Pre-B?tzinger neurons with preinspiratory discharges "in vivo" express NK1 receptors in the rat.

Substance P stimulates respiration, in part by a direct action on the pre-B?tzinger complex (preB?tC). This region of the medulla oblongata contains neurons that are strongly immunoreactive for the neurokinin-1 receptor (NK1R-ir), and a recent theory has postulated that these cells might be the adult form of excitatory interneurons that are essential for respiratory rhythmogenesis in neonates. Here we sought to determine whether preB?tC respiratory neurons are indeed NK1R-ir in the adult rat. Preinspiratory (pre-I) neurons were recorded in the preB?tC region of halothane-anesthetized rats. Most pre-I cells could be antidromically activated from the contralateral side of the medulla (7 of 10; latency 1.3 +/- 0.2 ms), suggesting that most of them were propriomedullary neurons rather than respiratory motoneurons or bulbospinal cells. Thirty-two pre-I neurons including seven cells with contralateral projection were labeled with biotinamide using the juxtacellular method. Eleven cells (34.4%) were NK1R-ir, including three of the seven pre-I cells that were antidromically activated from the contralateral side. In 3 control rats we labeled 20 preB?tC neurons with patterns of discharge other than pre-I and found that none were detectably NK1R-ir. In conclusion, some of the intensely NK1R-ir neurons of the adult preB?tC region are indeed respiratory interneurons as suggested by Gray et al. The subtype of NK1R identified by the antibody is detectable only in a small minority of preB?tC respiratory cells, most notably in pre-I interneurons. Given prior anatomical evidence, these NK1R-ir pre-I interneurons are most likely glutamatergic. The data are consistent with the possibility that the NK1R-ir pre-I interneurons of the adult preB?tC could be the adult form of a class of inspiratory neurons that are rhythmogenic in the neonate (either the pacemakers and/or an excitatory subtype of follower neurons).     

Hydrogen peroxide differentially affects activity in the pre-Bötzinger complex and hippocampus

Reactive oxygen species (ROS) modulate neuronal excitability. In the present study we examined the effects of hydrogen peroxide (H(2)O(2)), a well established ROS, on neuronal activity from two neonatal mouse brain regions, i.e., the pre-B?tzinger complex (preB?tC) within the ventral respiratory column (VRC) and the CA1 area of the hippocampus. In the preB?tC, 2.2 mM H(2)O(2) evoked a transient depression followed by augmentation of neuronal activity. The iron chelator deferoxamine (500 μM) did not prevent H(2)O(2)-mediated neuronal augmentation but prevented the initial depression. Combined application of Fe(2+) and H(2)O(2) only caused depression of the preB?tC rhythm. In contrast, H(2)O(2) suppressed neuronal activity in the CA1 region, and this effect was accentuated by coapplication of Fe(2+) and H(2)O(2), suggesting that hydroxyl radical generated by Fenton reaction mediates the effects of H(2)O(2) on CA1 neuronal activity. Malondialdehyde (MDA) levels were monitored as an index of lipid peroxidation in H(2)O(2)-treated preB?tC and CA1 areas. MDA levels were unaltered in H(2)O(2)-treated preB?tC, whereas MDA levels were markedly elevated in the CA1 region. These findings suggest that 1) exogenous administration of H(2)O(2) exerts differential effects on neuronal activities of preB?tC versus CA1 neuronal populations and 2) H(2)O(2) is a potent modulator of respiratory rhythmogenesis from the preB?tC without affecting global oxidative status.     

Functional imaging reveals respiratory network activity during hypoxic and opioid challenge in the neonate rat tilted sagittal slab preparation

In mammals, respiration-modulated networks are distributed rostrocaudally in the ventrolateral quadrant of the medulla. Recent studies have established that in neonate rodents, two spatially separate networks along this column-the parafacial respiratory group (pFRG) and the pre-B?tzinger complex (preB?tC)-are hypothesized to be sufficient for respiratory rhythm generation, but little is known about the connectivity within or between these networks. To be able to observe how these networks interact, we have developed a neonate rat medullary tilted sagittal slab, which exposes one column of respiration-modulated neurons on its surface, permitting functional imaging with cellular resolution. Here we examined how respiratory networks responded to hypoxic challenge and opioid-induced depression. At the systems level, the sagittal slab was congruent with more intact preparations: hypoxic challenge led to a significant increase in respiratory period and inspiratory burst amplitude, consistent with gasping. At opioid concentrations sufficient to slow respiration, we observed periods at integer multiples of control, matching quantal slowing. Consistent with single-unit recordings in more intact preparations, respiratory networks were distributed bimodally along the rostrocaudal axis, with respiratory neurons concentrated at the caudal pole of the facial nucleus, and 350 microns caudally, at the level of the pFRG and the preB?tC, respectively. Within these regions neurons active during hypoxia- and/or opioid-induced depression were ubiquitous and interdigitated. In particular, contrary to earlier reports, opiate-insensitive neurons were found at the level of the preB?tC.     

