Neuronal cholecystokinin release: morphine and naloxone effects on rat periaqueductal grey

Abstract

The periaqueductal grey area (PAG) of rat brain is strongly indicated as a major site of opiateand stimulation-induced analgesia. The PAG is thought to integrate perception and response to nociceptive stimuli; afferent projections include major imputs from limbic and sensory areas; efferent projections include those to the spinal cord and nucleus raphe magnus (Beitz, 1982). Apart from its content of opioid peptides, the PAG also contains cholecystokinin octapeptide (CCK), which may be involved in processing of nociceptive stimuli since it reverses morphine-, /I-endorphinand footshock-induced analgesia (Faris et al., 1983). Furthermore, at higher doses CCK is an antinociceptive agent in its own right (Jurna & Zetler, 1981; Sheehan & de Belleroche, 1984). A further indication of a possible interaction between CCK and opiate analgesia systems at the level of the PAG is the finding that the firing of CCK neurons in the EdingerWestphal nucleus of the PAG is reduced by noxious stimuli or intravenous morphine (Innis & Aghajanian, 1985). The following experiments were designed to ascertain whether there is a direct action of opiates on CCKcontaining neurons in the PAG as measured by CCK release. Male CFY rats were killed, their brains removed and the PAG dissected out and chopped (tissue slices 0.3mm thick). The slices were incubated at 37°C in Krebs-bicarbonate medium, pH 7.4, containing 0.5 mMbacitracin and gassed with 95% 0 , / 5 % CO,. After one 10 min preincubation the slices were sequentially transferred to drug-containing medium for a further IOmin, to evaluate basal release and then, for another 5 min, to a third chamber containing the drug plus high K + ion concentration (34 mM) to evoke neurotransmitter release. After incubation, tissue slices were placed in 90% CH30H/IO% H,O, O'C, and sonicated to extract low molecular weight forms of CCK. The methanolic extracts were dried under nitrogen and rehydrated before assay. The supernatants from the incubation vials were frozen until used for assay. Tissue and released levels of CCK were measured by radioimmunoassay with antisera Ab2717 (a generous gift from Professor J. Rehfeld) against monoiodinated human gastrin (2-17) as a tracer. Tissue slices were exposed to morphine hydrochloride ( 10-'0-10-' M) or naloxone hydrochloride ( l o ' 1 0 6 ~ ) . Two Caz+ ion concentrations were used for the experiments with morphine, 0.6 mM or 2.5 mM, to evaluate whether any morphine action was Ca2+ dependent.