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Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgb1) subunit is highly expressed in endothelial cells (EC). EC itgb1 is necessary for the formation of capillary networks during embryonic during development and its knockdown blunts the reactive hyperemia that manifests during ischemia reperfusion. We investigated the contribution of EC itgb1 in microcirculatory function and glucose uptake with emphasis in skeletal muscle. We hypothesized that loss of EC itgb1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. Mice with (itgb1 fl/fl SCLcre) and without (itgb1 fl/fl ) tamoxifen inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction had comparable exercise tolerance and pulmonary and cardiac functions. Using robust in vivo experimental platforms (i.e., intravital microscopy and hyperinsulinemic-euglycemic clamp), we show that itgb1 fl/fl SCLcre mice compared to itgb1 fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgb1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

Background: Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST) and Crh+ neurons in this region play a key role in chronic ethanol-induced increases in volitional intake, hypothesized to be driven by emergent negative affective behaviors. Excitatory N-methyl-d-aspartate receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. Specifically, GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior., Methods: Male and female mice underwent a home cage chronic drinking forced abstinence model (CDFA) to assess the impact of 1 day or 2 weeks of ethanol abstinence on BNST synaptic transmission and BNST Grin gene expression. Constitutive and conditional BNST GluN2D knockout mice were used to assess the impact of GluN2D deletion on continuous access ethanol intake as well as negative affect behaviors, using the open field, elevated zero maze, and forced swim tasks., Results: We report here that excitatory transmission undergoes time-dependent upregulation in BNST Crh+ cells. Further, knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in the dBNST or dBNST Crh+ neurons. Finally, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice., Conclusions: These data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking but not ethanol abstinence, highlighting sex differences and behavioral specificity. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior., (© 2024 Research Society on Alcohol.)

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (EC). EC itgβ1 is necessary for the formation of capillary networks during embryonic during development and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study we investigated the contribution of skeletal muscle EC itgβ1 in microcirculatory function and glucose uptake. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. We found that mice with (itgβ1 fl/fl SCLcre) and without (itgβ1 fl/fl ) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1 fl/fl SCLcre mice compared to itgβ1 fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

Nutritional fluctuations that occur early in life dictate metabolic adaptations that will affect susceptibility to weight gain and obesity later in life. The postnatal period in mice represents a time of dynamic changes in hypothalamic development and maternal consumption of a high fat diet during the lactation period (MHFD) changes the composition of milk and leads to enhanced susceptibility to obesity in offspring. Agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARH) react to changes in multiple metabolic signals and distribute neuroendocrine information to other brain regions, such as the paraventricular hypothalamic nucleus (PVH), which is known to integrate a variety of signals that regulate body weight. Development of neural projections from AgRP neurons to the PVH occurs during the lactation period and these projections are reduced in MHFD offspring, but underlying developmental mechanisms remain largely unknown. Microglia are the resident immune cells of the central nervous system and are involved in refinement of neural connections and modulation of synaptic transmission. Because high fat diet exposure causes activation of microglia in adults, a similar activation may occur in offspring exposed to MHFD and play a role in sculpting hypothalamic feeding circuitry. Genetically targeted axonal labeling and immunohistochemistry were used to visualize AgRP axons and microglia in postnatal mice derived from MHFD dams and morphological changes quantified. The results demonstrate regionally localized changes to microglial morphology in the PVH of MHFD offspring that suggest enhanced surveillance activity and are temporally restricted to the period when AgRP neurons innervate the PVH. In addition, axon labeling experiments confirm a significant decrease in AgRP innervation of the PVH in MHFD offspring and provide direct evidence of synaptic pruning of AgRP inputs to the PVH. Microglial depletion with the Colony-stimulating factor 1 receptor inhibitor PLX5622 determined that the decrease in AgRP innervation observed in MHFD offspring is dependent on microglia, and that microglia are required for weight gain that emerges as early as weaning in offspring of MHFD dams. However, these changes do not appear to be dependent on the degree of microglial mediated synaptic pruning. Together, these findings suggest that microglia are activated by exposure to MHFD and interact directly with AgRP axons during development to permanently alter their density, with implications for developmental programming of metabolic phenotype., Competing Interests: Competing Interest Statement: No competing interests

