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An intracerebral injection study on the role of striatal dopamine and 5-hydroxytryptamine in the production of tremor by harmine
Nuur~pharmacology,
1976. 15, 303-308.
Pergamon
Press.
Prmted
in Gt. Britain.
AN INTRACEREBRAL INJECTION STUDY ON THE ROLE OF STRIATAL DOPAMINE AND 5HYDROXYTRYPTAMINE IN THE PRODUCTION OF TREMOR BY HARMINE D. M. KELLY and R. J. NAYLOR Postgraduate
School of Studies in Pharmacology, University of Bradford, Bradford, West Yorkshire, U.K. (Accepted 3 October 1975)
Summary-The
intracerebral injection technique was used to investigate the role of striatal dopamine and serotonin in the production of tremor by harmine. When injected directly into the neostriatum, apomorphine and tetrahydroisoquinoline derivatives dopamine, noradrenaline, (+)-amphetamine, reduced the intensity of tremor produced by subcutaneous harmine but tetrahydronaphthalene derivatives were ineffective. Dopamine, noradrenaline and apomorphine were also effective after intrapallidal administration. Direct application of serotonin to the neo- but not the paleostriatum enhanced peripherally induced harmine tremor. The results suggest a relationship between dopamine and serotonin in the mediation of tremor and indicate that, whereas both pallidal and caudate dopamine functions are important for tremor antagonism, the site of a serotonin involvement with tremor is the neostriatum.
Evidence has been gained from both drug interaction studies (KELLY and NAYLOR, 1974) and from brain lesion work (COSTALL, KELLY and NAYLOR. 1976; KELLY and NA~OR, 1975) that the mechanisms by which harmine induces tremor in the rat involve both dopamine and 5-hydroxytryptamine. Further, the lesion studies have indicated that the striatum may be a site for the interaction between dopamine and 5-hydroxytryptamine. This possibility was investigated in the present studies using the intracerebral injection technique.
METHODS Animals
Male Sprague-Dawley (Caworth Strain) rats weighing 250 + 25 g were used throughout all experiments. Measurement of tremor Visual assessment. Rats were normally housed in groups of 8. For observation they were placed in individual, screened, perspex cages (30 x 20 x 15 cm) in a sound-proofed, diffusely illuminated room maintained at a temperature of 21 + 1°C. Rats were placed
in the observation cages at least 30min before injection of harmine to allow adaptation to the new environment. All observations were made between 09.00 and 21.00 hr. Previous studies have shown that the intensity of harmme tremor (assessed according to the scoring system shown in Table 1) is dose-related (KELLY and NAYLOR, 1974). A dose of 10 mg/kg administered sub-
cutaneously was selected as inducing a reproducible sub-maximal tremor response. Animals were observed in pairs, each rat receiving either drug or solvent intraeerebrally. Tremor was assessed at 1 min intervals for 20 min following intracerebral injection and subsequently at 15 min intervals for the duration of the drug effect. Harmine tremor has been shown to be differentiated into two components; 1) that occurring when the rat was undisturbed and at rest, i.e. “a resting tremor”, and 2) that which was only induced by increased activity or by mechanically shaking the cage, i.e. an “activity tremor” (KELLY and NAYLOR, 1974). It was impossible to differentiate resting tremor from activity tremor when tremor was assessed at 1 mm intervals, therefore, only activity tremor was assessed throughout these studies.
Table 1. Scoring system used for the estimation of the intensity of harmine tremor Score
Intensity of tremor
0
The appearance of the animals is the same as saline treated controls. Mild jaw and head tremor. Severe head tremor, but no body tremor. Mild whole body tremor. Severe whole body tremor.
1 2 3 4
303
D. M. KELLYand R. J. NAYLOR
304
33 .#a cc
CP
A’
&....,__
Fig. 1. Diagrammatic representation of the sites (@) of central administration of drugs. The diagrams were constructed from the histological data obtained from 10 rats for each area under investigation. CC, cerebral cortex; CP, caudate-putamen; GP, globus pallidus. Guide cannulae for. injections into the caudate-putamen were located at Anterior 8.0, Lateral, + 3.0, Vertical, + 3.0 (1.5); into the globus pallidus at Anterior 7.0, Lateral, k3.0, Vertical, + 1.0 (1.0); and into the cerebral cortex at Anterior 8.0, Lateral f3.0, Vertical, + 6.0 (1-O)(De GROOT,1959). Figures within parcnthesis indicate the extension (mm) of the injection cannuIX beyond the guides. These were selected for deposition ;II the “centre” of the area under investigation and, where practicable, at a point l.Omm beyond the location of the \tylet tip, allowing for termination of the guide within the area of investigation.
Electronic tremor recording. The technique for the electronic recording of tremor has been detailed elsewhere (KELLYand NAYLQR,1974). Briefly, the rat was placed in a perspcx cage which rested on a rubber support such that fine movements of the cage induced by tremor movements in the rat could be detected by a crystal phonocartridge positioned on a perspex block at the top of the cage. Cage movements caused stylus deflections and the resulting change in voltage output was recorded on a Grass Polygraph (Model No. 7B).
