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Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B ( Nmb ) or gastrin-releasing peptide ( Grp ). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs. The peptidergic neuronal circuit controlling sigh generation has been identified as ~200  Nmb- or Grp -expressing neurons in the RTN/pFRG breathing control centre of the medulla that project to ~200 receptor-expressing neurons in the respiratory rhythm generator, the preBötzinger Complex. Sigh centre neurons identified Although sighs are an integral part of breathing and respiratory physiology, little is known about the neuronal circuits controlling this behaviour. Here, Mark Krasnow and colleagues identify a small subset of genetically defined neurons in the medulla that project to the preBötzinger complex (preBötC), the respiratory rhythm generator, to drive sighing. Inhibition of this connection could completely eliminate sighs, while regular breathing was left intact. The authors propose a mechanism by which specific preBötC neurons may integrate physiological and possibly emotional inputs to turn regular breaths into sighs when appropriate.

A hypothalamic pulse generator located in the arcuate nucleus controls episodic release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) and is essential for reproduction. Recent evidence suggests this generator is composed of arcuate “KNDy” cells, the abbreviation based on coexpression of kisspeptin, neurokinin B, and dynorphin. However, direct visual evidence of KNDy neuron activity at a single-cell level during a pulse is lacking. Here, we use in vivo calcium imaging in freely moving female mice to show that individual KNDy neurons are synchronously activated in an episodic manner, and these synchronized episodes always precede LH pulses. Furthermore, synchronization among KNDy cells occurs in a temporal order, with some subsets of KNDy cells serving as “leaders” and others as “followers” during each synchronized episode. These results reveal an unsuspected temporal organization of activation and synchronization within the GnRH pulse generator, suggesting that different subsets of KNDy neurons are activated at pulse onset than afterward during maintenance and eventual termination of each pulse. Further studies to distinguish KNDy “leader” from “follower” cells is likely to have important clinical significance, since regulation of pulsatile GnRH secretion is essential for normal reproduction and disrupted in pathological conditions such as polycystic ovary syndrome and hypothalamic amenorrhea.

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B and dynorphin (KNDy cells) are essential for mammalian reproduction as key regulators of gonadotropin-releasing hormone (GnRH) secretion. Although multiple endogenous and exogenous signals act indirectly via KNDy neurons to regulate GnRH, the identity of upstream neurons that provide synaptic input to this subpopulation is unclear. We used rabies-mediated tract-tracing in transgenic Kiss1- Cre mice combined with whole-brain optical clearing and multiple-label immunofluorescence to create a comprehensive and quantitative brain-wide map of neurons providing monosynaptic input to KNDy cells, as well as identify the estrogen receptor content and peptidergic phenotype of afferents. Over 90% of monosynaptic input to KNDy neurons originated from hypothalamic nuclei in both male and female mice. The greatest input arose from non-KNDy ARC neurons, including proopiomelanocortin-expressing cells. Significant female-dominant sex differences in afferent input were detected from estrogen-sensitive hypothalamic nuclei critical for reproductive endocrine function and sexual behavior in mice, indicating KNDy cells may provide a unique site for the coordination of sex-specific behavior and gonadotropin release. These data provide key insight into the structural framework underlying the ability of KNDy neurons to integrate endogenous and environmental signals important for the regulation of reproductive function.

A. Kemal Topaloglu and colleagues report the identification of mutations in the neurokinin B receptor and its ligand in families with severe congenital gonadotropin deficiency and pubertal failure. These findings indicate that neurokinin B is a central regulator of human gonadal function. The timely secretion of gonadal sex steroids is essential for the initiation of puberty, the postpubertal maintenance of secondary sexual characteristics and the normal perinatal development of male external genitalia. Normal gonadal steroid production requires the actions of the pituitary-derived gonadotropins, luteinizing hormone and follicle-stimulating hormone. We report four human pedigrees with severe congenital gonadotropin deficiency and pubertal failure in which all affected individuals are homozygous for loss-of-function mutations in TAC3 (encoding Neurokinin B) or its receptor TACR3 (encoding NK3R). Neurokinin B, a member of the substance P–related tachykinin family, is known to be highly expressed in hypothalamic neurons that also express kisspeptin 1 , a recently identified regulator of gonadotropin-releasing hormone secretion 2 . These findings implicate Neurokinin B as a critical central regulator of human gonadal function and suggest new approaches to the pharmacological control of human reproduction and sex hormone-related diseases.

