Respiratory Motor Pattern Research Articles

Three brainstem areas involved in respiratory rhythm generation in bullfrogs.

Ventilation in mammals consists of at least three distinct phases: inspiration, post-inspiration and late-expiration. While distinct brainstem rhythm generating and pattern forming networks have long been assumed, recent data suggest the mammalian brainstem contains two coupled neuronal oscillators: one for inspiration and the other for active expiration. However, whether additional burst generating ability is required for generating other phases of ventilation in mammals is controversial. To investigate brainstem circuit architectures capable of producing multiphasic ventilatory rhythms, we utilized the isolated frog brainstem. This preparation produces two types of ventilatory motor patterns, buccal and lung bursts. Lung bursts can be divided into two phases, priming and powerstroke. Previously we identified two putative oscillators, the Buccal and Lung Areas. The Lung Area produces the lung powerstroke and the Buccal Area produces buccal bursts and - we assumed - the priming phase of lung bursts. However, here we identify an additional brainstem region that generates the priming phase. This Priming Area extends rostral and caudal of the Lung Area and is distinct from the Buccal Area. Using AMPA microinjections and reversible synaptic blockade, we demonstrate selective excitation and ablation (respectively) of priming phase activity. We also demonstrate that the Priming Area contains neurons active selectively during the priming phase. Thus, we propose that three distinct neuronal components generate the multiphase respiratory motor pattern produced by the frog brainstem: the buccal, priming and powerstroke burst generators. This raises the possibility that a similar multi-burst generator architecture mediates the three distinct phases of ventilation in mammals.

Vocal development in dystonic rats.

Vocal production, which requires the generation and integration of laryngeal and respiratory motor patterns, can be impaired in dystonia, a disorder believed due to dysfunction of sensorimotor pathways in the central nervous system. Herein, we analyze vocal and respiratory abnormalities in the dystonic (dt) rat, a well-characterized model of generalized dystonia. The dt rat is a recessive mutant with haploinsufficiency of Atcay which encodes the neuronally restricted protein caytaxin. Olivocerebellar functional abnormalities are central to the dt rat's truncal and appendicular dystonia and could also contribute to vocal and respiratory abnormalities in this model system. Differences in vocal repertoire composition were found between homozygote and wild-type dt rat pups developing after 3 weeks of life. Those spectro-temporal differences were not paralleled by differences in vocal activity or maximum lung pressures during quiet breathing and vocalization. However, breathing rhythm was slower in homozygote pups. This slower breathing rhythm persisted into adulthood. Given that cerebellectomy eliminates truncal and appendicular dystonia in the dt rat, we hypothesize that the altered breathing patterns stem either from a disturbance in the maturation of respiratory pattern generators or from deficient extracerebellar caytaxin expression affecting normal respiratory pattern generation. The altered breathing rhythm associated with vocal changes in the murine model resembles aspects of vocal dysfunction that are seen in humans with sporadic dystonia.

Spontaneous swallowing occurs during autoresuscitation in the in situ brainstem preparation of rat

Previous studies report that upper airway reflexes are operational during autoresuscitation from respiratory arrest. We investigated swallowing/breathing interactions, measured by recording of vagal (VNA) and phrenic nerve activities (PNA), during autoresuscitation in the in situ perfused brainstem preparation of juvenile rats. During the initial surgery, respiratory arrest was induced by exsanguination and cooling. Reperfusion (i.e. re-oxygenation and re-warming) of the brainstem circuits was associated with frequent spontaneous swallowing before resumption of respiration (n=6, ‘stage 1 autoresuscitation’). When recovered, the respiratory pattern was transiently apneustic-like (‘stage 2 autoresuscitation’). Spontaneous swallowing often occurred at the end of the prolonged PNA (n=9/12). Successful autoresuscitation was characterised by re-establishment of the 3 phase respiratory motor pattern and no spontaneous swallowing. Pharmacological inhibition (isoguvacine, 10mM, 50–75nl; n=10) of the Kölliker-Fuse nucleus (KF) mimicked stage 2 autoresuscitation. However, the frequency of spontaneous swallowing after KF inhibition did not correlate with subsequent recovery of the eupneic respiratory motor pattern.

