Neurochemical regulation of oxytocin secretion in lactation.

0163-769X/92/1301-0033$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society

Vol. 13, No. 1 Printed in U.S.A.

Neurochemical Regulation of Oxytocin Secretion in Lactation* WILLIAM R. CROWLEY AND WILLIAM E. ARMSTRONG Department of Pharmacology (W.R.C.) and Anatomy and Neurobiology (W.E.A.), University of TennesseeMemphis, College of Medicine, Memphis, Tennessee 38163

6. Cholecystokinin (CCK) and CRF IV. Modulation of OT Release from the Neurohypophysis A. Anatomy and morphology of the neurohypophysis B. Neurochemical influences on OT release in the neurohypophysis 1. Coexpressed peptides a. Endogenous opioids b. CCK c. CRF 2. Afferent neural systems: catecholamines and GABA 3. PRL V. Conclusions and Future Perspectives A. Central neurochemical mechanisms B. Neurohypophysial mechanisms

I. Introduction: Physiological Patterns of Oxytocin Secretion A. Physiological actions of oxytocin B. Dynamics of OT secretion during lactation 1. Electrophysiological characteristics of OT neurons in lactation 2. Patterns of OT release during lactation 3. Membrane characteristics of OT neurons 4. The excitability of neurohypophysial axons and stimulus-secretion coupling 5. OT synthesis during lactation II. Neuroanatomy of the Milk Ejection Reflex A. Location of OT neurons B. Morphology of OT neurons C. Neuroanatomical pathway mediating suckling-induced release of OT III. Central Neurochemical Regulation of OT Secretion During Lactation A. Noradrenergic control of OT release 1. Neuroanatomical studies 2. Pharmacological studies on OT release 3. Pharmacological studies on OT neuronal activity B. Dopaminergic control of OT release 1. Neuroanatomical studies 2. Pharmacological studies on OT release 3. Pharmacological studies on OT neuronal activity C. Central actions of other neurotransmitters on OT secretion 1. Serotonin 2. Acetylcholine 3. Glutamate 4. 7-Aminobutyric acid D. Central actions of neuropeptides on OT secretion 1. OT 2. Endogenous opioid peptides 3. Activin 4. Vasoactive intestinal polypeptide 5. Angiotensin II

I. Introduction: Physiological Patterns of Oxytocin Secretion A. Physiological actions of oxytocin

T

HE nonapeptide oxytocin (OT) is an excellent example of a messenger molecule with diverse physiological actions as well as modes of delivery to its target cells (1, 2). After release from neurosecretory terminals in the neurohypophysis, OT exerts effects as a hormone carried by the systemic circulation to distant target organs, perhaps the most important of which are the mammary gland and uterus (1). In addition, OT may serve as a hypophyseotropic factor, released from nerve terminals in the median eminence into the pituitary portal vasculature, to affect anterior pituitary secretion (2-7), as a peptidergic neurotransmitter or neuromodulator acting within the central nervous system to influence a variety of neuroendocrine, behavioral, and autonomic functions (2, 8-13), and as a paracrine regulatory peptide in the ovary and testis (14-18). Although its most widely investigated physiological actions across all of these modalities are related to reproduction, including the processes of lactation and maternal care, OT also appears to participate in a variety of physiological responses to physical and metabolic challenges.

Address requests for reprints to: Dr. William R. Crowley, Department of Pharmacology, University of Tennessee-Memphis, Memphis, Tennessee 38163. * Work from the authors' laboratories and the preparation of this review were supported by NIH grants HD-20074 (to W.R.C.) and NS23941 (to W.E.A.). 33

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CROWLEY AND ARMSTRONG

The best understood physiological function of OT is its role as the endocrine effector of the milk-ejection response for the nutritional support of offspring during lactation (1, 19-21), and the major focus of this review is the neural mechanisms involved in transducing the suckling stimulus of the offspring into an OT-secretory response, based on studies done primarily in the rat. The reflex ejection of milk from the mammary glands is evoked by the stimulation of the offsprings' nursing, which in turn induces brief periods in which OT-secreting neurons dramatically increase their rate of firing, leading to discrete episodes of OT release from the neurohypophysis through stimulus-secretion coupling (22, 23; see Section I.B.I). These cells are concentrated in clusters within the hypothalamic paraventricular (PVN), supraoptic (SON), and associated accessory neurosecretory nuclei (Section II.A). After arriving via the systemic circulation, OT acts at specific receptors in the mammary gland to cause contraction of the myoepithelial cells that are localized on the surface of the mammary alveoli and along the mammary ducts. When those on the alveoli contract, the compression of the cells raises intraalveolar pressure, leading to the expulsion of milk from the alveoli into the duct system. Second, by contracting the myoepithelial cells along the ducts, the ducts are shortened and widened, which thereby reduces resistance to the flow of milk through the duct system (1, 20). It is well known that OT contracts uterine smooth muscle and that plasma concentrations of OT are elevated during parturition in many species (24-28). Although the contractile action of OT on the uterine myometrium may not be obligatory for the occurrence of parturition, it does appear to contribute primarily to the process of fetal ejection once the process of parturition has begun (24, 28-34), and in species such as the rat, may be important for the temporal spacing of successive fetal ejections (32, 33). The physiological importance of OT release into the systemic circulation in other contexts is less certain. For example, plasma OT concentrations are increased by nauseant [e.g. LiCl) and satiety-inducing {e.g. cholecystokinin administration; abdominal distention) stimuli (35, 36), but no information is available on the role of OT released under these conditions. Circulating OT is also elevated after application of several physical stresses [e.g. pain, exercise, cold, heat, immobilization (37-40)], as well as by challenges to cardiovascular and body fluid homeostasis, including increased plasma osmolality (4145) and hypovolemia (43, 45, 46). In these situations, OT may contribute to the mobilization of various adaptive responses, as evidenced, for example, by its depressor, natriuretic and glucagon-stimulating actions during periods of dehydration and hypovolemia (41, 46-48). It is intriguing to note that the OT secretory responses

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to many of these stress-related stimuli, including those related to the cardiovascular system, are blunted or abolished during lactation (49-52), suggesting that some type of conservation mechanism may be operating during lactation to ensure sufficient supplies of the peptide to maintain milk ejection. Even though vasopressin (VP) is released concomitantly with OT in response to challenges to osmotic and cardiovascular homeostasis, with the possible exception of parturition (24), the other stimuli provoking OT release are largely selective for OT release, at least in the rat (37, 38). B. Dynamics of OT secretion during lactation Measurements of suckling-induced OT release have been made in a variety of species including rat, rabbit, sheep, cow, pig, goat, nonhuman primates, and humans (1, 19). Although there are many differences across species in the frequency and duration of nursing episodes (1), in general, plasma levels of OT are elevated soon, but not necessarily immediately, after the initiation of suckling, and this response is transient and intermittent, rather than sustained, in its expression. That is, plasma levels of OT often return to basal between successive milk ejections even when the suckling stimulus is continually applied (19, 20). That the neural controls underlying this pattern of secretion are likely to be complex and subject to numerous regulatory influences is apparent from a consideration of the dynamics of suckling-induced OT secretion, as revealed by descriptions of the electrophysiological behavior of OT-secreting cells, and the pattern of changes in circulating levels of OT in the lactating animal. 1. Electrophysiological characteristics of OT neurons in

lactation. The successful antidromic identification of neurons projecting from the PVN and SON to the neurohypophysis (53) and the demonstration that OT release during lactation persists under urethane anesthesia (22) allowed the first studies correlating the electrical activity of hypothalamic neurosecretory neurons with release of a specific hormone (23). Before the development of RIAs suitable for measurements of OT in plasma, the pattern of OT release during lactation was inferred from the electrical activity of OT neurons in relationship to changes in intramammary pressure. The following summary of these events is based on a large literature reviewed in detail elsewhere (54-57). In the anesthetized lactating rat allowed to suckle a litter after a period of separation, the latency to first milk ejection ranges from 10-60 min, and milk ejections recur every 5-20 min. Fifteen to 20 sec before each milk ejection, a brief (2-4 sec) and explosive increase in the firing rate occurs in 40-50% of antidromically identified PVN and SON neurons (Refs. 23 and 58 and Fig. 1). By


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NEUROTRANSMITTERS AND OXYTOCIN SECRETION

FIG. 1. Activities of OT and VP cells during milk ejection. The different traces represent the intramammary pressure (index of OT release), the firing rate in spikes per second (sp/s) and the unit activity of paraventricular (PV) and supraoptic (SO) cells recorded simultaneously. Panel A, Paired supraoptic-paraventricular OT cells recorded during suckling; the number beside each burst represents the amplitude of the burst, i.e. the total number of spikes; the number below each burst indicates the delay in minutes since the previous burst. Both OT cells display bursts simultaneously before each milk ejection induced by suckling, and for each cell, the bursts recur at regular intervals with relatively constant amplitudes. Panel B, Paired supraoptic-paraventricular VP cells recorded during suckling; both cells display a typical phasic pattern, i.e. successive periods of activity and silence occurring more or less regularly. The activity periods never correlate with the milk ejections during suckling. Note also that the phasic activities of the paired cells are not synchronous. [Reproduced with permission from P. Richard et al.: Pulsatility in Neuroendocrine Systems (edited by G. Leng), CRC Press, Boca Raton, FL, 1988 (57).]

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A Paired oxytocin cell recordings intramamammary pressure Pv

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convention (54), we refer to these increases as "neurosecretory" or "milk ejection bursts" and to cells exhibiting such behavior as "OT-neurons," with the understanding that as of this writing, independent evidence {e.g. immunocytochemical labeling after intracellular recording and filling) has not been obtained, most likely due to the difficulty of obtaining intracellular recordings from neurosecretory cells in vivo long enough to characterize them more fully (59). Between the bursts of activity associated with milk ejection, OT neurons fire irregularly or continuously. Neuronal bursting activity also has been noted during parturition in the rat, and the bursts are temporally correlated with uterine contractions (25). OT neurons are activated in a different manner by other stimuli that release the peptide, however. For example, if a lactating rat is challenged osmotically or dehydated, the interburst (sometimes called background) firing activity increases linearly in accordance with the degree of physiological stimulation (54), even though the total secretory response may be attenuated (49-52). The remaining neurosecretory neurons whose activity is not correlated with milk ejection are considered to be VP-secreting. These cells are electrically silent or display irregular activity under basal conditions and adopt a phasic, bursting pattern of activity to dehydration, elevations of osmotic pressure, and hemorrhage (54). Phasic

activity also exists in in vitro preparations, and the majority of phasically active neurons labeled intracellularly stain positively for VP (60) or VP-associated neurophysin (61). Simultaneous recordings of extracellular action potentials from paired OT neurons in the SON and PVN indicate that they fire synchronously before milk ejection (58, 62, 63). The onset of bursts in pairs of neurons (one in the SON, one in contralateral PVN) differs by an average of approximately 200 msec, and the lag times between the periods of peak firing during the burst are even smaller (63). In paired recordings, the OT neuron reaching the highest amplitude most often begins and peaks in its firing rate before the one with the lower amplitude (63). The amplitude of a burst within a given neuron is positively associated with the amount of OT released (64). This synchronization between neurons in the PVN and SON strongly implies a neural mechanism for coordination of the bursts, either among the OT neurons themselves or from shared synaptic input. Synchronization per se is unaffected by a number of pharmacological manipulations or interhemispheric transection, which disrupt the periodicity and amplitude of the bursts (57, 65). Intracellular recording studies in which the dye Lucifer Yellow has been injected into magnocellular neu-


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rons have, however, provided intriguing evidence for dye coupling among homotypic (i.e. OT-OT or VP-VP) neurons (56). This phenomenon may reflect electrotonic interactions among magnocellular neurons that could contribute to their synchronization (56). It is well established that the continuous attachment of pups gives rise to and is necessary for the periodic release of OT (66). Similarly, in the anesthetized rat, the activation of individual OT neurons is also dependent upon the number of pups above a threshold of five to seven, with cells firing more spikes per burst with more pups suckling (66). Interestingly, neither the length of time between bursts, nor the background activity (i.e. firing rate between bursts) is sensitive to changes in the numbers of pups nursing. Occasionally, a more reflexive release of OT can be seen by adding an additional pup or stimulating the nipple (66), which quickly produces an extra burst, but only after other pups have been attached for some period. Therefore, the continuous attachment of the offspring not only sets in motion the bursting pattern, but also appears to sensitize the OT neurons to additional afferent input. Stimulation of the mammary nerve directly (67) or the spinal cord (68) can also elicit OT release, but even with continuous spinal cord stimulation in the lactating rat, the intermittent pattern of release was never produced (68). Continuous or intermittent electrical stimulation applied directly to the nipples of the lactating rat, however, produces periodic milk ejections that mimic those produced by attached pups (69). 2. Patterns of OT release during lactation. The changes in plasma concentrations of OT during lactation as measured by RIA are in close accord with what would be predicted from the electrophysiological evidence reviewed above. The temporal patterning of suckling-induced OT release is best known for the rat and has been described recently in great detail by Higuchi et al (26, 28, 70), who monitored both the plasma concentrations of OT by RIA, as well as the rise in intramammary pressure in urethane-anesthetized rats subjected to continual suckling by ten pups after a period of several hours' separation. Consistent with the different levels of neuronal activity recorded from OT cells during and between milk ejections, plasma concentrations of OT during suckling remain basal save for the brief episodes of increased release that are closely associated with the rise in intramammary pressure and milk ejection (and by implication, with the neurosecretory bursts of activity in PVN and SON neurons). During these periods, plasma concentrations of OT abruptly rise approximately 2- to 3-fold and return to baseline within 2-3 min, consistent with a plasma half-life of approximately 1.5 min. Similar

