A brainstem peptide system activated at birth protects postnatal breathing.

Among numerous challenges encountered at the beginning of extrauterine life, the most celebrated is the first breath that initiates a life-sustaining motor activity1. The neural systems that regulate breathing are fragile early in development, and it is not clear how they adjust to support breathing at birth. Here we identify a neuropeptide system that becomes activated immediately after birth and supports breathing. Mice that lack PACAP selectively in neurons of the retrotrapezoid nucleus (RTN) displayed increased apnoeas and blunted CO2-stimulated breathing; re-expression of PACAP in RTN neurons corrected these breathing deficits. Deletion of the PACAP receptor PAC1 from the pre-Bötzinger complex-an RTN target region responsible for generating the respiratory rhythm-phenocopied the breathing deficits observed after RTN deletion of PACAP, and suppressed PACAP-evoked respiratory stimulation in the pre-Bötzinger complex. Notably, a postnatal burst of PACAP expression occurred in RTN neurons precisely at the time of birth, coinciding with exposure to the external environment. Neonatal mice with deletion of PACAP in RTN neurons displayed increased apnoeas that were further exacerbated by changes in ambient temperature. Our findings demonstrate that well-timed PACAP expression by RTN neurons provides an important supplementary respiratory drive immediately after birth and reveal key molecular components of a peptidergic neural circuit that supports breathing at a particularly vulnerable period in life.

Contributions of carotid bodies, retrotrapezoid nucleus neurons and preBötzinger complex astrocytes to the CO2 -sensitive drive for breathing.

Current models of respiratory CO2 chemosensitivity are centred around the function of a specific population of neurons residing in the medullary retrotrapezoid nucleus (RTN). However, there is significant evidence suggesting that chemosensitive neurons exist in other brainstem areas, including the rhythm-generating region of the medulla oblongata - the preBötzinger complex (preBötC). There is also evidence that astrocytes, non-neuronal brain cells, contribute to central CO2 chemosensitivity. In this study, we reevaluated the relative contributions of the RTN neurons, the preBötC astrocytes, and the carotid body chemoreceptors in mediating the respiratory responses to CO2 in experimental animals (adult laboratory rats). To block astroglial signalling via exocytotic release of transmitters, preBötC astrocytes were targeted to express the tetanus toxin light chain (TeLC). Bilateral expression of TeLC in preBötC astrocytes was associated with ∼20% and ∼30% reduction of the respiratory response to CO2 in conscious and anaesthetized animals, respectively. Carotid body denervation reduced the CO2 respiratory response by ∼25%. Bilateral inhibition of RTN neurons transduced to express Gi-coupled designer receptors exclusively activated by designer drug (DREADDGi ) by application of clozapine-N-oxide reduced the CO2 response by ∼20% and ∼40% in conscious and anaesthetized rats, respectively. Combined blockade of astroglial signalling in the preBötC, inhibition of RTN neurons and carotid body denervation reduced the CO2 -induced respiratory response by ∼70%. These data further support the hypothesis that the CO2 -sensitive drive to breathe requires inputs from the peripheral chemoreceptors and several central chemoreceptor sites. At the preBötC level, astrocytes modulate the activity of the respiratory network in response to CO2 , either by relaying chemosensory information (i.e. they act as CO2  sensors) or by enhancing the preBötC network excitability to chemosensory inputs. KEY POINTS: This study reevaluated the roles played by the carotid bodies, neurons of the retrotrapezoid nucleus (RTN) and astrocytes of the preBötC in mediating the CO2 -sensitive drive to breathe. The data obtained show that disruption of preBötC astroglial signalling, blockade of inputs from the peripheral chemoreceptors or inhibition of RTN neurons similarly reduce the respiratory response to hypercapnia. These data provide further support for the hypothesis that the CO2 -sensitive drive to breathe is mediated by the inputs from the peripheral chemoreceptors and several central chemoreceptor sites.

Breathing matters.

Breathing is a well-described, vital and surprisingly complex behaviour, with behavioural and physiological outputs that are easy to directly measure. Key neural elements for generating breathing pattern are distinct, compact and form a network amenable to detailed interrogation, promising the imminent discovery of molecular, cellular, synaptic and network mechanisms that give rise to the behaviour. Coupled oscillatory microcircuits make up the rhythmic core of the breathing network. Primary among these is the preBötzinger Complex (preBötC), which is composed of excitatory rhythmogenic interneurons and excitatory and inhibitory pattern-forming interneurons that together produce the essential periodic drive for inspiration. The preBötC coordinates all phases of the breathing cycle, coordinates breathing with orofacial behaviours and strongly influences, and is influenced by, emotion and cognition. Here, we review progress towards cracking the inner workings of this vital core.

