Heather J. Billings

Neurokinin B Acts via the Neurokinin-3 Receptor in the Retrochiasmatic Area to Stimulate Luteinizing Hormone Secretion in Sheep

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Neurokinin 3 Receptor Immunoreactivity in the Septal Region, Preoptic Area and Hypothalamus of the Female Sheep: Colocalisation in Neurokinin B Cells of the Arcuate Nucleus but not in Gonadotrophin-Releasing Hormone Neurones

Recent evidence has implicated neurokinin B (NKB) in the complex neuronal network mediating the effects of gonadal steroids on the regulation of gonadotrophin‐releasing hormone (GnRH) secretion. Because the neurokinin 3 receptor (NK3R) is considered to mediate the effects of NKB at the cellular level, we determined the distribution of immunoreactive NK3R in the septal region, preoptic area (POA) and hypothalamus of the ewe. NK3R cells and/or fibres were found in areas including the bed nucleus of the stria terminalis, POA, anterior hypothalamic and perifornical areas, dopaminergic A15 region, dorsomedial and lateral hypothalamus, arcuate nucleus (ARC) and the ventral premammillary nucleus. We also used dual‐label immunocytochemistry to determine whether a neuroanatomical basis for direct modulation of GnRH neurones by NKB was evident. No GnRH neurones at any rostral‐caudal level were observed to contain NK3R immunoreactivity, although GnRH neurones and fibres were in proximity to NK3R‐containing fibres. Because NKB fibres formed close contacts with NKB neurones in the ARC, we determined whether these NKB neurones also contained immunoreactive NK3R. In luteal‐phase ewes, 64% ± 11 of NKB neurones colocalised NK3R. In summary, NK3R is distributed in areas of the sheep POA and hypothalamus known to be involved in the control of reproductive neuroendocrine function. Colocalisation of NK3R in NKB neurones of the ARC suggests a potential mechanism for the autoregulation of this subpopulation; however, the lack of NK3R in GnRH neurones suggests that the actions of NKB on GnRH neurosecretory activity in the ewe are mediated indirectly via other neurones and/or neuropeptides.

KNDy (Kisspeptin/Neurokinin B/Dynorphin) Neurons Are Activated during Both Pulsatile and Surge Secretion of LH in the Ewe

KNDy (kisspeptin/neurokinin B/dynorphin) neurons of the arcuate nucleus (ARC) appear to mediate the negative feedback actions of estradiol and are thought to be key regulators of pulsatile LH secretion. In the ewe, KNDy neurons may also be involved with the positive feedback actions of estradiol (E(2)) to induce the LH surge, but the role of kisspeptin neurons in the preoptic area (POA) remains unclear. The goal of this study was to identify which population(s) of kisspeptin neurons is (are) activated during the LH surge and in response to the removal of E(2)-negative feedback, using Fos as an index of neuronal activation. Dual-label immunocytochemistry for kisspeptin and Fos was performed on sections containing the ARC and POA from ewes during the luteal phase of the estrous cycle, or before or after the onset of the LH surge (experiment 1), and from ovary-intact, short-term (24 h) and long-term (>30 d) ovariectomized (OVX) ewes in anestrus (experiment 2). The percentage of kisspeptin neurons expressing Fos in both the ARC and POA was significantly higher during the LH surge. In contrast, the percentage of kisspeptin/Fos colocalization was significantly increased in the ARC, but not POA, after both short- and long-term E(2) withdrawal. Thus, POA kisspeptin neurons in the sheep are activated during, and appear to contribute to, E(2)-positive feedback, whereas ARC kisspeptin (KNDy) neurons are activated during both surge and pulsatile modes of secretion and likely play a role in mediating both positive and negative feedback actions of E(2) on GnRH secretion in the ewe.

