Neuroendocrine regulation of lactation and milk production.

Neuroendocrine Regulation of Lactation and Milk Production William R. Crowley*1 ABSTRACT Prolactin (PRL) released from lactotrophs of the anterior pituitary gland in response to the suckling by the offspring is the major hormonal signal responsible for stimulation of milk synthesis in the mammary glands. PRL secretion is under chronic inhibition exerted by dopamine (DA), which is released from neurons of the arcuate nucleus of the hypothalamus into the hypophyseal portal vasculature. Suckling by the young activates ascending systems that decrease the release of DA from this system, resulting in enhanced responsiveness to one or more PRL-releasing hormones, such as thyrotropin-releasing hormone. The neuropeptide oxytocin (OT), synthesized in magnocellular neurons of the hypothalamic supraoptic, paraventricular, and several accessory nuclei, is responsible for contracting the myoepithelial cells of the mammary gland to produce milk ejection. Electrophysiological recordings demonstrate that shortly before each milk ejection, the entire neurosecretory OT population fires a synchronized burst of action potentials (the milk ejection burst), resulting in release of OT from nerve terminals in the neurohypophysis. Both of these neuroendocrine systems undergo alterations in late gestation that prepare them for the secretory demands of lactation, and that reduce their responsiveness to stimuli other than suckling, especially physical stressors. The demands of milk synthesis and release produce a condition of negative energy balance in the suckled mother, and, in laboratory rodents, are accompanied by a dramatic hyperphagia. The reduction in secretion of the adipocyte hormone, leptin, a hallmark of negative energy balance, may be an important endocrine signal to hypothalamic systems C 2015 American that integrate lactation-associated food intake with neuroendocrine systems. Physiological Society. Compr Physiol 5:255-291, 2015.

Introduction Lactation can be defined basically as the physiological condition during which milk is produced for the purpose of providing nutrition to the offspring. As a defining physiological feature of class mammalia, lactation is a unique physiologic state characterized by numerous anatomical, molecular, biochemical, physiological, and behavioral adaptations, involving the central nervous system as well as the mammary gland. Lactation has been traditionally differentiated into the phases of lactogenesis, that is, the onset of milk production, and galactopoiesis, the maintenance of an established lactation, and is preceded by structural and functional changes in the mammary gland, sometimes termed mammogenesis, that occur mainly during gestation (253). It is clear that at the level of the mammary gland, all aspects of lactation are regulated by an array of interacting endocrine and paracrine messengers whose roles and actions are still being elucidated. Of these, however, prolactin (PRL), which is absolutely required for lactogenesis and galactopoiesis, and oxytocin (OT), primarily responsible for the ejection and removal of milk from the mammary gland, are the two hormones that appear to be most critical in this regard. Neville (253, p. 2993-4) has provided an elegant and succinct description of their actions in lactation: “During lactation, prolactin provides a comprehensive signal that fosters synthesis and secretion of milk components and

Volume 5, January 2015

the survival of the alveolar cell. The volume of milk produced is determined by milk removal from the gland, a function a dependent upon oxytocin secretion by the posterior pituitary and contraction of the myoepithelial cells to force milk out of the alveoli.”

The release of both hormones during lactation is well known to occur though neuroendocrine reflex arcs initiated by suckling and other exteroceptive (i.e., olfactory and auditory) stimuli provided by the offspring, and activation of ascending pathways into the hypothalamus, resulting in unique patterns of secretion. These processes have been investigated for decades and are the subject of numerous authoritative reviews (125, 139); the literature on OT neurosecretion is particularly extensive (cf. 59, 370 for recent reviews). Yet, our understanding remains incomplete in some important respects. This review will summarize our current knowledge of the mechanisms that mediate these neuroendocrine reflexes and will indicate some of the major research questions still outstanding. * Correspondence

to [email protected] of Pharmacology and Toxicology, College of Pharmacy, University of Utah Health Sciences Center, Salt Lake City, Utah Published online, January 2015 (comprehensivephysiology.com) DOI: 10.1002/cphy.c140029 C American Physiological Society. Copyright 1 Department

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Neuroendocrine Control of Lactation

Although investigation of the neural mechanisms that underlie suckling-induced release of these hormones has been the traditional focus in the neuroendocrinology of lactation, several additional features of lactation have emerged as active research areas in recent years, and will also be addressed in this review. For example, the neuroendocrine controls over both PRL and OT undergo important changes during late gestation that prepare these systems for the secretory demands of lactation. Second, inasmuch as milk production entails significant alterations in nutrient flow and energy homeostasis, the contributions of the neuroendocrine systems that regulate energy balance to the physiological adaptations in lactation have been a topic of recent investigations. In addition, a remarkable feature of lactation in laboratory species is the dramatic hyporesponsiveness to stress, also apparently set in place during late gestation. A particularly exciting development is the recent evidence that lactation may confer some protection on the mother from neurotoxic insults, a phenomenon that may have important translational significance.

The Hormones of Lactation: Actions and Patterns of Secretion PRL It has been frequently noted that the range of biological actions of PRL in diverse species is enormous and extends well beyond lactation to include, for example, osmoregulation, metabolism, and immune functions. Supporting the abovementioned description of PRL as a “comprehensive signal” (253), PRL participates in multiple aspects of lactation, both in the mammary gland and in the central nervous system (136). PRL is secreted from lactotrophs (or mammotrophs) of the anterior pituitary gland and also from a population that secretes both PRL and growth hormone (somatomammotrophs). Native PRL is a 23 K molecular weight protein, but PRL also circulates in many isoforms, including those having posttranslational modifications (e.g., phosphorylation and glycosylation) that can affect biological activity. In addition, there are smaller PRL proteins formed from proteolytic cleavage, as well as dimeric and even larger multimeric forms (“big” or “macro” PRL), whose actions are not fully understood. The chemistry of PRL has been thoroughly reviewed elsewhere (41, 125). The actions of PRL at the level of the mammary gland to promote lactogenesis have been reviewed extensively and are well understood (32, 41, 125, 136, 139, 253, 352). Briefly, PRL is essential for stages I and II of lactogenesis, both of which encompass structural changes in the mammary gland and the expression of milk proteins. Hypophysectomy, pharmacological inhibition of PRL secretion or gene deletions of PRL itself or of the PRL receptor all suppress lactation, demonstrating the indispensable role of this hormone. Particularly with respect to expression of milk protein constituents, PRL is assisted by glucocorticoids and insulin, while estradiol, progesterone, placental lactogens, and growth hormone

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also exert a variety of important actions, in a species-specific manner (253). The PRL receptor is a member of cytokine superfamily and the binding of PRL to its receptor and the subsequent activation of signal transduction is similar to that also displayed by growth hormone, as reviewed previously (41, 125, 139, 352). PRL binds in a sequential, step-wise fashion across two PRL receptor monomers, which then promotes their dimerization, and in turn, initiates signal transduction, propagated chiefly via Jak-Stat proteins that are coupled to gene expression. Lactation-related genes that are regulated by PRL in this fashion include those encoding most milk proteins and enzymes involved in synthesis of other milk components (253). Recently emerging areas of investigation are focusing on PRL’s effects in the brain that have been termed “pleiotropic,” which in this sense connotes diverse actions that appear to facilitate multiple aspects of lactation (136). These effects include induction of maternal behavior, stimulation of food intake, potentiation of OT secretion, stimulation of neurogenesis, suppression of stress responsiveness, and inhibition of the hypothalamic-pituitary-ovarian axis. Some of these effects are mediated by PRL of anterior pituitary origin that gains access to the brain, while others may be mediated by PRL synthesized within the brain itself. Several of these features of central PRL actions relevant for the neuroendocrine regulation of lactation are addressed in later sections of this review.

OT The nonapeptide OT has actions in lactation that are more limited than PRL, but no less essential, inasmuch as deletion of the OT gene results in total failure of milk ejection from the mammary gland, and hence, a functional failure of lactation (254). OT is synthesized in large neurons, termed magnocellular, residing in the paraventricular (PVN), supraoptic (SON), and several “accessory” hypothalamic nuclei, and is released by action potentials from nerve terminals in the neurohypophysis into the general circulation (15, 370; see below). Stimulation of OT receptors produces an increase in contractions of the myoepithelial cells that are localized on the surface of the alveoli and along the ducts of the mammary gland. When the myoepithelial cells on the alveoli contract, their compression increases intra-alveolar pressure. Contraction of the myoepithelial cells on the ducts results in duct shortening and widening, reducing resistance to the passage of milk (78). Like PRL, OT can also act centrally to facilitate additional aspects of lactation, although in a more limited fashion than PRL; such actions include facilitation of PRL secretion and of maternal behavior. The OT receptor is a 7-transmembrane spanning receptor, coupled via Gq/11 to phospholipase C and formation of inositol 1,4,5-trisphosphate. This intracellular messenger releases Ca2+ from intracellular stores, which in turn activates a number of processes leading to contraction of the myoepithelia. These signaling mechanisms and the structural, biochemical



Percent maximal response


100 80 Oxytocin pulse PRL EPI Milk yield

60 40 20 0 0

5

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Figure 1 Typical plasma profiles of oxytocin, PRL, an epinephrine (EPI) and milk yield in a standard suckling test (eight pups after 6-8 h separation) in female rats. Oxytocin release episodes are presented as black bars. For experimental details, see references in 78. Reproduced with permission.

and physiological bases of milk ejection at the level of the mammary gland have been authoritatively reviewed by Wakerley (370), and more recently, by Arrowsmith and Wray (19).

Differential patterns of PRL and OT release in response to suckling Although the mechanical stimulation of the mammary glands (as well as olfactory and auditory stimuli) provided by the suckling offspring evoke the release of both PRL and OT, the patterns of release of these two hormones are quite distinct, perhaps reflecting, at least in part, the different functions of these two hormones in lactation. As observed in the most widely used experimental model (See Fig. 1), when female rats are suckled by their offspring after a period of separation, plasma concentrations of PRL begin to increase from the low baseline almost immediately, and reach peak levels by 10 to 15 min (78, 141, 142). Although this elevated level is sustained throughout the suckling period, frequent blood samplings reveal that a pulsatile pattern of PRL release can be discerned, with distinct peaks and troughs; trough levels of the PRL pulses during suckling, however, remain higher than the low presuckling baseline (148, 248). Elevated PRL concentrations can be sustained after the cessation of suckling for a period of time, depending in part on the duration of the previous suckling episode. A similar response pattern has been observed in rabbits, goats, and humans (141). As described in detail by Freeman et al. (125), the pattern of PRL release in response to suckling differs significantly from PRL secretion in other physiological, and especially reproductive, states. For example, female rats display a surge in PRL secretion on the afternoon of proestrus, lasting several hours and with a time course similar to the preovulatory surge of luteinizing hormone. During the first 10 days of pregnancy in rats, two daily surges of PRL secretion are observed, each lasting several hours; a diurnal surge occurs


in the late afternoon-early evening, while a nocturnal surge occurs in the mid-dark period hours, with basal levels maintained in between. This unique pattern is most likely induced by the cervical stimulation experienced during mating, as it also occurs after artificial cervical stimulation or after a sterile mating; its major function in the rat appears to be in support the corpus luteum of early pregnancy (125). A series of investigations by Grosvenor and co-workers has revealed some interesting dynamics with respect to PRL content and secretion within the anterior pituitary during suckling (cf. 141,142). These studies showed that a marked depletion in radioimmunoassayable PRL content within the anterior pituitary occurs during the first few minutes of suckling in rats, but that this does not simply reflect increased release of the hormone into the systemic circulation. Rather, it appears that PRL undergoes some as yet undefined chemical change within the lactotrophs that alters its detectability by standard assays. These investigators proposed that PRL is transformed from a prereleasable, storage form to a releasable form by the suckling stimulus, mediated by actions of the hypothalamic hormones regulating PRL secretion (see below). Following this “depletion-transformation” phase, the pool of PRL in the preleasable, storage form is gradually repleted. In contrast to the rapid onset and sustained high level of PRL release from the anterior pituitary in response to suckling in the separation-reunion paradigm, the release of OT from the neurohypophysis may be delayed for 15 to 20 min in conscious lactating rats, and up to 60 min in anesthetized rats, despite vigorous suckling by the litter [(77, 78, 142); see Fig. 1]. There are species differences in this aspect of suckling-induced OT secretion, however, in that release of the peptide can be observed within a few minutes in cows, pigs, rabbits, and humans (141, 210). OT release in response to suckling is pulsatile in all of these species, but the OT pulsatility pattern differs significantly from that seen with PRL as described above. For example, blood levels of OT during suckling generally remain at baseline, but are interrupted by

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single pulses of release occurring at 5 to 10 min intervals, with each pulse consisting of an abrupt increase in blood concentrations of twofold to threefold, followed by a return to the low baseline after only several minutes (148, 149). As reviewed in detail below, each OT secretory pulse is generated by a brief “milk ejection burst,” in which virtually the entire OT neurohormonal population fires a burst of action potentials synchronously for a few seconds (77, 370). During each secretory episode, a bolus of OT is delivered to the mammary gland, resulting in a brief increase in intramammary pressure and milk ejection (141, 148, 149). Milk is thus available to the offspring only intermittently as a result of this episodic pattern of OT release (77, 370). In between the episodic milk ejections, levels of circulating OT and the firing pattern of OT neurons are at a low baseline level, even though the suckling is continually applied. This pattern of pulsatile OT release is observed only during lactation and parturition (contributing to intermittent uterine contractions during the latter condition) (58, 149, 370). Other physiologic stimuli for OT release, such as physical stressors, dehydration, or administration of cholecystokinin, all produce a more graded and sustained increase that is related to the strength of the stimulus and that persists throughout the stimulation period, without evidence of episodic activation of OT neurons (58, 77, 370). These differences in the patterns of release of PRL and OT evoked by suckling make physiological sense when one considers the major biological functions of these hormones at the mammary gland. Sustained release of a hormonal signal during suckling, as is the case for PRL, would seem to be appropriate to continually stimulate the synthesis of the various components of milk. In contrast, nursing offspring need to be fed intermittently, not continuously, and the episodic release of OT in amounts needed to produce discrete milk ejections would be appropriate to achieve this goal.

The suckling-activated afferent pathway to the neuroendocrine hypothalamus A central issue still remaining in the neuroendocrine regulation of lactation concerns the anatomical delineation of the neural pathway(s) activated by suckling that ultimately impinge upon the hypothalamic control systems that generate the distinct patterns of PRL and OT secretion described above, that contribute to other physiological and behavioral features of lactation as well. As a component of the parvicellular (small cell) neuroendocrine system that governs secretion of all anterior pituitary hormones, PRL secretion from lactotrophs is regulated by inhibitory and stimulatory hypothalamic (hypophysiotropic) hormones that are synthesized by neurons within the hypothalamus and that gain access to the anterior pituitary gland after release into the hypothalamic-hypophyseal vasculature in the basal hypothalamus (125, 139). As discussed below, the most important hypothalamic hormonal systems controlling PRL during lactation reside in the arcuate nucleus and in the parvocellular

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PVN of the hypothalamus, while OT-synthesizing neurons are localized mainly to the SON and magnocellular PVN, and to several accessory sites within the hypothalamus, and project their axons directly into the neurohypophysis. How does the suckling stimulus gain access to these final output systems? Studies employing lesion, stimulation, electrophysiological recording, anterograde and retrograde tract tracing, and other neuroanatomical imaging techniques, most notably, cFos expression, have attempted to map the suckling-activated afferent pathway(s) to these two neuroendocrine systems, and pharmacological and neurochemical approaches have complemented these efforts to identify neurotransmitter systems that are essential for expression of these neuroendocrine reflex arcs. However, our knowledge of these systems remains incomplete, and it is clear that suckling information is not conveyed to the neuroendocrine centers in the hypothalamus via an easily discerned discrete system or circuit (370). It seems likely that the neural networks activated by the suckling stimulus will be common to, that is, shared by, both PRLand OT-regulatory systems up to some level within the CNS, but at some point, the ascending pathways must diverge to influence the separate neuroendocrine control systems and differential secretion patterns for these two hormones; it is these final components of the ascending systems that remain incompletely characterized. The following discussion is an attempt to synthesize results from a variety of experimental approaches; the reader is also referred to an excellent and detailed recent summary of this literature (370). Several caveats should be mentioned. Most of the studies have been performed in rats with OT release as the hormonal endpoint, so that there are fewer studies on pathways that might be specific to PRL control systems. Brainstem regions that have been implicated in suckling activation are quite complex in their numerous interactions with multiple brain areas, complicating the task of identifying which interconnections are specifically important for suckling-induced hormone release. It also must be recognized that suckling affects physiological processes other than PRL and OT release, for example, parental behaviors, and also engages central reward pathways (118), so that interpretation of the literature can be complicated by a variety of methodological and conceptual considerations. Sensory information from mechanoreceptors in the nipples is carried by the mammary nerves into spinal cord dorsal horn and ascends ipsilaterally in the spino-cervical tract to the lateral cervical nucleus; this latter nucleus is known to be activated by suckling and to be essential for expression of the milk ejection reflex (113,114,329). Ascending from this level, most suckling-activated fibers decussate (although some remain uncrossed), pass through the ventrolateral medulla, and eventually reach lateral aspects of the mesencephalic tegmentum that borders with the diencephalon (351); destruction of this region prevents expression of the milk ejection reflex (113, 114, 175). Nuclei within the brainstem that appear to be particularly responsive to suckling, as evidenced from imaging techniques, include the A1 (ventrolateral medulla), the



