Brainstem Respiratory Neurons Research Articles

Exploring Serotonergic mechanisms in Opioid Induced Respiratory Depression

Opioids profoundly depress breathing which can be fatal in the case of overdose. Despite growing knowledge of the pontine and medullary respiratory control circuits that are inhibited by opioids, the circuit and cellular mechanisms of opioid induced respiratory depression are not fully understood. Serotonin neurons located in the caudal medullary raphe modulate breathing and possess mu opioid receptors. Therefore, we hypothesized that serotonin-producing raphe neurons contribute to opioid induced respiratory depression. To test this, we employed to experimental approaches: 1) arterially perfused in situ working heart brainstem preparations and 2) ex vivo acute brain slice electrophysiology to identify and characterize serotonin neuron inputs onto brainstem respiratory neurons. To specifically activate serotonin neurons, we used ePet-Cre::Ai32 mice which Cre-dependently express channelrhodopsin-2, a light-activated cation channel, in serotonin neurons. At the circuit level, using the in situ preparation, we found that activation of caudal medullary raphe serotonin neurons increased respiratory rate by decreasing inspiratory and expiratory duration. Fentanyl application blocked the respiratory effects induced by serotonin neuron activation which were subsequently restored using naloxone. These results suggest serotonin neurons in the caudal medullary raphe are inhibited by opioids. To characterize the serotonin-mediated mechanisms at the cellular level, we made acute brain slices containing dorsal raphe neurons and lateral parabrachial neurons from ePet-Cre::Ai32 mice. We verified that optical stimulation resulted in action potentials and serotonin release from dorsal raphe serotonin neurons. Optically evoked serotonin release caused 5HT1A receptor-mediated outward currents in dorsal raphe serotonin neurons. To locate and characterize serotonin neuron inputs onto lateral parabrachial neurons, we performed whole-cell voltage clamp recordings from lateral parabrachial neurons and delivered paired light pulses (10 ms duration, 50 ms interval). We observed optically evoked inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA-A receptor antagonist gabazine (1 μM). Optically evoked IPSCs had long latency (> 5 ms) and high jitter (> 1 ms), indicating stimulation of serotonin terminals resulted in polysynaptic GABA release onto lateral parabrachial neurons. Future work will examine the influence of opioids on this raphe-parabrachial circuit. Uncovering the role of serotonin neurons in opioid induced respiratory depression may reveal targets for restoring breathing in the presence of opioids or other conditions where life-threatening apneas occur. This work was supported by the National Institutes of Health (NIDA) R01DA047978-05 grant to ESL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inaugural Review Prize 2023: The exercise hyperpnoea dilemma: A 21st-century perspective.

During mild or moderate exercise, alveolar ventilation increases in direct proportion to metabolic rate, regulating arterial CO2 pressure near resting levels. Mechanisms giving rise to the hyperpnoea of exercise are unsettled despite over a century of investigation. In the past three decades, neuroscience has advanced tremendously, raising optimism that the 'exercise hyperpnoea dilemma' can finally be solved. In this review, new perspectives are offered in the hope of stimulating original ideas based on modern neuroscience methods and current understanding. We first describe the ventilatory control system and the challenge exercise places upon blood-gas regulation. We highlight relevant system properties, including feedforward, feedback and adaptive (i.e., plasticity) control of breathing. We then elaborate a seldom explored hypothesis that the exercise ventilatory response continuously adapts (learns and relearns) throughout life and ponder if the memory 'engram' encoding the feedforward exercise ventilatory stimulus could reside within the cerebellum. Our hypotheses are based on accumulating evidence supporting the cerebellum's role in motor learning and the numerous direct and indirect projections from deep cerebellar nuclei to brainstem respiratory neurons. We propose that cerebellar learning may be obligatory for the accurate and adjustable exercise hyperpnoea capable of tracking changes in life conditions/experiences, and that learning arises from specific cerebellar microcircuits that can be interrogated using powerful techniques such as optogenetics and chemogenetics. Although this review is speculative, we consider it essential to reframe our perspective if we are to solve the till-now intractable exercise hyperpnoea dilemma.

Transcription factors regulating the specification of brainstem respiratory neurons.

