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Long-term treatment with haloperidol decreases the mRNA levels of complexin I, but not complexin II, in rat prefrontal cortex, nucleus accumbens and ventral tegmental area
Neuroscience Letters 290 (2000) 29±32
www.elsevier.com/locate/neulet
Long-term treatment with haloperidol decreases the mRNA levels of complexin I, but not complexin II, in rat prefrontal cortex, nucleus accumbens and ventral tegmental area Tatsuo Nakahara a,*, Keisuke Motomura b, Kijiro Hashimoto b, Hiroshi Ueki b, Leo Gotoh a, Hisao Hondo b, Tetsuyuki Tsutsumi b, Toshihide Kuroki c, Makoto Hirano b, Hideyuki Uchimura b a
Department of Chemistry, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan Center for Emotional and Behavioral Disorder, Hizen National Mental Hospital, Kanzaki, Saga 842-0192, Japan c Department of Neuropsychiatry, Faculty of Medicine, Kyushu University, Maidashi, Fukuoka 812-8582, Japan
b
Received 13 June 2000; accepted 28 June 2000
Abstract The effect of long-term treatment with haloperidol on gene expression of the presynaptic protein complexins was investigated in the discrete brain regions of rats, using reverse transcription-polymerase chain reaction. Four-weektreatment with haloperidol decanoate (25 mg eq/kg) produced a signi®cant decrease in the mRNA levels of complexin I in the medial prefrontal cortex, nucleus accumbens and ventral tegmental area, but not in the striatum and substantia nigra. No signi®cant changes in complexin II mRNA levels were observed in any brain region examined here. The reduced expression of complexin I may be associated with the haloperidol-induced depolarization block of mesocorticolimbic dopamine neurons. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Complexin; Haloperidol; Synaptic protein; Frontal cortex; Nucleus accumbens; Ventral tegmental area
Release of neurotransmitter from axon terminals requires the docking and fusion of synaptic vesicles with presynaptic plasma membrane [17]. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins consisting synaptobrevin II, syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP25) play a critical role in mediating exocytosis of synaptic vesicles [15]. NSF-dependent hydrolysis of ATP activated by alphasoluble NSF attachment protein (a-SNAP) disassembles the SNARE complex [6,16]. Complexins [4,7,18], a family of the presynaptic proteins, compete with a-SNAP for binding to the SNARE complex [7], suggesting an important role in regulation of SNARE function [5,7,13]. Two complexin isoforms, complexins I and II, are highly enriched in the brain, where they colocalize with syntaxin and SNAP-25 [7]. Moreover, complexins I and II have been shown to predominantly express at the somatic and dendritic synapses, respectively [3,18,19]. The synaptic function * Corresponding author. Tel.: 181-92-726-4754; fax: 181-92726-4842. E-mail address: [email protected] (T. Nakahara).
may depend on the site of synapses on neurons; the dendritic or spiny synapses generate the excitatory output, whereas the somatic synapses link with the inhibitory one [14]. The expression of complexin proteins may therefore be related to the neural functions such as excitatory or inhibitory action. In this regard, Harrison and Eastwood [3] demonstrated that complexins I and II are primarily expressed in inhibitory and excitatory neurons, respectively of the human brain. Furthermore, they found a signi®cant loss of complexin II proteins and its mRNAs in the medial temporal lobe of schizophrenic patients [3]. We previously reported that long-term treatment with haloperidol decanoate produced a signi®cant decrease in the mRNA levels of synaptotagmin I and IV, synaptobrevin II, syntaxin 1A and Rab 3A in rat nucleus accumbens [9]. These changes in the synaptic proteins may contribute to the antipsychotic-induced synaptic plasticity, which may be the basis for the antipsychotic action. However, it remains to be determined which neurons are involved in the reduction of the synaptic protein expression following long-term haloperidol treatment. In the present study, we examined the mRNA levels of complexins I and II in the discrete brain
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 31 2- 4
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T. Nakahara et al. / Neuroscience Letters 290 (2000) 29±32
