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Alterations of calmodulin and its mRNA in rat brain after acute and chronic administration of methamphetamine
Brain Research 765 Ž1997. 247–258
Research report
Alterations of calmodulin and its mRNA in rat brain after acute and chronic administration of methamphetamine Yoshio Shimizu ) , Kazufumi Akiyama, Masafumi Kodama, Takeshi Ishihara, Takashi Hamamura, Shigetoshi Kuroda Department of Neuropsychiatry, Okayama UniÕersity Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan Accepted 1 April 1997
Abstract The effect of acute and chronic administration of methamphetamine ŽMETH. on the levels of calmodulin ŽCaM. and its mRNAs has been investigated in rat brain using antisense oligonucleotides to three distinct rat CaM genes ŽCaM I, CaM II, CaM III.. CaM I mRNA was reduced in the striatum and nucleus accumbens within 2 h of acute administration of 4 mgrkg METH, but returned to the control level by 6 h. The CaM content in both the cytosolic and membrane fractions of the striatum was reduced 0.5, 2, and 6 h after acute administration of METH. In the chronic experiments, rats were treated with either 4 mgrkg METH or saline once daily for 14 days. This was followed by a withdrawal period of 28 days, and thereafter, the animals were challenged with either METH Ž4 mgrkg, i.p.. or saline. All the animals were decapitated 6 h after this injection. There were four treatment groups: METH-METH ŽMM.; METH-saline ŽMS.; saline-METH ŽSM.; and saline-saline ŽSS.. There was a significant decrease in the mRNA for CaM I and CaM II in the striatum, and CaM II and CaM III in the nucleus accumbens in the MS group and the MS and MM groups, respectively, when compared to the SS group. The CaM content in the striatal membrane fraction decreased in both the SM and MS groups but not in the MM group. In contrast, the CaM content in the membrane fraction of the mesolimbic area showed a significant increase in the MM group. The CaM content in the cytosolic fraction of these brain areas decreased in both the SM and MM groups. The total CaM decreased significantly in the SM and MM groups of the striatum, but increased significantly in the MM group of the mesolimbic area. The mRNA for CaM I and CaM III decreased significantly in the MM group, and in the SM and MM groups, in the substantia nigra pars compacta ŽSNC. and ventral tegmental area ŽVTA., respectively. The CaM content in both the cytosolic and membrane fractions and total CaM content of the SNrVTA decreased significantly in the SM, MS and MM group as compared with the SS group. In the medial prefrontal cortex and hippocampus the significant increase of CaM content in the membrane fraction of the MM group was also found, but neither the CaM content in the cytosol fraction nor total CaM content changed. These results suggest that chronic METH administration leads to a translocation of CaM from the cytosolic to membrane fractions; these may underlie METH-induced behavioral sensitization. q 1997 Elsevier Science B.V. Keywords: Methamphetamine; Behavioral sensitization; Calmodulin; Hybridization, in situ; Radioimmunoassay; Dopamine
1. Introduction It is well documented that chronic abuse with the amphetamine-group of psychostimulants wamphetamine ŽAMPH. and methamphetamine ŽMETH.x can lead to a paranoid-hallucinatory state resembling the positive symptoms of schizophrenia. Once individuals have experienced such a state, they are more vulnerable to relapse after psychological stress and if they reuse METH. This vulnerability persists for a long time, and, indeed, a continual psychotic state has been reported even in the absence of )
Corresponding author. Fax: q81 Ž86. 225-7594.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 4 3 5 - 6
further abuse w16x. In rodents, repeated administration of AMPH and METH leads to progressive augmentation of stereotyped behavior, a phenomenon known as behavioral sensitization w1,14x. Behavioral sensitization is also a sustained phenomenon, since administration of subchronic doses of METH to rats results in an augmented responses to a METH challenge, even after a prolonged period of abstinence w1x. Given that both METH-induced psychosis and behavioral sensitization share the common properties of an augmented and sustained response, an investigation into putative neural substrates which may be important in METH-induced behavioral sensitization may provide an insight into the pathogenesis of METH psychosis.
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Calmodulin ŽCaM. is an ubiquitous calcium-binding protein with a molecular weight of approximately 17 kDa. It is highly expressed in the brain. CaM undergoes a conformational change following binding to Ca2q, which allow it to bind to an inactive molecule of an enzyme, and transforms the latter into the active form. It thus plays an important role as a cofactor regulating a wide variety of calcium-dependent proteins. In this way, it influences a number of critical calcium-dependent intracellular signalling processes, including the synthesis and release of neurotransmitters, assembly and disassembly of microtubules, and the rate of cell proliferation w2x. Several lines of evidence have implicated CaM in the regulation of dopaminergic neurotransmission in the brain. For example, CaM enhances D 1-receptor mediated adenylate cyclase activity w15x, and pharmacological manipulation of dopaminergic activity changes CaM levels in subcellular fractions w4,11x. It has been previously reported that CaM levels increase significantly in both the striatal membrane and cytosolic fractions, and in the limbic membrane fractions of rats which were chronically administered AMPH and withdrawn for 4 weeks w4x. Additionally, after a period of AMPH withdrawal for 4 weeks, even a low-dose AMPH challenge elicited the translocation of CaM from the membrane to the cytosolic compartment in the striatum and limbic forebrain w4x. The alteration in CaM levels in the different subcellular compartments after chronic AMPH administration requires further study, particularly in relation to behavioral sensitization, and to compare the CaM protein and mRNA levels. However, to the best of our knowledge, there has so far been no study which has investigated the effect of chronic administration of AMPHrMETH on CaM mRNA. In the present study, we have investigated the levels of CaM and its mRNA in the major dopamine-innervated brain areas Žthe dorsolateral striatum and nucleus accumbens., the areas containing dopamine neurons Žthe substantia nigra pars compacta and ventral tegmental area., the medial prefrontal cortex and the hippocampus after chronic administration of METH.
2. Materials and methods
2.2. Treatment protocol In the acute experiment, the animals were decapitated either 2, 6 and 24 h Žfor in situ hybridization, n s 8 for each time point. or 0.5, 2, 6 and 24 h Žfor radioimmunoassay, n s 6 for each time point. after a single i.p. injection of 4 mgrkg METH. In the chronic experiments, the animals were treated with either 4 mgrkg METH or an equal volume of saline ŽSAL. once daily for 14 days. This was subsequently withdrawn for 28 days following which the animals were challenged with a single dose of either METH Ž4 mgrkg i.p.. or SAL 6 h prior to being sacrificed. This resulted in 4 treatment groups Ž n s 8 in each group for both in situ hybridization and radioimmunoassay.: chronic treatment with METH followed by either a METH challenge ŽMM. or saline challenge ŽMS., and chronic treatment with saline followed by a METH challenge ŽSM. or saline challenge ŽSS.. 2.3. Northern blot analysis The oligonucleotide probes used in the present study were the same as previously reported by Gannon et al. w3x, and are shown in Table 1. Northern blot analysis to confirm the specificity of each probe was conducted according to the method described by Gannon et al. w3x. The probes were 5X-end-radiolabeled with w g- 32 PxATP Žspecific activity 3000 Cirmmol, Du Pont-NEN. using T4 polynucleotide kinase ŽPharmacia Biotech AB, Uppsala, Sweden., and then the labeled probe was purified using a Sephadex G-50 spin column ŽPharmacia Biotech AB.. Total RNA was extracted from brain tissue by acid guanidinium-phenol-chloroform method using ISOGEN ŽNippongene, Tokyo, Japan.. Samples were then resuspended in 0.01% diethylpyrocarbonate. The RNA content and purity of aliquots of samples were calculated from O.D. reading at 260 nm and the 260r280 nm ratio, respectively. Following precipitation and microcentrifugation, RNA samples were resuspended in loading buffer Ž45% formamide, 6% formaldehyde, 20 mM 3-Ž N-morpholino.propanesulfonic acid ŽMOPS., 5% glycerol with Bromphenol Blue as marker, pH 7.0., loaded onto 1% agarose gels Žcontaining 2.25% formaldehyde, 20 mM MOPS, pH 7.0. and run in
2.1. Animals Male Sprague–Dawley rats ŽCharles River, Yokohama, Japan. weighing 220–240 g were used. They were housed under a 12 h lightr12 h dark cycle Žlight on 07:00 h, light off 19:00 h. with constant temperature Ž258C. and humidity, and allowed free access to food and water. All the animals used were handled gently for 3 min once daily for one week before being subjected to drug treatment. All the animal use procedures were in strict accordance with the Guidelines for Animal Experiments of Okayama University Medical School.
Table 1 Oligonucleotide sequences of each CaM gene Žaccording to Gannon and McEwen w3x. CaM I Ž40 mer: X 5 dTACCATGGTGCCAGCGAAGGAAGGAAGAGCGGAGCAGGCG. CaM II Ž41 mer: X 5 dTTCAGTCAGTTGGTCAGCCATGCTGCAAGGGCTACCGGTTT. CaM III Ž30 mer: 5X dTATCCGGAGCTCGGGGATCGAGGTTACTCT.
