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Effects of p-chlorophenylalanine on striatal acetylcholinesterase activity and on biogenic amine levels in nuclei raphe and caudate-putamen during physostigmine-induced tremor in rats
Neuroscience Letters 299 (2001) 105±108
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Effects of p-chlorophenylalanine on striatal acetylcholinesterase activity and on biogenic amine levels in nuclei raphe and caudateputamen during physostigmine-induced tremor in rats Hina Mehta, Bobby Thomas, Kochupurackal P. Mohanakumar* Laboratory of Neurochemistry, Division of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Calcutta 700 032, India Received 3 November 2000; received in revised form 21 December 2000; accepted 21 December 2000
Abstract The biochemical mechanism underlying tremor is unknown. We investigated the effects of p-chlorophenylalanine (pCPA), a serotonin (5-HT) depletor, on the neurochemical processes in nuclei raphe dorsalis (NRD) and caudatus putamen (NCP) paralleling physostigmine-induced tremor in rats. Peak of physostigmine tremor correlated with increase in 5-HT in NRD and NCP, and a decrease in striatal dopamine (DA), as assayed employing high pressure liquid chromotography-electrochemistry. Administration of pCPA caused signi®cant decrease in DA, norepinephrine (NE) and 5-HT levels in both the nuclei, without affecting striatal NE content and acetylcholinesterase activity. pCPA pretreatment signi®cantly inhibited physostigmine-induced tremor and blocked corresponding increase in the levels of 5-HT in NRD and NCP. These results indicate involvement of central 5-HT, but not DA or NE, in the genesis of physostigmine tremor. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Serotonin (5-HT) mediation of tremor; Dopamine; Norepinephrine; Motor dysfunction
Physostigmine is a reversible acetylcholinesterase (AChE) inhibitor that produces tremor following systemic administration in laboratory animals [3,10,17]. This behavioural dysfunction has been attributed to an increased acetylcholine (ACh) level in the brain following AChE inhibition [5]. However, some of the anticholinesterase organophaphates have been shown to cause tremor in animals by altering striatal serotonin (5-HT) content [2]. We have recently reported augmentative effects of physostigmine tremor by 5-HT agonists and attenuating effects by 5-HT antagonists in rats [16], and suggested a direct involvement of serotoninergic receptors in this behaviour. A tryptophan hydroxylase inhibitor, p-chlorophenylalanine (pCPA), which signi®cantly depletes 5-HT in the brain [4] has been used in investigations to study the role of 5-HT in pharmacological and behavioral actions of drugs [6,9,14]. In the present study, we have investigated the effects of pCPA on physostigmine-induced tremor, and the related changes in striatal AChE activity and in the concentrations of dopa* Corresponding author. Tel.: 191-33-473-3491/473 6793, ext. 213; fax: 191-33-473-5197/473-0284. E-mail address: [email protected] (K.P. Mohanakumar).
mine (DA), norepinephrine (NE) and 5-HT in nucleus raphe dorsalis (NRD) and nucleus caudatus putamen (striatum, NCP) to explore the involvement of biogenic amines in this motor dysfunction. Sprague±Dawley rats (200±300 g) used in the study were housed under standard conditions of temperature (22 ^ 18C), humidity (60 ^ 5%) and illumination (12 h light/dark cycle). The experimental protocol met the National Guidelines on the `Proper Care and Use of Animals in Laboratory Research' (Indian National Science Academy, New Delhi, 1999) and was approved by the Animal Ethics Committee of the Institute. Drugs and vehicles (saline) were administered intraperitoneally in equal volumes of 1 ml/kg. Previously we have demonstrated a dose and time dependent generation of tremor following physostigmine in rats [10,16]. The tremor initiated at 2±3 min, peaked at 7±8 min and lasted for 30±35 min. In the present study we have analyzed animals at 8 min following physostigmine salicylate (0.75 mg/kg Boehringer, Germany) when the whole body tremor was at its peak. pCPA methyl ester (300 mg/kg; Sigma Chemical Co.) was administered once every day for 3 days and the experiments were conducted on the fourth day.
