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The nucleus tractus solitarii (NTS) is one of the morphologically and functionally defined centers that engage in the autonomic regulation of cardiovascular activity. Phenotypically-characterized NTS neurons have been implicated in the differential regulation of blood pressure (BP). Here, we investigated whether phenylethanolamine N-methyltransferase (PNMT)-expressing NTS (NTSPNMT) neurons contribute to the control of BP. We demonstrate that photostimulation of NTSPNMT neurons has variable effects on BP. A depressor response was produced during optogenetic stimulation of NTSPNMT neurons projecting to the paraventricular nucleus of the hypothalamus, lateral parabrachial nucleus, and caudal ventrolateral medulla. Conversely, photostimulation of NTSPNMT neurons projecting to the rostral ventrolateral medulla produced a robust pressor response and bradycardia. In addition, genetic ablation of both NTSPNMT neurons and those projecting to the rostral ventrolateral medulla impaired the arterial baroreflex. Overall, we revealed the neuronal phenotype- and circuit-specific mechanisms underlying the contribution of NTSPNMT neurons to the regulation of BP.

The locus coeruleus (LC) has been implicated in the control of breathing. Congenital central hypoventilation syndrome results from mutation of the paired-like homeobox 2b (Phox2b) gene that is expressed in LC neurons. The present study was designed to address whether stimulation of Phox2b-expressing LC (Phox2b(LC)) neurons affects breathing and to reveal the putative circuit mechanism. A Cre-dependent viral vector encoding a Gq-coupled human M3 muscarinic receptor (hM3Dq) was delivered into the LC of Phox2b-Cre mice. The hM3Dq-transduced neurons were pharmacologically activated while respiratory function was measured by plethysmography. We demonstrated that selective stimulation of Phox2b(LC) neurons significantly increased basal ventilation in conscious mice. Genetic ablation of these neurons markedly impaired hypercapnic ventilatory responses. Moreover, stimulation of Phox2b(LC) neurons enhanced the activity of preBötzinger complex neurons. Finally, axons of Phox2b(LC) neurons projected to the preBötzinger complex. Collectively, Phox2b(LC) neurons contribute to the control of breathing most likely via an LC–preBötzinger complex circuit.

The nucleus tractus solitarii (NTS) is implicated in the control of breathing, but the neuronal phenotype and circuit mechanism involved in such a physiological function remain incompletely understood. This study focused on the respiratory role of paired-like homeobox 2b gene (Phox2b)-expressing NTS neurons and sought to determine whether selective stimulation of this set of neurons activates breathing in male mice. A Cre-dependent vector encoding a Gq-coupled human M3 muscarinic receptor (hM3Dq) was microinjected into the NTS of Phox2b-Cre transgenic mice. The hM3Dq-transduced neurons were pharmacologically activated in conscious mice while respiratory effects were measured by plethysmography. We demonstrate that chemogenetic stimulation of Phox2b-expressing NTS neurons significantly increased baseline minute volume via an increase in respiratory frequency rather than tidal volume. Chemogenetic stimulation also synergized with moderate CO2stimulation to enhance pulmonary ventilatory response. Selective ablation of Phox2b-expressing NTS neurons notably attenuated a hypercapnic ventilatory response. Moreover, histological evidence revealed that stimulation of Phox2b-expressing NTS neurons increased neuronal activity of the preBötzinger complex. Finally, we presented the neuroanatomical evidence of direct projection of Phox2b-expressing NTS neurons to putative respiratory central pattern generator. Overall, these findings suggest that selective activation of Phox2b-expressing NTS neurons potentiates baseline pulmonary ventilation via an excitatory drive to respiratory central pattern generator and this group of neurons is also required for the hypercapnic ventilatory response.SIGNIFICANCE STATEMENTThe nucleus tractus solitarii (NTS) has been implicated in the control of breathing. The paired-like homeobox 2b gene (Phox2b) is the disease-defining gene for congenital central hypoventilation syndrome and is restrictively present in brainstem nucleus, including the NTS. Using a chemogenetic approach, we demonstrate herein that selective stimulation of Phox2b-expressing NTS neurons vigorously potentiates baseline pulmonary ventilation via an excitatory drive to respiratory central pattern generator in rodents. Genetic ablation of these neurons attenuates the hypercapnic ventilatory response. We also suggest that a fraction of Phox2b-expressing neurons exhibit CO2sensitivity and presumably function as central respiratory chemoreceptors. The methodology is expected to provide a future applicability to the patients with sleep-related hypoventilation or apnea.

