ω-Conotoxin GVIA inhibits the methylphenidate-induced but not methamphetamine-induced behavior

Neuroseience Letters, 165 (1994) 191 194 © 1994 Elsevier Science Ireland Ltd. All rights reserved 0304-3940/94/$ 07.00

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c0-Conotoxin GVIA inhibits the methylphenidate-induced but not methamphetamine-induced behavior K i y o f u m i Y a m a d a ~', T o m o m i T e r a o k a b, Seiji M o r i t a b, T a k a a k i H a s e g a w a *~, T o s h i t a k a N a b e s h i m a ~'* "D~7)artment o[ Neurops.vehopharmaeology and Hospital Pharma~'y. Nugoya University School o1' Medicine, Showa-ku. Na,~oya 466, Jupan i,Third Tokushhna Institute o/' New Drug Research. Otsuk~l Pharmaceutical. Toku.shima, Japan1 (Received 31 August 1993: Revised version received 21 October 1993: Accepled 21 October 1993) Ker words.

o)-Conotoxin GV1A; N-type calcium channel: Methylphenidate; Methamphetamine; Dopamine release

We investigated the effects of antagonists for co-conotoxin GVIA I(o-CTX)-sensitive N-type voltage-sensitive calcium channels (N-channels) on methylphenidate- and methamphetamine-induced behavior, l.c.v, injection of co-CTX or neomycin, both N-channel antagonists, caused a dosedependent inhibition of methylphenidate-induced hypermotility in mice but failed to inhibit methamphetamine-induced hyperactivity. Further, o)-CTX inhibited the circling behavior induced by methylphenidate in rats that had kainic acid-induced unilateral striatal lesions. These results suggest that calcium influx through w-CTX-sensitive N-channels pla>s an important role in methylphenidate-induced behavior.

Voltage-sensitive calcium channels (VSCC) have a widespread distribution in excitatory tissues, including those of the central nervous system, and Ca -'+ influx into nerve terminals through the VSCC is the trigger for the release of neurotransmitters [11]. Electrophysiological and pharmacological studies have indicated that there are two classes of VSCC: low-voltage-activated I LVA) and high-voltage-activated (HVA) [13]. HVA calcium channels could be further separated into three different components: 1,4-dihydropyridine (DHP)-sensitive l_,channels, 6o-conotoxin GVIA (o)-CTX)-sensitive Nchannels and DHP- and co-CTX-resistant channels [1,13,17]. Aminoglycoside antibiotics, such as neomycin, have been shown to be selective inhibitors of [125I]o)-CTX binding [9,21], suggesting that they act as co-CTX-sensitive N-channel blockers. In vitro studies have indicated that co-CTX is a potent inhibitor of the release of neurotransmitters, such as dopamine (DA), norepinephrine, serotonin and acethylcholine, as well as inhibiting C a 2+ influx into synaptosomes [5,11,18,21]. Similarily, it is demonstrated that neomycin inhibits the release of dopamine and noradrelarine and Ca -~+ influx [4,8,21]. In contrast, DHP-sensitive L-channel antagonists have little effect in the release of neurotransmitters n o r C a 2+ influx. [12,21]. These resuits suggest that co-CTX-sensitive N-channels play a *Corresponding author. Fax: (81 ) (52) 733 9415.

dominant role in the regulation of neurotransmitter release in the brain. In addition, recent studies, using the technique of in vivo brain dialysis, have indicated that co-CTX inhibits the release of serotonin in the hippocampus [16] and dopamine (DA) in the striatum [7]. Despite the significant role played by (o-CTX-sensitive N-channels in neurotransmitter release, little is known about the pharmacological effects of N-channel antagonists in vivo. In the present study, accordingly, we examined the behavioral effects of o)-CTX and neomycin in mice and rats. Male ICR mice (25 30 g) and male Wistar rats (250 300 g; Clea, Japan) were used. co-CTX (Research Biochemicals) and neomycin (Wako Pure Chemical Industries) dissolved in saline were injected i.c.v, to mice in a vol. of 10/,tl 30 min before the administration of methylphenidate (10 mg/kg s.c.: Ritalin, Ciba Geigy) or methamphetamine (3 mg/kg s.c.: Philopon, Dai-Nippon Pharmaceutical). Nicardipine (Research Biochemicals) and diltiazem (Research Biochemicals) were administered p.o. 1 h before the stimulants. Each mouse was put in a circular chamber (30 x 30 cm, i.d. x h) immediately after methylphenidate or methamphetamine was administered and the locomotor activity of the mouse was measured with a tilting-type ambulometer (AMB-10, Ohara) for 1 h at 10-min intervals. To produce a striatal lesion, kainic acid (KA; 6 nmol in a vol. of 1 #1) was injected into the right striatum of rat (A: + 0.2, L: + 3.0, H: -5.51 accord-

