The effects of dihydropyridine compounds in behavioural tests of dopaminergic activity

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1 Br. J. Pharmacol. (1989), 98, The effects of dihydropyridine compounds in behavioural tests of dopaminergic activity 'Anne Bourson, 2Alma J. Gower, 3Anis K. Mir & Paul C. Moser Merrell Dow Research Institute, 16, rue d'ankara, Strasbourg Cedex, France 1 The effects of the dihydropyridine calcium channel blocker nifedipine and the activator Bay K 8644 were investigated in different behavioural tests involving dopaminergic systems. These were the discriminative stimulus induced by amphetamine, rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions and apomorphine-induced yawning in rats. 2 The yawning induced by apomorphine (40 pg kg- s.c.) was significantly potentiated by nifedipine (5-10mgkg-'i.p.). Bay K 8644 ( mgkg-'i.p.) dose-dependently inhibited yawning induced by apomorphine (80upgkg-'s.c.) and, at 0.4mgkg-', inhibited the nifedipine potentiation of apomorphine-induced yawning. In contrast to their effects on apomorphine-induced yawning, nifedipine and Bay K 8644 had no effect on apomorphine-induced penile erection. 3 Bay K 8644 ( mgkg- i p.) and nifedipine (5-20mgkg- i.p.) had no dose-related effect on the discrimination performance of rats trained to discriminate amphetamine from saline. However, nifedipine dose-dependently reduced the response rate of amphetamine-treated rats. Bay K 8644 had no effect on this measure except at high doses that also caused disruption. 4 Neither nifedipine (5-10mgkg- i.p.) nor Bay K 8644 ( mgkg-' i.p.) affected the turning behaviour induced by amphetamine (1 mgkg' i.p.) in rats with unilateral 6-OHDA lesion of the medial forebrain bundle, and did not induce turning themselves. 5 As the dihydropyridine compounds affected apomorphine-induced yawning but not penile erection, and did not affect amphetamine-induced rotation or drug discrimination, it seems unlikely that they are affecting dopamine release in vivo. Introduction It has previously been shown that the dihydropyridine L-type calcium channel activator methyl-1,4- dihydro - 2,6 - dimethyl nitro (2 - trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) can increase dopamine turnover in the CNS of rats and mice (Pileblad & Carlsson, 1987; Bolger et al., 1988; Bourson et al., 1989). These authors have postulated that this is due to a direct effect on dopamine release as calcium is known to be important in neurotransmitter release mechanisms (Ashley et al., 1984; Augustine et al., 1987). Furthermore, in vitro studies using potassium-stimulated release from brain slices 'Author for correspondence. 2 Present address: UCB Secteur Pharmaceutique, Chemin du Foriest, Braine l'alleud, Belgium. I Present address: Preclinical Research Department, Sandoz AG, CH-4002 Basel, Switzerland. suggest that L-type calcium channels may play a role in this process (Middlemiss, 1985; Middlemiss & Spedding, 1985). In order to examine the functional significance of this interaction between the L-type calcium channel and CNS dopamine systems we have studied the effects of the dihydropyridine calcium channel blocker nifedipine and the activator Bay K 8644 in several behavioural tests of dopaminergic activity. Tests were chosen that may be expected to demonstrate an effect on release in vivo, such as rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions (Ungerstedt, 1971), apomorphine-induced yawning (Gower et al., 1984) and the discriminative stimulus induced by amphetamine. Rats with unilateral 6-OHDA lesions of the nigrostriatal pathway will turn towards the lesioned side when treated with a dopamine releaser, such as amphetamine, because transmitter can only t The Macmillan Press Ltd 1989

2 DIHYDROPYRIDINE COMPOUNDS AND DOPAMINERGIC BEHAVIOURS 1313 be released from the intact presynaptic sites on the non-lesioned side (Ungerstedt, 1971). In the case of apomorphine-induced yawning there is considerable evidence that the low doses of apomorphine required to induce yawning are acting on dopamine autoreceptors and thereby reducing dopamine release (Protais et al., 1983; Dourish & Hutson, 1985). Finally, the amphetamine discriminative stimulus seems to rely on the ability of amphetamine to release dopamine (Schechter & Cook, 1975). The drug discrimination technique also has the advantage that animals can continue to show a discrimination despite a moderate degree of disruption. This feature was considered important as Bay K 8644 produces a spasticity-like syndrome at high doses (i.e. above 0.5mgkg-1i.p.) that may interfere with tests relying on a measure of locomotor activity (Bourson et al., 1989). Some of these results have been presented in abstract form to the British Pharmacological Society (Bourson et al., 1986). Methods Animals Male Sprague-Dawley rats (Charles