Aminophylline modulation of the mouse respiratory network changes during postnatal maturation

Aminophylline is a respiratory stimulant commonly used for the treatment of central apnea. Experiences from clinical practice, however, revealed that aminophylline is not reliably effective in preterm infants, whereas it is normally effective in infants and mature patients. In an established animal model for postnatal development of respiratory control mechanisms, we therefore examined the hypothesis that the clinical observations reflect a developmental change in the sensitivity of the central respiratory network to methylxanthines. The medullary respiratory network was isolated at different postnatal ages ( postnatal days 1–13; P1–P13) in a transverse mouse brain stem slice preparation. This preparation contains the pre-Bötzinger complex (PBC), a region that is critical for generation of respiratory rhythm. Spontaneous rhythmic respiratory activity was recorded from the hypoglossal (XII) rootlets and from neurons in the PBC by using the whole cell patch clamp technique. Bath-applied aminophylline [20 μM] increased the frequency (+41%) in neonatal animals (P1–P6) without affecting the amplitude of respiratory burst activity in XII rootlets. The same concentration of aminophylline did not have any significant effect on the frequency of respiratory XII bursts but increased the amplitude (+31%) in juvenile animals (P7–P13). In the same age group, aminophylline also augmented the amplitude and the duration of respiratory synaptic drive currents in respiratory PBC neurons. The data demonstrate that augmentation of the respiratory output is due to direct enhancement of central respiratory network activity and increase of synaptic drive of hypoglossal motoneurons in juvenile, but not neonatal, animals. This indicates a developmental change in the efficacy of aminophylline to reinforce central respiratory network activity. Therefore, we believe that the variable success in treating respiratory disturbances in premature infants reflects maturational changes in the expression of receptors and/or intracellular signal pathways in the central respiratory network.

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Identification of Two Types of Inspiratory Pacemaker Neurons in the Isolated Respiratory Neural Network of Mice

Journal of Neurophysiology ◽

10.1152/jn.2001.86.1.104 ◽

2001 ◽

Vol 86 (1) ◽

pp. 104-112 ◽

Cited By ~ 122

Author(s):

Muriel Thoby-Brisson ◽

Jan-Marino Ramirez

Keyword(s):

Neural Network ◽

Respiratory Rhythm ◽

Network Activity ◽

Pacemaker Activity ◽

Patch Clamp Technique ◽

Population Activity ◽

Pacemaker Neurons ◽

Whole Cell Patch Clamp ◽

Respiratory Network ◽

Bursting Activity

In the respiratory network of mice, we characterized with the whole cell patch-clamp technique pacemaker properties in neurons discharging in phase with inspiration. The respiratory network was isolated in a transverse brain stem slice containing the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. After blockade of respiratory network activity with 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), 18 of 52 inspiratory neurons exhibited endogenous pacemaker activity, which was voltage dependent, could be reset by brief current injections and could be entrained by repetitive stimuli. In the pacemaker group ( n = 18), eight neurons generated brief bursts (0.43 ± 0.03 s) at a relatively high frequency (1.05 ± 0.12 Hz) in CNQX. These bursts resembled the bursts that these neurons generated in the intact network during the interval between two inspiratory bursts. Cadmium (200 μM) altered but did not eliminate this bursting activity, while 0.5 μM tetrodotoxin suppressed bursting activity. Another set of pacemaker neurons (10 of 18) generated in CNQX longer bursts (1.57 ± 0.07 s) at a lower frequency (0.35 ± 0.01 Hz). These bursts resembled the inspiratory bursts generated in the intact network in phase with the population activity. This bursting activity was blocked by 50–100 μM cadmium or 0.5 μM tetrodotoxin. We conclude that the respiratory neural network contains pacemaker neurons with two types of bursting properties. The two types of pacemaker activities might have different functions within the respiratory network.

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Imaging of respiratory-related population activity with single-cell resolution

AJP Cell Physiology ◽

10.1152/ajpcell.00253.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. C508-C516 ◽

Cited By ~ 22

Author(s):

Frank Funke ◽

Mathias Dutschmann ◽

Michael Müller

Keyword(s):

