Inspiratory Neurons Research Articles

Point:Counterpoint: Medullary pacemaker neurons are essential for both eupnea and gasping in mammals vs. medullary pacemaker neurons are essential for gasping, but not eupnea, in mammals

to the editor: Implicit in the commentaries concerning the possible role of medullary pacemakers in the neurogenesis of eupnea and gasping is one critical and recurring question: How are in vitro rhythms related to these classical patterns of automatic ventilatory activity? Suzue ([5][1]) considered

Identification of different types of respiratory neurones in the dorsal brainstem nucleus tractus solitarius of the rat

In Nembutal anaesthetised, spontaneously breathing rats, stereotaxic mapping of the nucleus tractus solitarius (NTS) for respiratory neuronal activity was undertaken. Eight different types of respiratory cells were found between 0.25 and 1.5 mm lateral to midline, extending 0.5 mm caudal to 1.5 mm rostral to obex, and 0.4–1.5 mm below the dorsal surface. A study of the respiratory motor (diaphragm EMG) and neuronal responses to excitatory amino acid (EAA) stimulation of the NTS areas was undertaken. Electrical stimulation of the vagus nerve was employed to study the NTS cellular responses to activation of pulmonary afferents. The effects of chemical activation of the midbrain periaqueductal grey (PAG) on NTS respiratory neuronal activity were investigated. EAA microinjections into the ventrolateral NTS rostral to the obex resulted in an increase in respiratory motor frequency along with increases to inspiratory cell discharge, whilst microinjections into the medial NTS caudal to the obex caused respiratory depression. EAA stimulation of calamus scriptorius produced apnea. NTS inspiratory neurones were inhibited following stimulation of ipsilateral vagus nerve, suggesting their involvement in the Hering–Breuer reflex pathway. PAG stimulation caused excitation of the NTS inspiratory cells indicating the presence of an excitatory respiratory pathway between the two nuclei. Following β-adrenergic antagonist pre-treatment of ventrolateral NTS, EAA microinjections into PAG did not evoke a cardiorespiratory effect. Based on the various findings the role of NTS in organising respiration in the rat is discussed.

Effect of inspired oxygen concentration on periodic breathing in methyl‐CpG‐binding protein 2 (Mecp2) deficient mice

Rett syndrome (RTT) is characterized by periodic breathing (PB). The goal of this study was to determine if moderate hyperoxia or hypoxia affected the incidence and/or characteristics of PB in Mecp2+/− mice. Individual mice were exposed for 30 min to air, 40% or 12% oxygen. Respiratory pattern was monitored by telemetric measurement of pleural pressure. PB was defined as 3 or more periods of 3 to 20 incrementing then decrementing amplitude breaths separated by apneas. Hyperoxia increased incidence of PB in Mecp2+/− mice from 115.3 ± 27.5 to 219 ± 58.9 s and number of PB episodes from 8.1 ± 1.6 to 12.3 ± 2.4. Episode duration was the same. 40% oxygen increased A/V from 0.44 ± 0.04 to 0.58 ± 0.05. Hypoxia did not significantly affect the incidence (71.3 ± 31 vs 115.3 ± 27.5 sec), number of episodes (4.6 ± 1.6 vs 8.1 ± 1.6), period length nor A/V in Mecp2+/− mice. The duration of PB episodes was decreased in 12 % oxygen (10.2 ± 1.6 vs 14.1 ± 1.5 sec). This paradoxical effect of inspired oxygen concentration suggests that the increased loop gain in Mecp2+/− mice is not due to an augmented hypoxic drive from peripheral chemoreceptors. Rather the results suggest that this mouse model of RTT has an unbalanced contribution from excitatory and inhibitory inputs to inspiratory central neurons such that hyperoxic modulation of the inhibitory influences exacerbates their breathing instability. Support: FY06-314 March of Dimes

Effects of midline sagittal sectioning of medulla on pre-inspiratory and inspiratory neuron activity in vitro

Effects of midline sagittal sectioning of medulla on pre-inspiratory and inspiratory neuron activity in vitro

Modulation of synaptic and channel activities in the respiratory network of the mice by NO/cGMP signalling pathways