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MOPS buffer Ž20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0. at 200 V for 2–4 h at 48C. Following overnight capillary transfer of RNA to nylon membrane Ž H ybond-N q , A m ersham . using 10 = sodium chloridersodium citrate ŽSSC. solution Ž1 = SSC s 0.15 M sodium chlorider0.015 M sodium citrate., RNA was fixed to the membrane by baking for 3 h at 808C. The nylon membrane was subsequently UV cross-linked Ž200 mJ at 302 nm for 30 s.. Hybridization to the nylon membrane was performed overnight at 608C in a solution containing 5 = SSC buffer, 0.5% SDS, 100 m grml denatured salmon sperm DNA, and 10 6 cpmrml of 32 P-labeled oligonucleotide probe. Following hybridization, the membrane was washed under high stringency conditions Ž2 = 15 min with 5 = SSC, 0.5% SDS at room temperature; 2 = 15 min with 1 = SSC, 0.5% SDS at room temperature and 2 = 15 min with 0.1 = SSC, 0.5% SDS at 608C.. Membranes were wrapped in plastic film and exposed to X-ray film.
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Fig. 2. Northern analysis of CaM mRNAs in the rat brain. Total RNA was extracted from the rat brain, resolved on a 1.0% agarose gel and transferred to a nylon membrane Ž15 m grlane.. The membrane was divided into equal parts and hybridized with radiolabeled oligonucleotide probes to CaM I, CaM II and CaM III mRNA, as described in Section 2. Equal RNA loading and transfer were confirmed by ethidium bromide staining of the gel.
2.4. In situ hybridization The oligonucleotide probes were radiolabeled at the 3X-end with w a-w 35 SxthioxdATP Žspecific activity 1200 Cirmmol, Du Pont-NEN. using terminal deoxyribonucleotidyl transferase Ž3X-end labeling system, Du PontNEN.. The rat brains were rapidly removed, frozen in powdered dry ice and stored at y808C. Thick coronal sections Ž10 m m. were cut in a cryostat at y208C and mounted on a 3-aminopropyltriethoxy-silane ŽSigma.coated glass slide and stored at y808C until use. The sections were fixed in 4% paraformaldehyde in 10 mM phosphate-buffered saline ŽPBS. for 20 min at room temperature, rinsed in 4 = SSC. After a brief rinse in distilled
Fig. 1. Standard curve for radioimmunoassay. Each sample was diluted in assay buffer. The CaM content always ranged from 0.2 ng to 10 ng per tube as indicated in the concentration range bordered by the two vertical lines.
water, they were immersed in 0.1 M triethanolaminer0.9% NaCl for 2 min, in 0.25% acetic anhydride in 0.1 M triethanolaminer0.9% NaCl for 10 min, rinsed in 2 = SSC and dehydrated through a series of graded ethanol solutions Ž50, 70, 95 and 100%.. Each section was overlaid with 100 m l of hybridization buffer which contained 40% deionized formamide, 0.6 M NaCl, 1 mM EDTA, 1 = Denhardt’s solution, 10% dextran sulfate, 125 m grml salmon sperm DNA, 250 m grml yeast tRNA, 10 mM Tris-HCl ŽpH 7.4., 1.2 mgrml heparin, 0.1 M dithiothreitol and 10 6 dpm of 35 S-labeled oligonucleotide probe. The sections were incubated at 378C overnight in a moist chamber and then washed at room temperature for 30 min in 2 = SSC, followed by 1 h at 458C in 1 = SSC and 1 h at 458C in 0.5 = SSC. The washed tissue sections were dehydrated through a series of graded ethanol solutions Ž50, 70, 95 and 100%., and dried with a gentle stream of air. In control experiments designed to assess definite evidence for the sequence specificity of the signal, the sections were incubated with the radiolabeled oligonucleotide probe in the presence of 50 : 1 molar excess of the unlabeled oligonucleotide probe. Slide-mounted tissue sections were exposed directly to an Amersham X-ray film ŽHyperfilm-3H. in an X-ray cassette. Brain paste standards which contained a series of known amounts of w 35 SxdATP were sectioned Ž10 m m thick., and mounted onto a 3aminopropyltriethoxy-silane-coated glass slide. Both the standard and experimental slides were exposed on the same film. The exposure was continued at 48C for 7 days until the film was developed. The quantitation of the mRNA signals on X-ray films was analyzed using Power Macintosh-assisted NIH Image analysis software ŽVersion 1.55.. The optical densities of
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the left and right sides of the dorsolateral striatum, nucleus accumbens shell, substantia nigra pars compacta ŽSNC., ventral tegmental area ŽVTA., medial prefrontal cortex and CA1 region of the hippocampus ŽCA1. were quantified. These densities were converted to dpmrmg tissue using a standard curve Žlog fit. generated from the brain paste standards. The results of the chronic administration of METH are shown as a percentage of the control ŽSS group.. 2.5. CaM radioimmunoassay The brain was quickly dissected into the striatum, mesolimbic area containing the nucleus accumbens and olfactory tubercle, substantia nigra ŽSN. plus VTA ŽSNrVTA., medial prefrontal cortex and hippocampus. The dissected brain tissues were frozen immediately in liquid N2 , weighed and kept at y808C until required. The tissue was thawed and homogenized in 10 vols. of homogenization buffer Ž40 mM Tris, pH 7.4 at 48C, containing 0.32 M sucrose, 3 mM MgCl 2 , 1 mM leupeptin, 1 mM pepstatin and 1 mM phenylmethylsulfonyl fluoride., and the homogenates were centrifuged at 100 000 = g for 60
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min to separate the particulate Žmembrane. and soluble Žcytosol. fractions. Both fractions were individually resuspended and solubilized in 10 vols. of the homogenization buffer supplemented with 1% Lubrol PX ŽSigma.. The samples were heated at 908C for 1 h to enhance antigenicity of CaM, cooled rapidly and stored at y808C until required for radioimmunoassay ŽRIA.. RIA of CaM was conducted according to the method described by Gnegy et al. w4x with a minor modifications. 125 I-Bolton-Hunter labeled purified CaM Žw 125 IxCaM, NEN Du Pont. and highly purified CaM ŽCalbiochem. which was used for preparing a standard curve were both heated at 908C for 1 h prior to their use to enhance their antigenicity. Each sample was diluted into concentrations approximately 2 m g of proteinrEppendorf tube in assay buffer Ž100 mM boric acid, 50 mM sodium borate, 1 mM EGTA, 15 mM sodium azide, 75 mM NaCl, 10 m grml bovine serum albumin and 0.003% Tween 20., and incubated for approximately 24 h at 48C with 25 000 cpm of w 125 IxCaM, rabbit anti-CaM antibody ŽIgG-CaM-1, 1 : 50 final dilution of serum, IgG corporation. and normal rabbit serum Ž1 : 100 final dilution. in a final assay volume of 300 m l. In parallel samples, purified CaM at concentrations
Fig. 3. In situ hybridization signals of CaM mRNAs in various areas of rat brain. CaM I ŽA, B, C, D., CaM II ŽE, F, G, H. and CaM III ŽI, J, K, L. were visualized by in situ hybridization in coronal sections using selective 35 S-labeled antisense oligonucleotide probes, as described in Section 2. MPF, medial prefrontal cortex; Str, dorsolateral striatum; NAc, nucleus accumbens shell; CA1, CA1 region of the hippocampus; SNC, substantia nigra pars compacta; VTA, ventral tegmental area. A 50-fold excess of unlabeled respective probes when added to the in situ hybridization buffer completely abolished the hybridization signal Ždata not shown..
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Fig. 4. CaM mRNAs ŽCaM I, CaM II and CaM III. after a single injection of METH. Rats were decapitated either 2, 6 and 24 h Ž n s 8 for each time point. after a single injection of 4 mgrkg METH Ži.p... The optical densities of the dorsolateral striatum ŽA,B. and nucleus accumbens shell ŽC,D. were converted to dpmrmg tissue using a standard curve. The values are expressed as mean " S.E.M. The statistical significance was determined by one-way ANOVA followed by a post-hoc test ŽFisher’s PSLD.. ) P - 0.05.
ranging from 0.01 to 100 ngrassay tube was incubated in exactly the same manner in order to prepare a standard curve. The incubation was terminated by adding 500 m l containing 20% polyethylene glycol ŽMW s 6000, Wako Pure Chemical., 4 mgrml rice starch solution ŽWako Pure Chemical. and goat anti-rabbit IgG antibody Ž1 : 100 dilution; Kirkegaard & Perry Laboratories Inc... The resultant mixture was kept at 48C for 16 h with constant shaking to precipitate the antigen-antibody complex. Samples were then centrifuged at 10 000 = g for 20 min, and the super-
natant was aspirated. The radioactivity in the pellets was counted in a 125 I gamma counter. Under our RIA conditions, the total binding determined in the presence of the rabbit anti-CaM antibody and in the absence of unlabeled purified CaM, and the blank value in the absence of the anti-CaM antibody were approximately 20%, and less than 1% of the total radioactivity added into each assay tube, respectively. Quantitation of the CaM content in the brain extracts was determined by using the standard curve ŽFig. 1.. CaM contents of the cytosol and membrane fractions
Fig. 5. CaM mRNAs ŽCaM I, CaM II and CaM III. after chronic treatment with METH. Rats were chronically treated with 4 mgrkg METH or saline once daily for 14 days; this was then withdrawn for 28 days, and thereafter the rats were challenged with a single dose of either METH or saline 6 h before being sacrificed. This resulted in 4 treatment groups, each comprising 8 rats, as described in Section 2: METH-METH ŽMM, black columns., METH-SAL ŽMS, hatched columns., SAL-METH ŽSM, horizontally striped columns. and SAL-SAL ŽSS, white columns.. The optical densities of the dorsolateral striatum ŽA., nucleus accumbens shell ŽB., substantia nigra pars compacta ŽC., ventral tegmental area ŽD., medial prefrontal cortex ŽE. and CA1 region of the hippocampus ŽF. were converted to dpmrmg tissue using a standard curve. The mean dpmrmg tissue value obtained in the control group ŽSS. was used as a reference and set as 100%, and CaM mRNA levels shown were expressed as percentages against respective reference values, and shown as mean " S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. ) P - 0.05, ) ) P - 0.005.
