Involvement of medullary A2 noradrenergic neurons in the activation of oxytocin neurons after conditioned fear stimuli Lingling Zhu and Tatsushi Onaka Department of Physiology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, Japan 329-0498 Keywords: footshocks, Fos, freezing behaviour, rat, retrograde tracers Abstract Fear-related stimuli activate oxytocin neurons in the hypothalamus and facilitate oxytocin release from the pituitary. Oxytocin neurons in the supraoptic nucleus receive direct noradrenergic innervations from the A1 and A2 cell groups in the medulla oblongata. In the present study, we investigated the role of hypothalamic-projecting noradrenergic neurons in controlling oxytocin cell activity following fear-related stimuli in rats. An unconditioned fear stimulus (intermittently applied footshock) or conditioned fear stimulus induced expression of Fos protein, a protein product of an immediate-early gene, in magnocellular oxytocin neurons in the supraoptic or paraventricular nucleus. A neurotoxin, 5-amino-2,4-dihydroxy-a-methylphenylethylamine, microinjected into the vicinity of the supraoptic nucleus, selectively depleted the noradrenaline contents of the nucleus and blocked the Fos expression in the supraoptic nucleus after the unconditioned or conditioned fear stimulus. In the medulla oblongata, the unconditioned fear stimulus induced expression of Fos protein in both A2/C2 and A1/C1 catecholaminergic neurons. On the other hand, the conditioned fear stimulus induced expression of Fos protein preferentially in the A2/C2 neurons. Furthermore, the unconditioned fear stimulus induced Fos expression in the A1/C1 and A2/C2 catecholaminergic neurons labelled with retrograde tracers previously injected into the supraoptic nucleus. The conditioned fear stimulus induced Fos expression preferentially in the A2/C2 catecholaminergic neurons labelled with the retrograde tracers. These data suggest that the conditioned fear-induced oxytocin cell activity is mediated by the A2 noradrenergic neurons projecting to oxytocin neurons, while the unconditioned fear response is mediated by both A2 and A1 noradrenergic neurons. Introduction In a state of fear, a multifaceted defence response, involving behavioural, autonomic nervous and neuroendocrine system changes is elicited (Fendt & Fanselow, 1999). Fear can be provoked in an innate or learned way. Experimentally, innate fear can be induced by noxious stimuli. Learned or conditioned fear can be produced by Pavlovian conditioning, where a subject is trained to associate a neutral stimulus with an aversive unconditioned stimulus, such as an electric footshock. After pairing a neutral environmental stimulus with aversive shocks, the environmental stimulus alone turns out to predict the occurrence of the shocks and acts as a conditioned stimulus, eliciting behavioural and neuroendocrine responses. A conditioned fear stimulus facilitates oxytocin release from the pituitary (Onaka & Yagi, 1990). Oxytocin is synthesized by magnocellular supraoptic and paraventricular neurosecretory neurons in the hypothalamus, transported to the axonal terminals in the posterior pituitary and released to the blood. The magnocellular oxytocin neurons receive noradrenergic innervation, which arises mainly from the A1 noradrenergic neurons in the ventrolateral medulla, and from the A2 noradrenergic neurons in the dorsomedial medulla (Sawchenko & Swanson, 1982). Stimulation of the A1/A2 regions or administration of an a 1 -adrenoreceptor agonist activates oxytocin neurons (Leng et al., 1999). Stressful stimuli, including emotional stress, activate noradrenergic neurons and facilitate noradrenaline release in several brain regions, especially in the hypothalamus and amygdala (Tanaka et al., 2000). Oxytocin release in response to fear-related stimuli is blocked by noradrenaline depletion or administration of an a 1 -adrenoceptor antagonist (Onaka, 2000). Thus, it is possible that noradrenergic neurons in the medullary A1/A2 regions mediate oxytocin release in response to fear-related stimuli in rats. However, it is not known whether direct noradrenergic projections to the hypothalamus are essential for the oxytocin cell activity after fear-related stimuli, or which medullary noradrenergic cell groups are responsible for oxytocin cell activity in response to fear-related stimuli. In the present study, we ®rst examined the expression of Fos protein in the medulla oblongata after fear-related stimuli in rats in order to investigate whether these noradrenergic neurons are activated in response to fear stimuli. Fos protein is an immediate- early gene c-fos product and at least in some systems has been proven to be a marker of neuronal activation (Sagar et al., 1988; Ceccatelli et al., 1989). Secondly, we examined effects of selective destruction of noradrenergic ®bers projecting to the supraoptic nucleus upon the expression of Fos protein in the supraoptic nucleus, in order to determine whether the noradrenergic projections to the oxytocin neurons mediate activation of oxytocin neurons in response to fear stimuli. Finally, we examined the expression of Fos protein in the brainstem catecholaminergic cells in rats, which had been injected Correspondence: Dr T. Onaka, as above. E-mail: tonaka@jichi.ac.jp Received 8 February 2002, revised 13 September 2002, accepted 16 September 2002 doi:10.1046/j.1460-9568.2002.02285.x European Journal of Neuroscience, Vol. 16, pp. 2186±2198, 2002 ã Federation of European Neuroscience Societies