Modulation of gasp frequency by activation of pre-Bötzinger complex in vivo

Under hyperoxic conditions, both chemical stimulation of neurons and focal hypoxia in the pre-B?tzinger complex (pre-B?tC) in vivo modify the eupneic pattern of inspiratory motor output by eliciting changes in the patterning and timing of phrenic bursts, which includes both phasic and tonic excitation. The influence of this region on the gasping pattern of phrenic motor output produced during severe brain hypoxia is unknown. We therefore examined the effects of chemical stimulation of neurons (DL-homocysteic acid; DLH; 10 mM; < or =20 nl) and focal hypoxia (sodium cyanide; NaCN; 1 mM; < or =20 nl) in the pre-B?tC on hypoxia-induced gasping in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Unilateral microinjection of DLH into the pre-B?tC during hypoxia-induced gasping increased phrenic burst frequency by approximately 630% (P < 0.01) over baseline frequency due predominantly to a reduction in T(E) (from 28.9 +/- 6.2 to 5.2 +/- 1.8 s; mean +/- SE; P < 0.01). No significant changes in T(I) or rate of rise between hypoxia-induced gasps and the DLH-induced bursts were observed; the effects on peak amplitude of integrated phrenic nerve discharge were variable. Similar responses were evoked by unilateral microinjection of NaCN into the pre-B?tC. These findings demonstrate that both activation of pre-B?tC neurons and focal hypoxia in the pre-B?tC not only influence the eupneic pattern of phrenic motor output but also modify the expression of hypoxia-induced gasping in vivo. These findings also provide additional support to the concept of intrinsic hypoxic chemosensitivity of the pre-B?tC.     

Modulation of expiratory motor output evoked by chemical activation of pre-Bötzinger complex in vivo

We have previously demonstrated that chemical stimulation of the pre-B?tzinger complex (pre-B?tC) in the anesthetized cat produces either phasic or tonic excitation of phrenic nerve discharge. This region is characterized by a mixture of inspiratory-modulated, expiratory-modulated, and phase-spanning (including pre-inspiratory (pre-I)) neurons; however, its influence on expiratory motor output is unknown. We, therefore, examined the effects of chemical stimulation of the pre-B?tC on expiratory motor output recorded from the caudal iliohypogastric (lumbar, L(2)) nerve. We found that unilateral microinjection of DL-homocysteic acid (DLH; 10 mM; 10-20 nl) into 16 sites in the pre-B?tC enhanced lumbar nerve discharge, including changes in timing and patterning similar to those previously reported for phrenic motor output. Both increased peak amplitude and frequency of phasic lumbar bursts as well as tonic excitation of lumbar motor activity were observed. In some cases, evoked phasic lumbar nerve activity was synchronized in phase with phrenic nerve discharge. These findings demonstrate that chemical stimulation of the pre-B?tC not only excites inspiratory motor activity but also excites expiratory motor output, suggesting a role for the pre-B?tC in generation and modulation of inspiratory and expiratory rhythm and pattern.     

Hypercapnic and hypoxic responses require intact neural transmission from the pre-Bötzinger complex

The central respiratory network that includes the pre-Bötzinger complex (pre-BötC), a region believed to contain rhythmogenic neurons, is capable of responding to fluctuations in CO₂ and pH. However, the role of inputs from this site in mediating ventilatory responses to hypercapnia and/or hypoxia in nonsedated animals is not well established. Therefore, in the present study we tested the hypothesis that altered transmission from the pre-BötC to its target sites would decrease chemosensory responsiveness to acute hypercapnia and modulate the ventilatory response to hypoxia. Colchicine was used to block axonal transport. At 48 h after bilateral microinjections of colchicine into the pre-BötC (100 μg/uL, 100 nL/site), but not saline, the baseline frequency of breathing decreased; however, rhythmicity was not altered. In addition, there was a significant fall in the ventilatory response to hypercapnia (5 and 12% CO₂) and hypoxia (8% O₂). These findings indicate that, inputs from pre-BötC neurons are of critical importance in providing the normal ventilatory response to both hypercapnia and hypoxia.     