regions of rats receiving long-term treatment with haloperidol decanoate. Male Wistar rats (Kyudo Animal Laboratory, Kumamoto, Japan) initially weighing 240±260 g were housed four per cage, maintained on a 12 h light/dark cycle and given access to food and water ad libitum. All procedures were done in accordance with the Animal Care Guidelines at Kyushu University. Sixteen animals received 25 mg eq/0.5 ml/kg to haloperidol of haloperidol decanoate (Dainippon Pharmaceutical Co., Osaka, Japan) intramuscularly or an equal volume of sesame oil vehicle containing 15 mg/ml benzyl alcohol. Animals were killed after 28 days, and the brain was removed. Serial slices of 300 mm were made in a cryostat at 2128C, and ®ve brain regions were dissected freehand with a microknife as described previously [10]. The isolated tissues were stored at 2808C. Total RNA was prepared from the brain tissues as described previously [9]. The levels of complexin mRNAs in discrete brain regions of control and haloperidol-treated rats were quanti®ed by reverse transcription-polymerase chain reaction (RT-PCR) with an endogenous internal standard, b-actin, as previously described [8]. RNA samples were treated with deoxyribonuclease (Life Technologies, Gaitherburg, MD) according to the manufacturer's protocol. RT was performed on 500 ng total RNA for 1 h at 428C in a 10 ml reaction mixture containing 25 mM Tris±HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 1 mM each deoxynucleotide (dNTP), 10 U AMV reverse transcriptase (Life Sciences Inc., St. Petersburg, FL), 10 U rebonuclease inhibitor (Boehringer, Mannheim, Germany) and 0.8 mg oligo (dT)15 primer (Boehringer). The RT was terminated by heating the sample at 958C for 2 min. The multiplexed PCR was carried out in a 20 ml reaction mixture containing 10 mM Tris±HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 2 % dimethyl sulfoxide, 0.2 mM each dNTP, 0.1 mM each of 5 0 and 3 0 b-actinspeci®c primers, 1 mM each of 5 0 and 3 0 complexin-speci®c primers, 20 or 25 ng of reverse-transcribed total RNA, and 0.5 U Taq DNA polymerase (Boehringer). The primer sequences used for ampli®cation of the coding regions of complexin I, II [4,7,18] and b-actin [11] were as follows: complexin I, 5 0 -CCACTGCAGGACATGTTCAA-3 0 (forward); 5 0 -TAAGATTGGTAGGGAGGGGG-3 0 (reverse); target sequence 310 bp; complexin II; 5 0 -TATCTACCCTGCTTGGTGCC-3 0 (forward); 5 0 -ATCTCGAATCTGCTGCCG-3 0 (reverse); target sequence 349 bp; bactin, 5 0 -TCATGCCATCCTGCGTCTGGAC-CT-3 0 (forward); 5 0 -CCGGACTCATCGTACTCCTGCTTG-3 0 (reverse); target sequence 582 bp. The PCR ampli®cation was performed for 30 cycles, consisting of denaturation (94 8C, 45 s), annealing (658C, 45 s), and extension (728C, 75 s). After eight cycles, 0.1 mM each of b-actin primer pair was added to the reaction mixture and PCR cycles were further continued. The complexin I or complexin II mRNA was coampli®ed with b-actin mRNA, and
complexin I and II mRNA levels were determined using b-actin mRNA levels as an internal standard. 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 complexin mRNAs were calculated as the ratios of optical density of complexin PCR products to that of the b-actin PCR product. Student's t-test was used to compare vehicle and haloperidol groups. Ethidium bromide staining of a polyacrylamide gel revealed a single band at the expected size of ampli®cation product for each of the b-actin and complexin cDNAs (data not shown). To determine the optimal ampli®cations, PCR was performed using different amount of reverse-transcribed total RNA and different numbers of cycles (data not shown). These results indicated that ampli®cation was exponential between 20 and 23 cycles for b-actin and between 28 and 31 cycles for complexin I and II mRNAs in all brain regions tested. The PCR products were proportional to RNA input over a range of 10 to 40 ng total RNA for b-actin and complexin I and II mRNAs. Twenty nanograms or 25 ng of reverse-transcribed RNA were ampli®ed for 30 cycles for the quantitation of relative amount of complexin mRNAs in the rat brain. The quantitative RT-PCR was used to measure the amount of synaptic protein mRNAs in different brain regions following long-term treatment with haloperidol decanoate (Fig. 1). The rank order of the density of complexin I mRNA was as follows: the ventral tegmental area . the substantia nigra . the medial prefrontal cortex and striatum . the nucleus accumbens. In