Type 2 cytokine responses promote parasitic immunity and initiate tissue repair; however, they can also result in immunopathologies when not properly restricted. Although basophilia is recognized as a common feature of type 2 inflammation, the roles basophils play in regulating these responses are unknown. Here, we demonstrate that helminth-induced group 2 innate lymphoid cell (ILC2) responses are exaggerated in the absence of basophils, resulting in increased inflammation and diminished lung function. Additionally, we show that ILC2s from basophil-depleted mice express reduced amounts of the receptor for the neuropeptide neuromedin B (NMB). Critically, NMB stimulation inhibited ILC2 responses from control but not basophil-depleted mice, and basophils were sufficient to directly enhance NMB receptor expression on ILC2s. These studies suggest that basophils prime ILC2s to respond to neuron-derived signals necessary to maintain tissue integrity. Further, these data provide mechanistic insight into the functions of basophils and identify NMB as a potent inhibitor of type 2 inflammation. Siracusa and colleagues reveal a regulatory role for basophils in the context of anti-helminth immunity and identify the neuropeptide neuromedin B as a potent inhibitor of type 2 inflammation.

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1 ) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1 -dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1 -floxed/ Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre . The Kiss1 -floxed/ Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1 -floxed controls. Notably, ovariectomized (OVX) Kiss1 -floxed/ Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1 -floxed/ Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1ARH) co-express Kiss1, NKB, dynorphin and glutamate and are postulated to provide an episodic, excitatory drive to gonadotropin-releasing hormone 1 (GnRH) neurons, the synaptic mechanisms of which are unknown. We characterized the cellular basis for synchronized Kiss1ARH neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1ARH neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1ARH neuronal firing. The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1ARH neurons. We propose that Kiss1ARH neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function. Puberty and fertility are necessary for survival of the species. An evolutionarily ancient region of the brain called the hypothalamus regulates these processes. The hypothalamus releases a chemical messenger called gonadotropin-releasing hormone (or GnRH for short), which is then transported from the brain to the pituitary gland. GnRH activates the pituitary gland, which in turn releases reproductive hormones that control ovulation in females and sperm production in males. For this process to work correctly in females, the hypothalamus must release GnRH in appropriately timed pulses and produce one massive release, or “surge”, of GnRH to trigger ovulation. Two populations of neurons within the hypothalamus produce a peptide molecule called Kisspeptin and drive the activity and subsequent release of GnRH. One population resides in an area called the arcuate nucleus and the other in the preoptic nucleus. Recent findings suggest that the arcuate nucleus is the “pulse generator” responsible for triggering the rhythmic release of GnRH by the hypothalamus, whereas the preoptic nucleus induces the surge of GnRH. However, how these brain regions do this remains unclear. Using a technique called optogenetics, Qiu, Nestor et al. explored whether kisspeptin-producing neurons in the arcuate nucleus are able to communicate with each other to drive pulses of GnRH release. The idea was to selectively activate a subset of kisspeptin neurons in mice and determine whether this would activate the remaining neurons at the same time. Qiu, Nestor et al. introduced a light-sensitive protein into the kisspeptin-producing neurons on one side of the arcuate nucleus, and then used light to activate those neurons. As predicted, this caused kisspeptin neurons throughout the arcuate nucleus to coordinate their activity. In addition to their namesake peptide, kisspeptin-producing neurons also make the neurotransmitter glutamate, the excitatory peptide neurokinin B, and the inhibitory peptide dynorphin. Light-induced stimulation of the arcuate nucleus caused its kisspeptin neurons to also release neurokinin B and dynorphin, which synchronized the firing of the kisspeptin neurons. The hypothalamus then translates this coordinated activity into pulses of GnRH release. The light-induced stimulation also triggered the release of glutamate, which caused kisspeptin neurons within the preoptic nucleus to fire in bursts. This in turn robustly excited the GnRH neurons, giving rise to the surge of GnRH. These findings show that peptide and classical neurotransmitters collaborate to control GnRH neuron activity and, consequently, fertility. The results obtained by Qiu, Nestor et al. can be used to further explore kisspeptin-GnRH neuronal circuits, and to obtain insights into the role of neuronal peptide signaling in healthy as well as diseased states.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state. Summary Sentence Ablation of NK3R-containing neurons in the arcuate delays puberty onset in female sheep. Graphical Abstract

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. Summary sentence Components of the KNDy system in the pig are characterized for the first time and pig-specific features such as a lack of immunopositive NK3R expression in the ARC and resistance to inhibition of kisspeptin protein expression by a progestin are identified.