The midbrain periaqueductal grey has no role in the generation of the respiratory motor pattern, but provides command function for the modulation of respiratory activity

It has previously been shown that stimulation of cell-columns in the periaqueductal grey (PAG) triggers site-specific cardiorespiratory effects. These are believed to facilitate changes in behaviour through coordinated changes in autonomic outflow. Here, we investigated whether PAG-evoked respiratory commands can be studied in situ using the decerebrate perfused brainstem preparation. Phrenic, vagus and abdominal iliohypogastric nerves were recorded before and after microinjection of l-glutamate (30–50nl, 10mM) or isoguvacine (GABA-receptor agonist, 30–50nl, 10mM) into the PAG. l-glutamate microinjection triggered a range of site-specific respiratory modulations (n=17 preparations). Subsequent microinjection of isoguvacine into the same PAG sites had no effect on the baseline respiratory motor pattern or rhythm. We conclude that while the PAG has no function in respiratory pattern generation, PAG-evoked respiratory modulations can be evoked in situ in the absence of higher brain centres and while homeostatic parameters that may affect respiratory drive are held static.

Disinhibition of the pontine and dorsal respiratory groups reveals postinspiratory pacemaker activity (714.2)

The ‘pontine respiratory group’ in dorsolateral pons overlaps with the Kolliker‐Fuse nucleus (KF) while the ‘dorsal respiratory group’ is associated with ventrolateral areas of the nucleus of the solitary tract (NTS). The contribution of both to the generation of the eupneic respiratory motor pattern remains controversial. This study describes the severe disruption to normal breathing following focal microinjection of bicuculline (GABAA receptor antagonist) into the KF and NTS in the juvenile in in situ preparation of rat. Disinhibition of the KF produced an increased variability in the sequential patterning of the eupneic 3‐phase respiratory motor pattern. The chaotic respiratory pattern after KF disinhibition is largely associated with instability of postinspiratory gating mechanisms. In contrast, disinhibition of the NTS caused repetitive barrages of rhythmic, non‐swallow related discharges in vagus nerve that suppressed inspiratory activity. These rhythmic discharges were likely pacemaker‐driven since they were abolished by the persistent sodium channel blocker riluzole. Moreover, riluzole microinjection into KF or NTS alone triggered delayed inspiratory off‐switch, suggesting that pacemaker currents indeed contribute to the timing of postinspiratory mediated inspiratory off‐switch and respiratory motor patterning.Grant Funding Source: ARC

Inhibition of protein kinase A activity depresses phrenic drive and glycinergic signalling, but not rhythmogenesis in anaesthetized rat

The cAMP-protein kinase A (PKA) pathway plays a critical role in regulating neuronal activity. Yet, how PKA signalling shapes the population activity of neurons that regulate respiratory rhythm and motor patterns in vivo is poorly defined. We determined the respiratory effects of focally inhibiting endogenous PKA activity in defined classes of respiratory neurons in the ventrolateral medulla and spinal cord by microinjection of the membrane-permeable PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) in urethane-anaesthetized adult Sprague Dawley rats. Phrenic nerve activity, end-tidal CO2 and arterial pressure were recorded. Rp-cAMPS in the preBötzinger complex (preBötC) caused powerful, dose-dependent depression of phrenic burst amplitude and inspiratory period. Rp-cAMPS powerfully depressed burst amplitude in the phrenic premotor nucleus, but had no effect at the phrenic motor nucleus, suggesting a lack of persistent PKA activity here. Surprisingly, inhibition of PKA activity in the preBötC increased phrenic burst frequency, whereas in the Bötzinger complex phrenic frequency decreased. Pretreating the preBötC with strychnine, but not bicuculline, blocked the Rp-cAMPS-evoked increase in frequency, but not the depression of phrenic burst amplitude. We conclude that endogenous PKA activity in excitatory inspiratory preBötzinger neurons and phrenic premotor neurons, but not motor neurons, regulates network inspiratory drive currents that underpin the intensity of phrenic nerve discharge. We show that inhibition of PKA activity reduces tonic glycinergic transmission that normally restrains the frequency of rhythmic respiratory activity. Finally, we suggest that the maintenance of the respiratory rhythm in vivo is not dependent on endogenous cAMP-PKA signalling.