A2 (nucleus tractus solitarius), and the A6 (locus coeruleus) noradrenergic cell groups (nomenclature of Dahlstrom and Fuxe, 91), the ventromedial area of the medulla, lateral parabrachial nucleus of the pons, the paralemniscal nucleus of the pons, and the periacqueductal gray into the mesencephalon, including the dorsal raphe nucleus (208, 209, 237). Because the three noradrenergic nuclei, the dorsal raphe, a site of serotonergic cell bodies (91), and the lateral parabrachial nucleus are known to project directly into neuroendocrine control areas of the hypothalamus (293, 324), these particular structures may generate relatively discrete suckling inputs from brainstem to hypothalamus. As discussed below, noradrenergic neurons are critical in the suckling activation of OT neurons, but not for PRL secretion; the noradrenergic neurons of the brainstem, therefore, could represent a point of divergence of the pathways controlling OT versus PRL release. At the mesencephalic-diencephalic transition, the posterior intralaminar complex of the thalamus (encompassing the subparafascicular nuclei, sometimes also referred to as peripeduncular area) contains suckling-activated neurons (113, 143, 208, 209) that project directly to medial basal hypothalamus (87). Sensory-related structures such as the inferior colliculus, medial and lateral geniculate of the posterior thalamus are strongly activated by suckling, but also by exposure to pups without suckling (208), and thus, may potentially play a role in hormone in response to auditory stimuli from the pups, but relatively little is known about such mechanisms. Suckling-activated afferents may enter the diencephalon via the zona incerta and Fields of Forel (113) and then join the medial forebrain bundle as they ascend (237, 346). The exact routes taken by nerve fibers of the afferent pathway to the hypothalamic OT and PRL regulatory centers have not been described in great detail. The available evidence suggests that some suckling-activated afferents, for example, noradrenergic fibers from the A1 and A2 nuclei, directly innervate OT neurons of the PVN and SON (293). In addition, Moos et al. (237) have presented evidence that suckling activates a population of neurons in the ventromedial medulla, of unknown neurotransmitter phenotype, that can provide bilateral innervation to the OT neurons of the SON and PVN; these investigators have described axons of these neurons as leaving the medial forebrain bundle dorsally, then coursing ventrally in the periventricular zone at the level of the PVN, crossing the midline through the optic chiasm and entering the SON via the chiasm or just dorsal to it. However, it seems likely that most suckling-activated nerve fibers innervate intrahypothalamic regions containing neurons that, in turn, directly contact magnocellular OT neurons and PRL control areas. These include extra-nuclear areas immediately adjacent to the PVN and SON (e.g., 45, 282), the dorsomedial nucleus (326), and mammillary body complex (376). Suckling-activated axons may also ascend to more rostral limbic forebrain areas, such as the septum and bed nucleus of the stria terminalis, which then send projections back caudally to neuroendocrine hypothalamus (192, 258). Figure 2 presents



Forebrain

LS / BNST

MPOA pnr (Glu/GABA) PVN

pnr (Glu/GABA) ARC

SON

DCA Hypothalamus MFB

DMN

ZI/FoF Mam B

PIL Midbrain PAG/DR (5HT)

Medulla

LC (A6NE)

PB

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LCN Mammary gland mechanoreceptors

Spino-cervical tract DHsc

Figure 2 Key structures in the suckling-activated afferent pathway to the neuroendocrine hypothalamus. See text for details on experimental approaches. Abbreviations: AP, anterior pituitary gland; ARC: arcuate nucleus of the hypothalamus; DCA: dorsochiasmatic area; DHsc: dorsal horn of the spinal cord; DMN: dorsomedial nucleus of the hypothalamus; LC: locus coreruleus; LCN: lateral cervical nucleus; LS/BNST: lateral septum/bed nucleus of the stria terminalis; Mam B: mammillary body complex; ME: median eminence; MFB: medial forebrain bundle; MPOA: medial preoptic area; NE: norepinephrine; NTS: nucleus tractus solitaries; PAG/DR: periacqueductal gray/dorsal raphe nucleus; PB; parabrachial nucleus; PIL: posterior intralaminar complex; PL: paralemniscal nucleus; pnr: perinuclear regions with glutamatergic (Glu) and GABAergic (GABA) neurons; PVN: paraventricular nucleus of the hypothalamus; SON: supraoptic nucleus; TIP39: tuberoinfundibular peptide of 39 residues; VLM: ventrolateral medulla; VMM: ventromedial medulla; ZI/FoF: zona incerta/Fields of Forel; 5-HT: serotonin

an overall summary of the key structures identified in the afferent pathways(s) to the neuroendocrine hypothalamus that are specifically activated by suckling; more detail will be covered in the separate sections on PRL and OT.

Neuroendocrine Regulation of PRL Secretion During Lactation Hypothalamic hormones Inhibition by dopamine: PRL is somewhat unique among the anterior pituitary hormones in exhibiting very high levels

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of release in the absence of hypothalamic control, for example, from pituitary fragments or cultured anterior pituitary cells in vitro (125, 221). Thus, the predominant mode of hypothalamic control over PRL secretion in vivo is inhibitory, rather than stimulatory as is the case for the gonadotropins, thyrotropin and adrenocorticotropin, or more balanced stimulatory/inhibitory, as for growth hormone. Overwhelming evidence indicates that the most important hypothalamic hormonal regulation over PRL secretion is accomplished by dopamine (DA), which provides a tonic inhibitory control that must be removed before PRL can be secreted in response to physiologic stimuli, including suckling during lactation (cf. 32-34, 125, 221 for reviews). While the actions of DA in inhibiting PRL secretion are perhaps the most extensively characterized of any of the hypophysiotropic hormones that govern anterior pituitary hormone secretion, some important questions remain to be answered. The anatomy of the hypothalamic DA systems that regulate PRL have been extensively characterized and reviewed (32, 33, 125, 204; See Fig. 4 in Ref. 125). Of the four clusters of dopaminergic neurons within the diencephalon, the A12 group (using the original nomenclature from the pioneering mapping study of Dahlstrom and Fuxe (91)) is located in the hypothalamic arcuate nucleus, while the A14 cells are located in the hypothalamic periventricular nucleus. Within the arcuate nucleus, one dorsomedially located subset of DA neurons primarily innervates the median eminence, with particularly dense innervation of its external zone in proximity to portal vessels, and is referred to as the tuberoinfundibular DA (or TIDA) system. It is well established that this TIDA system accomplishes the major hypothalamic inhibitory control over PRL secretion (32, 33, 125). Reflecting the role of DA as a hypothalamic hormone rather than a neurotransmitter, TIDA neurons differ in some regulatory respects from nigrostriatal or mesolimbic DA neurons, for example, in lacking presynaptic, inhibitory autoreceptors (233). A second subset of A12 DA cells located more rostrally in the arcuate nucleus primarily innervates the neurohypophysis and intermediate lobe in the rat; this tuberohypophyseal DA (THDA) system clearly affects the release of the neurohypophyseal hormones (see below), and there is some evidence that it might contribute to inhibition of PRL release as well via short vascular connections to the anterior lobe (32, 33). The A14 DA cells of the periventricular nucleus of the rat appear to innervate only the intermediate lobe and influence release of α-melanocyte stimulating hormone; a physiological role for this pathway in PRL secretion during lactation is uncertain but may contribute to basal PRL secretion (125, 204). Pharmacological agents that inhibit DA synthesis, deplete DA, or block the D2 subtype of DA receptor, which is the predominant subtype present on lactotrophs, rapidly increase in PRL secretion; conversely, DA agonists, especially at the D2 receptor, decrease PRL release in response to physiologic stimuli, including suckling (125). These effects have translational significance in that D2 antagonists used clinically (e.g., typical neuroleptics and atypical neuroleptics related

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to risperidone for treatment of psychosis and bipolar disorder) can produce hyperprolactinemia, mainly presenting with issues related to infertility. Conversely, D2 agonists, such as bromocriptine and cabergoline, are commonly used to lower PRL hypersecretion from prolactinomas of the anterior pituitary gland. There is evidence that the various physiological stimuli for PRL secretion, including suckling, reduce the release of DA from the TIDA system. This withdrawal of DA has been inferred from measurements of DA content, turnover (an index of synthesis and release), or release in the median eminence, DA concentrations in portal blood, and DA content in the anterior pituitary gland, all of which show a reduction in response to suckling, or mammary nerve stimulation, which mimics suckling (60, 61, 84, 94-96, 101, 103, 247, 275, 276, 284, 296, 299, 373). That this reduction in DA release is itself a stimulus for increased PRL release has been demonstrated not only by the above-mentioned pharmacological studies, but also in in vitro preparations, for example, in static incubations of anterior pituitary cells or in more dynamic superfusion systems with anterior pituitary cell preparations, in which PRL release is stimulated by the withdrawal of DA after a period of exposure (220-222, 374). It is also interesting to note that upon removal of DA from superfused anterior pituitary cells, the resultant increase in PRL release also exhibits pulsatility (222), similar to suckling-induced PRL release in vivo. Further, and very importantly, DA withdrawal enhances the lactotroph response to PRL releasing hormone(s) and this interaction constitutes the final output from the brain for the increased release of PRL in response to suckling (see below). Signal transduction mechanisms in the lactotroph affected by DA or DA withdrawal have been investigated extensively (cf. Fig. 5, ref. 139), but our understanding of DA actions in lactotrophs remains incomplete. The D2 receptor in lactotrophs is negatively coupled to adenylate cyclase and formation of cyclic adenosine monophosphate (cAMP) (33, 220). From observations that DA can inhibit PRL secretion from anterior pituitary cells even when the intracellular levels of cAMP are elevated (99, 100), some authors have inferred that DA actions on the cAMP messenger system are not of primary importance in inhibition of PRL secretion. However, activation of adenylyl cyclase and increased cAMP formation in lactotrophs can be observed following DA withdrawal from anterior pituitary cells in vitro (220), so that the conclusion that inhibition of the cAMP messenger system is not important for DA inhibition of PRL may not be warranted. DA clearly has inhibitory effects on various aspects of Ca2+ signaling in lactotrophs, and this has been the subject of numerous investigations. Addition of DA to isolated lactotrophs in vitro produces a rapid onset membrane hyperpolarization, via activation of one or more subtypes of K+ channel, which in turn results in decreased entry of extracellular Ca2+ via voltage-regulated Ca2+ channels (reviewed in 139, 323). While this reduction in the entry of extracellular Ca2+ into the lactotroph is perhaps the most prominent and best characterized, it may not be the only, action of DA on Ca2+ signaling.




Addition of DA to isolated lactotrophs in vitro does not appear to directly inhibit the activity of phospholipase C and the subsequent generation of intracellular messengers that mobilize intracellular Ca2+ (i.e., inositol 1,4,5-trisphosphate, IP3) or that activate protein kinase C (PKC) (i.e., diacylglycerol), which is linked to entry of extracellular Ca2+ (220); it is possible that this is due to an already low level of activity in this messenger system under basal conditions. On the other hand, DA withdrawal from lactotrophs does result in the activation of phospholipase C and PKC (220). DA also inhibits PRL release and the increases in intracellular Ca2+ evoked by putative PRL-releasing hormones that activate this messenger system (see below). Hence, there is good agreement that a major, if not exclusive, mechanism underlying DA inhibition of PRL secretion is inhibition of Ca2+ signaling essential for endocrine exocytosis, especially the entry of extracellular Ca2+ , and that DA withdrawal from the lactotroph, which is likely to be the actual physiological signal in response to suckling, results in activation of various aspects of Ca2+ signaling [(33,140,222); see Fig. 3)]. However, it is also apparent that DA withdrawal alone does not produce the high rate of PRL secretion observed in vivo in response to suckling or to other physiologic stimuli (222). An important caveat to the findings cited above indicating that DA withdrawal occurs during suckling (or a stimulus that mimics suckling) is that the decrease in DA concentrations in portal blood reported in these studies has

DA

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PRL exocytosis

Figure 3 Hypothesized signal transduction mechanisms for the potentiation of TRH stimulation of PRL release by DA withdrawal. See text for details. The reduction in DA secretion into the portal vasculature in response to suckling activates phospholipase C, via disinhibition, resulting in increased formation of inositol 1,4,5-trisphosphate (IP3), which increases mobilization of Ca2+ from intracellular sources, and of diacylglycerol (DAG), which activates protein kinase C (PKC), resulting in increased entry of Ca2+ via voltage-regulated calcium channels. DA withdrawal per se most likely also leads to increased extracellular Ca2+ entry. TRH receptors are positively coupled to generation of these intracellular messengers, and the increased release of TRH in response to suckling thus enhances the rise in intracellular Ca2+ concentrations ([Ca2+ ]i ) resulting from DA withdrawal, leading to the exocytosis of PRL.


consistently been quite transient, on the order of several minutes (e.g., 94-96, 275, 276). A more complete understanding of DA mechanisms and actions during lactation must, therefore, take into account the dynamic interplay of DA withdrawal with one or more PRL-releasing hormones, as reviewed below. Finally, an intriguing, and still unexplained, feature of DA inhibition of PRL secretion is the ability of DA to inhibit PRL release evoked by secretagogues that most likely act within the lactotroph, that is, beyond the membrane D2 receptor; these include the Ca2+ ionophore A23187, which mimics IP3 in mobilizing intracellular Ca2+ , and phorbol esters, which activate PKC and increase entry of extracellular Ca2+ (99). This would seem to imply that DA might act, at least in part, on processes downstream from these initial signaling events, in addition to receptor-messenger coupling leading to the increases in [Ca2+ ]i . In this regard it is interesting to note that electron microscopic studies of lactotrophs exposed to DA have revealed some changes in cellular ultrastructure that are suggestive of some physical barrier to PRL release induced by DA (280). The TIDA system also serves as a target for PRL short loop inhibitory feedback (33, 125, 233). Unlike the “tropins” secreted by the anterior pituitary gland, PRL does not stimulate release of hormones from a peripheral endocrine gland that in turn act in hypothalamus and pituitary to regulate their own secretion in a homeostatic “long loop” feedback mechanism. Rather, PRL secretion is governed in part by a “short loop” feedback, in which PRL activates the TIDA neurons, generally assessed from indices of DA turnover or release, thereby inhibiting its own secretion. Indeed, pharmacological studies in which circulating PRL levels were raised or lowered have revealed that PRL is the major endocrine regulator of TIDA neuronal activity, and that this inhibitory autoregulation is an important feature of the neuroendocrine regulation of PRL secretion (9, 138, 233, 234). It remains to be conclusively established how the PRL protein gains access to TIDA neurons, but it is clear that these neurons express PRL receptors (cf. references in 136). PRL increases expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in DA biosynthesis, and also activates the enzyme via phosphorylation (12, 298), and also activates TIDA neurons electrophysiologically (218, 283). In lactating rats, however, PRL does not increase DA release, thus allowing for sustained elevations in PRL in response to suckling (13, 101, 138, 368). Recent observations by Romano and coworkers (283) have provided important new information that TIDA neurons of lactating mice actually continue to respond to elevations of PRL with an increase in electrophysiological activity, yet DA release is markedly suppressed. These investigators have suggested that this “uncoupling” of DA release from TIDA neuronal activity may result from altered signaling that affects tyrosine hydroxylase activity. This adaptation would seem to be physiologically significant in allowing for sustained levels of PRL release and milk synthesis in response to suckling, unimpeded by short

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loop negative feedback, and, as reviewed below, is set in place during late gestation. In some in vitro and in vivo preparations, very low concentrations of DA can stimulate PRL secretion directly from lactotrophs, an effect mediated by the D2 receptor and associated with an increase in cytosolic Ca 2+ . The physiological significance of these observations remains unclear, and methodological concerns have been discussed by Freeman (125) and Gregerson (139). It is conceivable, for example, that DA withdrawal, in addition to reducing DA inhibition, results in concentrations reaching the lactotroph that contribute to stimulation of PRL, together with stimulatory hypophysiotropic hormones. Neuropeptide Y (NPY) as a “co-inhibitor”: Although the primary role of the TIDA system in inhibition of PRL secretion is well substantiated, there is also good agreement that this system alone does not account for the profound level of suppression of PRL secretion observed in vivo, and it has been suggested that other chemical messengers are very likely to participate in the inhibitory regulation of PRL secretion, perhaps in augmenting the action of DA via direct actions on the lactotroph (125, 139). There are a number of candidates for this role (125, 378), and this section will highlight the role of NPY, which exerts a number of important effects during lactation, including hypophysiotropic regulation of PRL (76, 81). NPY is perhaps the most widely distributed neuropeptide in the brain with a number of prominent actions in neuroendocrine regulation (76, 177). Of particular relevance is the major NPY cell group in the hypothalamic arcuate nucleus, which provides innervation to the median eminence as well as to other intrahypothalamic structures. NPY is also detectable in physiologically relevant nanomolar concentrations in hypophyseal portal blood, suggestive of a role at the anterior pituitary gland (321). The first indication that hypothalamic NPY might influence PRL secretion during lactation came from observations that NPY is expressed in a subgroup of arcuate TIDA neurons in lactating rats and mice, which do not coexpress this peptide in other conditions (67, 68). Moreover, this novel expression pattern requires the presence of the suckling stimulus, inasmuch as NPY immunoreactivity disappears from TIDA cells upon removal of the litters (67, 68). Further investigations have revealed upregulation of NPY gene expression throughout the (non-TIDA) arcuate nucleus NPY system during lactation as well, indicating a broader involvement than only the TIDA system (e.g., 304). Indeed, this system exerts multiple effects on the neuroendocrine, metabolic and behavioral features of lactation, as will be reviewed below. Studies in the author’s laboratory (374) have demonstrated that NPY produces a dose-dependent reduction in PRL secretion from rat anterior pituitary cells in vitro, in physiologically relevant concentrations and with an efficacy comparable to DA. Moreover, a combination of DA plus NPY produces an additive inhibition of PRL release. Similar to withdrawal of DA, removal of NPY after period of exposure in vitro results

