- Email: [email protected]
Effect of chronic haloperidol treatment on synaptic protein mRNAs in the rat brain
Molecular Brain Research 61 Ž1998. 238–242
Short communication
Effect of chronic haloperidol treatment on synaptic protein mRNAs in the rat brain Tatsuo Nakahara a,) , Kaoru Nakamura b, Tetsuyuki Tsutsumi b, Kijiro Hashimoto b, Hisao Hondo b, Shinji Hisatomi b, Keisuke Motomura b, Hideyuki Uchimura b a
b
Department of Chemistry, Faculty of Science, Kyushu UniÕersity, Ropponmatsu, Fukuoka 810-8560, Japan Laboratory of Neurochemistry, Center for Emotional and BehaÕioral Disorder, Hizen National Mental Hospital, Kanzaki, Saga 842-0192, Japan Accepted 18 August 1998
Abstract Chronic haloperidol treatment caused significant decreases in the levels of synaptotagmin I and IV, synaptobrevin II, syntaxin 1A and Rab 3A mRNAs in the nucleus accumbens but not in the prefrontal cortex medial field, striatum, substantia nigra and ventral tegmental area. No significant changes in SNAP 25 and synaptophysin mRNA levels were observed in any brain region examined. The reduced expression of synaptic proteins may be related to haloperidol-induced depolarization block of mesolimbic dopamine neurons. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Haloperidol; Synaptobrevin; Synaptotagmin; Syntaxin; Rab 3A; Nucleus accumbens
Neurotransmitter release from nerve terminals requires the docking and fusion of synaptic vesicles with presynaptic plasma membrane w30x. The interaction of vesicular membrane proteins termed v-SNAREs, synaptobrevin and synaptotagmin, with target membrane proteins termed tSNAREs, SNAP 25 and syntaxin, plays a critical role for the process of vesicular docking and fusion w29x. Recent evidence suggests that synaptic proteins are involved in synaptic plasticity. The expression of syntaxin 1B has been reported to be enhanced after induction of long-term potentiation w28x, during kindling w14x and during learning w6x. Synaptotagmin has also been shown to modulate short-term synaptic plasticity w18x. Synapsin I is involved in amphetamine-induced sensitization w13x, and knocking out Rab 3A abolishes mossy fibre long-term potentiation w4x. Chronic treatment with antipsychotics induces depolarization block of dopamine cell firing in the midbrain w3,33x and reduces dopamine release w15,34x and metabolism w17,26x in the terminal regions. Although the molecular mechanism underlying the neuronal and synaptic alterations produced by antipsychotics is unclear, synaptic proteins may be involved in the antipsychotic-induced synap-
) Corresponding author. [email protected]
Fax:
q81-092-726-4842;
E-mail:
tic changes. Indeed, Eastwood et al. w8,9x showed that the expression of gene encoding synaptophysin is increased after chronic haloperidol treatment. In view of the critical role played by the v- and t-SNARE complex in synaptic vesicle exocytosis, it is of interest to determine whether synaptic proteins such as SNAREs are involved in antipsychotic-induced synaptic plasticity. To elucidate the longterm effect of antipsychotics on synaptic vesicle exocytosis, we examined synaptic protein mRNA levels in various regions of rat brain after chronic haloperidol treatment. Male Wistar rats ŽKyudo Animal Laboratory, Kumamoto, Japan. initially weighing 240–260 g were housed four per cage, maintained on a 12 h lightr12 h dark cycle and given unlimited access to food and water. Sixteen animals received 25 mg eqr0.5 mlrkg to haloperidol of haloperidol decanoate ŽDainippon Pharmaceutical, Osaka, Japan. intramuscularly or an equal volume of sesame oil vehicle. Animals were killed after 28 days, and the brain was removed. Serial slices of 300 mm were made in a cryostat at y128C, and five brain regions were dissected freehand with a microknife as described previously w19x. The isolated tissues were stored at y808C. Total RNA was prepared from the brain tissues by the method of Chomczynski and Sacchi w5x. The levels of synaptic protein mRNAs in discrete brain regions of control and haloperidol-treated rats were quanti-
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 3 0 - 7
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
fied by reverse transcription-polymerase chain reaction ŽRT-PCR. with an endogenous internal standard, b-actin w7x. RT was performed on 500 ng total RNA for 1 h at 428C after 10 min incubation at 258C in a 10 ml reaction mixture containing 25 mM Tris–HCl ŽpH 8.3., 50 mM KCl, 5 mM MgCl 2 , 2 mM dithiothreitol, 1 mM each deoxynucleotide ŽdNTP., 10 U AMV reverse transcriptase ŽLife Sciences, St. Petersburg, FL., 10 U rebonuclease inhibitor ŽBoehringer. and 0.8 mg oligo ŽdT.15 primer ŽBoehringer.. The RT was terminated by heating the sample at 998C for 5 min. The multiplexed PCR was carried out in a 20 ml reaction mixture containing 10 mM Tris–HCl ŽpH 8.3., 50 mM KCl, 1.5 mM MgCl 2 , 2% dimethyl sulfoxide, 0.2 mM each dNTP, 0.075 mM each of 5X and 3X b-actin-specific primers, 1 mM each of 5X and 3X synaptic protein specific primers, 20 or 25 ng of reverse-transcribed total RNA, and 0.5 U Taq DNA polymerase ŽBoehringer.. The PCR primers used for amplification of b-actin and synaptic protein mRNAs are shown in Table 1. The PCR amplification was performed for 22 or 26 Žsyntaxin 1A. cycles, consisting of denaturation Ž948C, 45 s., annealing Ž568C, 45 s., and extension Ž728C, 75 s.. For the amplification of syntaxin 1A mRNA, 0.075 mM each of b-actin primer pairs was added to the reaction mixture after 6 cycles and PCR cycles were further continued. The PCR products were analyzed on a 10% polyacrylamide gel electrophoresis. Gels were stained with ethidium bromide, visualized with UV trans-illumination, photographed, and submitted to image analysis. Quantitative image analysis of the PCR fragments was performed using the NIH image program. The levels of synaptic protein mRNAs were calculated as the ratios of optical density of synaptic protein PCR products to that of the b-actin PCR product. Student’s t-test was used to compare vehicle and haloperidol groups.