We examined signalling pathways which can involve NO as a second messenger in the respiratory network. In the functional slice preparation, NO donors depressed the respiratory motor output and enhanced its depression after brief episodes of hypoxia. In the inspiratory neurons, NO donors suppressed spontaneous excitatory and inhibitory synaptic currents, activated single K ATP channels and inhibited L-type Ca 2+ channels. NO scavengers, PTIO and hemoglobin, and the blocker of NO synthase, N-monomethyl- l-arginine, induced effects opposite to those of NO donors and indicated the role of endogenously generated NO in the modulation of the respiratory activity. Using fluorescent dyes DAF-2 and DCF, we imaged NO and reactive oxygen species (ROS). Concentrations of NO and ROS increased during brief episodes of hypoxia and they both contributed to the activation of K ATP channels due to oxygen withdrawal. The oxidizing agent t-butyl-hydroperoxide acted similarly to NO donors but it did not interfere with the effects of NO. Increase in cGMP levels with 8-Br-cGMP reproduced the actions of NO donors and occluded the effects of their subsequent applications. We propose that in the respiratory neurons, a constitutive production of NO is responsible for a tonic activation of cGMP-coupled signalling pathways and changes in NO levels modulate the respiratory motor output by altering the activity of K ATP and L-type Ca 2+ channels.

ER vesicles and mitochondria move and communicate at synapses

Endoplasmic reticulum (ER) and mitochondria are multifunctional cell organelles and their involvement in Ca2+ handling is important in various neural activities. In the respiratory neurons, we observed ER as continuous reticulum in the soma and as isolated vesicles in dendrites. The vesicles moved bidirectionally with intermittent stops and decreased their velocity near exocytotic sites. ER vesicles and mitochondria that resided in these regions changed lumenal Ca2+ and mitochondrial potential in concert with synaptic activity. Ca2+ release from ER or mitochondria evoked exocytosis. ER vesicles and mitochondria bidirectionally exchanged Ca2+, the efficacy of which depended on the distance between organelles. Depolarisation-evoked exocytosis had different kinetics, depending on whether functional ER vesicles and mitochondria were present in perisynaptic regions and able to exchange Ca2+ or only one organelle type was available. Transfer of Ca2+ from ER to mitochondria produced long-lasting elevations of residual Ca2+ that increased the duration of exocytosis. In slice preparations, synaptic currents in inspiratory neurons were suppressed after disengagement of ER vesicles and mitochondria, and the activity was potentiated after stimulation of Ca2+ exchange between the organelles. We propose that communication between perisynaptic ER vesicles and mitochondria can shape intracellular Ca2+ signals and modulate synaptic and integrative neural activities.

Early postnatal changes in respiratory activity in rat in vitro and modulatory effects of substance P

Developmental changes in the respiratory activity and its modulation by substance P (SP) were studied in the neonatal rat brainstem-spinal cord preparation from the day of birth to day 3 (P0-P3). The respiratory network activity in the ventrolateral medulla was represented by two types of bursts: basic regular bursts with typical decrementing shape and biphasic bursts appearing after augmented biphasic discharges in inspiratory neurons. With advancing postnatal age the respiratory output was considerably modified; the basic rhythm became faster by 20%, whereas the biphasic burst rate, which was originally 15 times slower, declined further by 180% and the C4 burst duration significantly decreased by 20% due to reduced decay time without preceding changes in the central inspiratory drive. SP had an age-dependent excitatory effect on respiratory activity. In the basic rhythm, SP could induce transient rhythm cessations on P0-P2 but not on P3. For the biphasic burst frequency, the sensitivity to SP significantly decreased from P0 to P3, whereas the range of SP-induced changes increased. In both types of bursts, SP prolonged C4 burst duration due to increasing decay time. This effect was three times greater on P3 and did not depend on the central inspiratory drive. Our results suggest that the potency of SP to regulate the respiratory activity elevates during the early postnatal period. The developmental changes in the respiratory activity appear to represent the transient stage in the maturation of rhythm and pattern generation mechanisms facilitating adaptive behavior of a quickly growing organism.