River, France) weighing g were used throughout. Before experimental use they were housed in wire cages in groups of 6 in temperature (22 + PC) and humidity (55% R.H.) controlled animal quarters on a 12h light-dark cycle with lights on between 6h 00min and 18h 00min. All animals had free access to food and tap water, except those used for the drug discrimination experiments which were maintained on a restricted diet as detailed below. Apomorphine-induced yawning and penile erection All experiments were carried out between 8 h 30min and 12 h 30 min. The rats were observed in groups of 4, each rat having received a different pretreatment. Immediately following the s.c. injection of apomorphine, the rats were placed in individual clear glass beakers (18cm high x 14.5cm diameter) containing a layer of sawdust. A mirror was positioned behind the beakers to permit all round observation of the rats. The number of yawns was counted by direct observation for 30min from the time the rats were placed in the beakers. In some experiments the incidence of penile erection was also recorded. Each rat was tested once only. Amphetamine drug discrimination Apparatus Testing was carried out using five standard operant cages (LI 830, Letica Scientific Instruments, Barcelona) with a food dispenser centred between two levers. The cages were housed separately in sound and light attenuating chambers and illuminated by a house light. The apparatus was programmed using solid state logic modules driven by an Apple Ile computer. Training The training and testing procedures have been described in detail previously (Tricklebank et al., 1987). Rats were deprived of food by 75-80% of their free-feeding weight and trained to press the right or left levers for food reinforcement (45 mg dustless Precision Pellets, Bioserv. Inc., New Jersey). Once the lever pressing response was established by use of continuous reinforcement, the conditions were changed to a fixed ratio (FR 10) schedule. Fifteen minutes before daily (Monday-Friday) test sessions lasting 14 min, the rats were injected with either amphetamine (0.8 mg kg1- i.p.) or saline vehicle. Half the rats were trained to press the left lever and half the right lever for food reward following amphetamine treatment. In both cases pressing on the alternate lever was reinforced following saline administration. Drug (D) or saline (S) was given to each rat according to one of two repeating sequences (DSSDSSDDSD or SSDDSDDSSD). Each day the total number of responses made on both levers before the first reward and the total number of responses on each lever during the session were recorded. The criterion for correct discrimination was that the total number of responses before the first reward did not exceed 14. Test of stimulus generalisation and antagonism Experiments were carried out once a week and started once the rats had achieved the discrimination criterion in at least 80% of the preceeding 15 training sessions. On experimental days the sessions lasted 3min and no reinforcement was given. The total number of responses on each lever was recorded and also the total number of responses at the time when a score of 10 was obtained on one of the levers. Generalisation to amphetamine occurred when the rat pressed 10 times on the amphetamineappropriate lever before pressing more than 4 times on the saline-appropriate lever. Experimental groups consisted of not less than six rats and the same rats were used to study different test drugs over a number of experimental sessions. Unilateral 6-OHDA lesion of the medialforebrain bundle Rats were pretreated with desmethylimipramine (25mg kg- i.p.) approximately 30 min before the injection of 6-OHDA in order to block the uptake of the neurotoxin into the noradrenergic fibres and thus

3 1314 A. BOURSON et al. produce a selective degeneration of the dopaminergic neurones. The rats were subsequently anaesthetized with pentobarbitone sodium (Clin-Midy, France; 30mgkg1 i.p.) and placed in a stereotaxic frame. 6-OHDA was dissolved in saline containing ascorbic acid (0.2mg ml -1) to a concentration of 4pg MP1-l, and 2 MI of this solution were slowly injected into the right medial forebrain bundle (MFB) at the coordinates AP = -2.0mm, L = mm from the bregma and 8 mm below the dura according to the atlas of Pellegrino & Cushman (1967). The injection was made over a period of 5 min 30 s, the needle was left in place for a further 30s to allow diffusion of compound away from the injection site. Recording of rotational behaviour The rat was placed in a rotometer similar to that described by Ungerstedt & Arbuthnott (1970). The rotational behaviour was recorded automatically as the number of 3600 turns towards or away from the lesioned side very 5 min during the 60 min following administration of amphetamine. Drugs and solutions The following compounds were