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Differential antagonistic effects of haloperidol on excitatory responses of cortical neurones to phenylephrine, noradrenaline and dopamine
Neuropharmacology Vol. 22, No. 8, pp. 945-952, 1983 Printed in Great Britain. All rights reserved
0028-3908/83 $03.00 + 0.00 Copyright ;() 1983 Pergamon Press Ltd
DIFFERENTIAL ANTAGONISTIC EFFECTS OF HALOPERIDOL ON EXCITATORY RESPONSES OF CORTICAL NEURONES TO PHENYLEPHRINE, NORADRENALINE AND DOPAMINE EVIDENCE
FOR
THREE
EXCITATORY CATECHOLAMINE-SENSITIVE RECEPTORS
C. M. BRADSHAW,R. Y. K. PUN*, N. T. SLATER~,M. J. STOKER3~and E. SZABADI§ Department of Psychiatry, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, England (Accepted 13 January 1983) Summary--The technique of microelectrophoresis was used in order to compare the effects of phenylephrine with those of noradrenaline and dopamine on single neurones in the somatosensory cerebral cortex of the rat. Phenylephrine evoked only excitatory responses on cortical neurones. The phenylephrine-sensitive cells, however, could either be excited or depressed by noradrenaline and dopamine. Phenylephrine appeared to be more potent than either noradrenaline or dopamine in evoking excitatory responses. Responses to phenylephrine had shorter latencies to onset and longer recovery times than excitatory responses to either noradrenaline or dopamine. When the absolute mobilities of phenylephrine, noradrenaline and dopamine were compared using an in vitro method, no significant differences were found between the mobilities of the three ionic species, suggesting that the three drugs have similar transport numbers. Thus, the difference in potency between phenylephrine and the other two drugs, and the difference in the time-courses of responses to phenylephrine and the other two drugs, are presumably of biological origin. The dopamine-receptor antagonist, haloperidol, discriminated between responses to phenylephrine and noradrenaline, the response to phenylephrine being more susceptible to antagonism by haloperidol than the response to noradrenaline; excitatory responses to acetylcholine were not affected. Haloperidol also discriminated between responses to phenylephrine and dopamine, the response to dopamine being more susceptible to antagonism by haloperidol than the response to phenylephrine; excitatory responses to acetylcholine were not affected. The ct-adrenoceptor antagonist, phenoxybenzamine, failed to discriminate between responses to phenylephrine and noradrenaline: responses to both agonists were equally antagonized, while responses to acetylcholine were not affected. It is concluded that the excitatory responses of cortical neurones to phenylephrine, noradrenaline and dopamine are likely to be mediated by at least three different receptors. It is suggested that the response to phenylephrine may reflect, primarily, the activation of cq-adrenoceptors, and the response to dopamine the activation of excitatory dopamine receptors.
Single cortical neurones are sensitive to noradrenaline and dopamine applied by microelectrophoresis; both excitatory and depressant reponses can be evoked by both catecholamines (Bevan, Bradshaw and Szabadi, 1977; Bevan, Bradshaw, Pun, Slater and Szabadi, 1978a). It is possible to discriminate between excitatory responses to the two catecholamines by the use of selective antagonists; while the response to noradrenaline is more
*Present address: Laboratory of Developmental Neurobiology, National Institute of Health, Bethesda, MD 20205, U.S.A. tPresent address: Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201, U.S.A. :~Present address: Astra CNS-Respiratory, Division of Astra Pharmaceuticals Ltd, St Peter's House, 2 Bricket Road, St Albans, Hertfordshire ALl 3JW, England. §Address correspondence to: E. Szabadi at the above address. 945
susceptible to antagonism by adrenoceptor blocking agents (e.g. phenoxybenzamine), the response to dopamine is more susceptible to antagonism by dopamine-receptor blocking agents (e.g. haloperidol, ct-flupenthixol). It has been suggested, therefore, that the excitatory responses of cortical neurones to the catecholamines are mediated by at least two separate receptors: the response to noradrenaline mainly by ct-adrenoceptors and the response to dopamine mainly by excitatory dopamine-receptors (Bevan et al., 1978a). Since both kinds of excitatory catecholamine receptors may occur on the same neurones, it is possible that the response to dopamine contains a component which reflects the activation of ct-adrenoceptors and that the response to noradrenaline is " c o n t a m i n a t e d " by the effects of dopamine-receptor activation. Indeed, it is well known that dopamine can stimulate ~-adrenoceptors in the periphery (Goldberg, 1975) and there is evidence that noradrenaline can stimulate dopamine
946
C.M. BRADSHAWet al.
receptors in endocrine and nervous tissues devoid of ~-adrenoceptors (Labrie, Beaulieu, Ferland, Raymond, Di Paolo, Caron, Veilleux, Denizeau, Euvard, Raynaud and Boissier, 1979; Watling and Dowling, 1981). A partial overlap between ~-adrenoceptor and dopamine-receptor activation by the two catecholamines may explain the observation that the discrimination between responses to noradrenaline and dopamine, using ~-adrenoceptor and dopaminereceptor antagonists, is usually only partial (see Bevan et al., 1978a). In an attempt to separate further the two excitatory catecholamine receptors pharmacologically, an agonist was chosen which is selective for one kind of receptor. Phenylephrine seemed to be the ideal choice since it is a potent stimulant of ~-adrenoceptors (Furchgott, 1972; Docherty and McGrath, 1980), with no affinity for dopamine-receptors (Labrie et al., 1979; Watling and Dowling, 1981). Furthermore, previous experiments in this laboratory have shown that phenylephrine was a potent excitant of cortical neurones (Bevan et al., 1977; Bradshaw, Stoker and Szabadi, 1982a; Bradshaw, Stoker and Szabadi, 1982b). In the present experiments, therefore, the effects of phenylephrine were compared with those of noradrenaline and dopamine, using haloperidol as a dopamine-receptor antagonist, and phenoxybenzamine as an ~-adrenoceptor antagonist. Some of the results presented here have been communicated to the British Pharmacological Society (Bradshaw, Pun, Slater, Stoker and Szabadi, 1981a).
METHODS
Pharmacological experiments
Male albino Wistar rats (250-350 g) were used in these experiments. The animals were anaesthetized with halothane (0.8-1.0~). The methods for the surgical preparation of the animals, for the manufacture of six-barrelled micropipettes, for the extracellular recording of action potentials, and for the microelectrophoretic application of drugs have been described elsewhere (Bradshaw, Roberts and Szabadi, 1973a; Bradshaw, Szabadi and Roberts, 1973b; Bradshaw, Roberts and Szabadi, 1974). Six-barrelled micropipettes of tip diameter of 3.0 to 5.0 ~tm were used. Two barrels of each micropipette contained 4.0 M NaC1, one for recording action potentials, and the other for current balancing. The remaining barrels contained solutions of drugs. The following drug solutions were used: (-)noradrenaline bitartrate (0.05 M, pH 3.04.0); (-)phenylephrine hydrochloride (0.05 M, pH 5.0 6.5); dopamine hydrochloride (0.05 M, pH 4.0~4.5); acetylcholine chloride (0.05 M, pH 4.5-5.5); phenoxybenzamine hydrochloride (0.01 M, pH 2.5-3.0); haloperidol (0.01 M dissolved in 0.01 M tartaric acid, pH 4.0).