contrast, the rank order of the density of complexin II mRNA was as follows: the medial prefrontal cortex . the nucleus accumbens . the striatum, substantia nigra and ventral tegmental area. Regression analysis of mRNA levels revealed that, in the control animals, the mRNA levels of complexin I highly correlated with those of SNAP25 or Rab 3A (data from [9]), although it did not reach a signi®cance (r 0:805, P 0:100 for SNAP25; r 0:820, P 0:089 for Rab 3A). Complexin II mRNA levels signi®cantly correlated with those of syntaxin 1A (r 0:935, P 0:020). Long-term treatment with haloperidol decanoate produced a signi®cant decrease in the mRNA levels of complexin I in the medial prefrontal cortex (223%, t 2:78, d:f: 25, P , 0:05), nucleus accumbens (237%, t 2:14, d:f: 30, P , 0:05), and ventral tegmental area (231%, t 2:86, d:f: 13, P , 0:01) (Fig. 1). The complexin I mRNA levels in the striatum (t 0:35, d:f: 25, P 0:73) and substantia nigra (t 20:30, d:f: 14, P 0:77) did not alter following long-term haloperidol treatment. There was no signi®cant difference in complexin II mRNA levels between vehicle and long-term haloperidol treatment groups in any brain region examined. The most striking ®nding of the present study was that
T. Nakahara et al. / Neuroscience Letters 290 (2000) 29±32
Fig. 1. Effects of long-term treatment with haloperidol decanoate on the levels of complexin I (A) and complexin II mRNAs (B) in discreate brain regions of rats. Rats received a single depot injection with haloperidol decanoate (25 mg eq/kg) or sesame oil vehicle and were killed after 4 weeks. Complexin mRNA levels of control (open columns) and haloperidol-treated groups (hatched columns) in the medial prefrontal cortex (mPFC), striatum (STR), nucleus accumbens (NAC), substantia nigra (SN) and ventral tegmental area (VTA) were determined by quantitative RT-PCR. The values represent the mean ^ SEM of 13±16 samples (mPFC, STR and NAC) and seven or eight samples (SN and VTA) derived from 16 animals per each treatment group. *P , 0:05, compared with controls using two-tailed Student's t-test.
long-term treatment with haloperidol decanoate decreased the mRNA levels of complexin I, but not complexin II, in rat prefrontal cortex, nucleus accumbens and ventral tegmental area. Since complexin proteins may play an inhibitory role in the release of neurotransmitters from presynaptic axon terminals [5,13], the decrease in the expression of complexin I proteins may increase the release of neurotransmitters at the inhibitory synaptic site, resulting in the inhibition of neural activity. Therefore, the decrease in complexin I mRNA levels in the prefrontal cortex, nucleus accumbens and ventral tegmental area may be associated with the depolarization block of mesolimbocortical dopamine neurons induced by long-term haloperidol [2]. The lack of the ability of haloperidol to alter complexin II mRNA expression may be attributed to the differential localization of dopamine receptor subtypes, as previously discussed for the synaptic protein gene expression [9]. We previously reported that haloperidol decanoate treatment caused a decrease in synaptic protein mRNAs in the
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nucleus accumbens, while leaving those in other brain regions unaffected [9]. The differential effect of long-term haloperidol on complexin I and synaptic protein mRNAs may be due to the differential localization of these proteins on neurons; complexin I is localized in axosomatic synapses [3,18,19], while synaptic proteins exist in all types of synapses. Recently, Eastwood et al. [1] have shown that chronic intermittent treatment with haloperidol (1 mg/kg, i.p./day) for 14 days had no effect on both comlexin I and II mRNA levels, but decreased the ratio of complexin II mRNA to complexin I mRNA, in the dorsolateral striatum and frontoparietal cortex. The discrepancy between their results and the present study 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 was lost, which may consequently mask the change in complexin mRNAs in the subregion of striatum such as the dorsolateral part. The differences in routes (chronic intermittent treatment vs. depot treatment) and doses (total dose: 1 mg/kg £ 14 days 14 mg/kg vs. 25 mg/kg) of haloperidol may also in¯uence the results. Pharmacokinetics and bioavailability of haloperidol have been reported to differ between the two treatment