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ing to the brain atlas of Paxinos and Watson [15], as described previously [20]. Two weeks after the striatal lesion was produced, methylphenidate (10 mg/kg i.p.) was administered (first challenge), 15 rain after the methylphenidate injection, the number of turns to the lesioned side was counted for 10 rain. Rats exhibiting > 40 turns in the 10-rain period were used in the following experiments. The number of turns made by each rat at the first challenge of methylphenidate was designated as the control value. Three days after the first challenge, (o-CTX (in a vol. of 10/tl) was injected intracisternally with a Hamilton microsyringe while rats were lightly anesthetized with ether. Methylphenidate was administered 1 h after the co-CTX injection (second challenge) and the number of turns in a 10-min period was counted. The effects of co-CTX on the circling behavior were assessed by comparing the number of turns evoked at the second methylphenidate challenge with the control number (first challenge). Statistical significance was assessed by one-way ANOVA followed by two-tailed Dunnett's test (multiple comparison). A two-tailed paired t-test was also utilized when assessing the effects ofog-CTX on circling behavior. W h e n co-CTX was injected i.c.v, in mice, no apparent changes in general behavior were observed at doses of < 1 pmol/brain. At doses of 3 and 10 pmol/brain, co-CTX caused slight sedation while, at 30 pmol/brain, persistent tremor was observed. Neomycin, at doses of > 100 nmol/ brain, also caused tremor in mice. When c0-CTX was injected intracisternally in rats, no apparent changes in [

general behavior were observed at the doses of up to 20 pmol/brain. At a dose of 50 pmol/brain, however, rats exhibited persistent tremor associated with abnormal posture. As shown in Figs. 1 and 2, both og-CrFX (0.3 and 1 pmol/brain) and neomycin (3 and 10 nmol/brain) caused dose-dependent inhibition of methylphenidate-induced hypermotility in mice at doses which had little effect on the stimulatory action of methamphetamine on locomotor activity. The stimulatory effects of both methylphenidate and methamphetamine were significantly inhibited by og-CTX at doses of >3 pmol/brain (data not shown). In contrast, nicardipine (3--30 mg/kg p.o.) and diltiazem (10-100 mg/kg p.o.) failed to inhibit the stimulatory effects of methyphenidate (data not shown). Pretreatment with co-CTX at a dose of 10 but not 5 pmol/brain significantly reduced the circling behavior caused by methylphenidate in the rats with the striatal lesion, the number of turns being reduced by -50% (Fig. 3). There is much evidence to indicate that neurotransmitter release is triggered by Ca ~+ influx through co-CTXsensitive N-channels [4,11,18,21]. However, little is known about the behavioral effects of co-CTX-sensitive N-channel antagonists in vivo. A study by Olivera et al. [14] indicated that direct injection of co-CTX into the mouse brain produced a persistent tremor. In the present study, we clearly demonstrated that og-CTX, as well as neomycin, had behavioral effects at doses less than those that produced tremors. Since both co-CTX and neomycin inhibited methylphenidate-induced hypermotility in mice

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Fig. 1. Effects of o~-CTX on methylphenidate- and methamphetamine-induced hypermotility in mice. co-CTX was administered i.c.v. 30 min before administration of methylphenidate (10 mg/kg s.c.) or methamphetamine (3 mg/kg s.c.). Left panel: ;>, control; e, methylphenidate + saline; A, methylphenidate + og-CTX (0.3 pmol/brain); II, methylphenidate + co-CTX (1 pmol/brain). Right panel: c,, control; e, methamphetamine + saline; A, methamphetamine + co-CTX (0.3 pmol/brain); I, methamphetamine + og-CTX (1 pmol/brain). Each point represents mean _+S.E. (n = 10). **P < 0.0t vs. control.