Spontaneously active neurones were studied in the cerebral cortex (stereotaxic co-ordinates, according to K6nig and Klippel (1968): A 4.8 6.5, L 0.9 2.4). The area of recording was prepared as described previously (Bradshaw and Szabadi, 1972), The dura was either incised with a hypodermic needle, or was penetrated directly with the micropipette. All the drugs were applied by microelectrophoresis. When a suitable unit was encountered, the agonists were applied in a regular cycle. Between successive applications of agonists retaining currents of - 10 nA were passed. Retaining currents of - 2 5 nA were used for the antagonists. Intervals between successive applications of the same agonists were kept constant in order to standardize the effects of the retaining currents upon release of the drug during the ejection period (Bradshaw et al., 1973a; Bradshaw et al., 1973b). The relative excitatory potencies of the three amines were evaluated by comparing responses to phenylephrine and noradrenaline, and to phenylephrine and dopamine on the same cells, using methods described elsewhere (Bradshaw et al., 1973b; Bevan, Bradshaw, Pun, Slater and Szabadi, 1978b). In order to compare the time-courses of excitatory responses to the amines, two time-course parameters were used: latency to onset and recovery time (Bradshaw et al., 1973b). These measures were taken on cells on which the two amines compared (i.e. phenylephrine and noradrenaline, and phenylephrine and dopamine) evoked responses of approximately equal size (Bevan et al., 1978b). The efl'ects of the antagonists were studied on cells which yielded consistent excitatory responses to phenylephrine, noradrenaline (or dopamine) and acetylcholine. The methods for conducting antagonism studies have been described in detail elsewhere (Bevan et al., 1977: Bevan et al.,1978a; Bradshaw et al., 1982). Measurement o f the release q[" noradrenaline J?om mieropipettes in vitro
The absolute mobilities of phenylephrine and noradrenaline, and phenylephrine and dopamine, were compared by the method of Bradshaw, Pun, Slater and Szabadi (1981c) (see also Bradshaw et al., 1982). Two experiments were conducted. In Experiment 1, three barrels of each six-barrelled micropipette were filled with 0.05 M [methylene-~4C]noradrenaline bitartrate plus 0.05 M noradrenaline hydrochloride; the remaining three barrels contained 0.05 M [methylene-~4C]noradrenaline bitartrate plus 0.05 M phenylephrine hydrochloride. In Experiment 2, three barrels of each micropipette were filled with 0.05 M [methylene-14C]noradrenaline bitartrate plus 0.05 M dopamine hydrochloride; the remaining three barrels contained 0.05 M [methylene-l~C]noradrenaline bitartrate plus 0.05M phenylephrine hydrochloride: D,L[methylene-~4C]noradrenaline bitartrate was obtained from the Radiochemical Centre, Amersham: the specific activity of the 0.05 M solution was
947
Haloperidol on cortical neurones Table 1. Comparison of time-course parameters of responses to phenylephrine and noradrenaline, and of responses to phenylephrine and dopamine Phenylephrine vs noradrenaline (n = 76) Phenylephrine Noradrenaline Phenylephrine vs dopamine (n = 26) Phenylephrine Dopamine
Latencyt
Recovery time~"
10.26 _+0.96 15.00 + 1.44**
156.24 _+6.88 108.58 _+5.06**
10.84 _+ 1.88 17.70_+3.66"*
175.08 -+-9.32 102.02_+8.01"*
tTime-course parameters are in seconds. Values are means + SEM. Asterisks denote statistically significant differences between values for phenylephrine and noradrenaline, and between values for phenylephrine and dopamine (t-test, paired comparison): *P < 0.02: **P < 0.001.
1.0 mCi/mmol. The pH of the combined solutions were 3.4 (0.05 M noradrenaline bitartrate plus 0.05 M noradrenaline hydrochloride); 3.4 (0.05M noradrenaline bitartrate plus 0.05 M phenylephrine hydrochloride); 3.4 (0.05 M noradrenaline bitartrate plus 0.05 M dopamine hydrochloride). The methods for the collection of samples, for the liquid scintillation counting and for the calculation of transport numbers have been described elsewhere (Bradshaw et al., 1973a; Bradshaw, Pun, Slater and Szabadi, 1981b; Bradshaw et al., 1981c).
RESULTS
Comparison of agonistic e.fjects of phenylephrine and noradrenaline Direction of responses to phenylephrine and noradrenaline. Out of 147 neurones responding to both phenylephrine and noradrenaline, all were excited by phenylephrine. On the other hand, noradrenaline excited 126 neurones (86~,), depressed 17 (11~o), and evoked biphasic responses (depression followed by excitation) on the remaining 4 neurones (3~o).
Apparent potencies of" phenylephrine and noradrenaline. The ejecting currents required to evoke approximately equal responses to phenylephrine and noradrenaline were compared on 73 neurones; smaller currents were required to apply phenylephrine than noradrenaline (t-test, paired comparison, P < 0.01). The sizes of responses evoked by identical ejecting currents were compared on 54 cells; phenylephrine evoked larger responses than did noradrenaline (t-test, paired comparison, P <0.001). Both comparisons indicated a greater potency of phenylephrine, the potency ratio (phenylephrine/ noradrenaline) being 1.230 + 0.066 (mean + SEM) and 1.182 +_ 0.038 (mean + SEM), respectively.
Time-courses of responses to phenylephrine and noradrenaline. Latencies and recovery times of responses to phenylephrine and noradrenaline were measured on 76 neurones yielding responses of approximately equal size to the two amines. The response to phenylephrine had a shorter latency and a longer recovery time than the response to noradrenaline (Table 1).
Comparison of agonistic effects of phenylephrine and dopamine Direction of responses to phenylephrine and dopamine. All the 51 neurones responding to both phenylephrine and dopamine were excited by phenylephrine. Dopamine excited 35 (69~o) of these, depressed 14 (27~) and evoked biphasic responses (depression followed by excitation) on the remaining 2 (4Vo).
Apparent potencies of phenylephrine and dopamine, The ejecting currents required to evoke approximately equal responses to phenylephrine and dopamine were compared on 23 neurones: smaller currents were required to apply phenylephrine than dopamine (t-test, paired comparison, P <0.001). The sizes of responses evoked by identical ejecting currents were compared on 11 neurones: phenylephrine evoked larger responses than did dopamine (t-test, paired comparison, P < 0.05), Both comparisons indicated a greater potency of phenylephrine, the potency ratio (phenylephrine/dopamine) being 2.159+0.452 ( m e a n + S E M ) and 1.191+ 0.077 (mean ± SEM), respectively.
Time-courses of responses to phenylephrine and dopamine. Latencies and recovery times of responses to phenylephrine and dopamine were compared on 26 neurones yielding responses of approximately equal size to the two amines. The response to phenylephrine had a shorter latency and a longer recovery time than the response to dopamine (Table I).
EffEcts of antagonists on excitatory responses to phenylephrine, noradrenaline and dopamine Effects of haloperidol on excitatory responses to phenylephrine and noradrenaline. The effect of haloperidol was examined on 14 neurones. On all the neurones tested haloperidol (5-25 nA) reversibly reduced the sizes of responses to phenylephrine and noradrenaline, while responses to acetylcholine were not affected. On 11 of the neurones haloperidol discriminated between the responses to phenylephrine and noradrenaline, the response to phenylephrine being antagonized to a greater degree than was the response to noradrenaline (Fig. 1). On the remaining 3 neurones responses to phenylephrine and
C . M . BRADSHAWet al.
948 (a)
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Fig. 1. Effect of haloperidol on excitatory responses to phenylephrine (PHE), noradrenaline (NA) and acetylcholine (ACh). Excerpts from the ratemeter recording of the firing rate of a single cortical neurone; ordinate: firing rate (spikes/sec): abscissa; running time (rain). Horizontal bars below the traces indicate microelectrophoretic drug applications; numbers refer to intensities of ejecting current (nA). Numbers above the traces indicate the sizes of the responses (total spike numbers, %), taking the size of the control response to each agonist as 100%. (a) Control responses to the agonists. (b) Responses to the agonists during the continuous application of haloperidol (5 nA). At the start of trace (b), haloperidol had been applied continuously for 7 rain. The response to phenylephrine was antagonized while the responses to noradrenaline and acetylcholine were only slightly reduced in size. (c) Recovery of responses to the agonists 29 min after the application of haloperidol had been terminated. noradrenaline were equally antagonized. The results from all the neurones are summarized in Fig. 4: in the presence of haloperidol, responses to phenylephrine were reduced in size to a greater extent than responses to noradrenaline (t-test, P <0.002); responses to acetylcholine were not significantly affected.
