Global cerebral ischemia in adult female rats interrupts estrous cyclicity and induces lasting changes in hypothalamic-pituitary-gonadal axis signaling peptides.

Persistent post-ischemic alterations to the hypothalamic-pituitary-adrenal (HPA) axis occur following global cerebral ischemia (GCI) in rodents. However, similar effects on hypothalamic-pituitary-gonadal (HPG) axis activation remain to be determined. Therefore, this study evaluated the effects of GCI in adult female rats (via four-vessel occlusion) on the regularity of the estrous cycle for 24-days post ischemia. A second objective aimed to assess persistent alterations of HPG axis activation through determination of the expression of estrogen receptor alpha (ERα), kisspeptin (Kiss1), and gonadotropin-inhibitory hormone (GnIH/RFamide-related peptide; RFRP3) in the medial preoptic area (POA), arcuate nucleus (ARC), dorsomedial nucleus (DMH) of the hypothalamus, and CA1 of the hippocampus 25 days post ischemia. Expression of glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN) and CA1 served as a proxy of altered HPA axis activation. Our findings demonstrated interruption of the estrous cycle in 87.5 % of ischemic rats, marked by persistent diestrus, lasting on average 11.86 days. Moreover, compared to sham-operated controls, ischemic female rats showed reduced Kiss1 expression in the hypothalamic ARC and POA, concomitant with elevated ERα in the ARC and increased GnIH in the DMH and CA1. Reduced GR expression in the CA1 was associated with increased GR-immunoreactivity in the PVN, indicative of lasting dysregulation of HPA axis activation. Together, these findings demonstrate GCI disruption of female rats' estrous cycle over multiple days, with a lasting impact on HPG axis regulators within the reproductive axis.

Acute restraint stress rapidly impacts reproductive neuroendocrinology and downstream gonad function in big brown bats (Eptesicus fuscus).

Animals face unpredictable challenges that require rapid, facultative physiological reactions to support survival but may compromise reproduction. Bats have a long-standing reputation for being highly sensitive to stressors, with sensitivity and resilience varying both within and among species, yet little is known about how stress affects the signaling that regulates reproductive physiology. Here, we provide the first description of the molecular response of the hypothalamic-pituitary-gonadal (HPG) axis of male big brown bats (Eptesicus fuscus) in response to short-term stress using a standardized restraint manipulation. This acute stressor was sufficient to upregulate plasma corticosterone and resulted in a rapid decrease in circulating testosterone. While we did not find differences in the mRNA expression of key steroidogenic enzymes (StAR, aromatase, 5-alpha reductase), seminiferous tubule diameter was reduced in stressed bats coupled with a 5-fold increase in glucocorticoid receptor (GR) mRNA expression in the testes. These changes, in part, may be mediated by RFamide-related peptide (RFRP) because fewer immunoreactive cell bodies were detected in the brains of stressed bats compared with controls - suggesting a possible increase in secretion - and increased RFRP expression locally in the gonads. The rapid sensitivity of the bat testes to stress may be connected to deleterious impacts on tissue health and function as supported by significant transcriptional upregulation of key pro-apoptotic signaling molecules (Bax, cytochrome c). Experiments like this broadly contribute to our understanding of the stronger ecological predictions regarding physiological responses of bats within the context of stress, which may impact decisions surrounding animal handling and conservation approaches.

Impact of Chronic Prenatal Stress on Maternal Neuroendocrine Function and Embryo and Placenta Development During Early-to-Mid-Pregnancy in Mice.

Psychological stress, both leading up to and during pregnancy, is associated with increased risk for negative pregnancy outcomes. Although the neuroendocrine circuits that link the stress response to reduced sexual motivation and mating are well-described, the specific pathways by which stress negatively impacts gestational outcomes remain unclear. Using a mouse model of chronic psychological stress during pregnancy, we investigated 1) how chronic exposure to stress during gestation impacts maternal reproductive neuroendocrine circuitry, and 2) whether stress alters developmental outcomes for the fetus or placenta by mid-pregnancy. Focusing on the stress-responsive neuropeptide RFRP-3, we identified novel contacts between RFRP-3-immunoreactive (RFRP-3-ir) cells and tuberoinfundibular dopaminergic neurons in the arcuate nucleus, thus providing a potential pathway linking the neuroendocrine stress response directly to pituitary prolactin production and release. However, neither of these cell populations nor circulating levels of pituitary hormones were affected by chronic stress. Conversely, circulating levels of steroid hormones relevant to gestational outcomes (progesterone and corticosterone) were altered in chronically-stressed dams across gestation, and those dams were qualitatively more likely to experience delays in fetal development. Together, these findings suggest that, up until at least mid-pregnancy, mothers appear to be relatively resilient to the effects of elevated glucocorticoids on reproductive neuroendocrine system function. We conclude that understanding how chronic psychological stress impacts reproductive outcomes will require understanding individual susceptibility and identifying reliable neuroendocrine changes resulting from gestational stress.