239
Ethidium bromide staining of a polyacrylamide gel revealed a single band at the expected size of amplification product for each of the b-actin and synaptic protein cDNAs Ždata not shown.. Since the b-actin, Rab 3A and synaptobrevin II cDNA sequences were amplified using the pair of primers derived from different exons of the genes, the contamination by genomic DNA did not interfere with the signals of PCR products of these cDNAs. To determine the optimal amplifications, PCR was performed using different amount of reverse-transcribed total RNA and different numbers of cycles Ždata not shown.. These results indicated that amplification was exponential between 20 and 23 cycles for b-actin and synaptic protein mRNAs except for syntaxin 1A in all brain regions tested. Amplification was also exponential between 22 and 28 cycles for syntaxin 1A mRNA. The PCR products were proportional to RNA input over a range of 10 to 40 ng total RNA for b-actin and synaptic protein mRNAs. Twenty nanograms or 25 ng of reverse-transcribed RNA were amplified for 22 cycles or 26 cycles Žsyntaxin 1A. for the quantitation of relative amount of synaptic protein mRNAs in the rat brain. The quantitative RT-PCR was used to measure the amount of synaptic protein mRNAs in different brain regions following chronic treatment with haloperidol ŽFigs. 1 and 2.. The highest level of synaptotagmin I mRNA was found in the frontal cortex, a high level in the substantia nigra, a moderate level in the striatum and nucleus accumbens, and the lowest level in the ventral tegmental area. Similar distributions of mRNA were observed for synaptotagmin IV and synaptobrevin II. Regression analysis of mRNA levels revealed that the levels of synaptotagmin I mRNA in the control animals significantly correlated with those of synaptotagmin IV Ž r s 0.890, P - 0.05. and synaptobrevin II Ž r s 0.972, P - 0.01. in the rat brain.
Table 1 Oligonucleotide primers of synaptic proteins and b-actin used for PCR Gene
Sequence
b-Actin
5 -ACACTGTGCCCATCTATGAGG-3 X X 5 -CAACGTCACACTTCATGATGG-3 X X 5 -TGTTCAAGATCCTGATCATTGG-3 X X 5 -TGCACTGCATTGAAGGACTC-3 X X 5 -CTGGAGGAGATGCAGAGGAG-3 X X 5 -ACTGGATTTAAGCTTGTTACAGGG-3 X X 5 -CTGCACCTCCTCCAAATCTTAC-3 X X 5 -ATGATGATGATGAGGATGATGG-3 X X 5 -TTGCTACGTGTGGCAGCTAC-3 X X 5 -TTCAGCCGACGAGGAGTAGT-3 X X 5 -CATCGGAGAGTTCAAAGTTCCTAT-3 X X 5 -GACTCGTTGTAGTAGGGGTTGAGTG-3 X X 5 -TTTGTGGTGAACATCAAGGAAG-3 X X 5 -TCTTGAGAACCTGTCAAAACTCAG-3 X X 5 -CTCAGTGAGATCGAGACCAGG-3 X X 5 -ATGATGCCCAGAATCACACA-3
Rab 3A SNAP 25 Synaptobrevin II Synaptophysin Synaptotagmin I Synaptotagmin IV Syntaxin 1A
X
X
X
X
Position
PCR product Žbp.
Refs.
482–502 841–861 145–166 402–421 194–213 434–457 71–92 323–344 145–164 322–341 1242–1265 1526–1550 747–768 966–989 566–586 806–825
380
w20x
277
w35x
264
w21x
274
w10x
197
w31x
309
w22x
243
w32x
260
w1x
The top and lower primers of each amplification pair are 5 sense and 3 antisense primers, respectively.
240
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
treatment with haloperidol. The Rab 3A mRNA levels in the ventral tegmental area was decreased by 24% after chronic haloperidol treatment, but the decrease in the
Fig. 1. Effects of chronic treatment with haloperidol on the levels of synaptobrevin II ŽA., synaptotagmin I ŽB. and synaptotagmin IV mRNA ŽC. in various regions of rat brain. Rats received a single depot injection with haloperidol decanoate Ž25 mg eqrkg. or sesame oil vehicle and were killed after 4 weeks. Synaptic protein mRNA levels from control Žopen columns. and haloperidol-treated groups Žhatched columns. in the frontal cortex medial field ŽFCM., striatum ŽSTR., nucleus accumbens ŽNAC., substantia nigra ŽSN. and ventral tegmental area ŽVTA. were analyzed by quantitative RT-PCR. The values represent the mean"SEM of 15 or 16 samples ŽFCM, STR and NAC. and 7 or 8 samples ŽSN and VTA. derived from 16 animals per treatment group. ) P - 0.05, )) P - 0.01, compared with controls using two-tailed Student’s t-test.
Chronic treatment with haloperidol caused significant decreases in the levels of synaptotagmin I Žy32%, t s 2.34, df s 30, P - 0.05., synaptotagmin IV Žy43%, t s 3.75, df s 30, P - 0.01., synaptobrevin II Žy24%, t s 2.68, df s 29, P - 0.05., syntaxin 1A Žy29%, t s 2.73, df s 28, P - 0.05. and Rab 3A Žy25%, t s 3.26, df s 28, P - 0.01. in the nucleus accumbens ŽFigs. 1 and 2.. In the nucleus accumbens, the SNAP 25 Ž t s 1.05, df s 30, P ) 0.05. and synaptophysin Ž t s 1.17, df s 29, P ) 0.05. mRNA levels were not significantly affected by chronic
Fig. 2. Effects of chronic treatment with haloperidol on the levels of syntaxin 1A ŽA., SNAP 25 ŽB., Rab 3A ŽC. and synaptophysin mRNAs ŽD. in various regions of rat brain. Drug treatment and quntitation of mRNA levels from control Žopen columns. and haloperidol-treated groups Žhatched columns. were performed as described in the legend to Fig. 1. FCM, frontal cortex medial field; STR, striatum; NAC, nucleus accumbens; SN, substantia nigra; VTA, ventral tegmental area. ) P - 0.05, )) P - 0.01, compared with controls using two-tailed Student’s t-test.
