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Differential effects of clozapine on ethanol-induced ascorbic acid release in mouse and rat striatum
Neuroscience Letters 380 (2005) 83–87
Differential effects of clozapine on ethanol-induced ascorbic acid release in mouse and rat striatum Yue Hou, Chun Fu Wu∗ , Jing Yu Yang, Ling Tu, Pei Fei Gu, Xiu Li Bi Department of Pharmacology, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China Received 10 November 2004; received in revised form 24 December 2004; accepted 8 January 2005
Abstract Previous studies have shown that acute systemic administration of ethanol-induced striatal ascorbic acid (AA) release in mice and rats. In the present study, in vivo brain microdialysis coupled with high performance liquid chromatography (HPLC) with electrochemical detection (ECD) was used to comparatively evaluate the effects of clozapine on ethanol-induced AA release in mouse and rat striatum. The results showed that clozapine, at the dose of 15 mg/kg i.p., had no effect on basal AA or ethanol-induced AA release in rat striatum. The potentiating effect of clozapine on ethanol-induced striatal AA release was still observed in rats, at the higher dose of 30 mg/kg. In contrast, clozapine significantly inhibited ethanol-induced AA release in mouse striatum, at the dose of 15 and 30 mg/kg, without affecting basal AA release. The present study suggested that clozapine differentially regulated ethanol-induced AA release in the mouse and rat striatum. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ascorbic acid; Clozapine; Striatum; Difference; Rat; Mouse
It has been shown recently that ascorbic acid (AA) acts not only as an antioxidant, but also as a neuromodulator in the central nervous system [3,7]. For example, AA can block the d-amphetamine-induced stereotypy and potentiate the cataleptic effect of haloperidol in rats [2,11]. Pre-treatment with AA potentiates ethanol-induced loss of righting reflex in mice [15]. Previous studies have shown that acute systemic administration of ethanol induced striatal AA release in mice and rats. Drugs acting on the different neuronal systems differentially modulated ethanol-induced striatal AA release. Activation of 5-HT1A receptors by 8-OH-DPAT [13] or inhibition of NMDA receptors by MK-801 [5] antagonizes, whereas, use of clozapine, an atypical neuroleptic, potentiates ethanolinduced AA release in the rat striatum [6]. Such effects of 8-OH-DPAT and MK-801 on ethanol-induced striatal AA release were observed in mice as well. However, it is reported the existence of the differentiation of rat and mouse brain in ∗
Corresponding author. Tel.: +86 24 23843567; fax: +86 24 23843567. E-mail addresses: [email protected], [email protected] (C.F. Wu). 0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.01.021
response to some of the centrally active drugs. For example, rats and mice showed different vulnerability of DA neurons to MPP+ toxicity. Striatal concentrations of MPP+ are greater in rats than in mice given an identical dose of MPTP, a dose that damages DA neurons in mice but not in rats [10]. Therefore, it is plausible to suggest to taking a caution when one evaluates a centrally active drug by using only the rat or mouse. In a preliminary study, we observed that clozapine could inhibit in mice, instead of increasing in rats, the striatal AA release induced by ethanol. In order to verify whether such discrepancy might be due to the difference in animal species and to provide more information about the species difference in response to the centrally active drugs, in the present study, the effects of clozapine on ethanol-induced AA release in rat and mouse striatum were studied under the same conditions. Female and male Wistar rats weighing 200–250 g and Swiss-Kunming mice weighing 25–30 g, were used in the experiments. The animals were provided by the Experimental Animal Center of Shenyang Pharmaceutical University. The animals were housed under standard conditions with food and water ad libitum and maintained on 12L:12D cycle (light on 06:30). All animal use procedures were in accordance with
