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Selective effects of buspirone and molindone on dopamine metabolism and function in the striatum and frontal cortex of the rat
Neuropharmacoloqy Vol. 22, No. 3A, pp. 273 278, 1983 Printed in Great Britain
0028-3908/83/3A0273-06803.00/0 Pergamon Press Ltd
SELECTIVE EFFECTS OF BUSPIRONE AND M O L I N D O N E ON DOPAMINE METABOLISM AND FUNCTION IN THE STRIATUM AND FRONTAL CORTEX OF THE RAT B. A. MCMILLEN* and CLAUDIAC. MCDONALD Department of Pharmacology, University of Texas Health Science Center, Dallas, TX 75235, U.S.A. (Accepted 22 Auoust 1982)
Summary--The hypothesis that the nerve endings of the dopamine projection of the frontal cortex lack autoreceptors for regulation of tyrosine hydroxylase was tested by using the preferential inhibitors of dopamine autoreceptors, molindole and buspirone. In contrast to haloperidol, which elevates dopamine metabolism in the striatum and frontal cortex, both molindone and buspirone elicited little change in dopamine metabolism in the frontal cortex at doses up to 3.0 mg/kg, which cause the same maximal response in the corpus striatum as does haloperidol. Thus, the lack of autoreceptors in the frontal cortex is of pharmacological importance. That preferential inhibition of striatal dopamine autoreceptors may reverse catalepsy by enhancing synthesis and release of dopamine was tested by first inducing catalepsy with different drugs and then administering molindone or buspirone. Only buspirone (1.0mg/kg) reversed catalepsy. This effect does not require presynaptic dopamine as catalepsy was reversed by buspirone in the dopamine-depleted rat (with 2.0mg/kg R04-1284) as well as after postsynaptic dopamine receptor blockade by haloperidol of cis-flupenthixol.Thus, the mechanism for the reversal of catalepsy appears to be located efferent from the dopamine neuron. Buspirone, a non-benzodiazepine anti-anxiety drug, may prove useful for treatment of extrapyramidal motor disorders of either iatrogenic or idiosyncratic origin.
Molindone and buspirone are known to possess preferential antagonist activity at dopamine autoreceptors of striatal nerve endings (Alander, GrabowskaAnd6n and And6n, 1980; McMillen, Sanghera, Matthews and German, 1981, 1982). Despite this common site of action these drugs are used clinically for different purposes: molindone is an antipsychotic drug and buspirone is an experimental anti-anxiety drug. Alander et al. (1980) demonstrated that molindone blocked more potently apomorphine-induced inhibition of the activation of tyrosine hydroxylase caused by cessation of dopamine impulse flow with 7-butyrolactone (GBL), a presynaptic receptor test, than it blocked apomorphine-induced turning, a postsynaptic receptor test. Buspirone blocked apomorphine in the ~'-butyrolactone presynaptic receptor test of Waiters and Roth (1976), potently increased dopamine synthesis, metabolism and impulse flow, but did not inhibit apomorphine-induced turning, did not cause catalepsy and, unlike molindone, was not efficacious as an anti-psychotic drug (McMillen et al., 1981; Stanton, Taylor and Riblet, 1981). Thus, both drugs share a similar preference for antagonism of the dopamine autoreceptor, even though their clinical usage is very different. The efficacy of buspirone for
reducing anxiety does not involve the benzodiazepine7-aminobutyric acid receptor complex and is mediated by an as yet unknown mechanism (Stanton et al., 1981; Taylor, Riblet and Stanton, 1982). Recently, Bannon, Michaud and Roth (1981) reported that dopaminergic nerve endings in the frontal cortex lack autoreceptors for the regulation of tyrosine hydroxylase. For example, cessation of impulse flow by injection of 7-butyrolaction resulted in a marked activation of striatal tyrosine hydroxylase, but not tyrosine hydroxylase in frontal cortex. If the dopamine projection of the frontal cortex lacks autoreceptors, molindone and buspirone should have a less substantial effect on dopamine metabolism in this brain area compared to their effects on striatal dopamine metabolism. Also, the effects of these drugs to increase synthesis and metabolism of dopamine in the striatum and thus increase release of dopamine at doses that do not appreciably inhibit postsynaptic dopamine receptors, suggests that buspirone and molindone may reduce the catalepsy (rigid paralysis) that normally occurs after a large dose of a classical antipsychotic drug by increasing competition for the postsynaptic receptor.
*Send correspondence to: Dr B. A. McMillen, Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC 27834, U.S.A. Key words: buspirone, dopamine, catalepsy, anti-anxiety, frontal cortex, corpus striatum.