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
mRNA levels was not significant Ž t s 1.82, df s 14, P ) 0.05.. There was no significant difference in the other synaptic protein mRNA levels between vehicle and chronic haloperidol treatments in any brain region examined. The synaptic protein mRNAs were selectively decreased in the nucleus accumbens but not in the striatum, frontal cortex and midbrain. Chronic neuroleptic treatment induces a delayed inactivation of dopamine neuron firing in the midbrain due to depolarization block w3,33x. The depolarization inactivation of dopamine cell in the substantia nigra and ventral tegmental area influences the neural activity in the striatum and nucleus accumbens. Dopamine release w15,34x and metabolism w17,26x are reduced in the terminal regions of nigrostriatal and mesolimbic dopamine systems after chronic treatment with haloperidol. The decrease in synaptic protein mRNA levels found in the nucleus accumbens after chronic haloperidol treatment may be due to the depolarization-induced inactivation of midbrain dopamine cell activity. In the present study, however, chronic haloperidol failed to induce a decrease in the synaptic protein mRNA levels in the striatum. Since mRNA is localized to the neuronal perikaryon, the different population of cell bodies received dopaminergic input in the striatum and nucleus accumbens may account for the lack of the striatal synaptic protein mRNA after haloperidol treatment. However, this does not seem unlikely because the neurons in the dorsal striatum have similar morphology with those in the nucleus accumbens w23x, and the ratio of RNA to DNA levels is similar in the striatum and nucleus accumbens w19x. Chronic haloperidol was reported to decrease GABA release w27x, substance P w16x, neurokinin A w16x, somatostatin mRNA w24x and opioid receptors w25x in the nucleus accumbens without affecting those in the striatum. These results suggest that the activity of GABA neurons within the nucleus accumbens is reduced by chronic haloperidol, because substance P coexists with GABA, and m-opioide receptors located on the GABA neurons w11x. The regional effect of haloperidol on the GABA neurons is probably related to the differential localization of dopamine receptor subtypes. Thus, D 3 dopamine receptors are abundant in the nucleus accumbens whereas D 2 receptors are located in the accumbens as well as the striatum w2x. The up regulation of D 3 receptors by chronic haloperidol has been observed in the nucleus accumbens but not in the striatum w12x. The mechanisms underlying the reduced levels of synaptic protein mRNAs found in the nucleus accumbens after chronic haloperidol are unknown, but it is possible to speculate that the expression of synaptic proteins in the GABA neurons within the nucleus accumbens might be reduced by chronic haloperidol treatment. The present results are inconsistent with previous studies which reported the enhanced expression of synaptophysin by haloperidol treatment. The treatment with haloperidol Ž2 mgrkgrday. for 2 weeks has shown to induce a significant increase in synaptophysin mRNA lev-
241
els in the striatum but not in the nucleus accumbens and frontal cortex w8x. Furthermore, it was recently shown that the treatment with haloperidol decanoate Ž25 mgrkgr3 weeks. for 16 weeks increased synaptophysin mRNA in the dorsolateral striatum and frontoparietal cortex but not in the hippocampus w9x. Our results showed a slight increase in the striatal synaptophysin mRNA levels of rats treated with haloperidol, but the increased level was not statistically significant compared with that of control rats. The discrepancy between the present study and the previous work may be attributed to the use of RT-PCR to quantify mRNA, as compared to in situ histochemistry. Since we used total RNA extracted from the whole striatum, anatomical resolution is lost, consequently the change in synaptophysin mRNA levels in the subregion of striatum, the dorsolateral striatum, may be masked.
References w1x M.K. Bennett, N. Calakos, R.H. Scheller, Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones, Science 257 Ž1992. 255–259. w2x M.-L. Bouthenet, E. Souil, M.-P. Martres, P. Sokoloff, B. Giros, J.-C. Schwartz, Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA, Brain Res. 564 Ž1991. 203–219. w3x B.S. Bunney, A.A. Grace, Acute and chronic haloperidol treatment: comparison of effects on nigral dopaminergic cell activity, Life Sci. 23 Ž1978. 1715–1728. w4x P.E. Castillo, R. Janz, T.C. Sudhof, T. Tzounopoulos, R.C. Malenka, ¨ R.A. Nicoll, Rab 3A is essential for mossy fibre long-term potentiation in the hippocampus, Nature 388 Ž1997. 590–593. w5x P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. w6x S. Davis, J. Rodger, A. Hicks, J. Mallet, S. Laroche, Brain structure and task-specific increase in expression of the gene encoding syntaxin 1B during learning in the rat: a potential molecular marker for learning-induced synaptic plasticity in neural networks, Eur. J. Neurosci. 8 Ž1996. 2068–2074. w7x K. Dukas, P. Sarfati, N. Vaysse, L. Pradayrol, Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction, Anal. Biochem. 215 Ž1993. 66–72. w8x S.L. Eastwood, P.W.J. Burnet, P.J. Harrison, Striatal synaptophysin expression and haloperidol-induced synaptic plasticity, NeuroReport 5 Ž1994. 677–680. w9x S.L. Eastwood, J. Heffernan, P.J. Harrison, Chronic haloperidol treatment differentially affects the expression of synaptic and neuronal plasticity-associated genes, Mol. Psychiatry 2 Ž1997. 322–329. w10x L.A. Elferink, W.S. Trimble, R.H. Scheller, Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system, J. Biol. Chem. 264 Ž1989. 11061–11064. w11x A.M. Graybiel, Neurotransmitters and neuromodulators in the basal ganglia, Trends Neurosci. 13 Ž1990. 244–254. w12x M.J. Hurley, C.M. Stubbs, P. Jenner, C.D. Marsden, Effect of chronic treatment with typical and atypical neuroleptics on the expression of D 2 and D 3 receptors in rat brain, Psychopharmacology 128 Ž1996. 362–370. w13x S. Iwata, G.H.K. Hewlett, S.T. Ferrell, A.J. Czernik, K.F. Meiri, M.E. Gnegy, Increased in vivo phosphorylation state of neuromodulin and synapsin I in striatum from rats treated with repeated amphetamine, J. Pharmacol. Exp. Ther. 278 Ž1996. 1428–1434.