α 2a -adrenergic receptor (α 2a -AR) agonists are candidate substance use disorder therapeutics due to their ability to recruit noradrenergic autoreceptors to dampen stress system engagement. However, we recently found that postsynaptic α 2a -ARs are required for stress-induced reinstatement of cocaine-conditioned behavior. Understanding the ensembles recruited by these postsynaptic receptors (heteroceptors) is necessary to understand noradrenergic circuit control. We utilized a variety of approaches in FosTRAP (Targeted Recombination in Active Populations) mice to define an ensemble of cells activated by the α 2a -AR partial agonist guanfacine ("Guansembles") in the bed nucleus of the stria terminalis (BST/BNST), a region key to stress-induced reinstatement of drug seeking. We define BNST "Guansembles" and show they differ from restraint stress-activated cells. Guanfacine produced inhibition of cAMP-dependent signaling in Guansembles, while chronic restraint stress increased cAMP-dependent signaling. Guanfacine both excited and inhibited aspects of Guansemble neuronal activity. Further, while some stressors produced overall reductions in Guansemble activity, active coping events during restraint stress and exposure to unexpected shocks were both associated with Guansemble recruitment. Using viral tracing, we define a BNST Guansemble afferent network that includes regions involved in the interplay of stress and homeostatic functions. Finally, we show that activation of Guansembles produces alterations in behavior on the elevated plus maze consistent with task-specific anxiety-like behavior. Overall, we define a population of BNST neurons recruited by α 2a -AR signaling that opposes the behavioral action of canonical autoreceptor α 2a -AR populations and which are differentially recruited by distinct stressors. Moreover, we demonstrate stressor-specific physiological responses in a specific neuronal population., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

The bed nucleus of the stria terminalis (BNST) is a critical mediator of stress responses and anxiety-like behaviors. Neurons expressing protein kinase C delta (BNST PKCδ ) are an abundant but understudied subpopulation implicated in inhibiting feeding, but which have conflicting reports about their role in anxiety-like behaviors. We have previously shown that expression of PKCδ is dynamically regulated by stress and that BNST PKCδ cells are recruited during bouts of active stress coping. Here, we first show that in vivo activation of this population is mildly aversive. This aversion was insensitive to prior restraint stress exposure. Further investigation revealed that unlike other BNST subpopulations, BNST PKCδ cells do not exhibit increased cfos expression following restraint stress. Ex vivo current clamp recordings also indicate they are resistant to firing. To elucidate their afferent control, we next used rabies tracing with whole-brain imaging and channelrhodopsin-assisted circuit mapping, finding that BNST PKCδ cells receive abundant input from affective, arousal, and sensory regions including the basolateral amygdala (BLA) paraventricular thalamus (PVT) and central amygdala PKCδ-expressing cells (CeA PKCδ ). Given these findings, we used in vivo optogenetics and fiber photometry to further examine BNST PKCδ cells in the context of stress and anxiety-like behavior. We found that BNST PKCδ cell activity is associated with increased anxiety-like behavior in the elevated plus maze, increases following footshock, and unlike other BNST subpopulations, does not desensitize to repeated stress exposure. Taken together, we propose a model in which BNST PKCδ cells may serve as threat detectors, integrating exteroceptive and interoceptive information to inform stress coping behaviors., (© 2023. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

The central melanocortin system is fundamentally important for controlling food intake and energy homeostasis. Melanocortin-3 receptor (MC3R) is one of two major receptors of the melanocortin system found in the brain. In contrast to the well-characterized melanocortin-4 receptor (MC4R), little is known regarding the organization of MC3R-expressing neural circuits. To increase our understanding of the intrinsic organization of MC3R neural circuits, identify specific differences between males and females, and gain a neural systems level perspective of this circuitry, we conducted a brain-wide mapping of neurons labeled for MC3R and characterized the distribution of their projections. Analysis revealed MC3R neuronal and terminal labeling in multiple brain regions that control a diverse range of physiological functions and behavioral processes. Notably, dense labeling was observed in the hypothalamus, as well as areas that share considerable connections with the hypothalamus, including the cortex, amygdala, thalamus, and brainstem. Additionally, MC3R neuronal labeling was sexually dimorphic in several areas, including the anteroventral periventricular area, arcuate nucleus, principal nucleus of the bed nucleus of the stria terminalis, and ventral premammillary region. Altogether, anatomical evidence reported here suggests that MC3R has the potential to influence several different classes of motivated behavior that are essential for survival, including ingestive, reproductive, defensive, and arousal behaviors, and is likely to modulate these behaviors differently in males and females., (© 2022 Wiley Periodicals LLC.)