METHODS
273
Female Sprague-Dawley rats (225-275g, Holtzman, Madison, WI) were housed in a room with a 12 hr light/dark cycle and maintained on standard
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B.A. MCMILLENand CLAUDIAC. MCDONALD
laboratory chow. Catalepsy was scored by the method phase was aspirated, the alumina washed with 1.0 ml of Shore and Dorris (1975). Each rat's forepaws were of 5 mM TRIS, and the amines and DOPAC eluted placed one at a time on a 3.0 cm peg and scored 1/2 with 1.0ml of 0.1 M HaPO4 and 20/(1 injected into point for each forepaw that remained in place 10 sec. the HPLC. Frontal cortices were homogenized in Then the rats were scored 1 point for each forepaw 3.0 ml 0.4 N HC103 and 2.0 ml of the supernate used that remained on a 9.0 cm peg for 10 sec. Finally, the for assay. Control concentrations of DOPAC in fronhomolateral limbs were crossed on one side and the tal cortex gave a 15-20~o response with the detector animal scored an additional point if the position was set at 2 nA full range. Alumina extraction was used to held for 10 sec. A score of around 1.0 indicated that separate DOPAC and internal standard from the the animals were akinetic while scores of 3.0-4.0 indi- tissue. cated a full cataleptic response to a drug. Drugs and sources For assay of the dopamine metabolite, dihydroxyApomorphine-HC1 (Sigma Chemical Co., St Louis, phenylacetic acid (DOPAC), the rats were killed by chloroform asphyxiation, the brains rapidly removed, MO), buspiron~HC1 (Mead/Johnson Pharmaceutical chilled in ice-cold saline, the olfactory tubercles re- Division, Evansville, IN), cis-flupenthixol-HC1 (H. moved and the brain then cut according to Glowinski Lundbeck and Co. A/S, Copenhagen), haloperidol and Iversen (1966). After removing the corpus stria- (McNeil Laboratories, Ft. Washington, PA), molinturn, the portion of the brain rostral to the cut done-HCl (Endo Laboratories, Garden City, N J) and through the anterior commissure was taken as the R04-1284 a 2-hydroxy-2-ethyl derivative of tetrabenafrontal cortex. In the experiments with buspirone and zine (Hoffman-LaRoche, Inc. Nutley, N J). All doses molindone, DOPAC was assayed by organic extrac- refer to the free base. tion and ftuorophor development (Murphy, Robinson and Sharman, 1969). The striata were homogenized in Statistical analyses Either Dunnett's t-test for multiple comparisons to 2.0ml of 0.4 N HCIO3 and the frontal cortices in 3.0 ml of 0.4 N HCIO 3 and the homogenates centri- a control or Wilcoxon test for non-parametric analyfuged at 12,000 g x 10 min. To 40 ml extraction tubes sis of two independent samples were used as indicated were added 1.0g KC1, 10 ml redistilled butylacetate in the Figures and Tables. All tests for significance and 1.5 or 2.5 ml of supernate. After shaking for were two tailed. 10 min and centrifuging at 1,000 rpm, 8.0 ml of butylacetate was transferred to a second extraction tube RESULTS containing 2.0 ml of an ethylenediamine solution (70 parts H20, 3 parts freshly redistilled ethylenediamine Effects on dopamine metabolism and 2 parts 0.1 N HC1). After shaking and centrifugFigure 1 shows the dose-reponse curves for the ing the tubes, the organic layer was aspirated and effect of haloperidol on DOPAC concentrations in fluorophor developed by placing 1.0ml of aqueous the striatum and frontal cortex. As others have phase in a test tube and heating in the dark for 20 min reported (Stawarz, Hill, Robinson, Setler, Dingell and at 55°C. The test tubes were then placed in ice water Sulser, 1975; Westerink, Lejeune, K0rf and van for 2 min and 0.2 ml of 6.0 N HC1 added and the test Praag, 1977), a larger response was observed in the tubes left on ice for 10 min. The tubes were removed striatum. A dose of 0.3 mg/kg (s.c.) of haloperidol profrom the ice bath, 0.2ml of 10~ ethylenediamine duced a maximal response in the striatum of 340°L added and 10 min allowed for the tubes to return to above control and in the frontal cortex of 270~o above room temperature. The samples were read in an Aminco-Bowman spectrophotoftuorometer at 385/ ~.101operidol Ul 485 nm (activation, fluorescence, uncorrected instru~J 4-1 +1 4.0 ( ~ ~ ( 6 ) 0.4 -~ ~, ment values). Control values for frontal cortex were only two to three times the blank in this assay. The assay procedure for the experiments with haloriO) peridol was switched to a high performance liquid 2.0 - 6 0.2 3 o ~o chromatographic (HPLC) method using a Beckman t.o. o.1 330 Isocratic HPLC equipped with a 5 ~ ultrasphere ODS column and a Bioanalytical Systems LC4A glassy carbon electrochemical detector. The mobile =0 o oo, o.o o; o; ,io phase was 15~0 methanol, 85~, 0.1 M KPO4, pH 3.0 u mg/kg containimg 2.0 mM octyl sodium sulfate and 0.1 mM Fig. 1. Effect of haloperidol on DOPAC concentrations in EDTA and pumped at 1.0 ml/min. Dihydroxybenzyl- the rat striatum and frontal cortex. Haloperidol was injected subcutaneously in various doses and the rats killed amine was used as internal standard. Striata were homogenized in 1.5 ml 0.4 N HC103, centrifuged and 90 rain later. Data are expressed as change from controls (shown at left in Figure) aginst dose plotted on a log scale. a 0.5 ml aliquot of the supernate added to a microfuge Number of rats in each group is shown in parentheses. tube containing 50 mg alumina and 0.5 ml of 0.5 M *P < 0.05 and tP < 0.01 (Dunnett's t-test) compared to TRIS. After shaking and centrifuging, the aqueous control.