242
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
w14x W. Kamphuis, T. Smirnova, A. Hicks, H. Hendriksen, J. Mallet, F.H. Lopes da Silva, The expression of syntaxin 1BrGR33 mRNA is enhanced in the hippocampal kindling model of epileptogenesis, J. Neurochem. 65 Ž1995. 1974–1980. w15x R.F. Lane, C.D. Blaha, Chronic haloperidol decreases dopamine release in striatum and nucleus accumbens in vivo: depolarization block as a possible mechanism of action, Brain Res. Bull. 18 Ž1987. 135–138. w16x N. Lindefors, E. Brodin, U. Ungerstedt, Neuroleptic treatment induces region-specific changes in levels of neurokinin A and substance P in rat brain, Neuropeptides 7 Ž1986. 265–280. w17x T. Matsumoto, H. Uchimura, M. Hirano, J.S. Kim, H. Yokoo, M. Shimomura, T. Nakahara, K. Inoue, K. Oomagari, Differential effects of acute and chronic administration of haloperidol on homovanillic acid levels in discrete dopaminergic areas of rat brain, Eur. J. Pharmacol. 89 Ž1983. 27–33. w18x T. Morimoto, X.-H. Wang, M.-M. Poo, Overexpression of synaptotagmin modulates short-term synaptic plasticity at developing neuromuscular junctions, Neuroscience 82 Ž1998. 969–978. w19x T. Nakahara, M. Hirano, T. Matsumoto, T. Kuroki, Y. Tatebayashi, T. Tsutsumi, K. Nishiyama, H. Ooboshi, K. Nakamura, H. Yao, A. Shiraishi, M. Waki, H. Uchimura, Regional distribution of DNA and RNA in rat brain: a sensitive determination using high-performance liquid chromatography with electrochemical detection, Neurochem. Res. 15 Ž1990. 609–611. w20x U. Nudel, R. Zakut, M. Shani, S. Neuman, Z. Levy, D. Yaffe, The nucleotide sequence of the rat cytoplasmic b-actin gene, Nucl. Acids Res. 11 Ž1983. 1759–1771. w21x G.A. Oyler, G.A. Higgins, R.A. Hart, E. Battenberg, M. Billingsley, F.E. Bloom, M.C. Wilson, The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations, J. Cell Biol. 109 Ž1989. 3039–3052. w22x M.S. Perin, V.A. Fried, G.A. Mignery, R. Jahn, T.C. Sudhof, ¨ Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C, Nature 345 Ž1990. 260– 263. w23x V.M. Pickel, A.C. Towle, T.H. Joh, J. Chan, Gamma-aminobutyric acid in the medial rat nucleus accumbens: ultrastructural localization in neurons receiving monosynaptic input from catecholaminergic afferents, J. Comp. Neurol. 272 Ž1988. 1–14.
w24x P. Salin, M. Mercugliano, M.-F. Chesselet, Differential effects of chronic treatment with haloperidol and clozapine on the level of preprosomatostatin mRNA in the striatum, nucleus accumbens, and frontal cortex of the rat, Cell. Mol. Neurobiol. 10 Ž1990. 127–144. w25x T. Sasaki, J.L. Kennedy, J.N. Nobrega, Autoradiographic mapping of m opioid receptor changes in rat brain after long-term haloperidol treatment: relationship to the development of vacuous chewing movements, Psychopharmacology 128 Ž1996. 97–104. w26x B. Scatton, Differential regional development of tolerance to increase in dopamine turnover upon repeated neuroleptic administration, Eur. J. Pharmacol. 46 Ž1977. 363–369. w27x R.E. See, M.A. Chapman, M.A. Klitenick, Chronic neuroleptic administration decreases extracellular GABA in the nucleus accumbens but not in the caudate-putamen of rats, Brain Res. 588 Ž1992. 177–180. w28x T. Smirnova, S. Laroche, M.L. Errington, A.A. Hicks, T.V.P. Bliss, J. Mallet, Transsynaptic expression of a presynaptic glutamate receptor during hippocampal long-term potentiation, Science 262 Ž1993. 433–436. w29x T. Sollner, S.W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. ¨ Geromanos, P. Tempst, J.E. Rothman, SNAP receptors implicated in vesicle targeting and fusion, Nature 362 Ž1993. 318–324. w30x T.C. Sudhof, The synaptic vesicle cycle: a cascade of protein–pro¨ tein interactions, Nature 375 Ž1995. 645–653. w31x T.C. Sudhof, F. Lottspeich, P. Greengard, E. Mehl, R. Jahn, The ¨ cDNA and derived amino acid sequences for rat and human synaptophysin, Nucl. Acids Res. 15 Ž1987. 9607. w32x L. Vician, I.K. Lim, G. Ferguson, G. Tocco, M. Baudry, H.R. Herschman, Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain, Proc. Natl. Acad. Sci. USA 92 Ž1995. 2164–2168. w33x F.J. White, R.Y. Wang, Comparison of the effects of chronic haloperidol treatment on A9 and A10 dopamine neurons in the rat, Life Sci. 32 Ž1983. 983–993. w34x S. Yamada, H. Yokoo, S. Nishi, Chronic treatment with haloperidol modifies the sensitivity of autoreceptors that modulate dopamine release in rat striatum, Eur. J. Pharmacol. 232 Ž1993. 1–6. w35x A. Zahraoui, N. Touchot, P. Chardin, A. Tavitian, Complete coding sequences of the ras related rab 3 and 4 cDNAs, Nucl. Acids Res. 16 Ž1988. 1204.
