Andrea E. Corcoran

Impaired Respiratory and Body Temperature Control Upon Acute Serotonergic Neuron Inhibition

Inducible neuron inhibition reveals essential roles for serotonergic neurons in respiratory and body temperature homeostasis. 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 (CO2) elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO2 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.

Medullary serotonin neurons and central CO2 chemoreception

Serotonergic (5-HT) neurons are putative central respiratory chemoreceptors, aiding in the brains ability to detect arterial changes in PCO2 and implement appropriate ventilatory responses to maintain blood homeostasis. These neurons are in close proximity to large medullary arteries and are intrinsically chemosensitive in vitro, characteristics expected for chemoreceptors. 5-HT neurons of the medullary raphé are stimulated by hypercapnia in vivo, and their disruption results in a blunted hypercapnic ventilatory response. More recently, data collected from transgenic and knockout mice have provided further insight into the role of 5-HT in chemosensitivity. This review summarizes current evidence in support of the hypothesis that 5-HT neurons are central chemoreceptors, and addresses arguments made against this role. We also briefly explore the relationship between the medullary raphé and another chemoreceptive site, the retrotrapezoid nucleus, and discuss how they may interact during hypercapnia to produce a robust ventilatory response.

Functional and Developmental Identification of a Molecular Subtype of Brain Serotonergic Neuron Specialized to Regulate Breathing Dynamics

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.

Serotonergic mechanisms are necessary for central respiratory chemoresponsiveness in situ

Evidence from in vivo and in vitro experiments conclude that serotonin (5-HT) neurons are involved in and play an important role in central respiratory CO2/H(+) chemosensitivity. This study was designed to assess the importance of 5-HT neurons and 5-HT receptor activation in the frequency and amplitude components of the hypercapnic response of the respiratory network in the unanesthetized perfused in situ juvenile rat brainstem preparation that exhibits patterns of phrenic nerve discharge similar to breathing in vivo. Exposure to a hypercapnic perfusate increased phrenic burst frequency and/or amplitude, the neural correlates of breathing frequency and tidal volume in vivo. Hypercapnic responses were also assessed during exposure to ketanserin (5-HT2 receptor antagonist), and 8-OH-DPAT (inhibiting 5-HT neurons via 5-HT1A autoreceptors). Neither of these drugs substantially altered baseline activity, however, both abolished hypercapnic responses of the respiratory network. These data illustrate that 5-HT neurons and 5-HT receptor activation are not required for respiratory rhythm generation per se, but are critical for CO2 responses in situ, supporting the hypothesis that 5-HT neurons play an important role in central ventilatory chemosensitivity in vivo.

Egr2-neurons control the adult respiratory response to hypercapnia.

`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).

Dual effects of 5-HT(1a) receptor activation on breathing in neonatal mice.

Inhibitory 5-HT1a receptors are located on serotonin (5-HT) neurons (autoreceptors) as well as neurons of the respiratory network (heteroreceptors). Thus, effects on breathing of 5-HT1a agonists, such as (R)-(+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), could either be due to decreased firing of 5-HT neurons or direct effects on the respiratory network. Mice in which the transcription factor LMX1B is genetically deleted selectively in Pet1-1-expressing cells (Lmx1bf/f/p) essentially have complete absence of central 5-HT neurons, providing a unique opportunity to separate the effect of activation of downstream 5-HT1a heteroreceptors from that of autoreceptors. We used rhythmically active medullary slices from wild-type (WT) and Lmx1bf/f/p neonatal mice to differentiate autoreceptor versus heteroreceptor effects of 8-OH-DPAT on hypoglossal nerve respiratory output. 8-OH-DPAT transiently increased respiratory burst frequency in Lmx1bf/f/p preparations, but not in WT slices. This excitation was abolished when synaptic inhibition was blocked by GABAergic/glycinergic receptor antagonists. Conversely, after 10 min of application, frequency in Lmx1bf/f/p slices was not different from baseline, whereas it was significantly depressed in WT slices. In WT mice in vivo, subcutaneous injection of 8-OH-DPAT produced similar biphasic respiratory effects as in Lmx1bf/f/p mice. We conclude that 5-HT1a receptor agonists have two competing effects: rapid stimulation of breathing due to excitation of the respiratory network, and delayed inhibition of breathing due to autoreceptor inhibition of 5-HT neurons. The former effect is presumably due to inhibition of inhibitory interneurons embedded in the respiratory network.