(sodium chloride). Control injections used solvent of equivalent pH and tonicity. Histological techniques
Upon completion of the experiments, rats were injected intracerebrally with trypan blue (1~1, 5% w/v aqueous solution). After l@-20min, they were anaesthetised with sodium pentobarbitone (80 mg/kg, i.p.) and exsanguinated. The brain was removed, sectioned at 25 pm and the intracerebral injection sites confirmed. Statistical analysis The effect of intracerebral administration of drugs upon the intensity of tremor induced by peripherally administered harmine was calculated by plotting the intensity of tremor against time after intracerebral injection for each rat and calculating the areas under the curves. Results for a group (minimum number of 4) of rats were combined and compared with results from a group of solvent-treated control animals using the Student’s t-test. Drugs
Harmine hydrochloride (Koch-Light) was dissolved in 1% lactic acid, 2-Amino-6,7-dihydroxy-1,2,3,4tetrahydronaphthalene hydrobromide, 2-amino6,7-dimethoxy-1,2,3,4_tetrahydronaphthalene hydrochloride, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrobromide, 6,7-dimethoxy-1,2,3,4_tetrahydroisoquinoline hydrochloride, dopamine hydrochloride (Koch-Light) and noradrenaline hydrogen tartrate (Hoechst) were dissolved in nitrogen bubbled distilled water. (+)-Amphetamine sulphate (Sigma) was dis-
Intracerebral injection technique
Stainless steel guide cannulae (a65 mm diameter) held on perspex holders were stereotaxically implanted into the brains of rats anaesthetized with chloral hydrate (300 mg/kg, i.p.). The perspex holders were fixed to the skull using retaining screws and acrylic cement. Stainless steel stylets (0.3 mm diam), extending 0.5 mm beyond the tip of the guide cannulae prevented occlusion of the lumen by blood and tissue. Guide cannulae were implanted bilaterally at the coordinates. shown in Figure 1. Animals were tist Jsed 1 week after cannulation. Rats were manually restrained, the stylet withdrawn and the inject& cannula (0.3 mm diameter) inserted. Drug solutions from an Agla micrometer syringe were delivered in a volume of 1~1 over a period of 5 set; the injection cannula remaining in position for a further 55 set before being withdrawn and replaced by the stylet. AI1 drug solutions were adjusted as closely as possible to pH 7.4 (sodium bicarbonate) and isotonic&y
Fig. 2. The effect of intrastriatal administration of dopamine, noradrenaline and dopaminergic agonists upon the intensity of tremor induced by harmine (10 mg/kg s.c.). Intensity of tremor was assessed according to the scoring system shown in Table 1. The intensity of tremor over a 20 min period following central administration was calculated by plotting the intensity of tremor against time for each rat and calculating the area under the curve. Histograms were constructed using the mean values for intensity (expressed as a percentage of solvent treated control values) of at least 4 rats. *P < 0.05; **P < 0.01; ***p < 0.001.
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by drugs
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20
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10
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Fig. 3. The effect of potassium chloride (1~1, 25% w/v solution) and procaine (1 ~1, 2% w/v solution) (stippled columns) injected into the caudate-putamen and globus pallidus upon the tremor induced by peripherally administered harmine (5, 10 and 20 mgkg SC.). Intensity of tremor was assessed according to the scoring system shown in Table 1. The intensity of tremor over a 20min period following central administration of drug was calculated by plotting the intensity of tremor against time for each rat and calculating the area under the curve in sq. mm. Histograms were constructed using the mean values for intensity over the 20min period obtained from at least 4 rats. *P < 0.05; **P < 0.01; ***P < 0.001. Results compared with those obtained from rats treated with harmine peripherally and drug solvents of equivalent pH and tonicity centrally administered (open columns).
solved in normal saline, apomorphine hydrochloride (Macfarlan Smith) in 0.1% sodium metabisulphite in normal saline and potassium chloride, procaine hydrochloride (B.D.H.) and 5hydroxytryptamine bimaleinate (Koch-Light) in distilled water. RESULTS
Histological
assessment of cannulae locations
The locations of guide cannulae for injections di-
rectly into the caudate-putamen, globus pallidus and cerebral cortex are shown in Figure 1. Locations in all the brains examined were found to be within the areas of investigation. Intracaudate mine
and intrapallidal
administration
of har-
Harmine, in doses up to 25Opg in 1% lactic acid or 10 pg in normal saline (2 ~1, injection vol), failed to induce tremor when injected into the caudate-putamen or globus pallidus. Occasional sniffing and biting behaviour, which was more intense after intrapallidal administration, was observed for up to 15-30 min after injection of either harmine or solvent. Modification of harmine tremor by intracaudate administration of pharmacological and neurochemical substances
Application of drug solvent into the caudate-putamen resulted in a transient (l-2min) reduction in the intensity of harmine tremor. After this period the intensity of tremor was indistinguishable from that observed in untreated harmine controls. Intrastriatal administration of dopamine, noradrenaline, apomorphine, (+)-amphetamine, 6,7-dihydroxy1,2,3,4-tetrahydroisoquinoline and 6,7-dimethoxy-1,2, 3,4-tetrahydroisoquinoline induced a dose-dependent reduction in the intensity of tremor induced by peripheral administration of harmine (Fig. 2). The reduction was immediate in onset and persisted for 20-35
min. Intrastriatal administration of 2-amino-6,7-dihyhydroxy-1,2,3,4_tetrahydronaphthalene or 2-amino-6, 7-dimethoxy-1,2,3,4_tetrahydronaphthalene failed to modify peripherally induced harmine tremor (Fig. 2). Intrastriatal administration of apomorphine induced an occasional biting behaviour and intrastriatal administration of (+)-amphetamine to harmine-pretreated rats resulted in a marked loss of motor control and coordination characterised by a flat body posture and a loss of use of the hind limbs. Intrastriatal administration of procaine or potassium chloride failed to modify harmine tremor (Fig. 3). Pretreatment of rats with intrastriatal 5-hydroxytryptamine (5-HT), 50 pg, administered 30 min before harmine (10 mg/kg) failed to modify tremor. However, pretreatment with 1OOpg 5-HT, resulted in an enhancement of tremor which was only evident 1 hr after harmine administration (Fig. 4). This enhancement of tremor was noted as a decrease in the periodicity of tremor rather than an increase in amplitude. Pretreatment with 5-HT (200,~g) resulted in a no greater increase in tremor than that observed after 5-HT (100 /Is). Modification of harmine tremor by intrapallidal administration of pharmacological and neurochemical substances
Injection of solvent into the globus pallidus resulted in a transient reduction in the intensity of tremor similar to that observed after injection of solvents into the caudate-putamen. Intrapallidal administration of dopamine, noradrenaline and apomorphine resulted in a dose-dependent reduction in the intensity of the tremor induced by peripheral administration of lOmg/kg harmine (Fig. 5). The reduction was immediate in onset and persisted for 20-35 min following injection.