Effect of haloperidol on excitatory responses to (Q)
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was examined on 10 neurones. On all the neurones tested, haloperidol (10-25 nA) reversibly reduced the sizes of responses to dopamine and phenylephrine, while responses to acetylcholine were not affected. On 8 of the neurones haloperidol discriminated between responses to dopamine and phenylephrine, the re-
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Fig. 2. Effect of haloperidol on excitatory responses to dopamine (DA), phenylephrine (PHE) and acetylcholine (ACh). Excerpts from the ratemeter recording of the firing rate of a single cortical neurone (conventions as in Fig. 1). (a) Control responses to the agonists. (b) Responses to the agonists during the continuous application of haloperidol (15 nA). At the start of trace (b), haloperidol had been applied continuously for 47 min. The response to dopamine was anatagonized while the responses to phenylephrine and acetylcholine were not affected. (c) Recovery of the response to dopamine 32 rain after the application of haloperidol had been terminated.
Haloperidol on cortical neurones
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Fig. 3. Effect of phenoxybenzamineon excitatory responses to phenylephrine (PHE), noradrenaline (NA) and acetylcholine (ACh). Excerpts from the ratemeter recording of the firing rate of a single cortical neurone (conventions as in Fig. 1). (a) Control responses to the agonists. (b) Responses to the agonists during the continuous application of phenoxybenzamine by diffusion (removal of the retaining current; 0 nA). At the start of trace (b), phenoxybenzamine had been applied continuously for 37 min. The responses to phenylephrine and noradrenaline were antagonized while the response to acetylcholine was not affected (c) Recovery of the responses to phenylephrine and noradrenaline 18 min after the application of phenoxybenzamine had been terminated.
sponse to dopamine being antagonized to a greater degree than the response to phenylephrine (Fig. 2). On the remaining 2 neurones, haloperidol failed to discriminate between responses to dopamine and phenylephrine. The results from all the neurones are summarized in Fig. 4: in the presence of haloperidol, responses to dopamine were reduced in size to a greater extent than responses to phenylephrine (ttest, P < 0.01); responses to acetylcholine were not significantly affected.
Effect of phenoxybenzamine on excitatory responses to phenylephrine and noradrenaline. The effect of phenoxybenzamine was examined on 11 neurones. Phenoxybenzamine (0~10nA) equally antagonized responses to both phenylephrine and noradrenaline, while responses to acetylcholine were not affected (Fig. 3). The results from all the neurones are summarized in Fig. 4.
Comparison ~1 the mobilities of the agonists in vitro Comparison of phenylephrine and noradrenaline. The effects of noradrenaline hydrochloride and phenylephrine hydrochloride on the release of labelled noradrenaline were compared using 11 micropipettes (Experiment 1, see Methods). In the case of both solutions, the rate of release of [~4C]noradrenaline was linearly related to the intensity of the ejecting current. The linear regression equations fitted by the least squares method were: y = 0.064x + 0.213 ([t4C]noradrenaline bitartrate + noradrenaline hydrochloride) (r = 0.999, P < 0.001), and y = 0.070x - 0.363 ([HC]noradrenaline bitartrate + phenylephrine hydrochloride) (r = 0.999, P < 0.001). In neither case did the intercept deviate
significantly from zero (t-test, P > 0.05, in both cases). The mean apparent transport number ( + SEM) of [t4C]noradrenaline was 0.110 + 0.006 in the presence of 0.05 M "cold" noradrenaline hydrochloride, and 0.101 +_0.004 in the presence of 0.05 M "cold" phenylephrine hydrochloride. The two values are not significantly different from one another (ttest, P > 0.1), indicating that equimolar concentrations of noradrenaline hydrochloride and phenylephrine hydrochloride caused similar reductions in the apparent transport number of [~4C]noradrenaline. This observation would suggest that noradrenaline and phenylephrine have similar absolute mobilities (see Bradshaw et al., 1981c). Comparison of phenylephrine and dopamine. The effects of phenylephrine hydrochloride and dopamine hydrochloride on the release of labelled noradrenaline were compared using 11 micropipettes (Experiment 2, see Methods). In the case of both solutions, the rate of release of [t4C]noradrenaline was linearly related to the intensity of the ejecting current. The linear regression equations fitted by the method of least squares were: y = 0 . 0 8 3 x - 0.304 ([14C]noradrenaline bitartrate + phenylephrine hydrochloride) (r = 0.999, P < 0.005), and y = 0.076x +0.010 ([~4C]noradrenaline bitartrate +dopamine hydrochloride) (r = 0.999, P < 0.001). In neither case did the intercept deviate significantly from zero (ttest, P > 0.05, in both cases). The mean apparent transport number ( + SEM) of [~4C]noradrenaline was 0.123 _+0.008 in the presence of 0.05 M "cold" phenylephrine hydrochloride, and 0.122 + 0.008 in the presence of 0.05 M "cold" dopamine hydrochloride. The two values are not significantly different
950
C . M . BRADSHAWet al. (a) PHE
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that phenylephrine and dopamine have similar absolute mobilities (see Bradshaw et al., 1981c).
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Fig. 4. Summary of effects of haloperidol and phenoxybenzamine on excitatory responses to phenylephrine (PHE), noradrenaline (NA), dopamine (DA) and acetylcholine (ACh). For each agonist, the length of the column represents the mean percentage change from control of the size of the response (total spike number) in the presence of the antagonist; vertical bars indicate SEM. (A) Effect of halopcridol on responses to phenylephrine, noradrenaline and acetylcholine on 14 cells. HaloperidoI significantly antagonized responses to phenylephrine and noradrenaline, but had no significant effect on responses to acetylcholine. Haloperidol had a significantly greater effect on responses to phcnylephrine than on responses to noradrenaline. (B) Effect of haloperidol on responses to phenylephrine, dopamine and acetylcholine on 10 cells. Haloperidol significantly antagonized responses to phenylephrine and dopamine, but had no significant effect on responses to acetylcholine. Haloperidol had a significantly greater effect on responses to dopamine than on responses to phenylephrine. (C) Effect of phenoxybenzamine on responses to phenylephrine, noradrenaline and acetylcholine on 11 cells. Phenoxybenzamine significantly antagonized responses to phenylephrine and noradrenaline, but had no significant effect on responses to acetylcholine. There was no significant difference between the degrees of antagonism of responses to the two amines.
from one another (t-test, P >0.1), indicating that equimolar concentrations of phenylephrine hydrochloride and dopamine hydrochloride caused similar reductions in the apparent transport number of [r4C]noradrenaline. This observation would suggest
In agreement with previous observations from this laboratory (Bevan et al., 1977: Bradshaw et al., 1981b) phenylephrine proved to be a potent excitant of cortical neurones. In fact, all the 396 phenylephrine-sensitive cells which have been studied, described both here and in previous papers (Bevan et al., 1977; Bradshaw et al., 1981b; Bradshaw et al., 1982a; Bradshaw e t a / . , 1982b), responded with excitation to phenylephrine. Since phenylephrine is a selective ~-adrenoceptor agonist (Furchgott, 1972; Besse and Furchgott, 1976; Docherty and McGrath, 1980), the observation of an exclusively excitatory effect of phenylephrine on cortical neurones supports the hypothesis that the activation of c~-adrenoceptors by sympathomimetic amines results in neuronal excitation in the cerebral cortex (Bevan et al., 1977; Szabadi, 1979). When compared on the same cells, phenylephrine appeared to be a more potent excitant then was either noradrenaline or dopamine. This difference in potency is likely to reflect a genuine biological difference between phenylephrine and the other two amines, since there was no significant difference between the electrophoretic mobilities of phenylephrine and noradrenaline, and of phenylephrine and dopamine. There are at least two factors which may contribute to the difference in potency between phenylephrine and noradrenaline. Firstly, the operation of an uptake mechanism for noradrenaline would tend to reduce the concentration of noradrenaline at the cellular receptor sites: this factor would have a smaller effect on the concentration of phenylephrine, since phenylephrine has only a very low affinity for the uptake mechanism (Iversen, 1967). Secondly, the size of the excitatory response to noradrenaline may be reduced by the concomitant activation of inhibitory fl-adrenoceptors (see Bevan et al., 1977; Szabadi, 1978); again, this factor would not make a significant contribution to the size of the response to phenylephrine, since phenylephrine has only a very low affinity for #-adrenoceptors (Furchgott, 1972). The observation that dopamine was a less potent excitant than was phenylephrine is not surprising, since it has been reported that dopamine is a less potent excitant of cortical neurones than noradrenaline (Bevan et al., 1978a). Thus, the order of potency of the three amines in evoking excitatory responses on cortical neurones is as follows: phenylephrine > noradrenaline > dopamine. When the time-course of the excitatory responses to phenylephrine and noradrenaline, and to phenylephrine and dopamine, were compared on the same cells, the responses to phenylephrine appeared to have shorter latencies and longer recovery times than the response to either noradrenaline or dopamine.
