Tonic excitation of nucleus reuniens decreases prefrontal-hippocampal coordination during slow-wave states.

The nucleus reuniens of the thalamus (RE) is an important node between the medial prefrontal cortex (mPFC) and the hippocampus (HPC). Previously, we have shown that its mode of activity and its influence in mPFC-HPC communication is dependent upon brain state. During slow-wave states, RE units are closely and rhythmically coupled to the ongoing mPFC-slow oscillation (SO), while during activated (theta) states, RE neurons fire in an arrhythmic and tonically active manner. Inactivating the RE selectively impoverishes coordination of the SO between mPFC and HPC and interestingly, both mPFC and RE stimulation during the SO cause larger responses in the HPC than during theta. It is unclear if the activity patterns within the RE across states may play a role in both phenomena. Here, we optogenetically excited RE neurons in a tonic fashion to assess the impact on mPFC-HPC coupling. This stimulation decreased the influence of mPFC stimulation in the HPC during SO states, in a manner similar to what is observed across state changes into theta. Importantly, this type of stimulation had no effect on evoked responses during theta. Perhaps more interestingly, tonic optogenetic excitation of the RE also decreased mPFC-HPC SO coherence. Thus, it may not be the integrity of the RE per se that is responsible for efficient communication between mPFC and HPC, but rather the particular state in which RE neurons find themselves. Our results have direct implications for how distant brain regions can communicate most effectively, an issue that is ultimately important for activity-dependent processes occurring during slow-wave sleep-dependent memory consolidation.

Expiratory activity during sleep in children.

Sleep irregularities and respiratory events (apnea, O2 desaturation or a combination thereof) are often present in the infant population. While inspiration is the main active process in the act of breathing, expiration is generally thought to occur passively. Although commonly considered as quiet during sleep, expiratory abdominal muscles have been proposed to be recruited to promote ventilation, facilitate gas exchange, and reduce the work of breathing during conditions of increased respiratory drive, exercise, or airway obstruction. In this study, we investigated the occurrence of expiratory abdominal muscle activity in polysomnographic studies of subjects (aged 0-2 years) suspected of sleep disordered breathing. Our results indicate that abdominal muscle activation occurs during sleep, most frequently during non-rapid eye movement and rapid-eye movement states compared to slow-wave sleep. Furthermore, abdominal muscle activity was present during regular breathing or associated with respiratory events (apneas or O2 desaturation). In the latter case, abdominal muscle recruitment more frequently followed the onset of respiratory events and terminated with recovery from blood O2 desaturation events. We conclude that expiratory abdominal muscle activity contributes to the pattern of respiratory muscle recruitment during sleep in infants and given its temporal relationship with respiratory events, we propose that its recruitment could facilitate proper ventilation by counteracting airway resistance and O2 desaturation in infancy across different stages of sleep.

The peptidergic control circuit for sighing.

Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.

Etonogestrel Administration Reduces the Expression of PHOX2B and Its Target Genes in the Solitary Tract Nucleus.

Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation.

Routine use of post-bronchodilator testing in pulmonary function testing labs.

PURPOSE: Pulmonary function tests (PFTs), including spirometry with and without post-bronchodilator (post-BD) testing, are frequently performed in the assessment of asthma, along with other obstructive airway disorders. Multiple publications over the past 15 years have noted that one in three physician-diagnosed asthma cases are not in fact asthma. In this quality assurance project, we assess whether PFT labs in Alberta have policies on post-BD testing, as extraneous and unnecessary use of post-BD testing can lead to wasted staff and patient time and unnecessary expenses to the health care system. METHODS: We reviewed, in collaboration with the College of Physicians and Surgeons of Alberta and Alberta Medical Association, all PFT labs in the province of Alberta (hospital-based private not-for-profit [NFP] and private for-profit [FP] labs). This health policy study of PFT labs involved identifying the proportions and regional distribution of NFP and private FP labs in the province of Alberta while assessing post-BD policies. Each PFT lab was asked for their policy regarding spirometry and asthma diagnosis from May 1 to August 31, 2017. RESULTS: A total of 92 PFT labs were identified in Alberta, 74 of which were private FP (independent) labs, while 18 were private NFP (public) hospital-based labs. Policies were as follows: (i) post-BD policy existed (and if so routinely performed / not routinely done); (ii) no post-BD policy; and (iii) lab chose not to participate. All 18 hospital labs responded: 10 had no policy; six had a policy or algorithm; one did not perform post-BD testing (exercise testing) and one had multiple testing sites. Of the private FP labs, three had relevant policies and/or algorithm and 10 had none. No information was provided from 61 labs. Access to PFT labs in Northern Alberta was limited. CONCLUSIONS: Lab policies surrounding post-BD testing were found to be heterogeneous in Alberta. Low response rates, despite the use of a systems approach and requests in writing and in person from FP labs, were notable. Development of a standardized policy across the province would be beneficial. Further higher-level review of the appropriateness of post-BD use in both FP and NFP PFT labs is needed.