Patterns of phrenic motor output evoked by chemical stimulation of neurons located in the pre-Bötzinger complex in vivo

The pre-B?tzinger complex (pre-B?tC) has been proposed to be essential for respiratory rhythm generation from work in vitro. Much less, however, is known about its role in the generation and modulation of respiratory rhythm in vivo. Therefore we examined whether chemical stimulation of the in vivo pre-B?tC manifests respiratory modulation consistent with a respiratory rhythm generator. In chloralose- or chloralose/urethan-anesthetized, vagotomized cats, we recorded phrenic nerve discharge and arterial blood pressure in response to chemical stimulation of neurons located in the pre-B?tC with DL-homocysteic acid (DLH; 10 mM; 21 nl). In 115 of the 122 sites examined in the pre-B?tC, unilateral microinjection of DLH produced an increase in phrenic nerve discharge that was characterized by one of the following changes in cycle timing and pattern: 1) a rapid series of high-amplitude, rapid rate of rise, short-duration bursts, 2) tonic excitation (with or without respiratory oscillations), 3) an integration of the first two types of responses (i.e., tonic excitation with high-amplitude, short-duration bursts superimposed), or 4) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a high-amplitude, short-duration burst). In 107 of these sites, the phrenic neurogram response was accompanied by an increase or decrease (>/=10 mmHg) in arterial blood pressure. Thus increases in respiratory burst frequency and production of tonic discharge of inspiratory output, both of which have been seen in vitro, as well as modulation of burst pattern can be produced by local perturbations of excitatory amino acid neurotransmission in the pre-B?tC in vivo. These findings are consistent with the proposed role of this region as the locus for respiratory rhythm generation.     

Inspiratory-phase short time scale synchrony in the brainstem slice is generated downstream of the pre-Bötzinger complex

Respiratory neurons are synchronized on a long time scale to generate inspiratory and expiratory-phase activities that are critical for respiration. Long time scale synchrony within the respiratory network occurs on a time scale of more than hundreds of milliseconds to seconds. During inspiration, neurons are synchronized on a short time scale to produce synchronous oscillations, which shape the pattern of inspiratory motor output. This latter form of synchrony within the respiratory network spans a shorter time range of tens of milliseconds. In the neonatal mouse rhythmically active medullary slice preparation, we recorded bilateral inspiratory activity from hypoglossal (XII) rootlets to study where in the slice synchronous oscillations are generated. Based on previous work that proposed the origin of these oscillations, we tested the pre-B?tzinger complex (PreB?tC) and the XII motor nucleus. Unilateral excitation of the PreB?tC, via local application of a perfusate containing high K(+), increased mean inspiratory burst frequency bilaterally (296+/-66%; n=10, P<0.01), but had no effect on the relative power of oscillations. In contrast, unilateral excitation of the XII nucleus increased both mean peak integrated activity bilaterally (ipsilateral: 41+/-10%, P<0.01; contralateral: 17+/-7%; P<0.05, n=10) and oscillation power in the ipsilateral (50+/-17%, n=7, P<0.05), but not in the contralateral rootlet. Cross-correlation analysis of control inspiratory activity recorded from the left and right XII rootlets produced cross-correlation histograms with significant peaks centered around a time lag of zero and showed no subsidiary harmonic peaks. Coherence analysis of left and right XII rootlet recordings demonstrated that oscillations are only weakly coherent. Together, the findings from local application experiments and cross-correlation and coherence analyses indicate that short time scale synchronous oscillations recorded in the slice are likely generated in or immediately upstream of the XII motor nucleus.     

Pre-Bötzinger complex functions as a central hypoxia chemosensor for respiration in vivo