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in a dramatic increase in PRL secretion; moreover, when cultured anterior pituitary cells were first exposed to both DA and NPY, the withdrawal of both resulted in a higher level of PRL release than removal of either one alone. These findings led us to propose that during lactation, NPY, and particularly NPY as expressed in TIDA neurons might act as a “cohormone” with DA to augment the inhibition of PRL release; further, we suggested that this might play a role in generating the pulsatile pattern of PRL during suckling (304). Such a “cohormone” role in regulation of PRL secretion is analogous to a similar action of NPY in augmenting the stimulatory effect of gonadotropin-releasing hormone on luteinizing hormone (83). Putative PRL-releasing hormones: Thyrotropinreleasing hormone and its interaction with DA: As alluded to above, withdrawal of DA alone (or even withdrawal of DA with coinhibitors such as NPY) does not appear to account totally for the physiological pattern of PRL release in response to suckling, indicating the necessity for stimulatory hypothalamic hormones. Unlike the other anterior pituitary hormones, however, no unique PRL-releasing hormone has been identified to date, and whether one exists at all remains a major area of uncertainty in PRL neuroendocrinology. A number of neuropeptides can stimulate PRL secretion directly from lactotrophs and can be detected in the hypophyseal portal vasculature, suggestive of a physiological role (125). Although there is not complete agreement, the tripeptide thyrotropinreleasing hormone (TRH) appears to have the most experimental support as a significant PRL-releasing factor during lactation, at least in rats. TRH-immunopositive neurons are found in several hypothalamic regions; the hypophysiotropic TRH system is primarily localized in the parvocellular regions of the PVN (119, 200, 201, 213), lesions of which impair PRL release in response to suckling (182). TRH is highly effective in physiological concentrations in stimulating PRL secretion from anterior pituitary cells as well as from PRL-secreting tumor cell lines (e.g., 131, 221, 374). The suckling-mimetic stimulus of mammary nerve stimulation in rats produces a significant increase in TRH concentrations in hypophyseal portal blood (95), while immunoneutralization of TRH inhibits suckling induced PRL release in rats (96). Interestingly, studies using either in vitro or in vivo models demonstrate that DA withdrawal, as occurs during suckling (see above), enhances the ability to TRH to stimulate PRL secretion (reviewed in 125, 222). The Grosvenor laboratory has also shown that a reduction of DA exposure enhances the ability of TRH to induce the depletion-transformation of pituitary PRL levels, as described above (141). Hence, the view has emerged that a dynamic interplay between DA withdrawal from, and TRH stimulation of, the lactotroph is the critical hypophysiotropic mechanism governing PRL secretion in response to suckling, and that the Ca2+ messenger system is the key signaling element in this interaction (cf. 222 for review). TRH receptors are positively coupled to activation of phospholipase C, and TRH is a classic activator of the



Ca2+ /inositol phosphate messenger system in the lactotroph (131). Studies in PRL-secreting tumor cells or in lactotrophs have shown that TRH produces two phases of PRL release, a rapid onset first phase, most likely mediated by the initial IP3-mediated mobilization of intracellular Ca2+ , followed by a second phase, which requires entry of extracellular Ca2+ , most likely mediated by PKC (131, 199, 300, 301, 374). Studies conducted in the author’s laboratory in superfused rat anterior pituitary cells have shown that DA can inhibit both phases of the PRL and Ca2+ responses to TRH, while our proposed cohormone, NPY, inhibits only the later phase release mediated by entry of extracellular Ca2+ (301, 374). Thus, taken together, much of the available evidence suggests that a decrease in TIDA activity in response to suckling results in the attenuation of tonic inhibition exerted by DA, primarily over the Ca2+ -inositol phosphate messenger system; as reviewed above, DA withdrawal is associated with activation of phospholipase C and subsequent messenger mechanisms that increase [Ca2+ ]i within the lactotroph. This may be the critical event in enhancing the ability of TRH to activate this messenger system, resulting in high levels of PRL stimulation (See Fig. 3). DA withdrawal-induced activation of the cAMP messenger system can add a further mechanism for PRL stimulation. Putative PRL-releasing hormones: Oxytocin: Several studies suggest that OT may serve as an additional PRLreleasing hormone, although this peptide has not been as thoroughly investigated in this role as has TRH. Early studies reported that OT can directly stimulate PRL release from lactotrophs, and that immunoneutralization of OT can prevent suckling-induced PRL release (288). Subsequent studies by Freeman and co-workers (180) showed that a prolonged infusion with an OT receptor antagonist also inhibited, but did not abolish, the PRL response to nursing. Some OT neurons in the parvocellular divisions of the PVN (see below) project axons to the median eminence, and OT is present in hypophyseal portal blood (133). Although it would seem to make physiological sense that the two hormones released in response to suckling could influence each other’s release, given the differences in the timing of OT versus PRL release episodes described above, the mechanistic details and physiological significance of this proposed interrelationship remain to be determined. Putative PRL-releasing hormones: Salsolinol: Based on the demonstration that removal of the neurohypophysis elevated basal levels of PRL, but prevented the suckling-induced release of the hormone, it has been hypothesized that the neural lobe may harbor a PRL-releasing factor that can gain access to the lactotrophs, most likely via vascular intercommunications (243). Further studies from this group (161,162) confirmed the presence of a direct PRL-releasing factor in this tissue, and ruled out several PRL secretagogues, including OT, as the neurohypophyseal PRL releaser. Subsequent studies have suggested that salsolinol ((R-1-methyl-6,7dihydroxy-1,2,3,4-tetrahydroquinoline), a DA metabolite, may be the active factor. In a series of studies, Toth et al.



(350) isolated this compound from neural lobe extracts and showed that it was effective in stimulating PRL release from lactotrophs in vitro. In lactating rats, concentrations of salsolinol in the neurohypophysis also decreased and increased, respectively, upon separation and reunion of the litters (350). It remains to be established how salsolinol interacts with other factors to the overall regulation of suckling-induced PRL secretion. Upstream regulators of PRL secretion in lactation: Classical and peptide neurotransmitters: As the final effectors from the brain to the anterior pituitary gland, hypophysiotropic hormones must integrate and respond to a variety of neuronal inputs that convey physiological signals, and there has been intense interest for many years in identifying and characterizing the actions of classical and peptide neurotransmitters that act as “upstream” regulators of anterior pituitary hormone secretion. With respect to the control of PRL secretion during lactation, it is particularly important to identify those systems that ultimately signal for the reduction in DA release from the TIDA system, in conjunction with activation of TRH release, which represents the final hypophysiotropic mechanism presented above. Although a large number of neurotransmitter and neuropeptide systems can influence PRL secretion in different physiological states (125, 378), our understanding of those systems that are important for suckling-activated PRL secretion remains incomplete. This section will focus on those systems for which the evidence for a physiological role in lactation seems most compelling. It is also important to evaluate whether any of these neurochemical systems might be a component of the suckling-activated afferent pathway, or whether they might be extrinsic to this pathway but exert a modulatory role on it. Monoamines: Neither of the catecholamine neurotransmitters, norepinephrine, and epinephrine, appear to be involved in any major way in regulation of PRL secretion, particularly during lactation (38, 63, 332). Pharmacological studies indicate a generally excitatory action of serotonin (5-hydroxytryptamine, 5-HT) on PRL secretion in different physiological conditions (75, 125, 167, 359), and this effect also appears to be important during lactation. For example, inhibition of serotonin synthesis inhibits suckling-induced PRL release in rats (187), as does blockade of 5-HT receptors with methysergide (130). Pharmacological studies in nonlactating female rats indicate that 5-HT or 5-HT agonists inhibit TIDA neuronal activity (223), and this interaction is supported by anatomical studies that reveal the presence of serotonergic nerve terminals innervating TIDA neuronal cell bodies (181). However, 5-HT neurons innervate TRH-positive neurons of the PVN as well (292), and destruction of serotonergic terminals in the PVN with a serotonin neurotoxin inhibits suckling-induced PRL release in rats (38). Serotonin may also affect anterior diencephalic structures linked to PRL control, as serotonin-specific depletion in the anterior hypothalamic nucleus inhibits suckling-induced PRL release (260), and suckling enhances 5-HT turnover in the medial preoptic nucleus (171). Injections of a serotonin neurotoxin into the

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AP Portal vasculature ME DA/NPY

MPOA

TRH

ARC

PVN

Dorsal Raphe (5-HT)

Hypothalamus

PIL Complex (TIP39)

Brainstem

Mesencephalon

Afferents

LCN Mammary gland afferents

DHsc

Figure 4

Some identified neurochemical inputs to the neuroendocrine hypothalamus activated by suckling to stimulate PRL secretion. See text for details. See Figure 2 for abbreviations.

dorsal raphe nucleus, which is situated in the mesencephalic periacqueductal gray and which contains 5-HT cell bodies that innervate the hypothalamus (91), also inhibits sucklinginduced PRL release (23). Taken together, these findings suggest that serotonergic neurons of the dorsal raphe may be activated by suckling as a component of the afferent pathway (see above), and may stimulate PRL secretion via effects exerted at several sites in the hypothalamus, including inhibitory actions on TIDA neurons and a concomitant excitatory effect on TRH neurons of the PVN (See Fig. 4). Glutamate: The major excitatory neurotransmitter in the neuroendocrine hypothalamus is glutamate (159, 358), and there is considerable for involvement of this excitatory amino acid in suckling-induced PRL secretion (246). Central administration of agonists at the N-methyl-d-aspartate (NMDA) subtype or at the AMPA (R,S-alpha-amino-3hydroxy-5-methyisoxazole-4-propionic acid) subtype of glutamate receptor increases PRL release in lactating and nonlactating female rats (5). Administration of AMPA antagonists into the third ventricle (264,389), PVN (37) or into the dorsal raphe nucleus (39), but not into the medial basal hypothalamus (37), inhibits suckling-induced PRL secretion. Inasmuch as glutamate-positive nerve terminals innervate TRH neurons of the PVN (381), glutamate’s stimulatory role on suckling-induced PRL release may be exerted primarily on the hypophysiotropic TRH neurons of the PVN and also on the

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serotonergic neurons of the dorsal raphe that in turn influence the hypothalamic centers (see above). γ-aminobutyric acid: As the major inhibitory neurotransmitter in the neuroendocrine hypothalamus (93), GABAergic neurons are in position to influence secretion of multiple anterior and posterior pituitary hormones. However, a clear physiologic role of γ-aminobutyric acid (GABA) in control of PRL release during lactation has not emerged. Most of the investigations of GABA effects on PRL secretion have been done in nonlactating animal models or on anterior pituitary cells in vitro, with considerable variability in results. However, as a general conclusion from such pharmacological studies, GABA may act centrally to stimulate PRL release by reducing TIDA activity (279); this effect could occur in the medial basal hypothalamus and/or preoptic area, and may be mediated by tuberoinfundibular GABAergic neurons present in the arcuate nucleus (194); see Fig. 5). Conversely, lactotrophs express GABA receptors, activation of which by GABA can directly inhibit PRL release, albeit in relatively high concentrations (224). Suckling increases the activity of the GABA-synthesizing enzyme glutamate decarboxylase in medial basal hypothalamus (279) and increases GABA turnover rate specifically within the ventrolateral preoptic area (188), but it is difficult to relate these neurochemical changes specifically to PRL secretory mechanisms. Endogenous opioid peptides: Opioid agonists, such as morphine, and the EOPs, ß-endorphin, the enkephalins and the dynorphins, exert stimulatory effects on PRL secretion via actions in the brain, and appear to play an important GABA

Arcuate nucleus

5-HT (Raphe)

EOP

DA/NPY

TIP39 (PIL)

Mesencephalon

Brainstem

Afferents

Figure 5 Local neurochemical circuitry in the arcuate nucleus regulating PRL secretion during lactation. Hypophysiotropic dopamine (DA) neurons in the arcuate nucleus that also express neuropeptide Y (NPY) are the major inhibitory regulators of PRL at the lactotroph. The activity of the DA/NPY cells can be inhibited by actions of GABAergic and endogenous opioid peptidergic (dynorphin, β-endorphin and metenkephalin) neurons present within the arcuate nucleus. Dynorphin neurons, in turn, are activated by neurons of the posterior intralaminar (PIL) complex, known to be activated by suckling and to express TIP39. Serotoninergic neurons (5-HT) of the dorsal raphe, also activated by suckling, also innervate and inhibit the DA/NPY cells. The inhibitory EOP influence on DA/NPY neurons is also mediated in part by enhanced 5-HT release.



obligatory role in lactation (reviewed in 75,125). Most studies have been performed in the rat, but the same effects occur in primates including humans (75). Increases in PRL secretion can be observed after central administration of each of the endogenous peptides, but these peptides or opioid agonists have no effect when applied to anterior pituitary cells in vitro, indicative of an exclusively central action (75). Suggesting a physiological role of EOP during lactation are the findings that the nonselective opioid receptor antagonist naloxone can inhibit suckling-induced PRL release in rats (14, 24, 122, 229, 289, 297, 306, 390). Antagonists more selective for the mu or kappa opioid receptor, but not the delta receptor, are also effective in attenuating suckling-induced PRL release (10, 24,60,330). Interestingly, specific immunoneutralization (via central injections) of each of the endogenous peptides also inhibits suckling-induced PRL release (60, 168), suggesting that each peptide may play a specific role. Neuroanatomical mapping studies (reviewed in 75) have revealed the presence of cell bodies and nerve terminals of all three families of EOP in areas implicated in the regulation of PRL secretion, including medial preoptic area, PVN nucleus and arcuate nucleus. A particularly important role for the dynorphin neurons of the arcuate nucleus is suggested by the observation that these cells provide the most abundant EOP innervation of TIDA neurons (123). In addition, suckling increases cFos expression in the ß-endorphin cells of the arcuate nucleus, suggestive of a role for this system (259). Finally, as described above for NPY, lactation is also associated with a novel expression of enkephalins in TIDA neurons as well (68, 227). It is, therefore, conceivable that each of these three families of EOP contributes to regulation of PRL secretion, but the specific mechanisms remain to be established. Interactions of EOP with the TIDA system have been a particular focus (Fig. 5), and, as might be predicted, studies in nonlactating and lactating rats consistently show that opioid peptides and agonists decrease TIDA neuronal activity (reviewed in 75, 125, 379). For example, immunoneutralization of either dynorphin or of the enkephalins prevented the suckling-induced inhibition of TIDA neurons as well as the release of PRL (61). Similar effects have been produced by specific mu or kappa opioid receptor antagonists (10). An EOP-5-HT interaction seems equally plausible, in that pharmacological studies in nonlactating rats show that opioid peptides and agonists can stimulate serotonergic activity in regions such as the medial preoptic and arcuate nuclei (170, 172, 360), and that pharmacological interference with 5-HT transmission blocks the stimulatory effect of morphine on PRL release (183,184,308). Indeed, there is also evidence that the effect of opioids to reduce TIDA activity described above may be mediated by an opioid activation of 5-HT release (102) (Fig. 5). To date, these interrelationships have not been examined in lactating rats, however. Tuberoinfundibular peptide of 39 residues (TIP39): TIP39 is a recently characterized neuropeptide with a relatively limited anatomical localization, but with potential for significant neuroendocrine regulatory actions, particularly in



lactation. This peptide, which is the endogenous ligand of the parathyroid hormone 2 receptor (PTH2R), is expressed by neurons in three sites: (i) the periventricular gray of the thalamus, (ii) the posterior intralaminar (PIL) complex of the thalamus, which encompasses the parvicellular subparafascicular nucleus, the posterior intralaminal nucleus, and a portion of the caudal zona incerta, and (iii) the pontine paralemniscal nucleus (108). As noted above, each of these areas has been implicated in the suckling-activated ascending pathway to the hypothalamus; moreover, anatomical studies reveal that TIP39 neurons of the PIL region project rostrally into the mediobasal hypothalamus, and into the SON and PVN nuclei and medial preoptic area (87, 88, 107, 108, 325) (Fig. 4). Recent reports using multidisciplinary approaches (87, 88) provide compelling evidence for an important action of TIP39 in suckling-induced PRL secretion. Thus, neurons of the PIL and paralemniscal nucleus (364) exhibit increased TIP39 gene and protein expression, and over 90% of these cells display increased cFos expression in response to suckling, indicative of activation, during lactation (363). Intraventricular injection of a PTH2 antagonist or injection of a lentiviral vector encoding a PTH2 antagonist into the medial basal hypothalamus blunted suckling-induced PRL secretion. Based on anatomical studies showing that TIP39positive fibers innervate dynorphin-positive cells of the arcuate nucleus (but not TIDA neurons directly) (325), and that dynorphin cells of the arcuate innervate TIDA neurons (123), as described above, the authors of these papers suggest that activation of the TIP39 neurons of the PIL complex by suckling may in turn excite the dynorphin cells of the arcuate nucleus that inhibit TIDA activity [(87, 88); see Fig. 5]. It is also interesting to note that in many hypothalamic areas the PTH2 receptor is localized on glutamate-positive nerve terminals and that TIP39 increases glutamate release, mechanism that can also play a role in promoting PRL release in response to suckling (87, 88, 108). It is tempting to conclude that this recently identified neuroendocrine peptide may represent a major component in the suckling-activated afferent pathway, linking areas in the mesencephalic-diencephalic border activated by suckling directly to the hypophysiotropic control centers in the hypothalamus affecting PRL secretion. Further, because TIP39 neurons innervate the magnocellular nuclei of the hypothalamus (87,88,107,108,325), and because excitotoxininduced lesions of the PIL area also inhibit the milk ejection reflex (143), these neurons may also be involved in control of OT secretion, but this has not yet been evaluated specifically.

