Central Respiratory Chemoreceptors Research Articles

Genetically-targeted Recordings from Respiratory Chemoreceptor Neurons in the Retrotrapezoid Nucleus of Freely Behaving Mice by Fiber Photometry

Central respiratory chemoreceptors regulate breathing homeostasis in relation to arterial pH/PCO2. Neurons located in the retrotrapezoid nucleus (RTN) are considered to function as central respiratory chemoreceptors and/or to be part of the neural network that drives the central chemoreflex. RTN neurons are sensitive to changes in pH in vitro under synaptic blockade and in anesthetized preparations with artificial ventilation. However, there is limited information about their activity in vivo in non-anesthetized conditions. Recent studies have shown that Nmb-expressing neurons in the RTN have acid-sensitive properties and are necessary for the respiratory response to CO2. In this study, we hypothesized that RTN- Nmb neurons are activated during hypercapnia in freely behaving condition. To test this hypothesis, we measured the dynamic activity of RTN neurons using a genetically encoded calcium indicator (GCaMP7s) via fiber photometry in non-anesthetized mice. Expression of GCaMP7s was genetically targeted to RTN neurons by microinjection of a Cre-dependent viral vector (AAV1-Syn-Flex-jGCAMP7s) in transgenic Nmb-Cre mice. One month later, the animals were placed in a plethysmography chamber to simultaneously record breathing and GCaMP7s fluorescence from RTN- Nmb neurons in freely behaving conditions. During hypercapnia, RTN neurons were markedly activated, and the effect size was graded to FICO2 (DF/F0 from baseline: 1.9 ± 1.0 % at 0.03, 8.0 ± 4.0% at 0.06 and 12.0 ± 5.0% at 0.09, n=6), and significantly different for each level of CO2 (RM One-way ANOVA, p=0.0012). Importantly, the increase in RTN neuron activity correlates with changes in the FICO2 (p<0.0001, R2=0.97) and respiratory frequency (p<0.0001, R2=0.85). This study shows that RTN neurons are potently activated by hypercapnia in freely behaving conditions, and the response of RTN neurons to CO2 aligns with changes in breathing. These results are consistent with the concept that RTN neurons monitor arterial PCO2/pH and are essential for the hypercapnic ventilatory response. This study was supported by National Institutes of Health (HL148004 ) to Stephen BG Abbott. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Galaninergic and hypercapnia-activated neuronal projections to the ventral respiratory column

In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate respiratory motor patterns. VRC microinjection of the neuropeptide galanin has been reported to dampen carbon dioxide (CO2)-mediated chemoreflex responses. Additionally, we previously demonstrated that galaninergic neurons in the retrotrapezoid nucleus (RTN) are implicated in the adaptive response to hypercapnic stimuli, suggesting a link between RTN neuroplasticity and increased neuronal drive to the VRC. VRC neurons express galanin receptor 1, suggesting potential regulatory action by galanin, however, the precise galaninergic chemoreceptor-VRC circuitry remains to be determined. This study aimed to identify sources of galaninergic input to the VRC that contribute to central respiratory chemoreception. We employed a combination of retrograde neuronal tracing, in situ hybridisation and immunohistochemistry to investigate VRC-projecting neurons that synthesise galanin mRNA. In an additional series of experiments, we used acute hypercapnia exposure (10% CO2, 1 h) and c-Fos immunohistochemistry to ascertain which galaninergic nuclei projecting to the VRC are activated. Our findings reveal that a total of 30 brain nuclei and 51 subnuclei project to the VRC, with 12 of these containing galaninergic neurons, including the RTN. Among these galaninergic populations, only a subset of the RTN neurons (approximately 55%) exhibited activation in response to acute hypercapnia. Our findings highlight that the RTN is the likely source of galaninergic transmission to the VRC in response to hypercapnic stimuli.