Breathing (or respiration) is an unconscious and complex motor behavior which neuronal drive emerges from the brainstem. In simplistic terms, respiratory motor activity comprises two phases, inspiration (uptake of oxygen, O2) and expiration (release of carbon dioxide, CO2). Breathing is not rigid, but instead highly adaptable to external and internal physiological demands of the organism. The neurons that generate, monitor, and adjust breathing patterns locate to two major brainstem structures, the pons and medulla oblongata. Extensive research over the last three decades has begun to identify the developmental origins of most brainstem neurons that control different aspects of breathing. This research has also elucidated the transcriptional control that secures the specification of brainstem respiratory neurons. In this review, we aim to summarize our current knowledge on the transcriptional regulation that operates during the specification of respiratory neurons, and we will highlight the cell lineages that contribute to the central respiratory circuit. Lastly, we will discuss on genetic disturbances altering transcription factor regulation and their impact in hypoventilation disorders in humans.

Activation of Brainstem mu‐Opioid Receptors (mORs) Increases Respiratory‐Related Motor Burst Amplitude in Neonatal Rat Brainstem‐Spinal Cord Preparations

Mu‐opioid receptors (mORs) located on brainstem respiratory neurons are part of an intricate peptidergic neuromodulatory system that regulates breathing. The endogenous role of mORs in respiratory motor control, however, is poorly understood. To explore this question, very low (nanomolar) concentrations of mOR agonist drugs were bath‐applied to neonatal rat (P1‐P3) brainstem‐spinal cord preparations while measuring respiratory‐related motor output from spinal C4‐C5 rootlets containing phrenic motor neuron axons. When DAMGO (synthetic mOR agonist) or endomorphin‐2 (endogenous mOR ligand) were bath‐applied for 15 min at 20‐100 nM, respiratory motor burst amplitude increased dramatically with a modest decrease in burst frequency. Burst amplitude facilitated by 45 ± 38% and 32 ± 24% (mean ± stdev) above baseline for 50 nM DAMGO (n=8) and 100 nM endomorphin‐2 (n=10), respectively (p<0.001). To test whether respiratory motor output on thoracic ventral roots was altered by mOR activation, brainstem‐spinal cord preparations were isolated to include spinal segments C1‐T8. Bath‐applied DAMGO (50 nM) induced burst amplitude facilitation on thoracic roots T4‐T6 and cervical roots C4‐C5 by 62 ± 19% and 38 ± 36%, respectively (p<0.012). To test whether mOR‐induced burst facilitation is due to brainstem or spinal mechanisms, a split‐bath chamber with a barrier at C1‐C2 was used to separately bathe the brainstem and spinal cord compartments. DAMGO (50 nM) application to the spinal cord (C2‐C8) had little effect on respiratory motor output measured from C4‐C5 rootlets, whereas DAMGO application to the brainstem increased C4‐C5 motor output by 34 ± 19% (p<0.004). These data suggest that mOR activation in the brainstem can transform the respiratory network to produce a motor pattern with decreased frequency balanced by increased amplitude, with relatively little change in overall neural output. Given that mOR‐induced facilitation was observed in both cervical and thoracic motor neuron pools, we hypothesize that burst amplitude facilitation is due to mOR activation on rhythm‐generating neurons or premotor neurons in the brainstem. This study reveals that endogenous mOR activation modulates the neural control of breathing in a manner that is potentially different from respiratory depression, which is more commonly associated with opioids.

Neonatal apneic phenotype in a murine congenital central hypoventilation syndrome model is induced through non-cell autonomous developmental mechanisms.

Congenital central hypoventilation syndrome (CCHS) represents a rare genetic disorder usually caused by mutations in the homeodomain transcription factor PHOX2B. Some CCHS patients suffer mainly from deficiencies in CO2 and/or O2 respiratory chemoreflex, whereas other patients present with full apnea shortly after birth. Our goal was to identify the neuropathological mechanisms of apneic presentations in CCHS. In the developing murine neuroepithelium, Phox2b is expressed in three discrete progenitor domains across the dorsal‐ventral axis, with different domains responsible for producing unique autonomic or visceral motor neurons. Restricting the expression of mutant Phox2b to the ventral visceral motor neuron domain induces marked newborn apnea together with a significant loss of visceral motor neurons, RTN ablation, and preBötzinger complex dysfunction. This finding suggests that the observed apnea develops through non‐cell autonomous developmental mechanisms. Mutant Phox2b expression in dorsal rhombencephalic neurons did not generate significant respiratory dysfunction, but did result in subtle metabolic thermoregulatory deficiencies. We confirm the expression of a novel murine Phox2b splice variant which shares exons 1 and 2 with the more widely studied Phox2b splice variant, but which differs in exon 3 where most CCHS mutations occur. We also show that mutant Phox2b expression in the visceral motor neuron progenitor domain increases cell proliferation at the expense of visceral motor neuron development. We propose that visceral motor neurons may function as organizers of brainstem respiratory neuron development, and that disruptions in their development result in secondary/non‐cell autonomous maldevelopment of key brainstem respiratory neurons.

