Actions and interactions of narcotic agonists and antagonists on brain stem neurones

Brain Research, 73 (1974) 167-170 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

167

Actions and interactions of narcotic agorfists and antagonists on brain stem neurones

G. J. BRAMWELL AND P. B. BRADLEY Department of Pharmacology (Preclinical), The Medical School, Birmingham, B15 2TJ (Great Britain)

(Accepted February 27th, 1974)

It has recently been demonstrated that spontaneously active neurones in the brain stem of urethane-anaesthetized rats are sensitive to iontophoretically applied morphine 4,5. Some of these neurones were excited (33/76) by morphine, applied with currents of 10-30 nA for 0.5-10.5 rain, and others were depressed (17/76). Other studies have shown that brain stem neurones responding to iontophoretically applied morphine are scattered in the region of the hypoglossal nerve 3, in an area containing cells sensitive to nociceptive stimuli 2. It was therefore decided to study more fully the responses of neurones in this region of the brain stem to morphine, and in particular, the relative sensitivity of morphine-induced neuronal excitation and depression to antagonism by the narcotic antagonists, nalorphine and naloxone. Male albino rats (Sprague-Dawley) were set up as previously described 3,4. Multibarrelled glass micropipettes were used to record neuronal activity and to eject drug ions from solutions of their salts; acetylcholine chloride (5 ~o, pH 4-5, Sigma); 5-hydroxytryptamine (5-HT) bimaleinate (5 ~ , pH 4, Koch-Light); morphine hydrochloride (1 ~o, pH 4.8, Macfarlan Smith Ltd.); nalorphine hydrochloride (1 ~ , pH 4.5, Burroughs Wellcome); naloxone hydrochloride (l ~, pH 4.5, Endo Laboratories). Only spontaneously active neurones were studied, many of which were identified histologically. Four different types of response were distinguished when morphine was applied to 99 brain stem neurones for periods of 60-120 sec with a current of 50 nA: excitation which developed slowly on switching on the applying current, but which recovered within 30 sec of terminating the application (38/99); slowly developing excitation which was followed by depression after the current was switched off (5/99); shortlasting depression which was difficult to distinguish from current artefacts (4/99); and long-lasting depression which developed slowly and outlasted the period of application by several minutes (18/99). In general morphine brought about these alterations in neuronal firing rate without affecting spike height or spike shape. Occasionally, however, excitation was accompanied by a reduction in spike height, due to overdepolarization of the cell, by prolonged iontophoretic applications. Where such changes occurred the results have not been included.

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superimposed on the spontaneous firing of a brain stem neurone. A 90 sec application of morphine (50 nA), which was insufficient to cause a significant increase in the firing rate, potentiated a subsequent application of acetylcholine. Control responsiveness to ACh was restored within 3 min of terminating the morphine application. The majority of cells excited by morphine were also excited by 5-HT (12/17) and ACh (34/36), and none were depressed by 5-HT or ACh, Similarly, the majority of cells responding to morphine with long-lasting depression were excited by 5-HT (10/! 1) and ACh (10/13), though two cells were depressed by ACh. Cells which responded to morphine by initial excitation followed by depression, responded to 5-HT and ACh in a similar way to cells which were excited, whereas cells exhibiting shortlasting depression responded to these two compounds inconsistently. Potentiation by morphine of ACh excitation was often observed (17/32) (Fig. 1), though antagonism of ACh excitation and potentiation or antagonism of 5-HT excitation or depression were not observed. Tachyphylaxis to the excitatory effects of morphine was observed on only 8 out of 23 occasions that morphine was applied more than 3 times to the same cell; in general the latency of onset decreased and the magnitude of the response increased with successive applications. Nalorphine failed to antagonise morphine excitation specifically (n ~ 4) when applied for 60-120 sec, prior to a morphine application, though on one occasion, both morphine and ACh excitations were antagonised. In fact, when applied together with morphine, clear potentiation of the excitatory effect was observed (n -- 3). Nalorphine did, however, antagonise long-lasting depression (5/6). However, it sometimes produced depressant effects o f its own (8/24), which were in some cases (3/8) qualitatively similar to responses to morphine. These depressant effects of nalorphine were all short-lasting and, like the corresponding short-lasting morphine depressions, were difficult to confirm. Naloxone was applied to 23 brain stem neurones for periods of 60-120 sec with