Haloperidol on cortical neurones The faster onset of the response to phenylephrine is likely to reflect the greater agonistic potency of phenylephrine (Szabadi and Bradshaw, 1974), and the longer recovery time of the response to phenylephrine is likely to be related to the slower rate of elimination of phenylephrine from the receptor sites due to the absence of operation of an active uptake mechanism for this amine (see above). Haloperidol was able to antagonize excitatory responses both to noradrenaline and phenylephrine. This observation is in agreement with previous findings that excitatory responses both to noradrenaline (Bevan et al., 1978a) and to phenylephrine (Bradshaw et al., 1982a) were susceptible to antagonism by haloperidol and is likely to reflect the e-adrenoceptor blocking property of the neuroleptic drug (Lavin, Hoffman and Lefkowitz, 1981; Atlas, Friedman, Litvin and Steer, 1982). The greater susceptibility to antagonism by haloperidol of responses to phenylephrine than of responses to noradrenaline was, however, an unexpected finding, In fact, it had been predicted that the responses to noradrenaline would be preferentially antagonized, since haloperidol has a higher affinity for dopamine receptors than for ~-adrenoceptors (Peroutka, U'Pritchard, Greenberg and Snyder, 1976: Barone, Corsico, Diena, Restelli, Glasser and Rodenghi, 1982) and the responses to noradrenaline may contain a component resulting from the activation of dopamine receptors (see Introduction). Therefore, it was necessary to examine the possibility that, in contrast to its action in the fish retina (Wafting and Dowling, 1981) and in the rat pituitary gland (Labrie et al., 1979), phenylephrine may stimulate dopamine receptors on cortical neurones. When, however, the effect of haloperidol on the excitatory responses to phenylephrine and dopamine was compared, the response to dopamine was preferentially antagonized, suggesting that the excitatory response to phenylephrine is unlikely to result from the activation of dopamine receptors. It is possible, therefore, that phenylephrine excites cortical neurones via a receptor which is different from the receptors activated by either noradrenaline or dopamine. In agreement with previous findings (Bevan et aL, 1977; Bevan et aL, 1978a; Bradshaw et aL, 1981b), phenoxybenzamine, an e-adrenoceptor blocking agent (Nickerson, 1967), effectively antagonized excitatory responses to both noradrenaline and phenylephrine. Phenoxybenzamine failed to discriminate between the excitatory responses to noradrenaline and phenylephrine, although it can discriminate between responses to noradrenaline and dopamine, the response to noradrenaline being more susceptible to antagonism by phenoxybenzamine than is the response to dopamine (Bevan et al., 1978a). The present experiment with phenoxybenzamine, therefore, does not provide any evidence in favour of a "dopamine component" in the response to noradrenaline (see above).
951
The effects of the antagonists observed in the present experiment and in previous experiments (Bevan et al., 1978a) suggest that the excitatory responses to phenylephrine, noradrenaline and dopamine may be mediated by at least three different receptors. Dopamine is likely to activate mainly specific dopamine receptors which are most sensitive to blockade by haloperidol and least sensitive to blockade by phenoxybenzamine. It is an intriguing possibility that the differential effect of haloperidol on responses to phenylephrine and noradrenaline reflects a dfscrimination between ~ and ~2-adrenoceptors. There is evidence that both et- and cq-adrenoceptors occur in brain tissue (U'Pritchard, Bechtel, Rouot and Snyder, 1979: Weinreich, Chiu, Warsh and Seeman, 1981) and that phenylephrine is a selective cq-adrenoceptor stimulant, whereas noradrenaline has an equal affinity for both types of c~-adrenoceptor (Langer, 1980). Furthermore, it has been reported recently that haloperidol has a significantly greater affinity for ~ - than for e2-adrenoceptors (Lavin et al., 1981; Atlas et al., 1982; Barone et al., 1982). The failure of phenoxybenzamine to discriminate between excitatory responses to phenylephrine and noradrenaline would also be compatible with this interpretation, since phenoxybenzamine shows only partial selectivity for ~t-adrenoceptors (See Langer, 1980). Acknowledgements--This work was supported by the Wellcome Trust and the Science Research Council. M.J.S. is an SRC/CASE Scholar in conjunction with ICI Ltd. We are grateful to Mr D. Praties for technical assistance. REFERENCES
Atlas D., Friedman Z., Litvin Y. and Steer M. L. (1982) Synthesis of new haloperidol analogues and characterization of their interactions with ~-adrenoceptors in rat parotid slices and human platelet membranes. Br. J. Pharmac. 75:213-217. Barone D., Corsico N., Diena A., Restelli A., Glasser A. and Rodenghi F. (1982) Biochemical and pharmacological activites of zetolidine (DL 308-IT): a new antidopaminergic agent. J. Pharm. Pharmac. 34: 129-132. Besse J. C. and Furchgott R. F. (1976) Dissociation constants and relative efficacies of agonists acting on alpha adrenergic receptors in rabbit aorta. J. Pharmac. exp. Ther. 197: 66-78. Bevan P., Bradshaw C. M., Pun R. Y. K., Slater N. T. and Szabadi E. (1978a) Responses of single cortical neurones to noradrenaline and dopamine. Neuropharmacology 17: 611-617. Bevan P., Bradshaw C. M., Pun R. Y. K., Slater N. T. and Szabadi E. (1978b) Comparison of the responses of single cortical neurones to tyramine and noradrenaline: effects of desipramine. Br. J. Pharmac. 63: 651-657. Bevan P., Bradshaw C. M. and Szabadi E. (1977) The pharmacology of adrenergic neuronal responses in the cerebral cortex: evidence for excitatory ~- and inhibitory //-receptors. Br. J. Pharmac. 59: 635-641. Bradshaw C. M., Pun R. Y. K., Slater N. T., Stoker M. J. and Szabadi E. (1981a) Comparison of the effect of haloperidol on excitatory responses of cortical neurones to dopamine, noradrenaline and phenylephrine. Br. J. Pharmac. 73: 243-244P. Bradshaw C. M., Pun R. Y. K., Slater N. T. and Szabadi
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E. (1981b) Comparison of the effects of methoxamine with those of noradrenaline and phenylephrine on single cortical neurones. Br. J. Pharmac. 73: 47-54. Bradshaw C. M., Pun R. Y. K., Slater N. T. and Szabadi E. (1981c) A procedure for comparing the mobilities of unlabelled drugs used in microelectrophoresis experiments. J. Pharmae. Meth. 5: 67-73. Bradshaw C. M., Roberts M. H. T. and Szabadi E. (1973a) Kinetics of the release of noradrenaline from micropipettes: interaction between ejecting and retaining current. Br. J. Pharmac. 49: 667-677. Bradshaw C. M., Roberts M. H. T. and Szabadi E. (19743 Effects of imipramine and desipramine on responses of single cortical neurones to noradrenaline and 5-hydroxytryptamine. Br. J. Pharmac. 52: 349-358. Bradshaw C. M., Stoker M. J. and Szabadi E. (1982a) Does 5-hydroxytryptamine excite cortical neurones by an action at c~-adrenoceptors? Br. J. Pharmae. 75: 36P. Bradshaw C. M., Stoker M. J. and Szabadi E. (1982b) The effect of microelectrophoretically applied clonidine on single cerebral cortical neurones in the rat: evidence for interaction with ~t-adrenoceptors. NaunynSchmiedebergs Arch. Pharmac. 320: 230-234. Bradshaw C. M. and Szabadi E. (1972) A technique for achieving greater stability of the brain for microiontophoretic studies of single cortical neurones. Br. J. Pharmac. 45: 184-186. Bradshaw C. M., Szabadi E. and Roberts M. H. T. (1973b) The reflection of ejecting and retaining currents in the time-course of neuronal responses to microelectrophoretically applied drugs. J. Pharm. Pharmac. 25: 513-520. Docherty J. R. and McGrath J. G. (1980) A comparison of pre- and post-junctional potencies of several alphaadrenoceptor agonists in the cardiovascular system and anococcygeus muscle of the rat. Naunyn-Schmiedebergs Arch. Pharmac. 312: 107-116. Furchgott R. F. (1972) The classification of adrenoceptors (adrenergic receptors) An evaluation from the standpoint of receptor theory. In: Handbook of Experimental Pharmacology Vol. 33, Catecholamines (Blaschko H. and Muscholl E., Eds), pp. 283 335. Springer-Verlag, Berlin. Goldberg L. I. (1975) The dopamine vascular receptor. Biochem. Pharmac. 24: 651-654.