Neuroendocrine Regulation of OT Secretion During Lactation Anatomy of the magnocellular OT neurosecretory system The magnocellular OT neuron present in the PVN and SON has been well-studied morphologically. As described by

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Armstrong (15), these cells have diameters of 20 to 35 μmol/L and typically have 1 to 3 dendrites, which can have a varicose appearance. SON OT neurons generally project their dendrites ventrally, into a zone termed the ventral glial lamina, where they receive axonal inputs. OT neurons of the PVN project dendrites into more dorsomedial zones of the nucleus (15). It is interesting to note that magnocellular OT somata and dendrites, as well as their nerve terminals in the neurohypophysis, contain dense core (i.e., peptide-containing) synaptic vesicles (277; see also Fig. 2 in reference 49), the significance of which will be discussed below. Immunocytochemical studies have revealed that subsets of magnocellular OT neurons also coexpress cholecystokinin, corticotropin-releasing hormone, galanin, proenkephalin A (precursor to both met- and leuenkephalin), and the EOP, dynorphin (116, 196, 205, 226). The neuroanatomical organization of the magnocellular OT neuroendocrine system is well established (cf. 15, 49, 58, 77, 370 for reviews), and the following discussion is based primarily on descriptions by Armstrong (cf., Fig. 3 in Ref. 15, and Fig. 5 in ref. 77). The SON is comprised almost exclusively of magnocellular OT and vasopressin neurons that project to the neurohypophysis; OT cells of the SON are generally localized in the more rostral and dorsal aspects of this nucleus. In the rat, this nucleus extends for a considerable distance in the anterior-posterior plane, with components that lie caudal to the optic chiasm, and contains the majority of OT neurons. It is well recognized that the PVN has both parvocellular and magnocellular subdivisions; magnocellular OT cells that project to the neurohypophysis are intermingled with magnocellular vasopressin neurons in the medial, lateral and caudal magocellular subdivisions. It is interesting to note that parvocellular OT and vasopressin neurons of the PVN project to other areas of the central nervous system, including hypothalamic and limbic structures, where they may influence parental and mating behaviors, and to the lower brainstem and spinal cord, where they play an important role in regulation of sympathetic outflow and cardiovascular function (324). It may not be as widely appreciated that up to one-third of the OT neurons contributing to the milk ejection reflex are localized in “accessory” sites, outside the PVN or SON proper. Notably, in the rat, many magnocellular OT neurons are present in the anterior commissural nucleus, which is immediately rostral to the PVN in approximately the same dorsal-ventral plane adjacent to the third ventricle, and are also found in a medially located zone along the third ventricle, termed the magnocellular periventricular nucleus (15). OT neurons are also scattered in the lateral hypothalamus between the SON and PVN. Axons from the SON course dorsomedially and form a supraopticohypophyseal tract that passes through the zona interna of the median eminence and then via the infundibular stalk into the neurohypophysis. These fibers are joined by axons of PVN OT neurons, some of which project laterally over the fornix and then ventrally to join the fibers from the SON, and with others that follow a more medial, periventricular route into the median eminence and infundibulum (15).

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OT-containing nerve terminals in the neurohypophysis have a varicose appearance and are enclosed by glial cells, known as pituicytes, which separate them physically from the capillaries (354). As reviewed below, some dynamic interactions between these glia and the OT nerve endings occur during lactation and in other conditions of heightened OT release, which may potentially modulate OT neurosecretion.

The Milk Ejection Reflex: Mechanisms of generation and synchronization The magnocellular OT system has been intensively investigated for decades and has provided a number of unique insights into processes such as electrophysiological mechanisms of bursting firing patterns, plasticity in neuronal-glial interrelationships, dendritic release of transmitters, and modulatory actions of neurosteroids, all of which contribute to the neurosecretion of OT in response to suckling. As noted above, the unique feature of OT release during lactation is its pattern of intermittent, discrete pulses in response to a continually applied suckling stimulus, in contrast to the sustained elevation of PRL, which results in periodic delivery of OT to the mammary gland to increase intramammary pressure and provide for milk ejection (58,77,370; Fig. 1). Underlying this episodic pattern of OT release is the bursting behavior of magnocellular OT neurons unique to lactation (and also parturition). As observed in the standard, separation-reunion paradigm, the onset of suckling does not result in an immediate release of OT or of increased OT neuronal activity in the rat; rather, the latency to first milk ejection may be as long as 15 to 20 min (78, 141), during which time OT neuronal firing is irregular (77, 370). However, approximately 15 to 20 s before each milk ejection, a brief and explosive increase in the firing rate of the magnocellular OT neurons occurs, lasting 2 to 4 s, followed by a short period of inhibition and then a return to irregular baseline activity (See Fig. 1 in Ref. 77 and Fig. 8 in Ref. 370). These “milk ejection bursts” recur at intervals of 5 to 10 min. The invasion of action potentials into the OT nerve terminals in the neurohypophysis releases the peptide into the capillaries via classic calcium-dependent exocytotic mechanisms, originally termed stimulus-secretion coupling by Douglas and Poisner (111). Periodic bursting and OT release also occurs during parturition to promote uterine contractions, but other physiological stimuli, for example, dehydration and other stresses, result in a sustained neuronal activation and release without such episodicity (58, 77, 281, 370). On the basis of classic electrophysiological studies in which simultaneous recordings were made from pairs of OT neurons in the SON and the contralateral PVN during suckling (30, 31, 211, 281, 371), it appears that virtually the entire magnocellular OT population, that is, cells in the magnocellular (and most likely the accessory) nuclei on both sides of the brain, are recruited into a burst firing mode synchronously, to deliver each bolus pulse of OT to the systemic circulation and thence, to the mammary gland (281, 370). It has been



emphasized (e.g., 49) that this synchronization of burst firing is somewhat loose in the sense that the time lag in the onset of bursting between two OT neurons can vary from instantaneous to upwards of 600 ms (30). Wakerley (370) has provided a calculation that the milk ejection burst generates approximately 500,000 action potentials from the approximately 9000 OT neurons of the rat, resulting in a bolus of approximately 1 ng of OT released into the systemic circulation to increase intramammary pressure for milk ejection. Two of the major outstanding research questions in the neuroendocrine regulation of lactation remain (i) how the continuous suckling stimulus generates this intermittent bursting behavior of magnocellular OT neurons and (ii) how this becomes synchronized across the entire population in several discrete locations. As noted by Leng et al. (202), the suckling stimulus to OT cells must be somewhat unique in promoting such intermittent activation, as other stimuli to this system such as stress or dehydration do not, and secondly, OT cells are most likely not induced to burst by excitatory inputs that in turn are simply activated in an intermittent manner by suckling. Rather, Wakerley (370) has summarized an overall view held by many in the field, which posits that suckling activates excitatory mechanisms that promote local interactions among OT neurons, which eventually, through a number of positive feedback interactions, leads to more coordination in their firing pattern. Increasing numbers of OT neurons are gradually recruited into this coordinated firing, and their responses to excitatory inputs are increased, until the entire population explodes with a burst of action potentials that lasts for a few seconds, to be followed by a brief period of inhibition, before the process begins anew. While many details of this phenomenon remain incompletely characterized, considerable progress has been made in recent years in identifying mechanisms that underlie this unique pattern of activation and neurosecretion; these include properties intrinsic to the OT neurons themselves, local interactions of OT cells with each other, with afferent inputs and with glial cells, and synchronization signals that may arise locally and/or from other brain regions.

Intrinsic properties of OT neurons A detailed analysis of the electrophysiological characteristics and behaviors of OT neurons is beyond the scope of this review (and of the author’s expertise), and the interested reader is referred to several excellent reviews (17, 18, 152, 370). Armstrong and coworkers (313-317, 333-335; reviewed in 17, 18) have described the electrophysiological properties of OT neurons that may facilitate the development of milk ejection bursts during lactation. As examples, OT neurons display spike broadening, that is, an increased duration of the nerve action potential, especially notable during repetitive firing, in late pregnant and lactating rats, as compared with virgin females, a property associated with increased Ca2+ influx. Another current in OT neurons that shows an increase in lactation is a K+ current, the sustained outward



rectifier. As described by Armstrong (18), when cell membrane hyperpolarization inactivates this current, OT neurons display rebound depolarizations that can generate a cluster of nerve action potentials. A third change, specific to OT neurons in pregnancy and lactation, is an increase in the Ca2+ dependent afterhyperpolarization. This current is suggested to regulate the duration and amplitude of milk ejection bursts, and, as described by Armstrong (18), could be viewed as a “braking mechanism” to help limit the duration of the milk ejection burst. It is generally agreed that these intrinsic properties of OT neurons alone do not provide a sufficient basis for generation of the synchronized milk ejection bursts, and it seems certain that afferent inputs activated by suckling are necessary to drive the reflex. Milk ejection-like bursts can be induced pharmacologically in OT neurons (by an α1 adrenergic agonist in low Ca2+ medium) in hypothalamic slices, but the bursts are asynchronous (375). However, synchronized bursts can be observed in organotypic hypothalamic cultures, most likely because local intrahypothalamic circuitry is preserved in this preparation (165, 173, 174); see below). These observations have led to the hypothesis that much of the synchronized OT milk ejection bursting in response to suckling is organized within the hypothalamus, but may be subjected to external inputs (370).

Regulation by neurotransmitter systems Glutamate: In the two decades since our earlier review on the neurochemical regulation of OT secretion in lactation (77), glutamate has been firmly established as a critical excitatory regulator of OT neuronal bursting in response to suckling. Pharmacological studies in lactating rats (264-266) demonstrated that central administration of AMPA or kainic acid to lactating rats readily increased OT release, while no effect was seen with NMDA or a metabotropic glutamate agonist given alone. However, a synergistic effect of NMDA and AMPA could be observed on OT release when both were coadministered in doses that were ineffective alone (266). This could be explained by the well-known phenomenon that glutamate may activate NMDA receptors upon membrane depolarization achieved via concurrent activation of AMPA receptors (164). That glutamate may play an obligatory role in sucklinginduced OT release is strongly suggested by the finding that an antagonist at AMPA/kainate receptors blocked sucklinginduced OT release (264). A critical role for glutamate in generating the milk ejection bursts has received abundant support from anatomical and electrophysiological studies. OT neurons in both magnocellular nuclei are heavily innervated by glutamate-positive nerve terminals, preferentially on dendritic processes (115, 358). The PVN itself contains glutamate-positive neurons, and also receives glutamatergic inputs from the perifornical region and anterior hypothalamus adjacent to the PVN, dorsomedial hypothalamic nucleus, and ventral premammillary and supramammillary nuclei, as well as from telencephalic sources that

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LS/BNST Glu PNR Glu/ GABA

PVN

SON

PNR Glu/ GABA

DCA

Glu

DMN Mam B

Brainstem

afferents

Figure 6 Intrahypothalamic circuitry and sources of excitatory and inhibitory amino acid inputs to OT neurons in the magnocellular nuclei. See Figure 2 for abbreviations. Glutamate neurons innervating the PVN and SON are located in the LS/BNST, DMN and Mam B, and also in perinuclear regions (PNR) adjacent to each magnocellular nucleus. GABA innervation to OT neurons also largely emanates from adjacent PNRs. Arrows also show bilateral inputs to PVN and SON from the DCA; reciprocal connections between the PVN and SON ipsilaterally, and bilateral PVN-PVN and SON-SON interconnections, which are believed to participate in synchronization of milk ejection bursts (see text for details).

include septum and bed nucleus of the stria terminalis (45, 85). Similarly, glutamate interneurons exist within the SON, and additional glutamate inputs emanate from a region just dorsal to the SON, termed the perinuclear zone, and from the hypothalamic PVN, the thalamic PVN, and, similar to PVN inputs, the dorsomedial, ventral premammillary and supramammillary nuclei, septum and bed nucleus of the stria terminalis (86). It is important to note the presence of glutamatergic neurons in some of the regions implicated in the suckling-activated afferent pathway (see Figs. 2 and 6). Electrophysiological studies clearly identify glutamate as the predominant excitatory transmitter in the magnocellular nuclei, with actions at both AMPA/kainate and NMDA receptors (316, 358; reviewed in 164). Armstrong and coworkers have provided evidence that AMPA receptor-mediated excitatory actions on OT neurons are increased during lactation (317). One experimental approach to assess the role of glutamate in milk ejection bursting has employed neuronal recordings from identified OT neurons in organotypic hypothalamic cultures generated from early postnatal rats, and has provided some important insights. Initial studies demonstrated that OT neurons in the cultures exhibit spontaneous, periodic bursting activity very reminiscent of milk ejection bursting in vivo (173, 174). Moreover, this behavior is dependent upon intermittent clusters of excitatory postsynaptic potentials that could be blocked by an antagonist at AMPA/kainate receptors,

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consistent with our pharmacological studies in lactating rats (264, 266), and leading these investigators to propose that the milk ejection bursting behavior of OT neurons during lactation is the product of a pulse generator organized in an intrahypothalamic glutamatergic circuit (174). As noted above, the anatomical substrate for such a local network clearly exists. Further studies from this laboratory recorded from pairs of OT neurons in the cultures and provided evidence that, as in vivo, OT bursting in vitro can occur synchronously in separate neurons, and in a glutamate-dependent manner (165). We will discuss this mechanism further below when considering neural mechanisms for the synchronization of milk ejection bursts. GABA: Local GABAergic inputs to magnocellular OT neurons also may contribute significantly to their bursting behavior, but in a complex manner. Pharmacological studies in lactating rats showed that central administration of either a GABA-A agonist or an antagonist disrupted milk ejection bursting and inhibited milk ejections in lactating rats, and it was therefore proposed by these investigators that these reflect an essential role of GABAergic transmission in generating milk ejection bursts that could be disrupted by either receptor stimulation or blockade (367). Electrophysiological studies support this concept. As might be expected, GABA represents the major inhibitory transmitter in the magnocellular nuclei (93), and GABA hyperpolarizes magnocellular OT



neurons in PVN and SON by activating Cl− conductance via classical GABA-A receptors (references in 77, 235). Studies by Moos (235) in lactating rats showed that administration of a GABA-A antagonist into the SON increased basal electrical activity of OT neurons but inhibited milk ejection bursting; conversely, application of GABA-A agonists decreased basal activity but facilitated bursting. She suggested that this could reflect inhibition by GABA of excitatory inputs not related to suckling as one mechanism (235); others have noted that membrane hyperpolarization can facilitate bursting via rebound depolarization (17). Whatever the mechanism, it is clear that GABAergic inhibitory transmission must participate in some manner to inducing or shaping the milk ejection bursts. As for glutamate, this GABAergic influence most likely derives from local intrahypothalamic sources. Anatomical studies show that while the SON itself lacks GABAergic interneurons, it receives a prominent GABAergic innervation from the perinuclear zone (282). The PVN does have GABAergic neurons in its anterior regions, and also receives inputs from the perifornical area and anterior hypothalamus adjacent to the PVN (282) (see Fig. 6). The NE/Glutamate interaction: In addition to glutamate, substantial evidence supports a major excitatory role for NE in suckling-induced OT release, and the noradrenergic innervation of the PVN and SON may represent sucklingactivated afferents that ascend from the medulla and interact with the local circuitry described above (See Fig. 7). As reviewed earlier (77, 293), OT neurons of the PVN, and also in the anterior commissural nucleus accessory site, receive a dense noradrenergic innervation. Dendrites of OT neurons in the SON are also contacted by ascending noradrenergic fibers (225). Imaging studies suggest strongly that the A1 and A2 noradrenergic cell groups, which innervate the magnocellular nuclei (77, 293), are directly activated by suckling (208, 209). Microinjection of the α1 agonist phenylephrine into the SON or PVN/anterior commissural nucleus evokes a brisk OT secretory response in lactating rats (263, 265). It is interesting to note that this agent also produces milk ejection-like bursting of SON OT neurons in a hypothalamic slice preparation from lactating rats (374). Parker and Crowley (263) also reported that an α-2 agonist or antagonist were without effect on OT release. Other in vivo approaches have demonstrated that the turnover rate of NE is increased in response to suckling in the SON and rostral PVN (probably including the anterior commissural nucleus) (84). Moreover, as measured with in vivo microdialysis, NE release is increased in PVN in response to suckling (26). That activation of these noradrenergic inputs is obligatory for suckling-induced OT release was shown by studies demonstrating its complete inhibition by systemic administration of an α-adrenergic antagonist (307) or by microinjection of the catecholamine neurotoxin 6-hydroxydopamine into the PVN and SON regions so as to destroy the noradrenergic nerve terminals innervating these structures (84).