306
D. M.
KELLYand R. J. NAYLOR noradrenaline which induced a brief period of marked muscular flaccidity, and (+)-amphetamine which induced weak stereotyped sniffing behaviour. Mod$cation of harmine tremor by dopamine, noradrenaline and apomorphine injected into the cerebral cortex
U
30 mm/min
Fig. 4. Effect of 5-HT (100,~g) administered into the caudate-putamen upon harmine tremor. Preliminary studies showed no difference between the tremor response of rats injected with solvent 30min before harmine, lOmg/kg S.C. (A) and untreated harmine controls. 5-HT was adminislered 30 min before harmine, 10 mg/kg S.C.(B). The record-
ings shown were obtained 1 hr after harmine administration using the procedure described in the Methods. Inhibition of pallidal function by direct application of procaine or by a spreading depression induced by potassium chloride also reduced the intensity of harmine tremor (Fig. 3). The tremor reduction after procaine lasted for approximately 30min, whereas the intensity of tremor returned to control values within 20 min of potassium chloride administration. lntrapallidal administration of 5-HT, 50, 100 or 200 pg failed to enhance harmine-induced tremor. Effects of the intrapallidal and intracaudate in the absence of any pretreatment
injections
Agents which were shown on bilateral intrapallidal and/or intrastriatal administration to modify harmine tremor, 10@400 pg dopamine and noradrenaline, 12.5-50 pg apomorphine, 25-50 pg (+)-amphetamine, 5&2OO,ug 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, 2-amino-6,7-dimethoxy-1,2,3,4-tetrahydronaphthalene, 6,7-dihydroxy-1,2,3,4_tetrahydroisoquinoline and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, failed to modify motor function when administered under similar conditions but in the absence of any pretreatment, with the exceptions of 100
g::
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amine
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Fig. 5. The effect of intrapallidal administration of dopamine and dopaminergic agonists upon the tremor induced by harmine (lOmg/kg, s.c.). Intensity of tremor was assessed according to the‘ scoring system shown in Table I. The intensity of tremor over a 20min period following central administration was calculated by plotting the intensity of tremor against time for each rat and calculating the area under the curve. Histograms were constructed using the mean values for intensity (expressed as a percentage of solvent treated control values) of at least 4 rats. *P < 0.05; **p < 0.01; ***p < 0.001.
Apart from an initial reduction in tremor (duration, l-2min) which could be attributed to an injection cortical artifact, administration of dopamine (5@200 pg), noradrenaline (50-200 pg) or apomorphine (12.5-50 pg) failed to modify the tremor induced by peripheral administration of harmine, lOmg/kg (P > 0.05). DISCUSSION The reduction in the tremorgenic activity of harmine in the rat by dopaminergic agonists would indicate that this tremor form may be mediated, at least in part, by an alteration in cerebral dopamine function. Dopamine is found in high concentrations in the striatal areas of the extrapyramidal system (BERTLER and ROSENGREN, 1959; BROCH and MARSDEN,1972) and electrolytic lesions of the paleostriatum have been shown to reduce harmine tremor and its reduction by dopaminergic agonists (KELLY and NAYLOR,1975). In the present studies, the reduction of harmine tremor by procaine and potassium chloride (spreading depression) in the globus pallidus but not the caudate-putamen emphasises the importance of the globus pallidus for tremor induction. The neostriatum is the most important source of pallidal input but electrolytic (KELLYand NAYLOR,1975) or temporary lesions (present studies) of the caudate-putamen failed to modify harmine tremor. Nevertheless, it would be incautious to suggest that the neostriatum cannot exert some influence upon harmine tremor for the remaining tissue may have retained some functional capacity and, more important, the present study shows that the direct injection of dopamine-like drugs into the caudate-putamen reduced the tremor induced by a peripheral injection of harmine. Apomorphine is generally considered to be a specific dopamine receptor stimulating agent (AND~~N, RUBENSON, FUXEand H&FELT, 1967) and upon injection into the neostriatum, in doses known to induce typical dopamine-like effects (contralateral asymmetries, stereotyped gnawing, biting and licking reactions) (COSTALL,NAYLORand PINDER, 1974, 1975), this agent was shown to reduce harmine tremor. Surprisingly, dopamine itself was found to be less active than apomorphine and the doses required to modify tremor were greater than those shown to be effective in previous studies (COSTALLet al., 1974, 1975) on dopamine-like activity in the striatum. Further, noradrenaline, which normally fails to modify the sys terns involved with asymmetry and stereotypy, and 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, which possesses only weak activity in such systems, were
Modification of harmine tremor by drugs both shown to be slightly more active than dopamine as antitremor agents. In addition, whilst methylation of the hydroxyl functions markedly reduces dopamine-like activity (COSTALL et al., 1975), the 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative remained effective as a tremor antagonist. Clearly, the striatal mechanisms involved with tremor antagonism are less specific than the dopamine systems analysed by COSTALLet al. (1974, 1975). It could be hypothesised that noradrenaline, amphetamine and the tetrahydroisoquinoline compounds were effective as antitremor agents by stimulating an unspecified dopamine mechanism (by release of dopamine or inhibition of reuptake processes or direct receptor stimulation) and that their differences in efficacy may have partly reflected their differential rates of metabolism by monoamine oxidase (BLASCHKO,RICHER and SCHLOSSMAN, 1937); but it is not understood why 2-amino-6,7-dihydroxy-tetrahydronaphthalene was inactive, since the conformation of this molecule is almost identical to that of the truns form of dopamine, and at the behavioural and biochemical level this agent is able to stimulate striatal and mesolimbic dopamine mechanisms (HORN, 1974; COSTALLet al., 1975; ELKHAWADand WOODRUFF,1975). The inactivity of 2-amino-6,7-dihydroxy-tetrahydronaphthalene particularly indicates that the antitremor activity of the above agents against harmine may be mediated at a different site to the other typical dopamine-induced effects. Although we have assumed that the neostriatum is the site of drug action after intrastriatal injections in the present studies, and the short latency of onset of effects would tend to support this assumption, the relatively large amounts of some drugs used and their abilities to diffuse rapidly, for example, apomorphine (BUITERWORTH,POIGNANTand BARBEAU,1975) and dopamine (BONDAREFF,ROUTTENBERG,NAROTZKY and MCLONE, 1970), necessitates a consideration of other sites of action. However, it should be noted that injections into the cerebral cortex were ineffective and this would indicate that drug diffusion may not be a critical factor. Brain lesion studies have indicated that the integrity of the neostriatum is non-essential for tremor induction and antagonism (KELLYand NAYLOR,1975), but the present intrastriatal injection studies emphasise that the caudate-putamen may still retain some modulatory control over harmine tremor. However, the importance of the globus pallidus for tremor control is indicated by the present studies and the finding of KELLY and NAYLOR (1975) that lesions of the globus pallidus reduce or abolish harmine tremor and that intrapallidal injections of dopamine and apomorphine also reduce harmine tremor. However, the specificity of this effect for dopamine agonists is questioned by the effectiveness of intrapallidal noradrenaline. Further, with the very close proximity of the pallidum to the caudate-putamen, it is possible that some diffusion may have occurred to striatal tissue.