Iversen L. L. (1967) The Uptake and Storage o/' Noradrenaline in Sympathetic Nerves. Cambridge University Press, Cambridge. K6nig J. F. R. and Klippel R. A. (1968) The Rat Brain: A Stereotaxie Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams & Wilkins, Baltimore. Labrie F., Beaulieu M., Ferland L., Raymond V., Di Paolo T., Caron M. G., Veilleux R., Denizeau P., Euvard C., Raynaud J. P. and Boissier P. R. (19793 Control of prolactin secretion at the pituitary level: a model for postsynaptic dopaminergic systems. In: Central Nert,ous System Effects of Hypothalamic Hormones and Other Peptides (Collu R., Barbeau A., Ducharne F. R. and Rochfort J. C., Eds), pp. 207 234. Raven Press, New York. Langer S. Z. 0980) Presynaptic regulation of the release of catecholamines. Pharmae. Rev. 32:337 363. Lavin T. N., Hoffman B. B. and Lefkowitz R. J. (19813 Determination of subtype selectivity of alpha-adrenergic antagonists: comparison of selective and non-selective radioligands. Molec. Pharmac. 20:28 34. Nickerson M. (19673 New developments in adrenergic blocking drugs. Ann. N.Y, Aead. Sei. 139:571 579. Peroutka S. J., U'Pritchard D. C., Greenberg D. A. and Snyder S. H. (19763 Neuroleptic drug interactions with norepinephrine alpha receptor binding sites in rat brain. Neuropharmaeology 16:549 556. Szabadi E. (19783 Functionally opposite receptors on neurones. L(lk" Sei. 23: 1889-1898. Szabadi E. (1979) Adrenoceptors on central neurones: microelectrophoretic studies. Neuropharmacology 18: 831-834. Szabadi E, and Bradshaw C. M. (I 974) The role of physical and biological factors in determining the time-course ot neuronal responses. Neuropharmaeology 13:537 545. U'Pritchard D. G., Bechtel W. D., Rouot B. M. and Snyder S. H. (1979) Multiple binding sites in rat brain: the effect of 6-hydroxydopamine. Molee. Pharmac. 16:47 61). Watling K. F. and Dowling F. E. (1981) Dopaminergic mechanisms in the teleost retina: effect of agonists, antagonists and ergots. J. Neurochem. 36: 559-568. Weinreich R., Chiu A., Warsh J. and Seeman P. (1981) Multiple binding sites for [3HJ-clonidine and [3H]-WB-410I in rat brain. Can. J. Physiol. Pharmae. 59: 1170-1177.
0028-3908/83 $03.00 + 0.00 Copyright ;() 1983 Pergamon Press Ltd
DIFFERENTIAL ANTAGONISTIC EFFECTS OF HALOPERIDOL ON EXCITATORY RESPONSES OF CORTICAL NEURONES TO PHENYLEPHRINE, NORADRENALINE AND DOPAMINE EVIDENCE
FOR
THREE
EXCITATORY CATECHOLAMINE-SENSITIVE RECEPTORS
C. M. BRADSHAW,R. Y. K. PUN*, N. T. SLATER~,M. J. STOKER3~and E. SZABADI§ Department of Psychiatry, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, England (Accepted 13 January 1983) Summary--The technique of microelectrophoresis was used in order to compare the effects of phenylephrine with those of noradrenaline and dopamine on single neurones in the somatosensory cerebral cortex of the rat. Phenylephrine evoked only excitatory responses on cortical neurones. The phenylephrine-sensitive cells, however, could either be excited or depressed by noradrenaline and dopamine. Phenylephrine appeared to be more potent than either noradrenaline or dopamine in evoking excitatory responses. Responses to phenylephrine had shorter latencies to onset and longer recovery times than excitatory responses to either noradrenaline or dopamine. When the absolute mobilities of phenylephrine, noradrenaline and dopamine were compared using an in vitro method, no significant differences were found between the mobilities of the three ionic species, suggesting that the three drugs have similar transport numbers. Thus, the difference in potency between phenylephrine and the other two drugs, and the difference in the time-courses of responses to phenylephrine and the other two drugs, are presumably of biological origin. The dopamine-receptor antagonist, haloperidol, discriminated between responses to phenylephrine and noradrenaline, the response to phenylephrine being more susceptible to antagonism by haloperidol than the response to noradrenaline; excitatory responses to acetylcholine were not affected. Haloperidol also discriminated between responses to phenylephrine and dopamine, the response to dopamine being more susceptible to antagonism by haloperidol than the response to phenylephrine; excitatory responses to acetylcholine were not affected. The ct-adrenoceptor antagonist, phenoxybenzamine, failed to discriminate between responses to phenylephrine and noradrenaline: responses to both agonists were equally antagonized, while responses to acetylcholine were not affected. It is concluded that the excitatory responses of cortical neurones to phenylephrine, noradrenaline and dopamine are likely to be mediated by at least three different receptors. It is suggested that the response to phenylephrine may reflect, primarily, the activation of cq-adrenoceptors, and the response to dopamine the activation of excitatory dopamine receptors.
Single cortical neurones are sensitive to noradrenaline and dopamine applied by microelectrophoresis; both excitatory and depressant reponses can be evoked by both catecholamines (Bevan, Bradshaw and Szabadi, 1977; Bevan, Bradshaw, Pun, Slater and Szabadi, 1978a). It is possible to discriminate between excitatory responses to the two catecholamines by the use of selective antagonists; while the response to noradrenaline is more
*Present address: Laboratory of Developmental Neurobiology, National Institute of Health, Bethesda, MD 20205, U.S.A. tPresent address: Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201, U.S.A. :~Present address: Astra CNS-Respiratory, Division of Astra Pharmaceuticals Ltd, St Peter's House, 2 Bricket Road, St Albans, Hertfordshire ALl 3JW, England. §Address correspondence to: E. Szabadi at the above address. 945
susceptible to antagonism by adrenoceptor blocking agents (e.g. phenoxybenzamine), the response to dopamine is more susceptible to antagonism by dopamine-receptor blocking agents (e.g. haloperidol, ct-flupenthixol). It has been suggested, therefore, that the excitatory responses of cortical neurones to the catecholamines are mediated by at least two separate receptors: the response to noradrenaline mainly by ct-adrenoceptors and the response to dopamine mainly by excitatory dopamine-receptors (Bevan et al., 1978a). Since both kinds of excitatory catecholamine receptors may occur on the same neurones, it is possible that the response to dopamine contains a component which reflects the activation of ct-adrenoceptors and that the response to noradrenaline is " c o n t a m i n a t e d " by the effects of dopamine-receptor activation. Indeed, it is well known that dopamine can stimulate ~-adrenoceptors in the periphery (Goldberg, 1975) and there is evidence that noradrenaline can stimulate dopamine
946
C.M. BRADSHAWet al.