procedures [12]. It should be noted that complexin I mRNA levels correlated with SNAP25 or Rab 3A mRNA levels, while complexin II mRNA levels correlated with syntaxin 1A mRNA levels. In line with the role of complexins in regulating SNARE function [4,6,11], the gene expression of complexins may thus correspond to that of synaptic proteins. Further studies on the microscopic distribution of these proteins are needed to con®rm this hypothesis. [1] Eastwood, S.L., Burnet, P.W.J. and Harrison, P.J., Expression of complexin I and II mRNAs and their regulation by antipsychotic drugs in the rat forebrain, Synapse, 36 (2000) 167±177. [2] Grace, A.A., Bunney, B.S., Moore, H. and Todd, C.L., Dopamine-cell depolarization block as a model for the therapeutic actions of antipsychotic drugs, Trends Neurosci., 20 (1997) 31±37. [3] Harrison, P.J. and Eastwood, S.L., Preferential involvement of excitatory neurons in medial temporal lobe in schizophrenia, Lancet, 352 (1998) 1669±1673. [4] Ishizuka, T., Saisu, H., Odani, S. and Abe, T., Synaphin: a protein associated with the docking/fusion complex in presynaptic terminals, Biochem. Biophys. Res. Commun., 213 (1995) 1107±1114. [5] Itakura, M., Misawa, H., Sekiguchi, M. and Takahashi, M., Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles, Biochem. Biophys. Res. Commun., 265 (1999) 691±696. [6] McMahon, H.T. and Sudhof, T.C., Synaptic core complex of synaptobrevin, syntaxin, and SNAP-25 forms high af®nity a-SNAP binding site, J. Biol. Chem., 270 (1995) 2213±2217. [7] McMahon, H.T., Missler, M., Li, C. and Sudhof, T.C., Complexins: cytosolic proteins that regulate SNAP receptor function, Cell, 83 (1995) 111±119. [8] Nakahara, T., Kuroki, T., Hashimoto, K., Hondo, H., Tsutsumi, T., Motomura, K., Ueki, H., Hirano, M. and Uchimura,
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[9]
[10]
[11] [12] [13]
T. Nakahara et al. / Neuroscience Letters 290 (2000) 29±32 H., Effect of atypical antipsychotics on phencyclidineinduced expression of arc in rat brain, NeuroReport, 11 (2000) 551±555. Nakahara, T., Nakamura, K., Tsutsumi, T., Hashimoto, K., Hondo, H., Hisatomi, S., Motomura, K. and Uchimura, H., Effect of chronic haloperidol treatment on synaptic protein mRNAs in the rat brain, Mol. Brain Res., 61 (1998) 238±242. Nakahara, T., Hirano, M., Matsumoto, T., Kuroki, T., Tatebayashi, Y., Tsutsumi, T., Nishiyama, K., Ooboshi, H., Nakamura, K., Yao, H., Shiraishi, A., Waki, M. and Uchimura, H., Regional distribution of DNA and RNA in rat brain: a sensitive determination using high-performance liquid chromatography with electrochemical detection, Neurochem. Res., 15 (1990) 609±611. Nudel, U., Zakut, R., Shani, M., Neuman, S., Levy, Z. and Yaffe, D., The nucleotide sequence of the rat cytoplasmic bactin gene, Nucl. Acids Res., 11 (1983) 1759±1771. Oh-e, Y., Miyazaki, H., Matsunaga, Y. and Hashimoto, M., Pharmacokinetics of haloperidol decanoate in rats, J. Pharmacobio.-Dyn., 14 (1991) 615±622. Ono, S., Baux, G., Sekiguchi, M., Fossier, P., Morel, N.F., Nihonmatsu, I., Hirata, K., Awaji, T., Takahashi, S. and Takahashi, M., Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals, Eur. J. Neurosci., 10 (1998) 2143±2152.
[14] Peters, A. and Palay, S.L., The morphology of synapses, J. Neurocytol., 25 (1996) 687±700. [15] Sollner, T., Whiteheart, S.W., Brunner, M., ErdjumentBromage, H., Geromanos, S., Tempst, P. and Rothman, J.E., SNAP receptors implicated in vesicle targeting and fusion, Nature, 362 (1993) 318±324. [16] Sollner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H. and Rothman, J.E., A protein assembly-disassembly pathway in vitro may correspond to sequential steps of synaptic vesicle docking, activation, and fusion, Cell, 75 (1993) 409± 418. [17] Sudhof, T.C., The synaptic vesicle cycle: a cascade of protein-protein interactions, Nature, 375 (1995) 645±653. [18] Takahashi, S., Yamamoto, H., Matsuda, Z., Ogawa, M., Yagyu, K., Taniguchi, T., Miyata, T., Kaba, H., Higuchi, T., Okutani, F. and Fujimoto, S., Identi®cation of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses, FEBS Lett., 368 (1995) 455±460. [19] Yamada, M., Saisu, H., Ishizuka, T., Takahashi, H. and Abe, T., Immunohistochemical distribution of the two isoforms of synaphin/complexin involved in neurotransmitter release: localization at the distinct central nervous system regions and synaptic types, Neuroscience, 93 (1999) 7±18.