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from the same brain tissue were individually expressed as ngrmg wet tissue weight, and their combined data were expressed as the total CaM. 2.6. Statistical analysis The differences among groups were analysed statistically by one-way ANOVA followed by Fisher’s protected least significance ŽPLSD. as a post-hoc test.
3. Results 3.1. Northern blot analysis Each probe recognizes molecular mRNA species of approximately 4.1 and 1.7 ŽCaM I mRNA., 1.4 ŽCaM II mRNA. and 2.3 and 1.0 kb ŽCaM III mRNA. in rat brain ŽFig. 2.. Northern analysis of total brain RNA revealed that the radiolabeled oligonucleotide probes detected species of CaM mRNAs which were previously identified using cDNA probes w8,9x. 3.2. Control experiments of in situ hybridization There was a similar pattern in the regional distribution of the mRNA species for CaM I, CaM II and CaM III. Thus, all the mRNAs were densely distributed in the pyramidal cell layers of the hippocampus, granule cell layers of dentate gyrus and cerebral cortex, while moderate levels were found in the striatum, nucleus accumbens shell region, substantia nigra compacta and ventral tegmental area ŽFig. 3.. This pattern of distribution was similar to that demonstrated by Gannon et al. w3x. Addition of a 50-fold excess of each unlabeled probe ŽCaM I, CaM II and CaM III. to the hybridization buffer completely abolished the hybridization signal Ždata not shown.. 3.3. CaM mRNA alterations in brain areas after acute and repeated METH administration The effect of acute METH administration on CaM mRNA levels was shown in Fig. 4. The mRNA for CaM I, but not CaM II or CaM III, in the striatum and nucleus accumbens, decreased at 2 h, but not 6 or 24 h, after the injection. The effect of chronic METH treatment and the effect of a challenge with METH following a period of
Fig. 6. The CaM content in cytosol and membrane fractions of the striatum after a single injection of METH. Rats were decapitated either 0.5, 2, 6 or 24 h Ž ns6 for each time point. after a single injection of 4 mgrkg METH Ži.p... The results are expressed as ng CaM per mg wet weight, and shown as mean"S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. ) P - 0.05, ) ) P - 0.005.
abstention on CaM mRNA levels are shown in Fig. 5. The mRNAs for CaM I and CaM II in the striatum, and CaM II and CaM III in the nucleus accumbens, showed a significant decrease in the MS group, and MS and MM groups, respectively. In contrast, there was no change in any of the CaM mRNA species in the striatum and nucleus accumbens in the SM group. In the SNC and VTA, there was a significant reduction in CaM I mRNA levels in the MM group, and CaM III mRNA in the SM and MM groups. The MS and MM groups were combined into a single group, and the mRNAs of this repeated METH treatment group were compared with those of the control ŽSS. group ŽTable 2.. There were significant reductions of CaM I mRNA in the striatum and VTA, CaM II mRNA in the striatum and nucleus accumbens, and CaM III mRNA in the nucleus accumbens, SNC and VTA. In contrast, none of the CaM mRNA species changed in the medial prefrontal cortex or CA1 region of the hippocampus after repeated METH treatment. 3.4. CaM protein alterations in brain areas after acute and repeated METH administration The antigenicity of CaM has been reported to increase after exposure to heat for 6 min w4x. This was confirmed in
Fig. 7. The CaM content of the cytosol ŽC. and membrane ŽM. fractions and the total CaM content from the different areas of rat brain after chronic treatment with METH. Rats were treated chronically with 4 mgrkg METH or saline once daily for 14 days, which was then withdrawn for 28 days, and thereafter, the rats were challenged with a single dose of either METH or saline 6 h before sacrifice so that 4 groups were formed with 8 rats in each group: METH-METH ŽMM, black columns., METH-SAL ŽMS, hatched columns., SAL-METH ŽSM, horizontally striped columns. and SAL-SAL ŽSS, white columns. as described in Section 2. The results for the striatum ŽA., mesolimbic area containing the nucleus accumbens and olfactory tubercle ŽB., substantia nigra plus ventral tegmental area ŽC., medial prefrontal cortex ŽD. and hippocampus ŽE. are expressed as ng CaM per mg wet weight, and shown as mean " S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. ) P - 0.05, )) P - 0.005.
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256 Table 2 Effect of repeated METH on CaM mRNA levels
Str NAc SNC VTA MPF CA1
CaM I
CaM II
CaM III
83.10"3.89 a 87.78"5.58 70.60"8.62 71.66"7.55 a 87.46"7.25 73.25"4.94
87.68"1.97 b 87.20q1.62 b 88.57"6.05 88.39"6.07 91.36"2.59 93.60"10.46
66.81"4.98 80.58"2.82 55.86"7.56 61.72"7.70 79.13"5.20 88.31"6.53
a a a
Rats were treated as described in the legend to Fig. 5. The mean dpmrmg tissue value obtained in the control group ŽSS. was used as a reference and set as 100%, and CaM mRNA levels of repeated METH-treated groups Žthe MM and MS combined. shown were expressed as percentages against respective control values, and shown as mean"S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. a P - 0.05, b P - 0.005.
our preliminary experiment which showed that boiling for 60 min increased the sensitivity of detecting CaM when compared to boiling for 6 min. Additionally, consistent with the technical note of NEN Du Pont, heat treatment for 1 h at 908C also increased the sensitivity of w 125 IxCaM. Therefore, all the samples, unlabeled purified CaM and w 125 IxCaM were all subjected to boiling at 908C for 1 h in subsequent experiments. The boiling did not affect the radioactivity of w 125 IxCaM Ždata not shown.. The effect of acute administration of METH on CaM content in the striatum is shown in Fig. 6. Thirty minutes after an injection of METH, CaM level in both the cytosol and membrane fractions of the striatum showed significant decrease. This reduction persisted at 2 h and 6 h after the METH injection, but returned to the control level within 24 h. The effect of chronic METH treatment and the effect of a challenge with METH on CaM content are shown in Fig. 7. The content of CaM in the cytosol of the striatum and mesolimbic area decreased significantly 6 h after acute administration of METH ŽSM group., returned to the control level after the withdrawal period of 28 days ŽMS group., but showed significant decrease after a challenge with METH ŽMM group. when compared to levels in the SS and MS groups. In contrast, the CaM content in the membrane fraction of the striatum decreased significantly 6 h after acute administration of METH ŽSM group., but this reduction persisted despite the withdrawal for 28 days ŽMS group.. The CaM content in the membrane fraction of the mesolimbic area did not change either after the acute administration of METH ŽSM group. or after the withdrawal period of 28 days ŽMS group., but showed a significant increase after the challenge with METH ŽMM group.. The total CaM decreased significantly in the striatum after acute administration of METH ŽSM group. and after challenge with METH ŽMM group., but showed a significant increase in the mesolimbic area after METH challenge ŽMM group.. In the SNrVTA, acute administration of METH ŽSM group. induced a significant decrease in CaM in both the cytosol and membrane fractions, which
persisted despite the withdrawal period of 28 days ŽMS group. and after challenge with METH ŽMM group.. The total CaM in the SNrVTA also decreased significantly after acute ŽSM group. and repeated ŽMS and MM groups. METH treatments. There was a significant increase in the CaM content of the membrane, but not of the cytosol, in the medial prefrontal cortex and hippocampus in the MM group. In these areas the total CaM content showed no significant change after acute and repeated METH treatments.