The state of somatic energy stores in metazoans is communicated to the brain, which regulates key aspects of behaviour, growth, nutrient partitioning and development 1 . The central melanocortin system acts through melanocortin 4 receptor (MC4R) to control appetite, food intake and energy expenditure 2 . Here we present evidence that MC3R regulates the timing of sexual maturation, the rate of linear growth and the accrual of lean mass, which are all energy-sensitive processes. We found that humans who carry loss-of-function mutations in MC3R, including a rare homozygote individual, have a later onset of puberty. Consistent with previous findings in mice, they also had reduced linear growth, lean mass and circulating levels of IGF1. Mice lacking Mc3r had delayed sexual maturation and an insensitivity of reproductive cycle length to nutritional perturbation. The expression of Mc3r is enriched in hypothalamic neurons that control reproduction and growth, and expression increases during postnatal development in a manner that is consistent with a role in the regulation of sexual maturation. These findings suggest a bifurcating model of nutrient sensing by the central melanocortin pathway with signalling through MC4R controlling the acquisition and retention of calories, whereas signalling through MC3R primarily regulates the disposition of calories into growth, lean mass and the timing of sexual maturation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2021.)

Ablation of hypothalamic AgRP (Agouti-related protein) neurons is known to lead to fatal anorexia, whereas their activation stimulates voracious feeding and suppresses other motivational states including fear and anxiety. Despite the critical role of AgRP neurons in bidirectionally controlling feeding, there are currently no therapeutics available specifically targeting this circuitry. The melanocortin-3 receptor (MC3R) is expressed in multiple brain regions and exhibits sexual dimorphism of expression in some of those regions in both mice and humans. MC3R deletion produced multiple forms of sexually dimorphic anorexia that resembled aspects of human anorexia nervosa. However, there was no sexual dimorphism in the expression of MC3R in AgRP neurons, 97% of which expressed MC3R. Chemogenetic manipulation of arcuate MC3R neurons and pharmacologic manipulation of MC3R each exerted potent bidirectional regulation over feeding behavior in male and female mice, whereas global ablation of MC3R-expressing cells produced fatal anorexia. Pharmacological effects of MC3R compounds on feeding were dependent on intact AgRP circuitry in the mice. Thus, the dominant effect of MC3R appears to be the regulation of the AgRP circuitry in both male and female mice, with sexually dimorphic sites playing specialized and subordinate roles in feeding behavior. Therefore, MC3R is a potential therapeutic target for disorders characterized by anorexia, as well as a potential target for weight loss therapeutics., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Puberty is the process whereby an individual acquires the ability to reproduce, and the attainment of puberty in a timely manner is critical for both humans and livestock. For livestock, the initiation of puberty at the appropriate time aids in increasing lifetime productivity, thus maximizing profitability for producers. For humans, particularly females, early or late puberty is associated with several adverse health outcomes, including polycystic ovary syndrome, obesity, metabolic syndrome, osteoporosis, and psychosocial distress. Therefore, characterizing the mechanisms responsible for puberty onset would have a significant impact on human and animal health. It has been postulated that a group of neurons in the arcuate nucleus of the hypothalamus may play a role in puberty onset. These neurons contain kisspeptin, neurokinin B (NKB), and dynorphin and are often called KNDy neurons. Although the role of kisspeptin in puberty onset has been heavily researched, the involvement of NKB and dynorphin is not well defined. This mini-review focuses on the role of NKB in the initiation of puberty in female sheep. Stimulation of the receptor for NKB, NK3R, elicits LH secretion in a GnRH-dependent manner in prepubertal ewes, and both functional and neuroanatomical changes to the NKB system, particularly within the preoptic area, appear to occur as female sheep transition from a prepubertal to an adult state. Thus, NKB is likely an important component of puberty onset in sheep, although its integration with other systems that impact the pubertal process, such as photoperiod and nutrition, remains to be elucidated., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neurokinin B (NKB) is critical for fertility in humans and stimulates gonadotrophin-releasing hormone/luteinising hormone (LH) secretion in several species, including sheep. There is increasing evidence that the actions of NKB in the retrochiasmatic area (RCh) contribute to the induction of the preovulatory LH surge in sheep. In the present study, we determined whether there are sex differences in the response to RCh administration of senktide, an agonist to the NKB receptor (neurokinin receptor-3 [NK3R]), and in NKB and NK3R expression in the RCh of sheep. To normalise endogenous hormone concentrations, animals were gonadectomised and given implants to mimic the pattern of ovarian steroids seen in the oestrous cycle. In females, senktide microimplants in the RCh produced an increase in LH concentrations that lasted for at least 8 hours after the start of treatment, whereas a much shorter increment (approximately 2 hours) was seen in males. We next collected tissue from gonadectomised lambs 18 hours after the insertion of oestradiol implants that produce an LH surge in female, but not male, sheep for immunohistochemical analysis of NKB and NK3R expression. As expected, there were more NKB-containing neurones in the arcuate nucleus of females than males. Interestingly, there was a similar sexual dimorphism in NK3R-containing neurones in the RCh, NKB-containing close contacts onto these RCh NK3R neurones, and overall NKB-positive fibres in this region. These data demonstrate that there are both functional and morphological sex differences in NKB-NK3R signalling in the RCh and raise the possibility that this dimorphism contributes to the sex-dependent ability of oestradiol to induce an LH surge in female sheep., (© 2020 British Society for Neuroendocrinology.)