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Long-term treatment with haloperidol decreases the mRNA levels of complexin I, but not complexin II, in rat prefrontal cortex, nucleus accumbens and ventral tegmental area Tatsuo Nakahara a,*, Keisuke Motomura b, Kijiro Hashimoto b, Hiroshi Ueki b, Leo Gotoh a, Hisao Hondo b, Tetsuyuki Tsutsumi b, Toshihide Kuroki c, Makoto Hirano b, Hideyuki Uchimura b a
Department of Chemistry, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan Center for Emotional and Behavioral Disorder, Hizen National Mental Hospital, Kanzaki, Saga 842-0192, Japan c Department of Neuropsychiatry, Faculty of Medicine, Kyushu University, Maidashi, Fukuoka 812-8582, Japan
b
Received 13 June 2000; accepted 28 June 2000
Abstract The effect of long-term treatment with haloperidol on gene expression of the presynaptic protein complexins was investigated in the discrete brain regions of rats, using reverse transcription-polymerase chain reaction. Four-weektreatment with haloperidol decanoate (25 mg eq/kg) produced a signi®cant decrease in the mRNA levels of complexin I in the medial prefrontal cortex, nucleus accumbens and ventral tegmental area, but not in the striatum and substantia nigra. No signi®cant changes in complexin II mRNA levels were observed in any brain region examined here. The reduced expression of complexin I may be associated with the haloperidol-induced depolarization block of mesocorticolimbic dopamine neurons. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Complexin; Haloperidol; Synaptic protein; Frontal cortex; Nucleus accumbens; Ventral tegmental area
Release of neurotransmitter from axon terminals requires the docking and fusion of synaptic vesicles with presynaptic plasma membrane [17]. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins consisting synaptobrevin II, syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP25) play a critical role in mediating exocytosis of synaptic vesicles [15]. NSF-dependent hydrolysis of ATP activated by alphasoluble NSF attachment protein (a-SNAP) disassembles the SNARE complex [6,16]. Complexins [4,7,18], a family of the presynaptic proteins, compete with a-SNAP for binding to the SNARE complex [7], suggesting an important role in regulation of SNARE function [5,7,13]. Two complexin isoforms, complexins I and II, are highly enriched in the brain, where they colocalize with syntaxin and SNAP-25 [7]. Moreover, complexins I and II have been shown to predominantly express at the somatic and dendritic synapses, respectively [3,18,19]. The synaptic function * Corresponding author. Tel.: 181-92-726-4754; fax: 181-92726-4842. E-mail address: [email protected] (T. Nakahara).
may depend on the site of synapses on neurons; the dendritic or spiny synapses generate the excitatory output, whereas the somatic synapses link with the inhibitory one [14]. The expression of complexin proteins may therefore be related to the neural functions such as excitatory or inhibitory action. In this regard, Harrison and Eastwood [3] demonstrated that complexins I and II are primarily expressed in inhibitory and excitatory neurons, respectively of the human brain. Furthermore, they found a signi®cant loss of complexin II proteins and its mRNAs in the medial temporal lobe of schizophrenic patients [3]. We previously reported that long-term treatment with haloperidol decanoate produced a signi®cant decrease in the mRNA levels of synaptotagmin I and IV, synaptobrevin II, syntaxin 1A and Rab 3A in rat nucleus accumbens [9]. These changes in the synaptic proteins may contribute to the antipsychotic-induced synaptic plasticity, which may be the basis for the antipsychotic action. However, it remains to be determined which neurons are involved in the reduction of the synaptic protein expression following long-term haloperidol treatment. In the present study, we examined the mRNA levels of complexins I and II in the discrete brain
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 31 2- 4
30
T. Nakahara et al. / Neuroscience Letters 290 (2000) 29±32