Pilocarpine-induced status epilepticus reduces chemosensory control of breathing

One of the possible causes of death in epilepsy is breathing disorders, especially apneas, which lead to an increase in CO2 levels (hypercapnia) and/or a decrease in O2 levels in arterial blood (hypoxemia). The respiratory neurons located in the ventral brainstem respiratory column are the main groups responsible for controlling breathing. Recent data from our group demonstrated respiratory changes in two experimental models of epilepsy, i.e. audiogenic epilepsy, and amygdala rapid kindling. Here, we aimed to evaluate respiratory changes in the classic model of temporal lobe epilepsy induced by intra-hippocampal injection of pilocarpine. Adult Wistar rats with stainless-steel cannulas implanted in the hippocampus region were used. The animals were submitted to pilocarpine injection (2.4 mg/μL, N = 12–15) or saline (N = 9) into the hippocampus. The respiratory parameters analyzed by whole-body plethysmography were respiratory rate (fR), tidal volume (VT) and ventilation (VE). Respiratory mechanics such as Newtonian airway resistance (Rn), viscance of the pulmonary parenchyma (G) and the elastance of the pulmonary parenchyma (H) were also investigated. No changes in baseline breathing were detected 15 or 30 days after pilocarpine-induced status epilepticus (SE). However, 30 days after pilocarpine-induced SE, a significant reduction in VE was observed during hypercapnic (7% CO2) stimulation, without affecting the hypoxia (8% O2) ventilatory response. We also did not observe changes in respiratory mechanics. The present results suggest that the impairment of the hypercapnia ventilatory response in pilocarpine-induced SE could be related to a presumable degeneration of brainstem respiratory neurons but not to peripheral mechanisms.

Mecp2 Disruption in Rats Causes Reshaping in Firing Activity and Patterns of Brainstem Respiratory Neurons

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations in the MECP2 gene, have breathing abnormalities manifested as periodical hypoventilation with compensatory hyperventilation, which are attributable to a high incidence of sudden death. Similar breathing abnormalities have been found in animal models with Mecp2 disruptions. Although RTT-type hypoventilation is believed to be due to depressed central inspiratory activity, whether this is true remains unknown. Here we show evidence for reshaping in firing activity and patterns of medullary respiratory neurons in RTT-type hypoventilation without evident depression in inspiratory neuronal activity. Experiments were performed in decerebrate rats in vivo. In Mecp2-null rats, abnormalities in breathing patterns were apparent in both decerebrate rats and awake animals, suggesting that RTT-type breathing abnormalities take place in the brainstem without forebrain input. In comparison to their wild-type counterparts, both inspiratory and expiratory neurons in Mecp2-null rats extended their firing duration, and fired more action potentials during each burst. No changes in inspiratory or expiratory neuronal distributions were found. Most inspiratory neurons started firing in the middle of expiration and changed their firing pattern to a phase-spanning type. The proportion of post-inspiratory neurons was reduced in the Mecp2-null rats. With the increased firing activity of both inspiratory and expiratory neurons in null rats, phrenic discharges shifted to a slow and deep breathing pattern. Thus, the RTT-type hypoventilation appears to result from reshaping of firing activity of both inspiratory and expiratory neurons without evident depression in central inspiratory activity.

