Naomi E. Rance

Coexpression of dynorphin and neurokinin B immunoreactivity in the rat hypothalamus: Morphologic evidence of interrelated function within the arcuate nucleus

Considerable evidence suggests that dynorphin and neurokinin B (NKB) neurons in the hypothalamic arcuate nucleus participate in the sex‐steroid regulation of reproduction. In the present study, we used dual‐label immunofluorescence to explore the distribution of prodynorphin and proNKB immunoreactivity in the rat hypothalamus. Additionally, we investigated whether arcuate prodynorphin‐ir (immunoreactive) neurons expressed the neurokinin 3 receptor (NK3R) or nuclear estrogen receptor‐α (ERα). We found that the majority of prodynorphin‐ir neurons in the rat arcuate nucleus expressed proNKB, whereas nearly all (99%) of the proNKB neurons were immunoreactive for prodynorphin. The arcuate nucleus was the only site in the hypothalamus where neuronal somata coexpressing prodynorphin and proNKB‐immunoreactivity were identified. A dense plexus of double‐labeled prodynorphin/proNKB‐ir fibers was found within the arcuate nucleus extending to the median eminence and throughout the periventricular zone of the hypothalamus. Prodynorphin/proNKB fibers were also identified in the paraventricular nucleus, anterior hypothalamic area, medial preoptic area, median preoptic nucleus, anteroventral periventricular nucleus, and bed nucleus of the stria terminalis in a distribution consistent with previously described arcuate nucleus projections. Interestingly, the majority of prodynorphin‐ir neurons in the arcuate nucleus expressed NK3R, and nearly 100% of the prodynorphin‐ir neurons contained nuclear ERα. Our results suggest that there is a close functional relationship between dynorphin and NKB peptides within the arcuate nucleus of the rat, which may include an autofeedback loop mediated through NK3R. The diverse hypothalamic projections of fibers expressing both prodynorphin and proNKB provide evidence that these neurons may participate in a variety of homeostatic and neuroendocrine processes. J. Comp. Neurol. 498:712–726, 2006.

Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback.

Menopause is characterized by depletion of ovarian follicles, a reduction of ovarian hormones to castrate levels and elevated levels of serum gonadotropins. Rather than degenerating, the reproductive neuroendocrine axis in postmenopausal women is intact and responds robustly to the removal of ovarian hormones. Studies in both human and non-human primates provide evidence that the gonadotropin hypersecretion in postmenopausal women is secondary to increased gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. In addition, menopause is accompanied by hypertrophy of neurons in the infundibular (arcuate) nucleus expressing KiSS-1, neurokinin B (NKB), substance P, dynorphin and estrogen receptor alpha (ERalpha) mRNA. Ovariectomy in experimental animals induces nearly identical findings, providing evidence that these changes are a compensatory response to ovarian failure. The anatomical site of the hypertrophied neurons, as well as the extensive data implicating kisspeptin, NKB and dynorphin in the regulation of GnRH secretion, provide compelling evidence that these neurons are part of the neural network responsible for the increased levels of serum gonadotropins in postmenopausal women. We propose that neurons expressing KiSS-1, NKB, substance P, dynorphin and ERalpha mRNA in the infundibular nucleus play an important role in sex-steroid feedback on gonadotropin secretion in the human.

Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence.

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual‐label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin‐releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB‐immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH‐ir somata exhibited NK3R staining. ProNKB and NK3R‐ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB‐ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis. J. Comp. Neurol. 489:372–386, 2005.