Buspirone and brain dopamine function
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control. Both molindone and buspirone required about 10 times this dose to produce maximal effects in the striatum. The two striatal dose-response curves shown in Figure 2 were almost super-imposable up to 3.0mg/kg, but then the response to molindone dropped off at 10mg/kg. While dramatic changes were occurring in the striatum, the response of the D O P A C concentrations in the frontal cortex to these two drugs was considerably less than that observed with haloperidol. Only 10mg/kg of buspirone produced a highly significant response. Thus, the apparent lack of presynaptic dopamine autoreceptors regulating tyrosine hydroxylation in the frontal cortex described by Bannon et al. (1981) was an important determinant for the ability of these drugs to increase dopamine metabolism in the frontal cortex. Effects on catalepsy Figure 3A shows that buspirone at 1.0 mg/kg (s.c.) reversed haloperidol-induced catalepsy. By 60min after a large (1.0 mg/kg s.c.) dose of haloperidol, the rats exhibited near maximal catalepsy, but those animals which received 1.0 mg/kg (s.c.) of buspirone exhibited a rapid reversal of catalepsy scores. In contrast, Figure 3B shows that 1.0 mg/kg (s.c.) molindone did not reverse haloperidol-induced catalepsy which suggested that inhibition of dopamine autoreceptors was not the site of action for buspirone. An alternative site of action could be the dopamine-stimulated adenylate "~,p. 2 2 , 3 A
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Fig. 3. Effect of buspirone or molindone on catalepsy. Haloperidot (1.0 mg/kg s.c.) was injected at 0 min and catalepsy allowed to develop for 60 min. Immediately after the catalepsy scoring at 60 min, half of the rats (e) received (arrow) (A) 1.0 mg/kg (s.c.) buspirone or (B) 1.0 mg/kg (s.c.) molindone. Buspirone caused a significant (tP < 0.01 ; Wilcoxon test) difference in catalepsy score. Number of rats in each group is shown in parentheses.
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Fig. 4. Effect of buspirone on catalepsy. Either cis-flupenthixol (l.0mg/kg s.c.) or R04-1284 (2.0mg/kg i.p.) was injected at 0 min and catalepsy allowed to develop for 60 min. Immediately after the catalepsy scoring at 60 min, half of the rats (O) received buspirone (1.0 mg/kg s.c.) designated by the arrow. Buspirone produced a significant (*P < 0.05, t P < 0.01 ; Wilcoxon test) difference in catalepsy scores. Number of rats in each group is shown in parentheses.
dose of cis-flupenthixol, which should near equally block both the dopamine-stimulated adenylate cyclase receptor (D~)and the 3H-neuroleptic binding site (D2) (cf. Seeman, 1980) was reversed by administration of buspirone. This result indicated that buspirone was not reducing catalepsy by stimulating adenylate cyclase activity in the striatum. In order to test whether this effect was due to the release of dopamine from presynaptic sites, dopamine was depleted with the tetrabenazine derivative, R04-1284 (see Methods), which rapidly decreases striatal dopamine concentrations by 90~o in 30min (Shore, 1976; McMillen and Shore, 1980) causing marked catalepsy. Again buspirone reduced catalepsy scores even though little dopamine was available for release (Fig. 4B).
Effects of route of administration Buspirone is known to exhibit a "first pass effect" and is rapidly and extensively metabolized in both rats and man (H. C. Stanton and R. Gammans, personal communication). To determine whether the effects of buspirone may be altered by metabolism, the drug was administered intraperitoneally (i.p.) and the results compared with the above results which used subcutaneous injections. Figure 5 shows that the switch to intraperitoneal injection shifted the buspirone dose-response curve for the increase in level of D O P A C to the right by 10-fold. Instead of a maximal effect of buspirone at 3.0 mg/kg (s.c.), the maximum was reached at 30mg/kg (i.p.). However, Table 1 shows that 1.0mg/kg (i.p.) buspirone still inhibited haloperidol-induced catalepsy as did the subcu-
taneous route of injection. Thus, switching to intraperitoneal administration markedly decreased the effects on dopamine metabolism, but not on catalepsy.
DISCUSSION
The data in Figures 2A and 2B provide pharmacological evidence to support the original report by Bannon et al. (1981) that the dopaminergic nerve endings in the frontal cortex lack autoreceptors. Compared to the effects of haloperidol, the increase in D O P A C concentrations in the frontal cortex showed a very flat response. Buspirone, in doses of 0.3 mg/kg or less, did not significantly increase concentrations of D O P A C in the frontal cortex and at 1.0mg/kg or more, small increases occurred. Molindone was without significant effect from 0.1 to 10 mg/kg. Buspirone (0.1-1.0mg/kg i.v.) increased the firing rate of the A-10 dopamine neurons in the ventral segmental area (Matthews and German, unpublished observations) so it is surprising that a larger increase of D O P A C concentrations was not observed. One possible explanation is that the cells from which impulse flow was Table 1. Effects of different doses of buspirone on haloperidol-induced catalepsy in rats Catalepsy score Buspirone 0.1 (s.c.) 1.0 (s.c.) 10.0 (s.c.) 1.0 (i.p.)
60 min 3.80 4 4 4
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4 0.75 0.50 1.25
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Haloperidol (l.0mg/kg s.c.) was injected into 4~6 rats per group and catalepsy allowed to develop for 60min. Immediately after scoring at 60rain, buspirone was injected and catalepsy scored at 90 and 120 min. There was no significant difference between route of administration at the 1.0 mg/kg dose (P > 0.05; Wilcoxon test).
