The pontine Kölliker-Fuse nucleus is important for reduced postinspiratory airflow elicited by stimulation of the ventral respiratory parafacial region.

Considering that the retrotrapezoid nucleus/respiratory parafacial region (RTN/pFRG) would be an important center in the central nervous system involved in the maintenance and modulation of respiratory activity, we hypothesized that neurons in this nucleus would also be involved in the postinspiratory (post-I) phase of the respiratory cycle through a connection with the pontine Kölliker-Fuse (KF) region. Here, we performed pharmacogenetic manipulation (AAV-hM3D(Gq)-mCherry or AAV-hM4D(Gi)-mCherry) in VGlut2-cre, Ai6 conscious mice to evaluate breathing parameters through whole body plethysmography under baseline conditions (normoxia: [Formula: see text] = 0.21) or under hypercapnia or hypoxia challenges ([Formula: see text] = 0.07 or [Formula: see text] = 0.08). Under normoxia, selective stimulation of RTN/pFRG resulted in a smaller increase in V̇e (1,272 ± 102.5, vs. RTN/pFRG stimulation: 1,878 ± 122.1 mL/kg/min), due to a smaller increase in VT (5.4 ± 0.35, vs. RTN/pFRG stimulation: 7.77 ± 0.21 mL/kg) without changing fR in a condition of KF inhibition. However, inhibition of the VGlut2 neurons in the KF did affect the TE1 produced by selective activation of RTN/pFRG (119.9 ± 2.53, vs. RTN/pFRG stimulation: 104 ± 2.46 ms). Both the hypercapnia and hypoxia ventilatory response were reduced after inhibition of VGlut2-expressing KF neurons. Therefore, consistent with anatomical projections RTN/pFRG neurons regulate lung ventilation by controlling all aspects of breathing, i.e., breathing frequency, inspiration, postinspiration, and active expiration. All the modulation seems to be dependent on the integrity of the glutamatergic neurons in the KF region.NEW & NOTEWORTHY Our research reveals specific roles and interactions between the retrotrapezoid nucleus/respiratory parafacial region (RTN/pFRG) and the pontine Kölliker-Fuse (KF) region in controlling respiratory phases. RTN/pFRG neurons are key in regulating all aspects of breathing, including frequency, inspiration, postinspiration, and active expiration. This regulation depends on the functional integrity of glutamatergic neurons in the KF region, aligning with anatomical projections.

Nucleus of the solitary tract neuronal degeneration and impaired hypoxia response in a model of Parkinson's disease.

Parkinson's disease (PD) involves the degeneration of dopaminergic neurons in the substantia nigra (SNpc) and manifests with both classic and non-classic motor symptoms, including respiratory failure. Our study aims to investigate the involvement of the commissural and intermediate nucleus of the solitary tract (cNTS and iNTS) in the attenuated respiratory response to hypoxia in PD. Using a PD rat model induced by bilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum of male Wistar rats, we explored potential alterations in the population of Phox2b neurons or hypoxia-activated neurons in the NTS projecting to the retrotrapezoid nucleus (RTN). Additionally, we explored neuronal connectivity between SNpc and cNTS. Projections pathways were assessed using unilateral injection of the retrograde tracer Fluorogold (FG) in the cNTS and RTN. Neuronal activation was evaluated by analyzing fos expression in rats exposed to hypoxia. In the PD model, the ventilatory response, measured through whole-body plethysmography, was impaired at both baseline and in response to hypoxia. A reduction in Phox2b-expressing neurons or hypoxia-activated neurons projecting to the RTN was observed. Additionally, we identified an indirect pathway linking the SNpc and cNTS, which passes through the periaqueductal gray (PAG). In conclusion, our findings suggest impairment in the SNpc-PAG-cNTS pathway in the PD model, explaining the loss of Phox2b-expressing neurons or hypoxia-activated neurons in the cNTS and subsequent respiratory impairment during hypoxic stimulation. We propose that the reduced population of Phox2b-expressing neurons in the NTS may include the same neurons activated by hypoxia and projecting to the RTN.