Elevated and sustained estradiol concentrations cause a gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) surge that is necessary for ovulation. In sheep, several different neural systems have been implicated in this stimulatory action of estradiol and this study focused on somatostatin (SST) neurons in the ventral lateral region of the ventral medial nucleus (vlVMN) which express c-Fos during the surge. First, we determined if increased activity of SST neurons could be related to elevated GnRH secretion by assessing SST synapses onto GnRH neurons and neurons coexpressing kisspeptin, neurokinin B, dynorphin (KNDy). We found that the percentage of preoptic area GnRH neurons that receive SST input increased during the surge compared with other phases of the cycle. However, since SST is generally inhibitory, and pharmacological manipulation of SST signaling did not alter the LH surge in sheep, we hypothesized that nitric oxide (NO) was also produced by these neurons to account for their activation during the surge. In support of this hypothesis we found that (1) the majority of SST cells in the vlVMN (>80%) contained neuronal nitric oxide synthase (nNOS); (2) the expression of c-Fos in dual-labeled SST-nNOS cells, but not in single-labeled cells, increased during the surge compared with other phases of the cycle; and (3) intracerebroventricular (ICV) infusion of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, completely blocked the estrogen-induced LH surge. These data support the hypothesis that the population of SST-nNOS cells in the vlVMN are a source of NO that is critical for the LH surge, and we propose that they are an important site of estradiol positive feedback in sheep., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recent evidence has implicated neurokinin B (NKB) signaling in the retrochiasmatic area (RCh) of the ewe in the LH surge. To test this hypothesis, we first lesioned NK3R neurons in this area by using a saporin conjugate (NK3-SAP). Three weeks after bilateral injection of NK3-SAP or a blank control (BLK-SAP) into the RCh, an LH surge was induced by using an artificial follicular-phase model in ovariectomized ewes. NK3-SAP lesioned approximately 88% of RCh NK3R-containing neurons and reduced the amplitude of the estrogen-induced LH surge by 58%, an inhibition similar to that seen previously with intracerebroventricular (icv) infusion of a KISS1R antagonist (p271). We next tested the hypothesis that NKB signaling in the RCh acts via kisspeptin by determining whether the combined effects of NK3R-SAP lesions and icv infusion of p271 were additive. Experiment 1 was replicated except that ewes received two sequential artificial follicular phases with infusions of p271 or vehicle using a crossover design. The combination of the two treatments decreased the peak of the LH surge by 59%, which was similar to that seen with NK3-SAP (52%) or p271 (54%) alone. In contrast, p271 infusion delayed the onset and peak of the LH surge in both NK3-SAP- and BLK-SAP-injected ewes. Based on these data, we propose that NKB signaling in the RCh increases kisspeptin levels critical for the full amplitude of the LH surge in the ewe but that kisspeptin release occurs independently of RCh input at the onset of the surge to initiate GnRH secretion., (Copyright © 2019 Endocrine Society.)