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.

Hyperoxia enhances slow-wave forebrain states in urethane-anesthetized and naturally sleeping rats.

Oxygen (O2) is a crucial element for physiological functioning in mammals. In particular, brain function is critically dependent on a minimum amount of circulating blood levels of O2 and both immediate and lasting neural dysfunction can result following anoxic or hypoxic episodes. Although the effects of deficiencies in O2 levels on the brain have been reasonably well studied, less is known about the influence of elevated levels of O2 (hyperoxia) in inspired gas under atmospheric pressure. This is of importance due to its typical use in surgical anesthesia, in the treatment of stroke and traumatic brain injury, and even in its recreational or alternative therapeutic use. Using local field potential (EEG) recordings in spontaneously breathing urethane-anesthetized and naturally sleeping rats, we characterized the influence of different levels of O2 in inspired gases on brain states. While rats were under urethane anesthesia, administration of 100% O2 elicited a significant and reversible increase in time spent in the deactivated (i.e., slow-wave) state, with concomitant decreases in both heartbeat and respiration rates. Increasing the concentration of carbon dioxide (to 5%) in inspired gas produced the opposite result on EEG states, mainly a decrease in the time spent in the deactivated state. Consistent with this, decreasing concentrations of O2 (to 15%) in inspired gases decreased time spent in the deactivated state. Further confirmation of the hyperoxic effect was found in naturally sleeping animals where it similarly increased time spent in slow-wave (nonrapid eye movement) states. Thus alterations of O2 in inspired air appear to directly affect forebrain EEG states, which has implications for brain function, as well as for the regulation of brain states and levels of forebrain arousal during sleep in both normal and pathological conditions. NEW & NOTEWORTHY We show that alterations of oxygen concentration in inspired air biases forebrain EEG state. Hyperoxia increases the prevalence of slow-wave states. Hypoxia and hypercapnia appear to do the opposite. This suggests that oxidative metabolism is an important stimulant for brain state.

Cholinergic modulation of the parafacial respiratory group.

KEY POINTS: This study investigates the effects of cholinergic transmission on the expiratory oscillator, the parafacial respiratory group (pFRG) in urethane anaesthetized adult rats. Local inhibition of the acetyl cholinesterase enzyme induced activation of expiratory abdominal muscles and active expiration. Local application of the cholinomimetic carbachol elicited recruitment of late expiratory neurons, expiratory abdominal muscle activity and active expiration. This effect was antagonized by local application of the muscarinic antagonists scopolamine, J104129 and 4DAMP. We observed distinct physiological responses between the more medial chemosensitive region of the retrotrapezoid nucleus and the more lateral region of pFRG. These results support the hypothesis that pFRG is under cholinergic neuromodulation and the region surrounding the facial nucleus contains a group of neurons with distinct physiological roles. ABSTRACT: Active inspiration and expiration are opposing respiratory phases generated by two separate oscillators in the brainstem: inspiration driven by a neuronal network located in the preBötzinger complex (preBötC) and expiration driven by a neuronal network located in the parafacial respiratory group (pFRG). While continuous activity of the preBötC is necessary for maintaining ventilation, the pFRG behaves as a conditional expiratory oscillator, being silent in resting conditions and becoming rhythmically active in the presence of increased respiratory drive (e.g. hypoxia, hypercapnia, exercise and through release of inhibition). Recent evidence from our laboratory suggests that expiratory activity in the principal expiratory pump muscles, the abdominals, is modulated in a state-dependent fashion, frequently occurring during periods of REM sleep. We hypothesized that acetylcholine, a neurotransmitter released in wakefulness and REM sleep by mesopontine structures, contributes to the activation of pFRG neurons and thus acts to promote the recruitment of expiratory abdominal muscle activity. We investigated the stimulatory effect of cholinergic neurotransmission on pFRG activity and recruitment of active expiration in vivo under anaesthesia. We demonstrate that local application of the acetylcholinesterase inhibitor physostigmine into the pFRG potentiated expiratory activity. Furthermore, local application of the cholinomimetic carbachol into the pFRG activated late expiratory neurons and induced long lasting rhythmic active expiration. This effect was completely abolished by pre-application of the muscarinic antagonist scopolamine, and more selective M3 antagonists 4DAMP and J104129. We conclude that cholinergic muscarinic transmission contributes to excitation of pFRG neurons and promotes both active recruitment of abdominal muscles and active expiratory flow.