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results have been obtained from RIA measurements in conscious lactating rats (71). The brevity of the individual OT release episodes and intra- as well as interindividual variability in their latency, timing, and magnitude make it difficult to design protocols for periodic blood samplings that allow accurate quantification of the increase in plasma OT concentrations induced by suckling in conscious rats. However, blood sampling in small volume (200-400 iA), at relatively frequent intervals (e.g. every 5 min) during a defined period of suckling after several hours of separation, can usually detect at least one, and perhaps several, OT secretory episodes in an individual animal (71). Other hormones are also released by the suckled female rat in the separation-reunion paradigm (Fig. 2). Within several minutes of the onset of suckling, the circulating concentrations of epinephrine, released from the adrenal medulla via sympathetic activation, reach a peak but return rapidly to their low baseline levels (72). Plasma levels of GH follow a similar pattern (73), while those of PRL increase quickly to reach a maximal level, typically by 15-30 min, that is maintained as long as the suckling stimulus is present (70, 71). However, frequent blood sampling indicates that PRL concentrations also fluctuate during suckling, with some temporal associations apparent between the pulses of PRL and OT (70). Bidirectional interactions between these two hormones of lactation are considered in Section V. There is also evidence for increased release of ACTH during suckling (74). In the rat, plasma concentrations of OT during parturition may also exhibit pulses, but this is superimposed upon a steadily increasing plasma level as successive offspring are delivered (28, 34). OT release in response to physical stresses or cardiovascular challenges appears HORMONAL RESPONSES TO SUCKLING AFTER SEPARATION

•— OXYTOCIN PULSE ••• P R L

• EPI = g MILK YIELD

15 MIN OF SUCKLING1

FiG. 2. Individual patterns of hormone release and milk yield in response to suckling in the lactating rat previously separated from its litter for several hours (schematized from data in Refs. 19-22, 407). Hormonal responses are normalized as the percent maximal plasma concentration, and relative milk yield is expressed as a percent of total milk available in the mammary glands. Plasma OT concentrations are elevated only briefly, in discrete episodes during the period of suckling.


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to occur mainly as a tonic, nonpulsatile increase, (41, 75), also similar to the pattern of electrical activity recorded from these cells in response to such treatments (54). 3. Membrane characteristics of OT neurons. The intracellular recordings necessary to study the membrane properties of hypothalamic neurosecretory neurons have thus far been accomplished mainly in in vitro preparations, due largely to mechanical instabilities present in vivo. Conclusive identification of intracellularly recorded neurons in vitro as oxytocinergic, however, requires filling the neuron with a marker molecule, such as Lucifer Yellow, followed by determination of its hormone type with immunohistochemistry. This is necessary at present because one cannot rely completely on electrophysiological characteristics to determine whether a neurosecretory cell studied in vitro secretes OT or VP. Although the presence of continous activity (1-10 Hz) in SON or PVN neurons in vitro, similar to that exhibited by OT neurons between milk ejections in vivo, has been the only electrophysiological criterion for identifying OT neurons in these preparations (e.g. Refs. 76 and 77), neurons exhibiting phasic activity in vivo (putative VP-neurons) may also exhibit extended periods (i.e. minutes) of continuous firing (78-80). As a further caution, a phasic bursting neuron will also occasionally show discharges correlated with milk ejection (54). Immunohistochemical labeling for OT in Lucifer Yellow-filled neurons in hypothalamic slices has been achieved (61, 81), but little information is available on the electrophysiological properties of these neurons as a group and in particular, how they might differ from VP neurons beyond the absence of phasic firing. OT and VP neurons in vitro appear to be indistinguishable in many of their membrane properties, including their high input resistance (~200 MQ,), membrane time constant (10-20 msec), and linear current-voltage relationship from —55 to -85 mV (55, 56, 82, 83). Other general, well characterized properties of SON neurons in vitro include a prominent spike hyperpolarizing afterpotential, produced largely by a transient outward potassium current (84), an apamin-sensitive, Ca++-dependent, afterhyperpolarization (AHP), which follows spike trains and gates firing rate (85, 86), and spike broadening with repetitive firing (87). Additional studies will be needed to determine the extent of similarity in these properities between OT and VP neurons. Modulation of these potentials by neurotransmitters or neuromodulators probably serves as the basis for some of the effects of these neuromessengers on magnocellular neuronal activity (55). Both OT and VP cells respond to osmotic challenge in vivo, and in vitro, virtually all SON neurons are depolarized by local increases in osmotic pressure (88-90). The

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basis of this depolarization within the physiological range appears to be activation of an inward current involving nonselective cation channels (90). During lactation, not only is the background firing rate of OT cells increased to osmotic stimulation, but the milk ejection burst amplitude is also enhanced (91). In addition, osmotic stimulation can facilitate the milk ejection reflex in rats by recruiting a larger number of cells into the responding population. This suggests that the level of depolarization of OT neuronal membranes is critical to the cells' responsiveness to whatever synaptic input drives the milk ejection bursts. Many magnocellular neurons also show a spike depolarizing afterpotential (DAP) after individual spikes or after a few spikes in succession (Fig. 3). As the summation of several DAPs is thought to give rise to the plateau potential underlying the bursts during the phasic activity of VP neurons (78, 92, 93), it seems reasonable that the OT and VP neurons may differ in the expression of this potential (90). However, studies combining intracellular recording with immunohistochemical staining have revealed that while the majority of VP neurons in rat exhibit a DAP, this potential is also present in some OT neurons (94) (Fig. 3), even though these neurons usually fail to exhibit phasic activity. In addition, the DAP has previously been associated with continuous firing (78) and can easily be masked with a strong stimulation by the AHP (86). Thus, the DAP per se is probably not a discriminating feature between OT and VP neurons. On the other hand, in contrast to VP bursts, OT bursts cannot be triggered by antidromic shocks (95). Further knowledge of the activation and inactivation kinetics of the conductance responsible for the plateau potential (78, 92, 93) and more studies of the slow depolarizing potential that may underlie intrinsic phasic activity (92, 96) may distinguish the two types of cells. The dramatic milk ejection bursting behavior of OT neurons during lactation is likely to primarily result from a change in synaptic activity, especially a brief, but strong, synaptic depolarization, during which the firing rate and burst duration would be regulated by the hyperpolarizing afterpotential and even more prominently by the AHP. At present, however, it is unknown to what extent the shape of the OT cell burst and its periodicity during suckling are determined by the intensity and time course of the assumed synchronizing input, and how this input might interact with intrinsic conductances. As well, the source of the synchronizing input is unknown, but some anatomical features of the SON and PVN could facilitate synchrony (see below). The problems hindering intracellular recordings from SON and PVN neurons in vivo will have to be overcome in order to understand these events.


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FIG. 3. Traces demonstrating electrophysiological properties shared by some immunocytochemically identified OT (panels A and C) and VP (panels B and D) neurons recorded intracellularly from the rat SON in vitro. In panels A and B, the voltage response of both neurons to similarly spaced, constant current steps is shown. The response in the hyperpolarizing direction was, for the most part, linear for both neurons, with a small degree of rectification at the most hyperpolarized level. Both neurons fired spikes to the depolarizing pulses and exhibited spike frequency adaptation, such that interspike intervals were longer during the latter part of the response. In panels C and D, the response to a small number of constantly spaced action potentials (inset) evoked by 5-msec depolarizing current pulses and delivered at 50 Hz is shown. Both neurons exhibited a hyperpolarizing afterpotential (AHP) followed by a depolarizing afterpotential (DAP). In general, the DAP is characteristic of VP neurons and is probably important for phasic activity, but it is also present in a minority of OT neurons (94). Spikes are clipped due to the sampling frequency. The inset is an expanded version of spikes in panel C at a higher resolution to show the response to each current pulse. Scale for inset: 20 mV/0.5 nA, 25 msec.

4. The excitability of neurohypophysial axons and stimulus-secretion coupling. Hormone release from the neurohypophysis is a Ca++-dependent event normally induced by the depolarization of secretory terminals by invading action potentials from magnocellular neurosecretory neurons (97,98). The bursts in the activity of OT neurons during lactation are translated into a highly efficient release of the peptide from the neurohypophysis. A correlation of hormone release with spike activity has shown that the amount of OT released per spike is much greater during a burst than during continuous activity (99). This facilitation of hormone release to closely spaced spikes holds true for both OT and VP when isolated neural lobes are stimulated in vitro (98, 100-102). OT release differs from that of VP, however, in showing continuing increases in efficiency to frequencies as high as 52 Hz, which approximates the frequency reached by bursting OT neurons, whereas VP release reaches maximal efficiency below 26 Hz, closer to the spike frequencies shown by VP neurons (103). OT release rates are also maintained at a relatively constant level for several minutes of continuous stimulation at a constant frequency, whereas VP release shows considerable fatigue (104). Release of both hormones recovers with periods of inactivity, such that intermittent bouts of activity also evoke

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VP neuron

AHP

more release than continuous stimulation with the same number of pulses (101, 102, 105). Thus, the nerve terminals of OT neurons are adapted to respond efficiently to either continuous or bursting spike activity, both of which are observed during OT release in vivo. Facilitation and fatigue are thus phenomena of hormone release dynamics that could be modified by neurotransmitters or neuromodulators that affect their underlying mechanisms (Section IV.B). Repetitive firing of neurohypophysial axons is associated with Ca++-dependent spike broadening, eventual failure of spike propagation (87,105-107), and enhanced neurohypophysial Ca++ uptake (101), some of which occurs in the excited neurohypophysial terminals themselves (108). Voltage-clamp studies show that frequencydependent broadening is accompanied by the inactivation of transient, voltage-sensitive K+ currents that normally repolarize the terminal after a spike (108, 109). Repetitive firing would thus delay repolarization of the membrane and allow more Ca++ to flow through voltagesensitive Ca++ channels, thereby facilitating hormone release. Intracellular Ca++ may also be elevated during repetitive firing by a mechanism independent of spike broadening (108). Although a sustained depolarization would lead to enhanced intracellular Ca++, in the absence


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of membrane repolarization at least one identified calcium current in neurohypophysial terminals (N type) would inactivate (110). The time course for the recovery of spike propagation and recovery from the inactivation of at least one of the involved K+ currents after repetitive firing is thought to take several seconds, and this could underlie the observed recovery from fatigue in hormone release (106, 109). Neurohypophysial terminals also have an action potential threshold of about —45 mV, or 10 mV more depolarized than the soma (106). The high spike threshold, coupled with the extensively branched and varicose morphology of neurohypophysial axons (Refs. 106 and 111 and Fig. 4) provides a means of electrical isolation of regions of the axonal arborization with even weak, local hyperpolarization (106, 112). Thus, neurochemical messengers operating within the neurohypophysis (Section IV.B) could regulate OT release through several mechanisms. These include actions on the currents responsible for the enhanced Ca++ influx during facilitation, such as transient K+ currents or the identified Ca++ currents directly, or other currents that affect membrane polarization and conductance. This, in turn, can influence the number of release sites (varicosities near blood vessels) that are effectively depolarized. In addition, the synchronous bursts of OT neurons during lactation are associated with a significant, transient rise in the concentration of extracellular K+ in the neural lobe to a level known to affect hormone release (113, 114). Thus, any mechanism altering K+ buffering in the extracellular space could also affect hormone release. 5. OT synthesis during lactation. The processes of neurohypophysial hormone synthesis, packaging into vesicles, and axonal transport to the neural lobe of the pituitary have been extensively reviewed (115-117). The successful cloning of the gene encoding the OT preprohormone (118) has allowed recent investigations into OT gene expression that extend the earlier approaches. The OT precursor consists of a signal sequence, followed by the OT nonapeptide, and the neurophysin sequence. Processing of the precursor to the mature OT and neurophysin peptides takes place within storage granules in transit to the neurohypophysis (117). Several groups have shown that the levels of OT prohormone messenger RNA (mRNA) in the hypothalamic magnocellular regions are elevated by approximately 2- to 3-fold during lactation in rats (119-121), presumably to allow stores of the peptide to keep pace with the demands of suckling-induced release. Extensive osmotic challenge similarly elevates hypothalamic OT mRNA approximately 2-fold (122, 123). The size of OT mRNA also increases in lactation, due to increased length of the polyadenylate tail (124), which may confer

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greater stability to the message. There is some disagreement regarding the time course for the increase of OT mRNA associated with lactation, with one laboratory reporting that the rise occurred during the latter third of gestation (121), while a second group found that the increase occurred immediately before parturition (120). The physiological signals that evoke increased OT synthesis during pregnancy-lactation are largely unknown. There is some evidence that at least for the early stage of lactation in rats, afferent stimulation by the offspring contributes to the maintenance of OT mRNA levels (125).