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the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China on November 14, 1988. The experiments were carried out under the approval of the Committee of Experimental Animal Administration of the University. Animals were anesthetized with chloral hydrate (350 mg/ kg i.p.) and implanted with Hospal AN 69 dialysis fibers (310 m i.d., Dasco, Bologna, Italy) transversally through either the rat striata (coordinates: A +1.5 mm from bregma, V −5.6 mm from occipital bone) or the mouse striata (coordinates: A +0.6 mm from bregma, V −3.5 mm from occipital bone). The procedure used to prepare and implant the dialysis probe was essentially the same as that described previously [12,16]. Brain dialysis was performed about 24 h after probe implantation in freely moving animals. Ringer’s solution (147 mM NaCl, 2.2 mM CaCl2 , and 4 mM KCl) was pumped through the dialysis probe at the constant rate of 5 l/min. After a 30-min washout, the dialysis samples were collected every 10 min and analyzed. AA contents in the samples were measured by HPLC with electrochemical detection as described before [14]. Test solutions (saline, ethanol or clozapine) were administered when the baseline of AA output was stable in last three samples. Clozapine (Changzhou Pharmaceutical Factory, Changzhou, China) was dissolved in 0.3 M HCl in saline, after which the pH was adjusted to pH 5–6 with sodium hydroxide and was intraperitoneally administered 10 min before saline or ethanol. Ethanol (Shenyang Reagents Co., China) was diluted with saline to 20% before use, and was injected intraperitoneally either at a dose of 3.0 g/kg for rats [6] or at a dose of 4.0 g/kg for mice [16]. The drug doses used were according to the previous experiments demonstrating their effects [6]. At the end of the experiments, the position of the dialysis fiber was verified, and the data were discarded if the fiber was positioned incorrectly. Statistical analysis was carried out by using SAS Software (SAS Institute, Cary, NC). To assess the significance of differences between groups, summed effects of drugs over the course of an experiment were used to compare treatment area under the curve (AUC) by multifactor analysis of variance (ANOVA) followed by Fisher’s least-significant difference post hoc tests. Two-way ANOVA was used to evaluate the interaction between drug treatment and ethanol groups. AA values are expressed as the percentage changes compared with the respective basal value, which was the mean of three consecutive samples within a variation of 10%. Basal AA release from rat striata was 19.6 ± 1.6 ng/ 50 l/10 min (n = 15), which was not rectified by retrieving rate of dialysis probe in vitro. Ethanol, at the dose of 3.0 g/kg i.p., induced a significant increase in striatal AA release in both male and female rats (for male: P < 0.001, Fig. 1a; for female: P < 0.001, Fig. 1b). Clozapine, at the dose of 15 mg/kg i.p., did not affect the basal levels of AA or the increasing effect of ethanol on AA release (Fig. 1a and
Fig. 1. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) rats. Clozapine was administered intraperitoneally, at the dose of 15 mg/kg, 10 min before ethanol administration (3.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–8 rats. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the corresponding control group. (--: saline; --: ethanol 3.0 g/kg; --: clozapine; -×-: clozapine + ethanol 3.0 g/kg).
b). When the dose of clozapine was increased to 30 mg/kg i.p., it markedly potentiated ethanol-induced AA release (for male: P < 0.001, Fig. 2a; for female: P < 0.001, Fig. 2b), without affecting the basal AA levels in the striatum. Two-way ANOVA analysis showed no significant interaction between clozapine 15 mg/kg and ethanol, but a significant interaction between clozapine 30 mg/kg and ethanol in the action on AA release (for male: clozapine 15 mg/kg: F(1,22) = 0.85, P = 0.3668, clozapine 30 mg/kg: F(1,22) = 15.67, P = 0.0007; for female: clozapine 15 mg/kg: F(1,22) = 0.44, P = 0.5151, clozapine 30 mg/kg: F(1,23) = 22.41, P = 0.0001). Basal AA release from mouse striata was 28.0 ± 2.5 ng/ 50 l/10 min (n = 15), which was not rectified by retrieving rate of dialysis probe in vitro. Ethanol, at the dose of 4.0 g/kg i.p., also induced a significant increase in striatal AA release in both male and female mice (for male: P < 0.001, Fig. 3a; for female: P < 0.001, Fig. 3b). Clozapine, at the dose of 15 mg/kg i.p., did not affect the basal levels of AA release, however, markedly inhibited ethanol-induced AA release (for
Y. Hou et al. / Neuroscience Letters 380 (2005) 83–87
Fig. 2. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) rats. Clozapine was administered intraperitoneally, at the dose of 30 mg/kg, 10 min before ethanol administration (3.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–8 rats. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the corresponding control group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the corresponding ethanol group. (--: saline; --: ethanol 3.0 g/kg; --: clozapine; -×-: clozapine + ethanol 3.0 g/kg).