Short communication
Effect of chronic haloperidol treatment on synaptic protein mRNAs in the rat brain Tatsuo Nakahara a,) , Kaoru Nakamura b, Tetsuyuki Tsutsumi b, Kijiro Hashimoto b, Hisao Hondo b, Shinji Hisatomi b, Keisuke Motomura b, Hideyuki Uchimura b a
b
Department of Chemistry, Faculty of Science, Kyushu UniÕersity, Ropponmatsu, Fukuoka 810-8560, Japan Laboratory of Neurochemistry, Center for Emotional and BehaÕioral Disorder, Hizen National Mental Hospital, Kanzaki, Saga 842-0192, Japan Accepted 18 August 1998
Abstract Chronic haloperidol treatment caused significant decreases in the levels of synaptotagmin I and IV, synaptobrevin II, syntaxin 1A and Rab 3A mRNAs in the nucleus accumbens but not in the prefrontal cortex medial field, striatum, substantia nigra and ventral tegmental area. No significant changes in SNAP 25 and synaptophysin mRNA levels were observed in any brain region examined. The reduced expression of synaptic proteins may be related to haloperidol-induced depolarization block of mesolimbic dopamine neurons. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Haloperidol; Synaptobrevin; Synaptotagmin; Syntaxin; Rab 3A; Nucleus accumbens
Neurotransmitter release from nerve terminals requires the docking and fusion of synaptic vesicles with presynaptic plasma membrane w30x. The interaction of vesicular membrane proteins termed v-SNAREs, synaptobrevin and synaptotagmin, with target membrane proteins termed tSNAREs, SNAP 25 and syntaxin, plays a critical role for the process of vesicular docking and fusion w29x. Recent evidence suggests that synaptic proteins are involved in synaptic plasticity. The expression of syntaxin 1B has been reported to be enhanced after induction of long-term potentiation w28x, during kindling w14x and during learning w6x. Synaptotagmin has also been shown to modulate short-term synaptic plasticity w18x. Synapsin I is involved in amphetamine-induced sensitization w13x, and knocking out Rab 3A abolishes mossy fibre long-term potentiation w4x. Chronic treatment with antipsychotics induces depolarization block of dopamine cell firing in the midbrain w3,33x and reduces dopamine release w15,34x and metabolism w17,26x in the terminal regions. Although the molecular mechanism underlying the neuronal and synaptic alterations produced by antipsychotics is unclear, synaptic proteins may be involved in the antipsychotic-induced synap-
) Corresponding author. [email protected]
Fax:
q81-092-726-4842;
E-mail:
tic changes. Indeed, Eastwood et al. w8,9x showed that the expression of gene encoding synaptophysin is increased after chronic haloperidol treatment. In view of the critical role played by the v- and t-SNARE complex in synaptic vesicle exocytosis, it is of interest to determine whether synaptic proteins such as SNAREs are involved in antipsychotic-induced synaptic plasticity. To elucidate the longterm effect of antipsychotics on synaptic vesicle exocytosis, we examined synaptic protein mRNA levels in various regions of rat brain after chronic haloperidol treatment. Male Wistar rats ŽKyudo Animal Laboratory, Kumamoto, Japan. initially weighing 240–260 g were housed four per cage, maintained on a 12 h lightr12 h dark cycle and given unlimited access to food and water. Sixteen animals received 25 mg eqr0.5 mlrkg to haloperidol of haloperidol decanoate ŽDainippon Pharmaceutical, Osaka, Japan. intramuscularly or an equal volume of sesame oil vehicle. Animals were killed after 28 days, and the brain was removed. Serial slices of 300 mm were made in a cryostat at y128C, and five brain regions were dissected freehand with a microknife as described previously w19x. The isolated tissues were stored at y808C. Total RNA was prepared from the brain tissues by the method of Chomczynski and Sacchi w5x. The levels of synaptic protein mRNAs in discrete brain regions of control and haloperidol-treated rats were quanti-
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 3 0 - 7
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
fied by reverse transcription-polymerase chain reaction ŽRT-PCR. with an endogenous internal standard, b-actin w7x. RT was performed on 500 ng total RNA for 1 h at 428C after 10 min incubation at 258C in a 10 ml reaction mixture containing 25 mM Tris–HCl ŽpH 8.3., 50 mM KCl, 5 mM MgCl 2 , 2 mM dithiothreitol, 1 mM each deoxynucleotide ŽdNTP., 10 U AMV reverse transcriptase ŽLife Sciences, St. Petersburg, FL., 10 U rebonuclease inhibitor ŽBoehringer. and 0.8 mg oligo ŽdT.15 primer ŽBoehringer.. The RT was terminated by heating the sample at 998C for 5 min. The multiplexed PCR was carried out in a 20 ml reaction mixture containing 10 mM Tris–HCl ŽpH 8.3., 50 mM KCl, 1.5 mM MgCl 2 , 2% dimethyl sulfoxide, 0.2 mM each dNTP, 0.075 mM each of 5X and 3X b-actin-specific primers, 1 mM each of 5X and 3X synaptic protein specific primers, 20 or 25 ng of reverse-transcribed total RNA, and 0.5 U Taq DNA polymerase ŽBoehringer.. The PCR primers used for amplification of b-actin and synaptic protein mRNAs are shown in Table 1. The PCR amplification was performed for 22 or 26 Žsyntaxin 1A. cycles, consisting of denaturation Ž948C, 45 s., annealing Ž568C, 45 s., and extension Ž728C, 75 s.. For the amplification of syntaxin 1A mRNA, 0.075 mM each of b-actin primer pairs was added to the reaction mixture after 6 cycles and PCR cycles were further continued. The PCR products were analyzed on a 10% polyacrylamide gel electrophoresis. Gels were stained with ethidium bromide, visualized with UV trans-illumination, photographed, and submitted to image analysis. Quantitative image analysis of the PCR fragments was performed using the NIH image program. The levels of synaptic protein mRNAs were calculated as the ratios of optical density of synaptic protein PCR products to that of the b-actin PCR product. Student’s t-test was used to compare vehicle and haloperidol groups.