Control of breathing by orexinergic signaling in the nucleus tractus solitarii

Orexin signaling plays a facilitatory role in respiration. Abnormalities in orexin levels correlate with disordered breathing patterns and impaired central respiratory chemoreception. Nucleus tractus solitarii (NTS) neurons expressing the transcription factor Phox2b contribute to the chemoreceptive regulation of respiration. However, the extent to which orexinergic signaling modulates respiratory activity in these Phox2b-expressing NTS neurons remains unclear. In the present study, the injection of orexin A into the NTS significantly increased the firing rate of the phrenic nerve. Further analysis using fluorescence in situ hybridization and immunohistochemistry revealed that orexin 1 receptors (OX1Rs) were primarily located in the ventrolateral subdivision of the NTS and expressed in 25% of Phox2b-expressing neurons. Additionally, electrophysiological recordings showed that exposure to orexin A increased the spontaneous firing rate of Phox2b-expressing neurons. Immunostaining experiments with cFos revealed that the OX1R-residing Phox2b-expressing neurons were activated by an 8% CO2 stimulus. Crucially, OX1R knockdown in these NTS neurons notably blunted the ventilatory response to 8% CO2, alongside an increase in sigh-related apneas. In conclusion, orexinergic signaling in the NTS facilitates breathing through the activation of OX1Rs, which induces the depolarization of Phox2b-expressing neurons. OX1Rs are essential for the involvement of Phox2b-expressing NTS neurons in the hypercapnic ventilatory response.

Loss-of-function of chemoreceptor neurons in the retrotrapezoid nucleus: What have we learned from it?

Central respiratory chemoreceptors are cells in the brain that regulate breathing in relation to arterial pH and PCO2. Neurons located at the retrotrapezoid nucleus (RTN) have been hypothesized to be central chemoreceptors and/or to be part of the neural network that drives the central respiratory chemoreflex. The inhibition or ablation of RTN chemoreceptor neurons has offered important insights into the role of these cells on central respiratory chemoreception and the neural control of breathing over almost 60 years since the original identification of acid-sensitive properties of this ventral medullary site. Here, we discuss the current definition of chemoreceptor neurons in the RTN and describe how this definition has evolved over time. We then summarize the results of studies that use loss-of-function approaches to evaluate the effects of disrupting the function of RTN neurons on respiration. These studies offer evidence that RTN neurons are indispensable for the central respiratory chemoreflex in mammals and exert a tonic drive to breathe at rest. Moreover, RTN has an interdependent relationship with oxygen sensing mechanisms for the maintenance of the neural drive to breathe and blood gas homeostasis. Collectively, RTN neurons are a genetically-defined group of putative central respiratory chemoreceptors that generate CO2-dependent drive that supports eupneic breathing and stimulates the hypercapnic ventilatory reflex.

Criteria for central respiratory chemoreceptors: experimental evidence supporting current candidate cell groups.

An interoceptive homeostatic system monitors levels of CO2/H+ and provides a proportionate drive to respiratory control networks that adjust lung ventilation to maintain physiologically appropriate levels of CO2 and rapidly regulate tissue acid-base balance. It has long been suspected that the sensory cells responsible for the major CNS contribution to this so-called respiratory CO2/H+ chemoreception are located in the brainstem-but there is still substantial debate in the field as to which specific cells subserve the sensory function. Indeed, at the present time, several cell types have been championed as potential respiratory chemoreceptors, including neurons and astrocytes. In this review, we advance a set of criteria that are necessary and sufficient for definitive acceptance of any cell type as a respiratory chemoreceptor. We examine the extant evidence supporting consideration of the different putative chemoreceptor candidate cell types in the context of these criteria and also note for each where the criteria have not yet been fulfilled. By enumerating these specific criteria we hope to provide a useful heuristic that can be employed both to evaluate the various existing respiratory chemoreceptor candidates, and also to focus effort on specific experimental tests that can satisfy the remaining requirements for definitive acceptance.