male: P < 0.001, Fig. 3a; for female: P < 0.001, Fig. 3b). Similarly, clozapine, at the dose of 30 mg/kg i.p., also markedly inhibited ethanol-induced AA release (for male: P < 0.001, Fig. 4a; for female: P < 0.001, Fig. 4b), without affecting the basal AA levels in the striatum. Two-way ANOVA analysis showed a significant interaction between clozapine 15 mg/kg, 30 mg/kg and ethanol in the action on AA release (for male: clozapine 15 mg/kg: F(1,18) = 9.44, P = 0.0066, clozapine 30 mg/kg: F(1,20) = 25.91, P = 0.0001; for female: clozapine 15 mg/kg: F(1,18) = 6.17, P = 0.0231, clozapine 30 mg/kg: F(1,17) = 13.40, P = 0.0019). In previous studies, female rats were used in studying the effect of clozapine on ethanol-induced striatal AA release [6]. The present results confirmed and extended previous observations, suggesting that there was no significant difference between female and male rats, either in the basal levels of AA
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Fig. 3. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) mice. Clozapine was administered intraperitoneally, at the dose of 15 mg/kg, 10 min before ethanol administration (4.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–7 mice. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the corresponding control group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the corresponding ethanol group. (--: saline; --: ethanol 4.0 g/kg; --: clozapine; -×-: clozapine + ethanol 4.0 g/kg).
or in the potentiating effect of clozapine on ethanol-induced striatal AA release. However, in mice of both genders, clozapine showed a significant inhibitory effect on ethanol-induced striatal AA release, which was opposite to its potentiating effect on ethanolinduced AA release in rat striatum. This is the first reported evidence that acute administration of clozapine differentially regulated ethanol-induced striatal AA release in mice and rats. In the present study, the doses of ethanol given to the rats and mice were comparable because they were calculated according to the body surface area of the animal species. Although the doses of clozapine were not such calculated, according to our observations, as the doses increased, clozapine showed its potentiating effect on ethanol-induced AA release in rats. However, in mice clozapine dose-dependently inhibited ethanol-induced AA release. At a dose higher than
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decrease in brain GPx activity [9]. Appearance of the banded expression of aldolase C during development differs between rat and mouse. This species difference is found in both mRNA and protein expression [8]. Species difference in the mechanism of 8-OH-DPAT-induced hypothermia is observed that a presynaptic 5-HT1A receptor agonist action is involved in the mouse, whereas the 5-HT1A receptor mediating hypothermia in the rat appears to be located postsynaptically with respect to 5-HT neurons [1]. Differential effects of cocaine on local cerebral glucose utilization are observed in the mouse and the rat. Cocaine increases glucose utilization in the nucleus accumbens of the rats, while it decreases metabolic rate in most of brain areas of mice [17]. Taking all above observations into account, it suggests that there exist some neurochemical and functional differences between rat and mouse brain, although in most situations the neurophysiological or neuropharmacological responses of these two species to the exogenous stimuli are very similar. Therefore, the present results implicated the need of caution for the explanation of the mechanism of action of a neuroactive drug by only using one species of the animal. In conclusion, the present study demonstrated for the first time that acute administration of clozapine differentially regulated ethanol-induced striatal AA release in mice and rats. However, the mechanism of species difference between mouse and rat striatum in response to the effect of clozapine on ethanol-induced AA release merits further study.
Fig. 4. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) mice. Clozapine was administered intraperitoneally, at the dose of 30 mg/kg, 10 min before ethanol administration (4.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–6 mice. ** P < 0.01, *** P < 0.001 compared with the corresponding control group. # P < 0.05, ## P < 0.01 compared with the corresponding ethanol group. (--: saline; --: ethanol 4.0 g/kg; --: clozapine; -×-: clozapine + ethanol 4.0 g/kg).