Recently, we identified a region located in the pre-B?tzinger complex (pre-B?tC; the proposed locus of respiratory rhythm generation) in which activation of ionotropic excitatory amino acid receptors using DL-homocysteic acid (DLH) elicits a variety of excitatory responses in the phrenic neurogram, ranging from tonic firing to a rapid series of high-amplitude, rapid rate of rise, short-duration inspiratory bursts that are indistinguishable from gasps produced by severe systemic hypoxia. Therefore we hypothesized that this unique region is chemosensitive to hypoxia. To test this hypothesis, we examined the response to unilateral microinjection of sodium cyanide (NaCN) into the pre-B?tC in chloralose- or chloralose/urethan-anesthetized vagotomized, paralyzed, mechanically ventilated cats. In all experiments, sites in the pre-B?tC were functionally identified using DLH (10 mM, 21 nl) as we have previously described. All sites were histologically confirmed to be in the pre-B?tC after completion of the experiment. Unilateral microinjection of NaCN (1 mM, 21 nl) into the pre-B?tC produced excitation of phrenic nerve discharge in 49 of the 81 sites examined. This augmentation of inspiratory output exhibited one of the following changes in cycle timing and/or pattern: 1) a series of high-amplitude, short-duration bursts in the phrenic neurogram (a discharge similar to a gasp), 2) a tonic excitation of phrenic neurogram output, 3) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a gasplike burst), or 4) an increase in frequency of phrenic bursts accompanied by small increases or decreases in the amplitude of integrated phrenic nerve discharge. Our findings identify a locus in the brain stem in which focal hypoxia augments respiratory output. We propose that the respiratory rhythm generator in the pre-B?tC has intrinsic hypoxic chemosensitivity that may play a role in hypoxia-induced gasping.     

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.     

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.     

Postnatal development differentially affects voltage-activated calcium currents in respiratory rhythmic versus nonrhythmic neurons of the pre-Bötzinger complex

The mammalian respiratory network reorganizes during early postnatal life. We characterized the postnatal developmental changes of calcium currents in neurons of the pre-B?tzinger complex (pBC), the presumed site for respiratory rhythm generation. The pBC contains not only respiratory rhythmic (R) but also nonrhythmic neurons (nR). Both types of neurons express low- and high-voltage-activated (LVA and HVA) calcium currents. This raises the interesting issue: do calcium currents of the two co-localized neuron types have similar developmental profiles? To address this issue, we used the whole cell patch-clamp technique to compare in transverse slices of mice LVA and HVA calcium current amplitudes of the two neuron populations (R and nR) during the first and second postnatal week (P0-P16). The amplitude of HVA currents did not significantly change in R pBC-neurons (P0-P16), but it significantly increased in nR pBC-neurons during P8-P16. The dehydropyridine (DHP)-sensitive current amplitudes did not significantly change during the early postnatal development, suggesting that the observed amplitude changes in nR pBC-neurons are caused by (DHP) insensitive calcium currents. The ratio between HVA calcium current amplitudes dramatically changed during early postnatal development: At P0-P3, current amplitudes were significantly larger in R pBC-neurons, whereas at P8-P16, current amplitudes were significantly larger in nR pBC-neurons. Our results suggest that calcium currents in pBC neurons are differentially altered during postnatal development and that R pBC-neurons have fully expressed calcium currents early during postnatal development. This may be critical for stable respiratory rhythm generation in the underlying rhythm generating network.     

Role of the Bötzinger complex in fastigial nucleus-mediated respiratory responses

We have reported that the phrenic neurogram (PN) is modulated by stimulation of the fastigial nucleus (FN) of the cerebellum. The present study was undertaken to search for brainstem site(s) involved in the FN efferent pathway to modulate phrenic nerve activities. Experiments were performed on 35 anesthetized, paralyzed, and ventilated cats, using the PN as the index of the respiratory motor output. Results showed that bilateral electrolytic lesions of the red nucleus (RN), the paramedian reticular nucleus (PRN), or the pontine respiratory group (PRG) had little effect on the ability of FN stimulation to modulate the respiratory output. However, the modulation was abolished by bilateral electrolytic lesions of the B?tzinger complex (B?tC). Further studies showed that bilateral chemical inactivation of B?tC neurons produced by topical microinjection of kainic acid or cobalt chloride failed to abolish the modulation. We concluded that fibers of passage, not synapses or cell bodies in the B?tC, were involved in the modulatory effect of FN stimulation on the PN. The RN, PRN, and PRG appear not to be important in the neural circuitry responsible for the FN modulation of the phrenic activity.     

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.     