Activation of the Medial Prefrontal Cortex Reverses Cognitive and Respiratory Symptoms in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2; Amir et al., 1999), a transcriptional regulatory protein (Klose et al., 2005). Mouse models of RTT (Mecp2 mutants) exhibit excitatory hypoconnectivity in the medial prefrontal cortex (mPFC; Sceniak et al., 2015), a region critical for functions that are abnormal in RTT patients, ranging from learning and memory to regulation of visceral homeostasis (Riga et al., 2014). The present study was designed to test the hypothesis that increasing the activity of mPFC pyramidal neurons in heterozygous female Mecp2 mutants (Hets) would ameliorate RTT-like symptoms, including deficits in respiratory control and long-term retrieval of auditory conditioned fear. Selective activation of mPFC pyramidal neurons in adult animals was achieved by bilateral infection with an AAV8 vector expressing excitatory hm3D(Gq) DREADD (Designer Receptors Exclusively Activated by Designer Drugs) (Armbruster et al., 2007) under the control of the CamKIIa promoter. DREADD activation in Mecp2 Hets completely restored long-term retrieval of auditory conditioned fear, eliminated respiratory apneas, and reduced respiratory frequency variability to wild-type (Wt) levels. Reversal of respiratory symptoms following mPFC activation was associated with normalization of Fos protein levels, a marker of neuronal activity, in a subset of brainstem respiratory neurons. Thus, despite reduced levels of MeCP2 and severe neurological deficits, mPFC circuits in Het mice are sufficiently intact to generate normal behavioral output when pyramidal cell activity is increased. These findings highlight the contribution of mPFC hypofunction to the pathophysiology of RTT and raise the possibility that selective activation of cortical regions such as the mPFC could provide therapeutic benefit to RTT patients.

Morphology of Dbx1 respiratory neurons in the preB\xf6tzinger complex and reticular formation of neonatal mice

The relationship between neuron morphology and function is a perennial issue in neuroscience. Information about synaptic integration, network connectivity, and the specific roles of neuronal subpopulations can be obtained through morphological analysis of key neurons within a microcircuit. Here we present morphologies of two classes of brainstem respiratory neurons. First, interneurons derived from Dbx1-expressing precursors (Dbx1 neurons) in the preBötzinger complex (preBötC) of the ventral medulla that generate the rhythm for inspiratory breathing movements. Second, Dbx1 neurons of the intermediate reticular formation that influence the motor pattern of pharyngeal and lingual movements during the inspiratory phase of the breathing cycle. We describe the image acquisition and subsequent digitization of morphologies of respiratory Dbx1 neurons from the preBötC and the intermediate reticular formation that were first recorded in vitro. These data can be analyzed comparatively to examine how morphology influences the roles of Dbx1 preBötC and Dbx1 reticular interneurons in respiration and can also be utilized to create morphologically accurate compartmental models for simulation and modeling of respiratory circuits.

Intraspinal microstimulation induced respiratory phase resetting

Electrical stimulation of the spinal cord can activate somatic motor circuits after spinal cord injury. In particular, stimulation of the dorsal spinal cord has translated into clinical use due to the relatively low invasiveness. Modeling studies indicate that dorsal activation of motor pathways occurs through excitation of afferent projections to spinal motor pools. However, because spinal stimulation does not always allow for activation of discrete neuronal populations, off target effects are often observed. In ongoing studies, our group is investigating intraspinal microstimulation as a tool to activate respiratory motoneurons with possible application to spinal cord injury. Currently it is unclear if and how intraspinal stimulation will alter the pattern of respiratory output produced by the brainstem. However, activation of ascending respiratory‐related afferent fibers in cervical dorsal white matter or spinobulbar projections from cervical interneurons has the potential to modulate the respiratory pattern. Here we tested the hypothesis that intraspinal microstimulation in the dorsal but not ventral mid‐cervical spinal cord would rapidly alter the timing of inspiratory discharge. Diaphragm, external intercostal, and sternocleidomastoid electromyography activity was recorded in N=6 anesthetized and ventilated spinal intact adult rats. Following unilateral laminectomy from C2‐T1 a 10×2 grid was established by dividing the rostrocaudal exposed cord into ten sites with the two stimulus tracts located 0.5 and 1mm lateral to midline. A tungsten electrode was gradually advanced from the dorsal spinal surface to depth of 1800–2400μm with 10–90 μA currents (0.3 ms per phase, biphasic pulses) delivered every 600 μm. Dorsal stimulation (average simulation depth of 478 μm) resulted in an abrupt increase in respiratory rate (breaths per minute) at~13% of tested sites. Of the sites that triggered increases in respiratory rate, 54% were located at the dorsal surface (i.e., depths of 0 μm). These initial results indicate that intraspinal stimulation of the dorsal cervical spinal cord can trigger rapid changes in respiratory rate, presumably via activation of ascending pathways which innervate brainstem respiratory neurons.Support or Funding InformationFUNDING: 1R01NS080180‐01A1 (DDF), NICHD: T32‐HD043730 (MDS), Department of Defense W81XWH‐14‐1‐0625 (PJR)