Neurokinin receptor-expressing pre-botzinger complex neurons in neonatal mice studied in vitro

Breathing in mammals depends on inspiratory-related neural activity generated in the pre-B?tzinger complex (preB?tC), where neurokinin receptor-expressing neurons (NKR(+)) have been hypothesized to play a critical rhythmogenic role. Currently, the extent to which the preB?tC is populated by rhythmogenic NKR(+) neurons and whether neurons without neurokinin receptor expression (NKR(-)) share similar electrical properties with NKR(+) neurons are not well understood. These interrelated problems must be resolved to understand the widespread excitatory effects of neuropeptides and the mechanism of respiratory rhythmogenesis. We recorded and imaged inspiratory neurons in neonatal mouse slices that isolate the preB?tC and generate respiratory motor output in vitro. Using tetramethylrhodamine conjugated to the endogenous NKR agonist substance P (TMR-SP) to tag neurons that express NKRs, we show that early inspiratory neurons with small whole cell capacitance (C(M)) are 36% TMR-SP(+) and 64% TMR-SP(-). Also, late inspiratory neurons with large C(M) are 67% TMR-SP(+) and 33% are TMR-SP(-). Thus NKR(+) and NKR(-) neurons exhibit the same phenotypic properties, which suggests that they may share functional roles also. Substance P (SP) alone evoked a voltage-insensitive inward current (I(SP)) that reversed at -19 mV and was associated with an increase in membrane conductance in both NKR(+) and NKR(-) neurons. Gap junctions may be needed to confer SP sensitivity to neurons that appear to lack NKR expression. We propose that cell death in NKR(+) preB?tC neurons, by targeted lesion or neurodegeneration, may impair breathing behavior by killing less than one half of the rhythmogenic preB?tC neurons and a large number of respiratory premotoneurons.     

Sleep-disordered breathing after targeted ablation of preBötzinger complex neurons

Ablation of preB?tzinger complex (preB?tC) neurons, critical for respiratory rhythm generation, resulted in a progressive, increasingly severe disruption of respiratory pattern, initially during sleep and then also during wakefulness in adult rats. Sleep-disordered breathing is highly prevalent in elderly humans and in some patients with neurodegenerative disease. We propose that sleep-disordered breathing results from loss of preB?tC neurons and could underlie death during sleep in these populations.     

Pharmacology of nicotinic receptors in preBötzinger complex that mediate modulation of respiratory pattern

Nicotine regulates respiratory pattern by modulating excitatory neurotransmission affecting inspiratory neurons within the preB?tzinger Complex (preB?tC). The nicotinic acetylcholine receptor (nAChR) subtypes mediating these effects are unknown. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve (XIIn) and patch-clamped inspiratory neurons in the preB?tC simultaneously. The alpha7 nAChR antagonists alpha-bungarotoxin or methyllycaconitine (MLA) had little effect on the actions of low concentrations of nicotine (0.5 microM), which included an increase in respiratory frequency; a decrease in amplitude of XIIn inspiratory bursts; a tonic inward current associated with an increase in membrane noise; an increase in the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), and; a decrease in the amplitude of inspiratory drive current in voltage-clamped preB?tC inspiratory neurons. These nicotinic actions were completely reversed by dihydro-beta-erythroidine (DH-beta-E) or hexamethonium and reduced by D-tubocurarine. Comparable concentrations of RJR-2403 (0.5-1 microM), an agonist selective for alpha4beta2 nAChRs, increased respiratory frequency to 186% and decreased the amplitude of XIIn inspiratory bursts to 83% of baseline. In voltage-clamped preB?tC inspiratory (including pacemaker) neurons, RJR-2403 induced a tonic inward current of -15.2 pA associated with an increase in membrane noise, increased the frequency to 157% and amplitude to 106% of spontaneous EPSCs, and decreased the amplitude of inspiratory drive current to 80% of baseline. MLA had little effect on RJR-2403 actions, while DH-beta-E completely reversed them. These results suggest that the predominant subtype of nAChRs in preB?tC in neonatal rats that mediates the modulation of respiratory pattern by low concentrations of nicotine is an alpha4beta2 combination and not an alpha7 subunit homomer. We do not exclude the possibility that co-assembly of alpha4beta2 with other subunits or other nAChR subtypes are also expressed in preB?tC neurons. The parallel changes in the cellular and systems level responses induced by different nicotinic agonists and antagonists support the idea that modulation of excitatory neurotransmission affecting preB?tC inspiratory neurons is a mechanism underlying the cholinergic regulation of respiratory pattern (). This study provides a useful model system for evaluating potential therapeutic cholinergic agents for their respiratory effects and side effects.     