CD40 is necessary for activation of naïve T cells by a dendritic cell line in vivo but not in vitro

The importance of CD40–CD40L interactions during CD4⁺ T‐cell activation has been extensively investigated over the years; however, it still remains questionable whether the interaction is a prerequisite for dendritic cell (DC)‐mediated antigen‐specific priming in vivo. Naïve CD4⁺ T cells require two signals for proper activation and induction of differentiation: signal 1 is provided by peptide antigens in the context of the major histocompatibility complex (MHC) class II, while signal 2 is delivered by costimulatory molecules such as CD80 or CD86 present on the antigen‐presenting cell (APC). It is well known that the expression of CD80/CD86 is upregulated after interaction between CD40 on APCs and CD40L expressed by at least partly activated T cells. We used a DC line, JawsII, to compare the importance of CD40 expression and downstream signalling in vitro and in vivo. JawsII cells represent pre‐immature bone marrow‐derived DCs expressing low levels of MHC molecules, low levels of B7 molecules and no CD40. We have previously shown that JawsII cells, despite the lack of CD40 expression, are capable of priming naïve allogeneic T cells in vitro. In correlation with the current literature, we present data showing that constitutive expression of CD40 significantly increases the priming capacity of JawsII cells in vitro. In addition, we show that CD40 expression is required for JawsII cell‐dependent T‐cell priming in vivo.     

Cholinergic neurotransmission in the preBötzinger Complex modulates excitability of inspiratory neurons and regulates respiratory rhythm

We investigated whether there is endogenous acetylcholine (ACh) release in the preBötzinger Complex (preBötC), a medullary region hypothesized to contain neurons generating respiratory rhythm, and how endogenous ACh modulates preBötC neuronal function and regulates respiratory pattern. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve roots (XIIn) and patch-clamped preBötC inspiratory neurons. Unilateral microinjection of physostigmine, an acetylcholinesterase inhibitor, into the preBötC increased the frequency of respiratory-related rhythmic activity from XIIn to 116±13% (mean±S.D.) of control. Ipsilateral physostigmine injection into the hypoglossal nucleus (XII nucleus) induced tonic activity, increased the amplitude and duration of the integrated inspiratory bursts of XIIn to 122±17% and 117±22% of control respectively; but did not alter frequency. In preBötC inspiratory neurons, bath application of physostigmine (10 μM) induced an inward current of 6.3±10.6 pA, increased the membrane noise, decreased the amplitude of phasic inspiratory drive current to 79±16% of control, increased the frequency of spontaneous excitatory postsynaptic currents to 163±103% and decreased the whole cell input resistance to 73±22% of control without affecting the threshold for generation of action potentials. Bath application of physostigmine concurrently induced tonic activity, increased the frequency, amplitude and duration of inspiratory bursts of XIIn motor output. Bath application of 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 2 μM), a M3 muscarinic acetylcholine receptor (mAChR) selective antagonist, increased the input resistance of preBötC inspiratory neurons to 116±9% of control and blocked all of the effects of physostigmine except for the increase in respiratory frequency. Dihydro-β-erythroidine (DH-β-E; 0.2 μM), an 4β2 nicotinic receptor (nAChR) selective antagonist, blocked all the effects of physostigmine except for the increase in inspiratory burst amplitude. In the presence of both 4-DAMP and DH-β-E, physostigmine induced opposite effects, i.e. a decrease in frequency and amplitude of XIIn rhythmic activity. These results suggest that there is cholinergic neurotransmission in the preBötC which regulates respiratory frequency, and in XII nucleus which regulates tonic activity, and the amplitude and duration of inspiratory bursts of XIIn in neonatal rats. Physiologically relevant levels of ACh release, via mAChRs antagonized by 4-DAMP and nAChRs antagonized by DH-β-E, modulate the excitability of inspiratory neurons and excitatory neurotransmission in the preBötC, consequently regulating respiratory rhythm.     