receptors in endocrine and nervous tissues devoid of ~-adrenoceptors (Labrie, Beaulieu, Ferland, Raymond, Di Paolo, Caron, Veilleux, Denizeau, Euvard, Raynaud and Boissier, 1979; Watling and Dowling, 1981). A partial overlap between ~-adrenoceptor and dopamine-receptor activation by the two catecholamines may explain the observation that the discrimination between responses to noradrenaline and dopamine, using ~-adrenoceptor and dopaminereceptor antagonists, is usually only partial (see Bevan et al., 1978a). In an attempt to separate further the two excitatory catecholamine receptors pharmacologically, an agonist was chosen which is selective for one kind of receptor. Phenylephrine seemed to be the ideal choice since it is a potent stimulant of ~-adrenoceptors (Furchgott, 1972; Docherty and McGrath, 1980), with no affinity for dopamine-receptors (Labrie et al., 1979; Watling and Dowling, 1981). Furthermore, previous experiments in this laboratory have shown that phenylephrine was a potent excitant of cortical neurones (Bevan et al., 1977; Bradshaw, Stoker and Szabadi, 1982a; Bradshaw, Stoker and Szabadi, 1982b). In the present experiments, therefore, the effects of phenylephrine were compared with those of noradrenaline and dopamine, using haloperidol as a dopamine-receptor antagonist, and phenoxybenzamine as an ~-adrenoceptor antagonist. Some of the results presented here have been communicated to the British Pharmacological Society (Bradshaw, Pun, Slater, Stoker and Szabadi, 1981a).
METHODS
Pharmacological experiments
Male albino Wistar rats (250-350 g) were used in these experiments. The animals were anaesthetized with halothane (0.8-1.0~). The methods for the surgical preparation of the animals, for the manufacture of six-barrelled micropipettes, for the extracellular recording of action potentials, and for the microelectrophoretic application of drugs have been described elsewhere (Bradshaw, Roberts and Szabadi, 1973a; Bradshaw, Szabadi and Roberts, 1973b; Bradshaw, Roberts and Szabadi, 1974). Six-barrelled micropipettes of tip diameter of 3.0 to 5.0 ~tm were used. Two barrels of each micropipette contained 4.0 M NaC1, one for recording action potentials, and the other for current balancing. The remaining barrels contained solutions of drugs. The following drug solutions were used: (-)noradrenaline bitartrate (0.05 M, pH 3.04.0); (-)phenylephrine hydrochloride (0.05 M, pH 5.0 6.5); dopamine hydrochloride (0.05 M, pH 4.0~4.5); acetylcholine chloride (0.05 M, pH 4.5-5.5); phenoxybenzamine hydrochloride (0.01 M, pH 2.5-3.0); haloperidol (0.01 M dissolved in 0.01 M tartaric acid, pH 4.0).
Spontaneously active neurones were studied in the cerebral cortex (stereotaxic co-ordinates, according to K6nig and Klippel (1968): A 4.8 6.5, L 0.9 2.4). The area of recording was prepared as described previously (Bradshaw and Szabadi, 1972), The dura was either incised with a hypodermic needle, or was penetrated directly with the micropipette. All the drugs were applied by microelectrophoresis. When a suitable unit was encountered, the agonists were applied in a regular cycle. Between successive applications of agonists retaining currents of - 10 nA were passed. Retaining currents of - 2 5 nA were used for the antagonists. Intervals between successive applications of the same agonists were kept constant in order to standardize the effects of the retaining currents upon release of the drug during the ejection period (Bradshaw et al., 1973a; Bradshaw et al., 1973b). The relative excitatory potencies of the three amines were evaluated by comparing responses to phenylephrine and noradrenaline, and to phenylephrine and dopamine on the same cells, using methods described elsewhere (Bradshaw et al., 1973b; Bevan, Bradshaw, Pun, Slater and Szabadi, 1978b). In order to compare the time-courses of excitatory responses to the amines, two time-course parameters were used: latency to onset and recovery time (Bradshaw et al., 1973b). These measures were taken on cells on which the two amines compared (i.e. phenylephrine and noradrenaline, and phenylephrine and dopamine) evoked responses of approximately equal size (Bevan et al., 1978b). The efl'ects of the antagonists were studied on cells which yielded consistent excitatory responses to phenylephrine, noradrenaline (or dopamine) and acetylcholine. The methods for conducting antagonism studies have been described in detail elsewhere (Bevan et al., 1977: Bevan et al.,1978a; Bradshaw et al., 1982). Measurement o f the release q[" noradrenaline J?om mieropipettes in vitro
The absolute mobilities of phenylephrine and noradrenaline, and phenylephrine and dopamine, were compared by the method of Bradshaw, Pun, Slater and Szabadi (1981c) (see also Bradshaw et al., 1982). Two experiments were conducted. In Experiment 1, three barrels of each six-barrelled micropipette were filled with 0.05 M [methylene-~4C]noradrenaline bitartrate plus 0.05 M noradrenaline hydrochloride; the remaining three barrels contained 0.05 M [methylene-~4C]noradrenaline bitartrate plus 0.05 M phenylephrine hydrochloride. In Experiment 2, three barrels of each micropipette were filled with 0.05 M [methylene-14C]noradrenaline bitartrate plus 0.05 M dopamine hydrochloride; the remaining three barrels contained 0.05 M [methylene-l~C]noradrenaline bitartrate plus 0.05M phenylephrine hydrochloride: D,L[methylene-~4C]noradrenaline bitartrate was obtained from the Radiochemical Centre, Amersham: the specific activity of the 0.05 M solution was
947
Haloperidol on cortical neurones Table 1. Comparison of time-course parameters of responses to phenylephrine and noradrenaline, and of responses to phenylephrine and dopamine Phenylephrine vs noradrenaline (n = 76) Phenylephrine Noradrenaline Phenylephrine vs dopamine (n = 26) Phenylephrine Dopamine
Latencyt
Recovery time~"
10.26 _+0.96 15.00 + 1.44**
156.24 _+6.88 108.58 _+5.06**
10.84 _+ 1.88 17.70_+3.66"*
175.08 -+-9.32 102.02_+8.01"*
tTime-course parameters are in seconds. Values are means + SEM. Asterisks denote statistically significant differences between values for phenylephrine and noradrenaline, and between values for phenylephrine and dopamine (t-test, paired comparison): *P < 0.02: **P < 0.001.
1.0 mCi/mmol. The pH of the combined solutions were 3.4 (0.05 M noradrenaline bitartrate plus 0.05 M noradrenaline hydrochloride); 3.4 (0.05M noradrenaline bitartrate plus 0.05 M phenylephrine hydrochloride); 3.4 (0.05 M noradrenaline bitartrate plus 0.05 M dopamine hydrochloride). The methods for the collection of samples, for the liquid scintillation counting and for the calculation of transport numbers have been described elsewhere (Bradshaw et al., 1973a; Bradshaw, Pun, Slater and Szabadi, 1981b; Bradshaw et al., 1981c).
RESULTS
Comparison of agonistic e.fjects of phenylephrine and noradrenaline Direction of responses to phenylephrine and noradrenaline. Out of 147 neurones responding to both phenylephrine and noradrenaline, all were excited by phenylephrine. On the other hand, noradrenaline excited 126 neurones (86~,), depressed 17 (11~o), and evoked biphasic responses (depression followed by excitation) on the remaining 4 neurones (3~o).
Apparent potencies of" phenylephrine and noradrenaline. The ejecting currents required to evoke approximately equal responses to phenylephrine and noradrenaline were compared on 73 neurones; smaller currents were required to apply phenylephrine than noradrenaline (t-test, paired comparison, P < 0.01). The sizes of responses evoked by identical ejecting currents were compared on 54 cells; phenylephrine evoked larger responses than did noradrenaline (t-test, paired comparison, P <0.001). Both comparisons indicated a greater potency of phenylephrine, the potency ratio (phenylephrine/ noradrenaline) being 1.230 + 0.066 (mean + SEM) and 1.182 +_ 0.038 (mean + SEM), respectively.
Time-courses of responses to phenylephrine and noradrenaline. Latencies and recovery times of responses to phenylephrine and noradrenaline were measured on 76 neurones yielding responses of approximately equal size to the two amines. The response to phenylephrine had a shorter latency and a longer recovery time than the response to noradrenaline (Table 1).
