Neuropharma~ologv. 1975, 14. 259 263. PergamonPress, Printed in Gt. Britain.

THE I N F L U E N C E OF RAPHE LESIONS ON THE EFFECT OF M O R P H I N E ON N O C I C E P T I O N AND CORTICAL ACh O U T P U T L. GARAU,M. L. MULASand G. PEPEU*' Department of Pharmacology. School of Pharmacy. Cagliari University. Cagliari 09100. Italy.

(Accepted 20 June 1974)

Summary

The spontaneous release of acetylcholine (ACh) from the cerebral cortex of rats with raphe lesions and sham operated rats was investigated and no differences between the two groups were observed. In rats with a raphe lesion, morphine showed no antinociceptive action ~evaluated by determining the jump threshold to electric shocks) nor did it produce the decrease in the rate of spontaneous release of cortical ACh observed in sham operatcd animals. The possibility that morphine may influence the cortical cholinergic system through neurones originating in the raphe nuclei is envisaged.

(1970) a n d SAMANIN a n d BERNASCONI (1972) d e m o n the midbrain raphe, the antinociceptive effect of morphine was strongly reduced. The lesion of the midbrain raphe brings about a marked depletion in brain 5-hydroxytryptamine (5-HT) (JOUVET, BOBILLER,PUJOL and RENAULD, 1966; KOSTOWSKI, GIACALONE, GARATTINI and VALZELLI, 1968). A relationship between 5-HT depletion and the disappearance of the analgesic effect of morphine was therefore proposed. Such a hypothesis was supported by the finding that the inhibition of 5-HT synthesis also reduces the antinociceptive effect of morphine (TENEN, 1968). Morphine also affects brain cholinergic mechanisms. An increase in brain acetylcholine (ACh) content following morphine administration was observed in the rat (HERKEN, MAIBAUERand MULLER, 1957; GIARMANand PEPEU, 1962; MAYNERT, 1967) and in the mouse (HARRIS, 1970; RICHTERand GOLDSTEIN, 1970), and the possibility of a correlation between analgesia and levels of ACh was examined (HOwES, HARRIS, DEWEY and VOYDA, 1969). Although no clear relationship was found, all the findings suggested that a cholinergic mechanism was involved in the analgesia. Morphine brings about a decrease in ACh output from the cerebral cortex in unanaesthetized rabbits (BEANI, BIANCHI, SANTINOCETOand MARCHETTI, 1968), in anaesthetized cats (MITCFIELL, 1963) and in unanaesthetized rats transected at midpointine level (MATTHEWS, LABRECQUEand DOMINO, 1973). Narcotic antagonists inhibit both the increase in ACh content (HowEs et al. 1969) and the decrease in ACh output from the cerebral cortex (JHAMANDAS,PHILLISand PINSKY, 1971; JHAMANDASand SUTAK~1974). The purpose of this study was to investigate whether the lesion of the midbrain raphe abolishes not only the antinociceptive effect but also the depressant effect of morphine on ACh output from the cerebral cortex. SAMANIN, GUMULKA a n d VALZELLI s t r a t e d that, in rats with a lesion of

METHODS

Wistar male rats. weighing 15(~200 g before the lesions were made, were used. Under pentobarbital anaesthesia (40 mg/kg i.p.) the rats were placed in a stereotaxic apparatus (Stoelting Co. Chicago). The lesions in the midbrain raphe were made by electrocoagulation with a Grass L 4 Model delivering currents of 3 5 mA. The electrode was a fine stainless steel needle insulated with epoxylite, except for a 0.5-1 mm portion behind the tip, and was positioned according to coordinates from the KON1G and KLIPPEL(1963) stereotaxic atlas of the rat brain. * Present address: Istituto di Farmacologia, Universita degli Studi, Viale G.B. Morgagni, 65, 65-50134 Firenze. Italy. 259

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L. GARAU,M. L. MULASand G. PEPEU

Seven days after the operation, the antinociceptive effect of morphine was tested by determining the jump threshold to electric shocks delivered through an electrified floor grid according to the procedure described by EVANS (1961). Not less than a week after the evaluation of the nociceptlve threshold, ACh release from the cerebral cortex was investigated. The method of MITCHELL (1963) adapted for the rat by HEMSWORTH and NEAL (1968) was used. The rats were anaesthetized with urethane (1 g/kg, i.p.) and placed in a stereotaxic apparatus. The skull was opened, the dura removed and a small Perspex cylinder covering a surface of 0"20 cm 2 and containing 0"5 ml of eserinized Ringer was placed on the exposed cortex. Every 20 rain the solution was removed from the cylinder and immediately bioassayed for ACh on the dorsal muscle of the leech as described by BARTOLINI and PEPEU (1967). The body temperature of the rats was carefully maintained at 37 _+ 0"2°C throughout the experiments by means of an electric lamp. At the end of the collecting periods, the placement and extent of lesions was verified in each rat. The photographic method of SKINNER (1971) on frozen brain sections 100/~m thick was used. 1

2

5

3

4

6

Fig. 1. Schemating drawing of a frontal section of the brainstem in A 160/zm, according to the coordinates taken from the stereotaxic atlas of KONIG and KLIPPEt.(I963), showing the placement and size of the raphe lesions in seven rats. RESULTS

Figure 1 shows the placement and the size of the lesion in seven rats, in which morphine exerted no antinociceptive effect. In rats 1, 3, 6 and 7 not only the median but also the dorsal raphe nucleus was largely damaged. In rats 2, 4 and 5 the median nucleus was destroyed and the dorsal was partly damaged. In all seven rats the decussatio peduncoli cerebellaris superiores was also damaged. In rats 1 and 4 there was some damage to the reticular formation. After the operation, the rats showed a moderate hypermotility. In 16 rats, histological check showed an incorrect placement of the lesions which was either too lateral or too rostral. In the first case the peduncoli cerebellaris superiores and the reticular formation were partly damaged; in the second, the tractus tectus spinalis was also involved. The rats with an incorrectly placed lesion showed no gross behavioural changes and were considered as sham operated animals in the present investigation. In Table 1, the effect of morphine on the jump threshold is reported. It can be seen that morphine brought about a significant increase in the jump threshold of the unoperated and sham operated rats. The rats with the raphe lesion showed no increase in the jump threshold. The effect of morphine on cortical ACh output in sham operated rats and in rats with a raphe lesion is shown in Figure 2. Figure 2A shows how the administration of morphine to sham operated rats was followed by a decrease in ACh output. The time course of the decrease in ACh output was

Raphe lesions and effect of morphine

261

Table 1. Effect of morphine (10 mg/kg, s.c.) on the jump threshold in lesioned and control rats Jump threshold Mean ___ S.E. in mA before drug after drug

No. of rats

Conditions Unoperated control Sham operated Raphe lesions

P

6

2'2 _+ 0"1

5'8 _+ 0'3

The influence of raphe lesions on the effect of morphine on nociception and cortical ACh output.

Neuropharma~ologv. 1975, 14. 259 263. PergamonPress, Printed in Gt. Britain. THE I N F L U E N C E OF RAPHE LESIONS ON THE EFFECT OF M O R P H I N E...
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