II. Neuroanatomy of the Milk Ejection Reflex A. Location of OT neurons For many years it was widely believed that each magnocellular nucleus contained one or the other peptide (i.e. exclusively OT or VP neurons) (126). A short time after this division was called into question by the seminal electrophysiological investigations discussed above, immunohistochemical investigations clearly established that OT and VP neurons were mixed within both nuclei in rats (127, 128), and in many other mammals (129, 130). Detailed descriptions of the location of OT neurons in the SON, PVN, and accessory neurosecretory nuclei of rats are contained in the papers of Rhodes et al. (131) and Hou-Yu et al. (132). Figure 5 presents a schematic diagram of the distribution of OT neurons in the rat hypothalamus that project to the neurohypophysis relative to those projecting elsewhere and to the VP neurons (133, 134). OT neurosecretory cells are found throughout the SON but are distributed preferentially in the rostral and dorsal parts of the nucleus. The number of SON OT neurons is only one-half to two-thirds that of VP neurons, but this is still greater than the number of PVN OT neurons (127, 131). Within the PVN proper, OT neurons are mixed in approximately equal proportions with VP cells, although the total number of neurosecretory cells is less than that seen in the SON. Beginning rostrally, PVN OT neurons are preferentially found in the medial magnocellular division. At the level of the lateral magnocellular portion of the PVN, OT neurons form a shell around the densely clustered VP cells. In the extreme posterior portion of the PVN, magnocellular neurons are sparse, but the majority are oxytocinergic. Some of these project to the neurohypophysis, while others innervate the brainstem as a part of the extrahypothalamic OT system (135,136). In addition, many OT neurons, accounting for approximately one-third of their total population, are found in the various accessory neurosecretory cell groups, and most of these also project to the neurohypophysis. Ros-


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FIG. 4. Nerve fibers in the neurohypophysis of the rat. Panel A, Electron micrograph of a rapidly frozen, freeze-substituted neural lobe from a male rat, showing the neurohemal contact zone. The tissue was labeled for OT-neurophysin using a postembedding, immunogold technique (304). Gold particles are most densely located over the dense core vesicles in nerve profiles, (arrows, arrowheads), but a certain amount of background labeling is apparent. Nerve terminals (arrowheads, lower half of micrograph) with dense core vesicles and microvesicles are seen near a basal lamina, which is distinct from the basal lamina of the endothelial cell (En) of the capillary wall. The nerve fibers (arrows) in the upper half of the micrograph are surrounded by a pituicyte (P) and do not contact the basal lamina. Panel B, Photomicrograph of nerve fibers in the neural lobe after an iontophoretic injection of Neurobiotin in the SON of a rat. The tissue was reacted with avidin-biotin-peroxidase and then with diaminobenzidine after allowing a 24 h survival period for anterograde transport of the tracer into the neural lobe. Note the many, variably sized varicosities (arrowheads) along the axons.


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FIG. 5. The distribution of magnocellular neurosecretory neurons in the rat with major divisions of the SON, PVN, and accessory nuclei, their efferent projections, and their hormone type. Panels A, B, C, and D represent four levels from rostral to caudal through the hypothalamus. On the left half of each coronal tracing is the distribution of all OT and VP neurons reacting with a neurophysin antibody that cross-reacts with both cell types. On the right half of each coronal tracing, the major conglomerations of these neurons are marked to distinguish primarily OT- (horizontal lines) or VP-containing (vertical lines) groups. Hatched areas indicate mixing of the two types. Also on the right, the major efferent connections of the nuclei are shown, with distal targets enclosed by dashed lines. Abbreviations: AC, anterior commissural nucleus; BS, brain stem; BSTV, ventral division of bed nucleus of the stria terminalis; Cir, nucleus circularis; EME, external layer of the median eminence; f, fornix; ic, internal capsule; LH, lateral hypothalamus; LPO, lateral preoptic area; MFB, accessory nuclei of the medial forebrain bundle; opt, optic tract; ox, optic chiasm; PaDC paraventricular nucleus, dorsal medial cap; PaLM paraventricular nucleus, lateral magnocellular division; PaMM paraventricular nucleus, medial magnocellular division; PaMP, paraventricular nucleus, medial parvocellular division; PaV, paraventricular nucleus, ventral division; PeM, periventricular magnocellular nucleus; PoF, posterior fornical accessory nucleus; PPit, posterior pituitary; sm, stria medullaris; SpC, spinal cord; SO, supraoptic nucleus; SOR, supraoptic nucleus, retrochiasmatic division; 3V, third ventricle. [ Reproduced with permission from W. E. Armstrong: The Rat Nervous System (edited by G. Paxinos), Academic Press, Sydney, Australia, 1985, pp 119-128 (134).]

tral to the PVN proper, the anterior commissural nucleus (ACN) (137) consists almost exclusively of OT neurons, as does a group of magnocellular periventricular neurons lying between the ACN and the PVN (131). Mixed populations of OT and VP neurons are seen in most other accessory nuclei. It is not known whether the OT neurons that lie outside the SON and PVN and that also project to the neurohypophysis have some different function from those in the major nuclei. B. Morphology of OT neurons

Based upon Golgi staining or intracellular filling, magnocellular neurosecretory neurons in the SON and PVN have diameters of 25-30 fim, and typically, one to three

short dendrites (133, 138-144). To date, the specific dendritic field of OT neurons has only been ascertained with immunohistochemical means (138, 145). In the SON, OT neurons share with VP neurons prominent dendritic extentions into a ventral lamina. This subregion is also rich in axonal inputs as well as in dendrodendritic close appositions that show dynamic changes during lactation as well as other states (see below). These dendrites can occasionally be quite varicose, and electron microscopic studies suggest they are capable of the exocytotic release of peptide (146). Outside the SON, the dendritic field differs slightly for different groups of OT neurons (147). In particular, the dendrites of OT cells in the ACN and in the medial magnocellular portion of the PVN have a predominantly


42


medial orientation, and some extend to the subependymal region. Periventricular OT neurons are more vertically oriented, with dendrites extending dorsally and ventrally in the subependymal region. In the posterior region of the PVN, dendrites from bipolar neurons project laterally along the main axonal outflow, ventromedially toward the ventricle, and across the midline above the third ventricle. There is evidence that OT neurons may be interconnected. Terminals immunopositive for OT have been observed to synapse upon OT neurons (148); however, the source of these inputs is unknown. Retrograde tracing studies report afferents to PVN from accessory nuclei and SON (149, 150), and the crossing of dendrites and perhaps axons to the contralateral PVN has been noted (145, 149). Finally, as cited above, there may also be gap junctions among OT neurons (81). In addition, although complete interhemispheric transections reduce OT cell burst amplitudes, drastically disrupt the positive correlation in the burst amplitudes between SON and contralateral PVN neurons, and alter the regular periodicity of the bursts, they do not affect the degree of synchronization between recorded pairs of OT neurons (65). While such interconnections could be relevent to the milk ejection reflex, it is noteworthy that gross lesions of the PVN inhibit, but do not prevent, milk ejection (151). The densely packed magnocellular neurons in the SON and PVN often exhibit direct membrane appositions between adjacent somata or dendrites. The extent of these appositions increases during lactation (152-155), and it has been speculated that this might promote interactions between adjacent neurons by allowing elevation of extracellular K+ ([K+]o) from the increased firing of adjacent cells (56). Recent studies with ionsensitive electrodes have confirmed that such an elevation of extracellular [K+]o occurs in the vicinity of OT neurons during the milk ejection burst (156). Although [K+]o could not be measured in the 15-20 nm space between closely apposed neurons in that study, a simple model predicted that [K + ] o would more than double in this space for a few milliseconds after an action potential; this may be sufficient to briefly affect the adjacent neuron and perhaps nearby terminals. However, the elevations of [K+]o are well buffered, even over the space of a few microns, and would probably not be sufficient to affect neurons not in close apposition. Considerable attention has been paid to the presence of shared synapses onto OT neurons, the frequency of which also increases significantly after parturition and during lactation (152,153,155). These involve instances in which a single nerve terminal contacts two or more separate postsynaptic elements (152,153). It is intriguing to note that most of these synapses contain 7-aminobutyric acid (157), and immunocytochemical studies indi-

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cate that this type of plasticity during lactation occurs among OT, but not VP, neurons (158). An increase in shared synapses may be an additional basis by which synchronization is enforced. C. Neuroanatomical pathway mediating sucklinginduced release of OT

In the rat, the afferent somatosensory pathway necessary for milk ejection is currently thought to be transmitted through the spinal cord via the ipsilateral dorsolateral funiculus to the lateral cervical nucleus (LCN), from which ascending fibers cross in the medulla before ascending to the mesencephalon and thalamus (159,160). It is unknown whether mammary sensory fibers reach the LCN directly, but direct projections of mammary nerve to the dorsal horn and dorsal column nuclei have been noted (161). The dorsal columns and ventroposterior thalamus are unnecessary for the expression of milk ejection (160). However, lesions in the dorsolateral funiculus-LCN region completely block the reflex (160), as do lesions in the mesencephalic lateral tegmentum (159, 162). Such midbrain lesions interrupt a variety of somatosensory inputs to the intercollicular region, the external nucleus of the inferior colliculus, and the deep layers of superior colliculus, and at present it is unknown whether these areas, rather than or in addition to the midbrain lateral tegmentum, are important for the milk ejection reflex (160). Anterograde tracing from the region of effective lesions in the midbrain reveals only a sparse diencephalic projection, which is primarily to the zona incerta, the Fields of Forel, and the posterior hypothalamus, but not directly to the SON or PVN (159). Hence, the final links between the somatosensory input to the midbrain and the hypothalamus remain largely undetermined. At present, it is also difficult from the existing lesion studies to implicate neurochemically defined cell groups (i.e. those whose neurotransmitter phenotype is known) as integral components of the milk ejection reflex somatosensory pathway, despite the wealth of information that specific neurotransmitters are involved in this process. Milk ejection in rats is strictly associated with a state of slow-wave sleep, and is never expressed during electroencephalogram arousal (163, 164). The absence or inhibition of cortical output is permissive for the milkejection reflex (164, 165), and the inactivation of cortex may prevent the inhibition of the reflex associated with painful stimuli (166). The anatomical substrate of this inhibition is unknown, but efferent connections from infralimbic and prefrontal cortex to the lateral hypothalamus have been described (167-169). There are no reported direct cortical projections to neurosecretory neurons, but recent evidence points to a direct projection


February, 1992


to PVN from lateral hypothalamus (170) so that the cortical influence could be indirect via this region. Cortical influences over the hypothalamus in general could also be mediated through the amygdala (171) or the septum. While a role for the amygdala in milk ejection has not been examined in detail, septal stimulation inhibits the activity of OT neurons (172) and the milk ejection reflex (173). This pathway is probably not direct, as septal efferents massively surround, but seldom enter, the SON (174), and only a few axons synapse on dendrites in the extreme dorsal SON or perinuclear zone (175). Interestingly, septal stimulation also desynchronizes the cortical electroencephalogram, raising the possibility that its inhibition of the milk-ejection reflex may be caused by a different anatomical circuit than that influencing the background activity of OT neurons (173).

III. Central Neurochemical Regulation of OT Secretion During Lactation The specific neural systems driving the continuous background firing of OT neurons or that coordinate the intermittent and synchronous milk ejection bursting pattern in response to suckling remain incompletely characterized. However, as in other areas of neuroendocrinology, some important insights have been garnered from pharmacological studies in which specific neurotransmitters have been manipulated and the effects assessed on circulating OT concentrations and/or on the level of electrical activity in identified OT neurons. While several systems have been strongly implicated, information regarding the specific neural sites where their regulatory influences on secretion of OT take place is still incomplete. These could include the magnocellular OT-containing subdivisions of the hypothalamic neurosecretory nuclei, other brain areas that in turn project to the magnocellular neurons, as well as the neurohypophysis. These various possibilities will be considered in this section, which focuses on the central actions of neurotransmitters, and in the succeeding section, which reviews neurochemical regulatory mechanisms in the neurohypophysis. There is little definitive information on the postsynaptic actions of specific neurotransmitters that contribute to the firing of a milk ejection burst. Extensive descriptions of neurotransmitter action on the electrical activity of hypothalamic neurosecretory cells (both OT and VP) are contained in several recent reviews (55-57) and will be discussed below with the focus largely on studies relevent for lactation. In addition to discerning the cellular effect of a given neurotransmitter on the electrical behavior of OT cells, it is important to evaluate whether and how such actions could contribute to the suckling-induced periodic neurosecretory bursting of OT

