Sensory neurons initiate defensive reflexes that ensure airway integrity. Dysfunction of laryngeal neurons is life-threatening, causing pulmonary aspiration, dysphagia, and choking, yet relevant ...sensory pathways remain poorly understood. Here, we discover rare throat-innervating neurons (∼100 neurons/mouse) that guard the airways against assault. We used genetic tools that broadly cover a vagal/glossopharyngeal sensory neuron atlas to map, ablate, and control specific afferent populations. Optogenetic activation of vagal P2RY1 neurons evokes a coordinated airway defense program—apnea, vocal fold adduction, swallowing, and expiratory reflexes. Ablation of vagal P2RY1 neurons eliminates protective responses to laryngeal water and acid challenge. Anatomical mapping revealed numerous laryngeal terminal types, with P2RY1 neurons forming corpuscular endings that appose laryngeal taste buds. Epithelial cells are primary airway sentinels that communicate with second-order P2RY1 neurons through ATP. These findings provide mechanistic insights into airway defense and a general molecular/genetic roadmap for internal organ sensation by the vagus nerve.
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•Broad genetic access enables functional dissection of a vagal sensory neuron atlas•Rare P2RY1 sensory neurons guard the airways by engaging an airway defense program•P2RY1 neurons innervate the laryngeal epithelium and appose laryngeal taste buds•Epithelial cells detect laryngeal water/acid and communicate to P2RY1 neurons via ATP
Prescott and colleagues reveal a rare population of vagal sensory neurons that serve as a first-line defense in the upper airways to prevent infiltration and injury within the respiratory tract. The authors develop an expansive genetic toolkit that broadly covers a vagal/glossopharyngeal sensory neuron atlas and use genetic approaches to map, ablate, and control vagal sensory neuron populations, revealing sensory neurons that receive input from laryngeal epithelial cells through ATP and in response mediate critical aspects of airway defense.
Physiological homeostasis is essential for organism survival. Highly responsive neuronal networks are involved, but their constituent neurons are just beginning to be resolved. To query brain ...serotonergic neurons in homeostasis, we used a neuronal silencing tool, mouse RC::FPDi (based on the synthetic G protein—coupled receptor Di), designed for cell type—specific, ligand-inducible, and reversible suppression of action potential firing. In mice harboring Di-expressing serotonergic neurons, administration of the ligand clozapine-N-oxide (CNO) by systemic injection attenuated the chemoreflex that normally increases respiration in response to tissue carbon dioxide (CO 2 ) elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO 2 acidosis. Body thermoregulation at room temperature was also disrupted after CNO triggering of Di; core temperatures plummeted, then recovered. This work establishes that serotonergic neurons regulate life-sustaining respiratory and thermoregulatory networks, and demonstrates a noninvasive tool for mapping neuron function.
Serotonergic (5HT) neurons modulate diverse behaviors and physiology and are implicated in distinct clinical disorders. Corresponding diversity in 5HT neuronal phenotypes is becoming apparent and is ...likely rooted in molecular differences, yet a comprehensive approach characterizing molecular variation across the 5HT system is lacking, as is concomitant linkage to cellular phenotypes. Here we combine intersectional fate mapping, neuron sorting, and genome-wide RNA-seq to deconstruct the mouse 5HT system at multiple levels of granularity—from anatomy, to genetic sublineages, to single neurons. Our unbiased analyses reveal principles underlying system organization, 5HT neuron subtypes, constellations of differentially expressed genes distinguishing subtypes, and predictions of subtype-specific functions. Using electrophysiology, subtype-specific neuron silencing, and conditional gene knockout, we show that these molecularly defined 5HT neuron subtypes are functionally distinct. Collectively, this resource classifies molecular diversity across the 5HT system and discovers sertonergic subtypes, markers, organizing principles, and subtype-specific functions with potential disease relevance.
•5HT neuron RNA-seq across anatomy and sublineage at population and single-cell scales•Unbiased analyses identify 5HT neuron subtypes and organizing principles•Differential gene expression predicts subtype-specific functions and disease associations•In vitro drug responses, sensorimotor gating, and behaviors map to distinct subtypes
Serotonergic neurons are often considered a single neuron type; however, examples of diversity suggest otherwise. Okaty and Freret et al. use intersectional genetics, RNA-seq, and subtype-specific manipulations to reveal a functional organization rooted in sublineage and anatomy and comprised of neuron subtypes highly diverse at the molecular and functional levels.
Serotonergic neurons modulate behavioral and physiological responses from aggression and anxiety to breathing and thermoregulation. Disorders involving serotonin (5HT) dysregulation are ...commensurately heterogeneous and numerous. We hypothesized that this breadth in functionality derives in part from a developmentally determined substructure of distinct subtypes of 5HT neurons each specialized to modulate specific behaviors. By manipulating developmentally defined subgroups one by one chemogenetically, we find that the Egr2-Pet1 subgroup is specialized to drive increased ventilation in response to carbon dioxide elevation and acidosis. Furthermore, this subtype exhibits intrinsic chemosensitivity and modality-specific projections—increasing firing during hypercapnic acidosis and selectively projecting to respiratory chemosensory but not motor centers, respectively. These findings show that serotonergic regulation of the respiratory chemoreflex is mediated by a specialized molecular subtype of 5HT neuron harboring unique physiological, biophysical, and hodological properties specified developmentally and demonstrate that the serotonergic system contains specialized modules contributing to its collective functional breadth.