Comparison of agonistic effects of phenylephrine and dopamine Direction of responses to phenylephrine and dopamine. All the 51 neurones responding to both phenylephrine and dopamine were excited by phenylephrine. Dopamine excited 35 (69~o) of these, depressed 14 (27~) and evoked biphasic responses (depression followed by excitation) on the remaining 2 (4Vo).
Apparent potencies of phenylephrine and dopamine, The ejecting currents required to evoke approximately equal responses to phenylephrine and dopamine were compared on 23 neurones: smaller currents were required to apply phenylephrine than dopamine (t-test, paired comparison, P <0.001). The sizes of responses evoked by identical ejecting currents were compared on 11 neurones: phenylephrine evoked larger responses than did dopamine (t-test, paired comparison, P < 0.05), Both comparisons indicated a greater potency of phenylephrine, the potency ratio (phenylephrine/dopamine) being 2.159+0.452 ( m e a n + S E M ) and 1.191+ 0.077 (mean ± SEM), respectively.
Time-courses of responses to phenylephrine and dopamine. Latencies and recovery times of responses to phenylephrine and dopamine were compared on 26 neurones yielding responses of approximately equal size to the two amines. The response to phenylephrine had a shorter latency and a longer recovery time than the response to dopamine (Table I).
EffEcts of antagonists on excitatory responses to phenylephrine, noradrenaline and dopamine Effects of haloperidol on excitatory responses to phenylephrine and noradrenaline. The effect of haloperidol was examined on 14 neurones. On all the neurones tested haloperidol (5-25 nA) reversibly reduced the sizes of responses to phenylephrine and noradrenaline, while responses to acetylcholine were not affected. On 11 of the neurones haloperidol discriminated between the responses to phenylephrine and noradrenaline, the response to phenylephrine being antagonized to a greater degree than was the response to noradrenaline (Fig. 1). On the remaining 3 neurones responses to phenylephrine and
C . M . BRADSHAWet al.
948 (a)
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Fig. 1. Effect of haloperidol on excitatory responses to phenylephrine (PHE), noradrenaline (NA) and acetylcholine (ACh). Excerpts from the ratemeter recording of the firing rate of a single cortical neurone; ordinate: firing rate (spikes/sec): abscissa; running time (rain). Horizontal bars below the traces indicate microelectrophoretic drug applications; numbers refer to intensities of ejecting current (nA). Numbers above the traces indicate the sizes of the responses (total spike numbers, %), taking the size of the control response to each agonist as 100%. (a) Control responses to the agonists. (b) Responses to the agonists during the continuous application of haloperidol (5 nA). At the start of trace (b), haloperidol had been applied continuously for 7 rain. The response to phenylephrine was antagonized while the responses to noradrenaline and acetylcholine were only slightly reduced in size. (c) Recovery of responses to the agonists 29 min after the application of haloperidol had been terminated. noradrenaline were equally antagonized. The results from all the neurones are summarized in Fig. 4: in the presence of haloperidol, responses to phenylephrine were reduced in size to a greater extent than responses to noradrenaline (t-test, P <0.002); responses to acetylcholine were not significantly affected.
Effect of haloperidol on excitatory responses to (Q)
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was examined on 10 neurones. On all the neurones tested, haloperidol (10-25 nA) reversibly reduced the sizes of responses to dopamine and phenylephrine, while responses to acetylcholine were not affected. On 8 of the neurones haloperidol discriminated between responses to dopamine and phenylephrine, the re-
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Fig. 2. Effect of haloperidol on excitatory responses to dopamine (DA), phenylephrine (PHE) and acetylcholine (ACh). Excerpts from the ratemeter recording of the firing rate of a single cortical neurone (conventions as in Fig. 1). (a) Control responses to the agonists. (b) Responses to the agonists during the continuous application of haloperidol (15 nA). At the start of trace (b), haloperidol had been applied continuously for 47 min. The response to dopamine was anatagonized while the responses to phenylephrine and acetylcholine were not affected. (c) Recovery of the response to dopamine 32 rain after the application of haloperidol had been terminated.
Haloperidol on cortical neurones
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Fig. 3. Effect of phenoxybenzamineon excitatory responses to phenylephrine (PHE), noradrenaline (NA) and acetylcholine (ACh). Excerpts from the ratemeter recording of the firing rate of a single cortical neurone (conventions as in Fig. 1). (a) Control responses to the agonists. (b) Responses to the agonists during the continuous application of phenoxybenzamine by diffusion (removal of the retaining current; 0 nA). At the start of trace (b), phenoxybenzamine had been applied continuously for 37 min. The responses to phenylephrine and noradrenaline were antagonized while the response to acetylcholine was not affected (c) Recovery of the responses to phenylephrine and noradrenaline 18 min after the application of phenoxybenzamine had been terminated.
sponse to dopamine being antagonized to a greater degree than the response to phenylephrine (Fig. 2). On the remaining 2 neurones, haloperidol failed to discriminate between responses to dopamine and phenylephrine. The results from all the neurones are summarized in Fig. 4: in the presence of haloperidol, responses to dopamine were reduced in size to a greater extent than responses to phenylephrine (ttest, P < 0.01); responses to acetylcholine were not significantly affected.
Effect of phenoxybenzamine on excitatory responses to phenylephrine and noradrenaline. The effect of phenoxybenzamine was examined on 11 neurones. Phenoxybenzamine (0~10nA) equally antagonized responses to both phenylephrine and noradrenaline, while responses to acetylcholine were not affected (Fig. 3). The results from all the neurones are summarized in Fig. 4.
Comparison ~1 the mobilities of the agonists in vitro Comparison of phenylephrine and noradrenaline. The effects of noradrenaline hydrochloride and phenylephrine hydrochloride on the release of labelled noradrenaline were compared using 11 micropipettes (Experiment 1, see Methods). In the case of both solutions, the rate of release of [~4C]noradrenaline was linearly related to the intensity of the ejecting current. The linear regression equations fitted by the least squares method were: y = 0.064x + 0.213 ([t4C]noradrenaline bitartrate + noradrenaline hydrochloride) (r = 0.999, P < 0.001), and y = 0.070x - 0.363 ([HC]noradrenaline bitartrate + phenylephrine hydrochloride) (r = 0.999, P < 0.001). In neither case did the intercept deviate
significantly from zero (t-test, P > 0.05, in both cases). The mean apparent transport number ( + SEM) of [t4C]noradrenaline was 0.110 + 0.006 in the presence of 0.05 M "cold" noradrenaline hydrochloride, and 0.101 +_0.004 in the presence of 0.05 M "cold" phenylephrine hydrochloride. The two values are not significantly different from one another (ttest, P > 0.1), indicating that equimolar concentrations of noradrenaline hydrochloride and phenylephrine hydrochloride caused similar reductions in the apparent transport number of [~4C]noradrenaline. This observation would suggest that noradrenaline and phenylephrine have similar absolute mobilities (see Bradshaw et al., 1981c). Comparison of phenylephrine and dopamine. The effects of phenylephrine hydrochloride and dopamine hydrochloride on the release of labelled noradrenaline were compared using 11 micropipettes (Experiment 2, see Methods). In the case of both solutions, the rate of release of [t4C]noradrenaline was linearly related to the intensity of the ejecting current. The linear regression equations fitted by the method of least squares were: y = 0 . 0 8 3 x - 0.304 ([14C]noradrenaline bitartrate + phenylephrine hydrochloride) (r = 0.999, P < 0.005), and y = 0.076x +0.010 ([~4C]noradrenaline bitartrate +dopamine hydrochloride) (r = 0.999, P < 0.001). In neither case did the intercept deviate significantly from zero (ttest, P > 0.05, in both cases). The mean apparent transport number ( + SEM) of [~4C]noradrenaline was 0.123 _+0.008 in the presence of 0.05 M "cold" phenylephrine hydrochloride, and 0.122 + 0.008 in the presence of 0.05 M "cold" dopamine hydrochloride. The two values are not significantly different
950
C . M . BRADSHAWet al. (a) PHE
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that phenylephrine and dopamine have similar absolute mobilities (see Bradshaw et al., 1981c).