239
Ethidium bromide staining of a polyacrylamide gel revealed a single band at the expected size of amplification product for each of the b-actin and synaptic protein cDNAs Ždata not shown.. Since the b-actin, Rab 3A and synaptobrevin II cDNA sequences were amplified using the pair of primers derived from different exons of the genes, the contamination by genomic DNA did not interfere with the signals of PCR products of these cDNAs. To determine the optimal amplifications, PCR was performed using different amount of reverse-transcribed total RNA and different numbers of cycles Ždata not shown.. These results indicated that amplification was exponential between 20 and 23 cycles for b-actin and synaptic protein mRNAs except for syntaxin 1A in all brain regions tested. Amplification was also exponential between 22 and 28 cycles for syntaxin 1A mRNA. The PCR products were proportional to RNA input over a range of 10 to 40 ng total RNA for b-actin and synaptic protein mRNAs. Twenty nanograms or 25 ng of reverse-transcribed RNA were amplified for 22 cycles or 26 cycles Žsyntaxin 1A. for the quantitation of relative amount of synaptic protein mRNAs in the rat brain. The quantitative RT-PCR was used to measure the amount of synaptic protein mRNAs in different brain regions following chronic treatment with haloperidol ŽFigs. 1 and 2.. The highest level of synaptotagmin I mRNA was found in the frontal cortex, a high level in the substantia nigra, a moderate level in the striatum and nucleus accumbens, and the lowest level in the ventral tegmental area. Similar distributions of mRNA were observed for synaptotagmin IV and synaptobrevin II. Regression analysis of mRNA levels revealed that the levels of synaptotagmin I mRNA in the control animals significantly correlated with those of synaptotagmin IV Ž r s 0.890, P - 0.05. and synaptobrevin II Ž r s 0.972, P - 0.01. in the rat brain.
Table 1 Oligonucleotide primers of synaptic proteins and b-actin used for PCR Gene
Sequence
b-Actin
5 -ACACTGTGCCCATCTATGAGG-3 X X 5 -CAACGTCACACTTCATGATGG-3 X X 5 -TGTTCAAGATCCTGATCATTGG-3 X X 5 -TGCACTGCATTGAAGGACTC-3 X X 5 -CTGGAGGAGATGCAGAGGAG-3 X X 5 -ACTGGATTTAAGCTTGTTACAGGG-3 X X 5 -CTGCACCTCCTCCAAATCTTAC-3 X X 5 -ATGATGATGATGAGGATGATGG-3 X X 5 -TTGCTACGTGTGGCAGCTAC-3 X X 5 -TTCAGCCGACGAGGAGTAGT-3 X X 5 -CATCGGAGAGTTCAAAGTTCCTAT-3 X X 5 -GACTCGTTGTAGTAGGGGTTGAGTG-3 X X 5 -TTTGTGGTGAACATCAAGGAAG-3 X X 5 -TCTTGAGAACCTGTCAAAACTCAG-3 X X 5 -CTCAGTGAGATCGAGACCAGG-3 X X 5 -ATGATGCCCAGAATCACACA-3
Rab 3A SNAP 25 Synaptobrevin II Synaptophysin Synaptotagmin I Synaptotagmin IV Syntaxin 1A
X
X
X
X
Position
PCR product Žbp.
Refs.
482–502 841–861 145–166 402–421 194–213 434–457 71–92 323–344 145–164 322–341 1242–1265 1526–1550 747–768 966–989 566–586 806–825
380
w20x
277
w35x
264
w21x
274
w10x
197
w31x
309
w22x
243
w32x
260
w1x
The top and lower primers of each amplification pair are 5 sense and 3 antisense primers, respectively.
240
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
treatment with haloperidol. The Rab 3A mRNA levels in the ventral tegmental area was decreased by 24% after chronic haloperidol treatment, but the decrease in the
Fig. 1. Effects of chronic treatment with haloperidol on the levels of synaptobrevin II ŽA., synaptotagmin I ŽB. and synaptotagmin IV mRNA ŽC. in various regions of rat brain. Rats received a single depot injection with haloperidol decanoate Ž25 mg eqrkg. or sesame oil vehicle and were killed after 4 weeks. Synaptic protein mRNA levels from control Žopen columns. and haloperidol-treated groups Žhatched columns. in the frontal cortex medial field ŽFCM., striatum ŽSTR., nucleus accumbens ŽNAC., substantia nigra ŽSN. and ventral tegmental area ŽVTA. were analyzed by quantitative RT-PCR. The values represent the mean"SEM of 15 or 16 samples ŽFCM, STR and NAC. and 7 or 8 samples ŽSN and VTA. derived from 16 animals per treatment group. ) P - 0.05, )) P - 0.01, compared with controls using two-tailed Student’s t-test.
Chronic treatment with haloperidol caused significant decreases in the levels of synaptotagmin I Žy32%, t s 2.34, df s 30, P - 0.05., synaptotagmin IV Žy43%, t s 3.75, df s 30, P - 0.01., synaptobrevin II Žy24%, t s 2.68, df s 29, P - 0.05., syntaxin 1A Žy29%, t s 2.73, df s 28, P - 0.05. and Rab 3A Žy25%, t s 3.26, df s 28, P - 0.01. in the nucleus accumbens ŽFigs. 1 and 2.. In the nucleus accumbens, the SNAP 25 Ž t s 1.05, df s 30, P ) 0.05. and synaptophysin Ž t s 1.17, df s 29, P ) 0.05. mRNA levels were not significantly affected by chronic
Fig. 2. Effects of chronic treatment with haloperidol on the levels of syntaxin 1A ŽA., SNAP 25 ŽB., Rab 3A ŽC. and synaptophysin mRNAs ŽD. in various regions of rat brain. Drug treatment and quntitation of mRNA levels from control Žopen columns. and haloperidol-treated groups Žhatched columns. were performed as described in the legend to Fig. 1. FCM, frontal cortex medial field; STR, striatum; NAC, nucleus accumbens; SN, substantia nigra; VTA, ventral tegmental area. ) P - 0.05, )) P - 0.01, compared with controls using two-tailed Student’s t-test.