Postnatal development of persistent inward currents in rat XII motoneurons and their modulation by serotonin, muscarine and noradrenaline.

KEY POINTS: Persistent inward currents (PICs) in spinal motoneurons are long-lasting, voltage-dependent currents that increase excitability; they are dramatically potentiated by serotonin, muscarine, and noradrenaline (norepinephrine). Loss of these modulators (and the PIC) during sleep is hypothesized as a major contributor to REM sleep atonia. Reduced excitability of XII motoneurons that drive airway muscles and maintain airway patency is causally implicated in obstructive sleep apnoea (OSA), but whether XII motoneurons possess a modulator-sensitive PIC that could be a factor in the reduced airway tone of sleep is unknown. Whole-cell recordings from rat XII motoneurons in brain slices indicate that PIC amplitude increases ∼50% between 1 and 23 days of age, when potentiation of the PIC by 5HT2 , muscarinic, or α1 noradrenergic agonists peaks at <50%, manyfold lower than the potentiation observed in spinal motoneurons. α1 noradrenergic receptor activation produced changes in XII motoneuron firing behaviour consistent with PIC involvement, but indicators of strong PIC activation were never observed; in vivo experiments are needed to determine the role of the modulator-sensitive PIC in sleep-dependent reductions in airway tone. ABSTRACT: Hypoglossal (XII) motoneurons play a key role in maintaining airway patency; reductions in their excitability during sleep through inhibition and disfacilitation, i.e. loss of excitatory modulation, is implicated in obstructive sleep apnoea. In spinal motoneurons, 5HT2 , muscarinic and α1 noradrenergic modulatory systems potentiate persistent inward currents (PICs) severalfold, dramatically increasing excitability. If the PICs in XII and spinal motoneurons are equally sensitive to modulation, loss of the PIC secondary to reduced modulatory tone during sleep could contribute to airway atonia. Modulatory systems also change developmentally. We therefore characterized developmental changes in magnitude of the XII motoneuron PIC and its sensitivity to modulation by comparing, in neonatal (P1-4) and juvenile (P14-23) rat brainstem slices, the PIC elicited by slow voltage ramps in the absence and presence of agonists for 5HT2 , muscarinic, and α1 noradrenergic receptors. XII motoneuron PIC amplitude increased developmentally (from -195 ± 12 to -304 ± 19 pA). In neonatal XII motoneurons, the PIC was only potentiated by α1 receptor activation (5 ± 4%). In contrast, all modulators potentiated the juvenile XII motoneurons PIC (5HT2 , 5 ± 5%; muscarine, 22 ± 11%; α1 , 18 ± 5%). These data suggest that the influence of the PIC and its modulation on XII motoneuron excitability will increase with postnatal development. Notably, the modulator-induced potentiation of the PIC in XII motoneurons was dramatically smaller than the 2- to 6-fold potentiation reported for spinal motoneurons. In vivo measurements are required to determine if the modulator-sensitive, XII motoneuron PIC is an important factor in sleep-state dependent reductions in airway tone.

Respiratory motoneuron properties during the transition from gill to lung breathing in the American bullfrog.

Amphibian respiratory development involves a dramatic metamorphic transition from gill to lung breathing and coordination of distinct motor outputs. To determine whether the emergence of adult respiratory motor patterns was associated with similarly dramatic changes in motoneuron (MN) properties, we characterized the intrinsic electrical properties of American bullfrog trigeminal MNs innervating respiratory muscles comprising the buccal pump. In premetamorphic tadpoles (TK stages IX-XVIII) and adult frogs, morphometric analyses and whole cell recordings were performed in trigeminal MNs identified by fluorescent retrograde labeling. Based on the amplitude of the depolarizing sag induced by hyperpolarizing voltage steps, two MN subtypes (I and II) were identified in tadpoles and adults. Compared with type II MNs, type I MNs had larger sag amplitudes (suggesting a larger hyperpolarization-activated inward current), greater input resistance, lower rheobase, hyperpolarized action potential threshold, steeper frequency-current relationships, and fast firing rates and received fewer excitatory postsynaptic currents. Postmetamorphosis, type I MNs exhibited similar sag, enhanced postinhibitory rebound, and increased action potential amplitude with a smaller-magnitude fast afterhyperpolarization. Compared with tadpoles, type II MNs from frogs received higher-frequency, larger-amplitude excitatory postsynaptic currents. Input resistance decreased and rheobase increased postmetamorphosis in all MNs, concurrent with increased soma area and hyperpolarized action potential threshold. We suggest that type I MNs are likely recruited in response to smaller, buccal-related synaptic inputs as well as larger lung-related inputs, whereas type II MNs are likely recruited in response to stronger synaptic inputs associated with larger buccal breaths, lung breaths, or nonrespiratory behaviors involving powerful muscle contractions.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism.