277
Buspirone and brain dopamine function recorded did not project to the frontal cortex but to other limbic areas. Molindone, unlike other antipsychotic drugs, has very little effect on dopamine metabolism in the frontal cortex. This is in marked contrast to other antipsychotic drugs, especially the atypical drugs (clozapine, sulpiride and thioridazine) which have stronger effects on the mesocortical system than on the nigra-striatal system (Stawarz et al., 1975; Westerink et al., 1977; McMillen, 1981). Molindone is known to reverse apomorphine- or (+)amphetamine-induced inhibition of dopamine-induced impulse flow in the nigro (Bunney, Roth and Aghajanian, 1975). However, the presence or lack of increased metabolism of dopamine does not necessarily indicate the degree of inhibition of postsynaptic dopamine receptors which is presumably the therapeutic site of action for the treatment of schizophrenia by antipsychotic drugs (cf. Snyder, Banerjee, Yamamura and Greenburg, 1974; Seeman, 19801. The mechanism by which buspirone causes decreased catalepsy is obscure. The hypothesis tested was that the presynaptic effects of both buspirone and molindone to increase dopamine synthesis and impulse flow would result in more dopamine being released and available to compete with haloperidol at postsynaptic receptor sites. This effect could reduce the catalepsy caused by blockade of postsynaptic dopamine receptors. However, only buspirone reversed haloperidol-induced catalepsy. Then cis-flupenthixol was used to provide a strong blockade of both Dx (adenylate cyclase-linked) and D2 receptors and again buspirone decreased catalepsy scores. That release of dopamine from presynaptic sites is not involved is indicated in Figure 4B, where buspirone reversed the catalepsy caused by depletion of dopamine. Thus, buspirone may be having its effects outside the nigro-striatal-nigra loop. It is known that lesions of the cortico-striate pathway, the substantia nigra zona reticulata and the ventral medial nucleus of the thalamus will prevent or reduce haloperidolinduced catalepsy (cf. DiChiara, Morelli, lmperato and Porceddu, 1981). Buspirone could have an effect at a non-dopaminergic site along the extrapyramidal pathway. McMillen et al. (1981, 1982) showed that buspirone increased dopamine cell impulse flow in the nigra to higher rates than with haloperidol, even after administration of haloperidol to the same animal. This effect was interpreted to indicate a site of action in addition to inhibition of presynaptic dopamine receptors. The present results are in agreement with this earlier report. Buspirone does alter striatal acetycholine levels 25-30~o, but the effects were not attributed to a classical dopaminergic effect (Kolasa, Fusi, Garattini, Consolo and Ladinsky, 1982). Figure 5 shows that intraperitoneal injection of buspirone resulted in a marked diminution of the increase in DOPAC concentration in the striatum. This result suggests that the rapid metabolism of buspirone by the liver greatly blunts the dopaminergic effects of the drug. Therefore, much of the effects on the
dopamine system may be due to buspirone itself, and these are demonstrable in vitro or by iontophoresis onto the dopamine neurons (Stanton et al., 1981; McMillen et al., 1981, 1982). In contrast, the reversal of catalepsy caused by buspirone was similar after either route of administration: neither marked potentiation nor inhibition was observed. This observation may indicate that both buspirone and one or more of its metabolites may be effective agents for reducing catalepsy. Buspirone continues to be an enigmatic drug. It is equipotent to diazepam as an anti-anxiety drug, both in the conflict test and clinical trials (Goldberg and Finnerty, 1979; Stanton et al., 19811, but exhibits no affinity for, or interaction with, the benzodiazepine binding site in vitro (Stanton et al., 1981), or in t,ivo (Sanghera, McMillen and German, unpublished). Whether its effects in reversing catalepsy are related to the anxiolytic action is unclear. The dose of 1.0mg/kg (s.c. or i.p.) used in the current report is similar to effective doses found in the conflict test (1.(~5.0mg/kg p.o.) and clinical trials (20mg/day divided in three doses). Potentially, buspirone may be useful in either iatrogenic or idiosyncratic Parkinson's syndrome. The lack of side-effects such as sedation, muscle relaxation or potentiation of sedatives (Stanton et al., 1981) may make this drug very useful as an adjunct for treatment of extrapyramidal disorders.
Acknowledgements--The authors thank the pharmaceutical companies for their gifts of drugs, Janet Harkness for her technical assistance and Ruth Houser and Regina Keith for preparing the manuscript. This research was supported by USPHS grant MH-05831.