Central and peripheral mechanisms underlying respiratory deficits in a mouse model of accelerated senescence.

Aging invariably decreases sensory and motor stimuli and affects several neuronal systems and their connectivity to key brain regions, including those involved in breathing. Nevertheless, further investigation is needed to fully comprehend the link between senescence and respiratory function. Here, we investigate whether a mouse model of accelerated senescence could develop central and peripheral respiratory abnormalities. Adult male Senescence Accelerated Mouse Prone 8 (SAMP8) and the control SAMR1 mice (10 months old) were used. Ventilatory parameters were assessed by whole-body plethysmography, and measurements of respiratory input impedance were performed. SAMP8 mice exhibited a reduction in the density of neurokinin-1 receptor immunoreactivity in the entire ventral respiratory column. Physiological experiments showed that SAMP8 mice exhibited a decreased tachypneic response to hypoxia (FiO2 = 0.08; 10 min) or hypercapnia (FiCO2 = 0.07; 10 min). Additionally, the ventilatory response to hypercapnia increased further due to higher tidal volume. Measurements of respiratory mechanics in SAMP8 mice showed decreased static compliance (Cstat), inspiratory capacity (IC), resistance (Rn), and elastance (H) at different ages (3, 6, and 10 months old). SAMP8 mice also have a decrease in contractile response to methacholine compared to SAMR1. In conclusion, our findings indicate that SAMP8 mice display a loss of the NK1-expressing neurons in the respiratory brainstem centers, along with impairments in both central and peripheral respiratory mechanisms. These observations suggest a potential impact on breathing in a senescence animal model.

The latency to awake from induced-obstructive sleep apnea is reduced in rats with chronic epilepsy.

OSA is known to increase the risk for SUDEP in persons with epilepsy, but the relationship between these two factors is not clear. Also, there is no study showing the acute responses to obstructive apnea in a chronic epilepsy model. Therefore, this study aimed to characterize cardiorespiratory responses to obstructive apnea and chemoreceptor stimulation in rats. In addition, we analyzed respiratory centers in the brain stem by immunohistochemistry. Epilepsy was induced with pilocarpine. About 30-60 days after the first spontaneous seizure, tracheal and thoracic balloons, and electrodes for recording the electroencephalogram, electromyogram, and electrocardiogram were implanted. Intermittent apneas were made by inflation of the tracheal balloon during wakefulness, NREM sleep, and REM sleep. During apnea, respiratory effort increased, and heart rate fell, especially with apneas made during wakefulness, both in control rats and rats with epilepsy. Latency to awake from apnea was longer with apneas made during REM than NREM, but rats with epilepsy awoke more rapidly than controls with apneas made during REM sleep. Rats with epilepsy also had less REM sleep. Cardiorespiratory responses to stimulation of carotid chemoreceptors with cyanide were similar in rats with epilepsy and controls. Immunohistochemical analysis of Phox2b, tryptophan hydroxylase, and NK1 in brain stem nuclei involved in breathing and sleep (retrotrapezoid nucleus, pre-Bötzinger complex, Bötzinger complex, and caudal raphe nuclei) revealed no differences between control rats and rats with epilepsy. In conclusion, our study showed that rats with epilepsy had a decrease in the latency to awaken from apneas during REM sleep, which may be related to neuroplasticity in some other brain regions related to respiratory control, awakening mechanisms, and autonomic modulation.

Interaction between Kölliker-Fuse/A7 and the parafacial respiratory region on the control of respiratory regulation.