used: nifedipine, Bay K 8644 (Bayer AG, FRG), 6-hydroxydopamine hydrobromide, desmethylimipramine hydrochloride, apomorphine hydrochloride (all from Sigma, U.S.A.), D-amphetamine sulphate (Fabriques de Laire, France). All solutions were prepared immediately before use. Nifedipine and Bay K 8644 were dissolved in polyethyleneglycol 50% (PEG 50%) and were protected from light by wrapping the bottles in aluminium foil. All other drugs were dissolved in sterile 0.9% w/v NaCl solution. Apart from apomorphine, which was administered subcutaneously, and 6-OHDA which was administered into the medial forebrain bundle, all drugs were injected intraperitoneally. Statistics 20r- Data from yawning, penile erection and rotation experiments were analysed by the Kruskal-Wallis analysis of variance for non-parametric data. If this was significant, a Mann-Whitney U-test was used to compare control and test groups. Response rates from the drug discrimination experiments were analysed by Student's t test. The response rates were calculated as the mean of the number of lever presses min-' of those rats in each treatment group that made more than 10 presses on any one lever, irrespective of their discrimination performance. Differences between groups were considered as significant if P < Results Effect of nifedipine and Bay K 8644 on yawning and penile erection induced by apomorphine Administration of apomorphine (20, 40 and 80 Mg kg- s.c.) dose-dependently induced yawning ( ; ; yawns 30min-' respectively, mean + s.e.mean, n = 8). A higher dose (120Mgkg-1s.c.) was a less effective inducer of yawning ( yawns 30min-'; n = 8). Nifedipine (1.25 to 20mg kg- i.p.) dose-dependently potentiated yawning induced by apomorphine (40Mg kg-1 s.c.), whereas given alone nifedipine induced only a low incidence of yawning (Figure 1). In contrast to the clear potentiation of yawning behaviour by nifedipine, no potentiation of penile erections induced by the same dose of apomorphine ( erections 30min-1) was observed ( and erections 30min 1 for 2.5 and 10mgkg-1 nifedipine, respectively; n = 8). Nifedipine (lomgkg- i.p.) did not induce penile erections when given alone (PEG 50%: ; nifedipine erections 30 min- 1, n = 8). Following i.p. administration, Bay K 8644 ( mg kg- i.p.) dose-dependently inhibited apomorphine-induced yawning (Figure 2). Furthermore, CA B 10 0' PEG Dose nifedipine (mg kg-' i.p.) Effect of nifedipine on yawning induced by Figure 1 apomorphine (40pgkg-1s.c.). Nifedipine was administered intraperitoneally 30min before apomorphine. Each point represents the mean number of yawns for 8-16 rats observed over a 30min period immediately following apomorphine administration. Vertical lines show s.e.mean. The hatched columns represent the effects of nifedipine alone. *P < 0.05 compared to PEG 50%-treated controls by the Mann-Whitney U-test.

4 DIHYDROPYRIDINE COMPOUNDS AND DOPAMINERGIC BEHAVIOURS i Ii L) 51) a 100 r U) 3 10 * ** a) C - ā) E co 501-0' 0o PEG Dose Bay K 8644 (mg kg-1 i.p.) Figure 2 Effect of Bay K 8644 on yawning induced by apomorphine (80.g kg- 1 s.c.). Bay K 8644 was administered intraperitoneally 60min before apomorphine. Each point represents the mean number of yawns for 8 rats observed over a 30min period immediately following apomorphine administration. Vertical lines show s.e.mean. *P < 0.05, **P < 0.01 compared to PEG 50%-treated controls by the Mann-Whitney U-test. the potentiation of apomorphine-induced yawning by nifedipine (lomgkg-1) was reversed by prior administration of Bay K 8644 (0.4mg kg-1). Thus, apomorphine (40ygkg-') induced yawns 30min- 1 (n = 8) which was increased to following nifedipine pretreatment (P < 0.01 compared to vehicle pretreatment, Mann-Whitney U-test) and unaffected by administration of both nifedipine and Bay K 8644 ( yawns 30 min'; P < 0.01 compared to nifedipine pretreatment alone, Mann-Whitney U-test). Bay K 8644, at doses that significantly inhibited yawning, did not affect penile erections induced by apomorphine (PEG 50% pretreatment: erections 30min-1; BayK OSmgkg-1: ; 0.25 mgkg-': ; n = 8). Bay K 8644 (0.25mg kg- i.p.) did not induce penile erections when given alone. Effect ofnifedipine and Bay K 8644 on the discriminative stimulus effects ofamphetamine Amphetamine ( mgkg-1i.p.) dose-dependently increased the percentage of rats selecting the amphetamine appropriate lever and pretreatment with either PEG 50% or nifedipine (20mg kg-1 i.p.) had no significant effect on this dose-response ** 0 C0. U) CD a) b 50 r 25F 0I a I I Amphetamine (mg kg-1 i.p.) Figure 3 Effect of nifedipine on the dose-response relationship to amphetamine in rats trained to discriminate amphetamine (0.8 mgkg- i.p.) from saline. Nifedipine (20mg kg-'; U) or PEG 50% (A) was injected 15min before amphetamine which was administered 15 min before the test. (0) Show the doseresponse to amphetamine