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
mRNA levels was not significant Ž t s 1.82, df s 14, P ) 0.05.. There was no significant difference in the other synaptic protein mRNA levels between vehicle and chronic haloperidol treatments in any brain region examined. The synaptic protein mRNAs were selectively decreased in the nucleus accumbens but not in the striatum, frontal cortex and midbrain. Chronic neuroleptic treatment induces a delayed inactivation of dopamine neuron firing in the midbrain due to depolarization block w3,33x. The depolarization inactivation of dopamine cell in the substantia nigra and ventral tegmental area influences the neural activity in the striatum and nucleus accumbens. Dopamine release w15,34x and metabolism w17,26x are reduced in the terminal regions of nigrostriatal and mesolimbic dopamine systems after chronic treatment with haloperidol. The decrease in synaptic protein mRNA levels found in the nucleus accumbens after chronic haloperidol treatment may be due to the depolarization-induced inactivation of midbrain dopamine cell activity. In the present study, however, chronic haloperidol failed to induce a decrease in the synaptic protein mRNA levels in the striatum. Since mRNA is localized to the neuronal perikaryon, the different population of cell bodies received dopaminergic input in the striatum and nucleus accumbens may account for the lack of the striatal synaptic protein mRNA after haloperidol treatment. However, this does not seem unlikely because the neurons in the dorsal striatum have similar morphology with those in the nucleus accumbens w23x, and the ratio of RNA to DNA levels is similar in the striatum and nucleus accumbens w19x. Chronic haloperidol was reported to decrease GABA release w27x, substance P w16x, neurokinin A w16x, somatostatin mRNA w24x and opioid receptors w25x in the nucleus accumbens without affecting those in the striatum. These results suggest that the activity of GABA neurons within the nucleus accumbens is reduced by chronic haloperidol, because substance P coexists with GABA, and m-opioide receptors located on the GABA neurons w11x. The regional effect of haloperidol on the GABA neurons is probably related to the differential localization of dopamine receptor subtypes. Thus, D 3 dopamine receptors are abundant in the nucleus accumbens whereas D 2 receptors are located in the accumbens as well as the striatum w2x. The up regulation of D 3 receptors by chronic haloperidol has been observed in the nucleus accumbens but not in the striatum w12x. The mechanisms underlying the reduced levels of synaptic protein mRNAs found in the nucleus accumbens after chronic haloperidol are unknown, but it is possible to speculate that the expression of synaptic proteins in the GABA neurons within the nucleus accumbens might be reduced by chronic haloperidol treatment. The present results are inconsistent with previous studies which reported the enhanced expression of synaptophysin by haloperidol treatment. The treatment with haloperidol Ž2 mgrkgrday. for 2 weeks has shown to induce a significant increase in synaptophysin mRNA lev-
241
els in the striatum but not in the nucleus accumbens and frontal cortex w8x. Furthermore, it was recently shown that the treatment with haloperidol decanoate Ž25 mgrkgr3 weeks. for 16 weeks increased synaptophysin mRNA in the dorsolateral striatum and frontoparietal cortex but not in the hippocampus w9x. Our results showed a slight increase in the striatal synaptophysin mRNA levels of rats treated with haloperidol, but the increased level was not statistically significant compared with that of control rats. The discrepancy between the present study and the previous work may be attributed to the use of RT-PCR to quantify mRNA, as compared to in situ histochemistry. Since we used total RNA extracted from the whole striatum, anatomical resolution is lost, consequently the change in synaptophysin mRNA levels in the subregion of striatum, the dorsolateral striatum, may be masked.
References w1x M.K. Bennett, N. Calakos, R.H. Scheller, Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones, Science 257 Ž1992. 255–259. w2x M.-L. Bouthenet, E. Souil, M.-P. Martres, P. Sokoloff, B. Giros, J.-C. Schwartz, Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA, Brain Res. 564 Ž1991. 203–219. w3x B.S. Bunney, A.A. Grace, Acute and chronic haloperidol treatment: comparison of effects on nigral dopaminergic cell activity, Life Sci. 23 Ž1978. 1715–1728. w4x P.E. Castillo, R. Janz, T.C. Sudhof, T. Tzounopoulos, R.C. Malenka, ¨ R.A. Nicoll, Rab 3A is essential for mossy fibre long-term potentiation in the hippocampus, Nature 388 Ž1997. 590–593. w5x P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. w6x S. Davis, J. Rodger, A. Hicks, J. Mallet, S. Laroche, Brain structure and task-specific increase in expression of the gene encoding syntaxin 1B during learning in the rat: a potential molecular marker for learning-induced synaptic plasticity in neural networks, Eur. J. Neurosci. 8 Ž1996. 2068–2074. w7x K. Dukas, P. Sarfati, N. Vaysse, L. Pradayrol, Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction, Anal. Biochem. 215 Ž1993. 66–72. w8x S.L. Eastwood, P.W.J. Burnet, P.J. Harrison, Striatal synaptophysin expression and haloperidol-induced synaptic plasticity, NeuroReport 5 Ž1994. 677–680. w9x S.L. Eastwood, J. Heffernan, P.J. Harrison, Chronic haloperidol treatment differentially affects the expression of synaptic and neuronal plasticity-associated genes, Mol. Psychiatry 2 Ž1997. 322–329. w10x L.A. Elferink, W.S. Trimble, R.H. Scheller, Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system, J. Biol. Chem. 264 Ž1989. 11061–11064. w11x A.M. Graybiel, Neurotransmitters and neuromodulators in the basal ganglia, Trends Neurosci. 13 Ž1990. 244–254. w12x M.J. Hurley, C.M. Stubbs, P. Jenner, C.D. Marsden, Effect of chronic treatment with typical and atypical neuroleptics on the expression of D 2 and D 3 receptors in rat brain, Psychopharmacology 128 Ž1996. 362–370. w13x S. Iwata, G.H.K. Hewlett, S.T. Ferrell, A.J. Czernik, K.F. Meiri, M.E. Gnegy, Increased in vivo phosphorylation state of neuromodulin and synapsin I in striatum from rats treated with repeated amphetamine, J. Pharmacol. Exp. Ther. 278 Ž1996. 1428–1434.