Efferent network of Neuromedin B neurons in the retrotrapezoid nucleus in mice

In the parafacial region of the medulla, the peptide Neuromedin B (NMB) is expressed in retrotrapezoid nucleus (RTN) neurons that are proposed to be central respiratory chemoreceptors. The efferent network of RTN Nmb neurons has been established in rats, but has not been examined in mice in detail. To map the downstream connections of RTN Nmb neurons we performed genetically-targeted tracing using novel Nmb-Cre knock-in mice. Adult Nmb-Cre mice were injected in the RTN unilaterally with a Cre-dependent AAV producing expression of red fluorescent protein (AAV-Cre ON -mScarlet). Expression of Nmb was identified by fluorescent in situ hybridization (FISH). Injections of AAV-Cre ON -mScarlet in the RTN (N=3) transduced 58 ± 11 % of Nmb-expressing neurons with a selectivity for Nmb neurons of 98 ± 1 %. Intensely labelled axons and terminals were observed in regions overlapping with the medullary and pontine respiratory network. We observe an intra-RTN network of fibers with close appositions between RTN Nmb neurons, as well as moderate projections to areas dorso-medial to the facial nucleus and medially adjacent to the locus coeruleus. There was a modest contralateral projection to the medullary respiratory network, but none to the pontine respiratory network. No labelled fibers were found rostral of the parabrachial nucleus. Anterograde tracing of RTN Nmb neurons was complemented using retrograde transport of Cre-recombinase (AAVrg-Cre) in R26-loxSTOPlox-L10-GFP mice (eGFP-reporter) mice combined with labelling of transcript for Nmb by FISH. Small volumes (3-6 nL) of AAVrg-Cre were unilaterally injected in the Pre-Bötzinger complex (PreBöt), lateral parabrachial nucleus (LPB), caudal ventral respiratory group (cVRG), and the nucleus of the solitary tract (NTS). We determined the proportion of total RTN Nmb neurons that were retro-labelled from each target (PreBöt- 75 ± 3%, n=3; LPB- 65 ± 3%, n=4; cVRG- 54 ± 6%, n=3; NTS- 38 ± 3%, n=3). This result suggests that RTN neurons have a high degree of collateralization within the medullary and pontine respiratory network. Injections of AAVrg-Cre in the PreBöt labelled 16 ± 11% of RTN Nmb neurons contralateral to the injection site. Collectively, these neuroanatomical observations show that the efferent network of RTN Nmb neurons broadly innervate and selectively the central respiratory network in alignment with prior descriptions in rat. Furthermore, we provide evidence for novel connectivity between RTN Nmb neurons. This anatomy supports the idea that RTN neurons generate a coordinated widespread excitation of the respiratory rhythm and pattern generating network in order to promote alveolar ventilation. Funding from NIH-HLBI #HL148004 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Steady-state chemoreflex drive captures ventilatory acclimatization during incremental ascent to high altitude: Effect of acetazolamide.

Ventilatory acclimatization (VA) is important to maintain adequate oxygenation with ascent to high altitude (HA). Transient hypoxic ventilatory response tests lack feasibility and fail to capture the integrated steady-state responses to chronic hypoxic exposure in HA fieldwork. We recently characterized a novel index of steady-state respiratory chemoreflex drive (SSCD), accounting for integrated contributions from central and peripheral respiratory chemoreceptors during steady-state breathing at prevailing chemostimuli. Acetazolamide is often utilized during ascent for prevention or treatment of altitude-related illnesses, eliciting metabolic acidosis and stimulating respiratory chemoreceptors. To determine if SSCD reflects VA during ascent to HA, we characterized SSCD in 25 lowlanders during incremental ascent to 4240 m over 7 days. We subsequently compared two separate subgroups: no acetazolamide (NAz; n= 14) and those taking an oral prophylactic dose of acetazolamide (Az; 125 mg BID; n= 11). At 1130/1400 m (day zero) and 4240 m (day seven), steady-state measurements of resting ventilation (V̇I ; L/min), pressure of end-tidal (PET )CO2 (Torr), and peripheral oxygen saturation (SpO2 ; %) were measured. A stimulus index (SI; PET CO2 /SpO2 ) was calculated, and SSCD was calculated by indexing V̇I against SI. We found that (a) both V̇I and SSCD increased with ascent to 4240 m (day seven; V̇I : +39%, p< 0.0001, Hedges' g= 1.52; SSCD: +56.%, p< 0.0001, Hedges' g= 1.65), (b) and these responses were larger in the Az versus NAz subgroup (V̇I : p= 0.02, Hedges' g= 1.04; SSCD: p= 0.02, Hedges' g= 1.05). The SSCD metric may have utility in assessing VA during prolonged stays at altitude, providing a feasible alternative to transient chemoreflex tests.

Assessing central and peripheral respiratory chemoreceptor interaction in humans.

