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Inhibition of anticholinergic drug-induced locomotor stimulation in mice by α-methyltyrosine
Neuropharmacology,
1973,
12, 1179-l
185
Pergamon Press. Prillted inC;t.Britain
INHIBITION OF ANTICHOLINERGIC DRUG-INDUCED LOCOMOTOR STIMULATION IN MICE BY wMETHYLTYROSINE* J. E. THORNBURG and K. E. MOORE Department of Pharmacology, Michigan State University, East Lansing, Michigan 48823 (Accepted I July 1973)
S~mary-~-~ethyltyrosjne,
administered in the diet to mice, inhibited locomotor stimulation scopolamine and atropine. FLA-63, administeredin thesamemanner, did not alter any of the drug-stimulated activities. These results suggest that a dopaminergic system is involved in the pathway mediatinganticholinergicdrug-induced locomotor stimulation. induced by benztropine,
Benztropine is structurally and pharmacologically similar to the classical anticholinergics, scopolamine and atropine. In contrast to scopolamine and a&opine, however, benztropine is a potent inhibitor of dopamine uptake by rat brain synaptosomes (COYLE and SNYDER, 1969; FARNEBO, FUXE, HAMBERGERand LJUNGDAHL, 1970; HORN, CWLE and SNYDER, 1971), prompting Coyle and Snyder to propose that benztropine exerts its antiparkinson activity by direct inhibition of dopamine reuptake in the corpus striatum. Nevertheless, scopolamine and other anticholinergics also appear to interact indirectly with dopaminergic systems in the brain. For example, scopolamine and benztropine potentiate gnawing behaviour induced by apomorphine (SCHEEL-KRUGER, 1970). Benztropine, atropine and trihexyphenidyl antagonize the increase in brain homovanillic acid concentrations induced by chlorpromazine or haloperidol (O’KEEFE, SHARMAN and VOGT, 1970; BOWERS and ROTH, 1972; ANDI~N, 1972). The anticholinergics, including benztropine, may produce these effectsthrough primary actions at cholinergic synapses which are in reciprocal balance with dopaminergic neurones in the striatum (see COSTALL, NAYLOR and OLLEY, 1972a,b).
(+)-Amphetamine like benztropine, is a potent inhibitor of dopamine uptake by rat brain synaptosomes (COYLE and SNYDER, 1969; THORNBURGand MOORE, 1973a). (+)-Amphetamine-stimulated locomotor activity appears to be mediated through a dopaminergic mechanism (COSTA, GROPPETTIand NAIMZADA, 1972); inhibition of tyrosine hydroxylase by a-methyltyrosine but not inhibition of dopamine-/I-hydroxylase by FLA-63 antagonizes (+)-amphetamine-stimulated activity (THORNBURG,1973; THORNBURGand MOORE, 1973b). If benztropine produces locomotor stimulation primarily by blocking reuptake of dopamine, the stimulant actions of this drug should resemble those of (+)-amphetamine but not of scopolamine or atropine. On the other hand, if locomotor stimulation induced by benztropine is the primary result of its anticholinergic properties, the actions of this drug should be mimicked by atropine and scopo1amin.e. The results of the present study support the latter hypothesis. Nevertheless, a dopaminergic mechanism does appear to be involved * Supported
by USPHS grants NS09174 and MH 13174. 1179
J. E.
1180
THORNBURC;and K. E. MOORE
in the locomotor stimulation produced by all three drugs. by u-methyltyrosine but not by FLA-63.
since their actions
are blocked
METHODS
Male albino mice (25-30 g) obtained from Spartan Research Animals, Inc. (Haslett. Michigan) were used in all experiments. Locomotor activity was measured using Woodard actophotometer cages, which consist of a circular rumvay with a central light source and six photocells equally spaced around the outer wall of the runway. Mice were acclimated to a 4 hr feeding schedule for 4 days. On the test day. mice received either the control diet (ground Wayne Lab Blox. 30 g’six animals) or a diet containing 0.4’:; r-methyltyrosine or 0.05% FLA-63. Testing commenced at 13.00-16.00 hr, I hr after withdrawal of the food cup. Two mice were placed in the actophotometer for 20 min, removed, administered either saline (0.01 ml’g) or stimulant drug i.p. and then returned to the actophotometer for 60 or 120 min, with activity being recorded at 20 min intervals. Data are plotted as “drug-stimulated activity,” since the baseline activities of mice fed the control and r-methyltyrosine diets were significantly different (Fig. I). For each dietary regimen, the mean activity during 1200-
200 -
I
100 TIME
12:
(mln)
t Fig. 1. Effects of a 4 hr diet containing 0.4% a-methyltyrosine (a-MT) or 0.05 % FLA-63 on locomotor activity in mice. The control (triangles), or-MT (circles) or FLA-63 (squares) diet was presented to mice for 4 hr. One hour after food was withdrawn, pairs of mice were accomodated to circular actophotometers for 20 min, removed and injected with saline (0.01 ml/g), indicated by arrow at zero time, and returned to the actophotometer for 2 hr. Symbols represent mean values and vertical lines 1 S.E.M. of at least 12 determinations. Solid symbols indicate a significant inhibition of locomotor activity (P < 0.01).