KEY POINTS: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. ABSTRACT: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

Functional Oxygen Sensitivity of Astrocytes.

In terrestrial mammals, the oxygen storage capacity of the CNS is limited, and neuronal function is rapidly impaired if oxygen supply is interrupted even for a short period of time. However, oxygen tension monitored by the peripheral (arterial) chemoreceptors is not sensitive to regional CNS differences in partial pressure of oxygen (PO2 ) that reflect variable levels of neuronal activity or local tissue hypoxia, pointing to the necessity of a functional brain oxygen sensor. This experimental animal (rats and mice) study shows that astrocytes, the most numerous brain glial cells, are sensitive to physiological changes in PO2 . Astrocytes respond to decreases in PO2 a few millimeters of mercury below normal brain oxygenation with elevations in intracellular calcium ([Ca(2+)]i). The hypoxia sensor of astrocytes resides in the mitochondria in which oxygen is consumed. Physiological decrease in PO2 inhibits astroglial mitochondrial respiration, leading to mitochondrial depolarization, production of free radicals, lipid peroxidation, activation of phospholipase C, IP3 receptors, and release of Ca(2+) from the intracellular stores. Hypoxia-induced [Ca(2+)]i increases in astrocytes trigger fusion of vesicular compartments containing ATP. Blockade of astrocytic signaling by overexpression of ATP-degrading enzymes or targeted astrocyte-specific expression of tetanus toxin light chain (to interfere with vesicular release mechanisms) within the brainstem respiratory rhythm-generating circuits reveals the fundamental physiological role of astroglial oxygen sensitivity; in low-oxygen conditions (environmental hypoxia), this mechanism increases breathing activity even in the absence of peripheral chemoreceptor oxygen sensing. These results demonstrate that astrocytes are functionally specialized CNS oxygen sensors tuned for rapid detection of physiological changes in brain oxygenation. Significance statement: Most, if not all, animal cells possess mechanisms that allow them to detect decreases in oxygen availability leading to slow-timescale, adaptive changes in gene expression and cell physiology. To date, only two types of mammalian cells have been demonstrated to be specialized for rapid functional oxygen sensing: glomus cells of the carotid body (peripheral respiratory chemoreceptors) that stimulate breathing when oxygenation of the arterial blood decreases; and pulmonary arterial smooth muscle cells responsible for hypoxic pulmonary vasoconstriction to limit perfusion of poorly ventilated regions of the lungs. Results of the present study suggest that there is another specialized oxygen-sensitive cell type in the body, the astrocyte, that is tuned for rapid detection of physiological changes in brain oxygenation.

Neuroglia and their roles in central respiratory control; an overview.

While once viewed as mere housekeepers, providing structural and metabolic support for neurons, it is now clear that neuroglia do much more. Phylogenetically, they have undergone enormous proliferation and diversification as central nervous systems grew in their complexity. In addition, they: i) are morphologically and functionally diverse; ii) play numerous, vital roles in maintaining CNS homeostasis; iii) are key players in brain development and responses to injury; and, iv) via gliotransmission, are likely participants in information processing. In this review, we discuss the diverse roles of neuroglia in maintaining homeostasis in the CNS, their evolutionary origins, the different types of neuroglia and their functional significance for respiratory control, and finally consider evidence that they contribute to the processing of chemosensory information in the respiratory network and the homeostatic control of blood gases.