43

neurons that characteristically and specifically precedes release of the hormone for milk ejection. Unfortunately, the majority of studies in this area have not employed lactating females as the animal model for in vivo recordings or as the source of tissue for in vitro studies. Hence as a general caveat, some caution should be used in applying observations made in the nonlactating state to events that occur in lactating animals. A. Noradrenergic control of OT release 1. Neuroanatomical studies. The observation of a prominent innervation of the PVN and SON by catecholamine (mainly norepinephrine)-containing fibers was made in the earliest days of fluorescence histochemical mapping of brain neurotransmitter systems (176) and has subsequently received extensive attention (177-180). The relationship of noradrenergic fibers to the OT and VPpositive elements within the magnocellular regions of these nuclei was first described in detail by McNeill and Sladek (181). Substantial numbers of noradrenergic fibers were localized to the immediate vicinity of the magnocellular OT perikarya in the rostral PVN and in several of the accessory clusters of OT cells anterior to the PVN such as the ACN. Although more scattered noradrenergic fibers were found near the OT cell bodies in the antero-dorsal aspects of the SON, these investigators made the important observation that long dendritic processes of the dorsally located OT perikarya of the SON invade the most ventral aspects of the nucleus, the ventral glial lamina, which receives a dense noradrenergic projection. Thus, in this structure, examination of noradrenergic input solely to areas containing OT perikarya may miss some important norepinephrine (NE)OT interconnections (56). Similar observations were made by Hornby and Piekut (182), who reported that in the PVN,'.. .[noradrenergic] immunostained fibers are seen in close proximation to and often appear to surround oxytocin-containing cells' (Ref. 182, p. 244). No significant relationship between OT neurons and epinephrine-containing fibers was observed in these studies. Studies at the electron microscopic level (183-185) support the concept that both axosomatic and axodendritic synaptic contacts are characteristic of the noradrenergic innervation of the OT system, and estimate that at least 20-25% of the noradrenergic varicosities form typical synaptic specializations with OT-positive perikarya and dendrites. This is, in all likelihood, a significant underestimate, as extensive serial sectioning through a nerve ending is required in order to determine whether or not that ending contains a synaptic specialization. For example, even moderate numbers of serial sections have revealed that at least


44


50% of noradrenergic varicosities in the PVN form synapses (186). The source of the noradrenergic innervation specifically to the OT system remains incompletely characterized and the subject of some uncertainty. Early studies (150, 175, 187) using retrograde and anterograde tract tracing to identify the sources of afferent input to the magnocellular hypothalamic nuclei (without indicating either the neurochemical identity of the afferents or their specific relationship to OT or VP cells) described projections to both the PVN and SON from medullary and pontine structures known to contain noradrenergic cells, including the lateral reticular formation, which contains the Al cell group [nomenclature of Dahlstrom and Fuxe (188)], the nucleus tractus solitarii (NTS), containing the A2 cell group, and the locus coeruleus (A6 cell group). Although it was consistently observed in these studies that Al cells projected to both PVN and SON, while inputs from A6 cells were limited to the PVN, there was disagreement regarding whether A2 cells projected to both magnocellular nuclei (150, 175) or only to PVN (187). Uncertainities regarding the source (s) of noradrenergic input to the OT system also continued in studies employing tract tracing in combination with immunocytochemical identification of the noradrenergic fibers and their relationship to OT and VP neurons. Sawchenko and Swanson (189) initially reported that Al noradrenergic cells predominantly innervated the parvocellular aspects of the PVN, and that the projections from this cell group to magnocellular regions of PVN and to SON were preferentially made to the VP-containing regions. They also described A2 noradrenergic cells as exclusively innervating parvocellular PVN, but not magnocellular PVN or SON. Others (190,191), however, showed a clear projection to SON from A2 noradrenergic cells. Subsequent studies by Cunningham and Sawchenko (192), with improved sensitivity in their anterograde tracing, confirmed the distribution of Al-derived noradrenergic fibers preferentially to regions of the PVN and SON containing VP perikarya, but revised the earlier view regarding the A2 projections, which were observed to innervate both the magnocellular PVN and SON, and to be in proximity to both VP and OT cells in these areas. Both noradrenergic and nonadrenergic projections from NTS to SON were also observed by Raby and Renaud (193). These conclusions have been buttressed by electrophysiological approaches in which the effects of electrical stimulation of the Al or A2 regions have been assessed on the firing patterns of electrophysiologically defined, putative OT and VP neurons. In anesthetized male rats, stimulation of the NTS activated approximately 70% of the putative OT cells in the PVN and approximately

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20% of the putative OT cells in the SON (190, 193, 194). That this acceleration of OT neuronal activity was mediated by noradrenergic mechanisms was supported by the demonstration that it could be abolished by local destruction of the noradrenergic terminals with the catecholamine neurotoxin, 6-hydroxydopamine (194). Stimulation of the Al cell group activated a smaller number (4/15) of OT cells in the PVN (194), and none in the SON (195). Although consistent with the anatomical evidence cited above, the conclusions obtained with these electrophysiological approaches must remain tentative since OT neurons were not defined in lactating animals. Considered together, the available evidence suggests that OT cells in the hypothalamic magnocellular regions receive a substantial innervation from ascending noradrenergic fibers, the primary source of which is the A2 cell group present in the NTS region of the medulla. Moreover, based on the above-cited stimulation-recording studies, the influence of NE derived from the A2 cells on OT neurons appears to be predominantly excitatory (see below). Indeed, two preliminary reports (196, 197) indicate that stimulation of the NTS in lactating rats increases OT release as evidenced by an increase in intramammary pressure. 2. Pharmacological studies on OT release. Although both excitatory and inhibitory effects of NE or agonists have been reported on OT release, as measured by RIA or by its biological effect, to increase intramammary pressure, these differential effects can be accounted for by actions at separate adrenergic receptor subtypes. There is consistent evidence, for example, that the excitatory adrenergic effect on OT release and OT neuronal activity is mediated via an «i-adrenergic receptor. In studies using the intramammary pressure response in anesthetized lactating rats as the measure of OT release, intracerebroventricular (IVT) administration of NE or the ax-agonist phenylephrine stimulated OT release, while a /3-adrenergic agonist was ineffective (198). a-Agonists also stimulated the release of OT from male rats after IVT administration (199) and from a hypothalamic explant preparation in vitro (200). The suckling-induced release of OT was also blocked by drug treatments that disrupt anoradrenergic neurotransmission by a variety of means, including synthesis inhibition (198), and a-, but not j8adrenergic, receptor blockade (198, 201, 202). These latter findings strongly implicate the excitatory a-adrenergic influence as a physiological component of the milk ejection reflex. This notion is further supported by studies in which the effects of suckling were evaluated on the turnover rate of NE, used as an index of the state of activity in noradrenergic nerve terminals, in various hypothalamic nuclei in the lactating rat (203). Suckling increased NE



February, 1992

turnover in the rostral PVN/ACN region and in SON, suggesting that the noradrenergic innervation to the OTcontaining magnocellular regions is activated during suckling (Fig. 6). That this is critical for OT release was suggested by the findings that destruction of the noradrenergic terminals in these regions with 6-hydroxydopamine attenuated the suckling-induced release of OT (203). These recent results support the earlier pharmacological studies on the importance of the noradrenergic input to the OT neurosecretory cells for suckling-induced OT release and further suggest that the stimulatory aadrenergic tone over OT secretion is exerted, at least in part, within the magnocellular regions. On the other hand, activation of /3-adrenergic receptors inhibits OT release under some conditions. Tribollet et al. (201) first reported that the j8-adrenergic antagonists propranolol or oxprenolol did not affect the occurrence of milk ejections in lactating rats that were actively milk ejecting but facilitated the reflex when administered to animals previously not responsive to the suckling stimulus. Conversely, Moos and Richard (204) found that central administration of the /3-adrenergic agonist isoproterenol decreased the occurrence of suckling-induced milk ejections, suggesting suppression of OT release. These earlier observations have been confirmed and extended more recently with RIA measurements of OT in conscious lactating rats (202). In these studies, repeated iv bolus injections of isoproterenol inhibited sucklinginduced OT release, and this effect was prevented by the 0-adrenergic antagonist propranolol, which greatly augmented suckling-induced release of OT when given alone. This finding suggests that some inhibitory /3-adrenergic influence is present during suckling. The neural loci at which the inhibitory /3-adrenergic

rPVN/ ACN

SON

cPVN

ARC

45

effect is exerted have not been conclusively identified, and there is evidence for both central (205) and neural lobe sites (see Section IV.B.2). It is also unknown at present what specific function /3-adrenergic inhibition might have in the lactating rat. There is some evidence that the /3-adrenergic ligand responsible might be epinephrine, which is released briefly from the adrenal medulla during suckling {Section I.A.2). For example, the facilitatory effect of propranolol on suckling-induced OT release was mimicked by adrenal demedullation, and the excitatory effect of either manipulation was prevented by /3-adrenergic receptor stimulation (202). These studies imply that a catecholamine of adrenal origin has an inhibitory effect on OT release via an action at /3adrenergic receptors. 3. Pharmacological studies on OT neuronal activity. Despite the caveat that most of the studies assessing the cellular actions of adrenergic agents on the activity of magnocellular neurons have not conclusively identified the cells as oxytocinergic, there appears to be some agreement in the conclusions derived from electrophysiological experiments and those reached from studies on OT release. In early studies, predominantly inhibitory actions were reported when NE was microelectrophoretically applied to antidromically identified neurosecretory cells in SON or PVN (OT-VP distinctions were not made) in vivo (206-208), and these effects in part appeared to be mainly mediated via a 0-adrenergic mechanism. j8-Adrenergic-mediated inhibitory effects of NE have been confirmed more recently (205). These findings suggest that one locus for the /3-adrenergic inhibition of OT release during lactation might be at the level of the OT perikarya. In contrast, recent studies, in which recordings were made from SON neurons in vivo and in vitro, demonstrate the major effect of NE as excitation, mediated via the ai-receptor (55, 209-213). According to Randle et al. (209), NE and a r agonists induced membrane depolarization, shortened the hyperpolarizing afterpotential, and increased the magnitude of the late depolarizing afterpotential in unidentified SON neurons. On the basis of these observations, it was argued that the actions of NE might involve inhibition of a transient K+ current (55, 209), which might enhance OT neuronal excitability, thereby facilitating the high frequency discharge characteristic of OT neurons during a milk ejection burst.

ME

FiG. 6. Effects of suckling on the turnover rate of NE (picograms per Hg protein/h) in microdissected hypothalamic nuclei of lactating rats (based on data in Ref. 203). Turnover rate was calculated from the decline in NE after synthesis inhibition with a-methyltyrosine. Suckling significantly ('*, P < 0.01) increased NE turnover rate in the rostral PVN/ACN area (rPVN/ACN) and SON, but not in the caudal PVN (cPVN), arcuate nucleus (ARC), or median eminence (ME).

B. Dopaminergic control of OT release 1. Neuroanatomical studies. Because of the dense noradrenergic projection to magnocellular regions of the hypothalamus, the discovery of a dopaminergic innervation to these areas awaited the development of techniques with increased sensitivity and specificity for this


46


catecholamine. Even though the concentration of dopamine (DA) is low relative to NE (214), it is now clear that dopaminergic fibers occur throughout the SON and in the magnocellular and parvocellular subdivisions of the PVN (215, 216), with frequent synaptic contacts between DA-immunopositive fibers and neuronal cell bodies and processes of both OT and VP cells (185, 216). The source(s) of the DA innervation to PVN or SON has not been precisely defined but has been considered most likely to arise from the All, A13, and A14 cell groups (188) of the posterior and periventricular hypothalamus (215). 2. Pharmacological studies on OT release. The preponderance of evidence favors an excitatory role for DA in control of OT release during lactation. After IVT administration, DA and several DA agonists stimulated OT release in lactating rats, as determined from elevations in intramammary pressure, and these effects were blocked by selective DA antagonists (198, 217). DA also stimulated OT release after IVT administration to male rats (199). Suckling-induced milk ejections were blocked after systemic administration of nonselective DA antagonists (198, 218), suggesting the importance of this excitatory dopaminergic influence on the milk ejection reflex. More recent studies (219), using agonists and antagonists selective for DA receptor subtypes and RIA measurements of plasma OT concentrations, indicate that the stimulatory effect is mediated via the D-l (positively coupled to adenylate cyclase-cAMP messenger system), rather than the D-2 (negatively coupled to this messenger) subtype. Investigations employing intracerebral microinjections indicate that DA acts primarily within the magnocellular nuclei, with the SON particularly responsive to stimulation with a D-l DA agonist (Fig. 7). On the other hand, there have also been reports of inhibitory influences of DA on OT release in lactating rats (203) and in the in vitro hypothalamic-neurohypophysial preparation (220,221). In vivo studies by Crowley and co-workers (203, 219) demonstrated that sucklinginduced OT release was inhibited by systemic treatment with agonists at the D-2 DA receptor, while administration of D-2 antagonists increased plasma OT concentrations in lactating rats separated from their litters. Further studies (222), however, suggested that the actions of these agents are not reflective of a direct inhibitory dopaminergic regulation of OT release, but rather are secondary to actions of PRL, whose secretion from the anterior pituitary is inhibited by a D-2 agonist and stimulated by a D-2 antagonist in parallel with OT. Thus, the stimulatory effect of the D-2 antagonist domperidone on plasma OT concentrations was prevented by prior immunoneutralization of PRL, while the inhibitory ef-

0 S 10 Basal

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5 15 30 45 60 SKI" 3 8 3 9 3

1 5 10 30 Ang II

0 5 10 Basal

5 15 30 45 60 SKf 36393

1 5 10 30 Ang II

5 15 30 45 60 SKf 38393

1 5 10 30 Ang II

40-

20-

0 5 10 Basal

Time of collection in a period, min.