30 mg/kg, that was 60 mg/kg, the inhibitory effect was still significant, however, some mice died during the experiment due to the deep suppression of the central nervous system by the combination of clozapine and ethanol. Therefore, it was reasonable to assume that such different effects of clozapine come from the differences in animal species, but not from the dose regime. Studies have found the existence of the species difference between mouse and rat brain in response to some lesions or drug treatments [4,9]. Differential effect of dietary selenium was observed on the long-term neurotoxicity induced by MDMA in mice and rats. Selenium deficiency impairs the cellular antioxidant status of the mouse brain and that this effect not only exacerbates the dopaminergic toxicity induced by MDMA but also facilitates the appearance of damage to 5-HT containing neurons. In contrast, rats fed a seleniumdeficient diet do not show any change in MDMA-induced neurotoxicity of 5-HT nerve terminals in spite of showing a
References [1] D.J. Bill, M. Knight, E.A. Forster, A. Fletcher, Direct evidence for an important species difference in the mechanism of 8-OH-DPATinduced hypothermia, Br. J. Pharmacol. 103 (1991) 1857–1864. [2] R.L. Dorris, R.E. Dill, Potentiation of haloperidol-induced catalepsy by ascorbic acid in rats and nonhuman primates, Pharmacol. Biochem. Behav. 24 (1986) 781–783. [3] R.A. Grunewald, Ascorbic acid in the brain, Brain Res. Rev. 18 (1993) 123–133. [4] I. Kandish, T. Van Groen, Differences in lesion-induced hippocampal plasticity between mice and rats, Neuroscience 116 (2003) 499–509. [5] J. Liu, C.F. Wu, W. Liu, H.L. Zhang, C.L. Li, Involvement of corticostriatal glutamatergic pathway in regulation of ethanol-induced ascorbic acid release in rat striatum, Addict. Biol. 4 (1999) 272–282. [6] W. Liu, C.F. Wu, J. Liu, M. Huang, K. Xiao, Differential effects of acute administration of haloperidol and clozapine on ethanol-induced ascorbic acid release in rat striatum, Eur. J. Pharmacol. 398 (2000) 333–339. [7] G.V. Rebec, R.C. Pierce, A vitamin as neuromodulator: ascorbic acid release into the extracellular fluid of the brain regulates dopaminergic and glutamatergic transmission, Prog. Neurobiol. 43 (1994) 537–565. [8] A. Rivkin, K. Herrup, Development of cerebellar modules: extrinsic control of late-phase zebrin II pattern and the exploration of rat/mouse species differences, Mol. Cell Neurosci. 24 (2003) 887–901. [9] V. Sanchez, J. Camarero, E. O’Shea, A.R. Green, M.I. Colado, Differential effect of dietary selenium on the long-term neurotoxicity induced by MDMA in mice and rats, Neuropharmacology 44 (2003) 449–461. [10] R.G.W. Staal, K.A. Hogan, C.L. Liang, D.C. German, P.K. Sonsalla, In vitro studies of striatal vesicles containing the vesicular
Y. Hou et al. / Neuroscience Letters 380 (2005) 83–87 monoamine transporter (VMAT2): rat versus mouse differences in sequestration of 1-methyl-4-phenylpyridinium, J. Pharmacol. Exp. Ther. 293 (2000) 329–335. [11] L.K. White, M. Carpenter, M. Block, A. Basse-Tomusk, T.W. Gardiner, G.V. Rebec, Ascorbate antagonizes the behavioral effects of amphetamine by a central mechanism, Psychopharmacology 94 (1988) 284–287. [12] C.F. Wu, R. Bertorelli, M. Sacconi, G. Pepeu, S. Consolo, Decrease of brain acetylcholine release in aging freely moving rats detected by microdialysis, Neurobiol. Aging 9 (1988) 357–361. [13] C.F. Wu, J. Liu, S. Consolo, W. Liu, 5-HT1A receptors mediate inhibition of ethanol-induced ascorbic acid release in rat striatum studied by microdialysis, Neurosci. Lett. 250 (1998) 95–98.
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[14] C.F. Wu, W. Liu, J. Liu, C.H. Yeh, dl-Fenfluramine inhibits ethanolinduced ascorbic acid release in rat striatum studied by microdialysis, Addict. Biol. 3 (1998) 295–308. [15] C.F. Wu, H.L. Zhang, W. Liu, Potentiation of ethanol-induced loss of the righting reflex by ascorbic acid in mice: interaction with dopamine antagonists, Pharmacol. Biochem. Behav. 65 (2000) 413–418. [16] P.G. Yan, C.F. Wu, M. Huang, W. Liu, Role of nitric oxide in ethanol-induced ascorbic acid release in striatum of freely moving mice, Toxicol. Lett. 145 (2003) 69–78. [17] A. Zocchi, G. Conti, F. Orzi, Differential effects of cocaine on local cerebral glucose utilization in the mouse and in the rat, Neurosci. Lett. 306 (2001) 177–180.