4. Discussion There has to date been no study which has examined the temporal relationship between the acute administration of psychostimulants and the levels of CaM mRNA and protein in different brain areas. The present study shows that the decrease in CaM protein in both the cytosolic and membrane fractions of the striatum occurs for longer periods than the decrease in the mRNA species for CaM I. This suggests that the decrease in CaM protein is induced by an attenuation in the expression of its mRNA. Previous studies investigating the effect of several doses of AMPH on CaM protein content have produced contradictory results w4,11x. In this study, we have used several time points over 24 h after an acute administration of METH in order to optimize sampling times for the chronic study. An interval of 6 h was chosen between the challenge injection and sacrifice in the chronic experiment, since the mRNA level had otherwise returned to basal levels 6 h after acute METH administration. A reduction in the CaM content in both the cytosolic and membrane fractions of the striatum and SNrVTA, and the cytosolic fraction of the mesolimbic area in the SM group, is consistent with the acute experiment. To our knowledge, no study has investigated the effect of acute and repeated administration of METH on CaM mRNA levels. In the present study, we have determined CaM mRNA levels after METH administration using quantitative in situ hybridization in the major dopamine-innervated areas of the brain Žthe dorsolateral striatum and nucleus accumbens shell., the dopaminergic cell areas ŽSNC, VTA., the medial prefrontal cortex and the hippocampus. The transient reductions of mRNA species for CaM I may be associated with a unique 3X untranslated regions of AU-rich ‘destabilizer-like’ element which exists in 4.0 kb mRNA species for CaM I w10x. The major finding of the present study is that METH challenge after a 4-week withdrawal period induces translocation of CaM from the cytosolic to the membrane fractions in the mesolimbic area. Our finding of translocation of CaM from the cytosol to the membrane fractions in the mesolimbic area in the MM group is consistent the report of Popov et al. w11x, who showed a similar transloca-
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tion of CaM which persisted for 2 days after the last dose of AMPH. However, Gnegy et al. w4x reported that CaM increased significantly in both the striatal membrane and cytosolic fractions, and in the mesolimbic membranes of rats receiving repeated doses of AMPH and withdrawn for 4 weeks. The translocation of CaM from the membrane to the cytosol occurred 30 min after a challenge with a low dose of AMPH Žfollowing chronic AMPH.. It has been suggested that translocation of CaM between the cytosol and the membrane fraction after AMPH treatment is dosedependent w11x. Thus, Popov et al. w11x showed that translocation of CaM from the membrane to the cytosol of the striatum and hippocampus occurred 40 min after a single injection of low-dose AMPH Ž1.25 mgrkg., while translocation occurred in the opposite direction, i.e. from the cytosol to the membrane 40 min after a single injection of a high-dose AMPH Ž5 mgrkg.. The difference in the time periods between AMPHrMETH injection and sacrifice in the present study in comparison to the study of Gnegy et al. w4x is unlikely to account for the difference in translocation of CaM. Thus, the induction of the same alteration in the striatal cytosol and membrane fractions at 30 min and 6 h after acute METH administration observed in the present study would exclude the possibility that the CaM might have been translocated at 30 min after METM challenge ŽMM group. in the same direction, as was observed by Gnegy et al. w4x. Therefore, the discrepancies in the effect of AMPHrMETH on CaM translocation in the previous studies w4,11x and present study are likely to be due to the different doses of AMPHrMETH employed. Taken together, these results suggest that repeated administration of METH leads to translocation of CaM from the cytosolic to the membrane fraction. As there was no apparent alteration in the content of the membrane fraction in the MS group, this translocation is likely to be a reversible process. In addition to the translocation of CaM from the cytosol to the membrane fraction, the total CaM in the mesolimbic area increased significantly after challenge with METH. Differential localization of mRNAs and their corresponding proteins might be one of the factors accounting for the discrepancy between the decrease in CaM mRNAs and the total CaM content of the nucleus accumbens in the MM group. On the other hand, repeated METH treatment decreased CaM I and CaM III mRNA in the VTA, SNC and VTA, respectively ŽTable 2.. The total CaM content in the SNrVTA decreased significantly after acute and repeated METH treatments. The observed decrease of total CaM levels in the SNrVTA may indicate a decrease in the de novo synthesis of new protein resulting from the decrease of CaM mRNAs. We have shown that translocation of CaM to the membrane fraction is induced in the mesolimbic area by a METH challenge after a period of withdrawal of 1 month from chronic METH dosing. It has been suggested that the translocation of CaM from the membrane to the cytosol is
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correlated with a decrease in the responsiveness of adenylate cyclase to dopamine in the rat striatum w7x. In contrast, the translocation of CaM from the cytosol to the membrane induced by a METH challenge after withdrawal from chronic METH administration in the present study could potentiate dopamine-stimulated adenylate cyclase activity in the dopaminergic neurons. The other putative neural substrate which is regulated by CaM in the membrane is the dopamine transporter. It has been reported that enhancement of Ca 2q-dependent w 3 Hxdopamine uptake resulted from a stimulation of translocation of the dopamine transporter from the cytosol to the presynaptic membrane of synaptosomes, and that enhancement of w 3 Hxdopamine uptake was suppressed by the CaM antagonist W-7, the CaM kinase II inhibitor KN-62 and the myosin light chain kinase inhibitor wortmannin w18x. It is conceivable that translocation of CaM from the cytosol to the membrane which is induced by a METH challenge may lead to enhanced dopamine uptake by the dopamine transporter on the dopaminergic presynaptic terminal in the striatum and nucleus accumbens. Sato et al. w17x reported that w 14 CxMETH when administered in vivo accumulated to a greater extent in the brain of rats which had previously received chronic doses of METH than the controls. METH is known to be a substrate for the dopamine transporter, and thus enhanced accumulation of w 14 CxMETH in sensitized rats may be associated with METH challenge-induced translocation of CaM to the membrane. Clearly, further study is required to investigate the putative role of CaM in dopamine transport. In the present study, the increase in CaM content in the membrane fraction induced by METH challenge after withdrawal from chronic METH was also observed in the medial prefrontal cortex and hippocampus. AMPH has been reported to increase dopamine release in the medial prefrontal cortex of rats w6x. Also, stress-induced dopamine release w5x and dopamine utilization w13x have been reported to be increased in the prefrontal cortex of AMPHsensitized rats, indicating that the medial prefrontal cortex is involved in interchangeability between psychostimulants and stress. The dopaminergic system in the frontal cortex and hippocampus as well as the nucleus accumbens may play a pivotal role in the sensitization process by causing the increase of CaM in the membrane fraction. By analogy with behavioral sensitization, Popov et al. w12x reported that the translocation of hippocampal CaM from the cytosol to the membrane occurred immediately after induction of hippocampal long-term potentiation ŽLTP., which is another neural plasticity model. Although the decrease of cytosolic CaM in these brain areas was not obvious in the MM group, the finding that the total CaM content did not change significantly appears to support the possible translocation of CaM from the cytosol to the membrane. In conclusion, the present study shows that acute and chronic administration of METH results in a differential alteration of the CaM content and CaM mRNA in discrete
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areas of the rat brain. CaM levels in both the striatal cytosolic and membrane fractions showed a decrease for up to at least 6 h, but returned to the control level at 24 h, after acute administration of 4 mgrkg METH. In contrast, METH challenge following a period of chronic administration resulted in translocation of CaM from the cytosol to the membrane in the mesolimbic area, and possibly in the prefrontal cortex and hippocampus. Further studies aimed at elucidating the putative role of the translocated CaM in the membrane need to be performed.
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Acknowledgements w12x
The study was supported by a Research Grant for the Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan in 1996, entitled ‘Studies on Diagnosis and Treatment of Psychoactive Use Disorders’ Žrecipient, K.A...
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References w15x w1x K. Akiyama, A. Kanzaki, K. Tuchida, H. Ujike, Methamphetamineinduced behavioral sensitization and its implications for relapse of schizophrenia, Schizophr. Res. 12 Ž1994. 251–257. w2x W.Y. Cheung, Calmodulin plays a pivotal role in cellular regulation, Science 207 Ž1979. 19–27. w3x M.N. Gannon, B.S. McEwen, Distribution and regulation of calmodulin mRNAs in rat brain, Mol. Brain Res. 22 Ž1994. 186–192. w4x M.E. Gnegy, G.H. Hewlett, S.L. Yee, M.J. Welsh, Alterations in calmodulin content and localization in areas of rat brain after repeated intermittent amphetamine, Brain Res. 562 Ž1991. 6–12. w5x T. Hamamura, H.C. Fibiger, Enhanced stress-induced dopamine release in the prefrontal cortex of amphetamine-sensitized rats, Eur. J. Pharmacol. 237 Ž1993. 65–72. w6x I.M. Maisonneuve, R.W. Keller, S.D. Glick, Similar effects of
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D-amphetamine and cocaine on extracellular dopamine levels in medial prefrontal cortex of rats, Brain Res. 535 Ž1990. 221–226. M. Memo, W. Lovenberg, I. Hanbauer, Agonist-induced subsensitivity of adenylate cyclase coupled with a dopamine receptor in slices from rat corpus striatum, Proc. Natl. Acad. Sci. USA 79 Ž1982. 4456–4460. H. Nojima, Structural organization of multiple rat calmodulin genes, J. Mol. Biol. 208 Ž1989. 269–282. H. Nojima, H. Sokabe, Structure of a gene for rat calmodulin, J. Mol. Biol. 193 Ž1987. 439–445. B. Ni, R. Brown, Modulation of neuronal calmodulin mRNA species in the rat brain stem by reserpine, Neurochem. Res. 18 Ž1993. 185–192. N. Popov, H. Matthies, Influence of dopamine receptor agonists and antagonists on calmodulin translocation in different brain regions, Eur. J. Pharmacol. 172 Ž1989. 205–210. N. Popov, K.G. Reymann, K. Schulzeck, S. Schulzeck, H. Matthies, Alterations in calmodulin content in fractions of rat hippocampal slices during tetanic- and calcium-induced long-term potentiation, Brain Res. Bull. 21 Ž1988. 201–206. T.E. Robinson, J.B. Becker, C.J. Moore, E. Castenada, G. Mittleman, Enduring enhancement in frontal cortex dopamine utilization in an animal model of amphetamine psychosis, Brain Res. 343 Ž1985. 374–377. T.E. Robinson, J.B. Becker, Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev. 11 Ž1986. 157–198. P.H. Roseboom, G.H.K. Hewlett, M.E. Gnegy, Repeated amphetamine administration alters the interaction between D 1-stimulated adenylyl cyclase activity and calmodulin in rat striatum, J. Pharmacol. Exp. Ther. 255 Ž1990. 197–203. M. Sato, C.C. Chen, K. Akiyama, S. Otsuki, Acute exacerbation of paranoid psychotic state after long term abstinence in patients with previous methamphetamine psychosis, Biol. Psychiatry 18 Ž1983. 429–440. M. Sato, Y. Numachi, T. Hamamura, Relapse of paranoid psychotic state in methamphetamine model of schizophrenia, Schizophr. Bull. 18 Ž1992. 115–122. T. Uchiyama, Y. Kiuchi, A. Yura, N. Nakachi, Y. Yamazaki, C. Yokomizo, K. Oguchi, Ca2q-dependent enhancement of w 3 Hxdopamine uptake in rat striatum: possible involvement of calmodulin-dependent kinases, J. Neurochem. 65 Ž1995. 2065–2071.