Central injection of senktide, an NK3 receptor agonist, or neuropeptide Y inhibits LH secretion and induces different patterns of Fos expression in the rat hypothalamus

Arcuate neurokinin B (NKB) neurons express estrogen receptor-alpha and are strongly modulated by gonadal steroids. Although numerous studies suggest that NKB neurons participate in the reproductive axis, there is no information on the regulation of luteinizing hormone (LH) secretion by NKB or its receptor, NK3. In the present study, we determined if central injection of senktide, a selective NK3 receptor agonist, would alter serum LH in ovariectomized, estrogen-primed rats. The effects of senktide were compared to neuropeptide Y (NPY), a well-characterized modulator of LH secretion. Saline, senktide, or NPY was injected into the lateral ventricle of unanesthetized rats and serial blood samples were collected for LH radioimmunoassay. The rats were sacrificed 90 min after injection and the brains were removed and processed for Fos immunocytochemistry. A significant inhibition of serum LH was observed from 30 to 90 min after injection of senktide relative to saline controls. In the senktide-injected rats, the inhibition of serum LH was accompanied by increased Fos expression in the medial preoptic area and arcuate nucleus--two reproductive control centers. Senktide also induced Fos in the paraventricular nuclei (PVN) and supraoptic nuclei (SON). Injection of NPY also inhibited serum LH but increased Fos expression only in the PVN and SON. This study provides the first demonstration of alterations in LH secretion by an NK3 receptor agonist. These data, combined with the induction of Fos in medial preoptic and arcuate neurons, strongly support the hypothesis that NKB neurons play a role in the regulation of gonadotropin secretion.

Forebrain projections of arcuate neurokinin B neurons demonstrated by anterograde tract-tracing and monosodium glutamate lesions in the rat

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor alpha (ERalpha). Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKB-immunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: (1) local projections within the arcuate nucleus bilaterally, (2) projections to the median eminence including the lateral palisade zone, (3) projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and (4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tract-tracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERalpha and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH.

Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight.

Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK(3)R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK(3)R agonist conjugated to the ribosome-inactivating toxin, saporin (NK(3)-SAP). Stereotaxic injections of NK(3)-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK(3)R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E(2) on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E(2)) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E(2), and the E(2) modulation of body weight.

Neurokinin B gene expression is increased in the arcuate nucleus of ovariectomized rats.

Hypertrophy and increased gene expression of tachykinin neurons occur in the infundibular (arcuate) nucleus of postmenopausal women. We have hypothesized that the alterations in tachykinin gene expression in the hypothalami of postmenopausal women are secondary to ovarian failure and not due to age per se. In this study, in situ hybridization and computer-assisted microscopy were used to determine whether ovariectomy modulates neurokinin B (NKB), substance P (SP) or proopiomelanocortin (POMC) gene expression in the rat arcuate nucleus. Four groups were examined: proestrus; diestrous day 1; ovariectomized, and constant estrus induced by a single injection of 20 mg/kg estradiol valerate. Rats were sacrificed 2 months after treatment. Computer-assisted microscopy was used to determine the number of tachykinin neurons, cell areas, and the autoradiographic grain density of labeled neurons. We report marked changes in NKB gene expression in ovariectomized rats. The number of neurons containing NKB gene transcripts was significantly greater in ovariectomized rats (16.9 +/- 1.0 neurons/arcuate section) than all other groups. There was also a significant difference in the number of NKB neurons/arcuate section between proestrous (8.9 +/- 1.8 neurons) and diestrous (4.8 +/- 1.0 neurons) rats. The lowest number of neurons was detected in the estradiol valerate-injected rats (2.9 +/- 0.6 NKB neurons/arcuate section). Furthermore, the autoradiographic grain density of NKB neurons was doubled in the ovariectomized group compared to all other groups. In contrast, few SP neurons were identified in the rat arcuate nucleus and no changes were detected during the estrous cycle or in response to ovariectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of body temperature and LH secretion by hypothalamic KNDy (kisspeptin, neurokinin B and dynorphin) neurons: a novel hypothesis on the mechanism of hot flushes.

Despite affecting millions of individuals, the etiology of hot flushes remains unknown. Here we review the physiology of hot flushes, CNS pathways regulating heat-dissipation effectors, and effects of estrogen on thermoregulation in animal models. Based on the marked changes in hypothalamic kisspeptin, neurokinin B and dynorphin (KNDy) neurons in postmenopausal women, we hypothesize that KNDy neurons play a role in the mechanism of flushes. In the rat, KNDy neurons project to preoptic thermoregulatory areas that express the neurokinin 3 receptor (NK3R), the primary receptor for NKB. Furthermore, activation of NK₃R in the median preoptic nucleus, part of the heat-defense pathway, reduces body temperature. Finally, ablation of KNDy neurons reduces cutaneous vasodilatation and partially blocks the effects of estrogen on thermoregulation. These data suggest that arcuate KNDy neurons relay estrogen signals to preoptic structures regulating heat-dissipation effectors, supporting the hypothesis that KNDy neurons participate in the generation of flushes.