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Fig. 2. Effects of neomycin on methylphenidate- and methamphetamine-induced hypermotility in mice. Neomycin was administered i.c.v. B0 min before administration of methylphenidate (10 mg/kg s.c.) or m e t h a m p h e t a m i n e (3 mg/kg s.c.). Left panel: , control: O, methylphenidate + saline: at, methylphenidate+ neomycin (3 nmol/brain); II, methylphenidate + neomycin (10 nmol/brain). Right panel: , control; O, methamphetamine + saline: at, m e t h a m p h e t a m i n e + neomycon (3 nmol/brain): II, m e t h a m p h e t a m i n e + neomycin (10 nmol/brain). Each point represents mean _+ S.E. (n = 10). **P < 0.01 vs. control.

at doses which had little effect on the stimulatory action of methamphetamine, it would appear that the inhibitory effects of (o-CTX-sensitive N-channel antagonists o11 methylphenidate-induced hypermotility are not non-specific sedative effects. Rather, it is suggested that calcium influx through o)-CTX-sensitive N-channels may plays an important role in methylphenidate-induced behavior. The inhibition by o)-CTX of the methylphenidate-induced circling behavior in rats with the striatal lesion further supports this hypothesis. The mechanisms by which g0-CTX and neomycin inhibit methylphenidate-induced behavior are uncertain. A possible mechanism is that they may inhibit the release of neurotransmitters that are responsible for the behavior induced by methylphenidate. It is well known that DA is involved in the behaviors caused by psychostimulants, such as methylphenidate and methamphetamine [10]. Further, it has been demonstrated that methylphenidate facilitates the Ca>-dependent vesicular release of DA, which release is sensitive to reserpine, by inhibiting DA reuptake [3,10], while methamphetamine preferentially releases a newly synthesized DA via a Ca2+-independent carrier-mediated process [2,6]. We have measured the content of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum alter direct injection of (o-CTX into rat striatum. The results indicated that DA content in the striatum was significantly increased by o)-CTX (20 pmol/brain) and that DOPAC/DA and HVA/DA ratios in the striatum

were dose-dependently reduced by o)-CTX (2 20 pmol/ brain), suggesting the inhibitory efl'ect of o)-CTX on DA release (unpubl. data). Since o)-CTX inhibits the release of not only DA but also other neurotransmitters, such as norepinephrine, serotonin and acethylcholine, it is possible that it could inhibit both methylphenidate- and meth21111-

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Fig. 3. Effects o f w - C T X on methylphenidate-induced circling behavior in rats with striatal lesion produced with kainic acid. Two weeks after striatal lesion was produced, methylphenidate ( 10 mg/kg) was administered i.p. and n u m b e r of turns was recorded (control). Three days later, o)-CTX was administered intracisternally 1 h before methylphenidate administration and circling behavior was measured again (treated). Each column represents mean _+ S.E. (,7- 6). * * P < 0.01 vs. corresponding control.

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amphetamine-induced hypermotility at high doses. Alternatively, inhibition of calcium influx through (o-CTXsensitive N-channels may modulate the activity of enzymes that are related to the neurotransmitters. In fact, it has been demonstrated that o)-CTX inhibits the activation of tyrosine hydroxylase by potassium depolarization in vitro [19]. This study was supported by Grant-in-Aid for Drug Abuse Research 92-268 from the Ministry of Health and Welfare, Japan. l Aosaki, N. and Kasai, H., Characterization of two kinds of highvoltage Ca-channel currents in chick sensory neurons. Different sensitivity to dihydropyridines and co-conotoxin GVIA, Pfluegers Arch., 414 (1989) 150-156. 2 Butcher, S.P., Fairbrother, I.S., Kelly, J.S. and Arbuthnott, G.W.. Amphetamine-induced dopamine release in the rat striatum: an in vivo microdialysis study, J. Neurochem., 50 (1988) 346- 355. 3 Butcher, S.P., Liptrot, J. and Arbuthnon, G.W., Characterization of methylphenidate and nomifensine induced dopamine release in rat striatum using in vivo brain microdialysis, Neurosci. Letc, 122 (1991) 245 248. 4 Erausquin, G., Brooker, G. and Hanbauer, I., K*-evoked dopamine release depends on a cytosolic Ca 2÷ pool regulated by N-type Ca -,+ channels, Neurosci. Lett., 145 (1992) 121 125. 5 Feuerstein, T.J., Dooley, D.J. and Seeger, W., Inhibition of norepinephrine and acetylcholine release from human neocortex by (oconotoxin GVIA, J. Pharmacol. Exp. Then, 252 (1990) 778-785. 6 Fischer, J.F. and Cho, A.K., Chemical release of dopamine from striatal homogenates: evidence for an exchange diffusion model, J. Pharmacoh Exp. Then, 208 (1979) 203-209. 7 Kato, T., Otsu, Y., Furune, Y. and Yamamoto, T., Different effects of L-, N- and T-type calcium channel blockers on striatal dopamine release measured by microdialysis in freely moving rats, Neurochem. Int.. 21 (1992) 99-107. 8 Keith, R., Horn, M.B., Piser, T.M. and Mangano, T.J., Effects of stimulus intensity on the inhibition by co-conotoxin GVIA and neomycin of K+-evoked [3H]norepinephrine release from hippocampal brain slices and synaptosomal calcium influx, Biochem. Pharmacol.. 45 (1993) 165 171.