Research report
Alterations of calmodulin and its mRNA in rat brain after acute and chronic administration of methamphetamine Yoshio Shimizu ) , Kazufumi Akiyama, Masafumi Kodama, Takeshi Ishihara, Takashi Hamamura, Shigetoshi Kuroda Department of Neuropsychiatry, Okayama UniÕersity Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan Accepted 1 April 1997
Abstract The effect of acute and chronic administration of methamphetamine ŽMETH. on the levels of calmodulin ŽCaM. and its mRNAs has been investigated in rat brain using antisense oligonucleotides to three distinct rat CaM genes ŽCaM I, CaM II, CaM III.. CaM I mRNA was reduced in the striatum and nucleus accumbens within 2 h of acute administration of 4 mgrkg METH, but returned to the control level by 6 h. The CaM content in both the cytosolic and membrane fractions of the striatum was reduced 0.5, 2, and 6 h after acute administration of METH. In the chronic experiments, rats were treated with either 4 mgrkg METH or saline once daily for 14 days. This was followed by a withdrawal period of 28 days, and thereafter, the animals were challenged with either METH Ž4 mgrkg, i.p.. or saline. All the animals were decapitated 6 h after this injection. There were four treatment groups: METH-METH ŽMM.; METH-saline ŽMS.; saline-METH ŽSM.; and saline-saline ŽSS.. There was a significant decrease in the mRNA for CaM I and CaM II in the striatum, and CaM II and CaM III in the nucleus accumbens in the MS group and the MS and MM groups, respectively, when compared to the SS group. The CaM content in the striatal membrane fraction decreased in both the SM and MS groups but not in the MM group. In contrast, the CaM content in the membrane fraction of the mesolimbic area showed a significant increase in the MM group. The CaM content in the cytosolic fraction of these brain areas decreased in both the SM and MM groups. The total CaM decreased significantly in the SM and MM groups of the striatum, but increased significantly in the MM group of the mesolimbic area. The mRNA for CaM I and CaM III decreased significantly in the MM group, and in the SM and MM groups, in the substantia nigra pars compacta ŽSNC. and ventral tegmental area ŽVTA., respectively. The CaM content in both the cytosolic and membrane fractions and total CaM content of the SNrVTA decreased significantly in the SM, MS and MM group as compared with the SS group. In the medial prefrontal cortex and hippocampus the significant increase of CaM content in the membrane fraction of the MM group was also found, but neither the CaM content in the cytosol fraction nor total CaM content changed. These results suggest that chronic METH administration leads to a translocation of CaM from the cytosolic to membrane fractions; these may underlie METH-induced behavioral sensitization. q 1997 Elsevier Science B.V. Keywords: Methamphetamine; Behavioral sensitization; Calmodulin; Hybridization, in situ; Radioimmunoassay; Dopamine
1. Introduction It is well documented that chronic abuse with the amphetamine-group of psychostimulants wamphetamine ŽAMPH. and methamphetamine ŽMETH.x can lead to a paranoid-hallucinatory state resembling the positive symptoms of schizophrenia. Once individuals have experienced such a state, they are more vulnerable to relapse after psychological stress and if they reuse METH. This vulnerability persists for a long time, and, indeed, a continual psychotic state has been reported even in the absence of )
Corresponding author. Fax: q81 Ž86. 225-7594.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 4 3 5 - 6
further abuse w16x. In rodents, repeated administration of AMPH and METH leads to progressive augmentation of stereotyped behavior, a phenomenon known as behavioral sensitization w1,14x. Behavioral sensitization is also a sustained phenomenon, since administration of subchronic doses of METH to rats results in an augmented responses to a METH challenge, even after a prolonged period of abstinence w1x. Given that both METH-induced psychosis and behavioral sensitization share the common properties of an augmented and sustained response, an investigation into putative neural substrates which may be important in METH-induced behavioral sensitization may provide an insight into the pathogenesis of METH psychosis.
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Calmodulin ŽCaM. is an ubiquitous calcium-binding protein with a molecular weight of approximately 17 kDa. It is highly expressed in the brain. CaM undergoes a conformational change following binding to Ca2q, which allow it to bind to an inactive molecule of an enzyme, and transforms the latter into the active form. It thus plays an important role as a cofactor regulating a wide variety of calcium-dependent proteins. In this way, it influences a number of critical calcium-dependent intracellular signalling processes, including the synthesis and release of neurotransmitters, assembly and disassembly of microtubules, and the rate of cell proliferation w2x. Several lines of evidence have implicated CaM in the regulation of dopaminergic neurotransmission in the brain. For example, CaM enhances D 1-receptor mediated adenylate cyclase activity w15x, and pharmacological manipulation of dopaminergic activity changes CaM levels in subcellular fractions w4,11x. It has been previously reported that CaM levels increase significantly in both the striatal membrane and cytosolic fractions, and in the limbic membrane fractions of rats which were chronically administered AMPH and withdrawn for 4 weeks w4x. Additionally, after a period of AMPH withdrawal for 4 weeks, even a low-dose AMPH challenge elicited the translocation of CaM from the membrane to the cytosolic compartment in the striatum and limbic forebrain w4x. The alteration in CaM levels in the different subcellular compartments after chronic AMPH administration requires further study, particularly in relation to behavioral sensitization, and to compare the CaM protein and mRNA levels. However, to the best of our knowledge, there has so far been no study which has investigated the effect of chronic administration of AMPHrMETH on CaM mRNA. In the present study, we have investigated the levels of CaM and its mRNA in the major dopamine-innervated brain areas Žthe dorsolateral striatum and nucleus accumbens., the areas containing dopamine neurons Žthe substantia nigra pars compacta and ventral tegmental area., the medial prefrontal cortex and the hippocampus after chronic administration of METH.
2. Materials and methods
2.2. Treatment protocol In the acute experiment, the animals were decapitated either 2, 6 and 24 h Žfor in situ hybridization, n s 8 for each time point. or 0.5, 2, 6 and 24 h Žfor radioimmunoassay, n s 6 for each time point. after a single i.p. injection of 4 mgrkg METH. In the chronic experiments, the animals were treated with either 4 mgrkg METH or an equal volume of saline ŽSAL. once daily for 14 days. This was subsequently withdrawn for 28 days following which the animals were challenged with a single dose of either METH Ž4 mgrkg i.p.. or SAL 6 h prior to being sacrificed. This resulted in 4 treatment groups Ž n s 8 in each group for both in situ hybridization and radioimmunoassay.: chronic treatment with METH followed by either a METH challenge ŽMM. or saline challenge ŽMS., and chronic treatment with saline followed by a METH challenge ŽSM. or saline challenge ŽSS.. 2.3. Northern blot analysis The oligonucleotide probes used in the present study were the same as previously reported by Gannon et al. w3x, and are shown in Table 1. Northern blot analysis to confirm the specificity of each probe was conducted according to the method described by Gannon et al. w3x. The probes were 5X-end-radiolabeled with w g- 32 PxATP Žspecific activity 3000 Cirmmol, Du Pont-NEN. using T4 polynucleotide kinase ŽPharmacia Biotech AB, Uppsala, Sweden., and then the labeled probe was purified using a Sephadex G-50 spin column ŽPharmacia Biotech AB.. Total RNA was extracted from brain tissue by acid guanidinium-phenol-chloroform method using ISOGEN ŽNippongene, Tokyo, Japan.. Samples were then resuspended in 0.01% diethylpyrocarbonate. The RNA content and purity of aliquots of samples were calculated from O.D. reading at 260 nm and the 260r280 nm ratio, respectively. Following precipitation and microcentrifugation, RNA samples were resuspended in loading buffer Ž45% formamide, 6% formaldehyde, 20 mM 3-Ž N-morpholino.propanesulfonic acid ŽMOPS., 5% glycerol with Bromphenol Blue as marker, pH 7.0., loaded onto 1% agarose gels Žcontaining 2.25% formaldehyde, 20 mM MOPS, pH 7.0. and run in
2.1. Animals Male Sprague–Dawley rats ŽCharles River, Yokohama, Japan. weighing 220–240 g were used. They were housed under a 12 h lightr12 h dark cycle Žlight on 07:00 h, light off 19:00 h. with constant temperature Ž258C. and humidity, and allowed free access to food and water. All the animals used were handled gently for 3 min once daily for one week before being subjected to drug treatment. All the animal use procedures were in strict accordance with the Guidelines for Animal Experiments of Okayama University Medical School.