What is the central question of this study? We investigated the interaction between central and peripheral respiratory chemoreceptors in healthy, awake human participants by using a background of step increases in steady-state normoxic fraction of inspired carbon dioxide to alter central chemoreceptor activation and by using the transient hypoxia test to target the peripheral chemoreceptors. What is the main finding and its importance? Our data suggest that the interaction between central and peripheral respiratory chemoreceptors is additive in minute ventilation and respiratory rate, but hypo-additive in tidal volume. Our study adds important new data in reconciling chemoreceptor interaction in awake healthy humans and is consistent with previous reports of simple addition in intact rodents and humans. Arterial blood gas levels are maintained through respiratory chemoreflexes, mediated by central chemoreceptors in the CNS and peripheral chemoreceptors located in the carotid bodies. The interaction between central and peripheral chemoreceptors is controversial, and few studies have investigated this interaction in awake, healthy humans, owing, in part, to methodological challenges. We investigated the interaction between the central and peripheral chemoreceptors in healthy humans using a transient hypoxia test (three consecutive breaths of 100% N2 ; TT-HVR), which targets the temporal domain and stimulus specificity of the peripheral chemoreceptors. The TT-HVRs were superimposed upon three randomized background levels of steady-state inspired fraction of normoxic CO2 ( ; 0, 0.02 and 0.04). Chemostimuli [calculated oxygen saturation ( )] and respiratory variable responses [respiratory rate (RR ), inspired tidal volume (VTI ) and ventilation ( )] were averaged from all three TT-HVR trials at each level. Responses were assessed as: (1) a change (∆) from baseline; and (2) indexed against . Aside from a significantly lower ∆VTI response in 0.04 (P=0.01), the hypoxic rate responses (∆RR or ∆RR / ; P=0.46 and P=0.81), hypoxic tidal volume response ( ; P=0.08) and the hypoxic ventilatory responses ( and ; P=0.09 and P=0.31) were not significantly different across trials. Our data suggest simple addition between central and peripheral chemoreceptors in , which is mediated through simple addition in RR responses, but hypo-addition in VTI responses. Our study adds important new data in reconciling chemoreceptor interaction in awake, healthy humans and is consistent with previous reports of simple addition in intact rodents and humans.

Does breathing pattern affect cerebrovascular reactivity?

What is the central question of this study? Is cerebrovascular reactivity affected by isocapnic changes in breathing pattern? What is the main finding and its importance? Cerebrovascular reactivity does not change with isocapnic variations in tidal volume and frequency. Deviations of arterial carbon dioxide tension from resting values affect cerebral blood vessel tone and thereby cerebral blood flow. Arterial carbon dioxide tension also affects central respiratory chemoreceptors, adjusting respiratory drive. This coincidence raises the question: does respiratory drive also affect the cerebral blood flow response to carbon dioxide? A change in cerebral blood flow for a given change in the arterial carbon dioxide tension is defined as cerebrovascular reactivity (CVR). Two studies have reached conflicting conclusions on this question, using voluntary control of breathing as a disturbing factor during measurements of CVR. Here, we address some of the methodological limitations of both studies by using sequential gas delivery and targeted control of carbon dioxide and oxygen to enable a separation of the effects of carbon dioxide on CVR from breathing vigour. We confirm that there is no detectable superimposed effect of breathing efforts on CVR.

Rostral ventrolateral medulla, retropontine region and autonomic regulations

The rostral half of the ventrolateral medulla (RVLM) and adjacent ventrolateral retropontine region (henceforth RVLMRP) have been divided into various sectors by neuroscientists interested in breathing or autonomic regulations. The RVLMRP regulates respiration, glycemia, vigilance and inflammation, in addition to blood pressure. It contains interoceptors that respond to acidification, hypoxia and intracranial pressure and its rostral end contains the retrotrapezoid nucleus (RTN) which is the main central respiratory chemoreceptor. Acid detection by the RTN is an intrinsic property of the principal neurons that is enhanced by paracrine influences from surrounding astrocytes and CO2-dependent vascular constriction. RTN mediates the hypercapnic ventilatory response via complex projections to the respiratory pattern generator (CPG). The RVLM contributes to autonomic response patterns via differential recruitment of several subtypes of adrenergic (C1) and non-adrenergic neurons that directly innervate sympathetic and parasympathetic preganglionic neurons. The RVLM also innervates many brainstem and hypothalamic nuclei that contribute, albeit less directly, to autonomic responses. All lower brainstem noradrenergic clusters including the locus coeruleus are among these targets. Sympathetic tone to the circulatory system is regulated by subsets of presympathetic RVLM neurons whose activity is continuously restrained by the baroreceptors and modulated by the respiratory CPG. The inhibitory input from baroreceptors and the excitatory input from the respiratory CPG originate from neurons located in or close to the rhythm generating region of the respiratory CPG (preBötzinger complex).