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 50 7- 5
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H. Mehta et al. / Neuroscience Letters 299 (2001) 105±108
The animals were continuously monitored in a blind study by two examiners who have been trained in evaluating tremor responses in animals. Interrator reliability was found to be highly signi®cant since the data generated by these independent observers exhibited a high degree of correlation (r 0:86 for physostigmine and 0.90 for pCPA 1 physostigmine groups; P $ 0:0001). Tremor was quanti®ed every 2 min on a modi®ed intensity-score basis in a scale of 0±4 as described earlier [16]. In short, tremor intensity was ranked 0, no tremor; 1, occasional muscle twitches or slight tremor which is barely visible at the head region; 2, moderate, intermittent tremor which is restricted to the head region, 3, visible tremor with occasional quiescent periods affecting the anterior region; 4, continuous, gross, whole body tremor. Tremor scores obtained for 30 min following physostigmine were computed for obtaining the average tremor score for physostigmine as well as for pCPA-physostigmine groups. NCP (striata) and NRD were micropunched from 1 mm frozen sections. Tissues were sonicated in chilled HClO4 (0.1 M, containing 0.05% ethylenediaminetetraacetic acid (EDTA)) and centrifuged at 12 000 £ g for 5 min. Ten microlitres of the clear supernatant was injected directly into a high performance liquid chromatography (HPLC) system (Waters, MA) equipped with an electrochemical detector. The biogenic amines were separated on an Ultrasphere, Ion Pair, C18 Reverse Phase analytical column (5 mm, 4.6 £ 250 mm; Beckman, CA) following the method described earlier [18]. The electrodetection was performed at 10.74 V. The mobile phase contained 8.65 mM heptane sulfonic acid, 0.27 mM EDTA, 13% acetonitrile, 0.45% triethylamine and 0.20% phosphoric acid (v/v). Two percent homogenates of NCP prepared in 100 mM phosphate buffer (pH 8.0) were centrifuged very shortly (tap spins) and the supernatants were used for the estimation of AChE as described earlier [10]. The reaction mixture contained 0.1 M sodium phosphate buffer (pH 8.0), 5 0 5 0 dithio-bis-(2-nitrobenzoic acid) (10 mM), acetylthiocholine iodide (75 mM) and tissue homogenate (80 ml). The rate of increase of absorbance was recorded in an Hitachi U-2000 spectrophotometer at 412 nm. Unit of the enzyme activity was expressed as mmol anion produced per min per mg protein, taking the extinction coef®cient of the yellow anion formed as 1412 5.74 £ 10 24. Protein was estimated following the procedure of Lowry et al. [7] taking bovine serum albumin as standard. Neurochemical data were computed for signi®cance employing Student's t-test. P , 0:05 was considered significant. Tremor data were subjected to the Signed Rank test, a non-parametric analysis. Interrator reliability was tested by linear regression analysis followed by determination of Pearson's correlation coef®cient (r) employing a statistical package, EPISTAT. Physostigmine caused a consistent tremor with short gaps and medium intensity which reached its peak by 7±8 min. Treatment with pCPA produced no tremor and remarkably
reduced (85%) the tremorogenic action of physostigmine in rats (Fig. 1). Physostigmine administration caused a signi®cant inhibition (35%) of AChE activity in the striatum in normal (Fig. 2A), but not in pCPA pretreated rats (32%). pCPA treatment in rats failed to affect striatal AChE activity (223%). A signi®cant increase in the level of 5-HT was observed in NCP (65%, Fig. 2B) and NRD (40%; Table 1) at 8 min following physostigmine. The peak of tremor coincided with a signi®cant decrease and increase of DA respectively in NCP (Fig. 2C) and NRD (Table 1). A signi®cant decrease of 5-HT (67% in NCP; 90% in NRD), DA (60% in NCP; 65% in NRD) and NE (50% in NRD) levels were observed in animals treated with pCPA (Fig. 2B,C and Table 1). NE levels were unaffected following physostigmine or pCPA in the striatum (Fig. 2D). No signi®cant difference was found in any of the parameters studied between the pCPA, and pCPA 1 physostigmine group. The present study provides evidences that point to a direct involvement of central 5-HT, and not DA, in the genesis of physostigmine-tremor. The results also suggest that pCPA does not in¯uence the cholinergic system in the brain, since the drug failed to alter striatal AChE activity. The present study con®rms our earlier ®ndings on the failure of 5-HT de®cient rats to develop tremor following physostigmine treatment [16]. The study also support the earlier ®ndings that the action of pCPA on 5-HT is non-selective in nature and it has also deleterious effects on the central catecholamine system [13]. pCPA is known to cause severe depletion of 5-HT in the brain following inhibition of the rate limiting enzyme, tryptophan-5-hydroxylase, of the 5-HT synthetic pathway [4]
Fig. 1. Effects of pCPA on physostigmine (PHY; 0.75 mg/kg)induced tremor in rats. pCPA (300 mg/kg) was administered intraperitoneally daily for 3 days and the animals were tested 16 h after the last injections. Average tremor scores for 2 min measured over a period of 30 min by two independent evaluators are given. Control animals or pCPA treated animals exhibited no visible tremor. Results are given as mean ^ SEM, for eight animals in each group. *Signi®cantly different from the physostigmine group. P # 0:001.