FIG. 7. Effects of intracerebral microinjection of the D-l dopamine agonist SKF 38393 and Ang II into the third ventricle (3V), PVN/ACN area, or SON on plasma OT concentrations in lactating rats separated from their litters. Significant increases in circulating OT were produced by D-l stimulation at each injection site, with the most marked response seen in SON. Microinjection of the D-l agonist into sites dorsal to these areas or into the medial basal hypothalamus were ineffective. Ang II (100 ng) also significantly increased OT release after administration into 3V, PVN/ACN, or SON. [From Parker, S. L., and W. R. Crowley, unpublished observations.]

feet on OT release of a D-2 agonist was reversed by treatment with PRL. In addition to possibly clarifying discrepancies in the literature over the actions of OT, these studies have uncovered a potentially important humoral influence over OT secretion exerted by another hormone of lactation, PRL (see Section IV.B.3). 3. Pharmacobgical studies on OT neuronal activity. Studies evaluating the effects of DA or agonists on the activity of OT neurons generally support the view that DA excites OT release via an action in the magnocellular regions. IVT administration of DA or the agonist apomorphine increased the frequency of milk ejections and stimulated the activity of OT neurons in the PVN, as indicated by increased frequency and amplitude (total number of spikes) of the milk ejection bursts (217). Conversely, DA antagonists given IVT prevented the suckling-induced activation of OT neurons as well as milk ejections, providing strong support that DA exerts a physiologically significant role in the milk ejection reflex (217). An effect of DA to increase activity of continuously firing (possibly oxytocinergic) cells in slices of SON in vitro has also been observed, with indirect evidence that this was a D1 receptor-mediated action (76).


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C. Central actions of other neurotransmitters on OT secretion Although there are indications that other neurotransmitter systems may also participate in the regulation of OT neuronal activity and release, the combination of anatomical, pharmacological, and electrophysiological approaches that has been applied to the catecholamine systems has not been directed at other systems, and physiological evidence for their involvement during lactation is particularly scant. Despite this incomplete record, however, the available evidence does indicate potentially relevant actions of serotonergic, cholinergic, and both excitatory and inhibitory amino acid systems on OT secretion. 1. Serotonin (5-HT). Projections from serotonergic nuclei in the brainstem innervate both the PVN and SON (150, 175, 223), and according to Sawchenko and co-workers (223), 5-HT-immunopositive fibers are distributed throughout these regions and appear somewhat more concentrated in the subdivisions dominated by OT cells. Retrograde tracing studies by this group indicated that the B7, B8, and B9 cell groups in the mesencephalon [Dahlstrom and Fuxe nomenclature (188)] contribute this innervation. Disparate effects on OT secretion have been reported after manipulations of serotonergic neurotransmission. In an early study (224), systemic administration of 5-HT to lactating rats decreased the weight gain of the offspring, which was used as evidence of the occurrence of milk ejections. This was proposed to reflect central inhibition of OT release, rather than an action at the mammary gland, as 5-HT does not antagonize, but rather mimics, the contractile effect of OT on mammary myoepithelium. Consistent with these observations, Moos and Richard (225) subsequently reported that IVT administration of 5-HT in urethane-anesthetized rats interrupted the regularity of the milk ejection bursts of cells in the PVN. However, these investigators also found that 5-HT receptor antagonists or the synthesis inhibitor pchlorophenylalanine inhibited milk ejections (litter weight gains) in conscious lactating rats, suggestive of an excitatory influence of this transmitter that apparently is influenced by the presence of anesthesia. More recent pharmacological studies in conscious male rats (226) also point to a stimulatory action of 5-HT on OT release and suggest that the effect is mediated by the 5HT2 receptor subtype. 2. Acetylcholine (ACh). Consistent evidence exists for cholinergic stimulation of OT secretion in both lactating and nonlactating animals, but details on the receptor and postreceptor mechanisms involved and on the physiological significance of the effect are lacking. Cholinergic

47

nerve terminals (178), as well as nicotinic and muscarinic cholinergic receptors (227), are present throughout the hypothalamic magnocellular nuclei, although the source of the cholinergic fibers has not been established (55). Hayward and co-workers (228, 229) have reported an abundance of putative nicotinic binding sites (based on very high affinity a-bungarotoxin binding) in proximity to magnocellular neurosecretory cells throughout both of these nuclei, with a particularly dense concentration in the ventral glial lamina of the SON, which contains many OT-positive dendritic processes. In male and lactating female rats, IVT administration of ACh stimulates OT release (199, 230, 231), but the receptor pharmacology of this response is unclear. Clarke et al. (231) observed that muscarinic agonists, but not nicotine, mimicked ACh in stimulating OT release, yet nicotinic, rather than muscarinic, antagonists inhibited suckling-induced increases in intramammary pressure. Both nicotinic and muscarinic cholinergic receptors were also implicated in early work by Moss et al. (207) showing stimulation of PVN neurosecretory cell activity in response to microiontophoretically applied ACh, and in more recent studies by Honda et al. (232), in which PVN OT cells were identified electrophysiologically in lactating rats. In both cases, the effect of ACh was most effectively inhibited by a combination of muscarinic and nicotinic antagonists. Using either in vivo (233) or in vitro (234) recordings of SON neuronal activity, other investigators have detected either no effect or an inhibition by ACh on the activity of putative OT neurons; these latter studies, however, were not performed in lactating rats. 3. Glutamate. Intracellular recordings (234) indicate that much of the basal excitatory neurotransmission in the hypothalamic magnocellular nuclei is generated by an excitatory amino acid (EAA) neurotransmitter. Glutamate-immunopositive nerve terminals have been detected in the SON, and particularly in the ventral glial lamina (235, 236), and application of EAA agonists to SON or PVN neurons routinely increases their excitability (55, 210). In the PVN, 6-cyano-7-nitroquinoxoline2,3-dione (CNQX), which is an antagonist at non-Nmethyl-D-aspartate (NMDA) receptors (237), blocked almost all of the excitatory postsynaptic potentials (236), but Renaud and Bourque (55) have implicated the NMD A, as well as non-NMDA, subtypes of EAA receptors in this response. Surprisingly, to date, there have been no reports of critical physiological experiments to test whether EAA agonists or antagonists alter OT release in lactation, or whether an EAA contributes to driving the milk ejection bursts of activity in OT cells induced by suckling. 4. y-Aminobutyric acid (GABA). Similarly, there is com-


48


pelling anatomical and electrophysiological evidence pointing to a role for GABAergic neurons in control of OT neuronal activity, but no physiological studies addressing central GABAergic influences on OT release during lactation. Anatomical studies have revealed a GABAergic innervation to both the PVN and SON (238242). The sources of these fibers have not been established, but they could derive in part from GABA-positive perikarya dorsolateral to the SON (55, 157) and/or from several other forebrain regions, such as the median preoptic nucleus, that send afferents to the magnocellular nuclei (55). Synaptic contacts between GABAergic nerve terminals and OT-positive cell bodies and processes in SON have been documented (157, 242), and GABAergic synapses may account for approximately 50% of all synapses in the SON (240). As noted in Section II.B, these GABAergic-OT connections are among the ultrastructural elements in the SON that undergo dynamic organizational changes during lactation; thus, the incidence of double synapses (i.e. one terminal contacting two OT cells simultaneously) increases during lactation, and many of the terminals involved are GABA-positive (157, 242). These observations imply functional significance to these GABAergic connections. As expected, the available evidence from extra- and intracellular recordings indicates that GABA inhibits activity in neurosecretory cells of the PVN and SON (55, 210, 243-245). This action is mediated by hyperpolarization and activation of a Cl" conductance via the classical GABA-A receptor. In vitro recordings from SON neurons in hypothalamic slices or explants (243-245) suggest that a considerable proportion of the spontaneous inhibitory postsynaptic potentials observed in magnocellular neurons are mediated by GABA. D. Central actions of neuropeptides on OT secretion In addition to the "classical" neurotransmitters, several neuropeptides (OT itself, endogenous opioid peptides, and activin) have been implicated in the control of OT secretion during lactation, in part through actions exerted centrally. This section will also review the effects of four other peptides, vasoactive intestinal polypeptide, angiotensin II, cholecystokinin, and corticotropin-releasing factor (CRF), whose role in lactation is uncertain, but which may be important for OT release in other physiological contexts. 1. OT. Over the past decade considerable evidence has coalesced to indicate that OT, released locally within the PVN and SON from neuritic processes, and acting locally within these nuclei as a transmitter or modulator, exerts a physiologically significant, excitatory influence over the activity of OT neurons and the resultant release of the peptide during lactation (2). Initial studies by

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Freund-Mercier and Richard (246, 247) demonstrated that administration of OT via the third ventricle to anesthetized rats suckling their litters increased the frequency of milk ejections and the frequency and amplitude of the milk ejection bursts recorded in the PVN; similar treatment with VP was ineffective. Exogenously administered OT was ineffective in the absence of active suckling by the offspring (64, 246-248), leading these workers to suggest that locally released OT might act within the magnocellular regions in a modulatory manner to facilitate the OT cells' response to somatosensory afferents. Because third ventricular administration of an OT receptor antagonist decreased the frequency of milk ejections as well as the frequency and amplitudes of the neurosecretory bursts, it was proposed that the intracerebral facilitatory influence of OT on its own secretion is a critical component of the milk ejection reflex (247). Support for this seminal hypothesis has come from a number of further observations. Application of OT to an in vitro slice preparation of PVN or SON increases the firing rate of the majority of continuously active (putative OT) cells (77, 248). The presence of OT-immunoreactive processes that contact OT-positive perikarya and dendrites (148), and of specific OT binding sites (249, 250) have been detected in the PVN and SON. Morphological evidence has also been presented that OT may be released from dendritic processes rather than or in addition to recurrent axonal collaterals (146). Perhaps most critically, OT release within the magnocellular nuclei has been demonstrated using both in vitro (251-253) and in vivo (65, 254) approaches. The in vivo OT release in the SON, which was detected with a push-pull perifusion technique, was increased during milk ejection bursts, but did not occur in response to administration of hypertonic saline (254), suggesting that the intermittent OT neuronal activity characteristic of lactation, but not the tonic increase that occurs to hyperosmolar stimuli, may be critical for intranuclear OT release to occur. Intranuclear release of OT during lactation may play several important roles. Moos et al. (254) found that the initial increase in the intra-SON release of OT preceded the first milk ejection, suggesting that a critical level of central OT release must be achieved before the peptide is elevated in the systemic circulation. These investigators (57, 65) have also obtained evidence that local release of OT may play a role in recruitment of OT cells into the population responding to suckling. Administration of OT to one magnocellular nucleus facilitated the activity of OT cells in both PVN and SON on the side contralateral to the injection, suggesting that centrally released OT may also influence activity in internuclear connections. Finally, chronic central administration of OT to nonlactating animals resulted in increased incidences of somatic juxtapositions and shared synapses


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(255), which are characteristic of the magnocellular nuclei during lactation. Thus, chronic central release of OT may be a mechanism contributing to the induction of the well known plastic structural changes in this system associated with lactation. 2. Endogenous opioid peptides. There is general agreement that stimulation of opioid receptors with a variety of agonists, including endogenous peptides such as metenkephalin and jS-endorphin, inhibits OT release, particularly during stimulated states such as lactation (256261) and dehydration (262-264). Conversely, blockade of opioid receptors with the antagonist naloxone generally does not affect plasma OT concentrations under basal conditions, but enhances OT released by suckling, stress, hypertonic saline, and during parturition (33, 257, 262, 265-271). These findings imply the existence of an endogenous opioid tone that may be operative under specific conditions to restrain the amount of OT released in response to physiological stimuli. The observations that morphine and naloxone reciprocally altered plasma levels of OT but did not affect the firing patterns of OT cells during lactation or stimulation with hypertonic saline (257,271) led to the initial proposal that opioid inhibition of OT release was not exerted centrally but occurred proximal to the release process in the neurohypophysis

(Section I V.B.I). However, recent reports indicate that under some circumstances, opioid agonists may depress the activity of OT neurons through a central action. An enkephalin analog inhibited the activity of unidentified neurosecretory cells recorded intracellularly in guinea pig PVN and SON (272), while morphine, leu-enkephalin, and an analog, D-ala, D-leu-enkephalin, inhibited the firing of putative OT neurons in vivo (34, 210) and in vitro (273, 274). In contrast to its lack of effect in morphine-naive rats, naloxone markedly increased both OT release and the firing rate of OT neurons when administered to rats previously rendered tolerant to and physically dependent on morphine (275), suggesting that some centrally mediated opioid influences can be revealed under these nonphysiological conditions. How opioid effects in the magnocellular regions contribute to the regulation of OT secretion in the nondependent animal is unclear at present. The magnocellular nuclei, and OT-containing subdivisions in particular, receive innervation from enkephalin-positive (276, 277) and ACTH (by implication, /3-endorphin-containing)positive fibers (278). There is also evidence for involvement of spinal sites, as intrathecal morphine prevented suckling-induced rises in intramammary pressure (260). However, intrathecal naloxone did not alter OT release in these studies, suggesting the lack of tonic inhibitory opioid tone in this locus as well as more centrally.

