THE ROLE OF AFFERENT INPUTS TO SUPRAOPTIC NUCLEUS OXYTOCIN NEURONS DURING NALOXONE-PRECIPITATED MORPHINE WITHDRAWAL IN THE RAT N. P. MURPHY,*² T. ONAKA,*‡ C. H. BROWN§ and G. LENG*  *Laboratory for Neuroendocrinology, Babraham Institute, Cambridge CB2 4AT, U.K. ²Department of Physiology, University Medical School, Teviot Pla ce, Edinburgh EH8 9AG, U.K. Abstract––During prolonged exposure to morphine, oxytocin neurons of the rat supraoptic nucleus develop dependence, shown by hyperexcitation following morphine withdrawal. The present study investigated the role of afferent projections to the supraoptic nucleus in this withdrawal excitation. Rats were made morphine-dependent by continuous intracerebroventricular infusion of morphine at increasing doses (up to 50 μg/h). On the sixth day, rats were anaesthetized with pentobarbitone and morphine withdrawal was precipitated by intraperitoneal injection of naloxone (5 mg/kg). Fos-immunoreactivity in the supraoptic nucleus, and also in the median preoptic nucleus, organum vasculosum of the lamina terminalis and subfornical organ, which project to the supraoptic nucleus, increased following morphine withdrawal. However, retrograde tracing from the supraoptic nucleus showed that, of the neurons in these regions which project to the supraoptic nucleus, only 0.4–7.1% expressed Fos in response to morphine withdrawal. Following morphine withdrawal, Fos-immunoreactivity was present in 39.2% and 19.8% of the tyrosine hydroxylase-immunoreactive neurons of the A1/C1 and A2/C2 cell groups. Of the cells in these regions identified as projecting to the supraoptic nucleus, 11.3% in the region of the A2 cell group and 12.7% in the region of the A1 cell group expressed Fos after morphine withdrawal. In a second study, monoamine release was measured in the supraoptic nucleus of urethane-anaesthetized morphine- dependent and -naı ¨ve rats. Retrodialysis of naloxone (10 5 M) into the supraoptic nucleus induced a small increase in plasma oxytocin concentration in morphine-dependent rats (13.54.8 pg/ml increase) but not in naı ¨ve rats (1.25.9 pg/ml decrease), with no significant change in monoamine release in either morphine-dependent or -naı ¨ve rats. Intravenous injection of naloxone (5 mg/kg) 1 h later produced a further significant increase in plasma oxytocin concentration in morphine-dependent rats concomitant with a significant increase in noradrenaline release from the supraoptic nucleus. Thus, morphine-withdrawal excitation of supraoptic oxytocin neurons occurs concurrently with a modestly increased activity of their input from the brainstem, and very little activation in other known inputs.  1997 IBRO. Published by Elsevier Science Ltd. Key words: opioid, noradrenaline, A2 cell group, median preoptic nucleus, organum vasculosum of the lamina terminalis, subfornical organ. The oxytocin neurons of the supraoptic and para- ventricular nuclei are highly sensitive to inhibition by morphine. 21,23,24 During continuous i.c.v. infusion of morphine, the oxytocin system becomes tolerant to its inhibitory effects, and concurrently develops dependence upon morphine. Acute withdrawal of morphine, by administration of the opiate antagon- ist naloxone, induces a prolonged hyperexcitation of oxytocin neurons leading to hypersecretion of oxytocin. 2 This withdrawal hyperexcitation may reflect an intrinsic hyperexcitability of the oxytocin neuron, or an increased a fferent excitation, or a combination of both. Oxytocin neurons express only low levels of mRNA for the μ-opioid receptor through which morphine acts. 30 Despite this, there are indications that the oxytocin neurons themselves are a primary target for the actions of morphine. However, there are also functionally coupled μ-receptors located presynaptically on a fferent nerve endings within the supraoptic nucleus, 20 and morphine is also known to act at several of the sites of origin of these a fferent nerve endings which possess μ-opioid recep- tors. 15,18,19 Potentially, dependence in the oxytocin system could develop at any or all of these levels. ²Present address: Department of Psychiatry and Biobehav- ioral Sciences, Neuropsychiatric Institute, M.R.L., U.C.L.A., 760 Westwood Plaza, Los Angeles, CA 90024, U.S.A ²Present address: Department of Physiology, Jichi Medical School, Minamikawachi-Machi, Tochigi-Ken, 329-04, Japan. To whom correspondence should be addressed. A bbreviations: CCK, cholecystokinin; DAB, 3,3- diaminobenzidine; FITC, fluorescein isothiocyanate; MnPO, median preoptic nucleus; NTS, nucleus tractus solitarii; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ; TH, tyrosine hydroxylase. Pergamon N euroscience Vol. 80, No. 2, pp. 567–577, 1997 Copyright  1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+ 0.00 PII: S0306-4522(97)00142-5 567