H. Mehta et al. / Neuroscience Letters 299 (2001) 105±108
107
Fig. 2. Effects of PHY, pCPA and pCPA 1 PHY on (A) acetylcholinesterase (AChE) activity (n 4), and on the levels of (B) 5-HT, (C) DA and (D) NE in the striatum of rats (n 8). The animals were analyzed at the 8th minute when the tremor intensity was at its peak Results are given as mean ^ SEM. *Signi®cantly different from the control group. P # 0:05.
and thus it has been employed to study 5-HT mediated events [6,9,14]. The present study examined the status of the biogenic amines in pCPA treated rats to verify whether DA and/or NE also contributed to physostigmine-induced tremor. We demonstrate here a signi®cant decrease of DA, NE and 5-HT content in pCPA treated rats and no effect on the striatal AChE activity. While our results on depletion of catecholamines in chronic pCPA rats derive support from earlier ®ndings [8,13,14], the ®nding on AChE is novel and interesting. This result indicates that the cholinergic system in the brain is spared following repeated high doses of pCPA. In a recent in vivo brain microdialysis study it has been shown that pCPA does not affect the striatal release of acetylcholine [11]. This indicates that the inhibition of physostigmine-tremor by pCPA in the present study was not mediated via the cholinergic system. On the other hand, our study demonstrates a direct relationship between physostigmine-induced tremor and increase in 5-HT levels in NRD and NCP. pCPA treatment blocked both the rise in 5-HT and the genesis of physostigmine-tremor. Such an association for catecholamines with physostigmine-tremor was not observed in the present study. Interestingly, nucleus accumbens or tuberculum olfactorium failed to register any changes in the biogenic amine levels following physostig-
mine administration (our unpublished observations). These results indicate a direct involvement of raphe and striatal 5-HT, but not DA or NE, in the genesis of physostigminetremor in rats. The biochemical basis of tremorogenesis by physostigmine has been conceived as due to an increase in cerebral ACh content by reversible inhibition of AChE [5]. We have demonstrated that 5-HT content is increased in the mouse midbrain area following physostigmine. This correlated with the duration and intensity of physostigmine-tremor Table 1 Effect of PHY and/or pCPA on the levels of biogenic amines (pmol/mg tissue) in nucleus raphe dorsalis a Treatments
NE
DA
5-HT
Control (n 8) PHY (n 8) pCPA (n 4) pCPA 1 PHY (n 4)
4.06 ^ 0.40 3.37 ^ 0.30 2.05 ^ 0.36* 2.75 ^ 0.30*
0.58 ^ 0.05 0.91 ^ 0.07* 0.2 ^ 0.01* 0.22 ^ 0.06*
4.17 ^ 0.65 5.86 ^ 0.33* 0.36 ^ 0.12* 0.4 ^ 0.11*
a
Animals were analyzed 8 min following physostigmine (750 mg/kg) treatment. pCPA was administered (300 mg/kg) for 3 days and the experiments were conducted 16 h after the last injection. *Signi®cantly different as compared to the control, P 0:05:
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H. Mehta et al. / Neuroscience Letters 299 (2001) 105±108
[10]. Further pharmacological support for this observation was obtained by the use of 5-HT antagonists and agonists which modi®ed physostigmine-induced tremor [16]. Studies using various AChE inhibitors (soman, sarin, tabun, carbaryl, etc.) con®rm these ®ndings on physostigmineinduced tremor [2,12,15]. It is interesting that physostigmine caused signi®cant reduction of DA levels in the striatum, which may be interpreted as a compensatory change following increases in the levels of ACh following AChE inhibition [1]. Hence it might be suggested that tremor resulted from changes in the levels of striatal ACh and/or DA. In comparison with physostigmine, pCPA caused even further reductions in central DA, but the animals did not show tremor. Physostigmine, while moderately reducing DA, signi®cantly increased 5-HT levels. In pCPA treated rats, 5-HT was strongly depleted and physostigmine augmented this effect of pCPA on brain serotonin. As tremor was reduced or absent in pCPA 1 physostigmine treated rats, an involvement of DA in physostigmine-induced tremor is unlikely. Incidentally, tremor coincided with an increased level of 5-HT, and serotonin depletion signi®ed inhibition of tremor in rats in the present study. These observations, taken together with our earlier reports which demonstrated a positive relationship of 5-HT content with physostigminetremor in mice [10] as well as the ability of serotoninergic drugs to modify this tremor [16] strongly suggest a direct involvement of serotonin in the tremorogenic action of physostigmine. Since 5-HT depletion could not abolish physostigmine-tremor completely, it may indicate an involvement of other cerebral components in addition to 5-HT. Financed by the Department of Science and Technology, Govt. of India. B.T. is a Senior Research Fellow of the Council of Scienti®c and Industrial Research, Government of India. [1] Bradford, H.F., Chemical Neurobiology: An Introduction to Neurochemistry, W.H. Freeman, New York, 1986, p. 507. [2] Fernando, J.C.R., Hoskins, B.H. and Ho, I.K., A striatal serotonergic involvement in the behavioral effects of anticholinesterase organophosphates, Eur. J. Pharmacol., 98 (1984) 129±132. [3] Gothoni, P., Lehtinen, M. and Silen, L., Quanti®cation of tremor in rats induced by physostigmine, Psychopharmacology, 74 (1981) 275±279. [4] Grahame-Smith, D.G., Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with monoamine oxidase inhibitor and L-tryptophan, J. Neurochem., 18 (1971) 1053±1066.