a given 20 min interval of mice administered saline was subtracted from the activity of mice administered an anticholinergic drug. The effects of saline and the anticholinergic drugs were determined concurrently. Data were analyzed using Student’s t-test (GOLDSTEIN, 1964) and the level of statistical significance was chosen as P < 0.01. The drugs used were DL-C+methyltyrosine and FLA-63 [bis(4-methyl-l-homopiperazinylthiocarbonyl disulfide)] from Regis Chemical Co., Chicago, Ill. ; benztropine mesylate (Cogentin”) from Dr. C. Stone, Merck, Sharp and Dohme Research Laboratory, West
1181
Benztropine stimulation
Point, Pa.; atropine sulphate from S. B. Pennick Co., Chicago Ill.; scopolamine bromide from Sigma Chemical Co.. St. Louis, MO. Doses of drugs are reported respective salts.
hydroas their
RESULTS
Administration and formation
of xc-methyltyrosine in the diet effectively reduces the endogenous content from radiolabelled tyrosine of dopamine and norepinephrine in brain (THORNBURG,1973; THORNBURG and MOORF.. 1973b). Similar administration of the dopamine-/j-hydroxylase inhibitor. FLA-63. markedly reduces the endogenous content and synthesis of norepinephrine but not of dopamine. Importantly, administration of these drugs in the diet minimizes nonspecific “stress-like” effects associated with intraperitoneal administration (THORNBURG and MOORE, 1971; 1973b). Benztropine (5 and IO mg’kg) caused a significant stimulation of motor activity (Fig. 2). cr-Methyltyrosine did not alter benztropine-stimulated activity during the initial 40 min period but significantly reduced the stimulated activity between 40 and 120 min after benztropine administration. In contrast. FLA-63 did not alter benztropine-stimulated
cooi60054002 =200a u ? 0-h
40
60
80
loo
AFTER
BENZTROPINE
120
f
F : 1600-
IOmg/kg
400-
200-
TIME
(min
1
Fig. 2. Effects of a 4 hr diet containing 0.4 % a-methyltyrosine (cc-MT) or 0.05 % FLA-63 on benztropine-stimulated locomotor activity. The control (triangles), a-MT (circles), or FLA-63 (squares) diet was presented to mice for 4 hr. One hour after food was withdrawn, mice were placed in actophotometers for 20 min, removed, administered either saline or benztropine i.p. (5 or 10 mg/kg) and returned to the actophotometer for 2 hr. Benztropine-stimulated activity values represent mean differences in counts per 20 min interval between mice receiving saline and mice receiving benztropine. Symbols represent means and vertical lines 1 S.E.M. of lo-26 determinations. Solid symbols indicate a significant inhibition of benztropine-induced stimulation (P -: 0.01).
J. E. THORNBURG and K. E. MOORE
1182
activity at any time interval, suggesting that the inhibitory effect of cc-methyltyrosine was related to inhibition of synthesis of brain dopamine. Similarly, stimulated activity induced by I mg,‘kg scopolamine was inhibited by tlmethyltyrosine only after an initial 40 min period of no effect (Fig. 3). In combination with r 0.5 mg/kg
L
4
r
20
40
60
00
100
120
I.Omg/kg
O40 TIME
60 AFTER
SCOPOLAMlNE(min)
Fig. 3. Effects of a 4 hr diet containing 0.4 % a-methyltyrosine (K-MT) or 0.05 % FLA-63 on scopolamine-stimulated locomotor activity. The control (triangles), a-MT (circles), or FLA-63 (squares) diet was presented to mice for 4 hr. One hour after food was withdrawn, mice were placed in actophotometers for 20 min, removed, administered either saline or scopolamine i.p.(O.25, 05 or I.0 mg/kg) and returned to the actophotometer for 1 or 2 hr. Scopolaminestimulated activity values represent mean differences in counts per 20 min interval between mice receiving saline and mice receiving scopolamine. Symbols represent means and vertical lines 1 S.E.M. of 8-19 determinations. Solid symbols indicate a significant inhibition of scopolamineinduced stimulation (P < 0.01).
doses of 0.25 and 0.50 mg/kg of scopolamine, however, a-methyltyrosine effectively reduced stimulated activity during the initial 40 min as well. FLA-63 had no effect on scopolaminestimulated activity. cc-Methyltyrosine almost completely blocked stimulated activity following 4 mg/kg atropine (Fig. 4). A dose of 2 mg:‘kg atropine did not produce significant stimulation. Although not shown, 8 mgikg atropine produced less stimulation than did 4 mg/kg, presumably due to toxicity.