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•Functional specializations map to distinct 5HT neuron developmental lineages•Life-sustaining breathing reflex is specifically driven by Egr2-Pet1 5HT neurons•Egr2-Pet1 5HT neurons function as frontline PCO2/pH chemoreceptor•5HT neuronal system is found to have sensory and motor subdivisions
Brust et al. show that the life-sustaining respiratory CO2 chemoreflex is regulated by a specialized subtype of serotonergic neuron—the Egr2-Pet1 subtype—and reveal its unique physiological, biophysical, and hogological properties centered around chemosensory processing and likely specified by genetic lineage developmentally. Thus, specialized modules contribute to serotonergic functional breadth.
Islet1 (Isl1) has been proposed as a marker of cardiac progenitor cells derived from the second heart field and is utilized to identify and purify cardiac progenitors from murine and human specimens ...for ex vivo expansion. The use of Isl1 as a specific second heart field marker is dependent on its exclusion from other cardiac lineages such as neural crest.
Determine whether Isl1 is expressed by cardiac neural crest.
We used an intersectional fate-mapping system using the RC::FrePe allele, which reports dual Flpe and Cre recombination. Combining Isl1(Cre/+), a SHF driver, and Wnt1::Flpe, a neural crest driver, with Rc::FrePe reveals that some Isl1 derivatives in the cardiac outflow tract derive from Wnt1-expressing neural crest progenitors. In contrast, no overlap was observed between Wnt1-derived neural crest and an alternative second heart field driver, Mef2c-AHF-Cre.
Isl1 is not restricted to second heart field progenitors in the developing heart but also labels cardiac neural crest. The intersection of Isl1 and Wnt1 lineages within the heart provides a caveat to using Isl1 as an exclusive second heart field cardiac progenitor marker and suggests that some Isl1-expressing progenitor cells derived from embryos, embryonic stem cultures, or induced pluripotent stem cultures may be of neural crest lineage.
Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized ...neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the
(
) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of
and the serotonergic transcription factor gene
, referred to as the
neuron subtype. Transgenic cell labeling showed
soma resident largely in the caudal medulla. Chemogenetic clozapine
oxide (CNO)-hM4Di perturbation of
neuron activity blunted the ventilatory response of the respiratory CO
chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO
axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a
neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These
neurons may act downstream of
serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei.
Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using genetic tools, we characterize a 5-HT neuron subtype defined by expression of
and
(
neurons), mapping soma localization to the caudal medulla primarily and axonal projections to brainstem motor nuclei most prominently, and, when silenced, observed blunting of the ventilatory response to inhaled CO
neurons thus appear distinct from and contrast previously described
neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive.
Sudden infant death syndrome (SIDS) cases often have abnormalities of the brainstem raphe serotonergic (5-HT) system. We hypothesize that raphe dysfunction contributes to a failure to autoresuscitate ...from multiple hypoxic events, leading to SIDS. We studied autoresuscitation in two transgenic mouse models in which exocytic neurotransmitter release was impaired via conditional expression of the light chain from tetanus toxin (tox) in raphe neurons expressing serotonergic bacterial artificial chromosome drivers Pet1 or Slc6a4. These used recombinase drivers targeted different portions of medullary raphe serotonergic, tryptophan hydroxylase 2 (Tph2)(+) neurons by postnatal day (P) 5 through P12: approximately one-third in triple transgenic Pet1::Flpe, hβactin::cre, RC::PFtox mice; approximately three-fourths inSlc6a4::cre, RC::Ptox mice; with the first model capturing a near equal number of Pet1(+),Tph2(+) versus Pet1(+),Tph2(low or negative) raphe cells. At P5, P8, and P12, "silenced" mice and controls were exposed to five, ∼37 s bouts of anoxia. Mortality was 5-10 times greater in "silenced" pups compared with controls at P5 and P8 (p = 0.001) but not P12, with cumulative survival not differing between experimental transgenic models. "Silenced" pups that eventually died took longer to initiate gasping (p = 0.0001), recover heart rate (p = 0.0001), and recover eupneic breathing (p = 0.011) during the initial anoxic challenges. Variability indices for baseline breathing distinguished "silenced" from controls but did not predict mortality. We conclude that dysfunction of even a portion of the raphe, as observed in many SIDS cases, can impair ability to autoresuscitate at critical periods in postnatal development and that baseline indices of breathing variability can identify mice at risk.