9 Knaus, tt., Striessnig, J., Koza, A. and Glossmann, t1.. Ncurotoxic aminoglycoside antibiotics are potent inhibitors of [csl]-omegaconotoxin GVIA binding to guinea-pig cerebral cortex membranes. Naunyn Schmiedebergs Arch. Pharmacol.. 336 I1987) 583 586. 10 McMillen, B.A., CNS stimulants: two distincl mechanisms of aclion for amphetamine-like drugs, Trends Pharmacoi. Sci., 4 (1983) 429 432. I 1 Miller, R.J., Multiple calcium channels and neuronal function, Science, 235(1987) 46 52. 12 Miller, R.J. and Freedman, S.B., Are dihydropyridme binding sites voltage-sensitive calcium channels'? Life Sci., 34 (1984) 1205 1221. 13 Nowycky, M.C., Fox, A.R and Tsien, R.W., Three types of neuronal calcium channels with different calcium agonist sensitivity, Nature (London), 316 (1985) 440 443. 14 Olivera, B.M., Gray, W.R., Zeikus, R., Mclntosh, L.M., Varga, J., Rivier. J., Santos, V. and Cruz, L.J.. Peptide neurotoxins from fishhunting cone snails, Science, 230 (1985) 1338-1343. 15 Paxinos, G. and Watson, C., The Rat Brain in Stcreotaxic Coodinates, Academic Press, New York, NY, 1982. 16 Pullar, I.A. and Findlay, J.D., Effect ot voltage-sensitive calcium channel antagonists on the release of 5-hydroxytryptamine from rat hippocampus in vivo, J. Neurochem., 59 (1992) 553 459. 17 Regan, L.J., Sah, D.W.Y. and Bean, B.P., Ca > channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and w-conotoxin, Neuron, 6 (199l) 269 280. 18 Reynolds, l.J., Wagner, J.A., Snyder, S.H., Thayer, S.A., Olivera, B.M. and Miller, R.J., Brain voltage-sensitive calcium channel subtypes differentiated by co-conotoxin fraction GVIA, Proc. Natl. Acad. Sci. USA, 83 (1986) 8804--8807. 19 Rittenhouse, A.R. and Zigmond, R.E., o)-Conotoxin inhibits the acute activation of tyrosine hydroxylase and the stimulation of norepinephrine release by potassium depolarization of sympathetic nerve ending, J. Neurochem., 56 (1991) 615-622. 20 Yamada, K., Fuji, K., Nabeshima. T. and Kameyama, T., Neurotoxicity induced by continuous infusion of quinolinic acid into the lateral ventricle in rats, Neurosci. Lett., 118 (1990) 128 13t. 21 Yamada, K., Teraoka, T., Morita, S., Hasegawa, T. and Nabeshima, T., Neuropharmacological characterization of voltagesensitive calcium channels: possible existence of neomycin-sensitive, o)-conotoxin GVIA- and dihydropyridines-resistant calcium channels in the rat brain, Jpn. J. Pharmacol., (1993),