The Suprachiasmatic Nucleus: A Clock of Multiple Components

Although impressive progress has been made in understanding the molecular basis of pacemaker function in the suprachiasmatic nucleus (SCN), fundamental questions about cellular and regional heterogeneity within the SCN, andhowthis heterogeneity might contribute toSCNpacemaker function at a tissue level, have remained unresolved. To reexamine cellular and regional heterogeneity within the SCN, the authors have focused on two key questions: which SCN cells are endogenously rhythmic and/or directly light responsive? Observations of endogenous rhythms of electrical activity, gene/protein expression, and protein phosphorylation suggest that the SCN in mammals examined to dateis composed of anatomically distinct rhythmic and nonrhythmic components. Endogenously rhythmic neurons are primarily found in rostral, dorsomedial, and ventromedial portions of the nucleus; at mid and caudal levels, the distribution of endogenously rhythmic cells in the SCN has the appearance of a “shell.” The majority of nonrhythmic cells, by contrast, are located in a central “core” region of the SCN, which is complementary to the shell. The location of light-responsive cells, defined by direct retinohypothalamic input and light-induced gene expression, largely overlaps the location of nonrhythmic cells in the SCN core, although, in hamsters and mice light-responsive cells are also present in the ventral portion of the rhythmic shell. While the relative positions of rhythmic and light-responsive components of the SCN are similar between species, the precise boundaries of these components, and neurochemical phenotype of cells within them, are variable. Intercellular communication between these components may bea key featurer esponsiblefor theuniquepace maker properties of the SCN observed at a tissue and whole animal level.

Neural systems mediating seasonal breeding in the ewe.

Seasonal reproduction in ewes is caused by a dramatic increase in response to oestradiol (E2) negative feedback during the nonbreeding (anoestrous) season. Considerable evidence supports the hypothesis that A15 dopaminergic neurones in the retrochiasmatic area (RCh) play a key role in these seasonal changes. These A15 neurones are stimulated by E2 and inhibit gonadotrophin‐releasing hormone (GnRH) secretion in anoestrus, but not the breeding season. Because A15 neurones do not contain oestrogen receptors‐α (ERα), it is likely that E2‐responsive afferents stimulate their activity when circulating E2 levels increase during anoestrus. Retrograde tract tracing studies identified a limited set of ERα‐containing afferents primarily found in four areas [ventromedial preoptic area, RCh, ventromedial and arcuate (ARC) nuclei]. Pharmacological and anatomical data are consistent with GABA‐ and glutamate‐containing afferents controlling A15 activity in anoestrus, with E2 inhibiting GABA and stimulating glutamate release at this time of year. Tract tracing demonstrated that A15 efferents project posteriorly to the median eminence and the ARC, suggesting possible direct actions on GnRH terminals or indirect actions via kisspeptin neurones in the ARC to inhibit GnRH in anoestrus. Identification of this neural circuitry sets the stage for the development of specific hypotheses for morphological or transmitter/receptor expression changes that would account for seasonal breeding in ewes.

Temporal Requirements of Thyroid Hormones for Seasonal Changes in LH Secretion

The transition between breeding and anestrous seasons in ewes is driven by an endogenous rhythm in responsiveness to estradiol negative feedback. One stage of this rhythm, the transition to anestrus, requires the presence of thyroid hormone during a window of responsiveness that opens in the late breeding season. The primary goal of this study was to assess when ewes lose responsiveness to thyroid hormone (i.e. when the window closes). In addition, we investigated whether thyroid hormone influences aspects of seasonality other than the transition to anestrus. Ovariectomized ewes maintained in a simulated natural photoperiod were implanted with estradiol, thyroidectomized, and treated with T4 for 100 d beginning at progressively later dates during the anestrous season. Onset of neuroendocrine anestrus (decrease in LH), latency to anestrus, and time of onset of the subsequent neuroendocrine breeding season (rise in LH) were determined. Ewes gradually lost responsiveness to T4 during the latter half of the a...

The premammillary hypothalamic area of the ewe: anatomical characterization of a melatonin target area mediating seasonal reproduction.

