Role of central oxytocin in the control of the milk ejection reflex

Brain Research Bulletin, Vol. 20, pp. 737-141. @Pergamon Press plc, 1988.printed in the U.S.A.

0361-9230/88$3.00 + .OO

Role of Central Oxytocin in the Control of the Milk Ejection Reflex MARIE-JOSE

FRE~ND-~ERCIER,~ FRANCOISE MOOS,? DOMINIQUE A. POULAIN,* PHILIPPE RICHARD, I- FLOREAL RODRIGUEZ, * DIONYSIA T. THEODOSIS* AND JEAN-DIDIER VINCENT*’

“Unite’ de Neurobiologie des Comportements, INSERM U 176, Bordeaux and ?Lahoratoire de Physiologic, UA 309 CNRS, Strasbourg, France

FREUND-MERCIER, M.-J., F. MOOS, D. A. POULAIN, P. RICHARD, F. RODRIGUEZ, D. T. THEODOSIS AND J.-D. VINCENT. Role of central oxytocin in the control of the milk ejection rejlex. BRAIN RES BULL 20(6) 737-741, 1988.-‘The neuropeptide oxytocin, synthetized by magnocellular neurons in the hypothalamus, is well known for its peripheral action after it is released into the bioodstream from axons in the neurohypophysis. Less familiar is the notion that it is also released centrally to control the activity of oxytociuergic neurons themselves. When injected into the third ventricle of Iactating rats during suckling, oxytocin increases the basal firing rate of oxytocinergic neurons as well as their activity at the time of each reflex milk ejection. On the other hand, centrally administered oxytocin engenders the neuronal-glial and synaptic plasticity characteristic of the oxytocin system when it is physiologically activated. From numerous in vivo and in vitro observations, it appears that central oxytocin is released in the hypothalamic nuclei themselves. For example, the use of push-pull cannulae inserted into one supraoptic nucleus of suckled rats shows that oxytocin is released inside the nucleus specifically during milk ejection. Moreover, ultrastructural immunocytochemistry reveals synaptic terminals in the supraoptic nucleus where both the pre- and postsynaptic elements are oxytocinergic. Nevertheless, the mech~ism of the central release of the neuropeptide has still to be determined, especially in view of electrophysiological observations indicating that the release process in the hypothalamus is different from that within the neurohypophysis. Central release Oxytocin Milk ejection reflex

Supraoptic nucleus

Paraventricular

OXY’l’OCIN, as many other neuropeptides synthetized by the brain, shows both peripheral and central actions (see [201). In addition to its well documented systemic effects, on the mammary gland, to induce milk ejection during lactation, and on the uterus, to bring on uterine contraction during parturition, it appears that oxytocin can also affect various functions of the central nervous system, as, for example, memory and maternal behaviour (see [4,71). Furthermore, as will be described in this review, there are now numerous electrophysiological and anatomical observations that suggest that oxytocin may act centrally to control its own central and peripheral release, at least during lactation. During lactation and parturition, oxytocin neurons display a characteristic intermittent synchronized electrical activity which permits pulsatile release of hormone from the neurohypophysis (see [131). At the same time, the oxytocinergic system undergoes important morphological changes that modify the relationship of its neurones to adjacent glial cells and their synaptic input (1191, see also Mon-

nucleus

Structural placticity

tagnese et af., this volume). Two lines of evidence suggest that central oxytocin may play an important role in these phenomena. On the one hand, when oxytocin is injected into the third ventricle of lactating rats, it has a strong facilitatory effect on the electrical activity of oxytocin neurones and thus, on the pulsatile release of hormone into the general circulation [.5&l. On the other hand, prolonged intracerebroventricular infusions of oxytocin in nonlactating, nongestating female rats induces the structural reorganization characteristic of the oxytocinergic system when it is physiologically activated [18]. These observations would strongly suggest, therefore, that a central neuropeptide can induce both anatomical and electrophysiological effects in the adult mammalian brain, Nevertheless, before we can conclude that oxytocin, at the level of the hypothalamus, truly affects the anatomy and electrical activity of the very neurons responsible for its secretion, we still need to determine its central source and mechanism of release. We here describe certain observa-

‘Requests for reprints should be addressed to Dr. J. D. Vincent, U I76 INSERM, Domaine de Carreire, 1 rue Camille Saint-Saens, Bordeaux Cbdex, France.