ATP sensitivity of preBötzinger complex neurones in neonatal rat in vitro: mechanism underlying a P2 receptor-mediated increase in inspiratory frequency

P2 receptor (R) signalling plays an important role in the central ventilatory response to hypoxia. The frequency increase that results from activation of P2Y₁Rs in the preBötzinger complex (preBötC; putative site of inspiratory rhythm generation) may contribute, but neither the cellular nor ionic mechanism(s) underlying these effects are known. We applied whole-cell recording to rhythmically-active medullary slices from neonatal rat to define, in preBötC neurones, the candidate cellular and ionic mechanisms through which ATP influences rhythm, and tested the hypothesis that putative rhythmogenic preBötC neurones are uniquely sensitive to ATP. ATP (1 m m ) evoked inward currents in all non-respiratory neurones and the majority of respiratory neurons, which included inspiratory, expiratory and putative rhythmogenic inspiratory neurones identified by sensitivity to substance P (1 μ m ) and DAMGO (50 μ m ) or by voltage-dependent pacemaker-like activity. ATP current densities were similar in all classes of preBötC respiratory neurone. Reversal potentials and input resistance changes for ATP currents in respiratory neurones suggested they resulted from either inhibition of a K⁺ channel or activation of a mixed cationic conductance. The P2YR agonist 2MeSADP (1 m m ) evoked only the latter type of current in inspiratory and pacemaker-like neurones. In summary, putative rhythmogenic preBötC neurones were sensitive to ATP. However, this sensitivity was not unique; ATP evoked similar currents in all types of preBötC respiratory neurone. The P2Y₁R-mediated frequency increase is therefore more likely to reflect activation of a mixed cationic conductance in multiple types of preBötC neurone than excitation of one, highly sensitive group.     

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.     

Sympathoexcitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Bötzinger complex of rats

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/B?tzinger/pre-B?tzinger complexes (RVLM/B?tC/pre-B?tC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/B?tC/pre-B?tC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/B?tC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 ± 0.02 vs. 1 ± 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 ± 2 vs. 82 ± 2%; P < 0.05; n = 8), but decreased postinspiratory (35 ± 3 vs. 12 ± 2%; P < 0.05) and late-expiratory (24 ± 4 vs. 2 ±1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 ± 6 vs. 40 ± 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 ± 8 vs. 26 ± 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/B?tC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-B?tC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/B?tC and RVLM/pre-B?tC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/B?tC is essential to the coupling of active expiration and sympathoexcitatory response.     

Models of respiratory rhythm generation in the pre-B?tzinger complex. I. Bursting pacemaker neurons.

A network of oscillatory bursting neurons with excitatory coupling is hypothesized to define the primary kernel for respiratory rhythm generation in the pre-B?tzinger complex (pre-B?tC) in mammals. Two minimal models of these neurons are proposed. In model 1, bursting arises via fast activation and slow inactivation of a persistent Na+ current INaP-h. In model 2, bursting arises via a fast-activating persistent Na+ current INaP and slow activation of a K+ current IKS. In both models, action potentials are generated via fast Na+ and K+ currents. The two models have few differences in parameters to facilitate a rigorous comparison of the two different burst-generating mechanisms. Both models are consistent with many of the dynamic features of electrophysiological recordings from pre-B?tC oscillatory bursting neurons in vitro, including voltage-dependent activity modes (silence, bursting, and beating), a voltage-dependent burst frequency that can vary from 0.05 to >1 Hz, and a decaying spike frequency during bursting. These results are robust and persist across a wide range of parameter values for both models. However, the dynamics of model 1 are more consistent with experimental data in that the burst duration decreases as the baseline membrane potential is depolarized and the model has a relatively flat membrane potential trajectory during the interburst interval. We propose several experimental tests to demonstrate the validity of either model and to differentiate between the two mechanisms.     

Models of respiratory rhythm generation in the pre-B?tzinger complex. III. Experimental tests of model predictions.