Abstract Recent evidence suggests that the ovine premammillary hypothalamic area (PMH) is an important target for the pineal hormone, melatonin, and its role in seasonal reproduction. In rodents, the PMH is a complex region consisting of several cell groups with differing neurochemical content and anatomical connections. Therefore, to obtain a better understanding of the potential neural targets for melatonin in this area of the sheep brain, we have undertaken a detailed anatomical characterization of the PMH, including its nuclear divisions and the location of neuropeptide/neurotransmitter cells within them. By combining immunocytochemistry for NeuN, a neuronal marker, with Nissl staining in anestrous, ovariectomized, estradiol-treated ewes, we identified three nuclei within the PMH: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus (PMv), and the ventral tuberomammillary nucleus (TMv). The cARC contained neurons that were immunoreactive for tyrosine hydroxylase, dynorphin, estrogen receptor α, cocaine- and amphetamine-regulated transcript peptide (CART), and nitric oxide synthase (NOS). The PMv was also characterized by the presence of cells that contained NOS and CART, although the size of these cells was larger than that of their corresponding phenotype in the cARC. By contrast, in the TMv, of the markers examined in the present study, only fibers immunoreactive for orexin were seen. Thus, the ovine PMH is a heterogeneous region comprised of three subdivisions, each with distinct morphological and neurochemical characteristics. This anatomical map of the PMH provides a basis for future studies to determine the functional contribution of each component to the influence of melatonin on seasonal reproduction.

Potential for polysialylated form of neural cell adhesion molecule-mediated neuroplasticity within the gonadotropin-releasing hormone neurosecretory system of the ewe

The GnRH neurosecretory system undergoes marked structural and functional changes throughout life. The initial goal of this study was to examine the neuroanatomical relationship between GnRH neurons and a glycoprotein implicated in neuroplasticity, the polysialylated form of neural cell adhesion molecule (PSA-NCAM). Using dual label immunocytochemistry in conjunction with confocal microscopy, we determined that fibers, terminals, and perikarya of GnRH neurons in adult ovariectomized ewes are intimately associated with PSA-NCAM. In the preoptic area, intense PSA-NCAM immunoreactivity was evident around the periphery of GnRH cell bodies. The second goal of this study was to determine whether PSA-NCAM expression associated with GnRH neurons varies in conjunction with seasonal changes in the activity of the GnRH neurosecretory system in ovariectomized ewes treated with constant release implants of estradiol. During the breeding season when reproductive neuroendocrine activity was enhanced, the expression of P...

Evidence that the Arcuate Nucleus Is an Important Site of Progesterone Negative Feedback in the Ewe

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.

IMMUNOCYTOCHEMICAL COLOCALIZATION OF GABA-B RECEPTOR SUBUNITS IN GONADOTROPIN-RELEASING HORMONE NEURONS OF THE SHEEP

GABA has been shown to play an important role in the control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone secretion in many mammals. In sheep, seasonal differences in the ability of GABA-B receptor antagonists to alter pulsatile luteinizing hormone secretion have led to the hypothesis that this receptor subtype mediates the increased inhibitory effects of estradiol on GnRH and luteinizing hormone pulse frequency seen during the non-breeding season (anestrus). The aim of the present study was to use multiple-label immunocytochemistry to determine if ovine GnRH neurons contain the GABA-B receptor subunits R1 and/or R2, and to determine whether there are seasonal differences in the colocalization of these subunits in GnRH neurons. A majority of GnRH cells in the preoptic area, anterior hypothalamic area, and medial basal hypothalamus of both breeding season and anestrous ewes contained either GABA-B R1 or R2 subunits; a subset of GnRH neurons in breeding season (42%) and anestrous ewes (60%) contained both subunits. In contrast to colocalization within cell bodies, GnRH fibers in the median eminence did not colocalize GABA-B receptor subunits. Although the percentage of GnRH neurons expressing GABA-B receptor subunits tended to be higher in anestrus than in the breeding season, there were no significant seasonal differences in R1 and R2 subunit colocalization in GnRH cell bodies. Thus, while GABA may act directly on GnRH cell bodies via GABA-B receptors in the sheep, any role that GABA-B receptors may play in seasonal reproductive changes is likely mediated by other neurons afferent to GnRH cells.