Responses of the Rat Medullary Neurons to Central Penicillin Microinjection

Background: The microinjection of penicillin in the medulla oblongata caused different alterations in the global respiratory output. The purpose of this paper was to study the involvement of impulse activity and the functional relationship of bulbar neurons in mechanisms underlying experimentally induced respiratory arrhythmias. Methods: Extracellular activity of neurons and diaphragmatic electromyogram activity were recorded and analyzed. Results: Penicillin microinjection increased the mean discharge rate of bulbar neurons and caused the prolongation of discharge of inspiratory neurons. Cross-correlation analysis revealed new excitatory connections between neurons and a high level of positive synchronization of neuronal activity. Conclusion: The local blockage of synaptic inhibition by penicillin within respiratory areas leads to drastic changes in the functioning of neuronal elements of the single respiratory network.

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

Fentanyl induced decrease in respiratory sinus arrhythmia may be reversed by activation of 5‐HT4a receptors

Opiate exposure induces a centrally mediated respiratory inhibition, bradycardia, and a reduction in respiratory sinus arrhythmia (RSA). Previous work has shown that RSA is primarily mediated by an increase in both inhibitory GABAergic and glycinergic activity to cardiac vagal neurons (CVNs) during inspiration, and the mu-opioid receptor agonist, fentanyl inhibits both inspiratory neurons and GABAergic neurotransmission to CVNs. 5-HT4a receptor agonists have been shown to reverse fentanyl-induced respiratory depression. This study examines whether the 5-HT4a agonist, BIMU-8, reverses the fentanyl induced decrease in inspiratory-evoked GABA neurotransmission to CVNs. CVNs were identified by the presence of a retrograde fluorescent tracer and inspiratory evoked GABAergic synaptic currents were examined using whole-cell patch-clamp techniques in an in-vitro preparation that spontaneously generates rhythmic respiratory activity. Inspiratory activity was recorded by monitoring the motor neuron population activity from hypoglossal nerve roolets. BIMU-8 was administrated at a dose of 10microM alone or 10 minutes after fentanyl application. Application of BIMU-8 alone increased inspiratory burst frequency by 50%. Co-application of BIMU-8 with fentanyl prevented the opioid induced decrease in inspiratory-evoked GABAergic neurotransmission to cardiac parasympathetic neurons. This work demonstrates 5-HT4a specific receptor agonists overcome mu-opioid receptor induced respiration and RSA depression and may provide a potential therapeutic target for opiate treatment. Supported by NIH grants HL59895 and 72006 to DM.

DAMGO increases CO2 sensitivity and disrupts respiratory rhythm and pattern when injected into the pre‐Botzinger Complex (pre‐Botz C) of awake goats

We investigated the ventilatory effects of the mu-opoiod agonist, DAMGO (1 nM, 500 nL), when injected into the pre-Botz C area of the intact, awake goat. DAMGO injected unilaterally or bilaterally into the pre-Botz C area via chronically implanted microtubules did not change pulmonary ventilation (VE), or respiratory rhythm and pattern during eupneic and hypoxic (PaO2 = 35 mmHg) breathing conditions (n = 4/4 goats), but did increase CO2 sensitivity (VE/PaCO2) (n = 4/4). This contrasts to the typical finding that opioid agonists administered globally depress breathing. Indeed, CO2 sensitivity decreased when DAMGO was injected into the raphe or parapyramidal area in a fifth goat. We also found that bilateral injection of DAMGO into the pre-Botz C periodically disrupts respiratory rhythm and pattern. In one goat breathing 5 and 7% CO2, periods of decreased inspiratory flow and frequency coincided with large, simultaneous diaphragm and abdominal muscle contractions that were 180° out of phase with flow. In a second goat breathing 5 and 7% CO2, periods of phasic abdominal muscle activity were twice the rate of the diaphragm activity. These data likely reflect the in vitro finding that DAMGO depresses inspiratory (I) neurons, disrupting the normal coupling between the pre-Botz C inspiratory rhythm generator and a rostrally located expiratory rhythm generator. Thus, our results not only provide in vivo evidence for the coupled oscillator concept, but also suggest that pre-Botz C I neurons can inhibit CO2 sensitivity. (Supported by NIH 25739 and the Veterans Administration)

Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats

There are at least two respiration-related rhythm generators in the medulla: the pre-Bötzinger complex, which produces inspiratory (Insp) neuron bursts, and the parafacial respiratory group (pFRG), which produces predominantly preinspiratory (Pre-I) neuron bursts. The pFRG Pre-I neuron activity has not been correlated with motor neuron activity in slice or block preparations of rostral medulla. In this study, we attempted to detect pFRG Pre-I activity as motor output in the rostral medulla. We recorded respiratory activity of the facial nerve in the brain stem-spinal cord preparation of 0- to 2-day-old rats. Facial nerve activity consisted of preinspiratory, Insp, and postinspiratory activity. Pre- and postinspiratory activity corresponded well with membrane potential trajectories of Pre-I neurons in the rostral ventrolateral medulla. In response to perfusion of 1 microM DAMGO (a mu-opiate agonist), fourth cervical ventral root (C4) Insp activity was depressed and facial nerve activity continued to synchronize with Pre-I neuron bursts. After transverse sectioning between the levels of the pre-Bötzinger complex and the pFRG, C4 Insp activity recovered within 15 min, but facial nerve activity was inhibited. When DAMGO was applied, C4 Insp activity was inhibited, and rhythmic facial nerve activity recovered. Subsequent elevation of K+ concentration reinduced C4 activity, but facial nerve activity was inhibited. Whole cell recordings in the rostral block revealed the presence of putative Pre-I neurons, the activity of which was synchronized with facial nerve activity. These results show that the rostral medulla, not including the pre-Bötzinger complex, produces Pre-I-like rhythmic activity that can be monitored as facial nerve motor output in newborn rat in vitro preparations.

Electrophysiological and morphological characteristics of GABAergic respiratory neurons in the mouse pre‐Bötzinger complex

The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre-Bötzinger complex. We used 67-kDa isoform of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) (Delta neo) knock-in (GAD67(GFP/+)) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre-Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre-Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP-positive neurons in the pre-Bötzinger complex were recorded in a perforated whole-cell patch-clamp mode. Six of 32 GFP-positive neurons were respiratory and the remaining 26 neurons were non-respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post-synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP-negative neurons were recorded in the pre-Bötzinger complex. GFP-positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP-positive inspiratory neurons had bipolar, triangular or crescent-shaped somata and GFP-negative inspiratory neurons had multipolar-shaped somata. The somata of GFP-positive inspiratory neurons were smaller than those of GFP-negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre-Bötzinger complex.

Dopamine desynchronizes the pace‐making neuronal activity of rat respiratory rhythm generation

In an excised Wistar rat medulla-spinal cord block preparation we previously found that dopamine slows respiratory rhythm by activation of dopamine D(4) receptors [Fujii et al., (2004)Neurosci. Res., 50, 355-359.] In the present paper, we investigated the effect of dopamine on pre-inspiratory (Pre-I) and inspiratory (I) neurons using the combination of an optical recording technique with a voltage-sensitive dye, unit recording and patch-clamp recording. Optical imaging of the ventral surface of the block preparation disclosed different locations and activity patterns of Pre-I and I neurons. In addition to slowing the rhythm, dopamine depressed respiratory activity of Pre-I neurons collectively but not that of I neurons. The dopaminergic suppression of Pre-I neurons was mimicked by a dopamine D(4) receptor agonist, PD168077. Unit recording and patch-clamp recording demonstrated that dopamine depolarizes Pre-I neurons, disperses Pre-I firing and depresses Pre-I phase postsynaptic potentials (PSPs) of I neurons. Immunohistological investigation revealed that Pre-I neurons express dopamine D(4) receptors. We found that approximately 60% of Pre-I neurons express dopamine D(4) receptors. These results show that dopaminergic respiratory rhythm depression is due to dispersion of synchronized Pre-I driving of I neurons caused by dopamine D(4) receptor stimulation of Pre-I neurons.

Norepinephrine Differentially Modulates Different Types of Respiratory Pacemaker and Nonpacemaker Neurons