Role of respiration in mind-body practices: concepts from contemporary science and traditional yoga texts.

The concept that interactions between the mind and body influence physical health has existed for a long time (1). These interactions form the basis for mind–body practices. They are considered bi-directional (“top-down” and “bottom-up”), with connections between the brain and peripheral tissues (2). Top-down mechanisms are initiated by the cerebral cortex and include clinical hypnosis, imagery, meditation, and breath awareness. Bottom-up mechanisms on the other hand, stimulate various somato-sensory, viscero-sensory, and chemo-sensory receptors (3, 4), which influence ascending pathways from the periphery to the brainstem and cerebral cortex. Mind-body practices are believed to act at multiple levels from gene expression at the cellular level to interaction between central brain regions (2). Taylor et al. (2) have proposed four mechanisms: (a) reorganization of cortical and subcortical structures and better interhemispheric balance; (b) improved central regulation of autonomic and immune functions; (c) re-patterning of primary interoceptive and higher order homeostatic mechanisms; and (d) modulation of epigenetic factors such as growth factors or hormones. These four effects are achieved by different ways including mental imagery, somatic relaxation, and deep breathing. Based on these effects, most mind–body practices are used in the treatment of psychosomatic illness. The ancient Indian practice of yoga is a mind–body practice. According to yoga texts, it is important to understand the origin of psychosomatic illness in order to treat it (Taittiriya Upanishad, circa 1200 B.C1) (5). This text continues to state that mental conflict, especially conflict between the intellect (called the vignanamaya kosha in Sanskrit) and the instinct (called the manomaya kosha in Sanskrit) causes a disturbance in the subtle, life energy, called prana in Sanskrit (5). This concept of an imbalance in the subtle life energy is mentioned in traditional descriptions of yoga but is not mentioned in contemporary medicine. Traditional yoga texts also suggest a solution for the imbalance in prana, through slow, deep breathing [Hatha Yoga Pradipika, circa 300 A.D.1] (6). It has been said that “when the mental state is disturbed, the life energy (prana) gets unbalanced and this leads to irregular breath; hence to regulate the mental state the yoga practitioner should regulate the breath” [Hatha Yoga Pradipika, circa 300 A.D.; Chapter 2, Verse 161] (6). Yoga regulated breathing acts as both a top-down and bottom-up mind-body practice. There is sufficient neuroanatomical evidence to support the idea that apart from the metabolic (chemoreceptor based) regulation of breathing internal and external factors influence breathing, which has been called behavioral breathing (7). Connections between cortical regions and brainstem respiratory neurons indicate that influences from higher centers can modify metabolic breathing (8, 9). A functional magnetic resonance study in normal subjects who had air hunger (with a low tidal volume) induced by mechanical-ventilation showed increased activity in limbic and paralimbic loci (10). Apart from these central connections, peripheral factors can also influence respiration. Nasal breathing activates olfactory cells; they activate the olfactory bulb and in turn the pre-piriform cortex and piriform cortex (11). Olfactory information ascends directly to limbic areas and hence olfactory information, which can be indirectly connected to breathing, influences emotions. Apart from being slow, deep and diaphragmatic, yoga breathing includes awareness of the movement of air in the nasal passages (12). Subjective awareness of inner sensations is referred to as interoception (13). An imaging study matched individuals’ subjective perception of their heartbeat with psychometric measures of their interoceptive awareness and emotionality (14). Their findings showed that the right anterior insula (rAI) is important for explicit subjective awareness. Conventional physiology has found benefits of deep breathing supporting the importance given to regulating the breath in yoga. Slow breathing had a balancing effect on the autonomic nervous system via enhanced parasympathetic activation (15). Slow and deep breathing stimulates inhibitory signals induced by stretch and hyperpolarizes cells, leading to synchronization of neural elements in the heart, lungs, limbic system, and cortex (16). Slow breathing also enhances vagal activity leading to reduced psychophysiological arousal, decreased sympathetic activity, and stress responses (17). Other effects include an increase in the antioxidant levels and hence deep breathing may help to reduce oxidative stress (18). In addition, deep breathing has been found to decrease cortisol and increase melatonin (19), possibly by the effects on the hypothalamic neuroendocrine regulation (15).