Benztropine stimulation
TIME
AFTER
1183
ATROPINE (min f
Fig. 4. Effects of a 4 hr diet containing 0.4 % cc-methyltyrosine (a-MT) on atropine-stimulated locomotor activity. The control (triangles) or a-MT (circles) diet was presented to mice for 4 hr. One hour after food was withdrawn, mice were placed in actophotometers for 20 min, removed, administered either saline or atropine i.p. (2 or 4 mgjkg) and returned to the actop~otometer for I hr. Atropine-stimulated activity values represent mean differences in counts per 20 min interval between mice receiving saline and mice receiving atropine. Symbols represent means and vertical lines 1 S.E.M. of IO-14 determinations. Solid symbols indicate a significant inhibition of atropine-induced stimulation (P < @01). DISCUSSION
The present data fail to distinguish between an anticholinergic or a dopaminergic mechanism of action for ben~trop~ne-stimulated locomotor activity. Nevertheless, this is the first demonstration that inhibition of dopamine synthesis in the brain causes a marked reduction in anticholinergic (antimuscarinic) stimulated locomotor activity. In a previous study, cc-methytyrosine blocked ( +)-amphetamine-improved shuttle box avoidance responding of rats, but did not alter similar effects of scopolamine (RECH and MOORE, 1968). The concept of a reciprocal balance between dopaminergic and cholinergic systems in the neostriatum is supported by considerable behavioural and biochemical evidence (SCHEELKRUGER, 1970; COSTALL et al., 1972a). Subthreshold doses of scopolamine and atropine potentiate amphetamine-induced increases in response rates of rats in an operant, shockavoidance situation (CARLTON, 1961; CARLTOE:and DIDAMO, 1961 j. Similarly, subthreshold doses of scopolamine potentiate amphetamine-induced locomotor activity in rats (FIBIGER, LYTLE and CAMPBELL, 1970). Scopolamine also decreases the threshold dose for amphetamine-induced stereotyped behaviour in rats (ARNFREDand RANDRUP. 196X; KLAWANS, RUBOWTS, PATEL and WEINER, 1972) and apomorphine-induced gnawing in mice (SCHEELKRUEGER, 1970). Moreover, unilateral administration of arecoline or haloperidol directly into the globus pallidus or caudate nucleus causes ipsilateral (toward the side of injection) circling, whereas atropine has the opposite effect (COSTALL et al., 1972b). Several investigators have examined biochemically the interactions of anticholinergic drugs and brain dopamine metabolism. In general, the anticholinergics tend to antagonize neuroleptic (dopamine receptor blockade)-induced increases in homovanillic acid contents (O’KEEFE et al., 1970; BOWERSand ROTH, 1972; AND~N, 1972) and changes in turnover rates
1184
J. E. THOK~BURG
and K. E. MOORE
of dopamine (AND~N and B~~IIAR~, 1971; CORR~DI, FUXE and LIDBRINK, 1972). HITZEMANN, LOH and DOMINO (1972) found that scopolamine. at doses which markedly increase locomotor activity, decreased the accumulation of “C-dopamine and 14C-norepinephrine in mouse brain after administration of either ‘“C-tyrosine or “C-DOPA. Whether the decreased accumulation of “C-catecholamines was due to decreased synthesis, increased release or increased intraneuronal degradation is unclear. GOODALE and MOORE (1973) and C~~ERAMY. GAUCHY, GLOWINSKI and BESSON (1973) found that benztropine and scopolamine increase the efflux of 3H-dopamine from the cat brain. Unfortunately, due to the difficulties in determination of acetylcholine synthesis and turnover rates, little is known about the effects of alterations of neostriatal dopamine metabolism on brain acetylcholine dynamics. Recent evidence obtained in our laboratory suggests that scopolamine and benztropine induce locomotor stimulation primarily by an anticholinergic mechanism. Amphetamine produced marked locomotor stimulation in the mouse on postnatal day IO, IO-15 days prior to the onset of significant scopolamineor benztropine-induced stimulation (THORNBURC; and MOORE, 1973~). CAMPBELL, LYTLH and Ftetw R (1969) had previously demonstrated similar developmental patterns for scopolamineand (+)-amphetamine-induced stimulation in the rat. The anatomical relationship between dopaminergic and cholinergic neurones in the basal ganglia remains unclear. Nevertheless, a normally functioning dopaminergic neuronal system appears to be required for anticholinergic drug-induced locomotor stimulation. REFERENCES N.-E. (1972). Dopamine turnover in the corpus striatum and the limbic system after treatment with neuroleptic and anti-acetylcholine drugs. J. Pharm. Pharmac. 24: 905-906. AND~N, N.