
decreased, precisely for 12–25% bellow control during the treatment, and furthermore it was decreased during the entire period of recovery (26, 24 and 13% after 7, 14 and 21 days of recovery, respectively). Contrary, the treatment with PCP revealed increased expression of GAP-43 in caudate-putamen region, for 37% after 2, 42% after 5 and 48% after 7 days of treatment (Fig. 3B). During the recovery period GAP-43 mRNA expression was consistently increased, from 52% after first, to 65% over the control level after second week of recovery. The effects of haloperidol on GAP-43 mRNA expression in caudate-putamen region were non-detectable (Fig. 3C).
Fig. 1. Northern blot analysis of GAP-43 mRNA expression after 2, 5 and 7 days of treatment and 7, 14 and 21 day of recovery after treatment with amphetamine (A), phencyclidine (B) and haloperidol (C). A%, B%, C%: ethidium bromide staining showing quality of RNA samples loading. D, E, F: Histograms representing GAP-43 mRNA/ethidium bromide ratio, after three repeated experiments, expressed as a percent of control (Tukey t-test, *P B0.05).
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Fig. 2. Representative photomicrographs of in situ hybridization with GAP-43 riboprobe in CA3 hippocampal region of rats treated with saline (A), amphetamine for 7 days (C) and after 2 weeks of recovery (D), and phencyclidine after 7 days of treatment (E) and 2 weeks of recovery (F). Hybridization with sense riboprobe resulted in absence of hybridization signal (B). Scale bar = 15 mm.
4. Discussion Both amphetamine and phencyclidine are used in developing animal models of psychosis. These different psychotomimetic drugs can mimic different symptoms of psychosis-like syndromes possibly acting through different neurotransmitter systems. Knowing that several recent publications reported alterations in GAP-43 gene and protein expression in postmortem human samples from patients suffered from different types of psychosis (mostly schizophrenia), in this study we wanted to compare possible differential regulation of GAP-43 gene expression in rat brains after treatment with either amphetamine or phencyclidine. Chronic
treatment with haloperidol was used to investigate changes on the level of GAP-43 gene expression in rats treated in a similar regiment like psychotic-suffering patients. Using this approach we wanted to compare changes induced by psychotomimetic drugs with morphological changes induced by most commonly used anti-psychotic treatment. Quantitative in situ hybridization was used in order to examine levels of GAP-43 mRNA in selective brain regions after chronic treatment with amphetamine or phencyclidine. The effects of chronic application of these psychotomimetic drugs on the expression of GAP-43 was different: amphetamine induced bi-phasic response of GAP-43 mRNA expression, with initial
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increase (up to 5 days of treatments) and subsequent decrease, lasting up to 3 weeks after treatment. Conversely, phencyclidine induced continual upregulation of GAP-43 mRNA expression. Chronic treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. Previous studies on postmortem samples of schizophrenic patients showed increased levels of GAP43 protein in selected brain areas and it was pointed out at GAP-43 as a potential marker for morphological changes specific for schizophrenia and resembling psychiatric syndromes (Sower et al., 1995; Perrone-Bizzozero et al., 1996). It was also shown for other neuronal proteins that their alterations participate in the morphological substrates of schizophrenia. Thompson et al. (1998) reported altered levels of another neuronal protein, synaptosomal associated protein
SNAP-25, in postmortem samples from schizophrenic patients. Interestingly, Karson et al. (1999) reported decreased levels of SNAP-25 and synaptophysine in prefrontal cortex of schizophrenic patients, but the levels of their mRNAs were not decreased. Following this line, it was of interest for us to compare the effects of two different psychotomimetic drugs, amphetamine and phencyclidine, that are widely abused in drug-addicts, on the expression of major neurite-outgrowth protein, GAP-43. To confirm specificity of detected changes, we have used another drug, haloperidol that is known to have opposite effects to those obtained by use of psychostimulants. Results obtained by Northern blot analysis showed different response of GAP-43 mRNA expression on amphetamine treatment (Fig. 1A, D). Using in situ hybridization we have shown that during 7 days of
Fig. 3. Histograms representing quantitative in situ hybridization after chronic treatment with amphetamine (A), phencyclidine (B) and haloperidol (C). Expression of GAP-43 is represented as a number of silver grains per cell in the CA3 region (CA3; white bars), fronto-parietal cortex (Cx; grey bars) and caudate-putamen (CPu; black bars). Tukey t-test, *PB 0.05.