Leptin, an adipocyte-derived peptide hormone, has been shown to facilitate breathing. However, the central sites and circuit mechanisms underlying the respiratory effects of leptin remain incompletely understood. The present study aimed to address whether neurons expressing leptin receptor b (LepRb) in the nucleus tractus solitarii (NTS) contribute to respiratory control. Both chemogenetic and optogenetic stimulation of LepRb-expressing NTS (NTS(LepRb)) neurons notably activated breathing. Moreover, stimulation of NTS(LepRb) neurons projecting to the lateral parabrachial nucleus (LPBN) not only remarkably increased basal ventilation to a level similar to that of the stimulation of all NTS(LepRb) neurons, but also activated LPBN neurons projecting to the preBötzinger complex (preBötC). By contrast, ablation of NTS(LepRb) neurons projecting to the LPBN notably eliminated the enhanced respiratory effect induced by NTS(LepRb) neuron stimulation. In brainstem slices, bath application of leptin rapidly depolarized the membrane potential, increased the spontaneous firing rate, and accelerated the Ca(2+) transients in most NTS(LepRb) neurons. Therefore, leptin potentiates breathing in the NTS most likely via an NTS–LPBN–preBötC circuit.

Reproduction is an energetically costly phenomenon. Therefore, to optimize reproductive success, male birds invest enough energetic resources for maintaining well-developed testes. The hypothalamic-pituitary-thyroid (HPT) axis in birds can crosstalk with the hypothalamic-pituitary-gonadal (HPG) axis, thus orchestrating both the reproduction and metabolism. However, until now, how the free-living birds timely optimize both the energy metabolism and reproduction via HPT-axis is not understood. To uncover this physiological mechanism, we investigated the relationships among body mass, testis size, plasma hormones including thyroid-stimulating hormone (TSH), thyroxine (T4), triiodothyronine (T3), metabolites including glucose (Glu), triglyceride (TG), total cholesterol (TC), uric acid (UA), diencephalon mRNA expressions of type 2 (Dio2) and 3 (Dio3) iodothyronine deiodinase enzymes, thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH), gonadotropin-releasing hormone I (GnRH-I), and gonadotropin-inhibitory hormone (GnIH) in a male Eurasian tree sparrow (ETS, Passer montanus). We found significantly larger testis size; elevated diencephalon Dio2 and TRH mRNA expressions, plasma T3, and UA levels; and significantly lowered Glu, TG, and TC levels during mating relative to the non-mating stages in male ETSs. However, Dio3, TSH, GnRH-I, and GnIH mRNA expression did not vary with the stage. Furthermore, life-history stage dependent variation in plasma T3 had both direct effects on the available energy substrates and indirect effects on body mass and testis size, indicating a complex regulation of metabolic pathways through the HPT- and HPG-axes. The identified differences and relationships in mRNA expression, plasma T3 and metabolites, and testis size in male ETSs contribute to our understanding how free-living birds adjust their molecular, endocrinal, and biochemical features to orchestrate their reproductive physiology and metabolism for the maintenance of well-developed testes.

Sleep-disordered breathing is characterized by disruptions of normal breathing patterns during sleep. Obesity is closely related to hypoventilation or apnea and becomes a primary risk factor for sleep-disordered breathing. Leptin, a peptide secreted by adipose tissue, has been implicated in central control of breathing. Activation of the retrotrapezoid nucleus (RTN) neurons, a critical central respiratory chemoreceptor candidate, potentiates a central drive to breathing. Here, we ask whether the disordered leptin signaling in the RTN is responsible for obesity-related hypoventilation. In a diet induced obesity (DIO) mouse model, the hypercapnic ventilatory response (HCVR) was assessed and the cellular leptin signaling in the RTN was examined. Our main findings demonstrate that DIO mice exhibit overweight, hypercapnia, high levels of serum and cerebrospinal leptin. During exposure to room air, DIO mice manifest basal hypoventilation with a rapid and shallow breathing pattern. Exposure to CO2 elicits the impaired HCVR in DIO mice. In addition, both the number of CO2-activated neurons and expression of TASK-2 channels in the RTN are dramatically reduced in DIO mice. Moreover, there is leptin signaling disorder in RTN neurons in DIO mice, including a significant decrease in leptin-activated RTN neurons, downregulation of phosphorylated STAT3 and upregulation of SOCS3. Altogether, we suggest that the disordered leptin/STAT3/SOCS3 signaling pathway in the RTN plays a role in obesity-related hypoventilation.