307
HANDLERand BAK (1968) have proposed that a balanced dopamine/5-HT system, operational within the striatum, is involved with the antagonism of harmaline-induced tremors by L-DOPA. A similar hypothesis may be advanced for harmine tremor. We have shown that lesions placed in the midbrain raphk nuclei, which cause selective depletions of telencephalit 5-HT, reduce harmine tremor (COSTALLet al., 1976). The present studies indicate that it may be the effects of such lesions on striatal 5-HT mechanisms that is most critical to tremor development, since intrastriatal injections of 5-HT were shown to enhance harmine tremor. However, the interaction of dopamine with 5-HT would not appear to involve the paleostriaturn, since intrapallidal injeo tions of 5-HT failed to modify harmine tremor. Although the present observations support a role for striatal dopamine/5-HT mechanisms in the modulation of harmine tremor, the studies give no indication as to the precise site of action of harmine itself. In an acute study, Cox and POTKONJAK(1971) have reported the occurrence of tremor after the injection of harmine into the caudate-putamen and globus pallidus. However, in the chronically prepared animals used in the present work both intrapallidal and intrastriatal harmine failed to induce tremor. Several studies have shown that harmine may have a significant effect on the cerebellar systems (LAMARREand ME.RCW, 1972). It is possible that cerebellar efferent systems and those from the striatum may interact at an unspecified common level. This may offer some explanation for the failure of paleostriatal lesions or intrastriatal dopamine to completely abolish harmine tremor. Nevertheless, whatever the complexity of the cerebral mechanisms for tremor modulation, the present observations are that, at the level of the striatum, tremor would appear to be associated with increased 5-HT and decreased dopamine function. Acknowledgements-This work was supported by a grant from the Medical Research Council. The authors are grateful to Dr. R. M. PINDERfor gifts of the tetrahydroisoquinoline and tetrahydronaphthalene derivatives. REFERENCES AND~N,N.-E., RUBENSON, A., FUXE, K. and HBKFELT,T. (1967). Evidence for dopamine receptor stimulation by hine J Pharm. Pharmac. 19: 627-629. and ROSENGREN, E. (1959). Occurrence and BEZZX, . distribution of dopamine in brain and other tissues. Experientia 15: 10-11. BLASCHKO,H., RICHTER,D. and SCHLOSSMAN, H. (1937). The oxidation of adrenaline and other amines. B&hem. J. 31: 2187-2196. BONDAREFF,W., ROUTTENBERG, A., NAROTZKY,R. and MCLONE, D. G. (1970). Intrastriatal spreading of biogenie amines. Expl Neurol. 28: 213229. BROCH,0. J. and MARSDEN,C. A. (1972). Regional distribution of monoamines in the corpus striatum of the rat. Brain Res. 187: 163173. BUTVZRWORTH, R. F., POIGNANT,J.-C. and BARBEALJ, A. (1975). Apomorphine and piribedil in rats: biochemical and pharmacologic studies. A& Neurology 9: 307-326.
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COSTALL,B., KELLY, D. M. and NA~LOR, R. J. (1976). The importance of 5hydroxytryptamine for the induction of harmine tremor and its antagonism by dopaminergic agonists assessed by lesions of the midbrain rapht nuclei. Eur. J. Pharmc. 35: 109-119. COSTALL,B., NAYLOR,R. J. and F’INDER,R. M. (1974). Design of agents for stimulation of neostriatal dopamine@ mechanisms. J. Pharm. Pharmac. 26: 753762. COSTALL,B., NAYLOR,R. J. and PINDER, R. M. (1975). Dyskinetic phenomena caused by the intrastriatal injectionofphenylethylamine,phenylpiperazine, tetrahydroisoquinoline and tetrahydronaphthalene derivatives in the guinea pig. Eur. .I. Pharmac. 31: 94-109. Cox, B. and POTKONJAK,D. (1971). An investigation of the tremorogenic actions of harmine in the rat. Eur. .r. Pharmac. 16: 39-45. DE GROOT,J. (1959). The rat brain in stereotaxic coordinates. Verh. K. Ned. Akad. Wet. 59: 1431.
ELKHAWAD,A. 0. and WOODRUFF,G. N. (1975). Studies on the behavioural pharmacology of a cyclic analogue of dopamine following its injection into the brains of conscious rats. Br. .I. Pharmac. 54: 107-114. HASSLER,R. and BAK, I. J. (1968). In: Third Symposium on Parkinson’s Disease (GILLINGHAM, F. J. and DONALDSON,I. M. L., Eds.), pp. 2932, Livingstone, Edinburgh. HORN, A. S. (1974). The conformation of dopamine at its uptake site: further studies with rigid analogues. J. Pharm. Pharmac. 26: 735-737. KELLY,D. M. and NAYLOR,R. J. (1974). Mechanisms of tremor induction by harmine. Eur. J. Pharmac. 27: 1424. KELLY,D. M. and NAYLOR,R. J. (1975). The importance of extrapyramidal function for the induction and antagonism of harmine tremor. Eur. J. Phaymac. 32: 76-86. LAMARRE,Y. and MERCIER,L.-A. (1972). Etude neurophysiologique du tremblement a I’harmaline chez le chat. Rev. can. Biol. 31: 181-191.