[5] Hallak, M. and Giacobini, E., Relation of brain regional physostigmine concentration to cholinesterase activity and acetylcholine and choline levels in rats, Neurochem. Res., 11 (1986) 1037±1048. [6] Lin-Shiau, S.Y. and Hsu, K.S., Modi®cation of 2,2 0 ,2 00 -tripyridine-induced tremor in mice by serotonergic agonists and antagonists and benzodiazepines, Pharmacol. Biochem. Behav., 48 (1994) 665±670. [7] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with folin phenol reagent, J. Biol. Chem., 193 (1951) 265±275. [8] Minabe, Y., Emori, K. and Ashby Jr., C.R., The depletion of brain serotonin levels by para-chlorophenylalanine administration signi®cantly alters the activity of midbrain dopamine cells in rats: an extracellular single cell recording study, Synapse, 22 (1996) 46±53. [9] Mohanakumar, K.P. and Ganguly, D.K., Tremorogenesis by LON-954 [N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride]: evidence for the involvement of 5-hydroxytryptamine, Brain Res. Bull., 22 (1989) 191±195. [10] Mohanakumar, K.P., Mitra, N. and Ganguly, D.K., Tremorogenesis by physostigmine is unrelated to acetylcholinesterase inhibition: evidence for serotoninergic involvement, Neurosci. Lett., 120 (1990) 91±93. [11] Nakai, K., Fujii, T., Fujimoto, K., Suzuki, T. and Kawashima, K., Effect of WAY-100135 on the hippocampal acetylcholine release potentiated by 8-OH-DPAT, a serotonin1A receptor agonist, in normal and p-chlorophenylalanine-treated rats as measured by in vivo microdialysis, Neurosci. Res., 31 (1998) 23±29. [12] Ray, S.K. and Poddar, M.K., Interaction of central serotonin and dopamine in the regulation of carbaryl-induced tremor, Eur. J. Pharmacol., 181 (1990) 159±166. [13] Reader, T.A. and Gauthier, P., Catecholamines and serotonin in the rat central nervous system after 6-OHDA, 5±7-DHT and p-CPA, J. Neural. Transm., 59 (1984) 207±227. [14] Riekkinen, M., Aroviita, L., Kivipelto, M., Taskila, K. and Riekkinen Jr., P., Depletion of serotonin, dopamine and noradrenaline in aged rats decreases the therapeutic effect of nicotine, but not of tetrahydroaminoacridine, Eur. J. Pharmacol., 308 (1996) 243±250. [15] Robinson, S.E. and Hambrecht, K.L., Effects of intrastriatal injections of soman on acetylcholine, dopamine and 5hydroxytryptamine metabolism, Life Sci., 42 (1988) 2331± 2339. [16] Sarkar, S., Thomas, B., Muralikrishnan, D. and Mohanakumar, K.P., Effects of serotoninergic drugs on tremor induced by physostigmine in rats, Behav. Brain Res., 109 (2000) 187±193. [17] Stanford, J.A. and Fowler, S.C., Scopolamine reversal of tremor produced by low doses of physostigmine in rats: Evidence for a cholinergic mechanism, Neurosci. Lett., 225 (1997) 157±160. [18] Thomas, B., Muralikrishnan, D. and Mohanakumar, K.P., In vivo hydroxyl radical generation in the striatum following systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine in mice, Brain Res., 852 (2000) 221±224.