Sex, not social behavior, predicts fecal glucocorticoid metabolite concentrations in a facultatively social rodent, the highland tuco-tuco (Ctenomys opimus).

Social relationships may influence circulating glucocorticoid levels, particularly in group-living species in which individuals regularly engage in interactions with conspecifics. The effects of such interactions appear to vary, with greater social contact being associated with increased glucocorticoid concentrations in some species but decreased concentrations in others. These distinct responses raise intriguing questions regarding relationships among social behavior, individual phenotypes, and glucocorticoid physiology. To explore such relationships in a free-living mammal with a dynamic social organization, we quantified variation in baseline glucocorticoids in a population of highland tuco-tucos (Ctenomys opimus) from Jujuy Province, Argentina. These subterranean rodents are facultatively social, with lone and group-living individuals regularly occurring within the same population. To assess potential endocrine correlates of this behavioral variability, we examined differences in baseline fecal glucocorticoid metabolite (fGCm) concentrations as a function of social group size and composition as well as several metrics of social behavior derived from social network analyses. Despite marked variability in social relationships among the 37 (12 male, 25 female) free-living tuco-tucos sampled, none of the measures of social behavior examined were significant predictors of variation in fGCm concentrations. In contrast, individual variation in glucocorticoid metabolites was best explained by sex, with males having higher fGCm concentrations than females. These analyses provide the first characterization of the glucocorticoid physiology of highland tuco-tucos and underscore the potential importance of intrinsic phenotypic factors (e.g., sex) in shaping glucocorticoid variation in free-living mammals.

Immunoreactive distribution of gonadotropin-inhibitory hormone precursor, RFRP, in a temperate bat species (Eptesicus fuscus).

Gonadotropin-inhibitory hormone (GnIH, also known RFRP-3 in mammals) is an important regulator of the hypothalamic-pituitary-gonadal axis and downstream reproductive physiology. Substantial species differences exist in the localization of cell bodies producing RFRP-3 and patterns of fiber immunoreactivity in the brain, raising the question of functional differences. Many temperate bat species exhibit unusual annual reproductive patterns. Male bats upregulate spermatogenesis in late spring which is asynchronous with periods of mating in the fall, while females have the physiological capacity to delay their reproductive investment over winter via sperm storage or delayed ovulation/fertilization. Neuroendocrine mechanisms regulating reproductive timing in male and female bats are not well-studied. We provide the first description of RFRP-precursor peptide of GnIH -expression and localization in the brain of any bat using a widespread temperate species (Eptesicus fuscus, big brown bat) as a model. RFRP mRNA expression was detected in the hypothalamus, testes, and ovaries of big brown bats. Cellular RFRP-immunoreactivity was observed within the periventricular nuclei, dorsomedial nucleus of the hypothalamus, arcuate nucleus (Arc), and median eminence (ME). As in other vertebrates, RFRP fiber immunoreactivity was widespread, with the greatest density observed in the hypothalamus, preoptic area, Arc, ME, midbrain, and thalamic nuclei. Putative interactions between RFRP-ir fibers and gonadotropin-releasing hormone (GnRH) cell bodies were observed in 16% of GnRH-immunoreactive cells, suggesting direct regulation of GnRH via RFRP signaling. This characterization of RFRP distribution contributes to a deeper understanding of bat neuroendocrinology, which serves as foundation for manipulative approaches examining changes in reproductive neuropeptide signaling in response to environmental and physiological challenges within, and among, bat species.

Hypothalamic Overexpression of Neurosecretory Protein GL Leads to Obesity in Male C57BL/6J Mice.