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FIG. 1. Effect of injection of oxytocin into the third ventricle. Simultaneous recording of the intram~mary pressure and rate of action potential discharge from one oxytocin cell in an anesthetized lactating rat during suckling. Prior to injection, suckling induced jnte~ittent activation of oxytocin cells and milk ejections; the number of spikes in the high frequency discahrge was 41, and the rise in intramammary pressure low. Following injection, the firing rate and total number of spikes (up to 84) in the high frequency discharge increased, as well as the amplitude of intramammary pressure, effects which became attenuated with time. A second injection reactivated the system. Numbers at the bottom show intervals between bursts in minutes. Adapted from 161. from in vitro and in vivo experiments may provide some answers to these questions.

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During suckling, oxytocin neurons evolve a synchronized electrical activity (high frequency discharge of action potentials) which permits a pulsatile release of hormone into the general circulation resulting in milk ejection 11, 13, 211. When oxytocin is injected into the third ventricle of lactating rats, it has a strong facilitatory effect on this neuronal activation [5&l. indeed, injections of 10 pg to 10 ng oxytocin into the third ventricle of suckied rats si~i~cantly augmented the number of spikes during each high frequency discharge prior to milk ejection (Fig. 1) and the frequency of reflex milk ejections (Fig. 2); these effects were dosedependent. Such injections were also capable of triggering the milk ejection reflex when it had failed to appear normally. In addition, such injections signi~cantly increased the background activity of slow firing oxytocin neurons but had no effect on fast firing oxytocin neurons or on nonneurosecretory neurons (Fig. 2). Injection of mesotocin or isotocin into the third ventricle had also a facilitatory effect on the milk ejection reflex. In contrast, vasopressin, vasotocin, MIF I (the pro-leu-gly-NH, terminal triplet of oxytocin) or neurophysin did not modify high frequency discharges or the milk ejection pattern [61. Central Oxytocin and the Anatomy System

oj' the Oxytacinergic

During lactation, glial coverage of oxytocinergic neurons in the supraoptic (SON) and paraventricular nuclei diminishes, so that large portions of their surface membrane

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FIG. 2. Top: Effect of intracerebroventricular injection of oxytocin (I ng) on the background electrical activity of oxytocin cells with a low basal firing rate in suckled rats. Immediately after injection, note the increase in basal tiring rate. Nonneurosecretory cells remained unresponsive. Bottom: Effect of central oxytocin on the intervals between successive milk ejections during suckling. Before oxytocin injection, milk ejection occurred every 14 min: oxytocin considerably reduced the following intervals. Oxytocin thus increases both the intensity of the high frequency discharges, and the frequency of milk ejections. Adapted from [61.

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nuclei in vitro. After dissection of the hypothalamus, magnocellular nuclei were incubated for 10 min in the absence (hatched bars) or presence (open bars) of increasing doses of exogenous oxytocin. Note the dose-dependent effect of oxytocin on its own release (value expressed as % of tissue content in hormone). Adapted from [IO].

become directly juxtaposed. Synaptic remodelling also associates pairs of oxytocinergic neurons through the formation of common presynaptic terminals. These structural changes, reversible after weaning, may be important functionally in facilitating the synchronization of neuronal firing noted at lactation and parturition. As described at length in the companion article by Montagnese et al. in this volume, infusion of oxytocin into the third ventricle continuously for 8 days, at doses similar to those that modify the electrical activity of the neurons 161, leads to the same structural changes as those seen during pa~urition and lactation [Igl. As for the electrophysiological effects, the anatomical consequences were also very specific to oxytocin since the hypothalamic magnocellular nuclei remained unmodified after similar infusions of cerebrospinal fluid alone or vasopressin. They did change on infusion of the synthetic peptide, 4-threonine oxytocin, which is closely analogous physiologically to oxytocin 1151.It appears, therefore, that both functional activation and structural remodelling in female rats can be specifically induced by central oxytocin or its analogues. II-ORIGIN

OF THE ENDOGENOUS OXYTOCIN ACTING ON THE OXYTOCINERGIC SYSTEM

A peripheral origin for the oxytocin present in the central nervous system can be excluded since oxytocin, as most other neuropeptides, does not cross the blood-brain barrier. Moreover, intravenous injections of large doses of oxytocin are without effect on the electrical activity of oxytocinergic

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FIG. 4. Release of oxytocin within the SON of a suckled lactating rat in vivo. A push-pull cannula inserted into the SON was used to perfuse the nucleus, before and during suckling and milk ejection. Each sample consisted of 0.3 ml perfusate over 15 min. Note the basal release of oxytocin within the nucleus before the pups were allowed to suckle. Oxytocin release started to increase before reflex milk ejections occurred and was maintained a certain time after the milk ejections ceased. Such an increase was not detected in the SON when the milk ejection reflex failed, nor when the push-pal cannula was placed outside the SON.