The nucleus of the solitary tract and the coordination of respiratory and sympathetic activities.

It is well known that breathing introduces rhythmical oscillations in the heart rate and arterial pressure levels. Sympathetic oscillations coupled to the respiratory activity have been suggested as an important homeostatic mechanism optimizing tissue perfusion and blood gas uptake/delivery. This respiratory-sympathetic coupling is strengthened in conditions of blood gas challenges (hypoxia and hypercapnia) as a result of the synchronized activation of brainstem respiratory and sympathetic neurons, culminating with the emergence of entrained cardiovascular and respiratory reflex responses. Studies have proposed that the ventrolateral region of the medulla oblongata is a major site of synaptic interaction between respiratory and sympathetic neurons. However, other brainstem regions also play a relevant role in the patterning of respiratory and sympathetic motor outputs. Recent findings suggest that the neurons of the nucleus of the solitary tract (NTS), in the dorsal medulla, are essential for the processing and coordination of respiratory and sympathetic responses to hypoxia. The NTS is the first synaptic station of the cardiorespiratory afferent inputs, including peripheral chemoreceptors, baroreceptors and pulmonary stretch receptors. The synaptic profile of the NTS neurons receiving the excitatory drive from afferent inputs is complex and involves distinct neurotransmitters, including glutamate, ATP and acetylcholine. In the present review we discuss the role of the NTS circuitry in coordinating sympathetic and respiratory reflex responses. We also analyze the neuroplasticity of NTS neurons and their contribution for the development of cardiorespiratory dysfunctions, as observed in neurogenic hypertension, obstructive sleep apnea and metabolic disorders.

Degeneration of Brainstem Respiratory Neurons in Dementia with Lewy Bodies

Respiratory dysfunction, including sleep disordered breathing, is characteristic of multiple system atrophy (MSA) and may reflect degeneration of brainstem respiratory nuclei involved in respiratory rhythmogenesis and chemosensitivity, including the pre-Bötzinger complex (preBötC), nucleus raphe pallidus (RPa), and nucleus raphe obscurus (ROb). However, impaired ventilatory responses to hypercapnia have also been reported in dementia with Lewy bodies (DLB), suggesting that these nuclei may also be affected in DLB. To determine whether there is involvement of the preBötC, RPa, and ROb in DLB. We applied stereological methods to analyze sections immunostained for neurokinin-1 receptor and tryptophan hydroxylase in neuropathologically confirmed cases of DLB, MSA, and controls. Reduction of neuronal density occurred in all three nuclei in DLB, as well as in MSA. The magnitude of neuronal depletion in ROb was similar in DLB and MSA (49% versus 56% respectively, compared to controls, P < 0.05), but neuronal loss in the preBötC and RPa was less severe in DLB than in MSA (40% loss in preBötC of DLB, P < 0.05 and 68% loss in MSA, P < 0.0001, compared to controls; 46% loss in RPa of DLB, P < 0.05 and 73% loss in MSA P < 0.0001, compared to controls). Medullary respiratory nuclei are affected in dementia with Lewy bodies but less severely than in multiple system atrophy. This may help explain differences in the frequency of sleep disordered breathing in these two disorders.

Rebuttal from Luc J. Teppema and Curtis A. Smith.