Respiration is regulated by various types of neurons located in the pontine-medullary regions. The Kölliker-Fuse (KF)/A7 noradrenergic neurons play a role in modulating the inspiratory cycle by influencing the respiratory output. These neurons are interconnected and may also project to brainstem and spinal cord, potentially involved in regulating the post-inspiratory phase. In the present study, we hypothesize that the parafacial (pF) neurons, in conjunction with adrenergic mechanisms originating from the KF/A7 region, may provide the neurophysiological basis for breathing modulation. We conducted experiments using urethane-anesthetized, vagotomized, and artificially ventilated male Wistar rats. Injection of L-glutamate into the KF/A7 region resulted in inhibition of inspiratory activity, and a prolonged and high-amplitude genioglossal activity (GGEMG). Blockade of the α1 adrenergic receptors (α1-AR) or the ionotropic glutamatergic receptors in the pF region decrease the activity of the GGEMG without affecting inspiratory cessation. In contrast, blockade of α2-AR in the pF region extended the duration of GG activity. Notably, the inspiratory and GGEMG activities induced by KF/A7 stimulation were completely blocked by bilateral blockade of glutamatergic receptors in the Bötzinger complex (BötC). While our study found a limited role for α1 and α2 adrenergic receptors at the pF level in modulating the breathing response to KF/A7 stimulation, it became evident that BötC neurons are responsible for the respiratory effects induced by KF/A7 stimulation.

TNFR1-mediated neuroinflammation is necessary for respiratory deficits observed in 6-hydroxydopamine mouse model of Parkinsons Disease.

Parkinson's Disease (PD) is characterized by classic motor symptoms related to movement, but PD patients can experience symptoms associated with impaired autonomic function, such as respiratory disturbances. Functional respiratory deficits are known to be associated with brainstem neurodegeneration in the mice model of PD induced by 6-hydroxydopamine (6-OHDA). Understanding the causes of neuronal death is essential for identifying specific targets to prevent degeneration. Many mechanisms can explain why neurons die in PD, and neuroinflammation is one of them. To test the influence of inflammation, mediated by microglia and astrocytes cells, in the respiratory disturbances associated with brainstem neurons death, we submitted wild-type (WT) and TNF receptor 1 (TNFR1) knockout male mice to the 6-OHDA model of PD. Also, male C57BL/6 animals were induced using the same PD model and treated with minocycline (45 mg/kg), a tetracycline antibiotic with anti-inflammatory properties. We show that degeneration of brainstem areas such as the retrotrapezoid nucleus (RTN) and the pre-Botzinger Complex (preBotC) were prevented in both protocols. Notably, respiratory disturbances were no longer observed in the animals where inflammation was suppressed. Thus, the data demonstrate that inflammation is responsible for the breathing impairment in the 6-OHDA-induced PD mouse model.

Update on vascular control of central chemoreceptors.

At least four mechanisms have been proposed to elucidate how neurons in the retrotrapezoid (RTN) region sense changes in CO2 /H+ to regulate breathing (i.e., function as respiratory chemosensors). These mechanisms include: (1) intrinsic neuronal sensitivity to H+ mediated by TASK-2 and GPR4; (2) paracrine activation of RTN neurons by CO2 -responsive astrocytes (via a purinergic mechanism); (3) enhanced excitatory synaptic input or disinhibition; and (4) CO2 -induced vascular contraction. Although blood flow can influence tissue CO2 /H+ levels, there is limited understanding of how control of vascular tone in central CO2 chemosensitive regions might contribute to respiratory output. In this review, we focus on recent evidence that CO2 /H+ -induced purinergic-dependent vasoconstriction in the ventral parafacial region near RTN neurons supports respiratory chemoreception. This mechanism appears to be unique to the ventral parafacial region and opposite to other brain regions, including medullary chemosensor regions, where CO2 /H+ elicits vasodilatation. We speculate that this mechanism helps to maintain CO2 /H+ levels in the vicinity of RTN neurons, thereby maintaining the drive to breathe. Important next steps include determining whether disruption of CO2 /H+ vascular reactivity contributes to or can be targeted to improve breathing problems in disease states, such as Parkinson's disease.