Anxiety-related mechanisms of respiratory dysfunction in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a severe neurological disorder that is associated with mutations in the methyl-CpG binding protein 2 (MECP2) gene. RTT patients suffer from mental retardation and behavioral disorders, including heightened anxiety and state-dependent breathing irregularities, such as hyperventilation and apnea. Many symptoms are recapitulated by the Mecp2-null male mice (Mecp2(-/y)). To characterize developmental progression of the respiratory phenotype and explore underlying mechanisms, we examined Mecp2(-/y) and wild-type (WT) mice from presymptomatic periods to end-stage disease. We monitored breathing patterns of unrestrained mice during wake-sleep states and while altering stress levels using movement restraint or threatening odorant (trimethylthiazoline). Respiratory motor patterns generated by in situ working heart-brainstem preparations (WHBPs) were measured to assess function of brainstem respiratory networks isolated from suprapontine structures. Data revealed two general stages of respiratory dysfunction in Mecp2(-/y) mice. At the early stage, respiratory abnormalities were limited to wakefulness, correlated with markers of stress (increased fecal deposition and blood corticosterone levels), and alleviated by antalarmin (corticotropin releasing hormone receptor 1 antagonist). Furthermore, the respiratory rhythm generated by WHBPs was similar in WT and Mecp2(-/y) mice. During the later stage, respiratory abnormalities were evident during wakefulness and sleep. Also, WHBPs from Mecp2(-/y) showed central apneas. We conclude that, at early disease stages, stress-related modulation from suprapontine structures is a significant factor in the Mecp2(-/y) respiratory phenotype and that anxiolytics may be effective. At later stages, abnormalities of brainstem respiratory networks are a significant cause of irregular breathing patterns and central apneas.

State-dependent modulation of breathing in urethane-anesthetized rats.

Respiratory activity is most fragile during sleep, in particular during paradoxical [or rapid eye movement (REM)] sleep and sleep state transitions. Rats are commonly used to study respiratory neuromodulation, but rodent sleep is characterized by a highly fragmented sleep pattern, thus making it very challenging to examine different sleep states and potential pharmacological manipulations within them. Sleep-like brain-state alternations occur in rats under urethane anesthesia and may be an effective and efficient model for sleep itself. The present study assessed state-dependent changes in breathing and respiratory muscle modulation under urethane anesthesia to determine their similarity to those occurring during natural sleep. Rats were anesthetized with urethane and respiratory airflow, as well as electromyographic activity in respiratory muscles were recorded in combination with local field potentials in neocortex and hippocampus to determine how breathing pattern and muscle activity are modulated with brain state. Measurements were made in normoxic, hypoxic, and hypercapnic conditions. Results were compared with recordings made from rats during natural sleep. Brain-state alternations under urethane anesthesia were closely correlated with changes in breathing rate and variability and with modulation of respiratory muscle tone. These changes closely mimicked those observed in natural sleep. Of great interest was that, during both REM and REM-like states, genioglossus muscle activity was strongly depressed and abdominal muscle activity showed potent expiratory modulation. We demonstrate that, in urethane-anesthetized rats, respiratory airflow and muscle activity are closely correlated with brain-state transitions and parallel those shown in natural sleep, providing a useful model to systematically study sleep-related changes in respiratory control.

Cholinergic projections to the preBötzinger complex.

Rhythmic inspiratory activity is generated in the preBötzinger complex (preBötC), a neuronal network located bilaterally in the ventrolateral medulla. Cholinergic neurotransmission affects respiratory rhythmogenic neurons and inhibitory glycinergic neurons in the preBötC. Acetylcholine has been extensively investigated given that cholinergic fibers and receptors are present and functional in the preBötC, are important in sleep/wake cycling, and modulate inspiratory frequency through its action on preBötC neurons. Despite its role in modulating inspiratory rhythm, the source of acetylcholine input to the preBötC is not known. In the present study, we used retrograde and anterograde viral tracing approaches in transgenic mice expressing Cre-recombinase driven by the choline acetyltransferase promoter to identify the source of cholinergic inputs to the preBötC. Surprisingly, we observed very few, if any, cholinergic projections originating from the laterodorsal and pedunculopontine tegmental nuclei (LDT/PPT), two main cholinergic, state-dependent systems long hypothesized as the main source of cholinergic inputs to the preBötC. On the contrary, we identified glutamatergic and GABAergic/glycinergic neurons in the PPT/LDT that send projections to the preBötC. Although these neurons contribute minimally to the direct cholinergic modulation of preBötC neurons, they could be involved in state-dependent regulation of breathing. Our data also suggest that the source of cholinergic inputs to the preBötC appears to originate from cholinergic neurons in neighboring regions of the medulla, the intermediate reticular formation, the lateral paragigantocellularis, and the nucleus of the solitary tract.