Table 1 Oligonucleotide sequences of each CaM gene Žaccording to Gannon and McEwen w3x. CaM I Ž40 mer: X 5 dTACCATGGTGCCAGCGAAGGAAGGAAGAGCGGAGCAGGCG. CaM II Ž41 mer: X 5 dTTCAGTCAGTTGGTCAGCCATGCTGCAAGGGCTACCGGTTT. CaM III Ž30 mer: 5X dTATCCGGAGCTCGGGGATCGAGGTTACTCT.
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MOPS buffer Ž20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0. at 200 V for 2–4 h at 48C. Following overnight capillary transfer of RNA to nylon membrane Ž H ybond-N q , A m ersham . using 10 = sodium chloridersodium citrate ŽSSC. solution Ž1 = SSC s 0.15 M sodium chlorider0.015 M sodium citrate., RNA was fixed to the membrane by baking for 3 h at 808C. The nylon membrane was subsequently UV cross-linked Ž200 mJ at 302 nm for 30 s.. Hybridization to the nylon membrane was performed overnight at 608C in a solution containing 5 = SSC buffer, 0.5% SDS, 100 m grml denatured salmon sperm DNA, and 10 6 cpmrml of 32 P-labeled oligonucleotide probe. Following hybridization, the membrane was washed under high stringency conditions Ž2 = 15 min with 5 = SSC, 0.5% SDS at room temperature; 2 = 15 min with 1 = SSC, 0.5% SDS at room temperature and 2 = 15 min with 0.1 = SSC, 0.5% SDS at 608C.. Membranes were wrapped in plastic film and exposed to X-ray film.
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Fig. 2. Northern analysis of CaM mRNAs in the rat brain. Total RNA was extracted from the rat brain, resolved on a 1.0% agarose gel and transferred to a nylon membrane Ž15 m grlane.. The membrane was divided into equal parts and hybridized with radiolabeled oligonucleotide probes to CaM I, CaM II and CaM III mRNA, as described in Section 2. Equal RNA loading and transfer were confirmed by ethidium bromide staining of the gel.
2.4. In situ hybridization The oligonucleotide probes were radiolabeled at the 3X-end with w a-w 35 SxthioxdATP Žspecific activity 1200 Cirmmol, Du Pont-NEN. using terminal deoxyribonucleotidyl transferase Ž3X-end labeling system, Du PontNEN.. The rat brains were rapidly removed, frozen in powdered dry ice and stored at y808C. Thick coronal sections Ž10 m m. were cut in a cryostat at y208C and mounted on a 3-aminopropyltriethoxy-silane ŽSigma.coated glass slide and stored at y808C until use. The sections were fixed in 4% paraformaldehyde in 10 mM phosphate-buffered saline ŽPBS. for 20 min at room temperature, rinsed in 4 = SSC. After a brief rinse in distilled
Fig. 1. Standard curve for radioimmunoassay. Each sample was diluted in assay buffer. The CaM content always ranged from 0.2 ng to 10 ng per tube as indicated in the concentration range bordered by the two vertical lines.
water, they were immersed in 0.1 M triethanolaminer0.9% NaCl for 2 min, in 0.25% acetic anhydride in 0.1 M triethanolaminer0.9% NaCl for 10 min, rinsed in 2 = SSC and dehydrated through a series of graded ethanol solutions Ž50, 70, 95 and 100%.. Each section was overlaid with 100 m l of hybridization buffer which contained 40% deionized formamide, 0.6 M NaCl, 1 mM EDTA, 1 = Denhardt’s solution, 10% dextran sulfate, 125 m grml salmon sperm DNA, 250 m grml yeast tRNA, 10 mM Tris-HCl ŽpH 7.4., 1.2 mgrml heparin, 0.1 M dithiothreitol and 10 6 dpm of 35 S-labeled oligonucleotide probe. The sections were incubated at 378C overnight in a moist chamber and then washed at room temperature for 30 min in 2 = SSC, followed by 1 h at 458C in 1 = SSC and 1 h at 458C in 0.5 = SSC. The washed tissue sections were dehydrated through a series of graded ethanol solutions Ž50, 70, 95 and 100%., and dried with a gentle stream of air. In control experiments designed to assess definite evidence for the sequence specificity of the signal, the sections were incubated with the radiolabeled oligonucleotide probe in the presence of 50 : 1 molar excess of the unlabeled oligonucleotide probe. Slide-mounted tissue sections were exposed directly to an Amersham X-ray film ŽHyperfilm-3H. in an X-ray cassette. Brain paste standards which contained a series of known amounts of w 35 SxdATP were sectioned Ž10 m m thick., and mounted onto a 3aminopropyltriethoxy-silane-coated glass slide. Both the standard and experimental slides were exposed on the same film. The exposure was continued at 48C for 7 days until the film was developed. The quantitation of the mRNA signals on X-ray films was analyzed using Power Macintosh-assisted NIH Image analysis software ŽVersion 1.55.. The optical densities of
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the left and right sides of the dorsolateral striatum, nucleus accumbens shell, substantia nigra pars compacta ŽSNC., ventral tegmental area ŽVTA., medial prefrontal cortex and CA1 region of the hippocampus ŽCA1. were quantified. These densities were converted to dpmrmg tissue using a standard curve Žlog fit. generated from the brain paste standards. The results of the chronic administration of METH are shown as a percentage of the control ŽSS group.. 2.5. CaM radioimmunoassay The brain was quickly dissected into the striatum, mesolimbic area containing the nucleus accumbens and olfactory tubercle, substantia nigra ŽSN. plus VTA ŽSNrVTA., medial prefrontal cortex and hippocampus. The dissected brain tissues were frozen immediately in liquid N2 , weighed and kept at y808C until required. The tissue was thawed and homogenized in 10 vols. of homogenization buffer Ž40 mM Tris, pH 7.4 at 48C, containing 0.32 M sucrose, 3 mM MgCl 2 , 1 mM leupeptin, 1 mM pepstatin and 1 mM phenylmethylsulfonyl fluoride., and the homogenates were centrifuged at 100 000 = g for 60
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min to separate the particulate Žmembrane. and soluble Žcytosol. fractions. Both fractions were individually resuspended and solubilized in 10 vols. of the homogenization buffer supplemented with 1% Lubrol PX ŽSigma.. The samples were heated at 908C for 1 h to enhance antigenicity of CaM, cooled rapidly and stored at y808C until required for radioimmunoassay ŽRIA.. RIA of CaM was conducted according to the method described by Gnegy et al. w4x with a minor modifications. 125 I-Bolton-Hunter labeled purified CaM Žw 125 IxCaM, NEN Du Pont. and highly purified CaM ŽCalbiochem. which was used for preparing a standard curve were both heated at 908C for 1 h prior to their use to enhance their antigenicity. Each sample was diluted into concentrations approximately 2 m g of proteinrEppendorf tube in assay buffer Ž100 mM boric acid, 50 mM sodium borate, 1 mM EGTA, 15 mM sodium azide, 75 mM NaCl, 10 m grml bovine serum albumin and 0.003% Tween 20., and incubated for approximately 24 h at 48C with 25 000 cpm of w 125 IxCaM, rabbit anti-CaM antibody ŽIgG-CaM-1, 1 : 50 final dilution of serum, IgG corporation. and normal rabbit serum Ž1 : 100 final dilution. in a final assay volume of 300 m l. In parallel samples, purified CaM at concentrations
Fig. 3. In situ hybridization signals of CaM mRNAs in various areas of rat brain. CaM I ŽA, B, C, D., CaM II ŽE, F, G, H. and CaM III ŽI, J, K, L. were visualized by in situ hybridization in coronal sections using selective 35 S-labeled antisense oligonucleotide probes, as described in Section 2. MPF, medial prefrontal cortex; Str, dorsolateral striatum; NAc, nucleus accumbens shell; CA1, CA1 region of the hippocampus; SNC, substantia nigra pars compacta; VTA, ventral tegmental area. A 50-fold excess of unlabeled respective probes when added to the in situ hybridization buffer completely abolished the hybridization signal Ždata not shown..