with no pretreatment. *P < 0.05 compared lo PEG 50%-treated controls by use of Student's t test. relationship (Figure 3). When different doses of nifedipine were tested in combination with a submaximal dose of amphetamine (0.4mg kg- i.p.), there was no dose-related effect on the ability of the animals to detect this dose of amphetamine, although a slight potentiation was obtained with 10mgkg-1 of nifedipine (Table 1). Further studies with a lower dose of amphetamine (0.2mgkg- i.p.) in combination with nifedipine (10rmgkg- i.p.) failed to confirm the potentiating effect of the dihydropyridine compound (treatment with either amphetamine alone or with amphetamine + nifedipine made 20% of rats select the amphetamine lever). Unlike its effect on discrimination performance, nifedipine consistently and dose-dependently

5 1316 A. BOURSON et al. Table I The effect of nifedipine or Bay K 8644 on the discriminative stimulus induced by amphetamine Treatment Dose (mg kg- ' i.p.) PEG 50% Nifedipine PEG 50% Bay K % amphetamine-lever responses Responses min-' * Rats were trained to discriminate amphetamine (0.8mgkg-'i.p.) from saline and the effects of nifedipine and Bay K 8644 were studied in combination with 0.4 and 0.2mg kg-' i.p. amphetamine respectively. Nifedipine was administered 15 min, and Bay K min, before amphetamine. n/n: number of animals responding (n) of the number tested (N). *P < 0.05 compared to PEG 50% treated control groups by means of Student's t test. n/n 13/13 9/13 12/14 14/14 13/13 7/7 10/12 10/12 1/6 reduced the response rate of amphetamine-treated rats (Table 1 and Figure 3). However, Bay K 8644 ( mg kg- i.p.) had no significant effect on either the discrimination performance, or on the response rates of rats receiving amphetamine (0.2mgkg-ti.p.; Table 1). At the highest dose used there was a clear disruption of lever pressing behaviour which corresponded to the appearance of the locomotor and postural effects of Bay K Effects ofnifedipine and Bay K 8644 on rotational behaviour Amphetamine dose-dependently induced ipsilateral rotation in rats with unilateral lesions of the MFB (0.25mgkg-1: ; 0.5mgkg-1: ; lmgkg-t: ; 2mgkg-': turns 60 min-1; n = 12-20). From these results a dose of mg kg- was chosen for further study with the dihydropyridine compounds, as it produced a reliable rotational response which was well below the maximum that could be obtained. Following pretreatment with PEG 50% amphetamine induced ipsilateral turns 60min-1 (mean + s.e.mean; n = 7) and this response was not significantly altered by 45min pretreatment with Bay K 8644 at either 0.06, 0.12, 0.25 or 0.5 mgkg- 1 ( , , and turns 60 min - 1 respectively; n = 5-7). Nifedipine (10 min pretreatment) also failed to affect the rotational behaviour induced by amphetamine (PEG 50% pretreatment: turns 60min'-; nifedipine 5mgkg-t: ; lomgkg-1: ; n = 5). Neither compound significantly induced rotations themselves over the same dose-ranges. Higher doses of Bay K 8644 could not be tested due to its inhibitory effects on locomotor activity. Discussion Although the involvement of the L-type calcium channel in neurotransmitter release is controversial, experiments with the dihydropyridine calcium channel activator Bay K 8644 consistently produce results suggesting that it increases either neurotransmitter release or turnover, both in vitro (Middlemiss, 1985; Middlemiss & Spedding, 1985) and in vivo (Bolger et al., 1988; Bourson et al., 1989). Results with the dihydropyridine calcium channel blockers are less clearcut; Middlemiss (1985), Middlemiss & Spedding (1985) and Bourson et al. (1989) found no effect on either release or metabolite levels, whereas others have demonstrated inhibition of neurotransmitter release (Nordstrbm et al., 1986; Pileblad & Carlsson, 1986) or even, in the case of dopamine, an increase (Nordstrom et al., 1986). The effects of Bay K 8644, referred to above, can be reversed by the dihydropyridine calcium channel blockers suggesting that when activated, L-type calcium channels can affect neurotransmitter release, but that normally they play a negligible role. Although the previous in vivo experiments do not differentiate between increased release and increased turnover, data from the in vitro studies suggest that it is neurotransmitter release that contributes to the raised metabolite levels. The experiments described in this paper were designed to investigate the functional interaction of the L-type calcium channel with dopamine release in behavioural models where dopamine release, or its modulation, is thought to play a major role. Although apomorphine-induced yawning was potentiated by nifedipine and inhibited by Bay K 8644, penile erection induced by the same compound, and both the stimulus effects of amphetamine and the rotational behaviour induced by amphetamine were unaffected by either compound.