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Fig. 4. Summary of effects of haloperidol and phenoxybenzamine on excitatory responses to phenylephrine (PHE), noradrenaline (NA), dopamine (DA) and acetylcholine (ACh). For each agonist, the length of the column represents the mean percentage change from control of the size of the response (total spike number) in the presence of the antagonist; vertical bars indicate SEM. (A) Effect of halopcridol on responses to phenylephrine, noradrenaline and acetylcholine on 14 cells. HaloperidoI significantly antagonized responses to phenylephrine and noradrenaline, but had no significant effect on responses to acetylcholine. Haloperidol had a significantly greater effect on responses to phcnylephrine than on responses to noradrenaline. (B) Effect of haloperidol on responses to phenylephrine, dopamine and acetylcholine on 10 cells. Haloperidol significantly antagonized responses to phenylephrine and dopamine, but had no significant effect on responses to acetylcholine. Haloperidol had a significantly greater effect on responses to dopamine than on responses to phenylephrine. (C) Effect of phenoxybenzamine on responses to phenylephrine, noradrenaline and acetylcholine on 11 cells. Phenoxybenzamine significantly antagonized responses to phenylephrine and noradrenaline, but had no significant effect on responses to acetylcholine. There was no significant difference between the degrees of antagonism of responses to the two amines.
from one another (t-test, P >0.1), indicating that equimolar concentrations of phenylephrine hydrochloride and dopamine hydrochloride caused similar reductions in the apparent transport number of [r4C]noradrenaline. This observation would suggest
In agreement with previous observations from this laboratory (Bevan et al., 1977: Bradshaw et al., 1981b) phenylephrine proved to be a potent excitant of cortical neurones. In fact, all the 396 phenylephrine-sensitive cells which have been studied, described both here and in previous papers (Bevan et al., 1977; Bradshaw et al., 1981b; Bradshaw et al., 1982a; Bradshaw e t a / . , 1982b), responded with excitation to phenylephrine. Since phenylephrine is a selective ~-adrenoceptor agonist (Furchgott, 1972; Besse and Furchgott, 1976; Docherty and McGrath, 1980), the observation of an exclusively excitatory effect of phenylephrine on cortical neurones supports the hypothesis that the activation of c~-adrenoceptors by sympathomimetic amines results in neuronal excitation in the cerebral cortex (Bevan et al., 1977; Szabadi, 1979). When compared on the same cells, phenylephrine appeared to be a more potent excitant then was either noradrenaline or dopamine. This difference in potency is likely to reflect a genuine biological difference between phenylephrine and the other two amines, since there was no significant difference between the electrophoretic mobilities of phenylephrine and noradrenaline, and of phenylephrine and dopamine. There are at least two factors which may contribute to the difference in potency between phenylephrine and noradrenaline. Firstly, the operation of an uptake mechanism for noradrenaline would tend to reduce the concentration of noradrenaline at the cellular receptor sites: this factor would have a smaller effect on the concentration of phenylephrine, since phenylephrine has only a very low affinity for the uptake mechanism (Iversen, 1967). Secondly, the size of the excitatory response to noradrenaline may be reduced by the concomitant activation of inhibitory fl-adrenoceptors (see Bevan et al., 1977; Szabadi, 1978); again, this factor would not make a significant contribution to the size of the response to phenylephrine, since phenylephrine has only a very low affinity for #-adrenoceptors (Furchgott, 1972). The observation that dopamine was a less potent excitant than was phenylephrine is not surprising, since it has been reported that dopamine is a less potent excitant of cortical neurones than noradrenaline (Bevan et al., 1978a). Thus, the order of potency of the three amines in evoking excitatory responses on cortical neurones is as follows: phenylephrine > noradrenaline > dopamine. When the time-course of the excitatory responses to phenylephrine and noradrenaline, and to phenylephrine and dopamine, were compared on the same cells, the responses to phenylephrine appeared to have shorter latencies and longer recovery times than the response to either noradrenaline or dopamine.
Haloperidol on cortical neurones The faster onset of the response to phenylephrine is likely to reflect the greater agonistic potency of phenylephrine (Szabadi and Bradshaw, 1974), and the longer recovery time of the response to phenylephrine is likely to be related to the slower rate of elimination of phenylephrine from the receptor sites due to the absence of operation of an active uptake mechanism for this amine (see above). Haloperidol was able to antagonize excitatory responses both to noradrenaline and phenylephrine. This observation is in agreement with previous findings that excitatory responses both to noradrenaline (Bevan et al., 1978a) and to phenylephrine (Bradshaw et al., 1982a) were susceptible to antagonism by haloperidol and is likely to reflect the e-adrenoceptor blocking property of the neuroleptic drug (Lavin, Hoffman and Lefkowitz, 1981; Atlas, Friedman, Litvin and Steer, 1982). The greater susceptibility to antagonism by haloperidol of responses to phenylephrine than of responses to noradrenaline was, however, an unexpected finding, In fact, it had been predicted that the responses to noradrenaline would be preferentially antagonized, since haloperidol has a higher affinity for dopamine receptors than for ~-adrenoceptors (Peroutka, U'Pritchard, Greenberg and Snyder, 1976: Barone, Corsico, Diena, Restelli, Glasser and Rodenghi, 1982) and the responses to noradrenaline may contain a component resulting from the activation of dopamine receptors (see Introduction). Therefore, it was necessary to examine the possibility that, in contrast to its action in the fish retina (Wafting and Dowling, 1981) and in the rat pituitary gland (Labrie et al., 1979), phenylephrine may stimulate dopamine receptors on cortical neurones. When, however, the effect of haloperidol on the excitatory responses to phenylephrine and dopamine was compared, the response to dopamine was preferentially antagonized, suggesting that the excitatory response to phenylephrine is unlikely to result from the activation of dopamine receptors. It is possible, therefore, that phenylephrine excites cortical neurones via a receptor which is different from the receptors activated by either noradrenaline or dopamine. In agreement with previous findings (Bevan et aL, 1977; Bevan et aL, 1978a; Bradshaw et aL, 1981b), phenoxybenzamine, an e-adrenoceptor blocking agent (Nickerson, 1967), effectively antagonized excitatory responses to both noradrenaline and phenylephrine. Phenoxybenzamine failed to discriminate between the excitatory responses to noradrenaline and phenylephrine, although it can discriminate between responses to noradrenaline and dopamine, the response to noradrenaline being more susceptible to antagonism by phenoxybenzamine than is the response to dopamine (Bevan et al., 1978a). The present experiment with phenoxybenzamine, therefore, does not provide any evidence in favour of a "dopamine component" in the response to noradrenaline (see above).
951
The effects of the antagonists observed in the present experiment and in previous experiments (Bevan et al., 1978a) suggest that the excitatory responses to phenylephrine, noradrenaline and dopamine may be mediated by at least three different receptors. Dopamine is likely to activate mainly specific dopamine receptors which are most sensitive to blockade by haloperidol and least sensitive to blockade by phenoxybenzamine. It is an intriguing possibility that the differential effect of haloperidol on responses to phenylephrine and noradrenaline reflects a dfscrimination between ~ and ~2-adrenoceptors. There is evidence that both et- and cq-adrenoceptors occur in brain tissue (U'Pritchard, Bechtel, Rouot and Snyder, 1979: Weinreich, Chiu, Warsh and Seeman, 1981) and that phenylephrine is a selective cq-adrenoceptor stimulant, whereas noradrenaline has an equal affinity for both types of c~-adrenoceptor (Langer, 1980). Furthermore, it has been reported recently that haloperidol has a significantly greater affinity for ~ - than for e2-adrenoceptors (Lavin et al., 1981; Atlas et al., 1982; Barone et al., 1982). The failure of phenoxybenzamine to discriminate between excitatory responses to phenylephrine and noradrenaline would also be compatible with this interpretation, since phenoxybenzamine shows only partial selectivity for ~t-adrenoceptors (See Langer, 1980). Acknowledgements--This work was supported by the Wellcome Trust and the Science Research Council. M.J.S. is an SRC/CASE Scholar in conjunction with ICI Ltd. We are grateful to Mr D. Praties for technical assistance. REFERENCES
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