Duffin & Mateika (2013) state that their rebreathing data could also be fitted to a parabola. If they consider a parabolic shape an indication of hyper-additive interaction, it is surprising that they did not compare the quality of the fits of linear regression vs. parabolic or hyperbolic fits in order to decide which interaction mode would fit their data best. Apart from this, we have doubts about several assumptions underlying the modified rebreathing technique: (1) the absence of carotid body activity in hyperoxia; (2) that the hypoxic response would be a modified acidic response; (3) due to variable changes in CBF during the manouevre, the tissue–arterial relationship cannot be constant (Battisti-Charbonney et al. 2011); and (4) absence of cortical influences on ventilation following 5 min of voluntary hyperventilation. As explained by Robbins (1988), a linear – relationship is not inconsistent with multiplicative interaction. As outlined previously (Teppema & Berendsen, 2012), we do not agree with the claim of simple addition in humans by Cui et al. (2012) because they ignored the O2–CO2 interaction within the carotid bodies. The fact that increasing carotid body output decreases the central CO2 threshold (Wilson & Day, 2013) is not inconsistent with (hyper)addition. By itself, it does not indicate hypoaddition. In the dog model, blood pressure changes in the carotid sinus did not change ventilation despite potential cross-talk between brainstem sympathetic and respiratory neurons (Saupe et al. 1995). There was no evidence of retrogradely perfused blood affecting ventilation via the brainstem. In dogs and goats, unilateral CBD has no functional implications (Smith et al. 1995). Reversing the stimulus order in steady-state conditions has no effect (Adams et al. 1978). The presentation order of changes in stimuli is not always peripheral and then central. Ischaemia or changes in CBF will first increase brain . During an apnoea, will rise first but thereafter it no longer depends on ventilation and tissue will rise faster than . Changes in metabolism may also cause changes in tissue /pH, to be followed later by changes in the arterial blood. The hybrid model (Wilson & Day, 2013) is inconsistent with data from dogs during the hyperventilation secondary to hypoxic exposure in non-REM sleep which showed an increased propensity for apnoea due to a steeper, not shallower, ventilatory response slope (Nakayama et al. 2002).

Very low levels of H2S in the blood are needed to affect the medullary respiratory neurons and the arterial chemoreceptors in‐vivo

Endogenous H2S has been proposed to be involved in the generation of breathing by the brainstem respiratory neurons (RN) and to transduce the effects of hypoxemia in the arterial chemoreceptors (CB). However, H2S at concentrations which should inhibit the cytochrome C oxidase (CCO) activity, i.e. above 30 μM, have been used in‐vitro to stimulate the CB (30–100 μM) or to affect the RN (200 μM). We determined the concentration of H2S dissolved in the arterial blood able to stimulate the CB and to stop breathing in 8 spontaneously‐breathing anesthetized rats. H2S partial pressure (PaH2S) and gaseous concentrations were determined in the arterial blood from alveolar PH2S during continuous venous infusion of NaHS. This approach overcomes most of the issues of other techniques that directly measure H2S dissolved in the blood. A stimulation of breathing occurred for a PaH2S of 0.007±0.003 mmHg, corresponding to a concentration of gaseous and total dissolved H2S of at most 0.63±0.31 μM and 1.89±0.93 μM. A rapid reduction in breathing occurred at a PaH2S of 0.042±0.020 mmHg, gaseous H2S of 3.68±1.72 μM, and total dissolved H2S of 11.04±5.16 μM. H2S affects the CB and RN in‐vivo via mechanisms independent of CCO. Whether these low levels of dissolved H2S are low enough to be compatible with the levels of H2S endogenously produced remains to be determined. Support provided by NIH grant 1R21NS080788–01.

Persistent activity and interactions in the brainstem respiratory network during hyperventilatory apnea

Brainstem respiratory neuron interactions during hyperventilatory apnea (HA) and the dissolution and reemergence of the motor pattern for breathing remain poorly understood. We used multi‐array recording, cross‐correlation, and gravity clustering methods to evaluate spike train data from the ventral respiratory column (VRC), pontine respiratory group, and medullary raphé nuclei of decerebrate vagotomized neuromuscularly‐blocked cats. Phrenic activity, blood pressure, end‐tidal CO2, and arterial blood gas measures of O2, CO2 and pH were monitored. During HA: (i) tonic activities persisted in subsets of VRC inspiratory (I) and expiratory (E) neurons; (ii) short‐time scale correlations indicated interactions among neurons that expressed E and non‐respiratory modulated discharge patterns during control conditions, including neurons with functional inhibitory actions on I neurons; (iii) selective stimulation of carotid chemoreceptors by close injection of CO2‐saturated saline transiently evoked rhythmic respiratory network activity and suppressed tonic E neuron activity; (iv) respiratory and slower (0.04 – 0.1 Hz) network oscillations were disrupted. We conclude that VRC tonic E neurons contribute to I drive inhibition during HA (cf. Sears et al. 1982) and document the loss of multiple rhythms during HA. Support: NIH grant NS19814

