Developmental changes in NMDA receptor subunits alter burst frequency of neonatal rat brainstem slices containing the 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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Organotypic slice cultures containing the preBötzinger complex generate respiratory-like rhythms

Journal of Neurophysiology ◽

10.1152/jn.00904.2015 ◽

2016 ◽

Vol 115 (2) ◽

pp. 1063-1070 ◽

Cited By ~ 7

Author(s):

Wiktor S. Phillips ◽

Mikkel Herly ◽

Christopher A. Del Negro ◽

Jens C. Rekling

Keyword(s):

Brain Stem ◽

Drug Exposure ◽

Fluorescent Dyes ◽

Calcium Oscillations ◽

Oscillatory Activity ◽

Slice Culture ◽

Burst Frequency ◽

Prebotzinger Complex ◽

Prebötzinger Complex

Study of acute brain stem slice preparations in vitro has advanced our understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. In the current study, we have developed an organotypic slice culture preparation containing the preBötzinger complex (preBötC), the core inspiratory rhythm generator of the ventrolateral brain stem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBötC) and dorsomedial regions of slice cultures at 7–43 days in vitro. These calcium oscillations appear to be driven by periodic bursts of inspiratory neuronal activity, because whole cell recordings from ventrolateral neurons in culture revealed inspiratory-like drive potentials, and no oscillatory activity was detected from glial fibrillary associated protein-expressing astrocytes in cultures. Acute slices showed a burst frequency of 10.9 ± 4.2 bursts/min, which was not different from that of brain stem slice cultures (13.7 ± 10.6 bursts/min). However, slice cocultures that include two cerebellar explants placed along the dorsolateral border of the brainstem displayed up to 193% faster burst frequency (22.4 ± 8.3 bursts/min) and higher signal amplitude (340%) compared with acute slices. We conclude that preBötC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended incubation (e.g., genetic transfection or chronic drug exposure) while simultaneously being amenable to imaging and electrophysiology at cellular, synaptic, and network levels.

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Inhibition of endoplasmic reticulum Ca2+ ATPase in preBötzinger complex of neonatal rat does not affect respiratory rhythm generation

Neuroscience ◽

10.1016/j.neuroscience.2012.08.016 ◽

2012 ◽

Vol 224 ◽

pp. 116-124 ◽

Cited By ~ 11

Author(s):

L. Beltran-Parrazal ◽

J. Fernandez-Ruiz ◽

R. Toledo ◽

J. Manzo ◽

C. Morgado-Valle

Keyword(s):

Endoplasmic Reticulum ◽

Respiratory Rhythm ◽

Neonatal Rat ◽

Rhythm Generation ◽

Respiratory Rhythm Generation ◽

Prebotzinger Complex ◽

Prebötzinger Complex

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Developmental changes in the expression of NMDA receptor subunits (NR1, NR2A, NR2B) in the cat visual cortex and the effects of dark rearing

Molecular Brain Research ◽

10.1016/s0169-328x(00)00076-0 ◽

2000 ◽

Vol 78 (1-2) ◽

pp. 196-200 ◽

Cited By ~ 52

Author(s):

Lu Chen ◽

Nigel G.F Cooper ◽

George D Mower

Keyword(s):

Visual Cortex ◽

Nmda Receptor ◽

Developmental Changes ◽

Nmda Receptor Subunits ◽

Cat Visual Cortex ◽

Dark Rearing

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Developmental Changes in the Role of Adenosine Clearance Mechanisms in Modulating the Activity of the preBötzinger Complex Inspiratory Network Under Basal and Hypoxic Conditions

The FASEB Journal ◽

10.1096/fasebj.2019.33.1_supplement.546.1 ◽

2019 ◽

Vol 33 (S1) ◽

Author(s):

Robert Jeffrey Reklow ◽

Tucaauê S. Alvares ◽

Sara M. Frangos ◽

Megan A. Hansen ◽

Gregory D. Funk

Keyword(s):

Developmental Changes ◽

Hypoxic Conditions ◽

Prebotzinger Complex ◽

Prebötzinger Complex

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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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