Stereotypic laryngeal and respiratory motor patterns generate different call types in rat ultrasound vocalization.

Rodents produce highly variable ultrasound whistles as communication signals unlike many other mammals, who employ flow-induced vocal fold oscillations to produce sound. The role of larynx muscles in controlling sound features across different call types in ultrasound vocalization (USV) was investigated using laryngeal muscle electromyographic (EMG) activity, subglottal pressure measurements and vocal sound output in awake and spontaneously behaving Sprague-Dawley rats. Results support the hypothesis that glottal shape determines fundamental frequency. EMG activities of thyroarytenoid and cricothyroid muscles were aligned with call duration. EMG intensity increased with fundamental frequency. Phasic activities of both muscles were aligned with fast changing fundamental frequency contours, for example in trills. Activities of the sternothyroid and sternohyoid muscles, two muscles involved in vocal production in other mammals, are not critical for the production of rat USV. To test how stereotypic laryngeal and respiratory activity are across call types and individuals, sets of ten EMG and subglottal pressure parameters were measured in six different call types from six rats. Using discriminant function analysis, on average 80% of parameter sets were correctly assigned to their respective call type. This was significantly higher than the chance level. Since fundamental frequency features of USV are tightly associated with stereotypic activity of intrinsic laryngeal muscles and muscles contributing to build-up of subglottal pressure, USV provide insight into the neurophysiological control of peripheral vocal motor patterns.

Descending control of the respiratory neuronal network by the midbrain periaqueductal grey in the rat in vivo

Emotional reactions such as vocalization take place during expiration, and thus expression of emotional behaviour requires a switch from inspiration to expiration. I investigated how the midbrain periaqueductal grey (PAG), a known behavioural modulator of breathing, influences the inspiratory-to-expiratory phase transition. Contemporary models propose that late inspiratory (late-I) and post-inspiratory (post-I) neurones found in the medulla, which are active during the inspiratory-to-expiratory phase transition are involved in converting inspiration to expiration. I examined the effect of excitatory amino acid (d,l-homocysteic acid; DLH) stimulation of the PAG on the discharge function of late-I and post-I neurones. The data show a topographical organization of DLH-induced late-I and post-I neuronal modulation within the PAG. Dorsal PAG stimulation induced tachypnoea and caused excitation of both the late-I and post-I neurones. Lateral PAG induced inspiratory prolongation and caused an excitation of late-I neurones but inhibition of post-I neurones. Ventrolateral PAG induced expiratory prolongation and caused a persistent activation of post-I neurones. As well, PAG stimulation modulated both the late-I and post-I cells for least two-three breaths even prior to the change in respiratory motor pattern. This indicates that the PAG influences the late-I and post-I cells independent of pulmonary or other sensory afferent feedback. I conclude that the PAG modulates the activity of the medullary late-I and post-I neurones, and this modulation contributes to the conversion of eupnoea into a behavioural breathing pattern.

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.

Bilateral coordination and the motor basis of female preference for sexual signals in canary song

The preference of female songbirds for particular traits in the songs of courting males has received considerable attention, but the relationship of preferred traits to male quality is poorly understood. Female domestic canaries (Serinus canaria, Linnaeus) preferentially solicit copulation with males that sing special high repetition rate, wide-band, multi-note syllables, called 'sexy' or A-syllables. Syllables are separated by minibreaths but each note is produced by pulsatile expiration, allowing high repetition rates and long duration phrases. The wide bandwidth is achieved by including two notes produced sequentially on opposite sides of the syrinx, in which the left and right sides are specialized for low or high frequencies, respectively. The emphasis of low frequencies is facilitated by a positive relationship between syllable repetition rate and the bandwidth of the fundamental frequency of notes sung by the left syrinx, such that bandwidth increases with increasing syllable repetition rate. The temporal offset between notes prevents cheating by unilaterally singing a note on the left side with a low fundamental frequency and prominent higher harmonics. The syringeal and respiratory motor patterns by which sexy syllables are produced support the hypothesis that these syllables provide a sensitive vocal-auditory indicator of a male's performance limit for the rapid, precisely coordinated interhemispheric switching, which is essential for many sensory and motor processes involving specialized contributions from each cerebral hemisphere.