regions of rats receiving long-term treatment with haloperidol decanoate. Male Wistar rats (Kyudo Animal Laboratory, Kumamoto, Japan) initially weighing 240±260 g were housed four per cage, maintained on a 12 h light/dark cycle and given access to food and water ad libitum. All procedures were done in accordance with the Animal Care Guidelines at Kyushu University. Sixteen animals received 25 mg eq/0.5 ml/kg to haloperidol of haloperidol decanoate (Dainippon Pharmaceutical Co., Osaka, Japan) intramuscularly or an equal volume of sesame oil vehicle containing 15 mg/ml benzyl alcohol. Animals were killed after 28 days, and the brain was removed. Serial slices of 300 mm were made in a cryostat at 2128C, and ®ve brain regions were dissected freehand with a microknife as described previously [10]. The isolated tissues were stored at 2808C. Total RNA was prepared from the brain tissues as described previously [9]. The levels of complexin mRNAs in discrete brain regions of control and haloperidol-treated rats were quanti®ed by reverse transcription-polymerase chain reaction (RT-PCR) with an endogenous internal standard, b-actin, as previously described [8]. RNA samples were treated with deoxyribonuclease (Life Technologies, Gaitherburg, MD) according to the manufacturer's protocol. RT was performed on 500 ng total RNA for 1 h at 428C in a 10 ml reaction mixture containing 25 mM Tris±HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 1 mM each deoxynucleotide (dNTP), 10 U AMV reverse transcriptase (Life Sciences Inc., St. Petersburg, FL), 10 U rebonuclease inhibitor (Boehringer, Mannheim, Germany) and 0.8 mg oligo (dT)15 primer (Boehringer). The RT was terminated by heating the sample at 958C for 2 min. The multiplexed PCR was carried out in a 20 ml reaction mixture containing 10 mM Tris±HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 2 % dimethyl sulfoxide, 0.2 mM each dNTP, 0.1 mM each of 5 0 and 3 0 b-actinspeci®c primers, 1 mM each of 5 0 and 3 0 complexin-speci®c primers, 20 or 25 ng of reverse-transcribed total RNA, and 0.5 U Taq DNA polymerase (Boehringer). The primer sequences used for ampli®cation of the coding regions of complexin I, II [4,7,18] and b-actin [11] were as follows: complexin I, 5 0 -CCACTGCAGGACATGTTCAA-3 0 (forward); 5 0 -TAAGATTGGTAGGGAGGGGG-3 0 (reverse); target sequence 310 bp; complexin II; 5 0 -TATCTACCCTGCTTGGTGCC-3 0 (forward); 5 0 -ATCTCGAATCTGCTGCCG-3 0 (reverse); target sequence 349 bp; bactin, 5 0 -TCATGCCATCCTGCGTCTGGAC-CT-3 0 (forward); 5 0 -CCGGACTCATCGTACTCCTGCTTG-3 0 (reverse); target sequence 582 bp. The PCR ampli®cation was performed for 30 cycles, consisting of denaturation (94 8C, 45 s), annealing (658C, 45 s), and extension (728C, 75 s). After eight cycles, 0.1 mM each of b-actin primer pair was added to the reaction mixture and PCR cycles were further continued. The complexin I or complexin II mRNA was coampli®ed with b-actin mRNA, and
complexin I and II mRNA levels were determined using b-actin mRNA levels as an internal standard. 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 complexin mRNAs were calculated as the ratios of optical density of complexin PCR products to that of the b-actin PCR product. Student's t-test was used to compare vehicle and haloperidol groups. Ethidium bromide staining of a polyacrylamide gel revealed a single band at the expected size of ampli®cation product for each of the b-actin and complexin cDNAs (data not shown). To determine the optimal ampli®cations, PCR was performed using different amount of reverse-transcribed total RNA and different numbers of cycles (data not shown). These results indicated that ampli®cation was exponential between 20 and 23 cycles for b-actin and between 28 and 31 cycles for complexin I and II mRNAs in all brain regions tested. The PCR products were proportional to RNA input over a range of 10 to 40 ng total RNA for b-actin and complexin I and II mRNAs. Twenty nanograms or 25 ng of reverse-transcribed RNA were ampli®ed for 30 cycles for the quantitation of relative amount of complexin mRNAs in the rat brain. The quantitative RT-PCR was used to measure the amount of synaptic protein mRNAs in different brain regions following long-term treatment with haloperidol decanoate (Fig. 1). The rank order of the density of complexin I mRNA was as follows: the ventral tegmental area . the substantia nigra . the medial prefrontal cortex and striatum . the nucleus accumbens. In contrast, the rank order of the density of complexin II mRNA was as follows: the medial prefrontal cortex . the nucleus accumbens . the striatum, substantia nigra and ventral tegmental area. Regression analysis of mRNA levels revealed that, in the control animals, the mRNA levels of complexin I highly correlated with those of SNAP25 or Rab 3A (data from [9]), although it did not reach a signi®cance (r 0:805, P 0:100 for SNAP25; r 0:820, P 0:089 for Rab 3A). Complexin II mRNA levels signi®cantly correlated with those of syntaxin 1A (r 0:935, P 0:020). Long-term treatment with haloperidol decanoate produced a signi®cant decrease in the mRNA levels of complexin I in the medial prefrontal cortex (223%, t 2:78, d:f: 25, P , 0:05), nucleus accumbens (237%, t 2:14, d:f: 30, P , 0:05), and ventral tegmental area (231%, t 2:86, d:f: 13, P , 0:01) (Fig. 1). The complexin I mRNA levels in the striatum (t 0:35, d:f: 25, P 0:73) and substantia nigra (t 20:30, d:f: 14, P 0:77) did not alter following long-term haloperidol treatment. There was no signi®cant difference in complexin II mRNA levels between vehicle and long-term haloperidol treatment groups in any brain region examined. The most striking ®nding of the present study was that
T. Nakahara et al. / Neuroscience Letters 290 (2000) 29±32
Fig. 1. Effects of long-term treatment with haloperidol decanoate on the levels of complexin I (A) and complexin II mRNAs (B) in discreate brain regions of rats. Rats received a single depot injection with haloperidol decanoate (25 mg eq/kg) or sesame oil vehicle and were killed after 4 weeks. Complexin mRNA levels of control (open columns) and haloperidol-treated groups (hatched columns) in the medial prefrontal cortex (mPFC), striatum (STR), nucleus accumbens (NAC), substantia nigra (SN) and ventral tegmental area (VTA) were determined by quantitative RT-PCR. The values represent the mean ^ SEM of 13±16 samples (mPFC, STR and NAC) and seven or eight samples (SN and VTA) derived from 16 animals per each treatment group. *P , 0:05, compared with controls using two-tailed Student's t-test.
long-term treatment with haloperidol decanoate decreased the mRNA levels of complexin I, but not complexin II, in rat prefrontal cortex, nucleus accumbens and ventral tegmental area. Since complexin proteins may play an inhibitory role in the release of neurotransmitters from presynaptic axon terminals [5,13], the decrease in the expression of complexin I proteins may increase the release of neurotransmitters at the inhibitory synaptic site, resulting in the inhibition of neural activity. Therefore, the decrease in complexin I mRNA levels in the prefrontal cortex, nucleus accumbens and ventral tegmental area may be associated with the depolarization block of mesolimbocortical dopamine neurons induced by long-term haloperidol [2]. The lack of the ability of haloperidol to alter complexin II mRNA expression may be attributed to the differential localization of dopamine receptor subtypes, as previously discussed for the synaptic protein gene expression [9]. We previously reported that haloperidol decanoate treatment caused a decrease in synaptic protein mRNAs in the
31
nucleus accumbens, while leaving those in other brain regions unaffected [9]. The differential effect of long-term haloperidol on complexin I and synaptic protein mRNAs may be due to the differential localization of these proteins on neurons; complexin I is localized in axosomatic synapses [3,18,19], while synaptic proteins exist in all types of synapses. Recently, Eastwood et al. [1] have shown that chronic intermittent treatment with haloperidol (1 mg/kg, i.p./day) for 14 days had no effect on both comlexin I and II mRNA levels, but decreased the ratio of complexin II mRNA to complexin I mRNA, in the dorsolateral striatum and frontoparietal cortex. The discrepancy between their results and the present study 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 was lost, which may consequently mask the change in complexin mRNAs in the subregion of striatum such as the dorsolateral part. The differences in routes (chronic intermittent treatment vs. depot treatment) and doses (total dose: 1 mg/kg £ 14 days 14 mg/kg vs. 25 mg/kg) of haloperidol may also in¯uence the results. Pharmacokinetics and bioavailability of haloperidol have been reported to differ between the two treatment procedures [12]. It should be noted that complexin I mRNA levels correlated with