From chemoreception to regulation: filling the gaps in understanding how insects control gas exchange.

Insects coordinate the opening and closing of spiracles with convective ventilatory movements to produce considerable intraspecific and interspecific variation in gas exchange patterns. But fundamental questions remain regarding how these movements are coordinated and modulated by central and peripheral respiratory chemoreceptors, and where these chemoreceptors are located and how they function. Recent findings have revealed regions of the CNS that generate coordinated respiratory motor activity, while peripheral neurons sensitive to respiratory gases have been identified in Drosophila. Importantly, plasticity in structure and function of neural elements of respiratory control indicate the need for caution when generalizing the mechanistic basis for breathing in insects, and an adaptive explanation for breathing pattern variability.

Acute hyperglycemia does not affect central respiratory chemoreflex responsiveness to CO2 in healthy humans

The central respiratory chemoreceptor complex (CCRC) is comprised of brainstem neurons and surrounding interoceptors, which collectively increase ventilation in response to elevated brainstem tissue CO2/[H+] (i.e., central chemoreflex; CCR). The extent that the CCRC detects/responds to other metabolically related chemostimuli is unknown. We aimed to test the effects of acute oral glucose ingestion on CCR reactivity in heathy human participants (n = 38). We instrumented participants with a pneumotachometer (minute ventilation) and a gas sample line connected to a dual gas analyzer (pressure of end-tidal CO2). Following a baseline (BL) period and capillary blood [glucose] (BG) sample, fasted (F) participants underwent a modified hyperoxic rebreathing test to assess CCR reactivity. Participants then consumed a 75 g standard glucose beverage (glucose loaded; GL). Following 30-min, they underwent a second BL, BG sample and hyperoxic rebreathing test. BG and metabolic rate were higher in GL, confirming the metabolic stimulus. However, the ventilatory recruitment threshold and the CCR responses were unchanged between F and GL states.

Astrocytic contribution to glutamate-related central respiratory chemoreception in vertebrates

Central respiratory chemoreceptors play a key role in the respiratory homeostasis by sensing CO2 and H+ in brain and activating the respiratory neural network. This ability of specific brain regions to respond to acidosis and hypercapnia is based on neuronal and glial mechanisms. Several decades ago, glutamatergic transmission was proposed to be involved as a main mechanism in central chemoreception. However, a complete identification of mechanism has been elusive. At the rostral medulla, chemosensitive neurons of the retrotrapezoid nucleus (RTN) are glutamatergic and they are stimulated by ATP released by RTN astrocytes in response to hypercapnia. In addition, recent findings show that caudal medullary astrocytes in brainstem can also contribute as CO2 and H+ sensors that release D-serine and glutamate, both gliotransmitters able to activate the respiratory neural network. In this review, we describe the mammalian astrocytic glutamatergic contribution to the central respiratory chemoreception trying to trace in vertebrates the emergence of several components involved in this process.

Patch‐to‐Seq Reveals Unique Transcriptomic Profiles of Chemosensitive Serotonergic Neurons

Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Prior data indicate that a sub-population of brainstem serotonin (5-HT) neurons are likely central respiratory chemoreceptors. However, it remains unclear which 5-HT neurons develop chemosensitivity and what molecular markers may identify this unique sub-population of 5-HT neurons. Here we employed the “patch-to-seq” technique to measure action potential firing rate responses of 5-HT neuron to hypercapnic acidosis using cell-attached patch-clamp electrophysiology followed by isolation of that neurons’ intracellular RNA content for single cell RNA Sequencing. Acute brainstem slices (200 µm) from young (P18-23) transgenic rats expressing eGFP in all 5-HT neurons (SSeGFP) were recorded while superfused with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D-AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). The Chemosensitivity Index (C.I.) determined cell phenotypes. pH-sensitive eGFP+ neurons (n=48) had an average C.I. of 169.8± 8.25 SEM whereas pH-insensitive eGFP+ neurons (n=44) had an average C.I. of 100.3 ± 1.4 (P < 0.0001; t-test). Cell extracts from 11 chemosensitive and 10 insensitive recorded cells were subjected to single cell RNA Sequencing (scRNA-Seq), from which an average of 48.3M reads were generated with a quality score of ~32.7 and ~75% mapping rate per sample with an average of ~8,000 genes detected. Purity of the isolated samples was confirmed by significantly more expression (Log2(FPKM+1)) of 6 serotonergic vs 7 neuronal and 8 glial gene markers, and more expression of 7 neuronal vs 8 known glial gene markers. There were 166differentially expressed genes (q < 0.05) between pH-sensitive and -insensitive 5-HT neuron populations none of which have a known contribution to pH regulation or sensing. Predicted upstream regulators of 352 DEGs (p<0.01) point to the importance of Egr2 activation, a developmental transcription factor previously shown to give rise to pH sensitive 5-HT neurons in mice. Machine learning algorithms identified unique markers of CO2/pH sensitive 5-HT neurons in rats, where supervised analyses (using Support Vector Machine learning and Recursive Feature Elimination (SVM-RFE)) yielded several genes in which expression levels significantly (P<0.001) and accurately (%) predict the predefined phenotype from the DEGs (CD46, 68.7%; Iba57, 71.2%; Chd5, 72.0%; and Maz, 74.0%) and among the 1,000s of detected genes (Hnmpk, 81.0%; Diablo, 87.9%) but the biological/function significance of these genes in CO2/pH sensing remains unclear. RNAScope confirmed 25% of Tph2+ neurons in the raphe midline co-express 4 or more RNA molecules of CD46 or Iba57 RNA. Together, these data suggest: 1) 5-HT pH sensitive neurons are transcriptionally distinct from insensitive 5-HT neurons, and 2) novel molecular markers of 5-HT pH sensitive rat neurons may be useful identifiers for this unique subpopulation.

Effects of Serotonin Terminal Lesions in the Retrotrapezoid Nucleus on Ventilatory Chemoreflexes

Brain chemoreceptors play a vital role in regulating blood gas homeostasis in response to acute changes in CO2 and/or pH. Serotonergic (5-HT) neurons, which also produce and release other neuromodulatory peptides, have properties consistent with being central respiratory chemoreceptors. In addition, recent in vitro studies have highlighted important contributions of G-protein-coupled 5-HT receptors (5-HT2 and/or 5-HT7 receptors) to the chemosensitivity of additional putative central respiratory chemoreceptors within the retrotrapezoid nucleus (RTN) marked by the expression of Phox2b. However, the importance of raphe-derived neuromodulation of the RTN on eupneic breathing as well as hypoxic and hypercapnic ventilatory chemoreflexes in vivo is unclear. Here we hypothesized that unilateral ablation of serotonergic terminals within the RTN cause a time-dependent attenuation of ventilatory chemoreflexes in vivo. To test this hypothesis, 8-week-old Sprague-Dawley rats received unilateral stereotaxic injections of the 5-HT pre-synaptic terminal toxin 5,7-Dihydroxytryptamine (5,7-DHT; 2 mg/ml, 2 µl vol.) with or without fluorescent microbeads (for injection site localization) targeted to the RTN after pretreatment with desipramine (30mg/kg; IP) to limit off-target effects on other aminergic neurons/terminals. A control group received an equal volume injection of vehicle in the RTN. Breathing was measured via whole body plethysmography while exposed to room air, hypercapnia (7% CO2 challenge), or hypoxia (12% O2 challenge) 2-3 days (d) before and 3, 5, 7, 10, and 14d post-injection. Preliminary data showed that RTN-targeted injections reduced ventilatory chemoreflexes 3, 5, and 7d post-injection with slightly greater effects in the 5,7-DHT injected group. Ventilatory chemoreflexes returned to control levels by 14d post-injection. To confirm the lesion site within the RTN and determine the extent of 5-HT-specific lesioning, we have also begun to quantify 5-HT using immunofluorescent staining. However, additional preliminary data showed no differences in 5-HT when comparing 5,7-DHT and control groups. Although these studies are ongoing, our data suggest that unilateral RTN-targeted 5-HT terminal lesions reduce ventilatory chemoreflexes.

Contribution of retrotrapezoid nucleus neurons to CO2 -amplified cardiorespiratory activity in spontaneously hypertensive rats.