-E. and B~DAKD, P. (1971). influences of cholinergic mechanisms on the function and turnover of brain dopamine. J. Pharm. Pharmac. 23: 460-462. ARNFRED, T. and RANDKUP, A. (1968). Cholinergic mechanism in brain inhibiting amphetamine-induced stereotyped behaviour. Ac,ta pharmac. 10s. 26: 3X4-394. BOWERS, M. B. and ROTH, R. H. (1972). Interaction of atropine-like drugs with dopamine-containing neurones in rat brain. Br. J. Pharmac. 44: 3OlL306. CAMPBELL, B. A., LYTLC, L. D. and FIBWER, H. C. (1969). Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Scirnce 166: 635-637. CARLTON, P. L. (1961). Augmentation of the behavioral effects of amphetamine by scopolamine. Psychopharmau~iqgia 2: 377-380. CARLTON, P. L. and DIDAMO, P. (1961). Augmentation of the behavioral effects of amphetamine by atropine. J. Pharmac. exp. Thw. 134: 91-96. CHERAMY, A., GAWHY, G., CI.OWINSKI, J. and BESSON, M.-J. (1973). In riw activation by benztropine of dopamine release and synthesis in the caudate nucleus. Eur. J. Pharmac. 21: 246-248. CORRODI, H., Fuxr, K. and LIDBRINK, P. (1972). Interaction between cholinergic and catecholaminergic neurones in rat brain. Bruin Res. 43: 397416. COSTA, E., GROPP~TTI, A. and NAIM~ADA, M. K. (1972). Effects of amphetamine on the turnover rate of brain catecholamines and motor activity. Br. J. Pharmac. 44: 742-751. COSTALL, B., NAYLOR, R. J. and OLLtr, J. E. (1972a). On the involvement of the caudate-putamen, globus pallidus and substantia nigra with neuroleptic and cholinergic modification of locomotor activity. Neuropharmacology I1 : 3 I 7-330. COSTALL, B., NAYLOR, R. J. and OLLEY, J. E. (1972b). Catalepsy and circling behaviour after intracerebral injections of neuroleptic, cholinergic and anticholinergic agents into the caudate-putamen, globus pallidus and substantia nigra of rat brain. Neuropharmacology 11: 645-663. COYLE, J. T. and SNYDER, S. H. (1969). Antiparkinsonian drugs: inhibition of dopamine uptake in the corpus striatum as a possible mechanism of action. Science 166: 899-901. FARNEBO, L.-O., FUXE, K., HAMBERGER, B. and LJUNGDAHL, H. (I 970). Effect of some antiparkinsonian drugs on catecholamine neurons. J. Pharm. Pharmac. 22: 733-737. FIBIGER, H. C., LYTLE, L. D. and CAMPBELL, B. A. (1970). Cholinergic modulation of adrenergic arousal in the developing rat. J. camp. ph.vsiol. P.s.vchol. 12: 384-389. New York. GOLDSTEIN, A. (I 964). Biostatistics: An Introductory Test , 272 pp. Macmillan, AND~N,
Benztropine
stimulation
1185
GCICDALE,D.
B. and MOORE,K. E. (1973). In t’jL’orelease of 3H-dopamine from cat brain by benztropine. ~~~~~?~~~~~~g~~~ 15: f 74. HETZEMANN, R. 3,, LCIH,H. H. and DOMINO, E. F. (1972). Effect of scopolamine on the cerebral accumulation of “C-catecholamines from ‘%-tyrosine. Pharmacology 8: 291-294. HORN, A. S., COYLE,J. T. and SNYDERS. H. (1971). Catecholamine uptake by sy~~ptosomes from rat brain: structure-activity relationships of drugs with differential effects on dopamine and norepinephrine neurons. Molec. Pharmac. 7: 66-80. KLAWANS, H. L., Rueovrrs, R., PATEL, B. C. and WEINER,W. J. (1972). Cholinergic and anticholinergic influences on amphetamine-jnduced stereotyped behavior. J. I\jerrrol. Sei. 17: W-308. O’KEEFE, R., SHARMAN, D. F. and VOW, M. (1970). Effect of drugs used in psychoses on cerebral dopamine metabolism. Br. 1. Pharmar. 38: 287-304. Racn, R. H. and MOORE, K. E. (1968). Interactions between ~-amphetamine and ff-methylt~r~s~ne in rat shuttle-box behavior. Brain Res. 8: 398-100. SCHEEL-KH~GER, J. (I970). Central effects of anticholinergic drugs measured by the apomorphine gnawing test in mice. Acra p~~~rnac. tax. 28: 1-16. TWQRNELJRG, J. E. (1973).Relative role of dopaminergic and noradrenergic neuron& systems for the stimulation of locomotor activity induced by amphetamine and other drugs. Fedn Proc. Fedn Am. Sors exp. Biol. 32: 753.
and MOORE, K. E. (1971). Stress-related effects of various inhibitors of catecholamine synthesis in the mouse. Archs i~t. Ph~~rn~c~~~~. Th&. 194: 158-167. THORNBURG,J. E. and MOORE,I(. E. (1973a). Dopamine and norep;nephrine uptake by rat brain synaptosomes: relative inhibitory potencies of I- and ~-ampheta~line and amantadjne. Res. Comm. &em.
THORNBURG, J. E.
Pathol. Pharmac. 5: 81-89.
THORNBURG, J. E. and MOORE, K. E. (1973b). The relative importance of dopaminergic and noradrenergic neuronai systems for the stimulation of locomotor activity induced by amphetamine and other drugs. ~e~rQp~a~rn~cff~~gy 12: 853-866.
and MOORE, K. E. (1973~). Postnatal development of ~n~tropine-induced locomotor evidence for an anticholinergic mechanism of action. Res. Cornin. &em. Pathal. Pharmac.