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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 <i>Mecp2</i>-null male mice (<i>Mecp2</i>^−/<i>y</i>). To characterize developmental progression of the respiratory phenotype and explore underlying mechanisms, we examined <i>Mecp2</i>^−/<i>y</i> 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 <i>in situ</i> 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 <i>Mecp2</i>^−/<i>y</i> 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 <i>Mecp2</i>^−/<i>y</i> mice. During the later stage, respiratory abnormalities were evident during wakefulness and sleep. Also, WHBPs from <i>Mecp2</i>^−/<i>y</i> showed central apneas. We conclude that, at early disease stages, stress-related modulation from suprapontine structures is a significant factor in the <i>Mecp2</i>^−/<i>y</i> 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 control of the respiratory pattern and coupled oscillators

The respiratory rhythm and motor pattern are generated by a spatially organized brain stem respiratory network with a rhythmogenic core comprising interacting neural populations within the pre-Botzinger (pre-BotC) and Botzinger (BotC) complexes controlled by drives and inputs from other brain stem compartments. Our previous large-scale computational model [3, 4] reproduced the behavior of this network under multiple natural and experimental conditions, but did not consider neural oscillations that were proposed to emerge within the retrotrapezoid nucleus / parafacial respiratory group (RTN/pFRG) and drive pre-inspiratory (or late-expiratory, late-E) oscillations in the abdominal motor output [2]. These late-E oscillations usually emerge with increasing metabolic demands (e.g., during hypercapnia or hypoxia) and couple with the respiratory oscillations generated by the BotC-pre-BotC core of the respiratory central pattern generator [1, 2]. In our experiments, performed in the arterially perfused in situ rat preparation, the respiratory motor activity was recorded simultaneously from abdominal, phrenic, hypoglossal and central vagus nerves. Under normal conditions (eupnea), the abdominal motor outflow showed only a low-amplitude post-inspiratory (post-I) activity. An expression of the abdominal late-E bursts, preceding and coupled to phrenic discharges, occurred during hypercapnia (7–10% CO2) and could be abolished by pharmacological inactivation of the RTN/pFRG region and by administration of riluzole, a persistent sodium current (I NaP ) blocker, hence confirming that the late-E oscillations originate in RTN/pFRG and involve intrinsic, I NaP -dependent mechanisms. Using these data we extended our computational model by incorporating in the RTN/pFRG compartment an additional late-E population (with neurons containing I NaP ) serving as a source of abdominal late-E activity. The proposed interactions between the BotC’s and pre-BotC’s populations and the late-E population of RTN/pFRG allowed the model to reproduce several experimentally observed behaviors, including quantal acceleration of abdominal late-E oscillations with progressive hypercapnia and quantal slowing of phrenic activity with progressive suppression of pre-BotC excitability (simulating the suppressing effects of opioids), as well as to predict a release of late-E oscillations by disinhibition of RTN/pFRG under normal conditions. The latter prediction has been confirmed in our experimentally studies. Our experimental and modeling studies suggest that under normal metabolic conditions the RTN/pFRG oscillator is inhibited by both the post-I population of BotC during inspiration and early-inspiratory (early-I) population of preBotC during inspiration. Therefore the late-E oscillations can be released by either a hypercapnia-evoked activation of chemosensitive RTN/pFRG neurons overcoming this inhibition or a hypoxia-dependent suppression of RTN/pFRG inhibition by BotC-pre-BotC circuits. Our model proposes mechanistic explanations for the emergence of RTN/pFRG oscillations and their interaction with the brain stem respiratory network. The emerging view is that the brain stem respiratory network has rhythmogenic capabilities at multiple hierarchical levels, which allows flexible, state-dependent expression of different rhythmogenic mechanisms under different physiological and metabolic conditions and enables a wide repertoire of respiratory behaviors.