SNAP25 or Rab 3A mRNA levels, while complexin II mRNA levels correlated with syntaxin 1A mRNA levels. In line with the role of complexins in regulating SNARE function [4,6,11], the gene expression of complexins may thus correspond to that of synaptic proteins. Further studies on the microscopic distribution of these proteins are needed to con®rm this hypothesis. [1] Eastwood, S.L., Burnet, P.W.J. and Harrison, P.J., Expression of complexin I and II mRNAs and their regulation by antipsychotic drugs in the rat forebrain, Synapse, 36 (2000) 167±177. [2] Grace, A.A., Bunney, B.S., Moore, H. and Todd, C.L., Dopamine-cell depolarization block as a model for the therapeutic actions of antipsychotic drugs, Trends Neurosci., 20 (1997) 31±37. [3] Harrison, P.J. and Eastwood, S.L., Preferential involvement of excitatory neurons in medial temporal lobe in schizophrenia, Lancet, 352 (1998) 1669±1673. [4] Ishizuka, T., Saisu, H., Odani, S. and Abe, T., Synaphin: a protein associated with the docking/fusion complex in presynaptic terminals, Biochem. Biophys. Res. Commun., 213 (1995) 1107±1114. [5] Itakura, M., Misawa, H., Sekiguchi, M. and Takahashi, M., Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles, Biochem. Biophys. Res. Commun., 265 (1999) 691±696. [6] McMahon, H.T. and Sudhof, T.C., Synaptic core complex of synaptobrevin, syntaxin, and SNAP-25 forms high af®nity a-SNAP binding site, J. Biol. Chem., 270 (1995) 2213±2217. [7] McMahon, H.T., Missler, M., Li, C. and Sudhof, T.C., Complexins: cytosolic proteins that regulate SNAP receptor function, Cell, 83 (1995) 111±119. [8] Nakahara, T., Kuroki, T., Hashimoto, K., Hondo, H., Tsutsumi, T., Motomura, K., Ueki, H., Hirano, M. and Uchimura,
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[9]
[10]
[11] [12] [13]
T. Nakahara et al. / Neuroscience Letters 290 (2000) 29±32 H., Effect of atypical antipsychotics on phencyclidineinduced expression of arc in rat brain, NeuroReport, 11 (2000) 551±555. Nakahara, T., Nakamura, K., Tsutsumi, T., Hashimoto, K., Hondo, H., Hisatomi, S., Motomura, K. and Uchimura, H., Effect of chronic haloperidol treatment on synaptic protein mRNAs in the rat brain, Mol. Brain Res., 61 (1998) 238±242. Nakahara, T., Hirano, M., Matsumoto, T., Kuroki, T., Tatebayashi, Y., Tsutsumi, T., Nishiyama, K., Ooboshi, H., Nakamura, K., Yao, H., Shiraishi, A., Waki, M. and Uchimura, H., Regional distribution of DNA and RNA in rat brain: a sensitive determination using high-performance liquid chromatography with electrochemical detection, Neurochem. Res., 15 (1990) 609±611. Nudel, U., Zakut, R., Shani, M., Neuman, S., Levy, Z. and Yaffe, D., The nucleotide sequence of the rat cytoplasmic bactin gene, Nucl. Acids Res., 11 (1983) 1759±1771. Oh-e, Y., Miyazaki, H., Matsunaga, Y. and Hashimoto, M., Pharmacokinetics of haloperidol decanoate in rats, J. Pharmacobio.-Dyn., 14 (1991) 615±622. Ono, S., Baux, G., Sekiguchi, M., Fossier, P., Morel, N.F., Nihonmatsu, I., Hirata, K., Awaji, T., Takahashi, S. and Takahashi, M., Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals, Eur. J. Neurosci., 10 (1998) 2143±2152.
[14] Peters, A. and Palay, S.L., The morphology of synapses, J. Neurocytol., 25 (1996) 687±700. [15] Sollner, T., Whiteheart, S.W., Brunner, M., ErdjumentBromage, H., Geromanos, S., Tempst, P. and Rothman, J.E., SNAP receptors implicated in vesicle targeting and fusion, Nature, 362 (1993) 318±324. [16] Sollner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H. and Rothman, J.E., A protein assembly-disassembly pathway in vitro may correspond to sequential steps of synaptic vesicle docking, activation, and fusion, Cell, 75 (1993) 409± 418. [17] Sudhof, T.C., The synaptic vesicle cycle: a cascade of protein-protein interactions, Nature, 375 (1995) 645±653. [18] Takahashi, S., Yamamoto, H., Matsuda, Z., Ogawa, M., Yagyu, K., Taniguchi, T., Miyata, T., Kaba, H., Higuchi, T., Okutani, F. and Fujimoto, S., Identi®cation of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses, FEBS Lett., 368 (1995) 455±460. [19] Yamada, M., Saisu, H., Ishizuka, T., Takahashi, H. and Abe, T., Immunohistochemical distribution of the two isoforms of synaphin/complexin involved in neurotransmitter release: localization at the distinct central nervous system regions and synaptic types, Neuroscience, 93 (1999) 7±18.