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 preBötzinger Complex (preBötC), 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 preBötC and on the respiratory-related motor activity from hypoglossal nerve (XIIn). Microinjection of nicotine into preBötC 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 μM, 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 preBötC 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 preBötC. 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.

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Pharmacology of Nicotinic Receptors in PreBötzinger Complex That Mediate Modulation of Respiratory Pattern

Journal of Neurophysiology ◽

10.1152/jn.2002.88.4.1851 ◽

2002 ◽

Vol 88 (4) ◽

pp. 1851-1858 ◽

Cited By ~ 38

Author(s):

Xuesi M. Shao ◽

Jack L. Feldman

Keyword(s):

Respiratory Frequency ◽

Respiratory Pattern ◽

Drive Current ◽

Inward Current ◽

Membrane Noise ◽

Inspiratory Neurons ◽

Low Concentrations ◽

Excitatory Neurotransmission ◽

Prebotzinger Complex ◽

Prebötzinger Complex

Nicotine regulates respiratory pattern by modulating excitatory neurotransmission affecting inspiratory neurons within the preBötzinger Complex (preBötC). 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 preBötC simultaneously. The α7 nAChR antagonists α-bungarotoxin or methyllycaconitine (MLA) had little effect on the actions of low concentrations of nicotine (0.5 μM), 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 preBötC inspiratory neurons. These nicotinic actions were completely reversed by dihydro-β-erythroidine (DH-β-E) or hexamethonium and reduced by d-tubocurarine. Comparable concentrations of RJR-2403 (0.5–1 μM), an agonist selective for α4β2 nAChRs, increased respiratory frequency to 186% and decreased the amplitude of XIIn inspiratory bursts to 83% of baseline. In voltage-clamped preBötC 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-β-E completely reversed them. These results suggest that the predominant subtype of nAChRs in preBötC in neonatal rats that mediates the modulation of respiratory pattern by low concentrations of nicotine is an α4β2 combination and not an α7 subunit homomer. We do not exclude the possibility that co-assembly of α4β2 with other subunits or other nAChR subtypes are also expressed in preBötC 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 preBötC inspiratory neurons is a mechanism underlying the cholinergic regulation of respiratory pattern ( Shao and Feldman 2001 ). This study provides a useful model system for evaluating potential therapeutic cholinergic agents for their respiratory effects and side effects.

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Acetylcholine Modulates Respiratory Pattern: Effects Mediated by M3-Like Receptors in PreBötzinger Complex Inspiratory Neurons

Journal of Neurophysiology ◽

10.1152/jn.2000.83.3.1243 ◽

2000 ◽

Vol 83 (3) ◽

pp. 1243-1252 ◽

Cited By ~ 70

Author(s):

X. M. Shao ◽

J. L. Feldman

Keyword(s):

Voltage Clamp ◽

Respiratory Rhythm ◽

Reversal Potential ◽

Motor Output ◽

Neonatal Rat ◽

Inward Current ◽

Inspiratory Neurons ◽

Bath Application ◽

Prebotzinger Complex ◽

Prebötzinger Complex

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 preBötzinger complex (preBötC), 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 μM 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 preBötC 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 preBötC 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 μM pirenzepine, 10 μM gallamine, or 10 μM himbacine had little effect on the CCh-induced current, whereas 10 μM 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-Vcurve 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 preBötC 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 Ca2+dependent. This inward cation current plays a major role in depolarizing preBötC 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.

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Emergence of Population Bursts from Simultaneous Activation of Small Subsets of preBotzinger Complex Inspiratory Neurons

Journal of Neuroscience ◽

10.1523/jneurosci.4574-12.2013 ◽

2013 ◽

Vol 33 (8) ◽

pp. 3332-3338 ◽

Cited By ~ 35

Author(s):

K. Kam ◽

J. W. Worrell ◽

C. Ventalon ◽

V. Emiliani ◽

J. L. Feldman

Keyword(s):

Inspiratory Neurons ◽

Simultaneous Activation ◽

Prebotzinger Complex ◽

Prebötzinger Complex

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Phase- and State-Dependent Modulation of Breathing Pattern by preBötzinger Complex Somatostatin Expressing Neurons

10.1101/2021.04.12.439520 ◽

2021 ◽

Author(s):

Raquel P. de Sousa Abreu ◽

Evgeny Bondarenko ◽

Jack L. Feldman

Keyword(s):

Breathing Pattern ◽

Pattern Generation ◽

Respiratory Pattern ◽

Phase Advance ◽

Breathing Frequency ◽

State Dependent ◽

Freely Behaving ◽

Freely Moving ◽

Prebotzinger Complex ◽

Prebötzinger Complex

AbstractAs neuronal subtypes are increasingly categorized, delineating their functional role is paramount. The preBötzinger Complex (preBötC) subpopulation expressing the neuropeptide somatostatin (SST) is classified as mostly excitatory, inspiratory-modulated and not rhythmogenic. We further characterized their phenotypic identity; 87% were glutamatergic and the balance were glycinergic and/or GABAergic. We then used optogenetics to investigate their modulatory role in both anesthetized and freely moving mice. In anesthetized mice, short photostimulation (100 ms) of preBötC SST+ neurons modulated breathing-related variables in a combinatory phase- and state-dependent manner; changes in inspiratory duration, inspiratory peak amplitude (Amp), and phase were different at higher (≥2.5 Hz) vs. lower (<2.5 Hz) breathing frequency. Moreover, we observed a biphasic effect of photostimulation during expiration that is probabilistic, i.e., photostimulation given at the same phase in consecutive cycles can evoke opposite responses (lengthening vs. shortening of the phase). This unexpected probabilistic state- and phase-dependent responses to photostimulation exposed properties of the preBötC that were not predicted and cannot be readily accounted for in current models of preBötC pattern generation. In freely moving mice, prolonged photostimulation decreased f in normoxia, hypoxia, or hypercapnia, and increased Amp and produced a phase advance, which was similar to the results in anesthetized mice when f≥2.5 Hz. We conclude that preBötC SST+ neurons are a key mediator of the extraordinary and essential lability of breathing pattern.Key points summaryWe transfected preBötzinger Complex (preBötC) somatostatin-expressing (SST+) neurons, which modulate respiratory pattern, but are not rhythmogenic, with channelrhodopsin to investigate phase- and state-dependent modulation of breathing pattern in anesthetized and freely behaving mice in normoxia, hypoxia, and hypercapnia.In anesthetized mice, photostimulation of preBötC SST+ neurons during inspiration increased inspiratory duration and amplitude regardless of baseline breathing frequency, f.In anesthetized mice with low f (<2.5 Hz), photostimulation of preBötC SST+ neurons during expiration evoked either phase advance or phase delay, whereas in anesthetized mice with high f (≥2.5 Hz) and in freely behaving mice in normoxia, hypoxia, or hypercapnia, photostimulation always evoked phase advance.Phase- and state-dependency is a function of overall breathing network excitability.The f-dependent probabilistic modulation of breathing pattern by preBötC SST+ neurons was unexpected, requiring reconsideration of current models of preBötC function, which neither predict nor can readily account for such responses.

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