Matthew J. Cunningham

KISSPEPTIN ACTIVATION OF GONADOTROPIN RELEASING HORMONE NEURONS AND REGULATION OF KISS-1 MRNA IN THE MALE RAT

The KiSS-1 gene codes for a family of neuropeptides called kisspeptins which bind to the G-protein-coupled receptor GPR54. To assess the possible effects of kisspeptins on gonadotropin secretion, we injected kisspeptin-52 into the lateral cerebral ventricles of adult male rats and found that kisspeptin-52 increased the serum levels of luteinizing hormone (p < 0.05). To determine whether the kisspeptin-52-induced stimulation of luteinizing hormone secretion was mediated by gonadotropin-releasing hormone (GnRH), we pretreated adult male rats with a GnRH antagonist (acyline), then challenged the animals with intracerebroventricularly administered kisspeptin-52. The GnRH antagonist blocked the kisspeptin-52-induced increase in luteinizing hormone. To examine whether kisspeptins stimulate transcriptional activity in GnRH neurons, we administered kisspeptin-52 intracerebroventricularly and found by immunocytochemistry that 86% of the GnRH neurons coexpressed Fos 2 h after the kisspeptin-52 challenge, whereas fewer than 1% of the GnRH neurons expressed Fos following injection of the vehicle alone (p < 0.001). To assess whether kisspeptins can directly act on GnRH neurons, we used double-label in situ hybridization and found that 77% of the GnRH neurons coexpress GPR54 mRNA. Finally, to determine whether KiSS-1 gene expression is regulated by gonadal hormones, we measured KiSS-1 mRNA levels by single-label in situ hybridization in intact and castrated males and found significantly higher levels in the arcuate nucleus of castrates. These results demonstrate that GnRH neurons are direct targets for regulation by kisspeptins and that KiSS-1 mRNA is regulated by gonadal hormones, suggesting that KiSS-1 neurons play an important role in the feedback regulation of gonadotropin secretion.

The Stimulatory Effect of Leptin on the Neuroendocrine Reproductive Axis of the Monkey

Leptin acts as a metabolic activator of the neuroendocrine reproductive axis in several rodent species, but whether leptin plays a similar role in primates is unknown. To explore this question, we examined the effects of leptin on gonadotropin and testosterone secretion in male rhesus monkeys that were fasted for 2 days. Mean plasma levels of LH and FSH, LH pulse frequency, and LH pulse amplitude were significantly higher in leptin-treated animals compared with saline-treated controls during the second day of the fast. To identify targets for leptin’s action, we used in situ hybridization and computerized imaging to map leptin receptor (Ob-R) messenger RNA (mRNA) distribution. Ob-R mRNA was observed in the anterior pituitary and several areas of the brain, including the arcuate and ventromedial nuclei of the hypothalamus. Ob-R mRNA was coexpressed in both POMC and neuropeptide Y neurons in the arcuate nucleus, whereas little or no coexpression of Ob-R mRNA was evident in GnRH neurons. These results sugges...

Galanin-Like Peptide (GALP) Is a Target for Regulation by Leptin in the Hypothalamus of the Rat

Galanin-like peptide (GALP), which was recently isolated from the porcine hypothalamus, shares sequence homology with galanin and binds with high affinity to galanin receptors. To study the distribution and regulation of GALP-expressing cells in the brain, we cloned a 120 base-pair cDNA fragment of rat GALP and produced an antisense riboprobe. In situ hybridization for GALP mRNA was then performed on tissue sections throughout the forebrain of adult ovariectomized female rats. We found GALP mRNA-containing cells in the arcuate nucleus (Arc), caudal dorsomedial nucleus, median eminence and the pituitary. Because GALP mRNA in the Arc appeared to overlap with the known distribution of leptin receptor mRNA, we tested the hypothesis that GALP expression is regulated by leptin. Using in situ hybridization, we compared the number of GALP mRNA-containing cells among groups of rats that were fed ad lib or fasted for 48 h and treated with either leptin or vehicle. Fasting reduced the number of identifiable cells co...