Ampakine CX614 increases respiratory rate in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra compacta (SNpc). In a mouse model of PD induced by the injection of 6-hydroxydopamine (6-OHDA) into the caudate putamen (CPu) dyspnea events are very common. Neuroanatomical and functional studies show that the number of glutamatergic neurons in the pre-Bötzinger Complex (preBötC) are reduced. We hypothesize that the neuronal loss, and consequently loss of glutamatergic connections in the respiratory network previously investigated, are responsible for the breathing impairment in PD. Here, we tested whether ampakines (CX614), a subgroup of AMPA receptor positive allosteric modulators, could stimulate the respiratory activity in PD-induced animals. CX614 (50 µM) injected intraperitoneally or directly into the preBötC region reduced the irregularity pattern and increased the respiratory rate by 37% or 82%, respectively, in PD-induced animals. CX614 also increased the respiratory frequency in healthy animals. These data suggest that ampakine CX614 could become a tool to restore breathing in PD.

Autoregulation of blood flow drives early hypotension in a rat model of systemic inflammation induced by bacterial lipopolysaccharide.

Uncontrolled vasodilation is known to account for hypotension in the advanced stages of sepsis and other systemic inflammatory conditions, but the mechanisms of hypotension in earlier stages of such conditions are not clear. By monitoring hemodynamics with the highest temporal resolution in unanesthetized rats, in combination with ex-vivo assessment of vascular function, we found that early development of hypotension following injection of bacterial lipopolysaccharide is brought about by a fall in vascular resistance when arterioles are still fully responsive to vasoactive agents. This approach further uncovered that the early development of hypotension stabilized blood flow. We thus hypothesized that prioritization of the local mechanisms of blood flow regulation (tissue autoregulation) over the brain-driven mechanisms of pressure regulation (baroreflex) underscored the early development of hypotension in this model. Consistent with this hypothesis, an assessment of squared coherence and partial-directed coherence revealed that, at the onset of hypotension, the flow-pressure relationship was strengthened at frequencies (<0.2 Hz) known to be associated with autoregulation. The autoregulatory escape to phenylephrine-induced vasoconstriction, another proxy of autoregulation, was also strengthened in this phase. The competitive demand that drives prioritization of flow over pressure regulation could be edema-associated hypovolemia, as this became detectable at the onset of hypotension. Accordingly, blood transfusion aimed at preventing hypovolemia brought the autoregulation proxies back to normal and prevented the fall in vascular resistance. This novel hypothesis opens a new avenue of investigation into the mechanisms that can drive hypotension in systemic inflammation.

The Pedunculopontine Tegmental Nucleus is not Important for Breathing Impairments Observed in a Parkinson's Disease Model.

Parkinson's disease (PD) is a motor disorder resulting from degeneration of dopaminergic neurons of substantia nigra pars compacta (SNpc), with classical and non-classical symptoms such as respiratory instability. An important region for breathing control, the Pedunculopontine Tegmental Nucleus (PPTg), is composed of cholinergic, glutamatergic, and GABAergic neurons. We hypothesize that degenerated PPTg neurons in a PD model contribute to the blunted respiratory activity. Adult mice (40 males and 29 females) that express the fluorescent green protein in cholinergic, glutamatergic or GABAergic cells were used (Chat-cre Ai6, Vglut2-cre Ai6 and Vgat-cre Ai6) and received bilateral intrastriatal injections of vehicle or 6-hydroxydopamine (6-OHDA). Ten days later, the animals were exposed to hypercapnia or hypoxia to activate PPTg neurons. Vglut2-cre Ai6 animals also received retrograde tracer injections (cholera toxin b) into the retrotrapezoid nucleus (RTN) or preBötzinger Complex (preBötC) and anterograde tracer injections (AAV-mCherry) into the SNpc. In 6-OHDA-injected mice, there is a 77% reduction in the number of dopaminergic neurons in SNpc without changing the number of neurons in the PPTg. Hypercapnia activated fewer Vglut2 neurons in PD, and hypoxia did not activate PPTg neurons. PPTg neurons do not input RTN or preBötC regions but receive projections from SNpc. Although our results did not show a reduction in the number of glutamatergic neurons in PPTg, we observed a reduction in the number of neurons activated by hypercapnia in the PD animal model, suggesting that PPTg may participate in the hypercapnia ventilatory response.