Modulation of Respiratory Activity by Hypocretin-1 (Orexin A) In Situ and In Vitro

Release of hypocretins (orexins) by neurons in the lateral hypothalamus is an important contributor to arousal state, thermoregulation, feeding behavior, and has recently been proposed to play a role in breathing and central chemosensitivity. Using the in situ arterially perfused juvenile rat preparation, we determined the effect of hypocretin-1 (hcrt-1) and SB-408124 (antagonist for hypocretin receptor subtype 1, hcrt-r1) on phrenic nerve activity, a neural correlate of breathing (neuroventilation), and the neuroventilatory sensitivity to CO(2). Application of hcrt-1 through the perfusate had little effect on baseline firing. Blocking hcrt-r1, however, prevented the phrenic burst frequency response normally associated with hypercapnia. These data suggest that endogenous hypocretinergic modulation enhances neuroventilatory chemosensitivity. Further studies using the in vitro medullary slice preparation explored the effect of hcrt-1 on hypoglossal nerve activity, a correlate of ventilation in vitro. Application of exogenous hcrt-1 failed to significantly alter hypoglossal burst output in neonatal rat slices, indicating that this portion of the neuroventilatory circuit is insensitive to hcrt-1. Taken together, these data suggest that hcrt-1 is a modulator of central chemosensitivity.

Respiratory rhythm of brainstem-spinal cord preparations: Effects of maturation, age, mass and oxygenation

We examined the effect of age, mass and the presence of the pons on the longevity (length of time spontaneous respiratory-related activity is produced) of brainstem-spinal cord preparations of neonatal rodents (rats and hamsters) and the level of oxygenation in the medulla respiratory network in these preparations. We found the longevity of the preparations from both species decreased with increasing postnatal age. Physical removal of the pons increased respiratory frequency and the longevity of the preparation. However, tissue oxygenation at the level of the medullary respiratory network was not affected by removal of the pons or increasing postnatal age (up to postnatal day 4). Taken together, these data suggest that the effect of removing the pons on respiratory frequency and the longevity of brainstem-spinal cord preparations with increasing postnatal age are primarily due to postnatal development and appear to be unrelated to mass or changes in levels of tissue oxygenation.

Activity of Tachykinin1-Expressing Pet1 Raphe Neurons Modulates the Respiratory Chemoreflex

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 Tachykinin-1 (Tac1) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1, referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine-N-oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2. Tac1-Pet1 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 Pet1 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 Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei. SIGNIFICANCE STATEMENT 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 Tachykinin1 and Pet1 (Tac1-Pet1 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 CO2. Tac1-Pet1 neurons thus appear distinct from and contrast previously described Egr2-Pet1 neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive.

CO2-inhibited neurons in the medullary raphé are GABAergic.

Previous studies have reported subsets of medullary raphé neurons that are either stimulated or inhibited by CO2/pH in vitro, in situ, and in vivo. We tested the hypothesis that medullary raphé CO2-inhibited neurons are GABAergic. Extracellular recordings in unanesthetized juvenile in situ rat preparations showed reversible hypercapnia-induced suppression of 19% (63/323) of medullary raphé neurons, and this suppression persisted after antagonism of NMDA, AMPA/kainate, and GABAA receptors. We stained a subset of CO2-inhibited cells and found that most (11/12) had glutamic acid decarboxylase 67 immunoreactivity (GAD67-ir). These data indicate that the majority of acidosis-inhibited medullary raphé neurons are GABAergic, and that their chemosensitivity is independent of major fast synaptic inputs. Thus, CO2-sensitive GABAergic neurons may play a role in central CO2/pH chemoreception.