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Fig. 2. Antagonism of morphine depression by naloxone. Morphine (50 nA) produced initial excitation, which was followed by depression when the applying current was switched off. ACh (25 nA) still caused excitation during the period of depression. Naloxone (50 nA) reversed the depresion and prevented its reoccurrence when morphine was reapplied 10 rain later. a current of 50 nA. In contrast with nalorphine, naloxone excited some cells (2/23) and depressed others (3/23). Specific antagonism o f morphine excitation was not observed (n ---- 11), though antagonism o f both morphine and ACh excitations was observed on one occasion. Specific antagonism of long-lasting depression (3/5) and of depression following excitation (2/2) was observed (Fig. 2). These results indicate that at least 3 different types of response to iontophoretically applied morphine can be distinguished in a part o f the brain implicated in analgesia as well as the dependence-producing action of morphineT,12; simple excitation, initial excitation followed by depression and long-lasting depression. Without performing more rigorous controls for current effects, including the use o f current balancing, the possibility o f short-lasting depressions as well cannot be confirmed. However, only simple excitation and long-lasting depression by iontophoretically applied morphine have previously been reported for this region of the brain 4,5. Furthermore, the pharmacology of morphine excitations and depressions appear to differ. Thus, only the depressions were antagonised by narcotic antagonists (10/13), whereas definite agonist or facilitatory effects were exerted by nalorphine and naloxone at the locus for the excitatory effects of morphine. Further experiments are at present being carried out in order to clarify the pharmacology of these morphine excitations. The possibility cannot be ruled out that insufficient antagonist was released from the micropipettes with the currents routinely employed (50 nA for 90-120 sec) to antagonise the sometimes marked excitatory effects o f morphine.

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The lower p r o p o r t i o n o f t a c h y p h y l a x i s t o w a r d s excitatory effects o f m o r p h i n e observed in the present study (35 ~ ) c o m p a r e d with t h a t previously quoted (77 ~ ) , can be explained in terms o f the m u c h longer a p p l i c a t i o n s and smaller currents (10-30 n A for u p to 10.5 min) e m p l o y e d previously. A l t h o u g h the present findings confirm the previously r e p o r t e d lack o f correlation between responses o f brain stem neurones to m o r p h i n e a n d the responses to 5 - H T a n d A C h (see refs. 4, 5), the possibility t h a t such a c o r r e l a t i o n exists c a n n o t be disc o u n t e d a l t o g e t h e r in view o f recent reports 1,6,8-1~. Thus the great m a j o r i t y o f cells excited by m o r p h i n e were also excited by 5 - H T a n d A C h in this and in previous studies4, 5. This w o r k was s u p p o r t e d by a g r a n t f r o m the M e d i c a l Research Council. The technical assistance o f Miss Elaine W a l k e r was much appreciated.

1 AKIL, H., AND MAYER, D. J., Antagonism of stimulation-produced analgesia by p-CPA, a sero-

tonin synthesis inhibitor, Brain Research, 44 (1972) 692-697. 2 BENJAMIN, R. M., Single neurons in the rat medulla responsive to nociceptive stimulation, Brain

Research, 24 (1970) 525-529. 3 BOAKES,R. J., BRAMWELL,G. J., BRIGGS,I., CANDY,J. M., AND TEMPESTA,E., Localisation with pontamine sky blue of neurones in the brain stem responding to microiontophoretically applied compounds, Neuropharmacology, (1974), in press. 4 BRADLEY, P. B., AND DRAY, A., Actions and interactions of microiontophoretically applied morphine with transmitter substances on brain stem neurones, Brit. J. Pharmacol., 47 (1973) 642P. 5 BRADLEY, P. B., AND DRAY, A., Morphine and neurotransmitter substances: microiontophoretic study in the rat brain stem, Brit. J. Pharmacol., 50 0974) 47-55. 6 DOMINO, E. F., AND WILSON, A. E., Enhanced utilization of brain acetylcholine during morphine withdrawal in the rat, Nature (Lond.), 243 (1973) 285-286. 7 HERE, A., TESCHEMACHER,HJ., ALBUS, K., AND ZIEGLG.~NSBERGER, W., Caudal brain stem structures mediating the precipitated morphine abstinence syndrome, Proc. X X V Int. Physiol., Cong., (1971) 246. 8 MATTHEWS, J. D., LABRECQUE, G., AND DOMINO, E. F., Effects of morphine, nalorphine and naloxone on neocortical release of acetylcholine in the rat, Psychopharmacologia (Berl.), 29 (1973) 113-120. 9 MULLIN, W. J., PHILLIS, J. W., AND PINSKY, C., Morphine enhancement of acetylcholine release from the brain in unanaesthetized cats, Europ. J. Pharmacol., 22 0973) 117-119. l0 PINSKY, C., FREDRICKSON, R. C. A., AND VAZQUEZ, A. J., Morphine withdrawal syndrome. Responses to cholinergic antagonists and to a partial cholinergic agonist, Nature (Lond.), 242 (1973) 59-60. I l SCHULZ, R., ANDGOLDSTEIN,A., Morphine tolerance and supersensitivity to 5-hydroxytryptamine in the mesenteric plexus of the guinea pig, Nature (Loner.), 244 (1973) 168-169. 12 TESCHEMACHER, HJ., SCHUBERT, P., AND HERZ, A., Autoradiographic studies concerning the supraspinal site of the antinociceptive action of morphine when inhibiting the hindleg flexor reflex in rabbits, Neuropharmacology, 12 (1973) 123-131.