Developmental Nicotine Exposure Alters AMPA Neurotransmission in the Hypoglossal Motor Nucleus and Pre-Bötzinger Complex of Neonatal Rats

Developmental nicotine exposure (DNE) impacts central respiratory control in neonates born to smoking mothers. We previously showed that DNE enhances the respiratory motor response to bath application of AMPA to the brainstem, although it was unclear which brainstem respiratory neurons mediated these effects (Pilarski and Fregosi, 2009). Here we examine how DNE influences AMPA-type glutamatergic neurotransmission in the pre-Bötzinger complex (pre-BötC) and the hypoglossal motor nucleus (XIIMN), which are neuronal populations located in the medulla that are necessary for normal breathing. Using rhythmic brainstem slices from neonatal rats, we microinjected AMPA into the pre-BötC or the XIIMN while recording from XII nerve rootlets (XIIn) as an index of respiratory motor output. DNE increased the duration of tonic activity and reduced rhythmic burst amplitude after AMPA microinjection into the XIIMN. Also, DNE led to an increase in respiratory burst frequency after AMPA injection into the pre-BötC. Whole-cell patch-clamp recordings of XII motoneurons showed that DNE increased motoneuron excitability but did not change inward currents. Immunohistochemical studies indicate that DNE reduced the expression of glutamate receptor subunits 2 and 3 (GluR2/3) in the XIIMN and the pre-BötC. Our data show that DNE alters AMPAergic synaptic transmission in both the XIIMN and pre-BötC, although the mechanism by which this occurs is unclear. We suggest that the DNE-induced reduction in GluR2/3 may represent an attempt to compensate for increased cell excitability, consistent with mechanisms underlying homeostatic plasticity.

How does CO2activate the neurons of the retrotrapezoid nucleus?

How does CO₂activate the neurons of the retrotrapezoid nucleus?

Brainstem Respiratory Oscillators Develop Independently of Neuronal Migration Defects in the Wnt/PCP Mouse Mutant looptail

The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2Lp/+ and Vangl2Lp/Lp embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function.

Functional and histopathological identification of the respiratory failure in a DMSXL transgenic mouse model of Myotonic Dystrophy

SUMMARYAcute and chronic respiratory failure is one of the major and potentially life-threatening features in individuals with myotonic dystrophy type 1 (DM1). Despite several clinical demonstrations showing respiratory problems in DM1 patients, the mechanisms are still not completely understood. This study was designed to investigate whether the DMSXL transgenic mouse model for DM1 exhibits respiratory disorders and, if so, to identify the pathological changes underlying these respiratory problems. Using pressure plethysmography, we assessed the breathing function in control mice and DMSXL mice generated after large expansions of the CTG repeat in successive generations of DM1 transgenic mice. Statistical analysis of breathing function measurements revealed a significant decrease in the most relevant respiratory parameters in DMSXL mice, indicating impaired respiratory function. Histological and morphometric analysis showed pathological changes in diaphragmatic muscle of DMSXL mice, characterized by an increase in the percentage of type I muscle fibers, the presence of central nuclei, partial denervation of end-plates (EPs) and a significant reduction in their size, shape complexity and density of acetylcholine receptors, all of which reflect a possible breakdown in communication between the diaphragmatic muscles fibers and the nerve terminals. Diaphragm muscle abnormalities were accompanied by an accumulation of mutant DMPK RNA foci in muscle fiber nuclei. Moreover, in DMSXL mice, the unmyelinated phrenic afferents are significantly lower. Also in these mice, significant neuronopathy was not detected in either cervical phrenic motor neurons or brainstem respiratory neurons. Because EPs are involved in the transmission of action potentials and the unmyelinated phrenic afferents exert a modulating influence on the respiratory drive, the pathological alterations affecting these structures might underlie the respiratory impairment detected in DMSXL mice. Understanding mechanisms of respiratory deficiency should guide pharmaceutical and clinical research towards better therapy for the respiratory deficits associated with DM1.