6 DIHYDROPYRIDINE COMPOUNDS AND DOPAMINERGIC BEHAVIOURS 1317 Apomorphine-induced yawning is thought to be the result of reduced dopamine release following stimulation of presynaptic dopamine receptors (Mogilnicka & Klimek, 1977; Protais et al., 1983; Gower et al., 1984; Stahle & Ungerstedt, 1984; Stoessl et al., 1987), and its potentiation by nifedipine and inhibition by Bay K 8644 are consistent with previous work suggesting an involvement of L-type calcium channels in dopamine release. The mutual antagonism between nifedipine and Bay K 8644 of their effects on yawning behaviour is also consistent with such an involvement, although the present data cannot distinguish between an inhibition by Bay K 8644 of the nifedipine potentiation or of the apomorphine response itself. However, apomorphine-induced penile erections are thought to be due to the same mechanism as that involved in the yawning response (Gower et al., 1984) and the inability of the dihydropyridine compounds to affect this parameter argues against an interaction with either apomorphine itself or dopamine release mechanisms. This conclusion is further supported by the lack of effect of either nifedipine or Bay K 8644 on amphetamine drug discrimination and on rotation in unilaterally lesioned rats, either alone or in combination with amphetamine. It is possible that the negative results we obtained with the dihydropyridine compounds in combination with amphetamine were due to the use of amphetamine itself, which releases dopamine from several storage pools (Chiueh & Moore, 1975) and. whose effect is not receptor-mediated as is the case with apomorphine. Such mechanisms may be less susceptible to modulation via dihydropyridine-sensitive calcium channels than those involved in apomorphine-induced changes in dopamine release, although several studies have suggested a role for calcium in amphetamine-induced behavioural changes. Fung & Uretsky (1980) have shown that intrastriatal injection of EGTA could inhibit amphetamine-induced rotation in mice. Also, Grebb (1986) has demonstrated that nifedipine can antagonise amphetamine-induced hyperactivity. However, the specificity of these effects to the dopamine releasing ability of amphetamine is questionable; the effects seen by Grebb (1986) occurred at doses higher than those we have used and are possibly due to other effects of calcium antagonists that are seen at high doses (De Feudis, 1987). References ARGIOLAS, A., MELIS, M.R. & GESSA, G.L. (1986). Oxytocin: an extremely potent inducer of penile erection and yawning in male rats. Eur. J. Pharmacol., 130, ASHLEY, RH., BRAMMER, M.J. & MARCHBANKS, R. (1984). Measurement of intrasynaptosomal free calcium by Nencini & Woolverton (1988) have recently demonstrated that nimodipine weakly inhibited the discriminative stimulus effects of amphetamine, but considered this to be a non-specific effect as it was not dose-related, of small magnitude and associated with a significant reduction in response rate. Our results are more clearcut in that neither nifedipine nor Bay K 8644 had an effect on the discrimination performance by rats, although both reduced the response rate. As both the discriminative effects and rotational behaviour induced by amphetamine have been well characterised as being mediated primarily by increased dopamine release (Ungerstedt, 1971; Schechter & Cook, 1975), our results would suggest that at these doses the dihydropyridine compounds do not affect amphetamine-induced dopamine release. It should be noted that the effects on dopamine turnover that we previously demonstrated for Bay K 8644 were seen using higher doses than were possible in the present study (Bourson et al., 1989), due to the appearance of interfering behaviours. This might also explain why the dihydropyridine compounds did not induce rotation themselves. These data suggest that at the doses we have used dihydropyridine compounds are not able to modify dopamine release in vivo and that the effects on apomorphine-induced yawning are specific to that behaviour. Clearly, further experiments are needed to determine the mechanisms responsible for the potentiation of apomorphine-induced yawning by nifedipine. 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