Many sudden infant death syndrome (SIDS) cases exhibit a partial (∼26%) brainstem serotonin deficiency. Using recombinase drivers, we targeted different fractions of serotonergic and raphe neurons in mice for tetanus toxin light chain expression, which prevented vesicular neurotransmitter release. In one model, approximately one-third of medullary Tph2(+) neurons are silenced by postnatal (P) days 5 and 12, along with some Pet1(+),Tph2(low or negative) raphe cells; in the other, approximately three-fourths of medullary Tph2(+) neurons, also with some Tph2(low or negative) cells. Both models demonstrated excessive mortality to anoxia (a postulated SIDS stressor) at P5 and P8. We demonstrated fatal vulnerability to anoxic stress at a specific time in postnatal life induced by a partial defect in raphe function. This models features of SIDS.
`The early growth response 2 transcription factor, Egr2, establishes a population of brainstem neurons essential for normal breathing at birth. Egr2-null mice die perinatally of respiratory ...insufficiency characterized by subnormal respiratory rate and severe apneas. Here we bypass this lethality using a noninvasive pharmacogenetic approach to inducibly perturb neuron activity postnatally, and ask if Egr2-neurons control respiration in adult mice. We found that the normal ventilatory increase in response to elevated tissue CO2 was impaired, blunted by 63.1±8.7% after neuron perturbation due to deficits in both respiratory amplitude and frequency. By contrast, room-air breathing was unaffected, suggesting that the drive for baseline breathing may not require those Egr2-neurons manipulated here. Of the multiple brainstem sites proposed to affect ventilation in response to hypercapnia, only the retrotrapezoid nucleus, a portion of the serotonergic raphé, and a portion of the A5 nucleus have a history of Egr2 expression. We recently showed that acute inhibition of serotonergic neurons en masse blunts the CO2 chemoreflex in adults, causing a difference in hypercapnic response of ∼50% after neuron perturbation through effects on respiratory amplitude only. The suppressed respiratory frequency upon perturbation of Egr2-neurons thus may stem from non-serotonergic neurons within the Egr2 domain. Perturbation of Egr2-neurons did not affect body temperature, even on exposure to ambient 4°C. These findings support a model in which Egr2-neurons are a critical component of the respiratory chemoreflex into adulthood. Methodologically, these results highlight how pharmacogenetic approaches allow neuron function to be queried in unanesthetized adult animals, reaching beyond the roadblocks of developmental lethality and compensation as well as the anatomical disturbances associated with invasive methods.
This article is part of a Special Issue entitled Optogenetics (7th BRES)
Löffler
et al
. highlight the important potential of designer receptors exclusively activated by a designer drug (DREADD)–based technologies to study cell type–specific functions but cautions that ...the triggering DREADD ligand, clozapine-
N
-oxide (CNO) might, through potential conversion products, have bioactivity outside of the synthetic DREADD receptor system and maintains that Ray
et al
. did not control for such activity. We recount controls used in our work that indicated no discernible DREADD-independent effects of CNO on the homeostatic assays employed and discuss in this regard murine studies reporting CNO bioneutrality in other assays, the rapid renal clearance of N-oxides like CNO, and evidence of negligible conversion to clozapine (CN) in mice and rats.
Mechanisms underlying Kv4 channel inactivation and recovery are presently unclear, although there is general consensus that the basic characteristics of these processes are not consistent with Shaker ...(Kv1) N- and P/C-type mechanisms. Kv4 channels also differ from Shaker in that they can undergo significant inactivation from pre-activated closed-states (closed-state inactivation, CSI), and that inactivation and recovery kinetics can be regulated by intracellular KChIP2 isoforms. To gain insight into the mechanisms regulating Kv4.3 CSI and recovery, we have analyzed the effects of increasing K+o from 2 mM to 98mM in the absence and in the presence of KChIP2b, the major KChIP2 isoform expressed in the mammalian ventricle. In the absence of KChIP2b, high K+o promoted Kv4.3 inactivated closed-states and significantly slowed the kinetics of recovery from both macroscopic and closed-state inactivation. Coexpression of KChIP2b in 2 mM K+o promoted non-inactivated closed-states and accelerated the kinetics of recovery from both macroscopic and CSI. In high K+o, KChIP2b eliminated or significantly reduced the slowing effects on recovery. Attenuation of CSI by the S4 charge-deletion mutant R302A, which produced significant stabilization of non-inactivated closed-states, effectively eliminated the opposing effects of high K+o and KChiP2b on macroscopic recovery kinetics, confirming that these results were due to alterations of CSI. Elevated K+o therefore slows Kv4.3 recovery by stabilizing inactivated closed-states, while KChIP2b accelerates recovery by destabilizing inactivated closed-states. Our results challenge underlying assumptions of presently popular Kv4 gating models and suggest that Kv4.3 possesses novel allosteric mechanisms, which are absent in Shaker, for coupling interactions between intracellular KChIP2b binding motifs and extracellular K+-sensitive regulatory sites.
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