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treatment amphetamine induced increase of GAP-43 mRNA expression in both CA3 and cortical regions, while only in the caudate-putamen region it was significantly decreased (Fig. 3A). The ability of repeated amphetamine treatment to alter patterns of synaptic connectivity in prefrontal cortex, increasing the lenght of dendrites of layer III pyramidal neurons, was previously shown by Robinson and Kolb (1997, 1999). Thus, the observed increased expression of GAP-43 mRNA in the cortical and CA3 regions during the repeated amphetamine treatment could be related to the similar morphological changes. However, during the recovery period, expression of GAP-43 was diminished after amphetamine treatment in all examined regions. Considering that caudate-putamen is the main location where amphetamine excerts its dopamine-stimulatory action in the brain, it can be suggested that this region was the first to show downregulation of GAP-43. This downregulation could be explained as a consequence of reorganization of neural connections in order to avoid and survive strong, amphetamine-induced, dopamine stimulation. On the other hand, downregulation of GAP-43 mRNA observed in recovery could be due to the fact that amphetamine was shown to be neurotoxic in higher doses or after longer treatment, as in our experiment, inducing axonolisis of dopaminergic projections (Gibb et al., 1997). Chronic exposure to D-amphetamine results in significant neurotoxicity in rat neocortical neurons in vitro and apoptotic pathways are involved in amphetamine-induced neurotoxicity (Stumm et al., 1999). Likewise, repeated treatment with amphetamine leads to alterations in phosphorylation/ dephosphorylation activities that can be detected in the incubated synaptosomes (Iwata et al., 1997). However, downregulation of GAP-43 mRNA expression detected after amphetamine treatment in our experiments could be also a result of neuronal loss. Phencyclidine is a psychotomimetic drug that acts as an NMDA antagonist, thus inhibiting glutamatergic transmission (Ellison, 1995; Steinpreis, 1996). Several studies indicated protective effects of PCP in rat brain ischemia (Barone et al., 1994), cortical lesion (Pohl et al., 1999) and other forms of brain injury (French, 1986). Several decades of research attempting to explain schizophrenia in terms of the dopamine hyperactivity hypothesis have produced disappointing results. A new hypothesis focusing on hypofunction of the NMDA glutamate transmitter system is emerging as a potentially more promising concept. Thus, in this study, we have analyzed changes of GAP-43 mRNA expression after chronic PCP treatment, with the idea to induce hypofunction of glutamatergic system in rat brain. It is known that repeated PCP administration to healthy subjects can induce psychotic symptoms. NMDA antagonists induce positive, negative, and cognitive schizophrenic-like symptoms in healthy volunteers and
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precipitate thought disorder and delusions in schizophrenia patients (Duncan et al., 1999). Thus, PCP was used in an attempt to develop an animal model of psychosis (Javitt and Zukin, 1991). It was also shown that PCP has properties of both indirect and direct dopaminergic agonist, increasing dopamine release and blocking the excitatory glutamatergic input on dopaminergic cells in striatal region (French, 1986). In this study we have shown that chronic application of PCP induced strong and continual upregulation of GAP-43 mRNA expression, detectable even after 3 weeks of recovery period. It can be suggested that aside from its NMDA antagonistic and dopamine-agonistic actions, PCP could possess some additional properties, resulting in increased sprouting and neurite outgrowth. Moreover, we have shown that given in doses of 5 mg/kg/day during 7 days, haloperidol does not significantly affect basic morphology that can be followed by the expression of GAP-43 gene, except for the small increase of GAP-43 mRNA expression observed in the cortical region, and small decrease in the CPu region. The significance of that finding is dual: first, it was shown that psychotomimetic drugs amphetamine and phencyclidine has opposite effects on GAP-43 mRNA expression, while haloperidol, the drug with different mode of action, did not induced long-lasting changes in GAP-43 mRNA expression. Second, using in this regiment, haloperidol has no toxic effects in rats. Given the relationship of GAP-43 expression to the establishment and remodeling of synaptic connections, here presented results support the hypothesis that chronic treatment with psychostimulant drugs, suggested to be used as a tool for development of animal model of schizophrenia, is associated with perturbations of synaptic connections in limbic and neocortical areas of the rat brain. Furthermore, our finding that GAP-43 levels were not affected in brains of saline and haloperidol treated rats suggest that these alterations are specific for the effect of amphetamine and phencyclidine.
Acknowledgements This work was supported by grant no. 03E16 from the Ministry of Science and Technology, Republic of Serbia, Yugoslavia. We are grateful to Dr E.E. Baetge for providing GAP-43 cDNA clone.
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