THORNBURG, J. E.
stimulation: 6: 313-320.
NP Vol. 12 No. 11-F
1973,
12, 1179-l
185
Pergamon Press. Prillted inC;t.Britain
INHIBITION OF ANTICHOLINERGIC DRUG-INDUCED LOCOMOTOR STIMULATION IN MICE BY wMETHYLTYROSINE* J. E. THORNBURG and K. E. MOORE Department of Pharmacology, Michigan State University, East Lansing, Michigan 48823 (Accepted I July 1973)
S~mary-~-~ethyltyrosjne,
administered in the diet to mice, inhibited locomotor stimulation scopolamine and atropine. FLA-63, administeredin thesamemanner, did not alter any of the drug-stimulated activities. These results suggest that a dopaminergic system is involved in the pathway mediatinganticholinergicdrug-induced locomotor stimulation. induced by benztropine,
Benztropine is structurally and pharmacologically similar to the classical anticholinergics, scopolamine and atropine. In contrast to scopolamine and a&opine, however, benztropine is a potent inhibitor of dopamine uptake by rat brain synaptosomes (COYLE and SNYDER, 1969; FARNEBO, FUXE, HAMBERGERand LJUNGDAHL, 1970; HORN, CWLE and SNYDER, 1971), prompting Coyle and Snyder to propose that benztropine exerts its antiparkinson activity by direct inhibition of dopamine reuptake in the corpus striatum. Nevertheless, scopolamine and other anticholinergics also appear to interact indirectly with dopaminergic systems in the brain. For example, scopolamine and benztropine potentiate gnawing behaviour induced by apomorphine (SCHEEL-KRUGER, 1970). Benztropine, atropine and trihexyphenidyl antagonize the increase in brain homovanillic acid concentrations induced by chlorpromazine or haloperidol (O’KEEFE, SHARMAN and VOGT, 1970; BOWERS and ROTH, 1972; ANDI~N, 1972). The anticholinergics, including benztropine, may produce these effectsthrough primary actions at cholinergic synapses which are in reciprocal balance with dopaminergic neurones in the striatum (see COSTALL, NAYLOR and OLLEY, 1972a,b).
(+)-Amphetamine like benztropine, is a potent inhibitor of dopamine uptake by rat brain synaptosomes (COYLE and SNYDER, 1969; THORNBURGand MOORE, 1973a). (+)-Amphetamine-stimulated locomotor activity appears to be mediated through a dopaminergic mechanism (COSTA, GROPPETTIand NAIMZADA, 1972); inhibition of tyrosine hydroxylase by a-methyltyrosine but not inhibition of dopamine-/I-hydroxylase by FLA-63 antagonizes (+)-amphetamine-stimulated activity (THORNBURG,1973; THORNBURGand MOORE, 1973b). If benztropine produces locomotor stimulation primarily by blocking reuptake of dopamine, the stimulant actions of this drug should resemble those of (+)-amphetamine but not of scopolamine or atropine. On the other hand, if locomotor stimulation induced by benztropine is the primary result of its anticholinergic properties, the actions of this drug should be mimicked by atropine and scopo1amin.e. The results of the present study support the latter hypothesis. Nevertheless, a dopaminergic mechanism does appear to be involved * Supported
by USPHS grants NS09174 and MH 13174. 1179
J. E.
1180
THORNBURC;and K. E. MOORE
in the locomotor stimulation produced by all three drugs. by u-methyltyrosine but not by FLA-63.
since their actions
are blocked
METHODS
Male albino mice (25-30 g) obtained from Spartan Research Animals, Inc. (Haslett. Michigan) were used in all experiments. Locomotor activity was measured using Woodard actophotometer cages, which consist of a circular rumvay with a central light source and six photocells equally spaced around the outer wall of the runway. Mice were acclimated to a 4 hr feeding schedule for 4 days. On the test day. mice received either the control diet (ground Wayne Lab Blox. 30 g’six animals) or a diet containing 0.4’:; r-methyltyrosine or 0.05% FLA-63. Testing commenced at 13.00-16.00 hr, I hr after withdrawal of the food cup. Two mice were placed in the actophotometer for 20 min, removed, administered either saline (0.01 ml’g) or stimulant drug i.p. and then returned to the actophotometer for 60 or 120 min, with activity being recorded at 20 min intervals. Data are plotted as “drug-stimulated activity,” since the baseline activities of mice fed the control and r-methyltyrosine diets were significantly different (Fig. I). For each dietary regimen, the mean activity during 1200-
200 -
I
100 TIME
12:
(mln)
t Fig. 1. Effects of a 4 hr diet containing 0.4% a-methyltyrosine (a-MT) or 0.05 % FLA-63 on locomotor activity in mice. The control (triangles), or-MT (circles) or FLA-63 (squares) diet was presented to mice for 4 hr. One hour after food was withdrawn, pairs of mice were accomodated to circular actophotometers for 20 min, removed and injected with saline (0.01 ml/g), indicated by arrow at zero time, and returned to the actophotometer for 2 hr. Symbols represent mean values and vertical lines 1 S.E.M. of at least 12 determinations. Solid symbols indicate a significant inhibition of locomotor activity (P < 0.01).