Pacemakers are found throughout the mammalian CNS. Yet, it remains largely unknown how these neurons contribute to network activity. Here we show that for the respiratory network isolated in transverse slices of mice, different functions can be assigned to different types of pacemakers and nonpacemakers. This difference becomes evident in response to norepinephrine (NE). Although NE depolarized 88% of synaptically isolated inspiratory neurons, this neuromodulator had differential effects on different neuron types. NE increased in cadmium-insensitive pacemakers burst frequency, not burst area and duration, and it increased in cadmium-sensitive pacemakers burst duration and area, but not frequency. NE also differentially modulated nonpacemakers. Two types of nonpacemakers were identified: "silent nonpacemakers" stop spiking, whereas "active nonpacemakers" spontaneously spike when isolated from the network. NE selectively induced cadmium-sensitive pacemaker properties in active, but not silent, nonpacemakers. Flufenamic acid (FFA), a blocker of ICAN, blocked the induction as well as modulation of cadmium-sensitive pacemaker activity, and blocked at the network level the NE-induced increase in burst area and duration of inspiratory network activity; the frequency modulation (FM) was unaffected. We therefore propose that modulation of cadmium-sensitive pacemaker activity contributes at the network level to changes in burst shape, not frequency. Riluzole blocked the FM of isolated cadmium-insensitive pacemakers. In the presence of riluzole, NE caused disorganized network activity, suggesting that cadmium-insensitive pacemakers are critical for rhythm generation. We conclude that different types of nonpacemaker and pacemaker neurons differentially control different aspects of the respiratory rhythm.

Distribution and medullary projection of respiratory neurons in the dorsolateral pons of the rat

The dorsolateral pons around the parabrachial nucleus including the Kölliker-Fuse nucleus is closely linked with the medullary respiratory center and plays an important role in respiratory control. We aimed to elucidate the firing properties, detailed distributions, and medullary projections of pontine respiratory neurons in pentobarbitone-anesthetized, paralyzed, and artificially ventilated rats with intact vagi. A total of 235 respiratory neurons were recorded from the dorsolateral pons in and around the Kölliker-Fuse nucleus. Six types of firing patterns were identified: inspiratory, expiratory–inspiratory phase spanning, inspiratory–expiratory phase spanning, decrementing expiratory, augmenting expiratory, and whole-phase expiratory patterns. Of these, the inspiratory neurons and the expiratory–inspiratory phase spanning neurons, which constituted the largest population (61%), were characterized most carefully by changing lung inflation levels, since under some conditions both showed similar firing patterns. Many (58%) of the 133 respiratory neurons examined were antidromically activated by electrical stimulation of the medulla. They were activated from the ventrolateral medulla around the ventral respiratory group and the Bötzinger complex and from the dorsomedial medulla around the nucleus tractus solitarii and the hypoglossal nucleus. The projections to the dorsomedial medulla were bilateral in many cases, and those to the ventrolateral medulla were unilateral. Of these medullary projections, two specific projections could be characterized in detail. First, many expiratory–inspiratory phase spanning neurons projected to the hypoglossal nucleus, suggesting that these pontine neurons are important premotor neurons of the hypoglossal motoneurons. This projection explains well the hypoglossal inspiratory activity, which is often dissociated from the phrenic inspiratory activity. Second, most whole-phase expiratory neurons that were distributed medially to the KF nucleus sent their axons toward the spinal cord via the midline medulla. These findings provide a new insight into the pontine control of medullary and spinal respiratory function.

Association of nicotinic acetylcholine receptors with central respiratory control in isolated brainstem-spinal cord preparation of neonatal rats

Nicotine exposure is a risk factor in several breathing disorders Nicotinic acetylcholine receptors (nAChRs) exist in the ventrolateral medulla, an important site for respiratory control. We examined the effects of nicotinic acetylcholine neurotransmission on central respiratory control by addition of a nAChR agonist or one of various antagonists into superfusion medium in the isolated brainstem-spinal cord from neonatal rats. Ventral C4 neuronal activity was monitored as central respiratory output, and activities of respiratory neurons in the ventrolateral medulla were recorded in whole-cell configuration. RJR-2403 (0.1-10 mM), alpha4beta2 nAChR agonist induced dose-dependent increases in respiratory frequency. Non-selective nAChR antagonist mecamylamine (0.1-100 mM), alpha4beta2 antagonist dihydro-beta-erythroidine (0.1-100 mM), alpha7 antagonist methyllycaconitine (0.1-100 mM), and a-bungarotoxin (0.01-10 mM) all induced dose-dependent reductions in C4 respiratory rate. We next examined effects of 20 mM dihydro-beta-erythroidine and 20mM methyllycaconitine on respiratory neurons. Dihydro-beta-erythroidine induces hyperpolarization and decreases intraburst firing frequency of inspiratory and preinspiratory neurons. In contrast, methyllycaconitine has no effect on the membrane potential of inspiratory neurons, but does decrease their intraburst firing frequency while inducing hyperpolarization and decreasing intraburst firing frequency in preinspiratory neurons. These findings indicate that alpha4beta2 nAChR is involved in both inspiratory and preinspiratory neurons, whereas alpha7 nAChR functions only in preinspiratory neurons to modulate C4 respiratory rate.