Silencing preBötzinger complex somatostatin-expressing neurons induces persistent apnea in awake rat

Delineating neurons that underlie complex behaviors is of fundamental interest. Using adeno-associated virus 2, we expressed the Drosophila allatostatin receptor in somatostatin (Sst)-expressing neurons in the preB?tzinger Complex (preB?tC). Rapid silencing of these neurons in awake rats induced a persistent apnea without any respiratory movements to rescue their breathing. We hypothesize that breathing requires preB?tC Sst neurons and that their sudden depression can lead to serious, even fatal, respiratory failure.     

Mechanisms underlying regulation of respiratory pattern by nicotine in preBötzinger complex

Cholinergic neurotransmission plays a role in regulation of respiratory pattern. Nicotine from cigarette smoke affects respiration and is a risk factor for sudden infant death syndrome (SIDS) and sleep-disordered breathing. The cellular and synaptic mechanisms underlying this regulation are not understood. Using a medullary slice preparation from neonatal rat that contains the preB?tzinger Complex (preB?tC), the hypothesized site for respiratory rhythm generation, and generates respiratory-related rhythm in vitro, we examined the effects of nicotine on excitatory neurotransmission affecting inspiratory neurons in preB?tC and on the respiratory-related motor activity from hypoglossal nerve (XIIn). Microinjection of nicotine into preB?tC increased respiratory frequency and decreased the amplitude of inspiratory bursts, whereas when injected into XII nucleus induced a tonic activity and an increase in amplitude but not in frequency of inspiratory bursts from XIIn. Bath application of nicotine (0.2--0.5 microM, approximately the arterial blood nicotine concentration immediately after smoking a cigarette) increased respiratory frequency up to 280% of control in a concentration-dependent manner. Nicotine decreased the amplitude to 82% and increased the duration to 124% of XIIn inspiratory bursts. In voltage-clamped preB?tC inspiratory neurons (including neurons with pacemaker properties), nicotine induced a tonic inward current of -19.4 +/- 13.4 pA associated with an increase in baseline noise. Spontaneous excitatory postsynaptic currents (sEPSCs) present during the expiratory period increased in frequency to 176% and in amplitude to 117% of control values; the phasic inspiratory drive inward currents decreased in amplitude to 66% and in duration to 89% of control values. The effects of nicotine were blocked by mecamylamine (Meca). The inspiratory drive current and sEPSCs were completely eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the presence or absence of nicotine. In the presence of tetrodotoxin (TTX), low concentrations of nicotine did not induce any tonic current or any increase in baseline noise, nor affect the input resistance in inspiratory neurons. In this study, we demonstrated that nicotine increased respiratory frequency and regulated respiratory pattern by modulating the excitatory neurotransmission in preB?tC. Activation of nicotinic acetylcholine receptors (nAChRs) enhanced the tonic excitatory synaptic input to inspiratory neurons including pacemaker neurons and at the same time, inhibited the phasic excitatory coupling between these neurons. These mechanisms may account for the cholinergic regulation of respiratory frequency and pattern.     

Acetylcholine modulates respiratory pattern: effects mediated by M3-like receptors in preBötzinger complex inspiratory neurons