1976. 15, 303-308.
Pergamon
Press.
Prmted
in Gt. Britain.
AN INTRACEREBRAL INJECTION STUDY ON THE ROLE OF STRIATAL DOPAMINE AND 5HYDROXYTRYPTAMINE IN THE PRODUCTION OF TREMOR BY HARMINE D. M. KELLY and R. J. NAYLOR Postgraduate
School of Studies in Pharmacology, University of Bradford, Bradford, West Yorkshire, U.K. (Accepted 3 October 1975)
Summary-The
intracerebral injection technique was used to investigate the role of striatal dopamine and serotonin in the production of tremor by harmine. When injected directly into the neostriatum, apomorphine and tetrahydroisoquinoline derivatives dopamine, noradrenaline, (+)-amphetamine, reduced the intensity of tremor produced by subcutaneous harmine but tetrahydronaphthalene derivatives were ineffective. Dopamine, noradrenaline and apomorphine were also effective after intrapallidal administration. Direct application of serotonin to the neo- but not the paleostriatum enhanced peripherally induced harmine tremor. The results suggest a relationship between dopamine and serotonin in the mediation of tremor and indicate that, whereas both pallidal and caudate dopamine functions are important for tremor antagonism, the site of a serotonin involvement with tremor is the neostriatum.
Evidence has been gained from both drug interaction studies (KELLY and NAYLOR, 1974) and from brain lesion work (COSTALL, KELLY and NAYLOR. 1976; KELLY and NA~OR, 1975) that the mechanisms by which harmine induces tremor in the rat involve both dopamine and 5-hydroxytryptamine. Further, the lesion studies have indicated that the striatum may be a site for the interaction between dopamine and 5-hydroxytryptamine. This possibility was investigated in the present studies using the intracerebral injection technique.
METHODS Animals
Male Sprague-Dawley (Caworth Strain) rats weighing 250 + 25 g were used throughout all experiments. Measurement of tremor Visual assessment. Rats were normally housed in groups of 8. For observation they were placed in individual, screened, perspex cages (30 x 20 x 15 cm) in a sound-proofed, diffusely illuminated room maintained at a temperature of 21 + 1°C. Rats were placed
in the observation cages at least 30min before injection of harmine to allow adaptation to the new environment. All observations were made between 09.00 and 21.00 hr. Previous studies have shown that the intensity of harmme tremor (assessed according to the scoring system shown in Table 1) is dose-related (KELLY and NAYLOR, 1974). A dose of 10 mg/kg administered sub-
cutaneously was selected as inducing a reproducible sub-maximal tremor response. Animals were observed in pairs, each rat receiving either drug or solvent intraeerebrally. Tremor was assessed at 1 min intervals for 20 min following intracerebral injection and subsequently at 15 min intervals for the duration of the drug effect. Harmine tremor has been shown to be differentiated into two components; 1) that occurring when the rat was undisturbed and at rest, i.e. “a resting tremor”, and 2) that which was only induced by increased activity or by mechanically shaking the cage, i.e. an “activity tremor” (KELLY and NAYLOR, 1974). It was impossible to differentiate resting tremor from activity tremor when tremor was assessed at 1 mm intervals, therefore, only activity tremor was assessed throughout these studies.
Table 1. Scoring system used for the estimation of the intensity of harmine tremor Score
Intensity of tremor
0
The appearance of the animals is the same as saline treated controls. Mild jaw and head tremor. Severe head tremor, but no body tremor. Mild whole body tremor. Severe whole body tremor.
1 2 3 4
303
D. M. KELLYand R. J. NAYLOR
304
33 .#a cc
CP
A’
&....,__
Fig. 1. Diagrammatic representation of the sites (@) of central administration of drugs. The diagrams were constructed from the histological data obtained from 10 rats for each area under investigation. CC, cerebral cortex; CP, caudate-putamen; GP, globus pallidus. Guide cannulae for. injections into the caudate-putamen were located at Anterior 8.0, Lateral, + 3.0, Vertical, + 3.0 (1.5); into the globus pallidus at Anterior 7.0, Lateral, k3.0, Vertical, + 1.0 (1.0); and into the cerebral cortex at Anterior 8.0, Lateral f3.0, Vertical, + 6.0 (1-O)(De GROOT,1959). Figures within parcnthesis indicate the extension (mm) of the injection cannuIX beyond the guides. These were selected for deposition ;II the “centre” of the area under investigation and, where practicable, at a point l.Omm beyond the location of the \tylet tip, allowing for termination of the guide within the area of investigation.
Electronic tremor recording. The technique for the electronic recording of tremor has been detailed elsewhere (KELLYand NAYLQR,1974). Briefly, the rat was placed in a perspcx cage which rested on a rubber support such that fine movements of the cage induced by tremor movements in the rat could be detected by a crystal phonocartridge positioned on a perspex block at the top of the cage. Cage movements caused stylus deflections and the resulting change in voltage output was recorded on a Grass Polygraph (Model No. 7B).
