Peptides, Vol. 12, pp. 1235-1238. © Pergamon Pressplc, 1991.Printed in the U.S.A. 0196-9781/91 $3.00 + .00 Naloxone Blocks the Release of Opioid Peptides in Periaqueductal Gray and N. Accumbens Induced by Intra-Amygdaloid Injection of Morphine Q. P. MA AND J. S. HAN Department of Physiology, Beijing Medical University, Beijing 100083, China Received 8 April 1991 MA, Q. P. AND J. S. HAN. Naloxone blocks the release of opioid peptides in periaqueductal gray and N. accumbens induced by intra-amygdaloid injection of morphine. PEPTIDES 12(6) 1235-1238, 1991.--The working hypothesis that the periaqueductal gray (PAG), N. accumbens and amygdala were connected serially in a unidirectionalloop for antinociception,in which Met- enkephalin and 13-endorphin were considered to be two important analgesic neurotransmitters,was examined by simultaneously perfusing the PAG and N. accumbensafter microinjection of morphine into the amygdala. Intra-amygdaloid injectionof morphine increased the release of enkephalinsand 13-endorphin in the PAG and N. accumbens.When the perfusionfluid contained 3 IxM of naloxone, the release of enkephalins and 13-endorphin was reduced in both the PAG and the N. accumbens.These results do not support the hypothesis of a unidirectional loop and its putative sequence. Opioid peptides Periaqueductal gray Nucleus accumbens Amygdala Morphine THE implication of central opioid peptides in the periaqueductal gray (PAG), nucleus accumbens and amygdala in mediating morphine analgesia has been well documented (7, 12, 19, 23, 25, 27). A unidirectional loop consisting of the three nuclei and others, with enkephalins (ENK) and [3-endorphin (13-EP) as im- portant analgesic neurotransmitters to be involved in pain modu- lation, has been postulated to explain the roles played by the three nuclei (9-11). Neurochemical studies recently conducted in this laboratory have confirmed that microinjection of morphine into the amygdala could increase the release of enkephalins and 13-endorphin in the PAG, and microinjection of morphine into the PAG could increase the release of ENK and [3-EP in the N. accumbens (14,15). The aim of the present study was to further examine the putative sequence (e.g., PAG, N. accumbens and amygdala) of the three nuclei in the induction of opioid peptide release in the PAG and N. accumbens by intra-amygdaloid in- jection of morphine. METHOD Animal Preparation Male rabbits, weighing 2.0°°3.0 kg, were anesthetized with pentobarbital (30 mg/kg) and implanted stereotaxically with 6 stainless steel cannulae directed to the three nuclei, PAG (P 9.5, L 1.0, H 12.5-13.0), N. accumbens (A 5.0-6.0, L 1.2, H 10.5-11.0) and amygdala (AP 0--A 1, L 5.5--6.0, H 15.00015.5) in both sides according to Sawyer et al. (20). The cannulae for injection were of 0.8 mm outer diameter with the lower end lo- cated 2.0 mm dorsal to the site of injection in the amygdala. The cannulae for perfusion were of 0.9 mm outer diameter with their lower end reaching the PAG or N. accumbens. Intracranial surgery was performed one week before the perfusion experi- ment. The animals were conscious and restrained in hammocks with eyes covered with blinders during the experiment. Intracerebral injection was performed through an injection tube of 0.4 mm outer diameter extending 2.0 mm beyond the tip of the cannula, the injection volume being 1 p,1 to be finished within 8 min via a slow injection apparatus (Palmer). Cerebral perfusion was per- formed by inserting an inner tube into the cannula, and protrud- ing beyond the cannula tip for 0.5 mm. Two peristaltic pumps were used for push-pull perfusion: one to push 37°(2 artificial cerebrospinal fluid (CSF) into the brain at a rate of 100 p,l/min, and another to pull the fluid synchronously (18). The perfusate was collected in tubes containing 200 pL1 in HC1. The outflow hose and the tube collecting perfusate were kept cold by ice wa- ter. At the beginning of the experiment, morphine (10 Ixg/Ixl or 20 Ixg/~l) or normal saline (NS) 1 Ixl was injected into the amygdala. At the same time, the PAG and N. accumbens were perfused simultaneously with artificial CSF or artificial CSF containing 3 IxM naloxone (i.e., naloxone was carried by perfu- sion fluid into the perfused nucleus). After a time lag of 20 min, the collection of perfusate was started and continued for 30 min. The perfusates were lyophilized and kept at -20°(2. Chemicals Used in the Experiment Morphine chloride was produced by Qinghai Drug Company, China. Leu-Enkephalin (LEK) and bacitracin were products of 1235