Perturbations of cholinergic neurotransmission in the brain stem affect respiratory motor pattern both in vivo and in vitro; the underlying cellular mechanisms are unclear. Using a medullary slice preparation from neonatal rat that spontaneously generates respiratory rhythm, we patch-clamped inspiratory neurons in the preB?tzinger complex (preB?tC), the hypothesized site for respiratory rhythm generation, and simultaneously recorded respiratory-related motor output from the hypoglossal nerve (XIIn). Most (88%) of the inspiratory neurons tested responded to local application of acetylcholine (ACh) or carbachol (CCh) or bath application of muscarine. Bath application of 50 microM muscarine increased the frequency, amplitude, and duration of XIIn inspiratory bursts. At the cellular level, muscarine induced a tonic inward current, increased the duration, and decreased the amplitude of the phasic inspiratory inward currents in preB?tC inspiratory neurons recorded under voltage clamp at -60 mV. Muscarine also induced seizure-like activity evident during expiratory periods in XIIn activity; these effects were blocked by atropine. In the presence of tetrodotoxin (TTX), local ejection of 2 mM CCh or ACh onto preB?tC inspiratory neurons induced an inward current along with an increase in membrane conductance under voltage clamp and induced a depolarization under current clamp. This response was blocked by atropine in a concentration-dependent manner. Bath application of 1 microM pirenzepine, 10 microM gallamine, or 10 microM himbacine had little effect on the CCh-induced current, whereas 10 microM 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the current. The current-voltage (I-V) relationship of the CCh-induced response was linear in the range of -110 to -20 mV and reversed at -11.4 mV. Similar responses were found in both pacemaker and nonpacemaker inspiratory neurons. The response to CCh was unaffected when patch electrodes contained a high concentration of EGTA (11 mM) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (10 mM). The response to CCh was reduced greatly by substitution of 128 mM Tris-Cl for NaCl in the bath solution; the I-V curve shifted to the left and the reversal potential shifted to -47 mV. Lowering extracellular Cl(-) concentration from 140 to 70 mM had no effect on the reversal potential. These results suggest that in preB?tC inspiratory neurons, ACh acts on M3-like ACh receptors on the postsynaptic neurons to open a channel permeable to Na(+) and K(+) that is not Ca(2+) dependent. This inward cation current plays a major role in depolarizing preB?tC inspiratory neurons, including pacemakers, that may account for the ACh-induced increase in the frequency of respiratory motor output observed at the systems/behavioral level.     

Normal breathing requires preBötzinger complex neurokinin-1 receptor-expressing neurons

The normal breathing rhythm in mammals is hypothesized to be generated by neurokinin-1 receptor (NK1R)-expressing neurons in the preB?tzinger complex (preB?tC), a medullary region proposed to contain the kernel of the circuits generating respiration. If this hypothesis is correct, then complete destruction of preB?tC NK1R neurons should severely perturb and perhaps even fatally arrest breathing. Here we show that specific and near complete bilateral (but not unilateral) destruction of preB?tC NK1R neurons results in both an ataxic breathing pattern with markedly altered blood gases and pH, and pathological responses to challenges such as hyperoxia, hypoxia and anesthesia. Thus, these approximately 600 neurons seem necessary for the generation of normal breathing in rats.     

Graded reductions in oxygenation evoke graded reconfiguration of the isolated respiratory network

Neurons depend on aerobic metabolism, yet are very sensitive to oxidative stress and, as a consequence, typically operate in a low O(2) environment. The balance between blood flow and metabolic activity, both of which can vary spatially and dynamically, suggests that local O(2) availability markedly influences network output. Yet the understanding of the underlying O(2)-sensing mechanisms is limited. Are network responses regulated by discrete O(2)-sensing mechanisms or, rather, are they the consequence of inherent O(2) sensitivities of mechanisms that generate the network activity? We hypothesized that a broad range of O(2) tensions progressively modulates network activity of the pre-B?tzinger complex (preB?tC), a neuronal network critical to the central control of breathing. Rhythmogenesis was measured from the preB?tC in transverse neonatal mouse brain stem slices that were exposed to graded reductions in O(2) between 0 and 95% O(2), producing tissue oxygenation values ranging from 20 ± 18 (mean ± SE) to 440 ± 56 Torr at the slice surface, respectively. The response of the preB?tC to graded changes in O(2) is progressive for some metrics and abrupt for others, suggesting that different aspects of the respiratory network have different sensitivities to O(2).     