INTRODUCTION: The mechanisms underlying obesity are not fully understood, necessitating the creation of novel animal models for the investigation of metabolic disorders. We have previously found that neurosecretory protein GL (NPGL), a newly identified hypothalamic neuropeptide, is involved in feeding behavior and fat accumulation in rats. However, the impact of NPGL on obesity remains unclear in any animal model. The present investigation sought to elucidate whether NPGL causes obesity in the obesity-prone mouse strain C57BL/6J. METHODS: We overexpressed the NPGL-precursor gene (Npgl) in the hypothalamus using adeno-associated virus in male C57BL/6J mice fed normal chow (NC) or a high-calorie diet (HCD). After 9 weeks of Npgl overexpression, we measured adipose tissues, muscle, and several organ masses in addition to food intake and body mass. To assess the effects of Npgl overexpression on peripheral tissues, we analyzed mRNA expression of lipid metabolism-related genes by quantitative RT-PCR. Whole body energy consumption was assessed using an O2/CO2 metabolism measurement before an apparent increase in body mass. RESULTS: Npgl overexpression increased food intake, body mass, adipose tissues and liver masses, and food efficiency under both NC and HCD, resulting in obesity observable within 8 weeks. Furthermore, we observed fat accumulation in adipose tissues and liver. Additionally, mRNA expression of lipid metabolism-related factors was increased in white adipose tissue and the liver after Npgl overexpression. Npgl overexpression inhibited energy expenditure during a dark period. CONCLUSION: Taken together, the present study suggests that NPGL can act as an obesogenic factor that acts within a short period of time in mice. As a result, this Npgl overexpression-induced obesity can be widely applied to study the etiology of obesity from genes to behavior.

Flexibility in an emergency life-history stage: acute food deprivation prevents sickness behaviour but not the immune response.

The emergency life-history stage (ELHS) can be divided into two subcategories that describe distinct, coordinated responses to disease- or non-disease-related physiological challenges. Whether an individual can simultaneously express aspects of both subcategories when faced with multiple challenges is poorly understood. Emergency life-history theory suggests that disease- and non-disease-related responses are coordinated at the level of the whole organism and therefore cannot be expressed simultaneously. However, the reactive scope and physiological regulatory network models suggest that traits can be independently regulated, allowing for components of both disease- and non-disease-related responses to be simultaneously expressed within a single organism. To test these ideas experimentally, we subjected female zebra finches to food deprivation, an immune challenge, both, or neither, and measured a suite of behavioural and physiological traits involved in the ELHS. We examined whether the trait values expressed by birds experiencing simultaneous challenges resembled trait values of birds experiencing a single challenge or if birds could express a mixture of trait values concurrently. We find that birds can respond to simultaneous challenges by regulating components of the behavioural and immune responses independently of one another. Modularity within these physio-behavioural networks adds additional dimensions to how we evaluate the intensity or quality of an ELHS. Whether modularity provides fitness advantages or costs in nature remains to be determined.

Food access modifies GnIH, but not CRH, cell number in the hypothalamus in a female songbird.

Food deprivation or restriction causes animals to mount a stereotypical behavioral and physiological response that involves overall increases in activity, elevated glucocorticoid production, and (often) inhibition of the reproductive system. Although there is increasing evidence that these responses can differ in their degree or covariation between the sexes, most studies to-date on food restriction/deprivation have focused on male songbirds. We therefore aimed to characterize the behavioral, physiological, and neuroendocrine response to acute food deprivation in a female songbird using a nomadic species, the zebra finch. We quantified behavior during a 6.5 h food deprivation and then measured physiological and neuroendocrine responses of female birds at the 6.5 h timepoint. Within 1 h of acute food deprivation, female zebra finches increased foraging behaviors, and after 6.5 h of food deprivation, females lost 5% of their body mass, on average. Change in body mass was positively associated with elevated corticosterone and (contrary to findings in male zebra finches) negatively related to the number of gonadotropin inhibitory hormone-immunoreactive cells in the hypothalamus. However, there was no effect of food deprivation on corticotropin releasing hormone-immunoreactive cells in the hypothalamus. There was also no relationship between corticotropin releasing hormone-immunoreactive cell number and circulating corticosterone. Our results are consistent with the hypothesis that neuroendocrine responses to food deprivation differ between male and female songbirds. Future studies should work to incorporate sex comparisons to evaluate sex-specific neuroendocrine responses to acute stress.

Neurosecretory protein GL induces fat accumulation in mice.

We recently discovered a novel gene encoding a small secretory protein, neurosecretory protein GL (NPGL), which stimulates feeding behavior in mice following acute administration. These findings suggest that dysregulation of NPGL contributes to obesity and metabolic disease. To explore this possibility, we investigated the impact of prolonged exposure to NPGL through 13 days of chronic intracerebroventricular (i.c.v.) infusion and examined feeding behavior, body composition, expressions of lipid metabolic factors, respiratory metabolism, locomotor activity, and food preference. Under standard chow diet, NPGL increased white adipose tissue (WAT) mass without affecting feeding behavior and body mass. In contrast, when fed a high-calorie diet, NPGL stimulated feeding behavior and increased body mass concomitant with marked fat accumulation. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that mRNA expressions for key enzymes and related factors involved in lipid metabolism were increased in WAT and liver. Likewise, analyses of respiratory metabolism and locomotor activity revealed that energy expenditure and locomotor activity were significantly decreased by NPGL. In contrast, selective feeding of macronutrients did not alter food preference in response to NPGL, although total calorie intake was increased. Immunohistochemical analysis revealed that NPGL-containing cells produce galanin, a neuropeptide that stimulates food intake. Taken together, these results provide further support for NPGL as a novel regulator of fat deposition through changes in energy intake and locomotor activity.

RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat.

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies.

Neurosecretory Protein GL Induces Fat Accumulation in Chicks.

We recently found a previously unidentified cDNA in chicken hypothalamus which encodes the precursor for neurosecretory protein GL (NPGL). A previous study showed that intracerebroventricular (i.c.v.) infusion of NPGL caused body mass gain in chicks. However, it was not clear which part(s) of the body gained mass. In the present study, we investigated which tissues increased in mass after chronic i.c.v. infusion of NPGL in chicks. We found that NPGL increased the masses of the liver, abdominal fat, and subcutaneous fat, while NPGL did not affect the masses of muscles, including pectoralis major, pectoralis minor, and biceps femoris. Oil Red O staining revealed that fat deposition had occurred in the liver. In addition, the size of the lipid droplets in the abdominal fat increased. Furthermore, we found an upregulation of lipogenesis and downregulation of lipolysis in the abdominal fat, but not in the liver. These results indicate that NPGL is involved in fat storage in chicks.

Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system†.

Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial-blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

Melatonin Administration Methods for Research in Mammals and Birds.

Endocrine research in animals often entails exogenous hormone administration. Special issues arise when developing administration protocols for hormones with circadian and seasonal periodicity. This article reviews various methods for the exogenous administration of hormones with such periodicities by focusing on melatonin. We discuss that methodological variations across studies can affect experimental results. Melatonin administration techniques used in vertebrates includes infusion pumps, beeswax pellets, oral administration, injections, SILASTIC capsules, osmotic pumps, transdermal delivery, beads, and sponges.

Pregnancy stage determines the effect of chronic stress on ovarian progesterone synthesis.

Although stress-induced glucocorticoid release is thought to be a primary driver by which maternal stress negatively impacts pregnancy outcomes, the downstream neuroendocrine targets mediating these adverse outcomes are less well understood. We hypothesized that stress-induced glucocorticoid secretion inhibits pituitary hormone secretion, resulting in decreased ovarian progesterone synthesis. Using a chronic restraint model of stress in mice, we quantified steroid hormone production, pituitary hormones, and expression of ovarian genes that support progesterone production at both early ( day 5) and midpregnancy ( day 10). Females subjected to daily restraint had elevated baseline glucocorticoids during both early and midpregnancy; however, lower circulating progesterone was observed only during early pregnancy. Lower progesterone production was associated with lower expression of steroidogenic enzymes in the ovary of restrained females during early pregnancy. There were no stress-related changes to luteinizing hormone (LH) or prolactin (PRL). By midpregnancy, circulating LH decreased regardless of treatment, and this was associated with downregulation of ovarian steroidogenic gene expression. Our results are consistent with a role for LH in maintaining steroidogenic enzyme expression in the ovary, but neither circulating PRL nor LH were associated with the stress-induced inhibition of ovarian progesterone production during early pregnancy. We conclude that chronic stress impacts endocrine networks differently in pregnant and nonpregnant mammals. These findings underscore the need for further studies exploring dynamic changes in endocrine networks participating in pregnancy initiation and progression to elucidate the physiological mechanisms that connect stress exposure to adverse pregnancy outcomes.

Brain-Derived Steroids, Behavior and Endocrine Conflicts Across Life History Stages in Birds: A Perspective.

Biological steroids were traditionally thought to be synthesized exclusively by the adrenal glands and gonads. Recent decades have seen the discovery of neurosteroid production that acts locally within the central nervous system to affect physiology and behavior. These actions include, for example, regulation of aggressive behavior, such as territoriality, and locomotor movement associated with migration. Important questions then arose as to how and why neurosteroid production evolved and why similar steroids of peripheral origin do not always fulfill these central roles? Investigations of free-living vertebrates suggest that synthesis and action of bioactive steroids within the brain may have evolved to regulate expression of specific behavior in different life history stages. Synthesis and secretion of these hormones from peripheral glands is broadcast throughout the organism via the blood stream. While widespread, general actions of steroids released into the blood might be relevant for regulation of morphological, physiological, and behavioral traits in one life history stage, such hormonal release may not be appropriate in other stages. Specific and localized production of bioactive steroids in the brain, but not released into the periphery, could be a way to avoid such conflicts. Two examples are highlighted. First, we compare the control of territorial aggression of songbirds in the breeding season under the influence of gonadal steroids with autumnal (non-breeding) territoriality regulated by sex steroid production in the brain either from circulating precursors such as dehydroepiandrosterone or local central production of sex steroids de novo from cholesterol. Second, we outline the production of 7α-hydroxypregnenolone within the brain that appears to affect locomotor behavior in several contexts. Local production of these steroids in the brain may provide specific regulation of behavioral traits throughout the year and independently of life history stage.