Teaching the Process of Science: Faculty Perceptions and an Effective Methodology

Most scientific endeavors require science process skills such as data interpretation, problem solving, experimental design, scientific writing, oral communication, collaborative work, and critical analysis of primary literature. These are the fundamental skills upon which the conceptual framework of scientific expertise is built. Unfortunately, most college science departments lack a formalized curriculum for teaching undergraduates science process skills. However, evidence strongly suggests that explicitly teaching undergraduates skills early in their education may enhance their understanding of science content. Our research reveals that faculty overwhelming support teaching undergraduates science process skills but typically do not spend enough time teaching skills due to the perceived need to cover content. To encourage faculty to address this issue, we provide our pedagogical philosophies, methods, and materials for teaching science process skills to freshman pursuing life science majors. We build upon previous work, showing student learning gains in both reading primary literature and scientific writing, and share student perspectives about a course where teaching the process of science, not content, was the focus. We recommend a wider implementation of courses that teach undergraduates science process skills early in their studies with the goals of improving student success and retention in the sciences and enhancing general science literacy.

Distribution and Regulation of Galanin-Like Peptide (GALP) in the Hypothalamus of the Mouse

Galanin-like peptide (GALP) is a newly discovered molecule whose expression in the brain is confined to the arcuate nucleus and median eminence. In the rat, cellular levels of GALP mRNA are reduced by fasting and reversed by peripheral administration of leptin. The purpose of this investigation was 1) to clone and map the distribution of GALP mRNA in the brain of the mouse; 2) to compare the pattern and magnitude of GALP mRNA expression in the leptin-deficient obese (ob/ob) mouse with that of wild-type controls; and 3) to examine the effects of leptin delivered into the brain on the expression of GALP mRNA in the ob/ob mouse. We report the sequence of a mouse GALP cDNA and show that GALP mRNA is expressed in the arcuate nucleus, median eminence, infundibular stalk, and the neurohypophysis of this species. The expression of GALP mRNA in the brain was markedly reduced in the ob/ob mice, compared with wild-type animals. Intracerebroventricular infusion of leptin to ob/ob mice increased both the number of GALP mRNA-expressing neurons and their content of GALP mRNA, compared with vehicle-treated controls. These observations demonstrate that GALP mRNA is induced by leptin through a direct action on the brain.

Galanin-Like Peptide as a Link Between Metabolism and Reproduction

Galanin‐like peptide (GALP) is a hypothalamic neuropeptide that binds and activates galanin receptors in vitro. Following the discovery of GALP, researchers have attempted to properly place it in the context of galanin receptor physiology. Central injections of GALP have revealed some common actions with galanin, such as acutely increased food intake and suppression of the thyroid axis. Other actions are unique to GALP, such as long‐term inhibition of food intake and stimulation of luteinizing hormone (LH) secretion in male rats. GALP and galanin also produce differential effects on expression of the immediate early gene product Fos in the brain. Determining which of these actions are dependent on galanin receptors (versus a putative GALP‐specific receptor), as well as which actions represent the authentic physiology of endogenous GALP will require continued experimentation. GALP gene expression is positively regulated by several hormones involved in the control of energy balance and metabolism, namely leptin, insulin and thyroid hormone. Based on current evidence, GALP neurones may serve as a hypothalamic relay, transmitting information from the periphery to circuits within the brain involved in the physiological control of metabolism and reproduction.

Regulation of Galanin-Like Peptide Gene Expression By Pituitary Hormones and Their Downstream Targets