Energetics and oxygen transport mechanisms in embryos

Abrupt, bilateral destruction of the pre-Bötzinger Complex (preBötC) leads to terminal apnea in unanesthetized goats and rats. In contrast, respiratory rhythm and pattern and arterial blood gases in goats during wakefulness and sleep are normal after incremental (over a month) destruction of >90% of the preBötC. Here, we tested the hypothesis that the difference in effects between abrupt and incremental destruction of the preBötC are a result of time-dependent plasticity, which manifests as anatomic changes at sites within the respiratory network. Accordingly, we report data from histological analyses comparing the brainstems of control goats, and goats that had undergone bilateral, incremental, ibotenic acid (IA)-induced preBötC lesioning. A major focus was on the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and the pontine respiratory group (PRG), which are sites thought to contribute to respiratory rhythmogenesis. We also studied the facial (FN), rostral nucleus ambiguus (NA), medullary raphé (MRN), hypoglossal (HN), and the dorsal motor vagal (DMV) nuclei. Neuronal counts, count region area (mm²), and neuronal densities were calculated using computer-assisted analyses and/or manual microscopy to compare control and preBötC-lesioned animals. We found that within the ventral and lateral medulla 2 mm rostral to the caudal pole of the FN (presumed pFRG/RTN), there were 25% and 65% more (P < 0.001) neurons, respectively, in preBötC-lesioned compared to control goats. Lesioned goats also showed 14% and 13% more (P < 0.001) neurons in the HN and medial parabrachialis nucleus, but 46%, 28%, 7%, and 17% fewer (P < 0.001) neurons in the FN, NA, DMV, and Kölliker-Fuse nuclei, respectively. In the remaining sites analyzed, there were no differences between groups. We conclude that anatomic changes at multiple sites within the respiratory network may contribute to the time-dependent plasticity in breathing following incremental and near-complete destruction of the preBötC.     

Isolation of the kernel for respiratory rhythm generation in a novel preparation: the pre-Bötzinger complex "island"

The pre-B?tzinger complex (pre-B?tC), a bilaterally distributed network of rhythmogenic neurons within the ventrolateral medulla, has been proposed to be the critical locus for respiratory rhythm generation in mammals. To date, thin transverse medullary slice preparations that capture the pre-B?tC have served as the optimal experimental model to study the region's inherent cellular and network properties. We have reduced the thin slices to isolated pre-B?tC "islands" to further establish whether the pre-B?tC has intrinsic rhythmicity and is the kernel for rhythmogenesis in the slice. We recorded neuron population activity locally in the pre-B?tC with macroelectrodes and fluorescent imaging of Ca(2+) activities with Calcium Green-1AM dye before and after excising the island. The isolated island remained rhythmically active with a population burst profile similar to the inspiratory burst in the slice. Rhythmic population activity persisted in islands after block of GABA(A)ergic and glycinergic synaptic inhibition. The loci of pre-B?tC Ca(2+) activity imaged in thin slices and islands were similar, and imaged pre-B?tC neurons exhibited synchronized flashing after blocking synaptic inhibition. Population burst frequency increased monotonically as extracellular potassium concentration was elevated, consistent with mathematical models consisting entirely of an excitatory network of synaptically coupled pacemaker neurons with heterogeneous, voltage-dependent bursting properties. Our results provide further evidence for a rhythmogenic kernel in the pre-B?tC in vitro and demonstrate that the islands are ideal preparations for studying the kernel's intrinsic properties.     

Central chemoreceptor modulation of breathing via multipath tuning in medullary ventrolateral respiratory column circuits

Ventrolateral respiratory column (VRC) circuits that modulate breathing in response to changes in central chemoreceptor drive are incompletely understood. We employed multielectrode arrays and spike train correlation methods to test predictions of the hypothesis that pre-B?tzinger complex (pre-B?tC) and retrotrapezoid nucleus/parafacial (RTN-pF) circuits cooperate in chemoreceptor-evoked tuning of ventral respiratory group (VRG) inspiratory neurons. Central chemoreceptors were selectively stimulated by injections of CO(2)-saturated saline into the vertebral artery in seven decerebrate, vagotomized, neuromuscularly blocked, and artificially ventilated cats. Among sampled neurons in the B?tzinger complex (B?tC)-to-VRG region, 70% (161 of 231) had a significant change in firing rate after chemoreceptor stimulation, as did 70% (101 of 144) of the RTN-pF neurons. Other responsive neurons (24 B?tC-VRG; 11 RTN-pF) had a change in the depth of respiratory modulation without a significant change in average firing rate. Seventy B?tC-VRG chemoresponsive neurons triggered 189 offset-feature correlograms (96 peaks; 93 troughs) with at least one responsive B?tC-VRG cell. Functional input from at least one RTN-pF cell could be inferred for 45 B?tC-VRG neurons (19%). Eleven RTN-pF cells were correlated with more than one B?tC-VRG target neuron, providing evidence for divergent connectivity. Thirty-seven RTN-pF neurons, 24 of which were chemoresponsive, were correlated with at least one chemoresponsive B?tC-VRG neuron. Correlation linkage maps and spike-triggered averages of phrenic nerve signals suggest transmission of chemoreceptor drive via a multipath network architecture: RTN-pF modulation of pre-B?tC-VRG rostral-to-caudal excitatory inspiratory neuron chains is tuned by feedforward and recurrent inhibition from other inspiratory neurons and from "tonic" expiratory neurons.     