Phox2b mutation mediated by Atoh1 expression impaired respiratory rhythm and ventilatory responses to hypoxia and hypercapnia.

Mutations in the transcription factor Phox2b cause congenital central hypoventilation syndrome (CCHS). The syndrome is characterized by hypoventilation and inability to regulate breathing to maintain adequate O2 and CO2 levels. The mechanism by which CCHS impact respiratory control is incompletely understood, and even less is known about the impact of the non-polyalanine repeat expansion mutations (NPARM) form. Our goal was to investigate the extent by which NPARM Phox2b mutation affect (a) respiratory rhythm; (b) ventilatory responses to hypercapnia (HCVR) and hypoxia (HVR); and (c) number of chemosensitive neurons in mice. We used a transgenic mouse line carrying a conditional Phox2bΔ8 mutation (same found in humans with NPARM CCHS). We crossed them with Atoh1cre mice to introduce mutation in regions involved with respiratory function and central chemoreflex control. Ventilation was measured by plethysmograph during neonatal and adult life. In room air, mutation in neonates and adult did not greatly impact basal ventilation. However, Phox2bΔ8, Atoh1cre increased breath irregularity in adults. The HVR and HCVR were impaired in neonates. The HVR, but not HCVR, was still partially compromised in adults. The mutation reduced the number of Phox2b+/TH--expressing neurons as well as the number of fos-activated cells within the ventral parafacial region (also named retrotrapezoid nucleus [RTN] region) induced by hypercapnia. Our data indicates that Phox2bΔ8 mutation in Atoh1-expressing cells impaired RTN neurons, as well as chemoreflex under hypoxia and hypercapnia specially early in life. This study provided new evidence for mechanisms related to NPARM form of CCHS neuropathology.

Cardiorespiratory alterations following intermittent photostimulation of RVLM C1 neurons: Implications for long-term blood pressure, breathing and sleep regulation in freely moving rats.

AIM: Sympathoexcitation and sleep-disordered breathing are common contributors for disease progression. Catecholaminergic neurons from the rostral ventrolateral medulla (RVLM-C1) modulate sympathetic outflow and have anatomical projections to respiratory neurons; however, the contribution of highly selective activation of RVLM-C1 neurons on long-term autonomic and breathing (dys)regulation remains to be understood. METHODS: To explore this relationship, a lentiviral vector carrying the light-sensitive cation channel channelrhodopsin-2 (LVV-PRSX8-ChR2-YFP) was unilaterally injected into the RVLM of healthy rats. On the contralateral side, LVV-PRSX8-ChR2-YFP was co-injected with a specific immunotoxin (DßH-SAP) targeted to eliminate C1 neurons. RESULTS: Intermittent photostimulation of RVLM-C1 in vivo, in unrestrained freely moving rats, elicited long-term facilitation of the sympathetic drive, a rise in blood pressure and sympatho-respiratory coupling. In addition, photoactivation of RVLM-C1 induced long-lasting ventilatory instability, characterized by oscillations in tidal volume and increased breathing variability, but only during non-rapid eye movement sleep. These effects were not observed when photostimulation of the RVLM was performed in the presence of DßH-SAP toxin. CONCLUSIONS: The finding that intermittent activation of RVLM-C1 neurons induces autonomic and breathing dysfunction suggest that episodic stimulation of RVLM-C1 may serve as a pathological substrate for the long-term development of cardiorespiratory disorders.

Respiratory deficits in a female rat model of Parkinson's disease.