(sodium chloride). Control injections used solvent of equivalent pH and tonicity. Histological techniques
Upon completion of the experiments, rats were injected intracerebrally with trypan blue (1~1, 5% w/v aqueous solution). After l@-20min, they were anaesthetised with sodium pentobarbitone (80 mg/kg, i.p.) and exsanguinated. The brain was removed, sectioned at 25 pm and the intracerebral injection sites confirmed. Statistical analysis The effect of intracerebral administration of drugs upon the intensity of tremor induced by peripherally administered harmine was calculated by plotting the intensity of tremor against time after intracerebral injection for each rat and calculating the areas under the curves. Results for a group (minimum number of 4) of rats were combined and compared with results from a group of solvent-treated control animals using the Student’s t-test. Drugs
Harmine hydrochloride (Koch-Light) was dissolved in 1% lactic acid, 2-Amino-6,7-dihydroxy-1,2,3,4tetrahydronaphthalene hydrobromide, 2-amino6,7-dimethoxy-1,2,3,4_tetrahydronaphthalene hydrochloride, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrobromide, 6,7-dimethoxy-1,2,3,4_tetrahydroisoquinoline hydrochloride, dopamine hydrochloride (Koch-Light) and noradrenaline hydrogen tartrate (Hoechst) were dissolved in nitrogen bubbled distilled water. (+)-Amphetamine sulphate (Sigma) was dis-
Intracerebral injection technique
Stainless steel guide cannulae (a65 mm diameter) held on perspex holders were stereotaxically implanted into the brains of rats anaesthetized with chloral hydrate (300 mg/kg, i.p.). The perspex holders were fixed to the skull using retaining screws and acrylic cement. Stainless steel stylets (0.3 mm diam), extending 0.5 mm beyond the tip of the guide cannulae prevented occlusion of the lumen by blood and tissue. Guide cannulae were implanted bilaterally at the coordinates. shown in Figure 1. Animals were tist Jsed 1 week after cannulation. Rats were manually restrained, the stylet withdrawn and the inject& cannula (0.3 mm diameter) inserted. Drug solutions from an Agla micrometer syringe were delivered in a volume of 1~1 over a period of 5 set; the injection cannula remaining in position for a further 55 set before being withdrawn and replaced by the stylet. AI1 drug solutions were adjusted as closely as possible to pH 7.4 (sodium bicarbonate) and isotonic&y
Fig. 2. The effect of intrastriatal administration of dopamine, noradrenaline and dopaminergic agonists upon the intensity of tremor induced by harmine (10 mg/kg s.c.). Intensity of tremor was assessed according to the scoring system shown in Table 1. The intensity of tremor over a 20 min period following central administration was calculated by plotting the intensity of tremor against time for each rat and calculating the area under the curve. Histograms were constructed using the mean values for intensity (expressed as a percentage of solvent treated control values) of at least 4 rats. *P < 0.05; **P < 0.01; ***p < 0.001.
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Fig. 3. The effect of potassium chloride (1~1, 25% w/v solution) and procaine (1 ~1, 2% w/v solution) (stippled columns) injected into the caudate-putamen and globus pallidus upon the tremor induced by peripherally administered harmine (5, 10 and 20 mgkg SC.). Intensity of tremor was assessed according to the scoring system shown in Table 1. The intensity of tremor over a 20min period following central administration of drug was calculated by plotting the intensity of tremor against time for each rat and calculating the area under the curve in sq. mm. Histograms were constructed using the mean values for intensity over the 20min period obtained from at least 4 rats. *P < 0.05; **P < 0.01; ***P < 0.001. Results compared with those obtained from rats treated with harmine peripherally and drug solvents of equivalent pH and tonicity centrally administered (open columns).
solved in normal saline, apomorphine hydrochloride (Macfarlan Smith) in 0.1% sodium metabisulphite in normal saline and potassium chloride, procaine hydrochloride (B.D.H.) and 5hydroxytryptamine bimaleinate (Koch-Light) in distilled water. RESULTS
Histological
assessment of cannulae locations
The locations of guide cannulae for injections di-
rectly into the caudate-putamen, globus pallidus and cerebral cortex are shown in Figure 1. Locations in all the brains examined were found to be within the areas of investigation. Intracaudate mine
and intrapallidal
administration
of har-
Harmine, in doses up to 25Opg in 1% lactic acid or 10 pg in normal saline (2 ~1, injection vol), failed to induce tremor when injected into the caudate-putamen or globus pallidus. Occasional sniffing and biting behaviour, which was more intense after intrapallidal administration, was observed for up to 15-30 min after injection of either harmine or solvent. Modification of harmine tremor by intracaudate administration of pharmacological and neurochemical substances
Application of drug solvent into the caudate-putamen resulted in a transient (l-2min) reduction in the intensity of harmine tremor. After this period the intensity of tremor was indistinguishable from that observed in untreated harmine controls. Intrastriatal administration of dopamine, noradrenaline, apomorphine, (+)-amphetamine, 6,7-dihydroxy1,2,3,4-tetrahydroisoquinoline and 6,7-dimethoxy-1,2, 3,4-tetrahydroisoquinoline induced a dose-dependent reduction in the intensity of tremor induced by peripheral administration of harmine (Fig. 2). The reduction was immediate in onset and persisted for 20-35
min. Intrastriatal administration of 2-amino-6,7-dihyhydroxy-1,2,3,4_tetrahydronaphthalene or 2-amino-6, 7-dimethoxy-1,2,3,4_tetrahydronaphthalene failed to modify peripherally induced harmine tremor (Fig. 2). Intrastriatal administration of apomorphine induced an occasional biting behaviour and intrastriatal administration of (+)-amphetamine to harmine-pretreated rats resulted in a marked loss of motor control and coordination characterised by a flat body posture and a loss of use of the hind limbs. Intrastriatal administration of procaine or potassium chloride failed to modify harmine tremor (Fig. 3). Pretreatment of rats with intrastriatal 5-hydroxytryptamine (5-HT), 50 pg, administered 30 min before harmine (10 mg/kg) failed to modify tremor. However, pretreatment with 1OOpg 5-HT, resulted in an enhancement of tremor which was only evident 1 hr after harmine administration (Fig. 4). This enhancement of tremor was noted as a decrease in the periodicity of tremor rather than an increase in amplitude. Pretreatment with 5-HT (200,~g) resulted in a no greater increase in tremor than that observed after 5-HT (100 /Is). Modification of harmine tremor by intrapallidal administration of pharmacological and neurochemical substances
Injection of solvent into the globus pallidus resulted in a transient reduction in the intensity of tremor similar to that observed after injection of solvents into the caudate-putamen. Intrapallidal administration of dopamine, noradrenaline and apomorphine resulted in a dose-dependent reduction in the intensity of the tremor induced by peripheral administration of lOmg/kg harmine (Fig. 5). The reduction was immediate in onset and persisted for 20-35 min following injection.