In vertebrates, myoglobin (Mb) and cytoglobin (Cygb) are closest relatives in the family of globins, which are heme-containing proteins that can bind gaseous molecules. Mb acts not only as an O2 transporter but also a nitric oxide (NO) scavenger in cardiac and striated muscle. Cygb has been suggested to play important functions in lipid-based signaling processes, defense against reactive oxygen species (ROS), and nitric oxide (NO) metabolism, and it is present in a variety of cell types. However, little information about the structures and functions of Mb and Cygb is known in birds. Here, we cloned the full-length open reading frames (ORFs) of the two globins in Eurasian Tree Sparrow (Passer montanus). The Mb ORF cDNA contains 465 base pairs (bp) encoding 154 amino acids (aa), and the Cygb ORF cDNA contains 540 bp encoding 179 aa. Our results show that the amino acid sequences and three-dimensional (3D) structures of Mb and Cygb are highly conserved in vertebrate species. Interestingly, two specific substitutions were detected in Cygb compared with other vertebrates, which resulted in slight variation of the 3D conformation (e.g., distance between Tyr H16 and Lys G8, the strength of hydrogen bonds, and angles between the G–H helices). Our results may contribute to further understanding the structures, properties, and functions of Mb and Cygb as well as the potential mechanisms of oxygen utilization pathways in vertebrates.

Life-history theory assumes that the fitness costs of immunity may have negative effects on reproductive success. Similarly, the immunocompetence handicap hypothesis is based on findings that testosterone (T) has immunosuppressive effects, although the basis of this hypothesis has recently been challenged. As much of the work examining the relationship between T levels and immune function has been carried out in captive-housed species, these results may not accurately reflect the situation of animals living in natural environments. To better understand the relationship between plasma T levels and immune function, studies focusing on free-living animals are needed. A previous study by our group determined the changes in both baseline and stress-induced T levels in free-living Eurasian Tree Sparrows (Passer montanus) across different annual cycle stages. In this study, we further report the phytohaemagglutinin skin-swelling (PHA) immune response in this multi-brooded species during different breeding sub-stages, and then determine the relationships between the PHA response and both baseline and stress-induced T levels. Our results show that the PHA response varied across the different sub-stages and differed significantly between the first and second brood stage. Furthermore, T levels in male Tree Sparrows are positively correlated with the PHA response during the breeding season, whereas this relationship is negative in females, suggesting that the biological function of T differs between the sexes. Therefore, our results suggest that free-living animals have evolved the ability to orchestrate trade-offs between reproduction and immune functions based on changes in physiology and the environment, which should provide further opportunities to study the flexibility and plasticity of physiological and ecological adaptations in natural environments.

Glucocorticoids mediate various essential biological functions by binding to and activating their specific receptors. In vertebrates, interactions between glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) play an important role in the feedback pathways of glucocorticoid regulation. In free-living birds, a number of studies have demonstrated seasonal regulation of the adrenocortical response to stress, but little is known about their downstream receptors, GR and MR. In this study, we successfully cloned the full-length open reading frames (ORFs) of the GR (eGR) and the MR (eMR) cDNAs from a passerine species, the Eurasian Tree Sparrow Passer montanus. The ORFs eGR contains 2,322 base pairs encoding 773 amino acids, and the eMR contains 2,952 base pairs encoding 983 amino acids. The eGR and eMR are highly conserved in their gene and amino acid sequences and in their secondary and 3D structures when compared with other species of birds and vertebrates.