Early work in ewes provided a wealth of information on the physiological regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by internal and external inputs. Identification of the neural systems involved, however, was limited by the lack of information on neural mechanisms underlying generation of GnRH pulses. Over the last decade, considerable evidence supported the hypothesis that a group of neurons in the arcuate nucleus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) are responsible for synchronizing secretion of GnRH during each pulse in ewes. In this review, we describe our current understanding of the neural systems mediating the actions of ovarian steroids and three external inputs on GnRH pulsatility in light of the hypothesis that KNDy neurons play a key role in GnRH pulse generation. In breeding season adults, estradiol (E 2 ) and progesterone decrease GnRH pulse amplitude and frequency, respectively, by actions on KNDy neurons, with E 2 decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E 2 inhibits GnRH pulse frequency by decreasing kisspeptin and increasing dynorphin release, actions that wane as the lamb matures to allow increased pulsatile GnRH secretion at puberty. Less is known about mediators of undernutrition and stress, although some evidence implicates kisspeptin and dynorphin, respectively, in the inhibition of GnRH pulse frequency by these factors. During the anoestrus, inhibitory photoperiod acting via melatonin activates A15 dopaminergic neurons that innervate KNDy neurons; E 2 increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release., (© 2018 Society for Reproduction and Fertility.)

Puberty is a process that integrates multiple inputs ultimately resulting in an increase in gonadotrophin-releasing hormone (GnRH) secretion. Although kisspeptin neurones play an integral role in GnRH secretion and puberty onset, other systems are also likely important. One potential component is nitric oxide (NO), a gaseous neurotransmitter synthesised by nitric oxide synthase (NOS). The present study aimed to neuroanatomically characterise neuronal NOS (nNOS) in prepubertal female sheep and determine whether oestradiol exerts effects on this system. Luteinising hormone secretion was reduced by oestradiol treatment in prepubertal ovariectomised ewes. Neurones immunoreactive for nNOS were identified in several areas, with the greatest number present in the ventrolateral portion of the ventromedial hypothalamus, followed by the ventromedial hypothalamus, preoptic area (POA) and arcuate nucleus (ARC). Next, we determined whether nNOS neurones contained oestrogen receptor (ER)α and could potentially communicate oestradiol (E 2 ) feedback to GnRH neurones. Neuronal NOS neurones contained ERα with the percentage of coexpression (12%-40%) depending upon the area analysed. We next investigated whether a neuroanatomical relationship existed between nNOS and kisspeptin or nNOS and GnRH neurones. A high percentage of kisspeptin neurones in the POA (79%) and ARC (98%) colocalised with nNOS. Kisspeptin close contacts were also associated with nNOS neurones. A greater number of close contacts were observed in the ARC than the POA. A high percentage of POA GnRH neurones (79%) also expressed nNOS, although no GnRH close contacts were observed onto nNOS neurones. Neither the numbers of nNOS neurones in the POA or hypothalamus, nor the percentage of nNOS coexpression with GnRH, kisspeptin or ERα were influenced by oestradiol. These experiments reveal that a neuroanatomical relationship exists between both nNOS and kisspeptin and nNOS and GnRH in prepubertal ewes. Therefore, nNOS may act both directly and indirectly to influence GnRH secretion in prepubertal sheep., (© 2017 British Society for Neuroendocrinology.)