Population calcium imaging of spontaneous respiratory and novel motor activity in the facial nucleus and ventral brainstem in newborn mice

The brainstem contains rhythm and pattern forming circuits, which drive cranial and spinal motor pools to produce respiratory and other motor patterns. Here we used calcium imaging combined with nerve recordings in newborn mice to reveal spontaneous population activity in the ventral brainstem and in the facial nucleus. In Fluo-8AM loaded brainstem-spinal cord preparations, respiratory activity on cervical nerves was synchronized with calcium signals at the ventrolateral brainstem surface. Individual ventrolateral neurons at the level of the parafacial respiratory group showed perfect or partial synchrony with respiratory nerve bursts. In brainstem-spinal cord preparations, cut at the level of the mid-facial nucleus, calcium signals were recorded in the dorsal, lateral and medial facial subnuclei during respiratory activity. Strong activity initiated in the dorsal subnucleus, followed by activity in lateral and medial subnuclei. Whole-cell recordings from facial motoneurons showed weak respiratory drives, and electrical field potential recordings confirmed respiratory drive to particularly the dorsal and lateral subnuclei. Putative facial premotoneurons showed respiratory-related calcium signals, and were predominantly located dorsomedial to the facial nucleus. A novel motor activity on facial, cervical and thoracic nerves was synchronized with calcium signals at the ventromedial brainstem extending from the level of the facial nucleus to the medulla–spinal cord border. Cervical dorsal root stimulation induced similar ventromedial activity. The medial facial subnucleus showed calcium signals synchronized with this novel motor activity on cervical nerves, and cervical dorsal root stimulation induced similar medial facial subnucleus activity. In conclusion, the dorsal and lateral facial subnuclei are strongly respiratory-modulated, and the brainstem contains a novel pattern forming circuit that drives the medial facial subnucleus and cervical motor pools.

Ventrolateral medullary functional connectivity and the respiratory and central chemoreceptor-evoked modulation of retrotrapezoid-parafacial neurons

The medullary ventral respiratory column (VRC) of neurons is essential for respiratory motor pattern generation; however, the functional connections among these cells are not well understood. A rostral extension of the VRC, including the retrotrapezoid nucleus/parafacial region (RTN-pF), contains neurons responsive to local perturbations of CO(2)/pH. We addressed the hypothesis that both local RTN-pF interactions and functional connections from more caudal VRC compartments--extending from the Bötzinger and pre-Bötzinger complexes to the ventral respiratory group (Böt-VRG)--influence the respiratory modulation of RTN-pF neurons and their responses to central chemoreceptor and baroreflex activation. Spike trains from 294 RTN-pF and 490 Böt-VRG neurons were monitored with multielectrode arrays along with phrenic nerve activity in 14 decerebrate, vagotomized cats. Overall, 214 RTN-pF and 398 Böt-VRG neurons were respiratory modulated; 124 and 95, respectively, were cardiac modulated. Subsets of these neurons were tested with sequential, selective, transient stimulation of central chemoreceptors and arterial baroreceptors; each cell's response was evaluated and categorized according to the change in firing rate (if any) following the stimulus. Cross-correlation analysis was applied to 2,884 RTN-pF↔RTN-pF and 8,490 Böt-VRG↔RTN-pF neuron pairs. In total, 174 RTN-pF neurons (59.5%) had significant features in short-time scale correlations with other RTN-pF neurons. Of these, 49 neurons triggered cross-correlograms with offset peaks or troughs (n = 99) indicative of paucisynaptic excitation or inhibition of the target. Forty-nine Böt-VRG neurons (10.0%) were triggers in 74 Böt-VRG→RTN-pF correlograms with offset features, suggesting that Böt-VRG trigger neurons influence RTN-pF target neurons. The results support the hypothesis that local RTN-pF neuron interactions and inputs from Böt-VRG neurons jointly contribute to respiratory modulation of RTN-pF neuronal discharge patterns and promotion or limitation of their responses to central chemoreceptor and baroreceptor stimulation.