A muscarinic, GIRK channel-mediated inhibition of inspiratory-related XII nerve motor output emerges in early postnatal development in mice.

In neonatal rhythmic medullary slices, muscarinic acetylcholine receptor (mAChR) activation of hypoglossal (XII) motoneurons that innervate the tongue has a net excitatory effect on XII inspiratory motor output. Conversely, during rapid eye movement sleep in adult rodents, XII motoneurons experience a loss of excitability partly due to activation of mAChRs. This may be mediated by activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Therefore, this study was designed to evaluate whether muscarinic modulation of XII inspiratory motor output in mouse rhythmic medullary slices includes GIRK channel-mediated inhibition and, if so, when this inhibitory mechanism emerges. Local pressure injection of the mAChR agonist muscarine potentiated inspiratory bursting by 150 ± 28% in postnatal day (P)0-P5 rhythmic medullary slice preparations. In the absence of muscarine, pharmacological GIRK channel block by Tertiapin-Q did not affect inspiratory burst parameters, whereas activation with ML297 decreased inspiratory burst area. Blocking GIRK channels by local preapplication of Tertiapin-Q revealed a developmental change in muscarinic modulation of inspiratory bursting. In P0-P2 rhythmic medullary slices, Tertiapin-Q preapplication had no significant effect on muscarinic potentiation of inspiratory bursting (a negligible 6% decrease). However, preapplication of Tertiapin-Q to P3-P5 rhythmic medullary slices caused a 19% increase in muscarinic potentiation of XII inspiratory burst amplitude. Immunofluorescence experiments revealed expression of GIRK 1 and 2 subunits and M1, M2, M3, and M5 mAChRs from P0 to P5. Overall, these data support that mechanisms underlying muscarinic modulation of inspiratory burst activity change postnatally and that potent GIRK-mediated inhibition described in adults emerges early in postnatal life.NEW & NOTEWORTHY Muscarinic modulation of inspiratory bursting at hypoglossal motoneurons has a net excitatory effect in neonatal rhythmic medullary slice preparations and a net inhibitory effect in adult animals. We demonstrate that muscarinic modulation of inspiratory bursting undergoes maturational changes from postnatal days 0 to 5 that include emergence of an inhibitory component mediated by G-protein-coupled inwardly rectifying potassium channels after postnatal day 3 in neonatal mouse rhythmic medullary slice preparations.

Prenatal fluoxetine has long-lasting, differential effects on respiratory control in male and female rats.

Serotonin (5-HT) is an important modulator of brain networks that control breathing. The selective serotonin reuptake inhibitor fluoxetine (FLX) is the first-line antidepressant drug prescribed during pregnancy. We investigated the effects of prenatal FLX exposure on baseline breathing, ventilatory and metabolic responses to hypercapnia and hypoxia as well as number of brainstem 5-HT and tyrosine hydroxylase (TH) neurons of rats during postnatal development (P0-82). Prenatal FLX exposure of males showed a lower baseline V̇e that appeared in juveniles and remained in adulthood, with no sleep-wake state dependency. Prenatal FLX exposure of females did not affect baseline breathing. Juvenile male FLX showed increased CO2 and hypoxic ventilatory responses, normalizing by adulthood. Alterations in juvenile FLX-treated males were associated with a greater number of 5-HT neurons in the raphe obscurus (ROB) and raphe magnus (RMAG). Adult FLX-exposed males showed greater number of 5-HT neurons in the raphe pallidus (RPA) and TH neurons in the A5, whereas reduced number of TH neurons in A7. Prenatal FLX exposure of female rats was associated with greater hyperventilation induced by hypercapnia at P0-2 and juveniles, whereas P12-14 and adult FLX (non-rapid eye movement, NREM sleep) rats showed an attenuation of the hyperventilation induced by CO2. FLX-exposed females had fewer 5-HT neurons in the RPA and reduced TH A6 density at P0-2; and greater number of TH neurons in the A7 at P12-14. These data indicate that prenatal FLX exposure affects the number of some monoaminergic regions in the brain and results in long-lasting, sex-specific changes in baseline breathing pattern and ventilatory responses to respiratory challenges.NEW & NOTEWORTHY Selective serotonin reuptake inhibitors (SSRIs) readily cross the placental and the fetal blood-brain barrier where it will affect 5-HT levels in the developing brain. Although SSRI is used during pregnancy, there are no studies showing SSRI exposure during late pregnancy and postnatal effects on breathing control in males and females. We demonstrated that fluoxetine exposure during late pregnancy in rats was associated with long-lasting, sex-specific effects on breathing and brainstem monoaminergic groups.