Galanin‐like peptide (GALP) mRNA is expressed in neurones of the hypothalamic arcuate nucleus and within pituicytes in the neurohypophysis. Several neuropeptides that are expressed in the arcuate nucleus participate in the neuroendocrine regulation of pituitary hormone secretion. Our objective was to determine the extent to which GALP might be a target for regulation by pituitary hormones or their downstream targets in the rat. The expression of GALP mRNA in the arcuate nucleus was reduced by hypophysectomy as determined by in situ hybridization. However, this did not appear to be attributable to the loss of either gonadal or adrenal steroids because castrated, ovariectomized and adrenalectomized rats had GALP mRNA expression that was indistinguishable from their respective controls. Next, we investigated the effects of growth hormone deficiency on GALP mRNA expression by studying dwarf rats and found that GALP gene expression was not different between dwarf rats and controls. We found that thyroidectomy led to a significant reduction in GALP mRNA expression compared to intact controls, and thyroidectomized rats implanted with thyroxine pellets had GALP mRNA expression that was similar to intact controls. Thus, the reduction of GALP mRNA expression seen in hypophysectomized animals may reflect, in part, a selective loss of thyroid hormone. We also found that the expression of GALP mRNA was increased in the neurohypophysis of lactating rats compared to nonlactating rats, whereas GALP mRNA expression in the arcuate nucleus was unaffected by lactation. This suggests that the induction of GALP gene expression in pituicytes is physiologically associated with activation of oxytocin and vasopressin secretion during lactation.

Galanin’s Functional Significance in the Regulation of the Neuroendocrine Reproductive Axis of the Monkey

Galanin stimulates the neuroendocrine reproductive axis in the rat, but whether galanin acts similarly in primate species is unknown. To test the hypothesis that galanin acts within the hypothalamo-hypophyseal axis to stimulate luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) secretion in the primate, galanin was administered either systemically or directly into the arcuate nucleus-median eminence of ovariectomized macaques (pigtailed or rhesus, respectively) that were maintained on estradiol. The mean plasma levels of LH were significantly elevated in pigtailed macaques after peripheral injection of galanin (2 mg) as compared with vehicle treatment. In rhesus monkeys, galanin (80 µM) administered by push-pull perfusion into the arcuate nucleus-median eminence did not significantly alter either GnRH or LH release. To determine whether in the monkey, as in the rat, subpopulations of medial forebrain GnRH neurons coexpress galanin mRNA, we used single- and double-label in situ hybridization and computerized imaging techniques. GnRH mRNA-containing cells were identified in both the medial and lateral forebrain of the female pigtailed macaque. No galanin mRNA expression was detectable in GnRH neurons located in either the medial preoptic area or mediobasal hypothalamus; however, within the substantia innominata a subset of GnRH mRNA-expressing neurons did coexpress galanin mRNA. Taken together, these results suggest that galanin induces LH release in primates, but galanin may not act directly on hypothalamic GnRH neurons. Presently, we have confirmed in another primate species the existence of GnRH gene expression in the lateral forebrain and discovered that a small subset of these neurons coexpress galanin. These particular cells may have a unique and as of yet undefined physiological function that is distinct from those GnRH neurons serving a hypophysiotropic function.

Galanin-Like peptide elicits a robust discharge of growth hormone in the rhesus monkey (Macaca mulatta).

Galanin-like peptide (GALP) stimulates the release of gonadotropin-releasing hormone in rodent and primate species. The widespread distribution of GALP fibers in the hypothalamus suggests that this neuropeptide may influence hypophysiotropic factors that control other aspects of adenohypophysial function. Here we studied the effects of intracerebroventricular administration of GALP on serum levels of growth hormone (GH) and prolactin (PRL) in adult male monkeys. The animals (n = 5) were orchidectomized and implanted with testosterone-containing Silastic capsules to maintain the circulating testosterone levels (∼9 ng/ml) within the physiological range. The animals were implanted with an intracerebroventricular cannula and venous catheter for continuous access to the cerebroventricular and the venous circulation, respectively. GALP (500 µg), or vehicle alone, was administered as a bolus intracerebroventricular injection, and sequential blood samples were collected at 20-min intervals for 3 h before and after the injections. Within 20 min following GALP injection, the GH concentrations increased 3.5-fold, and a peak level (12.9 ± 2.7 ng/ml) was observed 40 min after injection. The GH levels remained elevated until 60 min after injection and thereafter declined to values similar to those observed at 0 min. The GH concentrations were not changed by vehicle alone. A decline in PRL levels was observed following GALP administration, with significantly reduced concentrations occurring between 60 and120 min following the injection of the neuropeptide. We conclude that in the monkey GALP is a potent secretagogue for GH and an inhibitor of PRL secretion and that GALP may, therefore, interact with the hypothalamic circuitry involved in the regulation of these pituitary hormones.