NL

Acc

PVN

DCA

LC (A6 NE)

PB

SON

Hypothalamus

Pons

NTS (A2 NE)

VMM

VLM (A1 NE)

Medulla

LCN Mammary gland afferents

DHsc

Figure 7

Long-range, direct inputs from brainstem nuclei to the PVN and SON. Abbreviations: Acc: accessory magnocellular OT neurons; NL: neural lobe. See Figure 2 for other abbreviations. Noradrenergic neurons in the A1 (VLM) and A2 (NTS) cell groups directly innervate OT neurons in PVN and SON, and probably also the accessory OT populations. The PVN also receives noradrenergic input from the LC. Also depicted are the direct connections from neurochemically unidentified VMM neurons, passing through the DCA to PVN and SON.

Pharmacological and electrophysiological studies suggest that this “long-range” noradrenergic input interacts with the local excitatory glutamate circuitry described above. For example, we found that coinjection of phenylephrine and AMPA into the SON of lactating rats in doses that were submaximally effective doses when given alone, resulted in a synergistic increase in OT release; further, the release of OT in response to phenylephrine alone could be blocked by an AMPA antagonist, while conversely, AMPA stimulation of OT release was blocked by an α-1 antagonist (265). Electrophysiological studies from Tasker and co-workers support the concept of such an interaction between these two systems. For example, they found that magnocellular PVN neurons in an ex vivo slice preparation responded to application of NE or an α-1 agonist with an increase in excitatory postsynaptic potentials that could be blocked by ionotropic glutamate receptor antagonists, suggesting a presynaptic facilitatory action of NE on glutamate release (90). Further studies showed that

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this mechanism is operational in both the PVN and SON and involves localized glutamatergic interneurons (44). NE interactions with other systems: Our laboratories have shown that NE interacts with two other excitatory systems that contribute to stimulation of OT release during lactation. Many of the A1 and A2 noradrenergic neurons that innervate the magnocellular nuclei coexpress NPY (177); NPY neurons resident in the arcuate nucleus also provide innervation to the PVN, notable in the control of feeding and appetite (see below). Parker and Crowley (263) reported that administration of NPY or an agonist at the Y-1 receptor into the SON or PVN/anterior commissural nucleus of lactating rats stimulated OT release; in those studies NPY was also found to potentiate the OT response to phenylephrine. Thus, as in other brain regions, NPY could be coreleased with, and augment the response to, NE in the magnocellular nuclei. Central administration of histamine also stimulates OT release, while blockade of either H1 or H2 receptors inhibits suckling-induced OT release (294). Both magnocellular nuclei receive histaminergic inputs derived from a cell group in the tuberomammillary nucleus (257, 295), an area implicated in control of milk ejection bursting (376). Bealer and Crowley (27) showed that administration of histamine by retrodialysis into the PVN of nonlactating female rats stimulated OT release into the systemic circulation, an effect that could be blocked by the α-adrenergic antagonist, phentolamine. A subsequent study showed that suckling enhanced histamine release within the PVN, as determined with microdialysis (28). Taken together, these finding suggest that histaminergic neurons of the mammillary region may exert a presynaptic, stimulatory action on NE release within the magnocellular nuclei. Dopamine: OT neurons in both magnocellular nuclei also receive dopaminergic innervation, most likely deriving from intrahypothalamic DA systems (92, 212), but the contribution of this catecholamine to the intermittent milk ejection burst firing pattern has not been investigated as thoroughly as for NE or glutamate. In an early study, intraventricular injection of DA or the nonselective agonist apomorphine to anesthetized, lactating rats increased the frequency and amplitude of milk ejection bursts during suckling in PVN OT neurons, leading to increases in intramammary pressure responses; conversely, intraventricular administration of the nonselective antagonist haloperidol inhibited the suckling-induced activation of OT neurons (239). Pharmacological studies in the author’s laboratory have provided evidence for both excitatory and inhibitory dopaminergic effects mediated by different DA receptor subtypes and, to some extent, by differential actions in the magnocellular nuclei versus the neurohypophysis. Thus, we found that systemic administration of a selective D-1 agonist increased basal levels of OT in nonsuckled, lactating rats, while treatment with a selective D-1 antagonist inhibited suckling-induced OT release (79). Conversely, selective activation of the D2 receptor inhibited suckling-induced OT release (79, 84).

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Further studies using a microinjection approach localized the stimulatory D-1 effect to the SON of lactating rats, while the PVN appeared somewhat less responsive (262); a small component of this effect may also be exerted at the neurohypophysis (see below). In these studies only modest effects were seen on OT release after D-1 agonist microinjection into PVN. As reviewed below, we found that the effects of D-2 receptor stimulation or blockade, first believed to be exerted in the neurohypophysis, were actually secondary to their effects on PRL release, and that PRL has a facilitatory action on OT release from nerve terminals in the neural lobe (261).

Anatomical and neurochemical plasticity and reorganization within the magnocellular nuclei during lactation The magnocellular neuroendocrine system has served as a model for investigating anatomical plasticity in response to physiological demands, and the anatomical reorganizations that occur in the OT neurosecretory system during lactation are believed to contribute significantly to generation of the milk ejection bursting firing pattern (cf. 49, 58, 337, 342, 344, 370 for recent reviews). In the early 1980s, two laboratories independently reported that during lactation in rats, OT neurons of the SON exhibited many more somatic appositions than were seen in non-lactating animals; additionally, there were increased appearances of “double synapses” in the SON, in which presynaptic terminals were observed to contact more than one postsynaptic element, a phenomenon described as being rare in non-lactating animals. These changes were attributed to retractions of the fine astroglial processes that normally separate these neuronal elements from each other (146, 343). Hatton and co-workers subsequently reported that in lactating rats, there were more direct contacts among the dendrites in the ventral glial lamina area of the SON, to which the more dorsally located OT neurons project their dendrites, forming “dendritic bundles,” and more double synapses in these bundles, again attributed to retractions of glial processes (268). The phenomenon of glial retractions was also extended to the PVN (341), and to the neurohypophysis, as Tweedle and Hatton (355) reported that pituicyte (i.e., astroglial) ensheathments of neurosecretory axons were greatly reduced in lactating rats. This adaptation presumably removes a significant glial barrier between the neurosecretory axons and the vasculature, which would presumably facilitate release of the peptide into the bloodstream (342). Additional morphological changes noted in magnocellular OT neurons during lactation include hypertrophy of the cell bodies, shortening and decreased branching of the dendrites but enlargement and increased branching of the axons (reviewed in 337, 344). These seminal observations on morphological reorganizations affecting OT soma, dendrites and neurohemal terminals during lactation (203, 342, also see figures in 337), have led to the hypothesis that increased appositions among OT neurons within a nucleus might facilitate the development of coordinated firing for the milk ejection burst (343). The



subsequent demonstrations of increased dye coupling among OT neurons of the SON during lactation, implying the existence of gap junctions and electrical connectivity across OT neurons, was consistent with this view (147). However, an important caveat noted early on was that glial retractions and increased somatic and dendritic appositions were also seen in response to dehydration (232, 353), a condition in which milk ejection-like burst firing and episodic OT release are not observed. Nevertheless, the withdrawal of glial processes and the increased somatic and dendritic appositions only affect OT neurosecretory cells, and not vasopressin neurons (338, 339), and, as discussed below, begin to appear in late gestation and are in place during parturition, also characterized by OT neuronal bursting. Further, astroglial processes reinsert themselves between OT neurons after pup separation and after weaning (231, 340), likely indicating dependence upon the suckling stimulus during lactation. Hence, what has been termed a “neuronal-glial dialogue” (232, p. 681) appears to be specifically associated with states of heightened OT secretion, if not specifically to milk ejection bursting, and is especially marked during lactation, suggesting that such changes might be permissive or facilitatory to conditions of increased OT release. For example, it would seem logical that increased somatic appositions and double synapses would enhance the actions of excitatory neurotransmitters to create a stimulatory neurochemical microenvironment affecting multiple OT neurons, which might then facilitate coordinated activity among OT neurons within each magnocellular nucleus. It is therefore intriguing to note that many of the double synapses present during lactation are glutamatergic and noradrenergic (115, 228), which, as detailed above, interact with each other to provide the major excitatory drives to OT neurons in response to suckling. GABAergic double synapses also increase during lactation (134), which also contribute to shaping the milk ejection bursting behavior, as reviewed above. In addition, evidence has been presented for potentially important changes in glutamatergic and GABAergic interactions as a result of glial retractions. For example, glial retractions in the SON might increase the concentrations of glutamate in the extracellular space, as glutamate uptake by astrocytes would be decreased, and this can in turn activate inhibitory presynaptic receptors on adjacent GABAergic terminals (273, 274). Theodosis and co-workers have also reported that glial retractions result in changes in function of the NMDA and kainate subtypes of glutamate receptor that would most likely enhance excitatory neurotransmission (43, 256; see also 328 for review). Major questions remain regarding the anatomical reorganization in the magnocellular nuclei during lactation, one, for example, concerning the cellular mechanisms involved in glial retractions and reinsertions. These remain largely uncharacterized, and some potential mechanisms are discussed in a recent review (342). Perhaps a more important issue for the present context is whether or not they are required for suckling-induced OT release during lactation, and



some uncertainty surrounds this question at present. Results from the Theodosis laboratory, which first described the phenomenon, suggest that glial retractions are not necessary for the expression of OT burst firing or episodic OT release during parturition or lactation, on the basis of studies in which such retractions in both PVN and SON were prevented by a neurochemical approach during late gestation. Female rats so treated gave birth and exhibited normal patterns of sucklinginduced increases in intramammary pressure; likewise, OT neurons of the SON showed normal milk ejection bursts in response to suckling (65). These investigators concluded that while not obligatory for milk ejection bursting, glial retractions and the consequent reorganizations may be involved in a “fine tuning” of OT neuronal activity, for example, inhibiting inputs from other nonsuckling-related sources, so that OT neurons can respond only to suckling (65). It would also seem likely that these anatomical changes would serve to increase exposure of multiple OT neurons to excitatory transmitters activated by the suckling stimulus.

Intranuclear release of OT Following the discovery and detailed electrophysiological characterization of the milk ejection burst firing pattern of OT neurons, perhaps the most important advance in OT research in recent years has been the recognition that OT is released within the magnocellular nuclei, largely from dendrites and soma, and that OT exerts multiple paracrine and autocrine actions therein that are critical for expression of the milk ejection reflex. This mode of OT release has been commonly referred as intranuclear release or somatodendritic release, and a relatively large literature has been extensively reviewed elsewhere (49, 195, 202, 370). Some of the key findings are summarized as follows. Initial reports by Moos and coworkers (238, 240) showed that OT was released in the SON in response to suckling, as measured with push-pull perifusion, and, importantly, that this increased release began shortly before a milk ejection episode. Subsequently, Neumann et al., using in vivo microdialyis, observed OT release within both PVN and SON during lactation and parturition, but not in late pregnancy; vasopressin was not released in these areas during those times, but both magnocellular peptides were released in response to intranuclear hyperosmolality (251). It had been reported earlier (126) that intraventricular administration of OT to suckled rats increased the frequency and amplitude of milk ejection bursts recorded from PVN neurons, while central administration of an OT antagonist inhibited milk ejections and milk ejection burst firing, indicating an important facilitatory action of OT exerted within the magnocellular nuclei; interestingly, OT had no effect in the absence of suckling in these studies. That this release may occur primarily from dendrites and soma was suggested by the observations of exocytotic profiles in electron microscope images of dendrites in the SON (277), but some may derive also occur from axon terminals.

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Effects on milk ejection burst firing: Subsequent studies have provided more information on the mechanisms underlying intranuclear OT release and on how such release contributes ultimately to generation of the milk ejection burst. OT release from the somatodendritic compartments depends in part on entry of extracellular Ca2+ via voltage-dependent channels, particularly of the N-type (251, 348), although, as reviewed below, the Ca2+ -dependent mechanisms in the dendrite can be complex. Administration of an OT receptor antagonist into the SON inhibited both systemic and intranuclear OT release, leading to the important proposal that OT might act via a positive feedback mechanism to promote its own intranuclear release that, in turn, is required for release of OT from neurohypophyseal terminals into the systemic circulation (249). Perhaps not surprisingly, there is evidence that the two major excitatory neurotransmitters regulating sucklinginduced OT secretion, norepinephrine, and glutamate, also stimulate intranuclear release of the peptide (25). Thus, Bealer and Crowley reported that the suckling-induced release of OT in the PVN and SON could be attenuated by intranuclear administration via retrodialysis of an α-adrenergic antagonist (26). Histamine also stimulates intranuclear OT release via enhancement of NE release, as it does for systemic OT release (27, 28). Finally, patch clamp electrophysiological studies performed in ex vivo hypothalamic slices revealed that activation of the NMDA subtype of glutamate receptor can stimulate exocytotic OT release from SON neurons, and that this effect was more pronounced in slices from lactating rats (97). How does intranuclear OT release contribute to generation of the milk ejection burst firing pattern? In addition to being itself regulated by afferent neurotransmitter systems, the OT released within the magnocellular nuclei also interacts with NE, GABA, and glutamate inputs that are critical for generating these bursts. For example, in vivo microdialysis experiments (although in nonlactating rats) have shown that locally applied OT increases the release of NE within the SON (255). In addition, both pre- and postsynaptic modulations of GABAergic transmission by intranuclear OT have been demonstrated in electrophysiological studies. For example, Brussaard and co-workers reported that activation of OT neurons of the SON in hypothalamic slices (from nonlactating rats) decreased the release of GABA, and evidence was obtained for inhibitory retrograde signaling by somatodendritically released OT, and possibly by adenosine (97, 98). This group also reported that OT could exert an inhibitory modulation of postjunctional GABA-A receptor activation in the SON (57). On the other hand, Israel et al., using the hypothalamic organotypic culture preparation, found that low concentrations of OT applied to the cultures, suggested to be similar to those occurring in somatodendritic release, enhanced GABAergic transmission through a presynaptic mechanism (166). While seemingly contradictory to the studies of the Brussaard group (57, 97, 98), Israel et al. (166) also reported that this enhancement of GABA release actually promoted

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bursting in many OT neurons, and they proposed that this may be due to several mechanisms, including postinhibitory depolarization, somewhat reminiscent of the in vivo results of Moos (235), discussed above, who showed that GABA-A agonists inhibited basal activity of OT neurons, but facilitated milk ejection burst firing. Interactions of intranuclear OT with the critical local glutamatergic inputs have also been reported as contributing to milk ejection bursting. Using the organotypic hypothalamic culture system, Jourdain et al. (174) found that locally applied OT or an OT agonist enhanced the bursting activity of magnocellular OT neurons by decreasing the interburst interval, and also induced bursting in OT neurons that were in an irregular firing mode. An AMPA/kainate glutamate antagonist inhibited both spontaneous and OT-induced burst firing, and these authors concluded that “OT bursting activity is similar to spontaneous bursting activity and results from a patterned afferent drive . . .” (174 p. 6647), which they attributed to the local glutamate inputs. This has been supported by more recent studies on SON neurons in hypothalamic slices from lactating rats, in which AMPA/kainate blockade reduced the excitatory effects of very low doses of OT on OT neuronal firing (376). On the other hand, others have reported that OT reduced excitatory postsynaptic potentials in SON neurons in hypothalamic slices, and these investigators obtained evidence for mediation by endocannabinoids, which were released from OT neurons to act as inhibitory retrograde signals (154, 186). This mechanism has been proposed to contribute to the termination of bursts (284). A caveat to these studies is that the slices were obtained from male rats, raising the potential issue of relevance to the lactating state. It is also interesting to note the reports that central administration of OT enhances the excitatory responses of SON OT neurons to electrical stimulation of the ventral tegmentum (71) and dorsomedial hypothalamic nucleus (72) in suckled, lactating rats; recall that both of these regions have been implicated in the suckling-activated afferent pathway (see above). These findings support the concept that intranuclear OT might promote milk ejection burst firing of OT neurons by enhancing responses to “long range” as well as local afferent inputs. Adding to the complexity of intranuclear OT action in the magnocellular nuclei is the process referred to as “priming” (see Fig. 5 in Ref. 49). From in vivo pharmacological studies using microdialysis of the (nonlactating) rat SON, Ludwig, Leng and co-workers (217) demonstrated that mobilization of intracellular Ca2+ in dendrites can evoke somatodendritic OT release in the absence of increased electrical activity of the OT cell. More importantly, this initial effect was shown to potentiate somatodendritic OT release in response to neuronal spike activity. This “priming” effect was subsequently shown to be due to a Ca2+ -dependent translocation of peptidecontaining synaptic vesicles in the dendrites to a position in closer proximity to the cell membrane, presumably enhancing the likelihood of exocytotic release (202, 349). A critical



additional finding was that OT, known to mobilize intracellular Ca2+ upon receptor binding (191), could itself induce this priming action (217). Although these studies were not conducted in lactating rats, they have led to the widely held hypothesis (e.g., 49,202,285,370,377) that suckling inputs may evoke an initial intranuclear release of OT, which is stimulated by extracellular Ca2+ influx via voltage-regulated channels, and which occurs prior to the onset of milk ejection bursts (238). Via activation of OT receptors on the same (autocrine action) or adjacent (paracrine action) dendrite(s) (demonstrated by 127, 128), and via subsequent activation of phospholipase C and generation of IP3, intranuclear OT thus would prime the system by increasing release of Ca2+ from intracellular stores. As the OT neurons become progressively more active, the increase in nerve action potentials would produce greater somatodendritic OT release, as a result of the aforementioned priming effect. It is envisioned that the progressively greater amounts of intranuclear OT, in interactions with the local (e.g., glutamate) and longer range (e.g., NE) afferent systems described above, would foster progressively more excitation among multiple, adjacent OT neurons, until the entire population unites in firing the brief milk ejection burst. The development of this positive feedback mechanism may account in part for the latency to the initial milk ejections observed in the standard separation-reunion paradigm in rats. Recent evidence suggests that intranuclear OT is degraded by placental leucine aminopeptidase, which in the magnocellular nuclei could serve as an oxytocinase and terminate an action of intranuclear OT (347). Other actions of intranuclear OT: To the extent that the morphological reorganizations within the magnocellular nuclei contribute to the generation of milk ejection bursts (see above), this process also appears to be under control by intranuclear actions of OT. The Theodosis laboratory provided the initial demonstration that an 8-day infusion of OT into the third ventricle of female rats that had weaned a litter 1 month previously resulted in decreased glial coverage and increased somatic appositions and synaptic contacts on SON neurons; infusion of vasopressin was without effect (339). Subsequent studies showed that this action required the presence of physiological levels of estradiol, while high levels of progesterone appeared to be inhibitory (198, 230). Finally, detailed studies of the mechanisms that may be involved in this effect, performed in hypothalamic slices from female rats in late gestation, suggested that the effect of OT may occur through enhanced release of glutamate from local afferents (see above), which may prove to be the more proximal signal to the astroglia (198). Finally, intranuclear OT may also contribute to the up regulation of OT gene expression observed in the magnocellular nuclei during lactation (362,391). Studies in the author’s laboratory demonstrated that intraventricular administration of an OT receptor antagonist during early lactation decreased OT mRNA levels in the SON, but not PVN, while chronic third



ventricular infusion of OT attenuated the decrease in SON OT mRNA expression in response to litter separation during this time (309).