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Effects of p-chlorophenylalanine on striatal acetylcholinesterase activity and on biogenic amine levels in nuclei raphe and caudateputamen during physostigmine-induced tremor in rats Hina Mehta, Bobby Thomas, Kochupurackal P. Mohanakumar* Laboratory of Neurochemistry, Division of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Calcutta 700 032, India Received 3 November 2000; received in revised form 21 December 2000; accepted 21 December 2000
Abstract The biochemical mechanism underlying tremor is unknown. We investigated the effects of p-chlorophenylalanine (pCPA), a serotonin (5-HT) depletor, on the neurochemical processes in nuclei raphe dorsalis (NRD) and caudatus putamen (NCP) paralleling physostigmine-induced tremor in rats. Peak of physostigmine tremor correlated with increase in 5-HT in NRD and NCP, and a decrease in striatal dopamine (DA), as assayed employing high pressure liquid chromotography-electrochemistry. Administration of pCPA caused signi®cant decrease in DA, norepinephrine (NE) and 5-HT levels in both the nuclei, without affecting striatal NE content and acetylcholinesterase activity. pCPA pretreatment signi®cantly inhibited physostigmine-induced tremor and blocked corresponding increase in the levels of 5-HT in NRD and NCP. These results indicate involvement of central 5-HT, but not DA or NE, in the genesis of physostigmine tremor. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Serotonin (5-HT) mediation of tremor; Dopamine; Norepinephrine; Motor dysfunction
Physostigmine is a reversible acetylcholinesterase (AChE) inhibitor that produces tremor following systemic administration in laboratory animals [3,10,17]. This behavioural dysfunction has been attributed to an increased acetylcholine (ACh) level in the brain following AChE inhibition [5]. However, some of the anticholinesterase organophaphates have been shown to cause tremor in animals by altering striatal serotonin (5-HT) content [2]. We have recently reported augmentative effects of physostigmine tremor by 5-HT agonists and attenuating effects by 5-HT antagonists in rats [16], and suggested a direct involvement of serotoninergic receptors in this behaviour. A tryptophan hydroxylase inhibitor, p-chlorophenylalanine (pCPA), which signi®cantly depletes 5-HT in the brain [4] has been used in investigations to study the role of 5-HT in pharmacological and behavioral actions of drugs [6,9,14]. In the present study, we have investigated the effects of pCPA on physostigmine-induced tremor, and the related changes in striatal AChE activity and in the concentrations of dopa* Corresponding author. Tel.: 191-33-473-3491/473 6793, ext. 213; fax: 191-33-473-5197/473-0284. E-mail address: [email protected] (K.P. Mohanakumar).
mine (DA), norepinephrine (NE) and 5-HT in nucleus raphe dorsalis (NRD) and nucleus caudatus putamen (striatum, NCP) to explore the involvement of biogenic amines in this motor dysfunction. Sprague±Dawley rats (200±300 g) used in the study were housed under standard conditions of temperature (22 ^ 18C), humidity (60 ^ 5%) and illumination (12 h light/dark cycle). The experimental protocol met the National Guidelines on the `Proper Care and Use of Animals in Laboratory Research' (Indian National Science Academy, New Delhi, 1999) and was approved by the Animal Ethics Committee of the Institute. Drugs and vehicles (saline) were administered intraperitoneally in equal volumes of 1 ml/kg. Previously we have demonstrated a dose and time dependent generation of tremor following physostigmine in rats [10,16]. The tremor initiated at 2±3 min, peaked at 7±8 min and lasted for 30±35 min. In the present study we have analyzed animals at 8 min following physostigmine salicylate (0.75 mg/kg Boehringer, Germany) when the whole body tremor was at its peak. pCPA methyl ester (300 mg/kg; Sigma Chemical Co.) was administered once every day for 3 days and the experiments were conducted on the fourth day.