In this study, we investigated whether decreased sensitivity to estradiol negative feedback is associated with reduced estrogen receptor α (ESR1) expression in kisspeptin neurons as ewe lambs approach puberty. Lambs were ovariectomized and received no implant (OVX) or an implant containing estradiol (OVX+E). In the middle arcuate nucleus (mARC), ESR1 messenger RNA (mRNA) was greater in OVX than OVX+E lambs but did not differ elsewhere. Post hoc analysis of luteinizing hormone (LH) secretion from OVX+E lambs revealed three patterns of LH pulsatility: low [1 to 2 pulses per 12 hours; low frequency (LF), n = 3], moderate [6 to 7 pulses per 12 hours; moderate frequency (MF), n = 6], and high [>10 pulses per 12 hours; high frequency (HF), n = 5]. The percentage of kisspeptin neurons containing ESR1 mRNA in the preoptic area did not differ among HF, MF, or LF groups. However, the percentage of kisspeptin neurons containing ESR1 mRNA in the mARC was greater in HF (57%) than in MF (36%) or LF (27%) lambs and did not differ from OVX (50%) lambs. A higher percentage of kisspeptin neurons contained ESR1 protein in all regions of the arcuate nucleus (ARC) in OVX compared with OVX+E lambs. There were no differences in ESR1 protein among the HF, MF, or LF groups in the preoptic area or ARC. Contrary to our hypothesis, increases in LH pulsatility were associated with enhanced ESR1 mRNA abundance in kisspeptin neurons in the ARC, and absence of estradiol increased the percentage of kisspeptin neurons containing ESR1 protein in the ARC. Therefore, changes in the expression of ESR1, particularly in kisspeptin neurons in the ARC, do not explain the pubertal escape from estradiol negative feedback in ewe lambs., (Copyright © 2018 Endocrine Society.)

Background: Neuronal intermediates that communicate estrogen and progesterone feedback to gonadotropin-releasing hormone (GnRH) neurons are essential for modulating reproductive cyclicity. Individually, kisspeptin and nitric oxide (NO) influence GnRH secretion. However, it is possible these 2 neuronal intermediates interact with one another to affect reproductive cyclicity., Methods: We investigated the neuroanatomical relationship of one isoform of the enzyme that synthesizes NO, neuronal NO synthase (nNOS), to kisspeptin and GnRH in adult female rhesus monkeys and sheep using dual-label immunofluorescence. Additionally, we evaluated if the phase of the reproductive cycle would affect these relationships., Results: Overall, no effect of the stage of cycle was observed for any variable in this study. In the arcuate nucleus (ARC) of sheep, 98.8 ± 3.5% of kisspeptin neurons colocalized with nNOS, and kisspeptin close-contacts were observed onto nNOS neurons. In contrast to ewes, no colocalization was observed between kisspeptin and nNOS in the infundibular ARC of primates, but kisspeptin fibers were apposed to nNOS neurons. In the preoptic area of ewes, 15.0 ± 4.2% of GnRH neurons colocalized with nNOS. In primates, 38.8 ± 10.1% of GnRH neurons in the mediobasal hypothalamus colocalized with nNOS, and GnRH close-contacts were observed onto nNOS neurons in both sheep and primates., Conclusion: Although species differences were observed, this work establishes a neuroanatomical framework between nNOS and kisspeptin and nNOS and GnRH in adult female nonhuman primates and sheep., (© 2018 S. Karger AG, Basel.)

Two modes of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion are necessary for female fertility: surge and episodic secretion. However, the neural systems that regulate these GnRH secretion patterns are still under investigation. The neuropeptide somatostatin (SST) inhibits episodic LH secretion in humans and sheep, and several lines of evidence suggest SST may regulate secretion during the LH surge. In this study, we examined whether SST alters the LH surge in ewes by administering a SST receptor (SSTR) 2 agonist (octreotide) or antagonist [CYN154806 (CYN)] into the third ventricle during an estrogen-induced LH surge and whether endogenous SST alters episodic LH secretion. Neither octreotide nor CYN altered the amplitude or timing of the LH surge. Administration of CYN to intact ewes during the breeding season or anestrus increased LH secretion and increased c-Fos in a subset GnRH and kisspeptin cells during anestrus. To determine if these stimulatory effects are steroid dependent or independent, we administered CYN to ovariectomized ewes. This SSTR2 antagonist increased LH pulse frequency in ovariectomized ewes during anestrus but not during the breeding season. This study provides evidence that endogenous SST contributes to the control of LH secretion. The results demonstrate that SST, acting through SSTR2, inhibits episodic LH secretion, likely acting in the mediobasal hypothalamus, but action at this receptor does not alter surge secretion. Additionally, these data provide evidence that SST contributes to the steroid-independent suppression of LH pulse frequency during anestrus., (Copyright © 2017 Endocrine Society.)