306
D. M.
KELLYand R. J. NAYLOR noradrenaline which induced a brief period of marked muscular flaccidity, and (+)-amphetamine which induced weak stereotyped sniffing behaviour. Mod$cation of harmine tremor by dopamine, noradrenaline and apomorphine injected into the cerebral cortex
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30 mm/min
Fig. 4. Effect of 5-HT (100,~g) administered into the caudate-putamen upon harmine tremor. Preliminary studies showed no difference between the tremor response of rats injected with solvent 30min before harmine, lOmg/kg S.C. (A) and untreated harmine controls. 5-HT was adminislered 30 min before harmine, 10 mg/kg S.C.(B). The record-
ings shown were obtained 1 hr after harmine administration using the procedure described in the Methods. Inhibition of pallidal function by direct application of procaine or by a spreading depression induced by potassium chloride also reduced the intensity of harmine tremor (Fig. 3). The tremor reduction after procaine lasted for approximately 30min, whereas the intensity of tremor returned to control values within 20 min of potassium chloride administration. lntrapallidal administration of 5-HT, 50, 100 or 200 pg failed to enhance harmine-induced tremor. Effects of the intrapallidal and intracaudate in the absence of any pretreatment
injections
Agents which were shown on bilateral intrapallidal and/or intrastriatal administration to modify harmine tremor, 10@400 pg dopamine and noradrenaline, 12.5-50 pg apomorphine, 25-50 pg (+)-amphetamine, 5&2OO,ug 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, 2-amino-6,7-dimethoxy-1,2,3,4-tetrahydronaphthalene, 6,7-dihydroxy-1,2,3,4_tetrahydroisoquinoline and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, failed to modify motor function when administered under similar conditions but in the absence of any pretreatment, with the exceptions of 100
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Fig. 5. The effect of intrapallidal administration of dopamine and dopaminergic agonists upon the tremor induced by harmine (lOmg/kg, s.c.). Intensity of tremor was assessed according to the‘ scoring system shown in Table I. The intensity of tremor over a 20min period following central administration was calculated by plotting the intensity of tremor against time for each rat and calculating the area under the curve. Histograms were constructed using the mean values for intensity (expressed as a percentage of solvent treated control values) of at least 4 rats. *P < 0.05; **p < 0.01; ***p < 0.001.
Apart from an initial reduction in tremor (duration, l-2min) which could be attributed to an injection cortical artifact, administration of dopamine (5@200 pg), noradrenaline (50-200 pg) or apomorphine (12.5-50 pg) failed to modify the tremor induced by peripheral administration of harmine, lOmg/kg (P > 0.05). DISCUSSION The reduction in the tremorgenic activity of harmine in the rat by dopaminergic agonists would indicate that this tremor form may be mediated, at least in part, by an alteration in cerebral dopamine function. Dopamine is found in high concentrations in the striatal areas of the extrapyramidal system (BERTLER and ROSENGREN, 1959; BROCH and MARSDEN,1972) and electrolytic lesions of the paleostriatum have been shown to reduce harmine tremor and its reduction by dopaminergic agonists (KELLY and NAYLOR,1975). In the present studies, the reduction of harmine tremor by procaine and potassium chloride (spreading depression) in the globus pallidus but not the caudate-putamen emphasises the importance of the globus pallidus for tremor induction. The neostriatum is the most important source of pallidal input but electrolytic (KELLYand NAYLOR,1975) or temporary lesions (present studies) of the caudate-putamen failed to modify harmine tremor. Nevertheless, it would be incautious to suggest that the neostriatum cannot exert some influence upon harmine tremor for the remaining tissue may have retained some functional capacity and, more important, the present study shows that the direct injection of dopamine-like drugs into the caudate-putamen reduced the tremor induced by a peripheral injection of harmine. Apomorphine is generally considered to be a specific dopamine receptor stimulating agent (AND~~N, RUBENSON, FUXEand H&FELT, 1967) and upon injection into the neostriatum, in doses known to induce typical dopamine-like effects (contralateral asymmetries, stereotyped gnawing, biting and licking reactions) (COSTALL,NAYLORand PINDER, 1974, 1975), this agent was shown to reduce harmine tremor. Surprisingly, dopamine itself was found to be less active than apomorphine and the doses required to modify tremor were greater than those shown to be effective in previous studies (COSTALLet al., 1974, 1975) on dopamine-like activity in the striatum. Further, noradrenaline, which normally fails to modify the sys terns involved with asymmetry and stereotypy, and 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, which possesses only weak activity in such systems, were
Modification of harmine tremor by drugs both shown to be slightly more active than dopamine as antitremor agents. In addition, whilst methylation of the hydroxyl functions markedly reduces dopamine-like activity (COSTALL et al., 1975), the 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative remained effective as a tremor antagonist. Clearly, the striatal mechanisms involved with tremor antagonism are less specific than the dopamine systems analysed by COSTALLet al. (1974, 1975). It could be hypothesised that noradrenaline, amphetamine and the tetrahydroisoquinoline compounds were effective as antitremor agents by stimulating an unspecified dopamine mechanism (by release of dopamine or inhibition of reuptake processes or direct receptor stimulation) and that their differences in efficacy may have partly reflected their differential rates of metabolism by monoamine oxidase (BLASCHKO,RICHER and SCHLOSSMAN, 1937); but it is not understood why 2-amino-6,7-dihydroxy-tetrahydronaphthalene was inactive, since the conformation of this molecule is almost identical to that of the truns form of dopamine, and at the behavioural and biochemical level this agent is able to stimulate striatal and mesolimbic dopamine mechanisms (HORN, 1974; COSTALLet al., 1975; ELKHAWADand WOODRUFF,1975). The inactivity of 2-amino-6,7-dihydroxy-tetrahydronaphthalene particularly indicates that the antitremor activity of the above agents against harmine may be mediated at a different site to the other typical dopamine-induced effects. Although we have assumed that the neostriatum is the site of drug action after intrastriatal injections in the present studies, and the short latency of onset of effects would tend to support this assumption, the relatively large amounts of some drugs used and their abilities to diffuse rapidly, for example, apomorphine (BUITERWORTH,POIGNANTand BARBEAU,1975) and dopamine (BONDAREFF,ROUTTENBERG,NAROTZKY and MCLONE, 1970), necessitates a consideration of other sites of action. However, it should be noted that injections into the cerebral cortex were ineffective and this would indicate that drug diffusion may not be a critical factor. Brain lesion studies have indicated that the integrity of the neostriatum is non-essential for tremor induction and antagonism (KELLYand NAYLOR,1975), but the present intrastriatal injection studies emphasise that the caudate-putamen may still retain some modulatory control over harmine tremor. However, the importance of the globus pallidus for tremor control is indicated by the present studies and the finding of KELLY and NAYLOR (1975) that lesions of the globus pallidus reduce or abolish harmine tremor and that intrapallidal injections of dopamine and apomorphine also reduce harmine tremor. However, the specificity of this effect for dopamine agonists is questioned by the effectiveness of intrapallidal noradrenaline. Further, with the very close proximity of the pallidum to the caudate-putamen, it is possible that some diffusion may have occurred to striatal tissue.