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 50 7- 5
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H. Mehta et al. / Neuroscience Letters 299 (2001) 105±108
The animals were continuously monitored in a blind study by two examiners who have been trained in evaluating tremor responses in animals. Interrator reliability was found to be highly signi®cant since the data generated by these independent observers exhibited a high degree of correlation (r 0:86 for physostigmine and 0.90 for pCPA 1 physostigmine groups; P $ 0:0001). Tremor was quanti®ed every 2 min on a modi®ed intensity-score basis in a scale of 0±4 as described earlier [16]. In short, tremor intensity was ranked 0, no tremor; 1, occasional muscle twitches or slight tremor which is barely visible at the head region; 2, moderate, intermittent tremor which is restricted to the head region, 3, visible tremor with occasional quiescent periods affecting the anterior region; 4, continuous, gross, whole body tremor. Tremor scores obtained for 30 min following physostigmine were computed for obtaining the average tremor score for physostigmine as well as for pCPA-physostigmine groups. NCP (striata) and NRD were micropunched from 1 mm frozen sections. Tissues were sonicated in chilled HClO4 (0.1 M, containing 0.05% ethylenediaminetetraacetic acid (EDTA)) and centrifuged at 12 000 £ g for 5 min. Ten microlitres of the clear supernatant was injected directly into a high performance liquid chromatography (HPLC) system (Waters, MA) equipped with an electrochemical detector. The biogenic amines were separated on an Ultrasphere, Ion Pair, C18 Reverse Phase analytical column (5 mm, 4.6 £ 250 mm; Beckman, CA) following the method described earlier [18]. The electrodetection was performed at 10.74 V. The mobile phase contained 8.65 mM heptane sulfonic acid, 0.27 mM EDTA, 13% acetonitrile, 0.45% triethylamine and 0.20% phosphoric acid (v/v). Two percent homogenates of NCP prepared in 100 mM phosphate buffer (pH 8.0) were centrifuged very shortly (tap spins) and the supernatants were used for the estimation of AChE as described earlier [10]. The reaction mixture contained 0.1 M sodium phosphate buffer (pH 8.0), 5 0 5 0 dithio-bis-(2-nitrobenzoic acid) (10 mM), acetylthiocholine iodide (75 mM) and tissue homogenate (80 ml). The rate of increase of absorbance was recorded in an Hitachi U-2000 spectrophotometer at 412 nm. Unit of the enzyme activity was expressed as mmol anion produced per min per mg protein, taking the extinction coef®cient of the yellow anion formed as 1412 5.74 £ 10 24. Protein was estimated following the procedure of Lowry et al. [7] taking bovine serum albumin as standard. Neurochemical data were computed for signi®cance employing Student's t-test. P , 0:05 was considered significant. Tremor data were subjected to the Signed Rank test, a non-parametric analysis. Interrator reliability was tested by linear regression analysis followed by determination of Pearson's correlation coef®cient (r) employing a statistical package, EPISTAT. Physostigmine caused a consistent tremor with short gaps and medium intensity which reached its peak by 7±8 min. Treatment with pCPA produced no tremor and remarkably
reduced (85%) the tremorogenic action of physostigmine in rats (Fig. 1). Physostigmine administration caused a signi®cant inhibition (35%) of AChE activity in the striatum in normal (Fig. 2A), but not in pCPA pretreated rats (32%). pCPA treatment in rats failed to affect striatal AChE activity (223%). A signi®cant increase in the level of 5-HT was observed in NCP (65%, Fig. 2B) and NRD (40%; Table 1) at 8 min following physostigmine. The peak of tremor coincided with a signi®cant decrease and increase of DA respectively in NCP (Fig. 2C) and NRD (Table 1). A signi®cant decrease of 5-HT (67% in NCP; 90% in NRD), DA (60% in NCP; 65% in NRD) and NE (50% in NRD) levels were observed in animals treated with pCPA (Fig. 2B,C and Table 1). NE levels were unaffected following physostigmine or pCPA in the striatum (Fig. 2D). No signi®cant difference was found in any of the parameters studied between the pCPA, and pCPA 1 physostigmine group. The present study provides evidences that point to a direct involvement of central 5-HT, and not DA, in the genesis of physostigmine-tremor. The results also suggest that pCPA does not in¯uence the cholinergic system in the brain, since the drug failed to alter striatal AChE activity. The present study con®rms our earlier ®ndings on the failure of 5-HT de®cient rats to develop tremor following physostigmine treatment [16]. The study also support the earlier ®ndings that the action of pCPA on 5-HT is non-selective in nature and it has also deleterious effects on the central catecholamine system [13]. pCPA is known to cause severe depletion of 5-HT in the brain following inhibition of the rate limiting enzyme, tryptophan-5-hydroxylase, of the 5-HT synthetic pathway [4]
Fig. 1. Effects of pCPA on physostigmine (PHY; 0.75 mg/kg)induced tremor in rats. pCPA (300 mg/kg) was administered intraperitoneally daily for 3 days and the animals were tested 16 h after the last injections. Average tremor scores for 2 min measured over a period of 30 min by two independent evaluators are given. Control animals or pCPA treated animals exhibited no visible tremor. Results are given as mean ^ SEM, for eight animals in each group. *Signi®cantly different from the physostigmine group. P # 0:001.