a given 20 min interval of mice administered saline was subtracted from the activity of mice administered an anticholinergic drug. The effects of saline and the anticholinergic drugs were determined concurrently. Data were analyzed using Student’s t-test (GOLDSTEIN, 1964) and the level of statistical significance was chosen as P < 0.01. The drugs used were DL-C+methyltyrosine and FLA-63 [bis(4-methyl-l-homopiperazinylthiocarbonyl disulfide)] from Regis Chemical Co., Chicago, Ill. ; benztropine mesylate (Cogentin”) from Dr. C. Stone, Merck, Sharp and Dohme Research Laboratory, West
1181
Benztropine stimulation
Point, Pa.; atropine sulphate from S. B. Pennick Co., Chicago Ill.; scopolamine bromide from Sigma Chemical Co.. St. Louis, MO. Doses of drugs are reported respective salts.
hydroas their
RESULTS
Administration and formation
of xc-methyltyrosine in the diet effectively reduces the endogenous content from radiolabelled tyrosine of dopamine and norepinephrine in brain (THORNBURG,1973; THORNBURG and MOORF.. 1973b). Similar administration of the dopamine-/j-hydroxylase inhibitor. FLA-63. markedly reduces the endogenous content and synthesis of norepinephrine but not of dopamine. Importantly, administration of these drugs in the diet minimizes nonspecific “stress-like” effects associated with intraperitoneal administration (THORNBURG and MOORE, 1971; 1973b). Benztropine (5 and IO mg’kg) caused a significant stimulation of motor activity (Fig. 2). cr-Methyltyrosine did not alter benztropine-stimulated activity during the initial 40 min period but significantly reduced the stimulated activity between 40 and 120 min after benztropine administration. In contrast. FLA-63 did not alter benztropine-stimulated
cooi60054002 =200a u ? 0-h
40
60
80
loo
AFTER
BENZTROPINE
120
f
F : 1600-
IOmg/kg
400-
200-
TIME
(min
1
Fig. 2. Effects of a 4 hr diet containing 0.4 % a-methyltyrosine (cc-MT) or 0.05 % FLA-63 on benztropine-stimulated locomotor activity. The control (triangles), a-MT (circles), or FLA-63 (squares) diet was presented to mice for 4 hr. One hour after food was withdrawn, mice were placed in actophotometers for 20 min, removed, administered either saline or benztropine i.p. (5 or 10 mg/kg) and returned to the actophotometer for 2 hr. Benztropine-stimulated activity values represent mean differences in counts per 20 min interval between mice receiving saline and mice receiving benztropine. Symbols represent means and vertical lines 1 S.E.M. of lo-26 determinations. Solid symbols indicate a significant inhibition of benztropine-induced stimulation (P -: 0.01).
J. E. THORNBURG and K. E. MOORE
1182
activity at any time interval, suggesting that the inhibitory effect of cc-methyltyrosine was related to inhibition of synthesis of brain dopamine. Similarly, stimulated activity induced by I mg,‘kg scopolamine was inhibited by tlmethyltyrosine only after an initial 40 min period of no effect (Fig. 3). In combination with r 0.5 mg/kg
L
4
r
20
40
60
00
100
120
I.Omg/kg
O40 TIME
60 AFTER
SCOPOLAMlNE(min)
Fig. 3. Effects of a 4 hr diet containing 0.4 % a-methyltyrosine (K-MT) or 0.05 % FLA-63 on scopolamine-stimulated locomotor activity. The control (triangles), a-MT (circles), or FLA-63 (squares) diet was presented to mice for 4 hr. One hour after food was withdrawn, mice were placed in actophotometers for 20 min, removed, administered either saline or scopolamine i.p.(O.25, 05 or I.0 mg/kg) and returned to the actophotometer for 1 or 2 hr. Scopolaminestimulated activity values represent mean differences in counts per 20 min interval between mice receiving saline and mice receiving scopolamine. Symbols represent means and vertical lines 1 S.E.M. of 8-19 determinations. Solid symbols indicate a significant inhibition of scopolamineinduced stimulation (P < 0.01).
doses of 0.25 and 0.50 mg/kg of scopolamine, however, a-methyltyrosine effectively reduced stimulated activity during the initial 40 min as well. FLA-63 had no effect on scopolaminestimulated activity. cc-Methyltyrosine almost completely blocked stimulated activity following 4 mg/kg atropine (Fig. 4). A dose of 2 mg:‘kg atropine did not produce significant stimulation. Although not shown, 8 mgikg atropine produced less stimulation than did 4 mg/kg, presumably due to toxicity.