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3. Activin. The gonadal peptide activin, which is a dimer of two inhibin-j8A subunits (279), is expressed in the central nervous system, including a cluster of peptidergic, nonadrenergic cells in the NTS that project to the PVN and SON, where they contact OT cells (277, 280). According to Sawchenko and co-workers (277), many of these cells also express somatostatin and/or enkephalinlike immunoreactivities. In a preliminary report, Plotsky and co-workers (197) found that IVT administration of activin increased OT release in male rats, while infusion of an antiserum against this peptide into the PVN prevented suckling-induced OT release in lactating females. It is interesting to note that nonadrenergic as well as adrenergic afferents to the magnocellular hypothalamus emanate from the NTS (193), and it is conceivable that the stimulatory effect of NTS stimulation on OT release (196, 197) is due to action of both NE and activin that are present in separate ascending systems. 4. Vasoactive intestinal poly peptide. Vasoactive intestinal polypeptide also stimulates OT release in male rats after central injection (281). However, the specific role of this peptide in lactating females and its sites and mechanisms of action are unknown. 5. Angiotensin II (Ang II). Ang II is among the most effective stimulants of OT release and the firing of magnocellular neurons (putative OT cells) and is active via either the systemic (282, 283) or central (284-288) routes. There is electrophysiological evidence for multiple central sites for the Ang II stimulation of OT release, including the subfornical organ (282, 283, 289) and anteroventral third ventricle region (289), both of which are considered targets for circulating Ang II and which project to the hypothalamic magnocellular nuclei (290-292), as well as the magnocellular nuclei themselves (289, 293). The presence of nerve fibers immunopositive for renin (294) and for Ang II (291, 295) and of specific Ang II binding sites (296) in the PVN and SON further implicate these regions as a site of action for this peptide in OT release. Consistent with this view, discrete microinjection of Ang II into the PVN/ACN or SON markedly stimulates OT release in lactating rats (Fig. 7). Ang II also stimulates OT neuronal activity in lactating rats (289), but its physiological importance for the milk ejection reflex has not been determined. On the other hand, central treatment with saralasin, an Ang II receptor blocker, prevented the activation of PVN neurosecretory cells (both oxytocinergic and vasopressinergic) in response to hypertonic saline or hemorrhage (288). Because OT release in response to perturbations of the cardiovascular system is likely to be mediated by neural pathways separate from those activated by suckling (290), a central Ang II neuronal system, possibly deriving from the circumventricular organs and acting in


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the magnocellular nuclei, may be important for the release of OT specifically in response to hyperosmolality and hypovolemia. 6. Cholecystokinin (CCK) and CRF. As reviewed in Section IV.B.l, populations of magnocellular OT cells in the PVN coexpress CCK or CRF, and both of these peptides modulate OT release from the neurohypophysis (297). CCK also activates the firing of OT cells (298, 299) through a peripheral action involving increased vagal activity (35), whose influence is transmitted via an as yet undefined central pathway. Leng and co-workers (299) have shown that the peripheral CCK effect is highly selective for OT, as opposed to VP, cells and therefore may be used as a reliable criterion for defining magnocellular neurons as oxytocinergic in electrophysiological experiments. Some indirect evidence that OT neurons may respond directly to CCK is found in the report that CCK binding sites are present in the OT-containing subdivisions of the magnocellular and accessory neurosecretory nuclei, and that the numbers of these sites increase during hypertonic saline challenge (300). Central administration of CRF also increases OT release in male and female rats (39, 301). This effect may be exerted, in part, within the magnocellular nuclei, in view of the abundance of CRF-immunopositive fibers in these areas, probably arising from the CRF perikarya in parvocellular PVN (302) as well as the magnocellular OT-CRF cells. Although not extensively characterized, it seems likely that the CRF stimulation of OT release is an important component of the OT response to stress. Interestingly, Lightman and colleagues (301) have recently shown that the OT response to CRF is abolished during lactation, suggesting that one basis for the decreased effectiveness of stress to stimulate OT release during lactation (49-52) might be a reduction in the responsiveness of the OT system to CRF.

IV. Modulation of OT Release from the Neurohypophysis In addition to the centrally exerted controls reviewed above, OT release from neurosecretory terminals is subject to local regulation by neurochemical messengers acting in the neurohypophysis. These include peptides coexpressed in the OT- and VP-secreting cells, other centrally derived neurotransmitter systems that innervate the neural and intermediate lobes of the pituitary, and the anterior pituitary hormone, PRL. Although many of these effects undoubtedly will be shown to have physiological significance, their specific contributions to the characteristic patterns of OT release during lactation, parturition, or in response to other provocative stimuli remain undefined.

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A. Anatomy and morphology of the neurohypophysis The axons of magnocellular neurosecretory neurons project into the neurohypophysis and form terminals near fenestrated capillaries (303). Although there is considerable overlap in their localization, OT fibers are preferentially distributed in the perimeter of the lobe, while VP fibers tend to localize more centrally (304,305). The extent of branching of individual fibers within the neurohypophysis is not well studied, but it is known that the individual axons have numerous, perhaps thousands, of swellings (106, 111, 306), some of which are large Herring bodies, which may function primarily as storage rather than release sites (307). At present, there are no data on differences between the terminal arborization patterns of OT and VP neurons, a topic that warrants examination. The highly varicose nature of the terminal spray may provide a morphological basis for modulation of stimulus-secretion coupling within the neurohypophysis. At the neurohemal contact zone, neurohypophysial terminals appose the basal lamina via synapse-like (synaptoid) specializations. These formations house accumulations of microvesicles, which are thought to sequester Ca++, and dense core vesicles, which contain the secreted peptides (Fig. 4). An individual axon or axonal branch may make several such contacts (111). The endings and axons also have a close association with pituicytes, the endogenous glia of the neurohypophysis (308), many of which are astrocytic (309, 310), or microglia (311). Numerous electron microscopic studies, reviewed elsewhere (56, 312, 313), have established that pituicytes often surround neurosecretory axons and interpose processes between neurosecretory endings and the basal lamina of the contact zone. Figure 4 depicts such arrangements. During lactation in rats, there is an increased contact of neurosecretory terminals with the basal lamina (and a complementary decrease in pituicyte-basal lamina appositions) and a decrease in the engulfment of neurosecretory axons by pituicytes, probably due to their active movement away from the axon-lamina interface (reviewed in Refs. 56 and 313). These data suggest that pituicyte-axon interactions in the neurohypophysis are dynamic, and that glia may take an active role in secretion. According to Hatton (313), pituicytes could modulate secretion as removable diffusion barriers, or by altering the extracellular microenvironment of neurosecretory fibers, thereby influencing ion flux and spike propagation within the terminal arborizations. Occasionally, pituicytes are also the recipient of synaptoid contacts from other neuronal systems, and as discussed below, neurotransmitters may also affect the pituicyte-


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axonal relationships, providing an indirect means of modifying hormone release. B. Neurochemical influences on OT release in the neurohypophysis 1. Coexpressed peptides a. Endogenous opioids: Since the initial suggestion by Clarke et al. (257) that the preeminent site for the inhibitory opioid influence over OT release might be the neurohypophysis, numerous observations, largely performed in studies with the isolated stalk-neurohypophysis preparation in vitro, have supported this view. It has been consistently demonstrated, for example, that blockade of opioid receptors with the generalized receptor antagonists naloxone or naltrexone enhance electrically evoked release of OT (314-323), while stimulation of opioid receptors with morphine reduces the evoked release of OT (324) in this system. These findings imply that an endogenous opioid suppresses the release of OT during the activated secretory process. Considerable attention has been paid to establishing the identity and source of the endogenous opioid peptide(s) responsible for such regulation and their sites and mechanisms of action within the neurohypophysis. Controversy surrounds the initial claim from immunocytochemical studies that magnocellular OT neurons coexpress the opioid peptide met-enkephalin (325-327), as others (328, 329) have failed to observe this material in OT cells. In addition, enkephalin mRNA was not detectable in magnocellular neurons unless neurohypophysial hormone secretion was stimulated by hypertonic saline (119), suggesting that this peptide may be co-expressed only under particular conditions. Also arguing against involvement of an enkephalin-like peptide are the findings that neither met- nor leu-enkephalin, nor several of their analog agonists affect electrically evoked OT release in vitro, under conditions where other opioid agonists are effective (317, 321, 330-332). There is also evidence that /3-endorphin, released from intermediate lobe, is not involved in the opioid inhibition of OT release (315). On the other hand, several forms of the opioid peptide dynorphin readily suppress OT release in vitro (319, 333, 334). This peptide is released by electrical stimulation of the neurohypophysis (316) and appears to act preferentially at the /c-subtype of opioid receptor (335); K-agonists are the most effective opioids in inhibiting OT release (321, 323, 334), while selective /(-antagonists are as effective as naloxone in elevating OT release both in vivo (336) and in vitro (337). Furthermore, the major and perhaps only subtype of opioid receptor in the neurohypophysis is the K-receptor (338-341). There is consistent and convincing evidence from immunocytochemical co-

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localization studies that the dynorphin innervation of the neurohypophysis is contained exclusively within the vasopressinergic neurons (123, 342, 343). These findings have led to the proposal that VP cells release dynorphin, which acts at K-receptors to "cross-inhibit" OT release under specific conditions (267, 344, 345). At present, relatively little is known concerning the cellular mechanisms affected by dynorphin to inhibit OT release. As discussed previously (Section I.B.4), the inactivation of K+ currents in neurohypophysial nerve terminals during repetitive firing may account for spike broadening, leading to enhanced Ca++ entry, and frequency-dependent facilitation of OT release. In isolated neural lobes, K+ channel blockade shifts the frequencydependent OT release curve to the left, such that the maximal hormone release is reached at lower frequencies (346). Under conditions of K+ channel blockade, naloxone fails to further enhance OT release at high (15 Hz) frequency stimulation, although some enhancement occurs at lower frequency (3 Hz) (347, 348). Further work is needed to determine whether opioid inhibition acts on the K+ currents controlling spike duration, on voltagesensitive Ca++ currents, which would normally be enhanced during spike broadening, or on some other mechanism. The K-receptor has been localized to neurosecretory endings (334, 339, 349), and in addition, fibers immunoreactive for leu-enkephalin (possibly dynorphinergic) have been observed to 'surround the neurohypophysial glial cells (pituicytes) and make synaptoid contacts upon their soma and processes' (Ref. 350, p. 229). A substantial amount of K-like opioid binding in the neurohypophysis can be attributed to pituicytes (339, 351). The functional significance of this finding is not established since it is not known what the role of the pituicyte is in influencing OT release or how this might be altered by an opioid peptide. However, the finding that /c-agonists inhibit OT release from a nerve terminal preparation devoid of pituicytes (334) argues against these cells as the major site of opioid action in the neurohypophysis. It is also possible that dynorphin influences the activity of other OT-regulatory systems present within the neurohypophysis. For example, naloxone increases the electrical stimulation-induced release of DA in this tissue (352), and, as reviewed below {Section IV.B.2), there is evidence for stimulatory effects of DA on OT release. Experiments by Zhao et al. (321, 322) have also implicated the noradrenergic innervation to the neurohypophysis. In these studies, the enhancement of stimulated OT release by naloxone was attenuated by adrenergic receptor blockers, leading these workers to suggest that release of NE was inhibited by opioids and that noradrenergic nerve endings were interposed between the opioid peptide and the OT terminal. Thus, removal of


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opioid inhibition with naloxone would disinhibit the release of NE, which could then act in turn upon the OT nerve terminals to stimulate release. However, a subsequent study by this group (353) found that naloxoneenhanced OT release was normal despite nearly total depletions of NE, indicating that an opioid-noradrenergic interaction was not of major importance. b. CCK: Immunohistochemical findings suggest that many of the magnocellular OT cells in PVN and some of those in the SON coexpress CCK (326, 354-356). On the basis of relative content in the neurohypophysis and the presence of specific CCK binding sites localized to nerve terminals in this tissue, Bondy, Gainer and coworkers (297, 357) have argued this peptide's role in the autoregulation of OT release. This group has demonstrated that CCK-8 sulfate enhances basal release of OT from an in vitro stalk-neurohypophysis preparation, and that neither electrical stimulation nor influx of extracellular Ca++ was required for this effect. The additional observation that the stimulatory effect of CCK-8 on OT release was blocked by an inhibitor of protein kinase C suggests that CCK receptors on OT nerve terminals might be positively coupled to the Ca++/inositol phosphate messenger system. The neurohypophysial concentrations of OT and CCK are decreased in parallel during lactation (354) and by salt loading (123, 354, 358), suggesting that the positive autoregulatory influence of CCK as a co-peptide may be of importance under several physiological conditions in which OT release is elevated. c. CRF: CRF is a third example of a peptide coexpressed in subsets of magnocellular neurons with effects on OT release in the neurohypophysis. Evidence from several immunocytochemical studies indicates that approximately one-third of the magnocellular OT cells express CRF (359, 360) and that the two peptides are localized in the same secretory granules (361). Similar to CCK, CRF also stimulates basal OT release in vitro, but unlike the effect of CCK, the action of CRF is indirect (297, 362) and involves first the release of a-MSH from the intermediate lobe (363), which then acts in the neurohypophysis to promote OT release. Bondy and Gainer (362) also obtained evidence that activation of the inositol phosphate messenger system mediates the response to a-MSH. 2. Afferent neural systems: catecholamines and GABA. The morphology of tuberohypophysial DA system that innervates the neurohypophysis has been extensively investigated, but the nature of its regulatory influence on OT secretion is far from clear. DA-containing fibers derived from anterior aspects of the arcuate nucleus and adjacent periventricular nucleus in the medial-basal hypothalamus descend through the pituitary stalk and end in proximity to neurosecretory axons and pituicytes