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Fig. 4. CaM mRNAs ŽCaM I, CaM II and CaM III. after a single injection of METH. Rats were decapitated either 2, 6 and 24 h Ž n s 8 for each time point. after a single injection of 4 mgrkg METH Ži.p... The optical densities of the dorsolateral striatum ŽA,B. and nucleus accumbens shell ŽC,D. were converted to dpmrmg tissue using a standard curve. The values are expressed as mean " S.E.M. The statistical significance was determined by one-way ANOVA followed by a post-hoc test ŽFisher’s PSLD.. ) P - 0.05.
ranging from 0.01 to 100 ngrassay tube was incubated in exactly the same manner in order to prepare a standard curve. The incubation was terminated by adding 500 m l containing 20% polyethylene glycol ŽMW s 6000, Wako Pure Chemical., 4 mgrml rice starch solution ŽWako Pure Chemical. and goat anti-rabbit IgG antibody Ž1 : 100 dilution; Kirkegaard & Perry Laboratories Inc... The resultant mixture was kept at 48C for 16 h with constant shaking to precipitate the antigen-antibody complex. Samples were then centrifuged at 10 000 = g for 20 min, and the super-
natant was aspirated. The radioactivity in the pellets was counted in a 125 I gamma counter. Under our RIA conditions, the total binding determined in the presence of the rabbit anti-CaM antibody and in the absence of unlabeled purified CaM, and the blank value in the absence of the anti-CaM antibody were approximately 20%, and less than 1% of the total radioactivity added into each assay tube, respectively. Quantitation of the CaM content in the brain extracts was determined by using the standard curve ŽFig. 1.. CaM contents of the cytosol and membrane fractions
Fig. 5. CaM mRNAs ŽCaM I, CaM II and CaM III. after chronic treatment with METH. Rats were chronically treated with 4 mgrkg METH or saline once daily for 14 days; this was then withdrawn for 28 days, and thereafter the rats were challenged with a single dose of either METH or saline 6 h before being sacrificed. This resulted in 4 treatment groups, each comprising 8 rats, as described in Section 2: METH-METH ŽMM, black columns., METH-SAL ŽMS, hatched columns., SAL-METH ŽSM, horizontally striped columns. and SAL-SAL ŽSS, white columns.. The optical densities of the dorsolateral striatum ŽA., nucleus accumbens shell ŽB., substantia nigra pars compacta ŽC., ventral tegmental area ŽD., medial prefrontal cortex ŽE. and CA1 region of the hippocampus ŽF. were converted to dpmrmg tissue using a standard curve. The mean dpmrmg tissue value obtained in the control group ŽSS. was used as a reference and set as 100%, and CaM mRNA levels shown were expressed as percentages against respective reference values, and shown as mean " S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. ) P - 0.05, ) ) P - 0.005.
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from the same brain tissue were individually expressed as ngrmg wet tissue weight, and their combined data were expressed as the total CaM. 2.6. Statistical analysis The differences among groups were analysed statistically by one-way ANOVA followed by Fisher’s protected least significance ŽPLSD. as a post-hoc test.
3. Results 3.1. Northern blot analysis Each probe recognizes molecular mRNA species of approximately 4.1 and 1.7 ŽCaM I mRNA., 1.4 ŽCaM II mRNA. and 2.3 and 1.0 kb ŽCaM III mRNA. in rat brain ŽFig. 2.. Northern analysis of total brain RNA revealed that the radiolabeled oligonucleotide probes detected species of CaM mRNAs which were previously identified using cDNA probes w8,9x. 3.2. Control experiments of in situ hybridization There was a similar pattern in the regional distribution of the mRNA species for CaM I, CaM II and CaM III. Thus, all the mRNAs were densely distributed in the pyramidal cell layers of the hippocampus, granule cell layers of dentate gyrus and cerebral cortex, while moderate levels were found in the striatum, nucleus accumbens shell region, substantia nigra compacta and ventral tegmental area ŽFig. 3.. This pattern of distribution was similar to that demonstrated by Gannon et al. w3x. Addition of a 50-fold excess of each unlabeled probe ŽCaM I, CaM II and CaM III. to the hybridization buffer completely abolished the hybridization signal Ždata not shown.. 3.3. CaM mRNA alterations in brain areas after acute and repeated METH administration The effect of acute METH administration on CaM mRNA levels was shown in Fig. 4. The mRNA for CaM I, but not CaM II or CaM III, in the striatum and nucleus accumbens, decreased at 2 h, but not 6 or 24 h, after the injection. The effect of chronic METH treatment and the effect of a challenge with METH following a period of
Fig. 6. The CaM content in cytosol and membrane fractions of the striatum after a single injection of METH. Rats were decapitated either 0.5, 2, 6 or 24 h Ž ns6 for each time point. after a single injection of 4 mgrkg METH Ži.p... The results are expressed as ng CaM per mg wet weight, and shown as mean"S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. ) P - 0.05, ) ) P - 0.005.
abstention on CaM mRNA levels are shown in Fig. 5. The mRNAs for CaM I and CaM II in the striatum, and CaM II and CaM III in the nucleus accumbens, showed a significant decrease in the MS group, and MS and MM groups, respectively. In contrast, there was no change in any of the CaM mRNA species in the striatum and nucleus accumbens in the SM group. In the SNC and VTA, there was a significant reduction in CaM I mRNA levels in the MM group, and CaM III mRNA in the SM and MM groups. The MS and MM groups were combined into a single group, and the mRNAs of this repeated METH treatment group were compared with those of the control ŽSS. group ŽTable 2.. There were significant reductions of CaM I mRNA in the striatum and VTA, CaM II mRNA in the striatum and nucleus accumbens, and CaM III mRNA in the nucleus accumbens, SNC and VTA. In contrast, none of the CaM mRNA species changed in the medial prefrontal cortex or CA1 region of the hippocampus after repeated METH treatment. 3.4. CaM protein alterations in brain areas after acute and repeated METH administration The antigenicity of CaM has been reported to increase after exposure to heat for 6 min w4x. This was confirmed in
Fig. 7. The CaM content of the cytosol ŽC. and membrane ŽM. fractions and the total CaM content from the different areas of rat brain after chronic treatment with METH. Rats were treated chronically with 4 mgrkg METH or saline once daily for 14 days, which was then withdrawn for 28 days, and thereafter, the rats were challenged with a single dose of either METH or saline 6 h before sacrifice so that 4 groups were formed with 8 rats in each group: METH-METH ŽMM, black columns., METH-SAL ŽMS, hatched columns., SAL-METH ŽSM, horizontally striped columns. and SAL-SAL ŽSS, white columns. as described in Section 2. The results for the striatum ŽA., mesolimbic area containing the nucleus accumbens and olfactory tubercle ŽB., substantia nigra plus ventral tegmental area ŽC., medial prefrontal cortex ŽD. and hippocampus ŽE. are expressed as ng CaM per mg wet weight, and shown as mean " S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. ) P - 0.05, )) P - 0.005.
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256 Table 2 Effect of repeated METH on CaM mRNA levels
Str NAc SNC VTA MPF CA1
CaM I
CaM II
CaM III
83.10"3.89 a 87.78"5.58 70.60"8.62 71.66"7.55 a 87.46"7.25 73.25"4.94
87.68"1.97 b 87.20q1.62 b 88.57"6.05 88.39"6.07 91.36"2.59 93.60"10.46
66.81"4.98 80.58"2.82 55.86"7.56 61.72"7.70 79.13"5.20 88.31"6.53
a a a
Rats were treated as described in the legend to Fig. 5. The mean dpmrmg tissue value obtained in the control group ŽSS. was used as a reference and set as 100%, and CaM mRNA levels of repeated METH-treated groups Žthe MM and MS combined. shown were expressed as percentages against respective control values, and shown as mean"S.E.M. Statistical significance was determined by one-way ANOVA followed by post-hoc test ŽFisher’s PSLD.. a P - 0.05, b P - 0.005.
our preliminary experiment which showed that boiling for 60 min increased the sensitivity of detecting CaM when compared to boiling for 6 min. Additionally, consistent with the technical note of NEN Du Pont, heat treatment for 1 h at 908C also increased the sensitivity of w 125 IxCaM. Therefore, all the samples, unlabeled purified CaM and w 125 IxCaM were all subjected to boiling at 908C for 1 h in subsequent experiments. The boiling did not affect the radioactivity of w 125 IxCaM Ždata not shown.. The effect of acute administration of METH on CaM content in the striatum is shown in Fig. 6. Thirty minutes after an injection of METH, CaM level in both the cytosol and membrane fractions of the striatum showed significant decrease. This reduction persisted at 2 h and 6 h after the METH injection, but returned to the control level within 24 h. The effect of chronic METH treatment and the effect of a challenge with METH on CaM content are shown in Fig. 7. The content of CaM in the cytosol of the striatum and mesolimbic area decreased significantly 6 h after acute administration of METH ŽSM group., returned to the control level after the withdrawal period of 28 days ŽMS group., but showed significant decrease after a challenge with METH ŽMM group. when compared to levels in the SS and MS groups. In contrast, the CaM content in the membrane fraction of the striatum decreased significantly 6 h after acute administration of METH ŽSM group., but this reduction persisted despite the withdrawal for 28 days ŽMS group.. The CaM content in the membrane fraction of the mesolimbic area did not change either after the acute administration of METH ŽSM group. or after the withdrawal period of 28 days ŽMS group., but showed a significant increase after the challenge with METH ŽMM group.. The total CaM decreased significantly in the striatum after acute administration of METH ŽSM group. and after challenge with METH ŽMM group., but showed a significant increase in the mesolimbic area after METH challenge ŽMM group.. In the SNrVTA, acute administration of METH ŽSM group. induced a significant decrease in CaM in both the cytosol and membrane fractions, which
persisted despite the withdrawal period of 28 days ŽMS group. and after challenge with METH ŽMM group.. The total CaM in the SNrVTA also decreased significantly after acute ŽSM group. and repeated ŽMS and MM groups. METH treatments. There was a significant increase in the CaM content of the membrane, but not of the cytosol, in the medial prefrontal cortex and hippocampus in the MM group. In these areas the total CaM content showed no significant change after acute and repeated METH treatments.