OT actions in other brain areas and the milk ejection reflex Several reports suggest sites of action of OT in facilitating milk ejection bursts in addition to the magnocellular nuclei, most notably the ventrolateral septum and adjacent bed nucleus of the stria terminalis (Fig. 6). This region receives an oxytocinergic input from the parvocellular PVN, anterior commissural nucleus and perifornical areas (163), and neurons in these two areas express OT receptors (236). Sucklinginduced release of OT has been demonstrated in the lateral septum in response to suckling (250), and local administration of OT into this area facilitates the milk ejection burst firing of PVN and SON neurons during suckling (192, 236). Moreover, Lambert et al. (192) have described a population of neurons in this region that exhibit a burst firing pattern temporally linked to milk ejection bursts of the magnocellular nuclei. It is interesting to note that in our studies on suckling-induced effects on noradrenergic activity, the bed nucleus of the stria terminalis was the only nucleus other than the magnocellular nuclei showing an increase in turnover of NE in response to suckling (84). Lesions of this area alter milk ejection firing in the magnocellular nuclei but do not prevent burst synchronization (376). A second area receiving attention in this regard is the supramammillary nucleus in the ventro-posterior aspect of the hypothalamus, an area implicated as a possible synchronizing center (see below); this region also receives an OT projection, and OT facilitates the milk ejection reflex when applied here (89). These are intriguing observations, and the exact role(s) of these putative “extranuclear” OT target sites in milk ejection burst generation remain to be determined.

Synchronization of the OT population: Local versus long-range mechanisms From the discussion above, it is clear that multiple mechanisms within and in close proximity to the magnocellular and accessory nuclei are activated during suckling and promote the progressive excitation of the OT neuron population into a more coordinated firing pattern, leading eventually to the milk ejection burst. Recent reviews (e.g., 49,285,370) ascribe important roles in this process to intranuclear OT, via (i) direct excitatory paracrine and autocrine actions on multiple OT neurons, (ii) indirect excitatory effects mediated by enhancing noradrenergic, glutamatergic and GABAergic transmission, and (iii) in inducing glial retractions that can facilitate interneuronal interactions. It seems straightforward to envision how these actions can also promote synchrony in bursting among OT neurons within a single nucleus, but microinjection of OT into a single PVN or SON also enhances milk ejection bursting by OT cells in the contralateral nuclei, while

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microinjections of an OT antagonist into either of these nuclei can inhibit milk ejection burst firing by neurons within the injected nucleus as well as in a contralateral nucleus, suggesting that intranuclear OT also is involved in synchronization of the entire OT neurosecretory population (193, 238, 240). So, how does one account for the synchronization of OT neurons among the four magnocellular nuclei plus the accessory sites, such as the anterior commissural nucleus, in response to suckling? In interpreting what can be a complex literature, it is important to keep in mind that synchronization refers specifically to the close onset of milk ejection bursts across multiple OT neurons (typically between 0 s, i.e., a simultaneous onset, and onsets up to about 600 ms apart), while desynchronization means that bursting occurs, but the time lag in the onset of bursting among individual neurons is much longer. Experimental manipulations that interrupt bursting per se, will also affect synchronization, while the converse is not the case. At present, there appears to be two schools of thought regarding potential internuclear synchronization mechanisms, one emphasizing locally organized interconnections among the magnocellular nuclei, and a second focusing on synchronization signals that emanate from more distant structures and that could be imposed on the local internuclear controls. Arguing in favor of locally organized synchronization are the above-cited observations that synchronous burst firing of OT neurons can be observed in organotypic hypothalamic cultures (173, 174), evidently dependent upon excitatory glutamatergic inputs from local sources (165). This group of investigators has proposed that each local glutamatergic neuron might “govern” a cluster of OT neurons, and that overlapping afferent inputs from multiple glutamate neurons could synchronize firing of different groups of OT cells within a single nucleus. They also envision that unilateral internuclear synchronization, that is, between a PVN and SON (and accessory sites) on the same side of the hypothalamus, could be achieved by a “common pool” of glutamate neurons that innervate one or both of the magnocellular nuclei (165,173,174). Electrophysiological studies do support this concept in demonstrating the existence of ipsilateral communications between PVN and SON (290, 291) (see Fig. 6). As for the phenomenon of contralateral synchronization, there is electrophysiological evidence for (most likely) multisynaptic interconnections between the two SONs (327), and anatomical evidence that the two PVNs connect dorsally over the top of the third ventricle (the supraventricular gray commissure (302). Finally, Thellier et al. (336) reported that microinjection of retrograde tracers into any of the magnocellular nuclei labeled a small population of neurons dorsal to the optic chiasm in the ventral anterior hypothalamus, which they termed the dorsochiasmatic area, and which they speculated could serve as a local synchronization structure (Fig. 6). However, there is inconsistent evidence that these local interconnections might play a role in synchronization. For example, while interhemispheric sections that interrupted the dorsal inter-PVN connections did reduce milk ejection

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bursting, the bursts that occurred were synchronized; on the other hand, intranuclear OT no longer influenced activation of contralateral OT neurons in those studies (240). In contrast, lesions in the dorsochiasmatic area inhibit milk ejection bursts and especially, contralateral burst synchronization (237,376), further highlighting the importance of this area. It may also be the case that these local interconnections among the magnocellular nuclei could relay synchronizing signals that originate in more distal structures, depicted in Figure 7. For example, as discussed above in the section on the milk ejection afferent pathway, the Moos laboratory has identified neurons in the ventromedial medulla that are activated by suckling and that project bilaterally to SON, and most likely PVN, passing in part through the above-mentioned dorsochiasmatic area that seems involved in internuclear synchronization (237). Another candidate as a synchronizing center is the dorsomedial nucleus of the hypothalamus (DMH). On the basis of lesion and electrophysiological studies, Higuchi and colleagues provided evidence that the DMH is a component of the milk ejection afferent pathway with direct projections to SON; further, they reported the existence of DMH neurons that fire off brief bursts of action potentials immediately prior to each milk ejection (327). Subsequent studies showed that projections from DMH to SON were largely excitatory and target ipsilateral, contralateral or bilateral SON (155-157). While suggestive, the role of the DMH in synchronization as assessed with the definitive electrophysiological tests has not yet been reported. Wang and co-workers have proposed that the mammillary body and adjacent structures may serve as a synchronization center, based on their observations that discrete midline lesions in this structure markedly inhibited milk ejections and inhibited burst synchronization in recordings from paired SON OT neurons (376,388). These authors also discuss the extensive connections between magnocellular nuclei, and especially between the SON and mammillary body, including glutamate afferents.

Modulation at the neurohypophysis: Glial plasticity and neurotransmitters Although the vast majority of attention has been directed at mechanisms within the magnocellular nuclei that promote milk ejection bursting, the release of OT from nerve terminals may also be modulated in the neurophypophysis. To date, such neurohypophyseal mechanisms have not been integrated into an overall framework for the milk ejection reflex, but may be more significant for OT secretion during parturition or in response to dehydration (e.g., 362). The ultrastructure of the neurohypophysis has been well described in a number of reviews, and, as noted above, is comprised of neurosecretory endings of both types of magnocellular neurons (cf. 40, 145, 380 for reviews). Under basal conditions, pituicytes of the neurohypophysis interpose processes along the basal lamina, thereby separating nerve endings from the fenestrated capillaries, and enclose the peptidergic nerve



endings, thereby creating a considerable barrier to the vascular contact zone (354, 356). Analogous to the glial retractions in the magnocellular nuclei described above, pituicytes of the neurohypophysis also withdraw from neurosecretory endings during periods of stimulated release, including parturition, lactation and dehydration, increasing the proximity of neurosecretory endings to the capillaries (354, 356). Although not specific to OT neurons, or to lactation, this glial adaptation clearly permits closer neurovascular contacts to facilitate neurohormone release into the systemic circulation. As reviewed above, the THDA system, with cell bodies in the arcuate nucleus, innervates the neural and intermediate lobes of the rat (32, 125, 204). Early pharmacological studies suggested that DA might exert an inhibitory effect on OT release via a presynaptic action in the neural lobe (e.g., 366); consistent with this view, studies in our laboratories showed that suckling decreased the turnover rate of DA in the neurointermediate lobe (84). Based on our in vivo pharmacological studies in lactating rats, we predicted that this inhibitory influence of DA in the neurohypophysis would be mediated by the D2 receptor (84). However, this was not supported by direct tests of this hypothesis, using an in vitro preparation of isolated rat neurointermediate lobes obtained from lactating rats and subjected to an electrical stimulation paradigm designed to mimic milk ejection bursts, in that a D2 agonist had no effect on OT release, while a D1 agonist had a small stimulatory effect (84). This negative result led us to the unexpected finding that the effects of D2 agonists and antagonists on OT release in lactating rat reviewed above (inhibition by agonist, stimulation by antagonist) may actually be secondary to their effects on PRL secretion (see above). Thus, we found that passive immunoneutralization of PRL prevented suckling-induced or D2 antagonist-induced increases in OT release in lactating rats; moreover, iv administration of rat or ovine PRL increased basal OT release in vivo, and enhanced electrically evoked OT release from neurointermediate lobes in vitro (261). We suggested that PRL released by suckling could exert an important facilitatory action on OT release, at least in part, via direct action in the neurohypophysis. Evidence that OT can stimulate PRL release was reviewed above, and these findings suggest that each hormone of lactation may facilitate secretion of the other, although details remain to be established. An inhibitory action of opioid peptides on OT release has been demonstrated in many studies, and the general conclusion is that the major locus of this effect is at the neurohypophyseal nerve endings (cf. 40, 117 for reviews); for example, morphine can inhibit OT release evoked by electrical stimulation of the neurohypophysis, but not milk ejection burst firing of SON neurons, in lactating rats (70). The presence of an inhibitory EOP tone has been inferred from the demonstration that antagonists, such as naloxone (e.g., 42) or naltrexone (e.g., 261), enhance electrically evoked OT release from isolated neural lobes. The source of the opioid peptides could be from either of the magnocellular neurons themselves, in view of the coexpression of several EOP in



OT and vasopressin cells, and/or from independent EOP systems from the hypothalamus (40, 117). Whether or not opioid inhibition over OT plays a physiological role during lactation is not clear, but, as reviewed below, both central and neural lobe actions of opioid peptides may become more prominent during late pregnancy.

Preparation for Lactation: Adaptations in the PRL and OT Neuroendocrine Systems During Late Gestation Adaptations in the PRL neuroendocrine system As elegantly noted by Armstrong and Hatton (16, p. R27), “. . . the nervous system of lactating females has been carefully prepared and readied during pregnancy to preferentially deliver, during lactation, this behavioral response to suckling.” Although they were referring specifically to the unique pulsatile pattern of OT secretion seen in lactation, the neuroendocrine regulation of PRL also undergoes an important adaptive change during late pregnancy. As discussed above, the activity of the TIDA system that provides the tonic inhibition over PRL secretion is itself regulated by PRL, in a homeostatic, short loop, feedback mechanism in which elevations of PRL reliably increase TIDA activity (cf. 9, 138, 233, 234 for reviews). In lactating rats, however, PRL does not increase DA release, thus allowing for sustained elevations in PRL in response to suckling (13, 101, 283). Several studies suggest this reduced responsiveness to PRL inhibitory feedback is set in place during late gestation (11, 135, 138). PRL signals in TIDA neurons via specific components of the Jak/Stat pathway, leading ultimately to altered gene expression. In particular, the effect of PRL to induce phosphorylation of Stat5 and nuclear translocation of Stat5b in arcuate neurons is diminished in lactation; this may be due to increased arcuate expression of several SOCS (suppressors of cytokine signaling) proteins, which serve to inhibit Jak/Stat signal transduction (7, 8). Further studies from the Grattan laboratory (318) point to an important role of the changing ovarian steroid milieu in late pregnancy, which were initially described in detail in a classic paper by Bridges (46). Titers of both estradiol and progesterone gradually increase from mid-late gestation in rats, and while estradiol remains high through parturition, progesterone levels abruptly fall in the last 1 to 2 days prepartum. Steyn et al. (318) have reported that the high levels of estradiol and PRL characteristic of late gestation may be responsible for the upregulation of SOCS proteins, facilitated also by the sharp decrease in secretion of progesterone, which inhibits expression of these proteins.

Adaptations in the OT neuroendocrine system A substantial literature documents a number of changes in the OT neurosecretory system during pregnancy, and suggests

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that these may prepare this system for the demands of parturition and lactation, and in particular, that may be important for the development of the intermittent burst firing pattern unique to parturition and lactation. Indeed, evidence of occasional burst firing and pulsatile OT release shortly prior to parturition exists (320); our laboratories have observed, for example, pulsatile release of OT, in the absence of suckling, on gestation day 20, two days before parturition, but not on gestation day 15 or in ovariectomized virgin females (214). Even though this release was not as regular as seen in response to suckling during lactation, the finding that OT pulses are occasionally present in late gestation is consistent with an earlier report by Jiang and Wakerley that some OT neurons of the SON are capable of burst firing at this time in response to suckling (169), and with reports of increased basal levels of circulating OT during late pregnancy (109, 189). It may not be possible to detect suckling-induced OT release at earlier times in gestation due to technical reasons, that is, offspring are unable to attach to nipples (319). A critical question is whether the ability of OT neurons to burst fire is “acquired” de novo during gestation, or, as argued by Russell and co-workers in a thoughtful review (287), may be intrinsically present, but under very strong restraint (in their view by EOP), so that it is the presence of the unique stimuli associated with parturition and lactation that drives the intermittent bursting pattern. Arguing for some degree of alteration in OT neuronal functioning during pregnancy are the electrophysiological studies of Teruyama and Armstrong (333, 335) showing that OT neurons of rats in late gestation (day 19-22), but not earlier in pregnancy, display some of the features of OT neurons during lactation that may contribute to bursting behavior, that is, increased incidence of depolarizing afterpotentials, which are believed to contribute to OT neuronal excitability, and of several afterhyperpolarizing potentials, which may help limit burst duration. As reviewed below, additional changes occur in the hypothalamus and in the OT neurons during gestation that seem likely to play an important role in their preparation for parturition and lactation (cf. 25, 51-53, 58, 214, 287 for reviews). OT gene expression: A number of studies have reported that OT mRNA expression in the magnocellular nuclei is increased in late gestation, when compared with cycling rats or with rats in early gestation (73, 158, 310, 311, 362, 391), although a number of uncertainties remain regarding the magnitude and timing of this increase (287). More consistent evidence demonstrates that this upregulation of OT gene expression persists well into lactation (362, 391) and is maintained in part by the suckling stimulus via intranuclear OT and by a central action of PRL, at least in the early postpartum period (132, 310, 311). During mid-late gestation, other factors, as yet unidentified, may assume more importance (132). An attractive hypothesis is that the rapidly changing ovarian hormone milieu of late pregnancy might be responsible for the upregulation in OT gene expression. Supportive of this view are the findings that treatment of nonlactating, ovariectomized rats with estradiol and progesterone for 14 days,