Differential effects of clozapine on ethanol-induced ascorbic acid release in mouse and rat striatum Yue Hou, Chun Fu Wu∗ , Jing Yu Yang, Ling Tu, Pei Fei Gu, Xiu Li Bi Department of Pharmacology, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China Received 10 November 2004; received in revised form 24 December 2004; accepted 8 January 2005
Abstract Previous studies have shown that acute systemic administration of ethanol-induced striatal ascorbic acid (AA) release in mice and rats. In the present study, in vivo brain microdialysis coupled with high performance liquid chromatography (HPLC) with electrochemical detection (ECD) was used to comparatively evaluate the effects of clozapine on ethanol-induced AA release in mouse and rat striatum. The results showed that clozapine, at the dose of 15 mg/kg i.p., had no effect on basal AA or ethanol-induced AA release in rat striatum. The potentiating effect of clozapine on ethanol-induced striatal AA release was still observed in rats, at the higher dose of 30 mg/kg. In contrast, clozapine significantly inhibited ethanol-induced AA release in mouse striatum, at the dose of 15 and 30 mg/kg, without affecting basal AA release. The present study suggested that clozapine differentially regulated ethanol-induced AA release in the mouse and rat striatum. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ascorbic acid; Clozapine; Striatum; Difference; Rat; Mouse
It has been shown recently that ascorbic acid (AA) acts not only as an antioxidant, but also as a neuromodulator in the central nervous system [3,7]. For example, AA can block the d-amphetamine-induced stereotypy and potentiate the cataleptic effect of haloperidol in rats [2,11]. Pre-treatment with AA potentiates ethanol-induced loss of righting reflex in mice [15]. Previous studies have shown that acute systemic administration of ethanol induced striatal AA release in mice and rats. Drugs acting on the different neuronal systems differentially modulated ethanol-induced striatal AA release. Activation of 5-HT1A receptors by 8-OH-DPAT [13] or inhibition of NMDA receptors by MK-801 [5] antagonizes, whereas, use of clozapine, an atypical neuroleptic, potentiates ethanolinduced AA release in the rat striatum [6]. Such effects of 8-OH-DPAT and MK-801 on ethanol-induced striatal AA release were observed in mice as well. However, it is reported the existence of the differentiation of rat and mouse brain in ∗
Corresponding author. Tel.: +86 24 23843567; fax: +86 24 23843567. E-mail addresses: [email protected], [email protected] (C.F. Wu). 0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.01.021
response to some of the centrally active drugs. For example, rats and mice showed different vulnerability of DA neurons to MPP+ toxicity. Striatal concentrations of MPP+ are greater in rats than in mice given an identical dose of MPTP, a dose that damages DA neurons in mice but not in rats [10]. Therefore, it is plausible to suggest to taking a caution when one evaluates a centrally active drug by using only the rat or mouse. In a preliminary study, we observed that clozapine could inhibit in mice, instead of increasing in rats, the striatal AA release induced by ethanol. In order to verify whether such discrepancy might be due to the difference in animal species and to provide more information about the species difference in response to the centrally active drugs, in the present study, the effects of clozapine on ethanol-induced AA release in rat and mouse striatum were studied under the same conditions. Female and male Wistar rats weighing 200–250 g and Swiss-Kunming mice weighing 25–30 g, were used in the experiments. The animals were provided by the Experimental Animal Center of Shenyang Pharmaceutical University. The animals were housed under standard conditions with food and water ad libitum and maintained on 12L:12D cycle (light on 06:30). All animal use procedures were in accordance with