REFERENCES
Alander, T., Grabowska-And~n, M. and And~n, N.-E. (1980). Physiological significance of dopamine autoreceptors as studied following their selective blockade by molindone, d. Pharm. Pharmac. 32: 780-782. Bannon, M. J., Michaud, R. L. and Roth, R. H. (1981). Mesocortical dopamine neurons: lack of autoreceptors modulating dopamine synthesis. Molec. Pharmac. 19: 270-275. Bunney, B. S., Roth, R. H. and Aghajanian, G. K. (1975). Effects of molindone on central dopaminergic neuronal activity and metabolism: similarity to other neuroleptics. Psychopharmac. Commun. 1: 349-358. DiChiara, G., Morelli, M., Imperato, A. and Porceddu, M. L. (1981). Substantia nigra as an efferent station for dopaminergic behavioural syndromes arising in the striatum. In: Apomorphine and Other Dopaminomimetics (Gessa, G. L. and Corsini, G. U., Eds), Vol. 1, pp. 41 64. Raven Press, New York. Glowinski, J. and lversen, L. L. (1966). Regional studies of catecholamines in the rat brain. J. Neurochem. 13: 655-669. Goldberg, H. L. and Finnerty, R. J. (1979). The comparative efficacy of buspirone or diazepam in the treatment of anxiety. Am. J. Psychiat. 136: 1184-1187. Kolasa, K., Fusi, R., Garattini, S., Consolo, S. and Ladin-
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Shore, P. A. and Dorris, R. L. (1975). On a prime role for newly synthesized dopamine in striatal function. Eur. J. Pharmac. 30: 315--318. Snyder, S. H., Banerjee, S. P., Yamamura, H. I. and Greenberg, D. (1974). Drugs, neurotransmitters and schizophrenia. Science 184: 1243-1253. Stanton, H. C., Taylor, D. P. and Riblet, L. A. (1981). Buspirone--an anxio-selective drug with dopaminergic action. In: The Neurobiology of the Nucleus Accumbens (Chronister, R. B. and DeFrance, R. B., Eds), pp. 316-319. Haer Institute, Brunswick, Maine. Stawarz, R. J., Hill, H., Robinson, S. E., Setler, P., Dingell, J. V. and Sulser, F. (1975). On the significance of the increase in HVA caused by antipsychotic drugs in corpus striatum and limbic forebrain. Psychopharmacology 43: 125-130. Taylor, D. P., Riblet, L. A. and Stanton, H. C. (1982). Dopamine and anxiolytics. In: Anxiolytics: Neurochemical, Behavioral and Clinical Perspectives (Malick, J. 8 , Enna, S. J. and Yamamura, H. I., Eds) Raven Press, New York (in press). Waiters, J. R. and Roth, R. H. (1976). Dopaminergic neurons: an in vivo system for measuring drug interactions with presynaptic receptors. Naunyn-Schrniedebergs Arch. Pharmac. 296: 5-14. Westerinck, B. H. C., Lejeune, B., Korf, J. and Van Praag, H. M. (1977). On the significance of regional dopamine metabolism in the rat brain for the classification of centrally acting drugs. Eur. J. Pharmac. 42: 179-190.
0028-3908/83/3A0273-06803.00/0 Pergamon Press Ltd
SELECTIVE EFFECTS OF BUSPIRONE AND M O L I N D O N E ON DOPAMINE METABOLISM AND FUNCTION IN THE STRIATUM AND FRONTAL CORTEX OF THE RAT B. A. MCMILLEN* and CLAUDIAC. MCDONALD Department of Pharmacology, University of Texas Health Science Center, Dallas, TX 75235, U.S.A. (Accepted 22 Auoust 1982)
Summary--The hypothesis that the nerve endings of the dopamine projection of the frontal cortex lack autoreceptors for regulation of tyrosine hydroxylase was tested by using the preferential inhibitors of dopamine autoreceptors, molindole and buspirone. In contrast to haloperidol, which elevates dopamine metabolism in the striatum and frontal cortex, both molindone and buspirone elicited little change in dopamine metabolism in the frontal cortex at doses up to 3.0 mg/kg, which cause the same maximal response in the corpus striatum as does haloperidol. Thus, the lack of autoreceptors in the frontal cortex is of pharmacological importance. That preferential inhibition of striatal dopamine autoreceptors may reverse catalepsy by enhancing synthesis and release of dopamine was tested by first inducing catalepsy with different drugs and then administering molindone or buspirone. Only buspirone (1.0mg/kg) reversed catalepsy. This effect does not require presynaptic dopamine as catalepsy was reversed by buspirone in the dopamine-depleted rat (with 2.0mg/kg R04-1284) as well as after postsynaptic dopamine receptor blockade by haloperidol of cis-flupenthixol.Thus, the mechanism for the reversal of catalepsy appears to be located efferent from the dopamine neuron. Buspirone, a non-benzodiazepine anti-anxiety drug, may prove useful for treatment of extrapyramidal motor disorders of either iatrogenic or idiosyncratic origin.
Molindone and buspirone are known to possess preferential antagonist activity at dopamine autoreceptors of striatal nerve endings (Alander, GrabowskaAnd6n and And6n, 1980; McMillen, Sanghera, Matthews and German, 1981, 1982). Despite this common site of action these drugs are used clinically for different purposes: molindone is an antipsychotic drug and buspirone is an experimental anti-anxiety drug. Alander et al. (1980) demonstrated that molindone blocked more potently apomorphine-induced inhibition of the activation of tyrosine hydroxylase caused by cessation of dopamine impulse flow with 7-butyrolactone (GBL), a presynaptic receptor test, than it blocked apomorphine-induced turning, a postsynaptic receptor test. Buspirone blocked apomorphine in the ~'-butyrolactone presynaptic receptor test of Waiters and Roth (1976), potently increased dopamine synthesis, metabolism and impulse flow, but did not inhibit apomorphine-induced turning, did not cause catalepsy and, unlike molindone, was not efficacious as an anti-psychotic drug (McMillen et al., 1981; Stanton, Taylor and Riblet, 1981). Thus, both drugs share a similar preference for antagonism of the dopamine autoreceptor, even though their clinical usage is very different. The efficacy of buspirone for
reducing anxiety does not involve the benzodiazepine7-aminobutyric acid receptor complex and is mediated by an as yet unknown mechanism (Stanton et al., 1981; Taylor, Riblet and Stanton, 1982). Recently, Bannon, Michaud and Roth (1981) reported that dopaminergic nerve endings in the frontal cortex lack autoreceptors for the regulation of tyrosine hydroxylase. For example, cessation of impulse flow by injection of 7-butyrolaction resulted in a marked activation of striatal tyrosine hydroxylase, but not tyrosine hydroxylase in frontal cortex. If the dopamine projection of the frontal cortex lacks autoreceptors, molindone and buspirone should have a less substantial effect on dopamine metabolism in this brain area compared to their effects on striatal dopamine metabolism. Also, the effects of these drugs to increase synthesis and metabolism of dopamine in the striatum and thus increase release of dopamine at doses that do not appreciably inhibit postsynaptic dopamine receptors, suggests that buspirone and molindone may reduce the catalepsy (rigid paralysis) that normally occurs after a large dose of a classical antipsychotic drug by increasing competition for the postsynaptic receptor.