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(364-367). This system may be distinct from that which innervates the OT-containing regions of the PVN and SON (215, 216). Both D-l and D-2 DA binding sites are present in the neurohypophysis (368, 369), but whether they are localized on both types of cellular elements, i.e. terminals or pituicytes, has not yet been determined. In contrast to the relatively consistent reports that DA excites OT release through its central actions (Section HI. B), there is disagreement concerning the effect of DA on OT release at the level of the nerve terminals in the neurohypophysis. For example, inhibitory effects of DA on multiunit activity in neurosecretory axons of the pituitary stalk in vivo were reported by Passo et al. (370), and on OT release from isolated neurohypophysis in vitro by Barnes and Dyball (371) and Vizi and Volbekas (372). In contrast, stimulatory effects of this catecholamine on OT release were observed by Bridges et al. (199) and Crowley et al. (219), both using isolated stalk-neurohypophysial preparations, in vitro. In the latter study, the electrically evoked release of OT was enhanced by agonists at the D-l DA receptor while D-2 agonists were ineffective. Conflicting views of the role of tuberohypophysial DA have also emerged from studies in which the level of activity of this system has been monitored in correlation with physiologically induced changes in OT secretion. For example, dehydration increased both the synthesis (373) and release (374) of DA in neurohypophysis, but suckling was associated with a transient decrease of DA turnover in this structure (203). One potentially complicating factor is that the dopaminergic innervation of the neurohypophysis has been implicated in the regulation of PRL secretion from the anterior lobe of the pituitary gland (375), and as reviewed in the next section, this hormone can exert modulatory effects on the release of OT. Adding to the complexity of the neurochemical modulatory mechanisms in the neurohypophysis are the recent observations that contained within the tuberohypophysial dopaminergic axons is the inhibitory amino acid transmitter GABA (376, 377). The release of GABA in neurohypophysial tissue (378), and the presence of GABAergic fibers in the neurohypophysis in the vicinity of pituicytes and neurosecretory axons (241, 379, 380), had been described earlier. Studies conducted with neurohypophysial preparations indicate that GABA decreases the compound action potential in neurosecretory axons (381, 382), and the release of OT (383, 384), both via an action at the GABA-A type receptor. However, there is one report of a stimulatory effect of the GABAA agonist isoguvacine in this system (385). Studies examining the potential pre- and/or postsynaptic interactions of DA and GABA on OT release have not been reported to date. A small noradrenergic innervation of the neurohypo-


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physis is supplied in part from fibers of the sympathetic superior cervical ganglion and in part from axons ascending from the lower brainstem, particularly the A2 cell group (386, 387), which provides innervation to the magnocellular OT cells of the PVN and SON (192-195). The predominant subtype of adrenergic receptor in the neurohypophysis is /32-like and localized in large part on pituicytes (388). Opposite effects of adrenergic receptor stimulation or blockade on OT release from the neurohypophysis have been reported. In one study (68), administration of the /3-adrenergic antagonist propranolol to lactating rats facilitated the release of OT in response to in vivo pituitary stalk stimulation, a finding consistent with the demonstrations of 0-adrenergic receptor-mediated adrenergic inhibition over OT release, reviewed in an earlier section (Section III.A.2), and suggestive of the neurohypophysis as one site for this influence. On the other hand, both f}- and a-adrenergic receptor stimulation augmented electrically induced OT release from isolated neurohypophysis in vitro (321, 322). It is difficult to reconcile these disparate results, although it may be significant that in studies demonstrating an excitatory effect of adrenergic agonists, the neurohypophysial tissue was obtained from male animals (321, 322), while /?- adrenergic inhibitory effects have largely been observed in lactating animals (68, 201, 202, 204). It is possible that the state of lactation in some way alters the direction of the neurohypophysial adrenergic influence over OT release. /?-Adrenergic mechanisms have also been implicated in the morphological reorganization seen in the neurohypophysis when the magnocellular neurosecretory systems are activated. Incubation of neural lobes in vitro in the presence of the /3-adrenergic agonist isoproterenol decreases the pituicyte coverage and increases nerve ending proximity to the pericapillary basal lamina (389, 390). Bicknell et al. (391) and Hatton et al. (392) have reported that endogenous agonists at the /32-receptor, NE and epinephrine, as well as isoproterenol changed the shape of pituicytes in culture from the predominant large, flattened, and irregular form to a smaller, rounded, and more stellate structure, and it was suggested that this may be the structural basis for the uncovering of the perivascular space to OT neurosecretory endings in times of increased neurosecretion. Thus, it is possible that stimuli activating the hypothalamic neurosecretory system also increase release of a ligand at /3-adrenergic receptors on pituicytes, which induces the morphological changes that facilitate the access of secreted OT into the systemic circulation. As noted by Hatton et al. (392), this ligand could be NE derived from central neurons or the sympathetic nervous system, or circulating epinephrine after release from the adrenal medulla.

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3. PRL. Recent evidence indicates that PRL can enhance OT secretion through actions exerted both centrally (393) and in the neurohypophysis (222). Biologically active preparations of ovine or rat PRL did not affect basal release, but significantly enhanced the electrically evoked release of OT from an isolated stalk-neurohypophysial preparation from lactating rats (222). The cellular mechanism of this effect is unknown at present and its precise role and importance during lactation has not been determined. However, because interference with the release or action of PRL prevents OT secretion in response to suckling (222), it is possible that PRL exerts a physiologically critical permissive function over OT secretion during lactation (see below).

V. Conclusions and Future Perspectives The suckling-induced release of OT in the rat is unique among neuroendocrine reflexes in its temporal pattern, and any hypotheses regarding underlying neural mechanisms must account for a number of salient features of the milk ejection response (54-57). These include 1) the minutes-long latency from the onset of suckling to the first milk ejection [the 'recruitment phase' (57)], 2) the periodicity of the milk ejection bursts in firing and OT release despite a continuous stimulus input, 3) the amplitude of the response, and 4) the apparent participation of most, if not the entire, population of OT neurosecretory cells in the milk ejection burst, implying one or more mechanisms for synchronization of these cells (57). To date, the characteristic bursting activity of OT cells, with periods on the order of minutes, has not been observed in vitro (55-57). However, few recordings have been made in identified OT neurons and in lactating rats, so it remains possible that the membrane properties of OT neurons, particularly in lactation, could contribute to synchronous bursting activity. The mechanisms underlying the periodic firing of OT neurons per se are unknown, and in particular are unexplained by our limited knowledge of the intrinsic conductances of OT neurons. The possible contribution of intrinsic mechanisms should not be ignored, however, since in other endocrine cells (e.g. pancreatic /3-cells), slow dynamics in the handling of intracellular Ca++ can contribute to bursting patterns on the order of 1-2 min (394). Some of the mechanisms thought to promote synchronous bursting in hippocampal neurons may apply to the OT neuronal organization during lactation as well (395). These would include excitatory synaptic interconnections among OT neurons (148), and an increase in close appositions between adjacent OT neurons, which may amplify the increase in extracellular K+, producing a small depolarization in neighboring neurons and thus increasing their likelihood of firing (156). Finally, a similar synchronizing


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influence could be provided by even weak electrotonic coupling among OT neurons (56). A. Central neurochemical mechanisms It seems very likely from the available evidence that the milk ejection burst is driven by a strong afferent input to the magnocellular OT cells that is activated by suckling, and as reviewed herein, a number of neurotransmitters and neuropeptides are candidates for this role. As of this writing, three neurochemical messengers, NE, DA, and OT itself, have been most strongly implicated in the central mechanisms that underlie sucklinginduced OT release. Electrophysiological studies suggest that the continuous neural activity that can be recorded from putative OT neurons in vivo and in vitro probably reflects a balance between excitatory neurotransmission mediated by an EAA, such as glutamate (55, 234, 237), and inhibitory transmission mediated by GABA (243245). The nature of the involvement of these systems in the neurosecretory bursting of OT cells is unknown at present, but should receive high priority in future investigations. For example, the fast onset depolarization of OT neurons by direct EAA receptor-operated opening of a cation channel (236, 396) would seem to be an ideal mechanism for the abrupt generation of an explosive increase in OT neuronal firing. Sections III.A and B reviewed the extensive and compelling evidence for important stimulatory actions of NE and DA on OT release, but a number of questions remain regarding their roles in suckling-induced OT release. One concerns whether and how the suckling-activated somatosensory afferents gain access to the NE and DA cells that innervate the OT neurons. No study to date has demonstrated whether neurons in the NTS, which contains the A2 noradrenergic cell group innervating PVN and SON (190, 192-194), become activated in response to suckling or whether destruction of this structure impairs milk ejection. It is intriguing to note that this region does appear to receive somatic information from ascending sensory pathways in the spinal cord (397,398), including spinal areas implicated in the milk ejection reflex (159-161). Similarly, the critical mesencephalic locus for the milk ejection reflex as identified in lesion studies does not appear to project directly to PVN or SON but to dorsal hypothalamic-ventral thalamic fields in which several DA cell groups are localized (159). It is therefore possible that these DA cells comprise one of the last links in the milk ejection pathway. If the effects of NE reflect inhibition of the outward K+ current as proposed (55, 209), it is conceivable that this could be achieved through second messenger-mediated alteration in K+ channel function (399). The involvement of the aradrenergic receptor subtype in the

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NE stimulation of OT secretion (198, 209-213), in turn, implicates the second messenger systems coupled to this receptor, i.e. either the generation of inositol phosphates and diacylglycerol (400, 401) and/or arachidonic acid metabolites (402, 403) in the cellular action of NE. One can also posit the involvement of a second messenger, rather than direct channel activation, in the action of DA, since the D-l receptor, which is positively coupled to the formation of cAMP and activation of the A-kinase (404), mediates the excitatory action of DA on OT release (219). Despite the involvement of NE and DA in the excitation of OT cells and their importance in the milk ejection reflex, it is difficult with our present understanding of their excitatory actions to link these transmitters to the explosive discharge of OT cells during a milk ejection burst. However, in the case of NE at least, the proposed mechanism of a reduction of the repolarizing transient K+ current could modify the amplitude (spike frequency and duration) of the burst. The pattern in which the excitatory afferents that drive the neurosecretory bursts arrive at the OT cells also remains to be determined. It is possible that these inputs are tonically activated by suckling stimulation and are presented to the OT cells in a continuous pattern. The intermittency of the OT cells' response could then be enforced by a strong, tonically exerted, inhibitory influence at the level of the OT soma and dendritic regions (e.g. predominantly by GABA) that must be either transiently removed or overridden for the burst to be expressed. Arguing against this, however, are the findings that the basal firing rate of OT neurons is similar in suckled and nonsuckled rats and that the background (interburst) activity of OT neurons in a lactating rat is not affected by the number of pups nursing (66). Alternatively, episodicity of OT release could be imposed on the OT cells by intermittent activation of the excitatory afferent input during continual suckling. In contrast to the episodic pattern of OT release in response to suckling, OT release can be repeatedly elicited each time the mammary nerve is stimulated (67, 71), provided there is a short (20-90 sec) rest period between stimulations. Continuous electrical stimulation of the mammary glands, however, does produce an intermittent pattern of milk ejection similar to that occurring in response to suckling (69), whereas continual stimulation of the spinal cord sensory tracts does not (68). These observations suggest that one factor imposing pulsatility in this system is sensory adaptation at the level of the mechanoreceptors in the mammary gland. In addition, there is evidence for gating mechanisms at spinal and particularly at supraspinal levels (405). Additional neurochemical mechanisms to promote in-


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termittent milk ejections operate in the mammary gland and may be integrated with the central controls. It is well established that epinephrine of adrenal origin antagonizes the actions of OT at the mammary myoepithelial cell via actions at the /3-adrenergic receptor (20, 406). Grosvenor and Mena (20) have reported that after a brief period of adrenergic-induced relaxation of myoepithelial contraction, the cells become hyperresponsive to OT. Epinephrine may also be released intermittently from the adrenal medulla during suckling to provide this modulation of OT responsiveness (72). A further consequence of neurotransmitter actions that directly promote the milk ejection burst is the activation of the central release of OT (reviewed in Section III.D.I). This process may be set in motion during the initial activations of individual OT neurons by suckling and may be viewed as a reinforcing mechanism to recruit OT cells into the responding population and also increase the amplitude of the individual neurosecretory bursts (57), both of which would increase OT release. This third neurochemically identified, excitatory mechanism apparently occurs only if the OT neurons fire episodically; as noted earlier, intranuclear release of OT is not observed in response to hypertonic saline treatment (254), which induces a continuous increase, rather than bursting activity in OT cells (54). B. Neurohypophysial mechanisms The modulation of OT release by various neurochemical messengers acting at the level of the neurohypophysis may represent means by which OT release is amplified or restrained within circumscribed limits. The physiological circumstances in which such local controls become important have in general not been defined, although the inhibition of OT release by an endogenous opioid and the stimulation of OT release by the anterior pituitary hormone PRL may have particular adaptive value during lactation. The most extensively characterized neurochemical regulatory influence in the neurohypophysis is the Kreceptor-mediated "cross-inhibition" (346) of OT release by dynorphin released from VP neurons (Section IV.B.l.a). The observation that blockade of opioid receptors, e.g. with naloxone, increases OT release in nonlactating, but not in lactating, rats suggests that endogenous opioids do not brake OT release in response to suckling (52). However, because naloxone does increase OT release in lactating and nonlactating rats after osmotic or volemic stimulation, Summy-Long and co-workers (Ref. 52, p. 542) have suggested that, 'when the lactating rat becomes dehydrated, the combination of decreased responsiveness of oxytocinergic neurons to osmotic and/or hypovolemic stimuli and inhibition of OT secretion by