Glia contribute to the purinergic modulation of inspiratory rhythm-generating networks.

Glia modulate neuronal activity by releasing transmitters in a process called gliotransmission. The role of this process in controlling the activity of neuronal networks underlying motor behavior is unknown. ATP features prominently in gliotransmission; it also contributes to the homeostatic ventilatory response evoked by low oxygen through mechanisms that likely include excitation of preBötzinger complex (preBötC) neural networks, brainstem centers critical for breathing. We therefore inhibited glial function in rhythmically active inspiratory networks in vitro to determine whether glia contribute to preBötC ATP sensitivity. Glial toxins markedly reduced preBötC responses to ATP, but not other modulators. Furthermore, since preBötC glia responded to ATP with increased intracellular Ca(2+) and glutamate release, we conclude that glia contribute to the ATP sensitivity of preBötC networks, and possibly the hypoxic ventilatory response. Data reveal a role for glia in signal processing within brainstem motor networks that may be relevant to similar networks throughout the neuraxis.

KCNQ Current Contributes to Inspiratory Burst Termination in the Pre-Bötzinger Complex of Neonatal Rats in vitro.

The pre-Bötzinger complex (preBötC) of the ventral medulla generates the mammalian inspiratory breathing rhythm. When isolated in explants and deprived of synaptic inhibition, the preBötC continues to generate inspiratory-related rhythm. Mechanisms underlying burst generation have been investigated for decades, but cellular and synaptic mechanisms responsible for burst termination have received less attention. KCNQ-mediated K+ currents contribute to burst termination in other systems, and their transcripts are expressed in preBötC neurons. Therefore, we tested the hypothesis that KCNQ channels also contribute to burst termination in the preBötC. We recorded KCNQ-like currents in preBötC inspiratory neurons in neonatal rat slices that retain respiratory rhythmicity. Blocking KCNQ channels with XE991 or linopirdine (applied via superfusion or locally) increased inspiratory burst duration by 2- to 3-fold. By contrast, activation of KCNQ with retigabine decreased inspiratory burst duration by ~35%. These data from reduced preparations suggest that the KCNQ current in preBötC neurons contributes to inspiratory burst termination.

Mapping of the excitatory, inhibitory, and modulatory afferent projections to the anatomically defined active expiratory oscillator in adult male rats.

The lateral parafacial region (pFL ; which encompasses the parafacial respiratory group, pFRG) is a conditional oscillator that drives active expiration during periods of high respiratory demand, and increases ventilation through the recruitment of expiratory muscles. The pFL activity is highly modulated, and systematic analysis of its afferent projections is required to understand its connectivity and modulatory control. We combined a viral retrograde tracing approach to map direct brainstem projections to the putative location of pFL , with RNAScope and immunofluorescence to identify the neurochemical phenotype of the projecting neurons. Within the medulla, retrogradely-labeled, glutamatergic, glycinergic and GABAergic neurons were found in the ventral respiratory column (Bötzinger and preBötzinger Complex [preBötC], ventral respiratory group, ventral parafacial region [pFV ] and pFL ), nucleus of the solitary tract (NTS), reticular formation (RF), pontine and midbrain vestibular nuclei, and medullary raphe. In the pons and midbrain, retrogradely-labeled neurons of the same phenotypes were found in the Kölliker-Fuse and parabrachial nuclei, periaqueductal gray, pedunculopontine nucleus (PPT) and laterodorsal tegmentum (LDT). We also identified somatostatin-expressing neurons in the preBötC and PHOX2B immunopositive cells in the pFV , NTS, and part of the RF. Surprisingly, we found no catecholaminergic neurons in the NTS, A5 or Locus Coeruleus, no serotoninergic raphe neurons nor any cholinergic neurons in the PPT and LDT that projected to the pFL . Our results indicate that pFL neurons receive extensive excitatory and inhibitory inputs from several respiratory and nonrespiratory related brainstem regions that could contribute to the complex modulation of the conditional pFL oscillator for active expiration.