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the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China on November 14, 1988. The experiments were carried out under the approval of the Committee of Experimental Animal Administration of the University. Animals were anesthetized with chloral hydrate (350 mg/ kg i.p.) and implanted with Hospal AN 69 dialysis fibers (310 m i.d., Dasco, Bologna, Italy) transversally through either the rat striata (coordinates: A +1.5 mm from bregma, V −5.6 mm from occipital bone) or the mouse striata (coordinates: A +0.6 mm from bregma, V −3.5 mm from occipital bone). The procedure used to prepare and implant the dialysis probe was essentially the same as that described previously [12,16]. Brain dialysis was performed about 24 h after probe implantation in freely moving animals. Ringer’s solution (147 mM NaCl, 2.2 mM CaCl2 , and 4 mM KCl) was pumped through the dialysis probe at the constant rate of 5 l/min. After a 30-min washout, the dialysis samples were collected every 10 min and analyzed. AA contents in the samples were measured by HPLC with electrochemical detection as described before [14]. Test solutions (saline, ethanol or clozapine) were administered when the baseline of AA output was stable in last three samples. Clozapine (Changzhou Pharmaceutical Factory, Changzhou, China) was dissolved in 0.3 M HCl in saline, after which the pH was adjusted to pH 5–6 with sodium hydroxide and was intraperitoneally administered 10 min before saline or ethanol. Ethanol (Shenyang Reagents Co., China) was diluted with saline to 20% before use, and was injected intraperitoneally either at a dose of 3.0 g/kg for rats [6] or at a dose of 4.0 g/kg for mice [16]. The drug doses used were according to the previous experiments demonstrating their effects [6]. At the end of the experiments, the position of the dialysis fiber was verified, and the data were discarded if the fiber was positioned incorrectly. Statistical analysis was carried out by using SAS Software (SAS Institute, Cary, NC). To assess the significance of differences between groups, summed effects of drugs over the course of an experiment were used to compare treatment area under the curve (AUC) by multifactor analysis of variance (ANOVA) followed by Fisher’s least-significant difference post hoc tests. Two-way ANOVA was used to evaluate the interaction between drug treatment and ethanol groups. AA values are expressed as the percentage changes compared with the respective basal value, which was the mean of three consecutive samples within a variation of 10%. Basal AA release from rat striata was 19.6 ± 1.6 ng/ 50 l/10 min (n = 15), which was not rectified by retrieving rate of dialysis probe in vitro. Ethanol, at the dose of 3.0 g/kg i.p., induced a significant increase in striatal AA release in both male and female rats (for male: P < 0.001, Fig. 1a; for female: P < 0.001, Fig. 1b). Clozapine, at the dose of 15 mg/kg i.p., did not affect the basal levels of AA or the increasing effect of ethanol on AA release (Fig. 1a and
Fig. 1. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) rats. Clozapine was administered intraperitoneally, at the dose of 15 mg/kg, 10 min before ethanol administration (3.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–8 rats. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the corresponding control group. (--: saline; --: ethanol 3.0 g/kg; --: clozapine; -×-: clozapine + ethanol 3.0 g/kg).
b). When the dose of clozapine was increased to 30 mg/kg i.p., it markedly potentiated ethanol-induced AA release (for male: P < 0.001, Fig. 2a; for female: P < 0.001, Fig. 2b), without affecting the basal AA levels in the striatum. Two-way ANOVA analysis showed no significant interaction between clozapine 15 mg/kg and ethanol, but a significant interaction between clozapine 30 mg/kg and ethanol in the action on AA release (for male: clozapine 15 mg/kg: F(1,22) = 0.85, P = 0.3668, clozapine 30 mg/kg: F(1,22) = 15.67, P = 0.0007; for female: clozapine 15 mg/kg: F(1,22) = 0.44, P = 0.5151, clozapine 30 mg/kg: F(1,23) = 22.41, P = 0.0001). Basal AA release from mouse striata was 28.0 ± 2.5 ng/ 50 l/10 min (n = 15), which was not rectified by retrieving rate of dialysis probe in vitro. Ethanol, at the dose of 4.0 g/kg i.p., also induced a significant increase in striatal AA release in both male and female mice (for male: P < 0.001, Fig. 3a; for female: P < 0.001, Fig. 3b). Clozapine, at the dose of 15 mg/kg i.p., did not affect the basal levels of AA release, however, markedly inhibited ethanol-induced AA release (for
Y. Hou et al. / Neuroscience Letters 380 (2005) 83–87
Fig. 2. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) rats. Clozapine was administered intraperitoneally, at the dose of 30 mg/kg, 10 min before ethanol administration (3.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–8 rats. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the corresponding control group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the corresponding ethanol group. (--: saline; --: ethanol 3.0 g/kg; --: clozapine; -×-: clozapine + ethanol 3.0 g/kg).
