Mapping of afferent and efferent connections of phenylethanolamine N-methyltransferase-expressing neurons in the nucleus tractus solitarii.

OBJECTIVE: Phenylethanolamine N-methyltransferase (PNMT)-expressing neurons in the nucleus tractus solitarii (NTS) contribute to the regulation of autonomic functions. However, the neural circuits linking these neurons to other brain regions remain unclear. This study aims to investigate the connectivity mechanisms of the PNMT-expressing neurons in the NTS (NTSPNMT neurons). METHODS: The methodologies employed in this study included a modified rabies virus-based retrograde neural tracing technique, conventional viral anterograde tracing, and immunohistochemical staining procedures. RESULTS: A total of 43 upstream nuclei projecting to NTSPNMT neurons were identified, spanning several key brain regions including the medulla oblongata, pons, midbrain, cerebellum, diencephalon, and telencephalon. Notably, dense projections to the NTSPNMT neurons were observed from the central amygdaloid nucleus, paraventricular nucleus of the hypothalamus, area postrema, and the gigantocellular reticular nucleus. In contrast, the ventrolateral medulla, lateral parabrachial nucleus, and lateral hypothalamic area were identified as the primary destinations for axon terminals originating from NTSPNMT neurons. Additionally, reciprocal projections were evident among 21 nuclei, primarily situated within the medulla oblongata. CONCLUSION: Our research findings demonstrate that NTSPNMT neurons form extensive connections with numerous nuclei, emphasizing their essential role in the homeostatic regulation of vital autonomic functions.

Acid-sensing ion channel 1 in nucleus tractus solitarii neurons contributes to the enhanced CO2-stimulated cardiorespiratory effect in spontaneously hypertensive rats.

AIMS: Activation of central respiratory chemoreceptors provides excitatory drive to both respiratory and sympathetic outputs. The enhanced respiratory-sympathetic coupling contributes to the onset and development of hypertension. However, the specific central targets and molecular mechanisms involved in this process remain elusive. This study aimed to investigate the role of acid-sensing ion channel 1 (ASIC1) in nucleus tractus solitarii (NTS) neurons in CO2-stimulated cardiorespiratory effects in spontaneously hypertensive rats (SHRs). MAIN METHODS: Respiration and blood pressure of conscious rats were recorded by whole-body plethysmography and telemetry, respectively. Western blot was used to detect the expression difference of ASIC1 protein in NTS region between Wistar-Kyoto (WKY) rats and SHRs. Excitability of NTS neurons were assessed by extracellular recordings. KEY FINDINGS: Compared to WKY rats, the enhanced CO2-stimulated cardiopulmonary effect and up-regulation of ASIC1 in the NTS were already observed in 4-week-old prehypertensive SHRs. Furthermore, specific blockade of ASIC1 effectively attenuated the CO2-stimulated increase in firing rate of NTS neurons in anesthetized adult SHRs. Intracerebroventricular injections of the ASIC1a blocker PcTx1 or knockdown Asic1 in NTS neurons significantly reduced the heightened CO2-stimulated ventilatory response, and diminished the CO2-stimulated increase in arterial pressure and heart rate in adult SHRs. SIGNIFICANCE: These findings showed that dysregulated ASIC1 signaling in the NTS contribute to the exaggerated CO2-stimulated cardiorespiratory effects observed in SHRs.

Microbiota-derived acetate attenuates neuroinflammation in rostral ventrolateral medulla of spontaneously hypertensive rats.

BACKGROUND: Increased neuroinflammation in brain regions regulating sympathetic nerves is associated with hypertension. Emerging evidence from both human and animal studies suggests a link between hypertension and gut microbiota, as well as microbiota-derived metabolites short-chain fatty acids (SCFAs). However, the precise mechanisms underlying this gut-brain axis remain unclear. METHODS: The levels of microbiota-derived SCFAs in spontaneously hypertensive rats (SHRs) were determined by gas chromatography-mass spectrometry. To observe the effect of acetate on arterial blood pressure (ABP) in rats, sodium acetate was supplemented via drinking water for continuous 7 days. ABP was recorded by radio telemetry. The inflammatory factors, morphology of microglia and astrocytes in rostral ventrolateral medulla (RVLM) were detected. In addition, blood-brain barrier (BBB) permeability, composition and metabolomics of the gut microbiome, and intestinal pathological manifestations were also measured. RESULTS: The serum acetate levels in SHRs are lower than in normotensive control rats. Supplementation with acetate reduces ABP, inhibits sympathetic nerve activity in SHRs. Furthermore, acetate suppresses RVLM neuroinflammation in SHRs, increases microglia and astrocyte morphologic complexity, decreases BBB permeability, modulates intestinal flora, increases fecal flora metabolites, and inhibits intestinal fibrosis. CONCLUSIONS: Microbiota-derived acetate exerts antihypertensive effects by modulating microglia and astrocytes and inhibiting neuroinflammation and sympathetic output.

Inhibition of SOCS3 signaling in the nucleus tractus solitarii and retrotrapezoid nucleus alleviates hypoventilation in diet-induced obese male mice.