307
HANDLERand BAK (1968) have proposed that a balanced dopamine/5-HT system, operational within the striatum, is involved with the antagonism of harmaline-induced tremors by L-DOPA. A similar hypothesis may be advanced for harmine tremor. We have shown that lesions placed in the midbrain raphk nuclei, which cause selective depletions of telencephalit 5-HT, reduce harmine tremor (COSTALLet al., 1976). The present studies indicate that it may be the effects of such lesions on striatal 5-HT mechanisms that is most critical to tremor development, since intrastriatal injections of 5-HT were shown to enhance harmine tremor. However, the interaction of dopamine with 5-HT would not appear to involve the paleostriaturn, since intrapallidal injeo tions of 5-HT failed to modify harmine tremor. Although the present observations support a role for striatal dopamine/5-HT mechanisms in the modulation of harmine tremor, the studies give no indication as to the precise site of action of harmine itself. In an acute study, Cox and POTKONJAK(1971) have reported the occurrence of tremor after the injection of harmine into the caudate-putamen and globus pallidus. However, in the chronically prepared animals used in the present work both intrapallidal and intrastriatal harmine failed to induce tremor. Several studies have shown that harmine may have a significant effect on the cerebellar systems (LAMARREand ME.RCW, 1972). It is possible that cerebellar efferent systems and those from the striatum may interact at an unspecified common level. This may offer some explanation for the failure of paleostriatal lesions or intrastriatal dopamine to completely abolish harmine tremor. Nevertheless, whatever the complexity of the cerebral mechanisms for tremor modulation, the present observations are that, at the level of the striatum, tremor would appear to be associated with increased 5-HT and decreased dopamine function. Acknowledgements-This work was supported by a grant from the Medical Research Council. The authors are grateful to Dr. R. M. PINDERfor gifts of the tetrahydroisoquinoline and tetrahydronaphthalene derivatives. REFERENCES AND~N,N.-E., RUBENSON, A., FUXE, K. and HBKFELT,T. (1967). Evidence for dopamine receptor stimulation by hine J Pharm. Pharmac. 19: 627-629. and ROSENGREN, E. (1959). Occurrence and BEZZX, . distribution of dopamine in brain and other tissues. Experientia 15: 10-11. BLASCHKO,H., RICHTER,D. and SCHLOSSMAN, H. (1937). The oxidation of adrenaline and other amines. B&hem. J. 31: 2187-2196. BONDAREFF,W., ROUTTENBERG, A., NAROTZKY,R. and MCLONE, D. G. (1970). Intrastriatal spreading of biogenie amines. Expl Neurol. 28: 213229. BROCH,0. J. and MARSDEN,C. A. (1972). Regional distribution of monoamines in the corpus striatum of the rat. Brain Res. 187: 163173. BUTVZRWORTH, R. F., POIGNANT,J.-C. and BARBEALJ, A. (1975). Apomorphine and piribedil in rats: biochemical and pharmacologic studies. A& Neurology 9: 307-326.
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COSTALL,B., KELLY, D. M. and NA~LOR, R. J. (1976). The importance of 5hydroxytryptamine for the induction of harmine tremor and its antagonism by dopaminergic agonists assessed by lesions of the midbrain rapht nuclei. Eur. J. Pharmc. 35: 109-119. COSTALL,B., NAYLOR,R. J. and F’INDER,R. M. (1974). Design of agents for stimulation of neostriatal dopamine@ mechanisms. J. Pharm. Pharmac. 26: 753762. COSTALL,B., NAYLOR,R. J. and PINDER, R. M. (1975). Dyskinetic phenomena caused by the intrastriatal injectionofphenylethylamine,phenylpiperazine, tetrahydroisoquinoline and tetrahydronaphthalene derivatives in the guinea pig. Eur. .I. Pharmac. 31: 94-109. Cox, B. and POTKONJAK,D. (1971). An investigation of the tremorogenic actions of harmine in the rat. Eur. .r. Pharmac. 16: 39-45. DE GROOT,J. (1959). The rat brain in stereotaxic coordinates. Verh. K. Ned. Akad. Wet. 59: 1431.
ELKHAWAD,A. 0. and WOODRUFF,G. N. (1975). Studies on the behavioural pharmacology of a cyclic analogue of dopamine following its injection into the brains of conscious rats. Br. .I. Pharmac. 54: 107-114. HASSLER,R. and BAK, I. J. (1968). In: Third Symposium on Parkinson’s Disease (GILLINGHAM, F. J. and DONALDSON,I. M. L., Eds.), pp. 2932, Livingstone, Edinburgh. HORN, A. S. (1974). The conformation of dopamine at its uptake site: further studies with rigid analogues. J. Pharm. Pharmac. 26: 735-737. KELLY,D. M. and NAYLOR,R. J. (1974). Mechanisms of tremor induction by harmine. Eur. J. Pharmac. 27: 1424. KELLY,D. M. and NAYLOR,R. J. (1975). The importance of extrapyramidal function for the induction and antagonism of harmine tremor. Eur. J. Phaymac. 32: 76-86. LAMARRE,Y. and MERCIER,L.-A. (1972). Etude neurophysiologique du tremblement a I’harmaline chez le chat. Rev. can. Biol. 31: 181-191.