We used the testable predictions of mathematical models proposed by Butera et al. to evaluate cellular, synaptic, and population-level components of the hypothesis that respiratory rhythm in mammals is generated in vitro in the pre-B?tzinger complex (pre-B?tC) by a heterogeneous population of pacemaker neurons coupled by fast excitatory synapses. We prepared thin brain stem slices from neonatal rats that capture the pre-B?tC and maintain inspiratory-related motor activity in vitro. We recorded pacemaker neurons extracellularly and found: intrinsic bursting behavior that did not depend on Ca(2+) currents and persisted after blocking synaptic transmission; multistate behavior with transitions from quiescence to bursting and tonic spiking states as cellular excitability was increased via extracellular K(+) concentration ([K(+)](o)); a monotonic increase in burst frequency and decrease in burst duration with increasing [K(+)](o); heterogeneity among different cells sampled; and an increase in inspiratory burst duration and decrease in burst frequency by excitatory synaptic coupling in the respiratory network. These data affirm the basis for the network model, which is composed of heterogeneous pacemaker cells having a voltage-dependent burst-generating mechanism dominated by persistent Na(+) current (I(NaP)) and excitatory synaptic coupling that synchronizes cell activity. We investigated population-level activity in the pre-B?tC using local "macropatch" recordings and confirmed these model predictions: pre-B?tC activity preceded respiratory-related motor output by 100-400 ms, consistent with a heterogeneous pacemaker-cell population generating inspiratory rhythm in the pre-B?tC; pre-B?tC population burst amplitude decreased monotonically with increasing [K(+)](o) (while frequency increased), which can be attributed to pacemaker cell properties; and burst amplitude fluctuated from cycle to cycle after decreasing bilateral synaptic coupling surgically as predicted from stability analyses of the model. We conclude that the pacemaker cell and network models explain features of inspiratory rhythm generation in vitro.     

Persistent sodium current,membrane properties and bursting behavior of pre-bötzinger complex inspiratory neurons in vitro

We measured persistent Na(+) current and membrane properties of bursting-pacemaker and nonbursting inspiratory neurons of the neonatal rat pre-B?tzinger complex (pre-B?tC) in brain stem slice preparations with a rhythmically active respiratory network in vitro. In whole-cell recordings, slow voltage ramps ( g(NaP)/g(Leak) in nonpacemaker cells (P < 0.0002). We conclude that I(NaP) is ubiquitously expressed by pre-B?tC inspiratory neurons and that bursting pacemaker behavior within the heterogeneous population of inspiratory neurons is achieved with specific ratios of these two conductances, g(NaP) and g(Leak).     

Models of respiratory rhythm generation in the pre-B?tzinger complex. II. Populations Of coupled pacemaker neurons.

We have proposed models for the ionic basis of oscillatory bursting of respiratory pacemaker neurons in the pre-B?tzinger complex. In this paper, we investigate the frequency control and synchronization of these model neurons when coupled by excitatory amino-acid-mediated synapses and controlled by convergent synaptic inputs modeled as tonic excitation. Simulations of pairs of identical cells reveal that increasing tonic excitation increases the frequency of synchronous bursting, while increasing the strength of excitatory coupling between the neurons decreases the frequency of synchronous bursting. Low levels of coupling extend the range of values of tonic excitation where synchronous bursting is found. Simulations of a heterogeneous population of 50-500 bursting neurons reveal coupling effects similar to those found experimentally in vitro: coupling increases the mean burst duration and decreases the mean burst frequency. Burst synchronization occurred over a wide range of intrinsic frequencies (0.1-1 Hz) and even in populations where as few as 10% of the cells were intrinsically bursting. Weak coupling, extreme parameter heterogeneity, and low levels of depolarizing input could contribute to the desynchronization of the population and give rise to quasiperiodic states. The introduction of sparse coupling did not affect the burst synchrony, although it did make the interburst intervals more irregular from cycle to cycle. At a population level, both parameter heterogeneity and excitatory coupling synergistically combine to increase the dynamic input range: robust synchronous bursting persisted across a much greater range of parameter space (in terms of mean depolarizing input) than that of a single model cell. This extended dynamic range for the bursting cell population indicates that cellular heterogeneity is functionally advantageous. Our modeled system accounts for the range of intrinsic frequencies and spiking patterns of inspiratory (I) bursting cells found in the pre-B?tzinger complex in neonatal rat brain stem slices in vitro. There is a temporal dispersion in the spiking onset times of neurons in the population, predicted to be due to heterogeneity in intrinsic neuronal properties, with neurons starting to spike before (pre-I), with (I), or after (late-I) the onset of the population burst. Experimental tests for a number of the model's predictions are proposed.