Late-Expiratory Activity: Emergence and Interactions With the Respiratory CPG

The respiratory rhythm and motor pattern are hypothesized to be generated by a brain stem respiratory network with a rhythmogenic core consisting of neural populations interacting within and between the pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes and controlled by drives from other brain stem compartments. Our previous large-scale computational model reproduced the behavior of this network under many different conditions but did not consider neural oscillations that were proposed to emerge within the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) and drive preinspiratory (or late-expiratory, late-E) discharges in the abdominal motor output. Here we extend the analysis of our previously published data and consider new data on the generation of abdominal late-E activity as the basis for extending our computational model. The extended model incorporates an additional late-E population in RTN/pFRG, representing a source of late-E oscillatory activity. In the proposed model, under normal metabolic conditions, this RTN/pFRG oscillator is inhibited by BötC/pre-BötC circuits, and the late-E oscillations can be released by either hypercapnia-evoked activation of RTN/pFRG or by hypoxia-dependent suppression of RTN/pFRG inhibition by BötC/pre-BötC. The proposed interactions between BötC/pre-BötC and RTN/pFRG allow the model to reproduce several experimentally observed behaviors, including quantal acceleration of abdominal late-E oscillations with progressive hypercapnia and quantal slowing of phrenic activity with progressive suppression of pre-BötC excitability, as well as to predict a release of late-E oscillations by disinhibition of RTN/pFRG under normal conditions. The extended model proposes mechanistic explanations for the emergence of RTN/pFRG oscillations and their interaction with the brain stem respiratory network.

Evidence for distributed processing during hypoxic stimulation by the brainstem cardiorespiratory network

Brainstem neural networks play an important integrative role in the regulation of respiratory and cardiovascular responses to hypoxia, but their organization remains poorly understood. We addressed the hypothesis that a distributed brainstem network, including rostral medullary neurons (RMNs) in the region of the retrotrapezoid nucleus, dorsolateral pons, raphé, and ventrolateral cardiorespiratory column (VRC), integrate central and peripheral chemoreceptor inputs. Data were obtained from decerebrate adult cats. We used multi‐electrode arrays to record from up to 118 neurons simultaneously. Firing rate responses were measured during hypoxia and stimulation of central chemoreceptors. Spike trains were also analyzed with respiratory and cardiac cycle triggered histograms and cross correlation. Results include: 1. Multiple cardiorespiratory rhythms in neurons responsive to hypoxic stimulation. 2. Co‐expression of distributed eupneic network activity present together with gasp patterns during recovery from severe brain hypoxia. 3. Reciprocal effective RMN‐VRC connectivity of neurons responsive to both hypoxia and hypercapnia. The results suggest a brainstem network architecture with connectivity that shapes the respiratory motor pattern via overlapping circuits that modulate central and peripheral chemoreceptor‐mediated influences on breathing.Supported by NS19814, NS46062

Central and peripheral chemoreceptors evoke distinct responses in simultaneously recorded neurons of the raphé-pontomedullary respiratory network.

The brainstem network for generating and modulating the respiratory motor pattern includes neurons of the medullary ventrolateral respiratory column (VRC), dorsolateral pons (PRG) and raphé nuclei. Midline raphé neurons are proposed to be elements of a distributed brainstem system of central chemoreceptors, as well as modulators of central chemoreceptors at other sites, including the retrotrapezoid nucleus. Stimulation of the raphé system or peripheral chemoreceptors can induce a long-term facilitation of phrenic nerve activity; central chemoreceptor stimulation does not. The network mechanisms through which each class of chemoreceptor differentially influences breathing are poorly understood. Microelectrode arrays were used to monitor sets of spike trains from 114 PRG, 198 VRC and 166 midline neurons in six decerebrate vagotomized cats; 356 were recorded during sequential stimulation of both receptor classes via brief CO(2)-saturated saline injections in vertebral (central) and carotid arteries (peripheral). Seventy neurons responded to both stimuli. More neurons were responsive only to peripheral challenges than those responsive only to central chemoreceptor stimulation (PRG, 20 : 4; VRC, 41 : 10; midline, 25 : 13). Of 16 474 pairs of neurons evaluated for short-time scale correlations, similar percentages of reference neurons in each brain region had correlation features indicative of a specific interaction with at least one target neuron: PRG (59.6%), VRC (51.0%) and raphé nuclei (45.8%). The results suggest a brainstem network architecture with connectivity that shapes the respiratory motor pattern via overlapping circuits that modulate central and peripheral chemoreceptor-mediated influences on breathing.