This study examined the relationship of prenatal transportation stress (PNS) with exogenous GnRH-induced LH and testosterone secretion in sexually mature Brahman bulls. Brahman cows (n = 96; 48 were stressed by transportation at 5 stages of gestation and 48 were controls) produced a calf crop of 85 calves. All bulls (n = 46) from this calf crop were electroejaculated every 2 wk beginning at a scrotal circumference of 24 cm until sexual maturity (SM; i.e., 500 million sperm/ejaculate). The initial 11 control and 12 PNS bulls to reach SM were selected for the experiment. Within 7-21 d after reaching SM, bulls were fitted with jugular cannulas, from which blood samples were collected at 15-min intervals for 6 h prior to exogenous GnRH administration (10 ng/kg BW; i.v.) and for 6 h after GnRH. Serum concentrations of LH, testosterone, and cortisol were determined by RIA. Age and body weight did not differ ( > 0.1) between PNS and control bulls at the time of the experiment. All bulls responded similarly to exogenous GnRH, indicating no influence of PNS on LH or testosterone response to GnRH. More ( < 0.01) PNS (9 of 11) than control (3 of 12) bulls exhibited an endogenous pre-GnRH LH pulse, and more ( = 0.02) PNS (9 of 11) than control bulls (4 of 12) exhibited a pre-GnRH testosterone response to LH. The average concentration of testosterone during the 60 min (time -60, -45, -30, -15, and 0 min relative to GnRH) immediately preceding GnRH, tended to be greater ( = 0.07) in PNS (1.46 ± 0.30 ng/mL) than control (0.68 ± 0.28 ng/mL) bulls. During that time span serum cortisol was lower ( < 0.01) in PNS (4.00 ± 0.91 ng/mL) than control (7.8 ± 0.87 ng/mL) bulls. A treatment by time interaction ( = 0.03) affected testosterone concentrations from time -240 to 360 min relative to GnRH. Results from this study indicate that PNS did not affect pituitary responsiveness to GnRH or testicular responsiveness to GnRH-induced LH secretion.

Puberty onset involves increased gonadotrophin-release (GnRH) release as a result of decreased sensitivity to oestrogen (E 2 )-negative feedback. Because GnRH neurones lack E 2 receptor α, this pathway must contain interneurones. One likely candidate is KNDy neurones (kisspeptin, neurokinin B, dynorphin). The overarching hypothesis of the present study was that the prepubertal hiatus in luteinising hormone (LH) release involves reduced kisspeptin and/or heightened dynorphin input. We first tested the specific hypothesis that E 2 would reduce kisspeptin-immunopositive cell numbers and increase dynorphin-immunopositive cell numbers. We found that kisspeptin cell numbers were higher in ovariectomised (OVX) lambs than OVX lambs treated with E 2 (OVX+ E 2 ) or those left ovary-intact. Very few arcuate dynorphin cells were identified in any group. Next, we hypothesised that central blockade of κ-opioid receptor (KOR) would increase LH secretion at a prepubertal (6 months) but not postpubertal (10 months) age. Luteinising hormone pulse frequency and mean LH increased during infusion of a KOR antagonist, norbinaltorphimine, in OVX + E 2 lambs at the prepubertal age but not in the same lambs at the postpubertal age. We next hypothesised that E 2 would increase KOR expression in GnRH neurones or alter synaptic input to KNDy neurones in prepubertal ewes. Oestrogen treatment decreased the percentage of GnRH neurones coexpressing KOR (approximately 68%) compared to OVX alone (approximately 78%). No significant differences in synaptic contacts per cell between OVX and OVX + E 2 groups were observed. Although these initial data are consistent with dynorphin inhibiting pulsatile LH release prepubertally, additional work will be necessary to define the source and mechanisms of this inhibition., (© 2016 British Society for Neuroendocrinology.)