NEW FINDINGS: What is the central question of this study? How does the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease model affect the respiratory response in female rats? What effect does ovariectomy have on that response? What is the main finding and its importance? The results suggest a protective effect of ovarian hormones in maintaining normal neuroanatomical integrity of the medullary respiratory nucleus in females. It was observed that ovariectomy alone reduced neurokinin-1 density in the pre-Bötzinger complex and Bötzinger complex, and there was an incremental effect of 6-OHDA and ovariectomy on retrotrapezoid nucleus neurons. ABSTRACT: Emerging evidence indicates that the course of Parkinson's disease (PD) includes autonomic and respiratory deficiencies in addition to the classical motor symptoms. The prevalence of PD is lower in women, and it has been hypothesized that neuroprotection by ovarian hormones can explain this difference. While male PD animal models present changes in the central respiratory control areas, as well as ventilatory parameters under normoxia and hypercapnia, little is known about sex differences regarding respiratory deficits in this disease background. This study aimed to explore the neuroanatomical and functional respiratory changes in intact and ovariectomized (OVX) female rats subjected to chemically induced PD via a bilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA). The respiratory parameters were evaluated by whole-body plethysmography, and the neuroanatomy was monitored using immunohistochemistry. It was found that dopaminergic neurons in the substantia nigra and neurokinin-1 receptor density in the rostral ventrolateral respiratory group, Bötzinger and pre-Bötzinger complex were reduced in the chemically induced PD animals. Additionally, reduced numbers of Phox2b neurons were only observed in the retrotrapezoid nucleus of PD-OVX rats. Concerning respiratory parameters, in OVX rats, the resting and hypercapnia-induced tidal volume (VT ) is reduced, and ventilation ( V ̇ E ${\dot V_{\rm{E}}}$ ) changes independently of 6-OHDA administration. Notably, there is a reduction in the number of retrotrapezoid nucleus Phox2b neurons and hypercapnia-induced respiratory changes in PD-OVX animals due to a 6-OHDA and OVX interaction. These results suggest a protective effect induced by ovarian hormones in neuroanatomical changes observed in a female experimental PD model.

Regulation of blood vessels by ATP in the ventral medullary surface in a rat model of Parkinson's disease.

Parkinson's disease (PD) patients often experience impairment of autonomic and respiratory functions. These include conditions such as orthostatic hypotension and sleep apnea, which are highly correlated with dysfunctional central chemoreception. Blood flow is a fundamental determinant of tissue CO2/H+, yet the extent to which blood flow regulation within chemoreceptor regions contributes to respiratory behavior during neurological disease remains unknown. Here, we tested the hypothesis that 6-hydroxydopamine injection to inducing a known model of PD results in dysfunctional vascular homeostasis, biochemical dysregulation, and glial morphology of the ventral medullary surface (VMS). We show that hypercapnia (FiCO2 = 10%) induced elevated VMS pial vessel constriction in PD animals through a P2-receptor dependent mechanism. Similarly, we found a greater CO2-induced vascular constriction after ARL67156 (an ectonucleotidase inhibitor) in control and PD-induced animals. In addition, we also report that weighted gene correlational network analysis of the proteomic data showed a protein expression module differentially represented between both groups. This module showed that gene ontology enrichment for components of the ATP machinery were reduced in our PD-model compared to control animals. Altogether, our data indicate that dysfunction in purinergic signaling, potentially through altered ATP bioavailability in the VMS region, may compromise the RTN neuroglial vascular unit in a PD animal model.

The effect of central growth hormone action on hypoxia ventilatory response in conscious mice.

Growth hormone (GH)-responsive neurons regulate several homeostatic behaviors including metabolism, energy balance, arousal, and stress response. Therefore, it is possible that GH-responsive neurons play a role in other responses such as CO2/H+-dependent breathing behaviors. Here, we investigated whether central GH receptor (GHR) modulates respiratory activity in conscious unrestrained mice. First, we detected clusters of GH-responsive neurons in the tyrosine hydroxylase-expressing cells in the rostroventrolateral medulla (C1 region) and within the locus coeruleus (LC). No significant expression was detected in phox2b-expressing cells in the retrotrapezoid nucleus. Whole body plethysmography revealed a reduction in the tachypneic response to hypoxia (FiO2 = 0.08) without changing baseline breathing and the hypercapnic ventilatory response. Contrary to the physiological findings, we did not find significant differences in the number of fos-activated cells in the nucleus of the solitary tract (NTS), C1, LC and paraventricular nucleus of the hypothalamus (PVH). Our finding suggests a possible secondary role of central GH action in the tachypneic response to hypoxia in conscious mice.