4. Discussion There has to date been no study which has examined the temporal relationship between the acute administration of psychostimulants and the levels of CaM mRNA and protein in different brain areas. The present study shows that the decrease in CaM protein in both the cytosolic and membrane fractions of the striatum occurs for longer periods than the decrease in the mRNA species for CaM I. This suggests that the decrease in CaM protein is induced by an attenuation in the expression of its mRNA. Previous studies investigating the effect of several doses of AMPH on CaM protein content have produced contradictory results w4,11x. In this study, we have used several time points over 24 h after an acute administration of METH in order to optimize sampling times for the chronic study. An interval of 6 h was chosen between the challenge injection and sacrifice in the chronic experiment, since the mRNA level had otherwise returned to basal levels 6 h after acute METH administration. A reduction in the CaM content in both the cytosolic and membrane fractions of the striatum and SNrVTA, and the cytosolic fraction of the mesolimbic area in the SM group, is consistent with the acute experiment. To our knowledge, no study has investigated the effect of acute and repeated administration of METH on CaM mRNA levels. In the present study, we have determined CaM mRNA levels after METH administration using quantitative in situ hybridization in the major dopamine-innervated areas of the brain Žthe dorsolateral striatum and nucleus accumbens shell., the dopaminergic cell areas ŽSNC, VTA., the medial prefrontal cortex and the hippocampus. The transient reductions of mRNA species for CaM I may be associated with a unique 3X untranslated regions of AU-rich ‘destabilizer-like’ element which exists in 4.0 kb mRNA species for CaM I w10x. The major finding of the present study is that METH challenge after a 4-week withdrawal period induces translocation of CaM from the cytosolic to the membrane fractions in the mesolimbic area. Our finding of translocation of CaM from the cytosol to the membrane fractions in the mesolimbic area in the MM group is consistent the report of Popov et al. w11x, who showed a similar transloca-
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tion of CaM which persisted for 2 days after the last dose of AMPH. However, Gnegy et al. w4x reported that CaM increased significantly in both the striatal membrane and cytosolic fractions, and in the mesolimbic membranes of rats receiving repeated doses of AMPH and withdrawn for 4 weeks. The translocation of CaM from the membrane to the cytosol occurred 30 min after a challenge with a low dose of AMPH Žfollowing chronic AMPH.. It has been suggested that translocation of CaM between the cytosol and the membrane fraction after AMPH treatment is dosedependent w11x. Thus, Popov et al. w11x showed that translocation of CaM from the membrane to the cytosol of the striatum and hippocampus occurred 40 min after a single injection of low-dose AMPH Ž1.25 mgrkg., while translocation occurred in the opposite direction, i.e. from the cytosol to the membrane 40 min after a single injection of a high-dose AMPH Ž5 mgrkg.. The difference in the time periods between AMPHrMETH injection and sacrifice in the present study in comparison to the study of Gnegy et al. w4x is unlikely to account for the difference in translocation of CaM. Thus, the induction of the same alteration in the striatal cytosol and membrane fractions at 30 min and 6 h after acute METH administration observed in the present study would exclude the possibility that the CaM might have been translocated at 30 min after METM challenge ŽMM group. in the same direction, as was observed by Gnegy et al. w4x. Therefore, the discrepancies in the effect of AMPHrMETH on CaM translocation in the previous studies w4,11x and present study are likely to be due to the different doses of AMPHrMETH employed. Taken together, these results suggest that repeated administration of METH leads to translocation of CaM from the cytosolic to the membrane fraction. As there was no apparent alteration in the content of the membrane fraction in the MS group, this translocation is likely to be a reversible process. In addition to the translocation of CaM from the cytosol to the membrane fraction, the total CaM in the mesolimbic area increased significantly after challenge with METH. Differential localization of mRNAs and their corresponding proteins might be one of the factors accounting for the discrepancy between the decrease in CaM mRNAs and the total CaM content of the nucleus accumbens in the MM group. On the other hand, repeated METH treatment decreased CaM I and CaM III mRNA in the VTA, SNC and VTA, respectively ŽTable 2.. The total CaM content in the SNrVTA decreased significantly after acute and repeated METH treatments. The observed decrease of total CaM levels in the SNrVTA may indicate a decrease in the de novo synthesis of new protein resulting from the decrease of CaM mRNAs. We have shown that translocation of CaM to the membrane fraction is induced in the mesolimbic area by a METH challenge after a period of withdrawal of 1 month from chronic METH dosing. It has been suggested that the translocation of CaM from the membrane to the cytosol is
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correlated with a decrease in the responsiveness of adenylate cyclase to dopamine in the rat striatum w7x. In contrast, the translocation of CaM from the cytosol to the membrane induced by a METH challenge after withdrawal from chronic METH administration in the present study could potentiate dopamine-stimulated adenylate cyclase activity in the dopaminergic neurons. The other putative neural substrate which is regulated by CaM in the membrane is the dopamine transporter. It has been reported that enhancement of Ca 2q-dependent w 3 Hxdopamine uptake resulted from a stimulation of translocation of the dopamine transporter from the cytosol to the presynaptic membrane of synaptosomes, and that enhancement of w 3 Hxdopamine uptake was suppressed by the CaM antagonist W-7, the CaM kinase II inhibitor KN-62 and the myosin light chain kinase inhibitor wortmannin w18x. It is conceivable that translocation of CaM from the cytosol to the membrane which is induced by a METH challenge may lead to enhanced dopamine uptake by the dopamine transporter on the dopaminergic presynaptic terminal in the striatum and nucleus accumbens. Sato et al. w17x reported that w 14 CxMETH when administered in vivo accumulated to a greater extent in the brain of rats which had previously received chronic doses of METH than the controls. METH is known to be a substrate for the dopamine transporter, and thus enhanced accumulation of w 14 CxMETH in sensitized rats may be associated with METH challenge-induced translocation of CaM to the membrane. Clearly, further study is required to investigate the putative role of CaM in dopamine transport. In the present study, the increase in CaM content in the membrane fraction induced by METH challenge after withdrawal from chronic METH was also observed in the medial prefrontal cortex and hippocampus. AMPH has been reported to increase dopamine release in the medial prefrontal cortex of rats w6x. Also, stress-induced dopamine release w5x and dopamine utilization w13x have been reported to be increased in the prefrontal cortex of AMPHsensitized rats, indicating that the medial prefrontal cortex is involved in interchangeability between psychostimulants and stress. The dopaminergic system in the frontal cortex and hippocampus as well as the nucleus accumbens may play a pivotal role in the sensitization process by causing the increase of CaM in the membrane fraction. By analogy with behavioral sensitization, Popov et al. w12x reported that the translocation of hippocampal CaM from the cytosol to the membrane occurred immediately after induction of hippocampal long-term potentiation ŽLTP., which is another neural plasticity model. Although the decrease of cytosolic CaM in these brain areas was not obvious in the MM group, the finding that the total CaM content did not change significantly appears to support the possible translocation of CaM from the cytosol to the membrane. In conclusion, the present study shows that acute and chronic administration of METH results in a differential alteration of the CaM content and CaM mRNA in discrete
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areas of the rat brain. CaM levels in both the striatal cytosolic and membrane fractions showed a decrease for up to at least 6 h, but returned to the control level at 24 h, after acute administration of 4 mgrkg METH. In contrast, METH challenge following a period of chronic administration resulted in translocation of CaM from the cytosol to the membrane in the mesolimbic area, and possibly in the prefrontal cortex and hippocampus. Further studies aimed at elucidating the putative role of the translocated CaM in the membrane need to be performed.
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w8x w9x w10x
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Acknowledgements w12x
The study was supported by a Research Grant for the Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan in 1996, entitled ‘Studies on Diagnosis and Treatment of Psychoactive Use Disorders’ Žrecipient, K.A...
w13x
w14x
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