Pontine–Ventral Respiratory Column Interactions Through Raphé Circuits Detected Using Multi-Array Spike Train Recordings

Recently, Segers et al. identified functional connectivity between the ventrolateral respiratory column (VRC) and the pontine respiratory group (PRG). The apparent sparseness of detected paucisynaptic interactions motivated consideration of other potential functional pathways between these two regions. We report here evidence for "indirect" serial functional linkages between the PRG and VRC via intermediary brain stem midline raphé neurons. Arrays of microelectrodes were used to record sets of spike trains from a total of 145 PRG, 282 VRC, and 340 midline neurons in 11 decerebrate, vagotomized, neuromuscularly blocked, ventilated cats. Spike trains of 13,843 pairs of neurons that included at least one raphé cell were screened for respiratory modulation and short-time scale correlations. Significant correlogram features were detected in 7.2% of raphé-raphé (291/4,021), 4.3% of VRC-raphé (292/6,755), and 4.0% of the PRG-raphé (124/3,067) neuron pairs. Central peaks indicative of shared influences were the most common feature in correlations between pairs of raphé neurons, whereas correlated raphé-PRG and raphé-VRC neuron pairs displayed predominantly offset peaks and troughs, features suggesting a paucisynaptic influence of one neuron on the other. Overall, offset correlogram features provided evidence for 33 VRC-to-raphé-to-PRG and 45 PRG-to-raphé-to-VRC correlational linkage chains with one or two intermediate raphé neurons. The results support a respiratory network architecture with parallel VRC-to-PRG and PRG-to-VRC links operating through intervening midline circuits, and suggest that raphé neurons contribute to the respiratory modulation of PRG neurons and shape the respiratory motor pattern through coordinated divergent actions on both the PRG and VRC.

Noradrenergic modulation of the respiratory neural network

Noradrenergic dysregulation has been reported in human pathologies affecting the control of breathing, such as sudden infant death syndrome, congenital central hypoventilation syndrome and Rett syndrome. Noradrenergic neurons, located predominantly in pontine nuclei, are among the earliest to arise within the hindbrain and play an essential role in the maturation of the respiratory network. Noradrenergic neurons also play a major role in the modulation of the respiratory motor pattern from birth through adulthood. The critical importance of this signaling system in respiratory control is illustrated by the severe respiratory disturbances associated with gene mutations affecting noradrenergic neurons (Phox2 and Mecp2). Here, the role of catecholaminergic pontine nuclei in the control of breathing, the cellular effects of norepinephrine on the respiratory network and the pathological consequence to breathing of abnormalities in this signaling system will be discussed.

Postnatal emergence of synaptic plasticity associated with dynamic adaptation of the respiratory motor pattern

The shape of the three-phase respiratory motor pattern (inspiration, postinspiration, late expiration) is controlled by a central pattern generator (CPG) located in the ponto-medullary brainstem. Synaptic interactions between and within specific sub-compartments of the CPG are subject of intensive research. This review addresses the neural control of postinspiratory activity as the essential determinant of inspiratory/expiratory phase duration. The generation of the postinspiratory phase depends on synaptic interaction between neurones of the nucleus tractus solitarii (NTS), which relay afferent inputs from pulmonary stretch receptors, and the pontine Kölliker-Fuse nucleus (KF) as integral parts of the CPG. Both regions undergo significant changes during the first three postnatal weeks in rodents. Developmental changes in glutamatergic synaptic functions and its modulation by brain-derived neurotrophic factor may have implications in synaptic plasticity within the NTS/KF axis. We propose that dependent on these developmental changes, the CPG becomes permissive for short- and long-term plasticity associated with environmental, metabolic and behavioural adaptation of the breathing pattern.