Sally J. Krajewski

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.

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.

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.

Effects of ovariectomy on the neuroendocrine axes regulating reproduction and energy balance in young cynomolgus macaques

Degeneration of the ovary in middle‐aged women results in castrate levels of ovarian steroids and increased gonadotropin secretion from the anterior pituitary gland. Ageing in women is also accompanied by significant changes in energy homeostasis. We have observed alterations in hypothalamic morphology and gene expression in older women, including hypertrophy and increased gene expression of neurokinin B (NKB) neurones, elevated levels of gonadotropin releasing‐hormone (GnRH) mRNA and decreased numbers of neurones expressing pro‐opiomelanocortin (POMC) mRNA. To determine if loss of ovarian steroids could produce comparable changes in gene expression in young primates, we measured the effects of ovariectomy on NKB, GnRH and POMC gene expression in young cynomolgus monkeys. We also measured serum leptin and body weight to examine the consequences of ovariectomy on energy balance. NKB neurones in the infundibular nucleus of ovariectomized monkeys were larger, more numerous and displayed increased levels of NKB mRNA compared to those of intact controls. Moreover, ovariectomy increased the number of neurones expressing GnRH gene transcripts and elevated serum luteinizing hormone. By contrast, several parameters related to energy balance, including POMC gene expression, serum leptin and body weights, were unchanged by ovariectomy. Thus, the rise in NKB and GnRH gene expression in older women was simulated by ovariectomy in monkeys, but the changes in POMC gene expression and energy balance were not. This study provides strong support for the hypothesis that ovarian failure contributes to the increased NKB and GnRH gene expression observed in postmenopausal women.

Activation of Neurokinin 3 Receptors in the Median Preoptic Nucleus Decreases Core Temperature in the Rat

Estrogens have pronounced effects on thermoregulation, as illustrated by the occurrence of hot flushes secondary to estrogen withdrawal in menopausal women. Because neurokinin B (NKB) gene expression is markedly increased in the infundibular (arcuate) nucleus of postmenopausal women, and is modulated by estrogen withdrawal and replacement in multiple species, we have hypothesized that NKB neurons could play a role in the generation of flushes. There is no information, however, on whether the primary NKB receptor [neurokinin 3 receptor (NK(3)R)] modulates body temperature in any species. Here, we determine the effects of microinfusion of a selective NK(3)R agonist (senktide) into the rat median preoptic nucleus (MnPO), an important site in the heat-defense pathway. Senktide microinfusion into the rat MnPO decreased core temperature in a dose-dependent manner. The hypothermia induced by senktide was similar in ovariectomized rats with and without 17β-estradiol replacement. The hypothermic effect of senktide was prolonged in rats exposed to an ambient temperature of 29.0 C, compared with 21.5 C. Senktide microinfusion also altered tail skin vasomotion in rats exposed to an ambient temperature of 29.0 but not 21.5 C. Comparisons of the effects of senktide at different ambient temperatures indicated that the hypothermia was not secondary to thermoregulatory failure or a reduction in cold-induced thermogenesis. Other than a very mild increase in drinking, senktide microinfusion did not affect behavior. Terminal fluorescent dextran microinfusion showed targeting of the MnPO and adjacent septum, and immunohistochemical studies revealed that senktide induced a marked increase in Fos-activation in the MnPO. Because MnPO neurons expressed NK(3)R-immunoreactivity, the induction of MnPO Fos by senktide is likely a direct effect. By demonstrating that NK(3)R activation in the MnPO modulates body temperature, these studies support the hypothesis that hypothalamic NKB neurons could be involved in the generation of menopausal flushes.

Ambient temperature and 17β-estradiol modify Fos immunoreactivity in the median preoptic nucleus, a putative regulator of skin vasomotion.

Estrogen has pronounced effects on thermoregulation, but the anatomic sites of integration between the reproductive and thermoregulatory axes are unknown. In this study, we tested whether estradiol-17β (E(2)) treatment would alter the activity of thermoregulatory brain regions responding to mild changes in ambient temperature (T(AMBIENT)). Core and tail skin temperatures were recorded at the ambient temperatures of 20, 24, or 31 C in ovariectomized (OVX) rats with and without E(2). Neuronal activity was evaluated by counting the number of Fos-immunoreactive cells in the brains of rats killed 90 min after exposure to one of the three ambient temperatures. Of 14 brain areas examined, the median preoptic nucleus (MnPO) was the only site that exhibited increased Fos immunoreactivity at the high T(AMBIENT) of 31 C. At 24 C, OVX rats exhibited increased numbers of MnPO Fos-immunoreactive cells, compared with OVX + E(2) rats. Interestingly, tail skin vasomotion and MnPO Fos expression were affected in a similar manner by T(AMBIENT) and E(2) treatment. In the arcuate nucleus and anteroventral periventricular nucleus (AVPV), Fos immunoreactivity was highest at the low T(AMBIENT) of 20 C, with inhibitory (arcuate nucleus) and stimulatory (AVPV) effects of E(2). No other areas responded to both T(AMBIENT) and E(2) treatment. These results implicate the MnPO, the arcuate nucleus, and the AVPV as sites of integration between the reproductive and thermoregulatory axes. Combined with studies showing the importance of MnPO neurons in heat-defense pathways, the MnPO emerges as a likely site for E(2) modulation of thermoregulatory vasomotion.