male: P < 0.001, Fig. 3a; for female: P < 0.001, Fig. 3b). Similarly, clozapine, at the dose of 30 mg/kg i.p., also markedly inhibited ethanol-induced AA release (for male: P < 0.001, Fig. 4a; for female: P < 0.001, Fig. 4b), without affecting the basal AA levels in the striatum. Two-way ANOVA analysis showed a significant interaction between clozapine 15 mg/kg, 30 mg/kg and ethanol in the action on AA release (for male: clozapine 15 mg/kg: F(1,18) = 9.44, P = 0.0066, clozapine 30 mg/kg: F(1,20) = 25.91, P = 0.0001; for female: clozapine 15 mg/kg: F(1,18) = 6.17, P = 0.0231, clozapine 30 mg/kg: F(1,17) = 13.40, P = 0.0019). In previous studies, female rats were used in studying the effect of clozapine on ethanol-induced striatal AA release [6]. The present results confirmed and extended previous observations, suggesting that there was no significant difference between female and male rats, either in the basal levels of AA
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Fig. 3. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) mice. Clozapine was administered intraperitoneally, at the dose of 15 mg/kg, 10 min before ethanol administration (4.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–7 mice. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the corresponding control group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the corresponding ethanol group. (--: saline; --: ethanol 4.0 g/kg; --: clozapine; -×-: clozapine + ethanol 4.0 g/kg).
or in the potentiating effect of clozapine on ethanol-induced striatal AA release. However, in mice of both genders, clozapine showed a significant inhibitory effect on ethanol-induced striatal AA release, which was opposite to its potentiating effect on ethanolinduced AA release in rat striatum. This is the first reported evidence that acute administration of clozapine differentially regulated ethanol-induced striatal AA release in mice and rats. In the present study, the doses of ethanol given to the rats and mice were comparable because they were calculated according to the body surface area of the animal species. Although the doses of clozapine were not such calculated, according to our observations, as the doses increased, clozapine showed its potentiating effect on ethanol-induced AA release in rats. However, in mice clozapine dose-dependently inhibited ethanol-induced AA release. At a dose higher than
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decrease in brain GPx activity [9]. Appearance of the banded expression of aldolase C during development differs between rat and mouse. This species difference is found in both mRNA and protein expression [8]. Species difference in the mechanism of 8-OH-DPAT-induced hypothermia is observed that a presynaptic 5-HT1A receptor agonist action is involved in the mouse, whereas the 5-HT1A receptor mediating hypothermia in the rat appears to be located postsynaptically with respect to 5-HT neurons [1]. Differential effects of cocaine on local cerebral glucose utilization are observed in the mouse and the rat. Cocaine increases glucose utilization in the nucleus accumbens of the rats, while it decreases metabolic rate in most of brain areas of mice [17]. Taking all above observations into account, it suggests that there exist some neurochemical and functional differences between rat and mouse brain, although in most situations the neurophysiological or neuropharmacological responses of these two species to the exogenous stimuli are very similar. Therefore, the present results implicated the need of caution for the explanation of the mechanism of action of a neuroactive drug by only using one species of the animal. In conclusion, the present study demonstrated for the first time that acute administration of clozapine differentially regulated ethanol-induced striatal AA release in mice and rats. However, the mechanism of species difference between mouse and rat striatum in response to the effect of clozapine on ethanol-induced AA release merits further study.
Fig. 4. Effect of clozapine on ethanol-induced striatal ascorbic acid (AA) release in male (a) and female (b) mice. Clozapine was administered intraperitoneally, at the dose of 30 mg/kg, 10 min before ethanol administration (4.0 g/kg, i.p.). AA release is expressed as the percentage change from baseline. Data shown are means ± S.E.M. for 5–6 mice. ** P < 0.01, *** P < 0.001 compared with the corresponding control group. # P < 0.05, ## P < 0.01 compared with the corresponding ethanol group. (--: saline; --: ethanol 4.0 g/kg; --: clozapine; -×-: clozapine + ethanol 4.0 g/kg).
30 mg/kg, that was 60 mg/kg, the inhibitory effect was still significant, however, some mice died during the experiment due to the deep suppression of the central nervous system by the combination of clozapine and ethanol. Therefore, it was reasonable to assume that such different effects of clozapine come from the differences in animal species, but not from the dose regime. Studies have found the existence of the species difference between mouse and rat brain in response to some lesions or drug treatments [4,9]. Differential effect of dietary selenium was observed on the long-term neurotoxicity induced by MDMA in mice and rats. Selenium deficiency impairs the cellular antioxidant status of the mouse brain and that this effect not only exacerbates the dopaminergic toxicity induced by MDMA but also facilitates the appearance of damage to 5-HT containing neurons. In contrast, rats fed a seleniumdeficient diet do not show any change in MDMA-induced neurotoxicity of 5-HT nerve terminals in spite of showing a
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