Benztropine stimulation
TIME
AFTER
1183
ATROPINE (min f
Fig. 4. Effects of a 4 hr diet containing 0.4 % cc-methyltyrosine (a-MT) on atropine-stimulated locomotor activity. The control (triangles) or a-MT (circles) diet was presented to mice for 4 hr. One hour after food was withdrawn, mice were placed in actophotometers for 20 min, removed, administered either saline or atropine i.p. (2 or 4 mgjkg) and returned to the actop~otometer for I hr. Atropine-stimulated activity values represent mean differences in counts per 20 min interval between mice receiving saline and mice receiving atropine. Symbols represent means and vertical lines 1 S.E.M. of IO-14 determinations. Solid symbols indicate a significant inhibition of atropine-induced stimulation (P < @01). DISCUSSION
The present data fail to distinguish between an anticholinergic or a dopaminergic mechanism of action for ben~trop~ne-stimulated locomotor activity. Nevertheless, this is the first demonstration that inhibition of dopamine synthesis in the brain causes a marked reduction in anticholinergic (antimuscarinic) stimulated locomotor activity. In a previous study, cc-methytyrosine blocked ( +)-amphetamine-improved shuttle box avoidance responding of rats, but did not alter similar effects of scopolamine (RECH and MOORE, 1968). The concept of a reciprocal balance between dopaminergic and cholinergic systems in the neostriatum is supported by considerable behavioural and biochemical evidence (SCHEELKRUGER, 1970; COSTALL et al., 1972a). Subthreshold doses of scopolamine and atropine potentiate amphetamine-induced increases in response rates of rats in an operant, shockavoidance situation (CARLTON, 1961; CARLTOE:and DIDAMO, 1961 j. Similarly, subthreshold doses of scopolamine potentiate amphetamine-induced locomotor activity in rats (FIBIGER, LYTLE and CAMPBELL, 1970). Scopolamine also decreases the threshold dose for amphetamine-induced stereotyped behaviour in rats (ARNFREDand RANDRUP. 196X; KLAWANS, RUBOWTS, PATEL and WEINER, 1972) and apomorphine-induced gnawing in mice (SCHEELKRUEGER, 1970). Moreover, unilateral administration of arecoline or haloperidol directly into the globus pallidus or caudate nucleus causes ipsilateral (toward the side of injection) circling, whereas atropine has the opposite effect (COSTALL et al., 1972b). Several investigators have examined biochemically the interactions of anticholinergic drugs and brain dopamine metabolism. In general, the anticholinergics tend to antagonize neuroleptic (dopamine receptor blockade)-induced increases in homovanillic acid contents (O’KEEFE et al., 1970; BOWERSand ROTH, 1972; AND~N, 1972) and changes in turnover rates
1184
J. E. THOK~BURG
and K. E. MOORE
of dopamine (AND~N and B~~IIAR~, 1971; CORR~DI, FUXE and LIDBRINK, 1972). HITZEMANN, LOH and DOMINO (1972) found that scopolamine. at doses which markedly increase locomotor activity, decreased the accumulation of “C-dopamine and 14C-norepinephrine in mouse brain after administration of either ‘“C-tyrosine or “C-DOPA. Whether the decreased accumulation of “C-catecholamines was due to decreased synthesis, increased release or increased intraneuronal degradation is unclear. GOODALE and MOORE (1973) and C~~ERAMY. GAUCHY, GLOWINSKI and BESSON (1973) found that benztropine and scopolamine increase the efflux of 3H-dopamine from the cat brain. Unfortunately, due to the difficulties in determination of acetylcholine synthesis and turnover rates, little is known about the effects of alterations of neostriatal dopamine metabolism on brain acetylcholine dynamics. Recent evidence obtained in our laboratory suggests that scopolamine and benztropine induce locomotor stimulation primarily by an anticholinergic mechanism. Amphetamine produced marked locomotor stimulation in the mouse on postnatal day IO, IO-15 days prior to the onset of significant scopolamineor benztropine-induced stimulation (THORNBURC; and MOORE, 1973~). CAMPBELL, LYTLH and Ftetw R (1969) had previously demonstrated similar developmental patterns for scopolamineand (+)-amphetamine-induced stimulation in the rat. The anatomical relationship between dopaminergic and cholinergic neurones in the basal ganglia remains unclear. Nevertheless, a normally functioning dopaminergic neuronal system appears to be required for anticholinergic drug-induced locomotor stimulation. REFERENCES N.-E. (1972). Dopamine turnover in the corpus striatum and the limbic system after treatment with neuroleptic and anti-acetylcholine drugs. J. Pharm. Pharmac. 24: 905-906. AND~N, N.-E. and B~DAKD, P. (1971). influences of cholinergic mechanisms on the function and turnover of brain dopamine. J. Pharm. Pharmac. 