Synaptic origin of the respiratory-modulated activity of laryngeal motoneurons

To determine the synaptic source of the respiratory-related activity of laryngeal motoneurons, spike-triggered averaging of the membrane potentials of laryngeal motoneurons was conducted using spikes of respiratory neurons located between the Bötzinger complex and the rostral ventral respiratory group as triggers in decerebrate, paralyzed cats. We identified one excitatory and two inhibitory sources for inspiratory laryngeal motoneurons, and two inhibitory sources for expiratory laryngeal motoneurons. In inspiratory laryngeal motoneurons, monosynaptic excitatory postsynaptic potentials were evoked by spikes of inspiratory neurons with augmenting firing patterns, and monosynaptic inhibitory postsynaptic potentials (IPSPs) were evoked by spikes of expiratory neurons with decrementing firing patterns and by spikes of inspiratory neurons with decrementing firing patterns. In expiratory laryngeal motoneurons, monosynaptic IPSPs were evoked by spikes of inspiratory neurons with decrementing firing patterns and by spikes of expiratory neurons with augmenting firing patterns. We conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of membrane potential of laryngeal motoneurons.

Ryanodine receptor/Ca 2+ release mechanisms in rhythmically active respiratory neurons of cats in vivo

The cytosolic Ca 2+ released from internal stores is important for distinctive cell functions. To assess the role of ryanodine/Ca 2+ releasing mechanisms in the rhythmic activity of respiratory neurons, effects of intracellular injection of ryanodine on the membrane potential trajectory of postinspiratory and augmenting inspiratory neurons were investigated in unanesthetized, decerebrate, paralyzed and artificially ventilated cats. Ryanodine injection hyperpolarized the membrane and decreased input resistance throughout the respiratory cycle in both types of respiratory neurons. Specifically, membrane repolarization during postinspiration was accelerated in postinspiratory neurons, and the large hyperpolarization at the onset of postinspiration was increased in augmenting inspiratory neurons. Spike-afterhyperpolarization consisting of a fast, early component and slow, late component increased in size after ryanodine, resulting in prolongation of inter-spike intervals and decrease of burst discharge. Intracellular injection of caffeine produced similar effects on these respiratory neurons, and Ruthenium Red, an antagonist of ryanodine receptors, had opposite effects. Immunoreactivity for ryanodine receptors was detected in all respiratory neurons labeled intracellularly with neurobiotin. These results demonstrate that ryanodine-sensitive Ca 2+ stores modulate the periodic membrane potential fluctuations and spike activity in respiratory neurons.

Optical recording from respiratory pattern generator of fetal mouse brainstem reveals a distributed network

Unfailing respiration depends on neural mechanisms already present in mammals before birth. Experiments were made to determine how inspiratory and expiratory neurons are grouped in the brainstem of fetal mice. A further aim was to assess whether rhythmicity arises from a single pacemaker or is generated by multiple sites in the brainstem. To measure neuronal firing, a fluorescent calcium indicator dye was applied to embryonic central nervous systems isolated from mice. While respiratory commands were monitored electrically from third to fifth cervical ventral roots, activity was measured optically over areas containing groups of respiratory neurones, or single neurones, along the medulla from the facial nucleus to the pre-Bötzinger complex. Large optical signals allowed recordings to be made during individual respiratory cycles. Inspiratory and expiratory neurones were intermingled. A novel finding was that bursts of activity arose in a discrete area intermittently, occurring during some breaths, but failing in others. Raised CO2 partial pressure or lowered pH increased the frequency of respiration; neurons then fired reliably with every cycle. Movies of activity revealed patterns of activation of inspiratory and expiratory neurones during successive respiratory cycles; there was no evidence for waves spreading systematically from region to region. Our results suggest that firing of neurons in immature respiratory circuits is a stochastic process, and that the rhythm does not depend on a single pacemaker. Respiratory circuits in fetal mouse brainstem appear to possess a high safety factor for generating rhythmicity, which may or may not persist as development proceeds.