Lack of respiratory coupling with neocortical and hippocampal slow oscillations.

Previous work has demonstrated an influence of the respiratory cycle and, more specifically, rhythmic nasal inspiration for the entrainment of slow oscillations in olfactory cortex during ketamine-xylazine anesthesia. This respiratory entrainment has been suggested to occur more broadly during slow-wave states (including sleep) throughout the forebrain, in particular in the frontal and parahippocampal and hippocampal cortices. Using multisite local field potential recording methods and spectral coherence analysis in the rat, we show here that no such broad forebrain coupling takes place during slow-wave activity patterns under either ketamine-xylazine or urethane anesthesia and, furthermore, that it also does not arise during natural slow-wave sleep. Therefore, respiratory-related oscillatory neural activities are likely limited to primary olfactory structures during slow-wave forebrain states.

Stimulation of Respiratory Motor Output and Ventilation in a Murine Model of Pompe Disease by Ampakines.

Pompe disease results from a mutation in the acid α-glucosidase gene leading to lysosomal glycogen accumulation. Respiratory insufficiency is common, and the current U.S. Food and Drug Administration-approved treatment, enzyme replacement, has limited effectiveness. Ampakines are drugs that enhance α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor responses and can increase respiratory motor drive. Recent work indicates that respiratory motor drive can be blunted in Pompe disease, and thus pharmacologic stimulation of breathing may be beneficial. Using a murine Pompe model with the most severe clinical genotype (the Gaa(-/-) mouse), our primary objective was to test the hypothesis that ampakines can stimulate respiratory motor output and increase ventilation. Our second objective was to confirm that neuropathology was present in Pompe mouse medullary respiratory control neurons. The impact of ampakine CX717 on breathing was determined via phrenic and hypoglossal nerve recordings in anesthetized mice and whole-body plethysmography in unanesthetized mice. The medulla was examined using standard histological methods coupled with immunochemical markers of respiratory control neurons. Ampakine CX717 robustly increased phrenic and hypoglossal inspiratory bursting and reduced respiratory cycle variability in anesthetized Pompe mice, and it increased inspiratory tidal volume in unanesthetized Pompe mice. CX717 did not significantly alter these variables in wild-type mice. Medullary respiratory neurons showed extensive histopathology in Pompe mice. Ampakines stimulate respiratory neuromotor output and ventilation in Pompe mice, and therefore they have potential as an adjunctive therapy in Pompe disease.

Optogenetic excitation of preBötzinger complex neurons potently drives inspiratory activity in vivo.

KEY POINTS: This study investigates the effects on ventilation of an excitatory stimulus delivered in a spatially and temporally precise manner to the inspiratory oscillator, the preBötzinger complex (preBötC). We used an adeno-associated virus expressing channelrhodopsin driven by the synapsin promoter to target the region of the preBötC. Unilateral optogenetic stimulation of preBötC increased respiratory rate, minute ventilation and increased inspiratory modulated genioglossus muscle activity. Unilateral optogenetic stimulation of preBötC consistently entrained respiratory rate up to 180 breaths min(-1) both in presence of ongoing respiratory activity and in absence of inspiratory activity. Unilateral optogenetic stimulation of preBötC induced a strong phase-independent Type 0 respiratory reset, with a short delay in the response of 100 ms. We identified a refractory period of ∼200 ms where unilateral preBötC optogenetic stimulation is not able to initiate the next respiratory event. ABSTRACT: Understanding the sites and mechanisms underlying respiratory rhythmogenesis is of fundamental interest in the field of respiratory neurophysiology. Previous studies demonstrated the necessary and sufficient role of preBötzinger complex (preBötC) in generating inspiratory rhythms in vitro and in vivo. However, the influence of timed activation of the preBötC network in vivo is as yet unknown given the experimental approaches previously used. By unilaterally infecting preBötC neurons using an adeno-associated virus expressing channelrhodopsin we photo-activated the network in order to assess how excitation delivered in a spatially and temporally precise manner to the inspiratory oscillator influences ongoing breathing rhythms and related muscular activity in urethane-anaesthetized rats. We hypothesized that if an excitatory drive is necessary for rhythmogenesis and burst initiation, photo-activation of preBötC not only will increase respiratory rate, but also entrain it over a wide range of frequencies with fast onset, and have little effect on ongoing respiratory rhythm if a stimulus is delivered during inspiration. Stimulation of preBötC neurons consistently increased respiratory rate and entrained respiration up to fourfold baseline conditions. Furthermore, brief pulses of photostimulation delivered at random phases between inspiratory events robustly and consistently induced phase-independent (Type 0) respiratory reset and recruited inspiratory muscle activity at very short delays (∼100 ms). A 200 ms refractory period following inspiration was also identified. These data provide strong evidence for a fine control of inspiratory activity in the preBötC and provide further evidence that the preBötC network constitutes the fundamental oscillator of inspiratory rhythms.