Role for kisspeptin/neurokinin B/dynorphin (KNDy) neurons in cutaneous vasodilatation and the estrogen modulation of body temperature

Estrogen withdrawal in menopausal women leads to hot flushes, a syndrome characterized by the episodic activation of heat dissipation effectors. Despite the extraordinary number of individuals affected, the etiology of flushes remains an enigma. Because menopause is accompanied by marked alterations in hypothalamic kisspeptin/neurokinin B/dynorphin (KNDy) neurons, we hypothesized that these neurons could contribute to the generation of flushes. To determine if KNDy neurons participate in the regulation of body temperature, we evaluated the thermoregulatory effects of ablating KNDy neurons by injecting a selective toxin for neurokinin-3 expressing neurons [NK3-saporin (SAP)] into the rat arcuate nucleus. Remarkably, KNDy neuron ablation consistently reduced tail-skin temperature (TSKIN), indicating that KNDy neurons facilitate cutaneous vasodilatation, an important heat dissipation effector. Moreover, KNDy ablation blocked the reduction of TSKIN by 17β-estradiol (E2), which occurred in the environmental chamber during the light phase, but did not affect the E2 suppression of TSKIN during the dark phase. At the high ambient temperature of 33 °C, the average core temperature (TCORE) of ovariectomized (OVX) control rats was significantly elevated, and this value was reduced by E2 replacement. In contrast, the average TCORE of OVX, KNDy-ablated rats was lower than OVX control rats at 33 °C, and not altered by E2 replacement. These data provide unique evidence that KNDy neurons promote cutaneous vasodilatation and participate in the E2 modulation of body temperature. Because cutaneous vasodilatation is a cardinal sign of a hot flush, these results support the hypothesis that KNDy neurons could play a role in the generation of flushes.

Localization of neurons expressing substance P and neurokinin B gene transcripts in the human hypothalamus and basal forebrain

In situ hybridization histochemistry was used to map the distribution of neurons expressing the substance P (SP) or neurokinin B (NKB) genes in the human hypothalamus and basal forebrain. Hypothalami from five adult males were frozen in isopentane at −30°C and serially sectioned at 20 μm thickness. Every 20th section was hybridized with [35S]‐labeled, 48‐base synthetic cDNA probes that were complementary to either SP or NKB mRNAs. Slides were dipped into nuclear emulsion for visualization of mRNAs at the single‐cell level. The location of labeled neurons (greater than ×5 background) was mapped by using an image‐combining computer microscope system. A distinct and complementary distribution pattern of SP and NKB neurons was observed in the human hypothalamus and basal forebrain. NKB was the predominant tachykinin in the rostral hypothalamus, whereas SP mRNA predominated in the posterior hypothalamus. Numerous NKB neurons were identified in the magnocellular basal forebrain, the bed nucleus of stria terminalis, and the anterior hypothalamic area. Scattered NKB neurons were present in the infundibular and paraventricular nuclei, paraolfactory gyrus, posterior hypothalamic area, lateral division of the medial mammillary nucleus, and amygdala. Numerous neurons expressing SP mRNAs were identified in the premammillary, supramammillary, and medial mammillary nuclei; the posterior hypothalamic area; and the corpus striatum. Scattered SP neurons were also observed in the preoptic area; the infundibular, intermediate, dorsomedial, and ventromedial nuclei; the infundibular stalk; the amygdala; the bed nucleus of stria terminalis; and the paraolfactory gyrus. These studies provide the first description of the location of neurons that express tachykinin gene transcripts in the human hypothalamus. J. Comp. Neurol. 384:429–442, 1997.