H. Mehta et al. / Neuroscience Letters 299 (2001) 105±108
107
Fig. 2. Effects of PHY, pCPA and pCPA 1 PHY on (A) acetylcholinesterase (AChE) activity (n 4), and on the levels of (B) 5-HT, (C) DA and (D) NE in the striatum of rats (n 8). The animals were analyzed at the 8th minute when the tremor intensity was at its peak Results are given as mean ^ SEM. *Signi®cantly different from the control group. P # 0:05.
and thus it has been employed to study 5-HT mediated events [6,9,14]. The present study examined the status of the biogenic amines in pCPA treated rats to verify whether DA and/or NE also contributed to physostigmine-induced tremor. We demonstrate here a signi®cant decrease of DA, NE and 5-HT content in pCPA treated rats and no effect on the striatal AChE activity. While our results on depletion of catecholamines in chronic pCPA rats derive support from earlier ®ndings [8,13,14], the ®nding on AChE is novel and interesting. This result indicates that the cholinergic system in the brain is spared following repeated high doses of pCPA. In a recent in vivo brain microdialysis study it has been shown that pCPA does not affect the striatal release of acetylcholine [11]. This indicates that the inhibition of physostigmine-tremor by pCPA in the present study was not mediated via the cholinergic system. On the other hand, our study demonstrates a direct relationship between physostigmine-induced tremor and increase in 5-HT levels in NRD and NCP. pCPA treatment blocked both the rise in 5-HT and the genesis of physostigmine-tremor. Such an association for catecholamines with physostigmine-tremor was not observed in the present study. Interestingly, nucleus accumbens or tuberculum olfactorium failed to register any changes in the biogenic amine levels following physostig-
mine administration (our unpublished observations). These results indicate a direct involvement of raphe and striatal 5-HT, but not DA or NE, in the genesis of physostigminetremor in rats. The biochemical basis of tremorogenesis by physostigmine has been conceived as due to an increase in cerebral ACh content by reversible inhibition of AChE [5]. We have demonstrated that 5-HT content is increased in the mouse midbrain area following physostigmine. This correlated with the duration and intensity of physostigmine-tremor Table 1 Effect of PHY and/or pCPA on the levels of biogenic amines (pmol/mg tissue) in nucleus raphe dorsalis a Treatments
NE
DA
5-HT
Control (n 8) PHY (n 8) pCPA (n 4) pCPA 1 PHY (n 4)
4.06 ^ 0.40 3.37 ^ 0.30 2.05 ^ 0.36* 2.75 ^ 0.30*
0.58 ^ 0.05 0.91 ^ 0.07* 0.2 ^ 0.01* 0.22 ^ 0.06*
4.17 ^ 0.65 5.86 ^ 0.33* 0.36 ^ 0.12* 0.4 ^ 0.11*
a
Animals were analyzed 8 min following physostigmine (750 mg/kg) treatment. pCPA was administered (300 mg/kg) for 3 days and the experiments were conducted 16 h after the last injection. *Signi®cantly different as compared to the control, P 0:05:
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[10]. Further pharmacological support for this observation was obtained by the use of 5-HT antagonists and agonists which modi®ed physostigmine-induced tremor [16]. Studies using various AChE inhibitors (soman, sarin, tabun, carbaryl, etc.) con®rm these ®ndings on physostigmineinduced tremor [2,12,15]. It is interesting that physostigmine caused signi®cant reduction of DA levels in the striatum, which may be interpreted as a compensatory change following increases in the levels of ACh following AChE inhibition [1]. Hence it might be suggested that tremor resulted from changes in the levels of striatal ACh and/or DA. In comparison with physostigmine, pCPA caused even further reductions in central DA, but the animals did not show tremor. Physostigmine, while moderately reducing DA, signi®cantly increased 5-HT levels. In pCPA treated rats, 5-HT was strongly depleted and physostigmine augmented this effect of pCPA on brain serotonin. As tremor was reduced or absent in pCPA 1 physostigmine treated rats, an involvement of DA in physostigmine-induced tremor is unlikely. Incidentally, tremor coincided with an increased level of 5-HT, and serotonin depletion signi®ed inhibition of tremor in rats in the present study. These observations, taken together with our earlier reports which demonstrated a positive relationship of 5-HT content with physostigminetremor in mice [10] as well as the ability of serotoninergic drugs to modify this tremor [16] strongly suggest a direct involvement of serotonin in the tremorogenic action of physostigmine. Since 5-HT depletion could not abolish physostigmine-tremor completely, it may indicate an involvement of other cerebral components in addition to 5-HT. Financed by the Department of Science and Technology, Govt. of India. B.T. is a Senior Research Fellow of the Council of Scienti®c and Industrial Research, Government of India. [1] Bradford, H.F., Chemical Neurobiology: An Introduction to Neurochemistry, W.H. Freeman, New York, 1986, p. 507. [2] Fernando, J.C.R., Hoskins, B.H. and Ho, I.K., A striatal serotonergic involvement in the behavioral effects of anticholinesterase organophosphates, Eur. J. Pharmacol., 98 (1984) 129±132. [3] Gothoni, P., Lehtinen, M. and Silen, L., Quanti®cation of tremor in rats induced by physostigmine, Psychopharmacology, 74 (1981) 275±279. [4] Grahame-Smith, D.G., Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with monoamine oxidase inhibitor and L-tryptophan, J. Neurochem., 18 (1971) 1053±1066.