*Send correspondence to: Dr B. A. McMillen, Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC 27834, U.S.A. Key words: buspirone, dopamine, catalepsy, anti-anxiety, frontal cortex, corpus striatum.
METHODS
273
Female Sprague-Dawley rats (225-275g, Holtzman, Madison, WI) were housed in a room with a 12 hr light/dark cycle and maintained on standard
274
B.A. MCMILLENand CLAUDIAC. MCDONALD
laboratory chow. Catalepsy was scored by the method phase was aspirated, the alumina washed with 1.0 ml of Shore and Dorris (1975). Each rat's forepaws were of 5 mM TRIS, and the amines and DOPAC eluted placed one at a time on a 3.0 cm peg and scored 1/2 with 1.0ml of 0.1 M HaPO4 and 20/(1 injected into point for each forepaw that remained in place 10 sec. the HPLC. Frontal cortices were homogenized in Then the rats were scored 1 point for each forepaw 3.0 ml 0.4 N HC103 and 2.0 ml of the supernate used that remained on a 9.0 cm peg for 10 sec. Finally, the for assay. Control concentrations of DOPAC in fronhomolateral limbs were crossed on one side and the tal cortex gave a 15-20~o response with the detector animal scored an additional point if the position was set at 2 nA full range. Alumina extraction was used to held for 10 sec. A score of around 1.0 indicated that separate DOPAC and internal standard from the the animals were akinetic while scores of 3.0-4.0 indi- tissue. cated a full cataleptic response to a drug. Drugs and sources For assay of the dopamine metabolite, dihydroxyApomorphine-HC1 (Sigma Chemical Co., St Louis, phenylacetic acid (DOPAC), the rats were killed by chloroform asphyxiation, the brains rapidly removed, MO), buspiron~HC1 (Mead/Johnson Pharmaceutical chilled in ice-cold saline, the olfactory tubercles re- Division, Evansville, IN), cis-flupenthixol-HC1 (H. moved and the brain then cut according to Glowinski Lundbeck and Co. A/S, Copenhagen), haloperidol and Iversen (1966). After removing the corpus stria- (McNeil Laboratories, Ft. Washington, PA), molinturn, the portion of the brain rostral to the cut done-HCl (Endo Laboratories, Garden City, N J) and through the anterior commissure was taken as the R04-1284 a 2-hydroxy-2-ethyl derivative of tetrabenafrontal cortex. In the experiments with buspirone and zine (Hoffman-LaRoche, Inc. Nutley, N J). All doses molindone, DOPAC was assayed by organic extrac- refer to the free base. tion and ftuorophor development (Murphy, Robinson and Sharman, 1969). The striata were homogenized in Statistical analyses Either Dunnett's t-test for multiple comparisons to 2.0ml of 0.4 N HCIO3 and the frontal cortices in 3.0 ml of 0.4 N HCIO 3 and the homogenates centri- a control or Wilcoxon test for non-parametric analyfuged at 12,000 g x 10 min. To 40 ml extraction tubes sis of two independent samples were used as indicated were added 1.0g KC1, 10 ml redistilled butylacetate in the Figures and Tables. All tests for significance and 1.5 or 2.5 ml of supernate. After shaking for were two tailed. 10 min and centrifuging at 1,000 rpm, 8.0 ml of butylacetate was transferred to a second extraction tube RESULTS containing 2.0 ml of an ethylenediamine solution (70 parts H20, 3 parts freshly redistilled ethylenediamine Effects on dopamine metabolism and 2 parts 0.1 N HC1). After shaking and centrifugFigure 1 shows the dose-reponse curves for the ing the tubes, the organic layer was aspirated and effect of haloperidol on DOPAC concentrations in fluorophor developed by placing 1.0ml of aqueous the striatum and frontal cortex. As others have phase in a test tube and heating in the dark for 20 min reported (Stawarz, Hill, Robinson, Setler, Dingell and at 55°C. The test tubes were then placed in ice water Sulser, 1975; Westerink, Lejeune, K0rf and van for 2 min and 0.2 ml of 6.0 N HC1 added and the test Praag, 1977), a larger response was observed in the tubes left on ice for 10 min. The tubes were removed striatum. A dose of 0.3 mg/kg (s.c.) of haloperidol profrom the ice bath, 0.2ml of 10~ ethylenediamine duced a maximal response in the striatum of 340°L added and 10 min allowed for the tubes to return to above control and in the frontal cortex of 270~o above room temperature. The samples were read in an Aminco-Bowman spectrophotoftuorometer at 385/ ~.101operidol Ul 485 nm (activation, fluorescence, uncorrected instru~J 4-1 +1 4.0 ( ~ ~ ( 6 ) 0.4 -~ ~, ment values). Control values for frontal cortex were only two to three times the blank in this assay. The assay procedure for the experiments with haloriO) peridol was switched to a high performance liquid 2.0 - 6 0.2 3 o ~o chromatographic (HPLC) method using a Beckman t.o. o.1 330 Isocratic HPLC equipped with a 5 ~ ultrasphere ODS column and a Bioanalytical Systems LC4A glassy carbon electrochemical detector. The mobile =0 o oo, o.o o; o; ,io phase was 15~0 methanol, 85~, 0.1 M KPO4, pH 3.0 u mg/kg containimg 2.0 mM octyl sodium sulfate and 0.1 mM Fig. 1. Effect of haloperidol on DOPAC concentrations in EDTA and pumped at 1.0 ml/min. Dihydroxybenzyl- the rat striatum and frontal cortex. Haloperidol was injected subcutaneously in various doses and the rats killed amine was used as internal standard. Striata were homogenized in 1.5 ml 0.4 N HC103, centrifuged and 90 rain later. Data are expressed as change from controls (shown at left in Figure) aginst dose plotted on a log scale. a 0.5 ml aliquot of the supernate added to a microfuge Number of rats in each group is shown in parentheses. tube containing 50 mg alumina and 0.5 ml of 0.5 M *P < 0.05 and tP < 0.01 (Dunnett's t-test) compared to TRIS. After shaking and centrifuging, the aqueous control.