Knockdown of PHOX2B in the retrotrapezoid nucleus reduces the central CO2 chemoreflex in rats.

PHOX2B is a transcription factor essential for the development of different classes of neurons in the central and peripheral nervous system. Heterozygous mutations in the PHOX2B coding region are responsible for the occurrence of Congenital Central Hypoventilation Syndrome (CCHS), a rare neurological disorder characterised by inadequate chemosensitivity and life-threatening sleep-related hypoventilation. Animal studies suggest that chemoreflex defects are caused in part by the improper development or function of PHOX2B expressing neurons in the retrotrapezoid nucleus (RTN), a central hub for CO2 chemosensitivity. Although the function of PHOX2B in rodents during development is well established, its role in the adult respiratory network remains unknown. In this study, we investigated whether reduction in PHOX2B expression in chemosensitive neuromedin-B (NMB) expressing neurons in the RTN altered respiratory function. Four weeks following local RTN injection of a lentiviral vector expressing the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of PHOX2B expression was observed in Nmb neurons compared to both naive rats and rats injected with the non-target shRNA. PHOX2B knockdown did not affect breathing in room air or under hypoxia, but ventilation was significantly impaired during hypercapnia. PHOX2B knockdown did not alter Nmb expression but it was associated with reduced expression of both Task2 and Gpr4, two CO2/pH sensors in the RTN. We conclude that PHOX2B in the adult brain has an important role in CO2 chemoreception and reduced PHOX2B expression in CCHS beyond the developmental period may contribute to the impaired central chemoreflex function.

Etonogestrel promotes respiratory recovery in an in vivo rat model of central chemoreflex impairment.

AIM: The central CO2 chemoreflex is a vital component of respiratory control networks, providing excitatory drive during resting conditions and challenges to blood gas homeostasis. The retrotrapezoid nucleus is a crucial hub for CO2 chemosensitivity; its ablation or inhibition attenuates CO2 chemoreflexes and diminishes restful breathing. Similar phenotypes characterize certain hypoventilation syndromes, suggesting underlying retrotrapezoid nucleus impairment in these disorders. Progesterone stimulates restful breathing and CO2 chemoreflexes. However, its mechanisms and sites of actions remain unknown and the experimental use of synthetic progestins in patients and animal models have been met with mixed respiratory outcomes. METHODS: We investigated whether acute or chronic administration of the progestinic drug, etonogestrel, could rescue respiratory chemoreflexes following selective lesion of the retrotrapezoid nucleus with saporin toxin. Adult female Sprague Dawley rats were grouped based on lesion size determined by the number of surviving chemosensitive neurons, and ventilatory responses were measured by whole body plethysmography. RESULTS: Ventilatory responses to hypercapnia (but not hypoxia) were compromised in a lesion-dependent manner. Chronic etonogestrel treatment improved CO2 chemosensitivity selectively in rats with moderate lesion, suggesting that a residual number of chemosensitive neurons are required for etonogestrel-induced CO2 chemoreflex recovery. CONCLUSION: This study provides new evidence for the use of progestins as respiratory stimulants under conditions of central hypoventilation and provides a new testable model for assessing the mechanism of action of progestins in the respiratory network.

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.

Active expiration induced by excitation of ventral medulla in adult anesthetized rats.