The central leptin signaling system has been found to facilitate breathing and is linked to obesity-related hypoventilation. Activation of leptin signaling in the nucleus tractus solitarii (NTS) and retrotrapezoid nucleus (RTN) enhances respiratory drive. In this study, we investigated how medullary leptin signaling contributes to hypoventilation and whether respective deletion of SOCS3 in the NTS and RTN could mitigate hypoventilation in diet-induced obesity (DIO) male mice. Our findings revealed a decrease in the number of CO2-activated NTS neurons and downregulation of acid-sensing ion channels in DIO mice compared to lean control mice. Moreover, NTS leptin signaling was disrupted, as evidenced by the downregulation of phosphorylated STAT3 and the upregulation of SOCS3 in DIO mice. Importantly, deleting SOCS3 in the NTS and RTN significantly improved the diminished hypercapnic ventilatory response in DIO mice. In conclusion, our study suggests that disrupted medullary leptin signaling contributes to obesity-related hypoventilation, and inhibiting the upregulated SOCS3 in the NTS and RTN can alleviate this condition.

Whole-brain monosynaptic inputs and outputs of leptin receptor b neurons of the nucleus tractus solitarii in mice.

The nucleus tractus solitarii (NTS) is the primary central station that integrates visceral afferent information and regulates respiratory, gastrointestinal, cardiovascular, and other physiological functions. Leptin receptor b (LepRb)-expressing neurons of the NTS (NTSLepRb neurons) are implicated in central respiration regulation, respiratory facilitation, and respiratory drive enhancement. Furthermore, LepRb dysfunction is involved in obesity, insulin resistance, and sleep-disordered breathing. However, the monosynaptic inputs and outputs of NTSLepRb neurons in whole-brain mapping remain to be elucidated. Therefore, the exploration of its whole-brain connection system may provide strong support for comprehensively understanding the physiological and pathological functions of NTSLepRb neurons. In the present study, we used a cell type-specific, modified rabies virus and adeno-associated virus with the Cre-loxp system to map monosynaptic inputs and outputs of NTSLepRb neurons in LepRb-Cre mice. The results showed that NTSLepRb neurons received inputs from 48 nuclei in the whole brain from five brain regions, including especially the medulla. We found that NTSLepRb neurons received inputs from nuclei associated with respiration, such as the pre-Bötzinger complex, ambiguus nucleus, and parabrachial nucleus. Interestingly, some brain areas related to cardiovascular regulation-i.e., the ventrolateral periaqueductal gray and locus coeruleus-also sent a small number of inputs to NTSLepRb neurons. In addition, anterograde tracing results demonstrated that NTSLepRb neurons sent efferent projections to 15 nuclei, including the dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus, which are involved in regulation of energy metabolism and feeding behaviors. Quantitative statistical analysis revealed that the inputs of the whole brain to NTSLepRb neurons were significantly greater than the outputs. Our study comprehensively revealed neuronal connections of NTSLepRb neurons in the whole brain and provided a neuroanatomical basis for further research on physiological and pathological functions of NTSLepRb neurons.

Circuit-Specific Control of Blood Pressure by PNMT-Expressing Nucleus Tractus Solitarii Neurons.

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.

A Neural Circuit Mechanism Controlling Breathing by Leptin in the Nucleus Tractus Solitarii.

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 (NTSLepRb) neurons notably activated breathing. Moreover, stimulation of NTSLepRb 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 NTSLepRb neurons, but also activated LPBN neurons projecting to the preBötzinger complex (preBötC). By contrast, ablation of NTSLepRb neurons projecting to the LPBN notably eliminated the enhanced respiratory effect induced by NTSLepRb neuron stimulation. In brainstem slices, bath application of leptin rapidly depolarized the membrane potential, increased the spontaneous firing rate, and accelerated the Ca2+ transients in most NTSLepRb neurons. Therefore, leptin potentiates breathing in the NTS most likely via an NTS-LPBN-preBötC circuit.

Respiratory Control by Phox2b-expressing Neurons in a Locus Coeruleus-preBötzinger Complex Circuit.

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 (Phox2bLC) 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 Phox2bLC neurons significantly increased basal ventilation in conscious mice. Genetic ablation of these neurons markedly impaired hypercapnic ventilatory responses. Moreover, stimulation of Phox2bLC neurons enhanced the activity of preBötzinger complex neurons. Finally, axons of Phox2bLC neurons projected to the preBötzinger complex. Collectively, Phox2bLC neurons contribute to the control of breathing most likely via an LC-preBötzinger complex circuit.

Hypothalamic-Pituitary-Thyroid Axis Crosstalk With the Hypothalamic-Pituitary-Gonadal Axis and Metabolic Regulation in the Eurasian Tree Sparrow During Mating and Non-mating Periods.

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.

Disordered Leptin signaling in the retrotrapezoid nucleus is associated with the impaired hypercapnic ventilatory response in obesity.

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.

Activation of Phox2b-Expressing Neurons in the Nucleus Tractus Solitarii Drives Breathing in Mice.

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 CO2 stimulation 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 STATEMENT The 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 CO2 sensitivity 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.