Buspirone and brain dopamine function
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control. Both molindone and buspirone required about 10 times this dose to produce maximal effects in the striatum. The two striatal dose-response curves shown in Figure 2 were almost super-imposable up to 3.0mg/kg, but then the response to molindone dropped off at 10mg/kg. While dramatic changes were occurring in the striatum, the response of the D O P A C concentrations in the frontal cortex to these two drugs was considerably less than that observed with haloperidol. Only 10mg/kg of buspirone produced a highly significant response. Thus, the apparent lack of presynaptic dopamine autoreceptors regulating tyrosine hydroxylation in the frontal cortex described by Bannon et al. (1981) was an important determinant for the ability of these drugs to increase dopamine metabolism in the frontal cortex. Effects on catalepsy Figure 3A shows that buspirone at 1.0 mg/kg (s.c.) reversed haloperidol-induced catalepsy. By 60min after a large (1.0 mg/kg s.c.) dose of haloperidol, the rats exhibited near maximal catalepsy, but those animals which received 1.0 mg/kg (s.c.) of buspirone exhibited a rapid reversal of catalepsy scores. In contrast, Figure 3B shows that 1.0 mg/kg (s.c.) molindone did not reverse haloperidol-induced catalepsy which suggested that inhibition of dopamine autoreceptors was not the site of action for buspirone. An alternative site of action could be the dopamine-stimulated adenylate "~,p. 2 2 , 3 A
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MINUTES
Fig. 4. Effect of buspirone on catalepsy. Either cis-flupenthixol (l.0mg/kg s.c.) or R04-1284 (2.0mg/kg i.p.) was injected at 0 min and catalepsy allowed to develop for 60 min. Immediately after the catalepsy scoring at 60 min, half of the rats (O) received buspirone (1.0 mg/kg s.c.) designated by the arrow. Buspirone produced a significant (*P < 0.05, t P < 0.01 ; Wilcoxon test) difference in catalepsy scores. Number of rats in each group is shown in parentheses.
dose of cis-flupenthixol, which should near equally block both the dopamine-stimulated adenylate cyclase receptor (D~)and the 3H-neuroleptic binding site (D2) (cf. Seeman, 1980) was reversed by administration of buspirone. This result indicated that buspirone was not reducing catalepsy by stimulating adenylate cyclase activity in the striatum. In order to test whether this effect was due to the release of dopamine from presynaptic sites, dopamine was depleted with the tetrabenazine derivative, R04-1284 (see Methods), which rapidly decreases striatal dopamine concentrations by 90~o in 30min (Shore, 1976; McMillen and Shore, 1980) causing marked catalepsy. Again buspirone reduced catalepsy scores even though little dopamine was available for release (Fig. 4B).
Effects of route of administration Buspirone is known to exhibit a "first pass effect" and is rapidly and extensively metabolized in both rats and man (H. C. Stanton and R. Gammans, personal communication). To determine whether the effects of buspirone may be altered by metabolism, the drug was administered intraperitoneally (i.p.) and the results compared with the above results which used subcutaneous injections. Figure 5 shows that the switch to intraperitoneal injection shifted the buspirone dose-response curve for the increase in level of D O P A C to the right by 10-fold. Instead of a maximal effect of buspirone at 3.0 mg/kg (s.c.), the maximum was reached at 30mg/kg (i.p.). However, Table 1 shows that 1.0mg/kg (i.p.) buspirone still inhibited haloperidol-induced catalepsy as did the subcu-
taneous route of injection. Thus, switching to intraperitoneal administration markedly decreased the effects on dopamine metabolism, but not on catalepsy.