Data from perinatal and juvenile rodents support our hypothesis that the preBötzinger complex generates inspiratory rhythm and the retrotrapezoid nucleus-parafacial respiratory group (RTN/pFRG) generates active expiration (AE). Although the role of the RTN/pFRG in adulthood is disputed, we hypothesized that its rhythmogenicity persists but is typically silenced by synaptic inhibition. We show in adult anesthetized rats that local pharmacological disinhibition or optogenetic excitation of the RTN/pFRG can generate AE and transforms previously silent RTN/pFRG neurons into rhythmically active cells whose firing is correlated with late-phase active expiration. Brief excitatory stimuli also reset the respiratory rhythm, indicating strong coupling of AE to inspiration. The AE network location in adult rats overlaps with the perinatal pFRG and appears lateral to the chemosensitive region of adult RTN. We suggest that (1) the RTN/pFRG contains a conditional oscillator that generates AE, and (2) at rest and in anesthesia, synaptic inhibition of RTN/pFRG suppresses AE.

Prefrontal-Hippocampal Pathways Through the Nucleus Reuniens Are Functionally Biased by Brain State.

Circuit-level communication between disparate brain regions is fundamental for the complexities of the central nervous system operation. Co-ordinated bouts of rhythmic activity between the prefrontal cortex (PFC) and hippocampus (HPC), in particular, are important for mnemonic processes. This is true during awake behavior, as well as during offline states like sleep. We have recently shown that the anatomically interposed thalamic nucleus reuniens (RE) has a role in coordinating slow-wave activity between the PFC and HPC. Here, we took advantage of spontaneous brain state changes occurring during urethane anesthesia in order to assess if PFC-HPC communication was modified during activated (theta) vs. deactivated (slow oscillation: SO) states. These forebrain states are highly similar to those expressed during rapid eye movement (REM) and non-REM stages of natural sleep, respectively. Evoked potentials and excitatory current sinks in the HPC were consistently larger during SO states, regardless of whether PFC or RE afferents were stimulated. Interestingly, PFC stimulation during theta appeared to preferentially use a cortico-cortical pathway, presumably involving the entorhinal cortex as opposed to the more direct RE to HPC conduit. Optogenetic and chemogenetic manipulations of the RE suggested that this state-dependent biasing was mediated by responding in the RE itself. Finally, the phase of both ongoing rhythms also appeared to be an important factor in modulating HPC responses, with maximal field excitatory postsynaptic potentials (EPSPs) occurring during the negative-going phase of both rhythms. Thus, forebrain state plays an important role in how communication takes place across the PFC and HPC, with the RE as a determining factor in how this is shaped. Furthermore, ongoing sleep-like rhythms influence the coordination and perhaps potentiate excitatory processing in this extended episodic memory circuit. Our results have direct implications for activity-dependent processes relevant to sleep-dependent memory consolidation.

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.

Neural crest-specific deletion of Bmp7 leads to midfacial hypoplasia, nasal airway obstruction, and disordered breathing modelling Obstructive Sleep Apnea.

Pediatric obstructive sleep apnea (OSA), a relatively common sleep-related breathing disorder (SRBD) affecting approximately 1-5% of children, is often caused by anatomical obstruction and/or collapse of the nasal and/or pharyngeal airways. The resulting sleep disruption and intermittent hypoxia lead to various systemic morbidities. Predicting the development of OSA from craniofacial features alone is currently not possible and a controversy remains if upper airway obstruction facilitates reduced midfacial growth or vice-versa. Currently, there is no rodent model that recapitulates both the development of craniofacial abnormalities and upper airway obstruction to address these questions. Here, we describe that mice with a neural crest-specific deletion of Bmp7 (Bmp7ncko) present with shorter, more acute angled cranial base, midfacial hypoplasia, nasal septum deviation, turbinate swelling and branching defects, and nasal airway obstruction. Interestingly, several of these craniofacial features develop after birth during periods of rapid midfacial growth and precede the development of an upper airway obstruction. We identified that in this rodent model, no single feature appeared to predict upper airway obstruction, but the sum of those features resulted in a reduced breathing frequency, apneas and overall reduced oxygen consumption. Metabolomics analysis of serum from peripheral blood identified increased levels of hydroxyproline, a metabolite upregulated under hypoxic conditions. As this model recapitulates many features observed in OSA, it offers unique opportunities for studying how upper airway obstruction affects breathing physiology and leads to systemic morbidities.