SOCS3 Ablation in Leptin Receptor-Expressing Cells Causes Autonomic and Cardiac Dysfunctions in Middle-Aged Mice despite Improving Energy and Glucose Metabolism.

Leptin resistance is a hallmark of obesity. Treatments aiming to improve leptin sensitivity are considered a promising therapeutical approach against obesity. However, leptin receptor (LepR) signaling also modulates several neurovegetative aspects, such as the cardiovascular system and hepatic gluconeogenesis. Thus, we investigated the long-term consequences of increased leptin sensitivity, considering the potential beneficial and deleterious effects. To generate a mouse model with increased leptin sensitivity, the suppressor of cytokine signaling 3 (SOCS3) was ablated in LepR-expressing cells (LepR∆SOCS3 mice). LepR∆SOCS3 mice displayed reduced food intake, body adiposity and weight gain, as well as improved glucose tolerance and insulin sensitivity, and were protected against aging-induced leptin resistance. Surprisingly, a very high mortality rate was observed in aging LepR∆SOCS3 mice. LepR∆SOCS3 mice showed cardiomyocyte hypertrophy, increased myocardial fibrosis and reduced cardiovascular capacity. LepR∆SOCS3 mice exhibited impaired post-ischemic cardiac functional recovery and middle-aged LepR∆SOCS3 mice showed substantial arhythmic events during the post-ischemic reperfusion period. Finally, LepR∆SOCS3 mice exhibited fasting-induced hypoglycemia and impaired counterregulatory response to glucopenia associated with reduced gluconeogenesis. In conclusion, although increased sensitivity to leptin improved the energy and glucose homeostasis of aging LepR∆SOCS3 mice, major autonomic/neurovegetative dysfunctions compromised the health and longevity of these animals. Consequently, these potentially negative aspects need to be considered in the therapies that increase leptin sensitivity chronically.

5-HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity.

A brainstem homeostatic system senses CO2 /H+ to regulate ventilation, blood gases and acid-base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO2 /H+ sensitivity of RTN neurons is mediated indirectly, by raphe-derived serotonin acting on 5-HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT-PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb+ /Phox2b+ ) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These dorsolateral Nmb+ /Htr7+ neurons tended to express Nmb at high levels and the intrinsic RTN proton detectors Gpr4 and Kcnk5 at low levels. In mouse brainstem slices, CO2 -stimulated firing in RTN neurons was mostly unaffected by a 5-HT7 receptor antagonist, SB269970 (n = 11/13). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co-injected LP-44, a 5-HT7 receptor agonist, but had no effect on CO2 -stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5-HT7 receptors have negligible effects on CO2 -evoked firing activity in RTN neurons or on CO2 -stimulated breathing in mice. KEY POINTS: Neurons of the retrotrapezoid nucleus (RTN) are intrinsic CO2 /H+ chemosensors and serve as an integrative excitatory hub for control of breathing. Serotonin can activate RTN neurons, in part via 5-HT7 receptors, and those effects have been implicated in conferring an indirect CO2  sensitivity. Multiple single cell molecular approaches revealed low levels of 5-HT7 receptor transcript expression restricted to a limited population of RTN neurons. Pharmacological experiments showed that 5-HT7 receptors in RTN are not required for CO2 /H+ -stimulation of RTN neuronal activity or CO2 -stimulated breathing. These data do not support a role for 5-HT7 receptors in respiratory chemosensitivity mediated by RTN neurons.