This study demonstrates that both CO2 -induced respiratory and cardiovascular responses are augmented in spontaneously hypertensive rats (SHRs). Genetic ablation of the retrotrapezoid nucleus (RTN) neurons depresses enhanced hypercapnic ventilatory response and eliminates CO2 -stimulated increase in arterial pressure and heart rate in SHRs. SHRs have a high protein level of pH-sensitive channels in the RTN, including the TASK-2 channel, Kv12.1 channel and acid-sensing ion channel 3. The inhibition of putative TASK-2 channel activity by clofilium diminishes amplified hypercapnic ventilatory and cardiovascular responses, and reduces the number of CO2 -activated RTN neurons in SHRs. These results indicate that RTN neurons contribute to enhanced CO2 -stimulated respiratory and cardiovascular responses in SHRs. The respiratory regulation of cardiovascular activity is essential for maintaining an efficient ventilation and perfusion ratio. Activation of central respiratory chemoreceptors not only elicits a ventilatory response but also regulates sympathetic nerve activity and arterial blood pressure (ABP). The retrotrapezoid nucleus (RTN) is the most completely characterized cluster of central respiratory chemoreceptors. We hypothesize that RTN neurons contribute to augmented CO2 -stimulated respiratory and cardiovascular responses in adult spontaneously hypertensive rats (SHRs). Our findings indicate that SHRs exhibit more enhanced hypercapnic cardiorespiratory responses than age-matched normotensive Wistar-Kyoto rats. Genetic ablation of RTN neurons notably depresses an enhanced hypercapnic ventilatory response (HCVR) and eliminates a CO2 -stimulated greater increase in ABP and heart rate in SHRs. In addition, SHRs have a higher protein level of pH-sensitive channels in the RTN, including TASK-2 channels, Kv12.1 channels and acid-sensing ion channel 3. Administration of clofilium (i.p.), an unselective inhibitor of TASK-2 channels, not only significantly reduces the enhanced HCVR but also inhibits CO2 -amplified increases in ABP and heart rate in SHRs. Moreover, clofilium significantly decreases the number of CO2 -activated RTN neurons in SHRs. Taken together, we suggest that RTN neurons play an important role in enhanced hypercapnic ventilatory and cardiovascular responses in SHRs and the putative mechanism involved is associated with TASK-2 channel activity in the RTN.

Prior oxygenation, but not chemoreflex responsiveness, determines breath-hold duration during voluntary apnea.

Central and peripheral respiratory chemoreceptors are stimulated during voluntary breath holding due to chemostimuli (i.e., hypoxia and hypercapnia) accumulating at the metabolic rate. We hypothesized that voluntary breath‐hold duration (BHD) would be (a) positively related to the initial pressure of inspired oxygen prior to breath holding, and (b) negatively correlated with respiratory chemoreflex responsiveness. In 16 healthy participants, voluntary breath holds were performed under three conditions: hyperoxia (following five normal tidal breaths of 100% O2), normoxia (breathing room air), and hypoxia (following ~30‐min of 13.5%–14% inspired O2). In addition, the hypoxic ventilatory response (HVR) was tested and steady‐state chemoreflex drive (SS‐CD) was calculated in room air and during steady‐state hypoxia. We found that (a) voluntary BHD was positively related to initial oxygen status in a dose‐dependent fashion, (b) the HVR was not correlated with BHD in any oxygen condition, and (c) SS‐CD magnitude was not correlated with BHD in normoxia or hypoxia. Although chemoreceptors are likely stimulated during breath holding, they appear to contribute less to BHD compared to other factors such as volitional drive or lung volume.

Differential Contribution of the Retrotrapezoid Nucleus and C1 Neurons to Active Expiration and Arousal in Rats.