neurones 181. Rather, oxytocin appears to be released within the CNS, as shown by numerous observations, in vitro and in vivo. Release of O~t~ci~ by Hypothalamic IfI Vitro

Magno&eIl~la~ Nuclei

A strong argument for the central release of oxytocin is that it is normally found in the cerebrospinal fluid [14]. More questionable is where this central oxytocin comes from, especially in view of the fact that oxytocin-secreting neurons occur in many areas of the CNS, other than the hypothalamic magnocellular nuclei (see 1161). Nevertheless, recent evidence strongly suggests that oxytocin is released within the supraoptic and paraventricular nuclei themselves. The release of oxytocin, as well as vasopressin, by paraventricular and supraoptic nuclei perifused in vitro can be detected and measured by radioimmunoassay 13, 9, 101. When oxytocin (0.4 to 4 nM) was added to the incubation medium bathing such preparations, the release of endogenous oxytocin was increased in a dose-dependent manner (Fig. 3). Isotocin had a similar but less potent effect whereas arginine vasopressin left the release of oxytocin unaffected. On the other hand, an oxytocin antagonist [d(CH,)SOVTI significantly reduced basal oxytocin release and blocked the stimulatory effect normally evoked by exogenous oxytocin 1101. In no case was vasopressin release modified. Release of Oxytocin by Hypothalamic In Vivo

Magnocellular

Nuclei

In order to demonstrate such an endogenous release of oxytocin in vivo, we used push-pull cannulae to determine whether oxytocin was released from the SON of anaesthetized rats during suckling. It was thus seen that during control periods, there is a release of low level of oxytocin, which varies from one rat to another but which is constant for one given animal. A few minutes before the milk ejection reflex, that is, before the occurrence of high frequency discharges at regular intervals, the release of oxytocin inside the SON significantly increased (Fig. 4). No such increase was observed in suckled rats that either failed to display a milk ejection reflex, or when the cannula was placed outside the SON.

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Release of oxytocin within the hypothalamic magnocellutar nuclei appears to be c~cium-dependent since it can be blocked in vitro by Ca”+ channel blockers or by omitting Ca2+ from the perfusion medium [lo]. On the other hand, repetitive electrical stimulation of the magnocellular nuclei, maintained in vitro, with short pulses (1 msec) were ineffective even if the pulse intensity was raised up to 20 mA and the frequency up to 80 Hz. Only electrical stimulation with long duration pulses (10 msec, 10 Hz, 4 mA) increased oxytocin release significantly. Moreover, such release was insensitive to tetrodotoxin [3]. The failure of electrical stimulation with short pulses to release oxytocin from the magnocellular nuclei, as well as the failure of tetrodotoxin which suppresses action potentials to reduce the electrically evoked neuropeptide release, suggests that the mechanism of release of this peptide within the magnocellular nuclei differs from that described within the neurohypophysis [12]. One possibility is that oxytocin may be released within the hypothalamus from the dendrites of neurosecreto~ cells, as has been described for dopamine in the substantia nigra [2]. Nevertheless, a more classical type of release from axonal boutons is also possible since axonal synaptic terminals immunoreactive to antisera against oxytocin and its related neurophysin do occur in the SON and synapse onto oxytocin-immunoreactive somata and dendrites (1171, also Montagnese et al., this volume). Thus, one cannot exclude the possibility that oxytocin is also released from axon collaterals of oxytocin neurons from within or just outside the SON.

ET AL.

IV-CONCLUSION

The release of endogenous oxytocin within the hypothalamic magno~ellular nuclei may play a major role in the regulation of the activation of the oxytocinergic system responsible for reflex milk ejection during suckling. On the one hand, oxytocin affects the anatomy of the oxytocinergic system, inducing a mo~hological organization such that synchronization of neuronal firing, set off by afferent drive or other mechanisms, is further facilitated within each nucleus. On the other hand, oxytocin release from axon collaterals and/or dendrites within the magnocellular nuclei in response to suckling may be responsible for the regulation of the electrical activity of these cells during milk ejection reflex. Indeed, oxytocin has been shown to excite magnocellular neurones when applied microiontophoretically I1 1I. Oxytocin can thus be considered the initial and final condition for the activation of oxytocin neurons by suckling during lactation. The mechanisms by which this neuropeptide exerts such actions remain to be elucidated.

ACKNOWLEDGEMENTS We arc

very grateful to Dr. M. Manning for his generous gifts of agonists and antagonists of oxytocin. We also thank Mme. D. Heckenauer for typing the manu&ipt. This study was supported by grants from the UniversitB de Bordeaux II, Universiti: de Strasbourg I, INSERM, CNRS and Fondation de la Recherche Medicale.

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