DISCUSSION
The data in Figures 2A and 2B provide pharmacological evidence to support the original report by Bannon et al. (1981) that the dopaminergic nerve endings in the frontal cortex lack autoreceptors. Compared to the effects of haloperidol, the increase in D O P A C concentrations in the frontal cortex showed a very flat response. Buspirone, in doses of 0.3 mg/kg or less, did not significantly increase concentrations of D O P A C in the frontal cortex and at 1.0mg/kg or more, small increases occurred. Molindone was without significant effect from 0.1 to 10 mg/kg. Buspirone (0.1-1.0mg/kg i.v.) increased the firing rate of the A-10 dopamine neurons in the ventral segmental area (Matthews and German, unpublished observations) so it is surprising that a larger increase of D O P A C concentrations was not observed. One possible explanation is that the cells from which impulse flow was Table 1. Effects of different doses of buspirone on haloperidol-induced catalepsy in rats Catalepsy score Buspirone 0.1 (s.c.) 1.0 (s.c.) 10.0 (s.c.) 1.0 (i.p.)
60 min 3.80 4 4 4
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4 0.75 0.50 1.25
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Haloperidol (l.0mg/kg s.c.) was injected into 4~6 rats per group and catalepsy allowed to develop for 60min. Immediately after scoring at 60rain, buspirone was injected and catalepsy scored at 90 and 120 min. There was no significant difference between route of administration at the 1.0 mg/kg dose (P > 0.05; Wilcoxon test).
277
Buspirone and brain dopamine function recorded did not project to the frontal cortex but to other limbic areas. Molindone, unlike other antipsychotic drugs, has very little effect on dopamine metabolism in the frontal cortex. This is in marked contrast to other antipsychotic drugs, especially the atypical drugs (clozapine, sulpiride and thioridazine) which have stronger effects on the mesocortical system than on the nigra-striatal system (Stawarz et al., 1975; Westerink et al., 1977; McMillen, 1981). Molindone is known to reverse apomorphine- or (+)amphetamine-induced inhibition of dopamine-induced impulse flow in the nigro (Bunney, Roth and Aghajanian, 1975). However, the presence or lack of increased metabolism of dopamine does not necessarily indicate the degree of inhibition of postsynaptic dopamine receptors which is presumably the therapeutic site of action for the treatment of schizophrenia by antipsychotic drugs (cf. Snyder, Banerjee, Yamamura and Greenburg, 1974; Seeman, 19801. The mechanism by which buspirone causes decreased catalepsy is obscure. The hypothesis tested was that the presynaptic effects of both buspirone and molindone to increase dopamine synthesis and impulse flow would result in more dopamine being released and available to compete with haloperidol at postsynaptic receptor sites. This effect could reduce the catalepsy caused by blockade of postsynaptic dopamine receptors. However, only buspirone reversed haloperidol-induced catalepsy. Then cis-flupenthixol was used to provide a strong blockade of both Dx (adenylate cyclase-linked) and D2 receptors and again buspirone decreased catalepsy scores. That release of dopamine from presynaptic sites is not involved is indicated in Figure 4B, where buspirone reversed the catalepsy caused by depletion of dopamine. Thus, buspirone may be having its effects outside the nigro-striatal-nigra loop. It is known that lesions of the cortico-striate pathway, the substantia nigra zona reticulata and the ventral medial nucleus of the thalamus will prevent or reduce haloperidolinduced catalepsy (cf. DiChiara, Morelli, lmperato and Porceddu, 1981). Buspirone could have an effect at a non-dopaminergic site along the extrapyramidal pathway. McMillen et al. (1981, 1982) showed that buspirone increased dopamine cell impulse flow in the nigra to higher rates than with haloperidol, even after administration of haloperidol to the same animal. This effect was interpreted to indicate a site of action in addition to inhibition of presynaptic dopamine receptors. The present results are in agreement with this earlier report. Buspirone does alter striatal acetycholine levels 25-30~o, but the effects were not attributed to a classical dopaminergic effect (Kolasa, Fusi, Garattini, Consolo and Ladinsky, 1982). Figure 5 shows that intraperitoneal injection of buspirone resulted in a marked diminution of the increase in DOPAC concentration in the striatum. This result suggests that the rapid metabolism of buspirone by the liver greatly blunts the dopaminergic effects of the drug. Therefore, much of the effects on the
dopamine system may be due to buspirone itself, and these are demonstrable in vitro or by iontophoresis onto the dopamine neurons (Stanton et al., 1981; McMillen et al., 1981, 1982). In contrast, the reversal of catalepsy caused by buspirone was similar after either route of administration: neither marked potentiation nor inhibition was observed. This observation may indicate that both buspirone and one or more of its metabolites may be effective agents for reducing catalepsy. Buspirone continues to be an enigmatic drug. It is equipotent to diazepam as an anti-anxiety drug, both in the conflict test and clinical trials (Goldberg and Finnerty, 1979; Stanton et al., 19811, but exhibits no affinity for, or interaction with, the benzodiazepine binding site in vitro (Stanton et al., 1981), or in t,ivo (Sanghera, McMillen and German, unpublished). Whether its effects in reversing catalepsy are related to the anxiolytic action is unclear. The dose of 1.0mg/kg (s.c. or i.p.) used in the current report is similar to effective doses found in the conflict test (1.(~5.0mg/kg p.o.) and clinical trials (20mg/day divided in three doses). Potentially, buspirone may be useful in either iatrogenic or idiosyncratic Parkinson's syndrome. The lack of side-effects such as sedation, muscle relaxation or potentiation of sedatives (Stanton et al., 1981) may make this drug very useful as an adjunct for treatment of extrapyramidal disorders.
Acknowledgements--The authors thank the pharmaceutical companies for their gifts of drugs, Janet Harkness for her technical assistance and Ruth Houser and Regina Keith for preparing the manuscript. This research was supported by USPHS grant MH-05831.
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