Collectively, the retrotrapezoid nucleus (RTN) and adjacent C1 neurons regulate breathing, circulation and the state of vigilance, but previous methods to manipulate the activity of these neurons have been insufficiently selective to parse out their relative roles. We hypothesize that RTN and C1 neurons regulate distinct aspects of breathing (e.g., frequency, amplitude, active expiration, sighing) and differ in their ability to produce arousal from sleep. Here we use optogenetics and a combination of viral vectors in adult male and female Th-Cre rats to transduce selectively RTN (Phox2b+/Nmb+) or C1 neurons (Phox2b+/Th+) with Channelrhodopsin-2. RTN photostimulation modestly increased the probability of arousal. RTN stimulation robustly increased breathing frequency and amplitude; it also triggered strong active expiration but not sighs. Consistent with these responses, RTN innervates the entire pontomedullary respiratory network, including expiratory premotor neurons in the caudal ventral respiratory group, but RTN has very limited projections to brainstem regions that regulate arousal (locus ceruleus, CGRP+ parabrachial neurons). C1 neuron stimulation produced robust arousals and similar increases in breathing frequency and amplitude compared with RTN stimulation, but sighs were elicited and active expiration was absent. Unlike RTN, C1 neurons innervate the locus ceruleus, CGRP+ processes within the parabrachial complex, and lack projections to caudal ventral respiratory group. In sum, stimulating C1 or RTN activates breathing robustly, but only RTN neuron stimulation produces active expiration, consistent with their role as central respiratory chemoreceptors. Conversely, C1 stimulation strongly stimulates ascending arousal systems and sighs, consistent with their postulated role in acute stress responses.SIGNIFICANCE STATEMENT The C1 neurons and the retrotrapezoid nucleus (RTN) reside in the rostral ventrolateral medulla. Both regulate breathing and the cardiovascular system but in ways that are unclear because of technical limitations (anesthesia, nonselective neuronal actuators). Using optogenetics in unanesthetized rats, we found that selective stimulation of either RTN or C1 neurons activates breathing. However, only RTN triggers active expiration, presumably because RTN, unlike C1, has direct excitatory projections to abdominal premotor neurons. The arousal potential of the C1 neurons is far greater than that of the RTN, however, consistent with C1's projections to brainstem wake-promoting structures. In short, C1 neurons orchestrate cardiorespiratory and arousal responses to somatic stresses, whereas RTN selectively controls lung ventilation and arterial Pco2 stability.

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.

Coupling Electrophysiology with Single Cell RNA Sequencing to Uncover Transcriptional Markers of Chemosensitive Serotonergic Neurons

Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Previous data support the hypothesis that a sub‐population of brainstem serotonin (5‐HT) neurons serve as central respiratory chemoreceptors, where they become sensitive to pH/CO2 on or after post‐natal day 12 (P12) in rats. However, to date it remains unclear which 5‐HT neurons develop chemosensitivity and what molecular markers may identify this unique sub‐population of 5‐HT neurons. To address these questions, we employed a “patch‐to‐seq” technique to first distinguish 5‐HT neurons as chemosensitive (or insensitive) using electrophysiologic recording, and then to isolate each neurons’ RNA for single cell RNA Sequencing to identify potential cellular sensing mechanisms and/or unique transcriptional markers in each phenotypically‐identified 5‐HT neuron population. Acute brainstem slices (200 μm) were made from young (P18–23) transgenic rats which express eGFP in all 5‐HT neurons (SSeGFP), and action potential firing rates measured via a cell‐attached patch configuration in eGFP+ 5‐HT neurons during superfusion with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). Cellular phenotypes were assessed by calculating a Chemosensitivity Index (C.I.). pH‐sensitive eGFP+ neurons (n=7) had an average C.I. of 177.0±14.6 whereas pH‐insensitive eGFP+ neurons (n=9) had an average C.I. of 98.0 ± 2.9 (P &lt; 0.0001). Cellular contents of each recorded cell were collected and subjected to single cell RNA Sequencing (scRNA‐Seq), from which an average of 19.3M reads were generated with a quality score of ~33.7 and ~83% mapping rate per sample (32,883 known genes quantified). Preliminary analyses indicate very few differentially expressed genes (q &lt; 0.05) between pH‐sensitive and ‐ insensitive 5‐HT neuron populations, and of those differentially expressed no gene obviously contributes to pH regulation or sensing. In addition, unbiased hierarchical clustering and principal component analyses failed to clearly separate samples by phenotype. However, a supervised analysis using Support Vector Machine learning and Recursive Feature Elimination (SVM‐RFE) identified several genes in which expression levels significantly predict the predefined phenotype, but the biological/functional significance of these genes in CO2/pH sensing remains unclear and instead these genes may provide a molecular signature to distinguish pH/CO2 sensitive vs insensitive 5‐HT neurons. We conclude from these preliminary data that pH/CO2 sensitivity in 5‐HT neurons is not a transcriptionally regulated phenotype.Support or Funding InformationParker B. Francis FoundationNHLBI ‐ R01 HL122358