23: 460-462. ARNFRED, T. and RANDKUP, A. (1968). Cholinergic mechanism in brain inhibiting amphetamine-induced stereotyped behaviour. Ac,ta pharmac. 10s. 26: 3X4-394. BOWERS, M. B. and ROTH, R. H. (1972). Interaction of atropine-like drugs with dopamine-containing neurones in rat brain. Br. J. Pharmac. 44: 3OlL306. CAMPBELL, B. A., LYTLC, L. D. and FIBWER, H. C. (1969). Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Scirnce 166: 635-637. CARLTON, P. L. (1961). Augmentation of the behavioral effects of amphetamine by scopolamine. Psychopharmau~iqgia 2: 377-380. CARLTON, P. L. and DIDAMO, P. (1961). Augmentation of the behavioral effects of amphetamine by atropine. J. Pharmac. exp. Thw. 134: 91-96. CHERAMY, A., GAWHY, G., CI.OWINSKI, J. and BESSON, M.-J. (1973). In riw activation by benztropine of dopamine release and synthesis in the caudate nucleus. Eur. J. Pharmac. 21: 246-248. CORRODI, H., Fuxr, K. and LIDBRINK, P. (1972). Interaction between cholinergic and catecholaminergic neurones in rat brain. Bruin Res. 43: 397416. COSTA, E., GROPP~TTI, A. and NAIM~ADA, M. K. (1972). Effects of amphetamine on the turnover rate of brain catecholamines and motor activity. Br. J. Pharmac. 44: 742-751. COSTALL, B., NAYLOR, R. J. and OLLtr, J. E. (1972a). On the involvement of the caudate-putamen, globus pallidus and substantia nigra with neuroleptic and cholinergic modification of locomotor activity. Neuropharmacology I1 : 3 I 7-330. COSTALL, B., NAYLOR, R. J. and OLLEY, J. E. (1972b). Catalepsy and circling behaviour after intracerebral injections of neuroleptic, cholinergic and anticholinergic agents into the caudate-putamen, globus pallidus and substantia nigra of rat brain. Neuropharmacology 11: 645-663. COYLE, J. T. and SNYDER, S. H. (1969). Antiparkinsonian drugs: inhibition of dopamine uptake in the corpus striatum as a possible mechanism of action. Science 166: 899-901. FARNEBO, L.-O., FUXE, K., HAMBERGER, B. and LJUNGDAHL, H. (I 970). Effect of some antiparkinsonian drugs on catecholamine neurons. J. Pharm. Pharmac. 22: 733-737. FIBIGER, H. C., LYTLE, L. D. and CAMPBELL, B. A. (1970). Cholinergic modulation of adrenergic arousal in the developing rat. J. camp. ph.vsiol. P.s.vchol. 12: 384-389. New York. GOLDSTEIN, A. (I 964). Biostatistics: An Introductory Test , 272 pp. Macmillan, AND~N,
Benztropine
stimulation
1185
GCICDALE,D.
B. and MOORE,K. E. (1973). In t’jL’orelease of 3H-dopamine from cat brain by benztropine. ~~~~~?~~~~~~g~~~ 15: f 74. HETZEMANN, R. 3,, LCIH,H. H. and DOMINO, E. F. (1972). Effect of scopolamine on the cerebral accumulation of “C-catecholamines from ‘%-tyrosine. Pharmacology 8: 291-294. HORN, A. S., COYLE,J. T. and SNYDERS. H. (1971). Catecholamine uptake by sy~~ptosomes from rat brain: structure-activity relationships of drugs with differential effects on dopamine and norepinephrine neurons. Molec. Pharmac. 7: 66-80. KLAWANS, H. L., Rueovrrs, R., PATEL, B. C. and WEINER,W. J. (1972). Cholinergic and anticholinergic influences on amphetamine-jnduced stereotyped behavior. J. I\jerrrol. Sei. 17: W-308. O’KEEFE, R., SHARMAN, D. F. and VOW, M. (1970). Effect of drugs used in psychoses on cerebral dopamine metabolism. Br. 1. Pharmar. 38: 287-304. Racn, R. H. and MOORE, K. E. (1968). Interactions between ~-amphetamine and ff-methylt~r~s~ne in rat shuttle-box behavior. Brain Res. 8: 398-100. SCHEEL-KH~GER, J. (I970). Central effects of anticholinergic drugs measured by the apomorphine gnawing test in mice. Acra p~~~rnac. tax. 28: 1-16. TWQRNELJRG, J. E. (1973).Relative role of dopaminergic and noradrenergic neuron& systems for the stimulation of locomotor activity induced by amphetamine and other drugs. Fedn Proc. Fedn Am. Sors exp. Biol. 32: 753.
and MOORE, K. E. (1971). Stress-related effects of various inhibitors of catecholamine synthesis in the mouse. Archs i~t. Ph~~rn~c~~~~. Th&. 194: 158-167. THORNBURG,J. E. and MOORE,I(. E. (1973a). Dopamine and norep;nephrine uptake by rat brain synaptosomes: relative inhibitory potencies of I- and ~-ampheta~line and amantadjne. Res. Comm. &em.
THORNBURG, J. E.
Pathol. Pharmac. 5: 81-89.
THORNBURG, J. E. and MOORE, K. E. (1973b). The relative importance of dopaminergic and noradrenergic neuronai systems for the stimulation of locomotor activity induced by amphetamine and other drugs. ~e~rQp~a~rn~cff~~gy 12: 853-866.
and MOORE, K. E. (1973~). Postnatal development of ~n~tropine-induced locomotor evidence for an anticholinergic mechanism of action. Res. Cornin. &em. Pathal. Pharmac.
THORNBURG, J. E.
stimulation: 6: 313-320.
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