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followed by selective withdrawal of progesterone, designed by Bridges (46) to mimic, at least to some extent, the changes in these steroids during mid- to late gestation, increased OT mRNA expression in the PVN and SON (74). Moreover, the reduction in progesterone secretion may be particularly critical inasmuch as maintenance of high levels of this steroid during late gestation prevents the increase in OT mRNA (74). Further work from the Amico laboratory (36, 344) has suggested that the inhibitory effect of progesterone on OT mRNA expression may be mediated by one of its active metabolites, specifically the neurosteroid allopregnanolone (3α-hydroxy5α-pregnan-20-one), which is a positive allosteric modulator of GABAergic transmission via binding to the Cl− ion channel of the GABA-A receptor (53). As reviewed below, a reduction in allopregnanolone formation and/or action on GABA receptors has been implicated in several other modifications of the OT system during late gestation. It seems likely that the contribution of estradiol to OT gene expression might be exerted directly on OT neurons via Estrogen Receptor ß (160), but there is disagreement on whether these cells also express the Progestin Receptor (121, 345). Morphological reorganization: The anatomical adaptations in the magnocellular nuclei that were discussed in detail above, such as the retractions in astrocytic processes that likely promote excitatory local interneuronal communications, are clearly in place by late gestation (146, 340), and several changes can be observed as early as gestation day 15 (340). Central infusions of OT in ovariectomized, steroidtreated rats also resulted in increased somatic appositions and double synapses among OT neurons of the SON (230); analogous to ovarian hormonal effects on OT gene expression discussed above, progesterone antagonized the effect of central OT on these parameters (198). Hence, it is possible that the ovarian steroids of late gestation contribute to the morphological adaptations of late gestation as well as to OT expression, but this has not been tested directly in late pregnant rats. Moreover, these studies have also suggested that intranuclear OT may be important in preparation of the OT system during gestation (see below). Effects of neurotransmitters: Also potentially contributory to preparation of the OT system for the demands of lactation are changes in effects of important neurotransmitter systems. For example, our pharmacological studies have shown that an enhanced systemic release of OT in response to central administration of an α1 adrenergic agonist is seen in late pregnant (day 18-20) versus mid-pregnant (day 12-14) or nonpregnant females; further, the intranuclear OT response to noradrenergic stimulation is increased by mid-gestation compared to nonpregnant animals (216). However, despite this and other evidence cited above that points to increased activity in the OT system in late gestation, it would also seem important that OT release be restrained to some degree during this time to allow for buildup of OT content within the neurohypophysis for subsequent release, as well as to prevent premature activation of the system that could interfere with parturition or lactation. Considerable evidence suggests



that important inhibitory effects are exerted by GABA and by EOP during gestation, and also that dynamic changes occur in these systems close to the time of parturition. As reviewed above, GABA provides the major inhibitory influence over OT neuronal activity, and some of the morphological reorganizations in magnocellular nuclei involve increased GABAergic inputs. An initial report by Fenelon and Herbison demonstrated a gradual increase in the expression of the mRNA encoding the α1 subunit of the postsynaptic GABA-A receptor in PVN and SON neurons during gestation, up through day 19, followed by a reduction on the day of parturition and into lactation (120). In a subsequent study (121), they presented evidence that estradiol may be primarily responsible for the increase in the α1 subunit expression during gestation. These authors noted that an increased number of GABA-A receptors containing α1 subunits in their pentamer composition would be predicted to confer an enhanced sensitivity to the neurosteroid progesterone metabolite, allopregnanolone, levels of which would be expected to parallel those of progesterone, that is, an increase until late gestation, followed by an abrupt decline. The decline in allopregnanolone, along with the diminished responsiveness to the neurosteroid, would be predicted to reduce the overall tone of GABAergic inhibition over the OT system in readiness for parturition and lactation (120). A series of landmark papers by Brussaard and co-workers provided subsequent tests of this hypothesis. These investigators confirmed the reduction in α1 subunit expression in magnocellular neurons of the SON in late pregnancy, and, with electrophysiological approaches, demonstrated that this is accompanied by a decrease in the facilitatory modulation of GABAergic inhibition by allopregnanolone, as predicted (56). Additional studies by this group demonstrated that the distinct neurosteroid-insensitive composition of the GABAA receptor and the decreased efficacy of allopregnanolone persisted through lactation, but reversed after weaning (55). However, further studies in α1-knockout mice indicated that the neurosteroid-resistance of the GABA-A receptor seen in late gestation is not due to the decreased α1 subunit expression, as originally hypothesized, but rather, is mediated by phosphorylation of the GABA-A receptor by PKC, subsequent to an action of intranuclear OT (185). Thus, it appears that a combination of the fall in progesterone in late gestation and reduction in allopregnanolone formation, together with an action of intranuclear OT to decrease sensitivity of the GABA-A receptors in OT neurons to allopregnanolone, reduces inhibitory GABAergic tone to this system. Evidence for changes in EOP inhibition over OT has been obtained in pharmacological studies by Douglas et al. (109), who showed that systemic administration of the nonselective opioid antagonist naloxone increased circulating OT concentrations only on days 15, 18, and 21 of pregnancy, but not in cycling, early pregnant or in lactating rats. From these observations, they inferred that an opioid restraint of OT secretion develops during gestation, but eventually wanes postpartum. Their further studies, using isolated, electrically stimulated



neural lobes, suggested that the inhibitory opioid influence at the OT nerve terminal becomes desensitized close to the time of parturition (109). This group also found that although the inhibitory effect of opioids at the nerve terminal may diminish in late gestation, an inhibitory effect of opioids on OT neuronal firing becomes apparent at this time, mainly exerted through presynaptic inhibition of NE release in the magnocellular nuclei, an action not demonstrable in mid-pregnant or nonpregnant females (110). It is possible that these effects are exerted by β-endorphin, from neurons of the arcuate nucleus, or by enkephalins expressed in brainstem afferents. Further, just as allopreganolone provides an enhanced GABAergic tone during pregnancy (see above), this neurosteroid has also been implicated in the development of the central opioid inhibition over OT, again possibly mediated by decreased NE release (50, 286). As reviewed below, this neurosteroid-opioid interaction has been implicated in adaptations of the OT system to stress stimuli. Intranuclear OT and OT receptor expression: In view of the above-mentioned observations of increased OT gene expression, morphological reorganizations, and occasional pulsatile release during late gestation, and from suggestions that actions of intranuclear OT may be important for development of some of these preparatory adaptations, our laboratories have investigated whether intranuclear OT might play an important role during gestation in readying the OT system for parturition and lactation (cf. 25, 214 for reviews). Several studies show that intranuclear OT release is detectable in the magnocellular nuclei during pregnancy in rats, but levels as measured with microdialysis remain constant over gestation, as compared with nonpregnant animals, until the onset of parturition, during which time an increase occurs (216, 252). However, we found that OT receptor binding, as assessed with autoradiography, is progressively increases from mid- to late gestation in the PVN and SON as compared with nonpregnant rats, suggestive of increased sensitivity of OT neurons to the peptide (29). We also obtained evidence that estradiol may be involved in this upregulation of OT receptor expression (29). Further, blockade of central OT receptors during late gestation has significant effects on OT secretion during lactation. For example, we found that a chronic third ventricular infusion of an OT antagonist from mid-gestation until parturition, while not interfering with parturition or the onset of maternal behavior, did affect the OT release pattern in response to suckling, primarily producing a significant delay in release, and significantly impairing litter growth rates, consistent with an overall reduction in milk ejections and milk delivery to the pups (215). Moreover, whole cell current clamp recordings of OT neurons in ex vivo slices from pregnant rats revealed that OT neurons from animals treated chronically with the OT antagonist exhibited decreased amplitudes of several afterhyperpolarization currents (335). We proposed, therefore, that intranuclear OT may be important during mid to late gestation in altering intrinsic properties of OT neurons, and ultimately, in influencing the response characteristics of the OT system in lactation (335).

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Reduction in stress responsiveness in the PRL and OT neuroendocrine systems The discussion above points to a balance of excitatory and inhibitory influences affecting the OT neuroendocrine system in place during mid-late gestation that interact dynamically with each other, and that, on the one hand, prepare OT neurons for the demands of parturition and lactation, but at the same time, provide for novel restraining mechanisms, seemingly to prevent release of OT before the time of its major physiological need. An additional example of such conservation involving both the PRL and OT neuroendocrine systems concerns the major suppression of neuroendocrine stress responses, a condition that develops during pregnancy, continues on throughout lactation and is reset after weaning. Both PRL and OT can be considered as classic stress hormones, along with adrenal steroids and catecholamines, in various animal species and humans, as, levels of both hypophyseal hormones increase in response to a variety of physical stressors, such as forced swim or immobilization in rats, for example (64, 125, 197). A substantial literature documents that responses of all of these neuroendocrine systems to physical stressors is markedly dampened during lactation, and recent work demonstrates that this hyporesponsiveness to stress is set in place during late gestation. Decreased responsiveness of the hypothalamic-pituitaryadrenal axis to stress in late gestation and lactation has been amply documented in the literature, and the interested reader is referred to several excellent reviews of this phenomenon and some of the proposed underlying mechanisms and physiological significance (52, 53, 303). With respect to the hormones of lactation, several studies show that application of an immobilization stress or inhalation of ether readily increases PRL secretion in nonlactating rats, but completely fails to do so in lactating females (22, 151, 179). It is important to note that this lack of response is not solely due to already high levels of PRL released from suckling because a lack of response is also seen in lactating rats that have been separated from their litters, in which PRL levels are at baseline (e.g., 22). Likewise, immobilization or forced swim stresses will increase OT release in nonlactating rats, but not in lactating rats (64, 150, 252). It has been commonly suggested in these studies that the hyporesponsiveness of both PRL and OT to stress stimuli during lactation that are clearly effective, for example, in cycling females, may conserve both of these hormones so that they can respond specifically and optimally to the stimuli associated with parturition and with lactation. Surprisingly little is known about exactly when and how the PRL system loses its responsiveness to stress during lactation, and this would be a fruitful area of further research. Likewise, the mechanisms that “gate” out OT responses to stress during lactation are largely unknown. It is clear, however, that hyporesponsiveness of OT neurons to activation by stress is set in place during pregnancy (e.g., 252). Using systemic administration of interleukin-1ß (IL-1ß) as a physical stressor that mimics a host response to infection, Russell and

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coworkers have shown that the OT neuronal and secretory response to this cytokine is diminished in late pregnant, compared to nonpregnant rats, and that this reduction in response is attributable to an action of EOP, inasmuch as administration of the opioid antagonist naloxone restored the OT response to IL-1ß in late pregnancy (54). Further, because inhibition of allopregnanolone synthesis has the same effect, i.e., to reverse the dampened OT response to IL-1ß (50, 54), these authors have proposed that allopregnanolone induces the central opioid inhibition of OT neurons in the magnocellular nuclei, resulting in decreased OT responsiveness to stress stimuli in late gestation. Pharmacological studies by Summy-Long and co-workers suggest that this inhibitory effect of opioids may continue in lactation and serves to preferentially inhibit the OT response to physiological challenges such as dehydration and hemorrhage in favor of OT release in response to suckling (144, 319). For example, Hartman et al. showed that OT release in response to an osmotic challenge or to hypovolemia was diminished in lactating rats compared to cycling rats, and that administration of the opioid antagonist naloxone to lactating rats augmented OT release in response to dehydration, but not to suckling, implying inhibitory opioid tone selectively affecting physiological signals other than suckling (144). In view of the evidence cited above that the inhibitory effect of opioids during lactation is most likely exerted on the neurohypophyseal nerve terminals (70), some process must be responsible for reinstating responsiveness to opioids at this level that was lost during late pregnancy (109, see above). More fundamentally, an important issue that remains to be resolved concerns how the neurochemical systems regulating OT can distinguish between suckling versus nonsuckling inputs. With regard to the OT system, it is important to note that although the OT secretory response to stressors is diminished during lactation, as compared with nonlactating rats, stress stimuli can impede milk ejections during lactation. This is due in large part to inhibitory actions of adrenal catecholamines directly on the mammary gland to inhibit milk ejection (references in 141, 307), but may also involve disruption of OT release (307), as well as interference with the slow wave sleep EEG pattern characteristic of nursing rats (370).

Lactation and neuroprotection Perhaps related to the overall phenomenon of hyporesponsiveness to stress in lactation are the recent observations that lactating rodents are provided with some level of protection against insults to the brain, and especially against the deleterious effects of excitotoxic stimuli. Pharmacological studies by the Smith laboratory that were primarily directed at investigating excitatory amino acid effects on anterior pituitary hormone secretion noted that the usual behavioral signs of hyperactivity upon central administration of the glutamate agonist NMDA were absent in lactating females (5). Additional studies by this group demonstrated that hippocampal and cortical



activation (as assessed by cFos expression) produced by central NMDA was also suppressed in lactating rats (4), and that this dampened effect of NMDA could be reversed by either blockade of progestin receptors or by removal of the litters, implying some essential role of the suckling stimulus and perhaps mediated by hormonal effects (3). The notion that lactation might constitute a neuroprotective state has been taken forward in recent years by Morales and coworkers (cf. 241 for review). For example, this group has employed a variety of imaging approaches to assess in detail hippocampal damage after treatment with the excitotoxin kainic acid in lactating rats in comparison with control female rats in diestrus, and demonstrated that the effects of kainate to induce seizures and damage to neurons in hippocampal areas CA1, CA3 and CA4 were markedly attenuated in lactating rats (361). Microglial and astrocytic activation in the hippocampus in response to kainic acid were also inhibited in lactation (59). In considering the possible hormonal mediation of this phenomenon, Vanoye-Carlo et al. (361) noted the abundant evidence for neuroprotective effects of estradiol and progesterone in various experimental models. In view of our discussion above on various adaptations during pregnancy that are mediated by allopregnanolone, the neuroprotective action of this steroid (e.g., 69), and the observations that some degree of neuroprotection is already in place during pregnancy with its high allopregnanalone levels (311,312), it is tempting to speculate that this neurosteroid may be critical in lactational neuroprotection. While this hypothesis has not yet been directly examined, the Morales group has obtained evidence for an action of either native PRL or a PRL isoform in the hippocampus to inhibit neurotoxicity induced by kainate (242, 331). Many details of the mechanisms underlying this action, as well as of potential contributions by the other hormones of lactation, remain to be established, but this exciting possibility may prove to have translational significance in humans as well.

Adaptations in the Neuroendocrine Regulation of Energy Balance During Lactation Lactation: Hyperphagia but negative energy balance In this section, we consider how hormones of energy metabolism, in particular leptin and ghrelin, act centrally and contribute to the overall neuroendocrine regulation of lactation. In many mammalian species, the synthesis and secretion of milk presents a significant metabolic challenge with respect to regulation of energy balance and fuel flow. In typical laboratory species such as rats and mice, the energy demand associated with milk synthesis for support of the suckling offspring may exceed the energy needs of all other physiological processes combined (365). Typical strategies to meet this need include mobilization of energy from lipid



stores, reduction of physical activity, and inhibition of growth, thyroid function and reproduction (106, 305, 365, 369, 383, 385). In a number of species, but especially in laboratory rodents, the most dramatic adaptation is hyperphagia, with daily food intake often increasing fourfold to fivefold over the nonlactating level (124, 384). Investigations of this phenomenon indicate that while the circadian entrainment of feeding to activity rhythms is maintained (104), both meal number and meal size are increased (124, 385). The hyperphagia of lactation depends upon the suckling stimulus (106,124), and thus, it is of great interest to understand how sucklingactivated afferent pathways (see above) might interact with the central appetite-regulating systems. A unique feature of lactational hyperphagia is that despite the markedly increased food intake, lactating rats appear to be in modest negative energy balance, and display some, but not all, neuroendocrine and metabolic features associated with this condition (305, 365, 369). For example, metabolic changes include hypoglycemia, and the associated hypoleptinemia and hypo-insulinemia (48, 82, 105, 272, 305, 384), which are also characteristic of nonlactating animals in negative energy balance (6, 21). One dissociation concerns the appetite-stimulatory, gastric hormone ghrelin, circulating levels of which are increased during food deprivation (6,21, 176), but not during lactation (2). Thus, an emerging component of the overall neuroendocrine regulation of lactation involves signaling by metabolic hormones to the appetite-regulatory networks in the brain during the unique metabolic state of lactation.