55

opioid peptides may conserve pituitary stores of the hormone needed for milk ejection.' Initial results from our laboratories indicate that one important site of action for the stimulatory action of the anterior pituitary hormone PRL on OT release is the neurohypophysis as PRL increased the electrically evoked, but not the basal, release of the peptide from an isolated stalk-neurointermediate lobe preparation in vitro (222). In further studies (Parker, S. L. and W. R. Crowley, unpublished observations), PRL failed to affect OT release after injections into the third ventricle or PVN/ACN region, suggesting a noncentral site of action and consistent with the neural lobe as a major target. The physiological importance of this effect of PRL is suggested by the findings that suckling-induced release of OT was inhibited when the concomitant release of PRL was prevented by the DA agonist bromocriptine or when the action of PRL was antagonized by immunoneutralization (222). This implies that whatever the mechanism affected by PRL, it may be obligatory in order for OT release to occur. It is noteworthy that in the separation-reunion paradigm employed with the lactating rat, by the time the first milk ejection occurs, typically 10-15 min after the onset of suckling, circulating PRL concentrations are markedly elevated, although generally not yet maximal (26, 70, 71). Moreover, unlike OT, PRL levels remain elevated as long as the suckling stimulus is present (70, 71). The neurohypophysis, then, is likely to be exposed to high levels of PRL throughout the duration of suckling. In view of the growing evidence that a population of OT neurons, most likely innervating the median eminence, acts as a PRL-releasing hormone in lactation (25), one could envision a bi-directional positive feedback relationship between these two hormones of lactation. Thus, suckling may activate a subset of hypophyseotropic OT neurons that contribute to the initiation of PRL secretion, which may then facilitate the release of OT for milk ejection. Such a mutual stimulation between OT and PRL might be of considerable significance in the maintenance of lactation. References 1. Wakerley JB, Clarke G, Summerlee AJS 1988 Milk ejection and its control. In: Knobil E, Neill J (eds) The Physiology of Reproduction. Raven Press Ltd., New York, pp 2283-2321 2. Richard P, Moos F, Freund-Mercier M-J 1991 Central effects of oxytocin. Physiol Rev 71:331-370 3. Samson WK, Lumpkin MD, McCann SM 1986 Evidence for a physiological role for oxytocin in the control of prolactin secretion. Endocrinology 119:554-560 4. Johnston CA, Negro-Vilar A 1988 Role of oxytocin on prolactin secretion during proestrus and in different physiological or pharmacological paradigms. Endocrinology 122:341-350 5. Arey BJ, Freeman ME 1989 Hypothalamic factors involved in the endogenous stimulatory rhythm regulating prolactin secretion. Endocrinology 124:878-883


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6. Evans JJ, Robinson G, Catt KJ 1989 Gonadotrophin-releasing activity of neurohypophyseal hormones. I. Potential for modulation of pituitary hormone secretion in rats. J Endocrinol 122:99106 7. Evans JJ, Catt KJ 1989 Gonadotrophin-releasing activity of neurohypophyseal hormones. II. The pituitary oxytocin receptor mediating gonadotrophin release differs from that of corticotrophes. J Endocrinol 122:107-116 8. Swanson LW, Sawchenko PE 1980 Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31:410-417 9. Buijs RM, deVries GJ, Van Leeuven FW, Swaab DF 1983 Vasopressin and oxytocin: distribution and putative function in the brain. Prog Brain Res 60:115-122 10. Gibbs DM 1986 Vasopressin and oxytocin: hypothalamic modulators of the stress response: a review. Psychoneuroendocrinology 11:131-140 11. van Wimersma Greidanus TB, Burbach JP, Veldhuis HD 1986 Vasopressin and oxytocin. Their presence in the central nervous system and their functional significance in brain processes related to behaviour and memory. Acta Endocrinol (Copenh) [Suppl] 276:85-94 12. Johnston CA, Lopez F, Samson WK, Negro-Vilar A 1990 Physiologically important role for oxytocin in the preovulatory release of luteinizing hormone. Neurosci Lett 120:256-258 13. Caldwell JD, Jirikowski GF, Greer ER, Pedersen CA 1989 Medial preoptic area oxytocin and female sexual receptivity. Behav Neurosci 103:655-662 14. Watkins WB, Choy VJ 1988 Identification of neurohypophyseal peptides in the ovaries of several mammalian and nonmammalian species. Peptides 9:27-32 15. Holtorf AP, Furuyu K, Ivell R, McArdle CA 1989 Oxytocin production and oxytocin messenger ribonucleic acid levels in bovine granulosa cells are regulated by insulin and insulin-like growth factor-1: dependence on developmental status of the ovarian follicle. Endocrinology 125:2612-2620 16. Viggiano M, Franchi AM, Zicari JL, Rettori V, Gimeno MA, Kozlowski GP, Gimeno AL 1989 Involvement of oxytocin in ovulation and in the outputs of cyclo-oxygenase and 5-lipoxygenase products from isolated rat ovaries. Prostaglandins 37:367-378 17. Plevrakis I, Clamagirand C, Pontonnier GG 1990 Oxytocin biosynthesis in serum-free cultures of human granulosa cells. J Endocrinol 124:R5-R8 18. Foo NC, Carter D, Murphy D, Ivell R 1991 Vasopressin and oxytocin gene expression in rat testis. Endocrinology 128:21182128 19. Mena F, Clapp C, Martinez-Escalera G, Pacheco P, Grosvenor CE 1986 Integrative regulation of milk ejection. In: Amico JA, Robinson AG (eds) Oxytocin: Clinical and Basic Studies. Elsevier Science Publishers, New York, pp 179-198 20. Grosvenor CE, Mena F 1982 Regulating mechanisms for oxytocin and prolactin secretion during lactation. In: Muller EE, MacLeod RM (eds) Neuroendocrine Perspectives. Elsevier Biomedical Press, New York, vol 1:69-10 21. Lincoln DW, Paisley AC 1982 Neuroendocrine control of milk ejection. J Reprod Fertil 65:571-586 22. Lincoln DW, Hill A, Wakerley JB 1973 The milk ejection reflex of the rat: an intermittent function not abolished by surgical levels of anaesthesia. J Endocrinol 57:459-476 23. Wakerley JB, Lincoln DW 1973 The milk ejection reflex of the rat: a 20- to 40-fold acceleration in the firing of paraventricular neurones during oxytocin release. J Endocrinol 57:477-493 24. Fuchs A-R 1978 Hormonal control of myometrial function during pregnancy and parturition. Acta Endocrinol (Copenh) [Suppl] 221:1-10 25. Summerlee AJS 1981 Extracellular recordings from oxytocin neurones during the expulsive phase of birth in unanaesthetized rats. J Physiol (Lond) 321:1-9 26. Higuchi T, Honda K, Fukuoka T, Negoro H, Wakabayashi K 1985 Release of oxytocin during suckling and parturition in the rat. J Endocrinol 105:339-346 27. Higuchi T, Uchide K, Honda K, Negoro H 1986 Oxytocin release

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Dynamic studies of growth hormone and prolactin in female rat. Neuroendocrinology 21:193-203 Voogt JL, Sar M, Meites J 1969 Influence of cycling, pregnancy, labor, and suckling on corticosterone and ACTH levels. Am J Physiol 216:655-658 Dogterom J, van Wimersma Greidanus TB, Swaab DF 1977 Evidence for release of vasopressin and oxytocin into cerebrospinal fluid: measurements in plasma and CSF of intact and hypophysectomized rats. Neuroendocrinology 24:108-113 Mason WT 1983 Excitation by dopamine of putative oxytocinergic neurones in the rat supraoptic nucleus in vitro: evidence for two classes of continuously firing neurones. Brain Res 267:113-121 Yamashita H, Okuya S, Inenaga K, Kasai M, Uesugi S, Kannan H, Kaneko T 1987 Oxytocin predominantly excites putative oxytocin neurons in the rat supraoptic nucleus in vitro. Brain Res 416:364-368 Andrew RD, Dudek FE 1984 Analysis of intracellularly recorded phasic bursting by mammalian neuroendocrine cells. J Neurophysiol 51:552-566 Brimble MJ, Dyball REJ 1977 Characterization of the responses of oxytocin- and vasopressin-secreting neurones in the supraoptic nucleus to osmotic stimulation. J Physiol (Lond) 271:253-271 Poulain DA, Wakerley JB, Dyball REJ 1977 Electrophysiological differentiation of oxytocin- and vasopressin-secreting neurones. Proc R Soc Lond 196:367-384 Cobbett P, Smithson KG, Hatton GI 1985 Dye-coupled magnocellular peptidergic neurons of the rat paraventricular nucleus show homotypic immunoreactivity. Neuroscience 16:885-895 Cobbett P, Inenaga K, Mason WT 1988 Mechanisms of phasic bursting in vasopressin cells. In: Leng G (ed) Pulsatility in Neuroendocrine Systems. CRC Press, Boca Raton, FL, pp 155-196 Bourque CW, Renaud LP 1990 Electrophysiology of mammalian magnocellular vasopressin and oxytocin neurosecretory neurons. Front Neuroendocrinol 11:183-212 Bourque CW 1988 Transient calcium-dependent potassium current in magnocellular neurosecretory cells of the rat supraoptic nucleus. J Physiol (Lond) 397:331-347 Andrew RD, Dudek FE 1984 Intrinsic inhibition in magnocellular neuroendocrine cells of rat hypothalamus. J Physiol (Lond) 353:171-185 Bourque CW, Brown DA 1987 Apamin and d-tubocurarine block the afterhyperpolarization of rat supraoptic neurosecretory neurons. Neurosci Lett 82:185-190 Bourque CW 1991 Activity-dependent modulation of nerve terminal excitation in a mammalian peptidergic system. Trends Neurosci 14:28-30 Abe H, Ogata N 1982 Ionic mechanism for the osmoticallyinduced depolarization in neurones of the guinea-pig supraoptic nucleus in vitro. J Physiol (Lond) 327:157-171 Mason WT 1980 Supraoptic neurones of rat hypothalamus are osmosensitive. Nature 287:154-157 Bourque CW 1989 Ionic basis for the intrinsic activation of rat supraoptic neurones by hyperosmotic stimuli. J Physiol (Lond) 417:263-277 Negoro H, Honda K, Uchide K, Higuchi T 1987 Facilitation of milk ejection-related activation of oxytocin-secreting neurones by osmotic stimulation in the rat. Exp Brain Res 65:312-316 Andrew RD 1987 Endogenous bursting by rat supraoptic neuroendocrine cells is calcium dependent. J Physiol (Lond) 384:451-465 Bourque CW 1986 Calcium-dependent spike after-current induces burst firing in magnocellular neurosecretory cells. Neurosci Lett 70:204-209 Armstrong WE, Smith BN, Tian M 1991 Electrophysiological differences between identified oxytocin and vasopressin neurons recorded intracellularly from rat supraoptic nucleus in vitro. Soc Neurosci Abstr 17:473.9, p. 1189 Dyball REJ, Leng G 1986 Regulation of the milk ejection reflex in the rat. J Physiol (Lond) 380:239-256 Dudek FE, Gribkoff VK 1987 Synaptic activation of slow depolarization in rat supraoptic nucleus neurones in vitro. J Physiol (Lond) 387:273-296 Douglas WW, Poisner AM 1964 Stimulus-secretion coupling in a


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Lactation abolishes corticotropin-releasing factor-induced oxytocin secretion in the conscious rat.

Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C.

Lactation and the physiology of prolactin secretion.

Atropinization decreases oxytocin secretion in dairy cows.

The role of oxytocin in cardiovascular regulation.

Neuroendocrine regulation of lactation and milk production.

Homologous regulation of brain oxytocin receptors.

Regulation of adipose tissue metabolism in support of lactation.

Excitation of tuberoinfundibular dopamine neurons by oxytocin: crosstalk in the control of lactation.

Nonpuerperal lactation and normal prolactin regulation.

Studies in human lactation: milk composition and daily secretion rates of macronutrients in the first year of lactation.

Serotonin and serotonin transport in the regulation of lactation.

Reflex control of magnocellular vasopressin and oxytocin secretion.

Luteal oxytocin: characteristics and control of synchronous episodes of oxytocin and PGF2 alpha secretion at luteolysis in ruminants.

Oxytocinase in the female rat hypothalamus: a novel mechanism controlling oxytocin neurones during lactation.

Effects of daily exogenous oxytocin on lactation milk yield and composition.

Glucose regulation of glucagon secretion.

Regulation of lung surfactant secretion.

Regulation of gastric acid secretion.

Regulation of parathyroid hormone secretion.

Fractalkine signaling in regulation of insulin secretion.

Regulation of gonadotropin secretion in man.

Regulation of aldosterone secretion in primary aldosteronism.

Approaches Mediating Oxytocin Regulation of the Immune System.