Serotonergic modulation of respiratory motoneurons and interneurons in brainstem slices of perinatal rats

Respiration-related membrane potential fluctuations were recorded in hypoglossal (XII) motoneurons and pre-B?tzinger complex (pre-B?tC) interneurons in medullary slices from perinatal rats. Bath application of serotonin (5-HT) evoked a ketanserine-sensitive depolarization (approximately 11 mV) and tonic spike discharge in XII motoneurons, whereas pre-B?tC neurons responded with a <6 mV depolarization and no tonic discharge. The membrane effects were accompanied by an increase in respiratory frequency by up to 260% in 64% of preparations. A frequency decrease leading to block of respiratory activity could also occur (20%) as well as an initial acceleration that turned into a frequency depression (16%). In contrast, iontophoresis of 5-HT into the pre-B?tC exclusively increased respiratory frequency by 30-220%, whereas iontophoresis into the XII nucleus did not change respiratory frequency but induced tonic nerve discharge. The effects of local iontophoretic administration of 5-HT on membrane properties of XII and pre-B?tC cells were very similar to those upon bath application. Bath application and iontophoresis of the 5-HT2 receptor agonist -methyl-hydroxytryptamine mimicked the effects of 5-HT. Bath application of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide did not affect XII nerve bursting or pre-B?tC neurons. Iontophoresis of 8-hydroxydipropylaminotetralin hydrobromide had almost no effect on respiratory frequency and induced in the interneurons either a depolarization or hyperpolarization (<5 mV) which was blocked by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)N-2-pyridinylcyclohexane carboxamide. In conclusion, 5-HT-evoked tonic excitation of respiratory XII motoneurons is mediated by postsynaptic 5-HT2 receptors. The excitatory effects on respiratory rhythm are also primarily attributable to postsynaptic 5-HT2 receptors of pre-B?tC neurons. Additional modulatory effects on the interneurons appear to be mediated by postsynaptic 5-HT1A receptors.     

Dynamics of neuromodulatory feedback determines frequency modulation in a reduced respiratory network: A computational study

Neuromodulators, such as amines and neuropeptides, alter the activity of neurons and neuronal networks. In this work, we investigate how neuromodulators, which activate G_q-protein second messenger systems, can modulate the bursting frequency of neurons in a critical portion of the respiratory neural network, the pre-Bötzinger complex (preBötC). These neurons are a vital part of the ponto-medullary neuronal network, which generates a stable respiratory rhythm whose frequency is regulated by neuromodulator release from the nearby Raphe nucleus. Using a simulated 50-cell network of excitatory preBötC neurons with a heterogeneous distribution of persistent sodium conductance and Ca²⁺, we determined conditions for frequency modulation in such a network by simulating interaction between Raphe and preBötC nuclei. We found that the positive feedback between the Raphe excitability and preBötC activity induces frequency modulation in the preBötC neurons. In addition, the frequency of the respiratory rhythm can be regulated via phasic release of excitatory neuromodulators from the Raphe nucleus. We predict that the application of a G_q antagonist will eliminate this frequency modulation by the Raphe and keep the network frequency constant and low. In contrast, application of a G_q agonist will result in a high frequency for all levels of Raphe stimulation. Our modeling results also suggest that high [K⁺] requirement in respiratory brain slice experiments may serve as a compensatory mechanism for low neuromodulatory tone.     

Identification of NO/sGC signalling in the bulbospinal respiratory pathway after C2-C3 hemisection of the spinal cord in rats

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylyl cyclase (β1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2-C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around β1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2-C3 hemisection by a loss of β1sGC positivity. The results confirm, that β1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.     

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 B?tzinger and pre-B?tzinger complexes to the ventral respiratory group (B?t-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 B?t-VRG neurons were monitored with multielectrode arrays along with phrenic nerve activity in 14 decerebrate, vagotomized cats. Overall, 214 RTN-pF and 398 B?t-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 B?t-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 B?t-VRG neurons (10.0%) were triggers in 74 B?t-VRG→RTN-pF correlograms with offset features, suggesting that B?t-VRG trigger neurons influence RTN-pF target neurons. The results support the hypothesis that local RTN-pF neuron interactions and inputs from B?t-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.