Orexigenic and anorexigenic neuropeptide expression in lactation We discussed above the observations that TIDA neurons of the arcuate nucleus express several peptides de novo during lactation, including NPY, and summarized evidence that this peptide may play a “cohormonal role” with DA in inhibition of PRL secretion. Subsequently, a number of studies have shown that one neurochemical feature of lactation in rats and mice is increased expression of the genes encoding NPY and its coexpressed peptide messenger, the agouti-related peptide (AgRP), in the non-TIDA arcuate neurons of the hypothalamus; additionally, the content of NPY peptide is increased in important nerve terminal regions, including the PVN and median eminence (66-68, 104, 206, 219, 304, 322, 386). NPY is also expressed in a population of DMH neurons specifically during lactation in rats (206). As with the novel expression of NPY in TIDA neurons, the increased expression of NPY and AgRP in the non-TIDA cells of the arcuate nucleus depends upon the suckling stimulus (206). A large literature attests to the important actions of these two coexpressed neuropeptides in stimulating appetite and food intake, especially in response to negative energy balance, via projections from their cell bodies in the arcuate nucleus to parvocellular PVN (cf. 1, 178, 382 for reviews). NPY acts on PVN neurons via its own receptors (most likely

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the Y1 and Y5 subtypes), while AgRP acts as an inverse agonist on the melanocortin 3/4 receptor in the PVN, functionally antagonizing the important anorexigenic messenger, α-melanotropin (62, 80-82), a peptide synthesized from the precursor proopiomelanocortin (POMC), which is expressed in a separate population of neurons in the arcuate nucleus that also project to PVN (178, 382). Interestingly, some (304, 323, 387), but not all (82), studies demonstrate a reduction in POMC gene expression during lactation in rats. Additionally, approximately one-third of the NPY/AgRP cells of the arcuate also utilize GABA as a third messenger, and these orexigenic neurons also promote food intake by inhibiting the activity of the adjacent POMC neurons (278), and very likely also engage other systems involved in regulation of food intake (35, 62). Because lactation is a condition of negative energy balance and hyperphagia, one seemingly obvious role for the upregulated NPY/AgRP system (and downregulated POMC expression) in lactation would be in mediating the lactational hyperphagia. Consistent with this hypothesis, third ventricular administration of an antisense oligonucleotide to knock down expression of the NPY Y5 receptor subtype significantly decreased food intake and also affected other maternal behaviors in lactating rats (190). However, tests of this hypothesis in the author’s laboratory have also indicated that food intake in lactating rats is generally quite resistant to pharmacological agents that decrease feeding in other physiological conditions. For example, in our studies, central administration of antiNPY IgG, which is effective in decreasing 24-hr food intake in male rats (112), had no effect on food intake in lactating rats (unpublished observations). Likewise, the NPY antagonist [D-tyr(27,36), D-thr(32)]NPY(27-36), which inhibits NPY-induced and deprivation-induced feeding behavior (244), did not affect food intake when infused into the third ventricle of lactating rats (80). Further, food intake appears to be normal in lactating NPY-knockout mice (153). Central administration of the anorexigenic peptide, α-melanotropin, which functionally antagonizes AgRP, also failed to decrease food intake when administered centrally to lactating rats in a regimen that was clearly effective in nonlactating rats (80). Our further studies found that a combination of the NPY antagonist analogue plus α-melanotropin significantly reduced food intake when infused centrally in lactating rats over several days, but even this combined treatment to antagonize both NPY and AgRP did not return food intake to the nonlactating level, and the inhibitory effect waned by the fourth day of infusion (80). On the other hand, Phillips and Palmiter employed a novel and much more robust pharmacogenomic approach, in which a total ablation of the NPY/AgRP population of the arcuate nucleus was achieved acutely by administration of diphtheria toxin to mice in which the human diphteria toxin receptor had been targeted to the AgRP gene locus; this more profound depletion of NPY and AgRP resulted in a dramatic decrease of food intake in lactating mice (269). Taken together, these findings support the concept that

280


an integrated action of both orexigenic comessenger peptides is essential in mediating lactational hyperphagia, although it is highly likely that other orexigenic systems are involved as well.

Regulation of NPY and AgRP expression by metabolic signals associated with lactation As for the hyperphagia of lactation itself (see above), the increased expression of NPY (and AgRP) genes in the arcuate nucleus and the increased NPY-like immunoreactive content and unique innervation pattern in median eminence during lactation depend upon the suckling stimulus, as these neurochemical changes are reversed by removal of the litters and are restored within hours of litter return (68, 206, 374). It is therefore interesting to note that some brain regions identified above as being a component of the suckling-activated pathway for PRL and OT secretion, such as the nucleus tractus solitarius and the lateral parabrachial nucleus, have also been implicated in the neural regulation of food intake via direct projections to the hypothalamus (e.g., 1, 35). It is plausible, for example, that suckling-activated afferents may innervate the orexigenic arcuate NPY/AgRP neurons and activate neurogenic stimulus-transcription coupling mechanisms that promote increased expression of these peptides. It has also been considered that the effect of suckling on NPY/AgRP expression could be mediated by an action of PRL, which is known to gain access to the arcuate nucleus (see above), and which exerts orexigenic actions during lactation (see below). However, several groups have failed to affect NPY gene expression in arcuate neurons by inhibiting or stimulating PRL release during lactation (207, 267). In contrast, PRL does seem to be important for the novel expression of NPY in DMH neurons during lactation (206), but at present there is disagreement on whether this NPY cell group participates in lactational hyperphagia (cf. 387 vs. 269). As noted above, one common feature of lactation and other periods of negative energy balance is a reduction in circulating leptin and insulin, hormones that are well established to inhibit NPY and AgRP gene expression and to stimulate POMC gene expression (178, 382). The reduction in leptin during lactation reflects in part negative energy balance, and depends in part upon the continual suckling stimulus (48, 106). To test the hypothesis that the reduction in leptin and/or insulin might underlie the upregulation of NPY and AgRP gene expression in the arcuate nucleus, we devised a repletion paradigm that restores circulating leptin or insulin to the higher circulating levels characteristic of cycling female rats by infusing these hormones via subcutaneously implanted Alzet Osmotic Minipumps. These studies demonstrated that selective leptin repletion in lactating rats reversed the upregulation of NPY mRNA and AgRP mRNA expression in arcuate neurons, and also decreased NPY immunoreactivity in the PVN and ME (82). Thus, as in other states of negative energy balance, a reduction in the inhibitory leptin signaling to the arcuate



NPY/AgRP neurons may be important for stimulating the expression of these orexigenic peptides. However, a consistent, and seemingly paradoxical, finding of these and other studies is that despite the readily observable inhibitory effects of leptin on orexigenic peptide expression, systemic leptin replacement has little or no inhibitory effect on food intake during lactation, as it does in other conditions (82, 322, 384, 387). This lack of effect on food intake may be due to several factors. It may be the case, for example, that some degree of resistance to leptin, which is known to develop during gestation (137), persists during lactation (47, 104, 105, 322). Increased secretion of the gastric hormone ghrelin is observed in response to negative energy balance, and under these conditions, ghrelin also contributes to the upregulation of NPY and AgRP expression in the arcuate nucleus and to increased food intake (1, 21, 178). Studies on a role for ghrelin in lactational hyperphagia have not yet been published; however, in unpublished studies from the author’s laboratory, we found that systemic treatment with a selective ghrelin receptor antagonist ([D-Lys3] growth hormone releasing peptide-6) decreased food intake in both cycling and lactating females over a 2-day treatment period. The expression of NPY and AgRP was also reduced in ghrelin antagonist-treated lactating rats. Unlike leptin, there do not appear to be any changes in circulating levels of ghrelin in lactating rats as compared with cycling female rats (2, 323). However, the recent observation that ghrelin receptor mRNA expression is increased in hypothalamus of lactating rats (323) suggests that orexigenic circuits might be more sensitive to this hormone in lactating animals. Hence, taken together, one can posit that, as in other conditions of negative energy balance, an interplay between a reduction in the anorexigenic tone of leptin, perhaps allowing for enhanced orexigenic ghrelin action, may be an important neuroendocrine mechanism underlying lactational hyperphagia. A major target for these hormonal actions would be the orexigenic and possibly anorexigenic neural systems in the arcuate-PVN circuit, and it is clear that systems must be involved as well. In addition, we have proposed elsewhere (76, 81) that the reduction in leptin secretion during lactation, dependent upon the suckling stimulus as reviewed above, and the consequent up-regulation of the arcuate NPY/AgRP system, also are important in mediating some of the other characteristic neuroendocrine adaptations common to conditions of negative energy balance including lactation (Fig. 8). Indeed, it is intriguing to note the levels of activity of the hypothalamic-pituitary gonadal, thyroid and adrenal axes are generally in the same direction during lactation as in a period of food deprivation (Table 1; cf. Refs. 76, 129, 305, 372). Moreover, Table 1 also shows that each of these neuroendocrine changes in lactation can be induced in nonlactating rats by leptin withdrawal and by increasing activity of the NPY and AgRP peptide systems. The NPY/AgRP system has been classically viewed as orchestrating the regulation of food intake with anterior pituitary responses to negative energy balance, with leptin reduction as a key metabolic



Suckling stimulation

PRL secretion

Hyperphagia of lactation

Increased arcuate NPY/AgRP

Milk synthesis/ secretion

Negative energy balance

Decreased leptin

ACTH secretion

TSH secretion LH secretion

Figure 8 Integration by arcuate NPY/AgRP neurons of hyperphagia and neuroendocrine adaptations to the negative energy balance in lactation. The negative energy balance due to the demands of milk synthesis and secretion, results in decreased leptin secretion from adipose tissue. Hypo-leptinemia provides an important signal to stimulate the expression of NPY and AgRP in arcuate neurons, which mediate in part the hyperphagia of lactation as well as increased secretion of ACTH and decrease in TSH and LH secretion. PRL released in response to suckling also contributes significantly to lactational hyperphagia. These mechanisms depend upon the suckling stimulus.

signal (1, 6), and our work suggests that this mechanism is an important neuroendocrine regulatory mechanism in lactation as well (76, 81) (Fig. 8).

PRL: Orexigenic actions during lactation Woodside and coworkers (383, 385) have reviewed evidence that PRL released by suckling may interact with these systems in contributing to the hyperphagia of lactation through central actions. For example, using an experimental model of lactating rats with cut galactophores, in which suckling still evokes PRL secretion, but milk removal is prevented, thereby eliminating the energy demand of milk, this laboratory showed that inhibition of PRL release with a DA agonist had a significant effect to reduce food intake, while intraventricular administration of PRL restored food intake in this preparation (383). It is well known that PRL receptors are expressed in many brain regions, including nuclei involved in food intake and energy balance, such as the arcuate and PVN nuclei (20, 270, 271). It is possible that upon gaining access to these regions, PRL might activate an orexigenic system such as the NPY cells of the DMH (206). Naef and Woodside (245) have also provided evidence that a central action PRL might contribute to leptin resistance during lactation.

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Table 1


Neuroendocrine Parallels in Lactation and Negative Energy Balance (e.g., Food Deprivation) in Female Rats

Hypothalamicpituitary axis

Activity in lactation

Activity in food deprivation

Effect of reduced leptin

Action of NPY

Action of AgRP

Ovarian

Decrease

Decrease

Decrease

Decrease∗

Decrease∗

Thyroid

Decrease

Decrease

Decrease

Decrease

Decrease

Adrenal

Increase

Increase

Increase

Increase

Increase

∗ in low ovarian hormone state. The decrease in leptin secretion associated with both negative energy balance (in nonlactating rats) and lactation is linked to the decrease in ovarian and thyroid function and activation of the adrenal axis, via activation of NPY and AgRP systems, in both conditions. See text for references.

Conclusions Lactation is a unique physiological state featuring a number of behavioral, endocrine and neural adaptations, all directed to facilitating nutritional support of the offspring. At least in laboratory rats, the primary experimental model used in this research, the suckling stimulus, and perhaps other afferent olfactory and auditory stimuli emanating from the offspring, activate neural pathways that that are still incompletely defined, but that ascend into neuroendocrine control areas of the hypothalamus to (i) increase PRL secretion from the anterior pituitary gland for milk synthesis and OT release from the posterior pituitary for milk ejection; (ii) induce a condition of hyperphagia and other changes in regulation of energy balance, associated with decreased leptin secretion from adipocytes and increased NPY and AgRP expression in arcuate neurons; (iii) produce a condition of hyporesponsiveness to stress; and (iv) activate mechanisms that promote neuroprotection from excitotoxic and perhaps other insults. A more complete anatomical and electrophysiological description of the suckling-activated neural pathways impinging on these various control systems will further our understanding of brain mechanisms that regulate multiple features of lactation. Suckling results in a sustained elevation in PRL secretion, although discrete pulses in PRL release are superimposed on the elevated basal levels. A considerable body of evidence supports the concept that decreased release of DA from nerve terminals of the TIDA neurons into the hypophyseal portal blood is the critical hypophysiotropic mechanism that results in elevated PRL release; such DA withdrawal results in an increase in intracellular Ca2+ signaling in the lactotroph, and is permissive to and synergistic with the stimulatory action of TRH on the Ca2+ -inositol phosphate messenger system. Whether other hypophysiotropic hormones also act directly on the lactotroph, either for inhibition or stimulation of PRL secretion in response to suckling, is still unclear. A number of neurotransmitter and neuropeptide systems have been strongly implicated in suckling-induced PRL secretion, with the strongest evidence favoring stimulatory effects of 5-HT, glutamate and EOP, most likely mediated via inhibition of the

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TIDA system. An exciting recent advance is the identification of TIP39, a neuropeptide expressed in neurons in several nuclei of the caudal diencephalon and mesencephalon that have been implicated in the direct suckling-activated pathway, as a stimulatory messenger for PRL secretion, also most likely via suppression of TIDA activity. It is probable that additional physiologically significant systems activated by suckling and affecting PRL secretion remain to be discovered. In recent years, particular emphasis has been placed on central actions of PRL as a “pleiotropic” hormone in support of lactation, that is, having actions beyond its role in milk synthesis (136), especially in maternal behavior, and also mediating increased food intake, stress hyporesponsiveness, and neuroprotection, all of which remain very fruitful areas for further investigation. The major preoccupations of oxytocinologists for the last several decades has centered around the issue of how, in the face of a continually present suckling stimulus, neurosecretory OT neurons of the SON and PVN respond with an intermittent milk ejection burst firing pattern, resulting in periodic bolus releases of the peptide that stimulate milk ejections. Considerable progress has been made in understanding the contributions of (i) intrinsic properties of magnocellular OT neurons; (ii) the actions of specific transmitter systems, and especially NE, glutamate, and GABA; (iii) reversible morphological reorganizations within the magnocellular nuclei that may promote coordinated firing within a nucleus; and (iv) the actions of OT released from dendrites within the magnocellular nuclei, which appear to be particularly critical in stimulating excitatory interactions among OT neurons and in coordinating and perhaps synchronizing the milk ejections bursts. A number of mechanistic details remain to be definitively established, especially regarding interactions among the various afferent neurochemical systems with intranuclear OT within the magnocellular nuclei. A very important question that awaits definitive resolution concerns how internuclear synchronization of burst firing is achieved among the OT neurons of the separate magnocellular and several additional accessory nuclei both ipsilaterally and contralaterally. In addition, although there is general agreement that OT mRNA levels are increased in the magnocellular nuclei during lactation,



more information is needed on the physiological factors that regulate OT gene expression during this period. Important changes in the PRL and OT neuroendocrine systems occur during gestation, particularly in late gestation, which help prepare these systems for the demands of lactation. These include a reduction in the normal excitatory response of TIDA neurons to PRL, thus diminishing PRL short-loop negative feedback during lactation. Late gestation is a period in which a delicate balance appears to develop in the OT system, between excitatory influences that increase OT synthesis and neuronal excitability in preparation for subsequent demands versus restraining mechanisms to prevent premature activation of the system and release of the peptide that could interfere with parturition or lactation. The changing ovarian hormone milieu of late pregnancy, and particularly the fall in progesterone, appears to be responsible, at least in part, for inducing many of these adaptations, and this also is an area that will require additional research to complete our understanding. In addition, allopregnanolone, a neurosteroid metabolite of progesterone, and modulator of GABA-A receptor function, has also been implicated in some of these adaptations. A particularly dramatic feature of lactation is hyporesponsiveness of multiple hormonal systems to stress, the mechanisms for which have been studied in great detail for the hypothalamic-pituitary-adrenal axis, but about which little is known for the OT and PRL systems. It is generally proposed that this adaptation serves to prevent PRL and OT release in response to stimuli other than suckling. Related to this, a particularly exciting area for future research is the further elucidation of mechanisms that afford some degree of protection against neurotoxic insults in a lactating mother, and whether this might have clinical relevance. It is evident that the energetic demands of milk production during lactation require alterations in the neuroendocrine regulation of energy balance. It is particularly noteworthy that, at least in rats, lactation is characterized by a profound hyperphagia, but coexisting with negative energy balance, and features many of the metabolic and neuroendocrine adaptations that are more commonly seen in response to food deprivation. Many of the details of this altered regulation await further research. For example, we still have an incomplete understanding of the hormonal and neurotransmitter systems that mediate the hyperphagia of lactation in laboratory rodents.


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Milk production and composition of mid-lactation cows consuming perennial ryegrass-and chicory-based diets.

Season and lactation number effects on milk production and reproduction of dairy cattle in Arizona.

Impact of alfalfa maturity and preservation method on milk production by cows in early lactation.

Effect of reserpine on milk production and serum prolactin of cows hormonally induced into lactation.

The effect of protein degradability on milk composition and production of early lactation, somatotropin-injected cows.

Lactation Curve Pattern and Prediction of Milk Production Performance in Crossbred Cows.

Effect of dry period length on milk production in subsequent lactation.

Cannabinoid receptor agonist disrupts behavioral and neuroendocrine responses during lactation.

Changes in milk fat phospholipids during lactation.

Neuroendocrine regulation and homeostasis.

Lactation and composition of milk in undernourished women.

Effects of protein level and forage source on milk production and composition in early lactation dairy cows.

Hormonal induction of lactation: estrogen and progesterone in milk.

Neuroendocrine Regulation of Metabolism.

Porcine Milk Oligosaccharides and Sialic Acid Concentrations Vary Throughout Lactation.

The effect of high and low levels of supplementation on milk production, nitrogen utilization efficiency, and milk protein fractions in late-lactation dairy cows.

Acupuncture stimulation and neuroendocrine regulation.

Nonpuerperal lactation and normal prolactin regulation.

Variance components and genetic parameters for milk production and lactation pattern in an ethiopian multibreed dairy cattle population.

Peptidomic profile of milk of Holstein cows at peak lactation.

Neuroendocrine regulation of plasma angiotensinogen.

Neurochemical regulation of oxytocin secretion in lactation.

Neuroendocrine regulation of maternal behavior.

Milk oligosaccharides over time of lactation from different dog breeds.