242
T. Nakahara et al.r Molecular Brain Research 61 (1998) 238–242
w14x W. Kamphuis, T. Smirnova, A. Hicks, H. Hendriksen, J. Mallet, F.H. Lopes da Silva, The expression of syntaxin 1BrGR33 mRNA is enhanced in the hippocampal kindling model of epileptogenesis, J. Neurochem. 65 Ž1995. 1974–1980. w15x R.F. Lane, C.D. Blaha, Chronic haloperidol decreases dopamine release in striatum and nucleus accumbens in vivo: depolarization block as a possible mechanism of action, Brain Res. Bull. 18 Ž1987. 135–138. w16x N. Lindefors, E. Brodin, U. Ungerstedt, Neuroleptic treatment induces region-specific changes in levels of neurokinin A and substance P in rat brain, Neuropeptides 7 Ž1986. 265–280. w17x T. Matsumoto, H. Uchimura, M. Hirano, J.S. Kim, H. Yokoo, M. Shimomura, T. Nakahara, K. Inoue, K. Oomagari, Differential effects of acute and chronic administration of haloperidol on homovanillic acid levels in discrete dopaminergic areas of rat brain, Eur. J. Pharmacol. 89 Ž1983. 27–33. w18x T. Morimoto, X.-H. Wang, M.-M. Poo, Overexpression of synaptotagmin modulates short-term synaptic plasticity at developing neuromuscular junctions, Neuroscience 82 Ž1998. 969–978. w19x T. Nakahara, M. Hirano, T. Matsumoto, T. Kuroki, Y. Tatebayashi, T. Tsutsumi, K. Nishiyama, H. Ooboshi, K. Nakamura, H. Yao, A. Shiraishi, M. Waki, H. Uchimura, Regional distribution of DNA and RNA in rat brain: a sensitive determination using high-performance liquid chromatography with electrochemical detection, Neurochem. Res. 15 Ž1990. 609–611. w20x U. Nudel, R. Zakut, M. Shani, S. Neuman, Z. Levy, D. Yaffe, The nucleotide sequence of the rat cytoplasmic b-actin gene, Nucl. Acids Res. 11 Ž1983. 1759–1771. w21x G.A. Oyler, G.A. Higgins, R.A. Hart, E. Battenberg, M. Billingsley, F.E. Bloom, M.C. Wilson, The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations, J. Cell Biol. 109 Ž1989. 3039–3052. w22x M.S. Perin, V.A. Fried, G.A. Mignery, R. Jahn, T.C. Sudhof, ¨ Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C, Nature 345 Ž1990. 260– 263. w23x V.M. Pickel, A.C. Towle, T.H. Joh, J. Chan, Gamma-aminobutyric acid in the medial rat nucleus accumbens: ultrastructural localization in neurons receiving monosynaptic input from catecholaminergic afferents, J. Comp. Neurol. 272 Ž1988. 1–14.
w24x P. Salin, M. Mercugliano, M.-F. Chesselet, Differential effects of chronic treatment with haloperidol and clozapine on the level of preprosomatostatin mRNA in the striatum, nucleus accumbens, and frontal cortex of the rat, Cell. Mol. Neurobiol. 10 Ž1990. 127–144. w25x T. Sasaki, J.L. Kennedy, J.N. Nobrega, Autoradiographic mapping of m opioid receptor changes in rat brain after long-term haloperidol treatment: relationship to the development of vacuous chewing movements, Psychopharmacology 128 Ž1996. 97–104. w26x B. Scatton, Differential regional development of tolerance to increase in dopamine turnover upon repeated neuroleptic administration, Eur. J. Pharmacol. 46 Ž1977. 363–369. w27x R.E. See, M.A. Chapman, M.A. Klitenick, Chronic neuroleptic administration decreases extracellular GABA in the nucleus accumbens but not in the caudate-putamen of rats, Brain Res. 588 Ž1992. 177–180. w28x T. Smirnova, S. Laroche, M.L. Errington, A.A. Hicks, T.V.P. Bliss, J. Mallet, Transsynaptic expression of a presynaptic glutamate receptor during hippocampal long-term potentiation, Science 262 Ž1993. 433–436. w29x T. Sollner, S.W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. ¨ Geromanos, P. Tempst, J.E. Rothman, SNAP receptors implicated in vesicle targeting and fusion, Nature 362 Ž1993. 318–324. w30x T.C. Sudhof, The synaptic vesicle cycle: a cascade of protein–pro¨ tein interactions, Nature 375 Ž1995. 645–653. w31x T.C. Sudhof, F. Lottspeich, P. Greengard, E. Mehl, R. Jahn, The ¨ cDNA and derived amino acid sequences for rat and human synaptophysin, Nucl. Acids Res. 15 Ž1987. 9607. w32x L. Vician, I.K. Lim, G. Ferguson, G. Tocco, M. Baudry, H.R. Herschman, Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain, Proc. Natl. Acad. Sci. USA 92 Ž1995. 2164–2168. w33x F.J. White, R.Y. Wang, Comparison of the effects of chronic haloperidol treatment on A9 and A10 dopamine neurons in the rat, Life Sci. 32 Ž1983. 983–993. w34x S. Yamada, H. Yokoo, S. Nishi, Chronic treatment with haloperidol modifies the sensitivity of autoreceptors that modulate dopamine release in rat striatum, Eur. J. Pharmacol. 232 Ž1993. 1–6. w35x A. Zahraoui, N. Touchot, P. Chardin, A. Tavitian, Complete coding sequences of the ras related rab 3 and 4 cDNAs, Nucl. Acids Res. 16 Ž1988. 1204.