Author Correction: Localization and function of neurosecretory protein GM, a novel small secretory protein, in the chicken hypothalamus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Seasonal Reproduction in Vertebrates: Melatonin Synthesis, Binding, and Functionality Using Tinbergen's Four Questions.

One of the many functions of melatonin in vertebrates is seasonal reproductive timing. Longer nights in winter correspond to an extended duration of melatonin secretion. The purpose of this review is to discuss melatonin synthesis, receptor subtypes, and function in the context of seasonality across vertebrates. We conclude with Tinbergen's Four Questions to create a comparative framework for future melatonin research in the context of seasonal reproduction.

Localization and function of neurosecretory protein GM, a novel small secretory protein, in the chicken hypothalamus.

Recently, we discovered a novel cDNA encoding the precursor of a small secretory protein, neurosecretory protein GL (NPGL), in the hypothalamic infundibulum of chickens. NPGL plays an important role in the regulation of growth and feeding. A database search indicated that the NPGL gene has a paralogous gene: neurosecretory protein GM (NPGM), also in chickens. We identified cDNA encoding the NPGM precursor in chickens. Morphological analysis showed that NPGM-containing cells are specifically localized in the medial mammillary nucleus (MM) and infundibular nucleus (IN) in the hypothalamus. In addition, we found that NPGM and NPGL are co-localized, especially in the MM. The expression levels of NPGM mRNA gradually decreased during post-hatch development, in contrast to those of NPGL mRNA. Moreover, we investigated the relationship between NPGM and other known factors. NPGM was found to be produced in histaminergic neurons in the MM. NPGM and histidine decarboxylase, a histamine-producing enzyme, displayed similar expression patterns during post-hatch development. Acute intracerebroventricular injection of NPGM decreased food intake, similar to the effect of histamine. To our knowledge, this is the first report of the localization and function of NPGM in the brain of vertebrates. These results will further advance the understanding mechanisms underlying energy homeostasis.

Chronic subcutaneous infusion of neurosecretory protein GM increases body mass gain in chicks.

Recently we discovered a small hypothalamic protein in the chicken, named neurosecretory protein GL (NPGL), which is associated with body growth and energy metabolism in birds and rodents. Genome database analysis suggested that the NPGL gene has a paralogous gene in vertebrates, named neurosecretory protein GM (NPGM). However, the biological action of NPGM remains unclear. In this study, we investigated whether NPGM affects body growth in chicks. We found that subcutaneous infusion of NPGM for six days increased body mass gain in a dose-dependent manner. Despite the observed increase in body mass, infusion of NPGM did not alter food and water intake. Of note, we observed tendency of mass increase of several peripheral tissues, specifically. When we compared several tissue types, NPGM seemed to induce the largest growth increase in white adipose tissue mass. These results suggest that NPGM may accelerate fat accumulation and body growth. In addition, we analyzed whether NPGM increases body growth through the action of pituitary hormones. However, we observed no significant changes in mRNA expression of pituitary hormones or plasma levels of growth hormone in NPGM-treated chicks. This is the first report describing the biological action of NPGM in vertebrates.

Effects of chronic intracerebroventricular infusion of neurosecretory protein GL on body mass and food and water intake in chicks.

Recently, we discovered a novel cDNA encoding the precursor of a small secretory protein, neurosecretory protein GL (NPGL), in the chicken mediobasal hypothalamus. In this study, immunohistochemical analysis revealed that NPGL was produced in the infundibular and medial mammillary nuclei of the mediobasal hypothalamus, with immunoreactive fibers also detected in the hypothalamus and the median eminence. As it is known that these regions are involved in feeding behavior in chicks, we surveyed the effects of chronic intracerebroventricular infusion of NPGL on feeding behavior and body mass for a period of two weeks. NPGL stimulated food and water intake, with a concomitant increase in body mass. However, NPGL did not influence mRNA expression of several hypothalamic ingestion-related neuropeptides. Our data suggest that NPGL may be a novel neuronal regulator involved in growth processes in chicks.