[5] Hallak, M. and Giacobini, E., Relation of brain regional physostigmine concentration to cholinesterase activity and acetylcholine and choline levels in rats, Neurochem. Res., 11 (1986) 1037±1048. [6] Lin-Shiau, S.Y. and Hsu, K.S., Modi®cation of 2,2 0 ,2 00 -tripyridine-induced tremor in mice by serotonergic agonists and antagonists and benzodiazepines, Pharmacol. Biochem. Behav., 48 (1994) 665±670. [7] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with folin phenol reagent, J. Biol. Chem., 193 (1951) 265±275. [8] Minabe, Y., Emori, K. and Ashby Jr., C.R., The depletion of brain serotonin levels by para-chlorophenylalanine administration signi®cantly alters the activity of midbrain dopamine cells in rats: an extracellular single cell recording study, Synapse, 22 (1996) 46±53. [9] Mohanakumar, K.P. and Ganguly, D.K., Tremorogenesis by LON-954 [N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride]: evidence for the involvement of 5-hydroxytryptamine, Brain Res. Bull., 22 (1989) 191±195. [10] Mohanakumar, K.P., Mitra, N. and Ganguly, D.K., Tremorogenesis by physostigmine is unrelated to acetylcholinesterase inhibition: evidence for serotoninergic involvement, Neurosci. Lett., 120 (1990) 91±93. [11] Nakai, K., Fujii, T., Fujimoto, K., Suzuki, T. and Kawashima, K., Effect of WAY-100135 on the hippocampal acetylcholine release potentiated by 8-OH-DPAT, a serotonin1A receptor agonist, in normal and p-chlorophenylalanine-treated rats as measured by in vivo microdialysis, Neurosci. Res., 31 (1998) 23±29. [12] Ray, S.K. and Poddar, M.K., Interaction of central serotonin and dopamine in the regulation of carbaryl-induced tremor, Eur. J. Pharmacol., 181 (1990) 159±166. [13] Reader, T.A. and Gauthier, P., Catecholamines and serotonin in the rat central nervous system after 6-OHDA, 5±7-DHT and p-CPA, J. Neural. Transm., 59 (1984) 207±227. [14] Riekkinen, M., Aroviita, L., Kivipelto, M., Taskila, K. and Riekkinen Jr., P., Depletion of serotonin, dopamine and noradrenaline in aged rats decreases the therapeutic effect of nicotine, but not of tetrahydroaminoacridine, Eur. J. Pharmacol., 308 (1996) 243±250. [15] Robinson, S.E. and Hambrecht, K.L., Effects of intrastriatal injections of soman on acetylcholine, dopamine and 5hydroxytryptamine metabolism, Life Sci., 42 (1988) 2331± 2339. [16] Sarkar, S., Thomas, B., Muralikrishnan, D. and Mohanakumar, K.P., Effects of serotoninergic drugs on tremor induced by physostigmine in rats, Behav. Brain Res., 109 (2000) 187±193. [17] Stanford, J.A. and Fowler, S.C., Scopolamine reversal of tremor produced by low doses of physostigmine in rats: Evidence for a cholinergic mechanism, Neurosci. Lett., 225 (1997) 157±160. [18] Thomas, B., Muralikrishnan, D. and Mohanakumar, K.P., In vivo hydroxyl radical generation in the striatum following systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine in mice, Brain Res., 852 (2000) 221±224.