Single Cell ◽

Neuronal Activity ◽

Single Cells ◽

Activity Patterns ◽

Respiratory Rhythm ◽

Network Activity ◽

Respiratory Neurons ◽

Ventrolateral Medulla ◽

Population Activity ◽

Respiratory Network

The pre-Bötzinger complex (PBC) in the rostral ventrolateral medulla contains a kernel involved in respiratory rhythm generation. So far, its respiratory activity has been analyzed predominantly by electrophysiological approaches. Recent advances in fluorescence imaging now allow for the visualization of neuronal population activity in rhythmogenic networks. In the respiratory network, voltage-sensitive dyes have been used mainly, so far, but their low sensitivity prevents an analysis of activity patterns of single neurons during rhythmogenesis. We now have succeeded in using more sensitive Ca2+ imaging to study respiratory neurons in rhythmically active brain stem slices of neonatal rats. For the visualization of neuronal activity, fluo-3 was suited best in terms of neuronal specificity, minimized background fluorescence, and response magnitude. The tissue penetration of fluo-3 was improved by hyperosmolar treatment (100 mM mannitol) during dye loading. Rhythmic population activity was imaged with single-cell resolution using a sensitive charge-coupled device camera and a ×20 objective, and it was correlated with extracellularly recorded mass activity of the contralateral PBC. Correlated optical neuronal activity was obvious online in 29% of slices. Rhythmic neurons located deeper became detectable during offline image processing. Based on their activity patterns, 74% of rhythmic neurons were classified as inspiratory and 26% as expiratory neurons. Our approach is well suited to visualize and correlate the activity of several single cells with respiratory network activity. We demonstrate that neuronal synchronization and possibly even network configurations can be analyzed in a noninvasive approach with single-cell resolution and at frame rates currently not reached by most scanning-based imaging techniques.

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Lack of the Kir4.1 Channel Subunit Abolishes K+ Buffering Properties of Astrocytes in the Ventral Respiratory Group: Impact on Extracellular K+ Regulation

Journal of Neurophysiology ◽

10.1152/jn.00996.2005 ◽

2006 ◽

Vol 95 (3) ◽

pp. 1843-1852 ◽

Cited By ~ 122

Author(s):

Clemens Neusch ◽

Nestoras Papadopoulos ◽

Michael Müller ◽

Iris Maletzki ◽

Stefan M. Winter ◽

...

Keyword(s):

Fluorescent Protein ◽

Respiratory Rhythm ◽

Brain Slices ◽

Physiological Role ◽

Functional Expression ◽

Network Activity ◽

Early Postnatal Development ◽

Ventral Respiratory Group ◽

Respiratory Network ◽

Green Fluorescent

Ongoing rhythmic neuronal activity in the ventral respiratory group (VRG) of the brain stem results in periodic changes of extracellular K+. To estimate the involvement of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in extracellular K+ clearance, we examined its functional expression in astrocytes of the respiratory network. Kir4.1 was expressed in astroglial cells of the VRG, predominantly in fine astrocytic processes surrounding capillaries and in close proximity to VRG neurons. Kir4.1 expression was up-regulated during early postnatal development. The physiological role of astrocytic Kir4.1 was studied using mice with a null mutation in the Kir4.1 channel gene that were interbred with transgenic mice expressing the enhanced green fluorescent protein in their astrocytes. The membrane potential was depolarized in astrocytes of Kir4.1−/− mice, and Ba2+-sensitive inward K+ currents were diminished. Brain slices from Kir4.1−/− mice, containing the pre-Bötzinger complex, which generates a respiratory rhythm, did not show any obvious differences in rhythmic bursting activity compared with wild-type controls, indicating that the lack of Kir4.1 channels alone does not impair respiratory network activity. Extracellular K+ measurements revealed that Kir4.1 channels contribute to extracellular K+ regulation. Kir4.1 channels reduce baseline K+ levels, and they compensate for the K+ undershoot. Our data indicate that Kir4.1 channels 1) are expressed in perineuronal processes of astrocytes, 2) constitute the major part of the astrocytic Kir conductance, and 3) contribute to regulation of extracellular K+ in the respiratory network.

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Functional Imaging Reveals Respiratory Network Activity During Hypoxic and Opioid Challenge in the Neonate Rat Tilted Sagittal Slab Preparation

Journal of Neurophysiology ◽

10.1152/jn.01056.2006 ◽

2007 ◽

Vol 97 (3) ◽

pp. 2283-2292 ◽

Cited By ~ 40

Author(s):

Benjamin J. Barnes ◽

Chi-Minh Tuong ◽

Nicholas M. Mellen

Keyword(s):

Functional Imaging ◽

Respiratory Rhythm ◽

Network Activity ◽

Respiratory Neurons ◽

Respiratory Network ◽

Burst Amplitude ◽

Single Unit Recordings ◽

Parafacial Respiratory Group ◽

Neonate Rat ◽

Respiratory Networks

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-Bötzinger complex (preBötC)—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 preBötC, 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 preBötC.

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Mechanisms Underlying Adaptation of Respiratory Network Activity to Modulatory Stimuli in the Mouse Embryo

Neural Plasticity ◽

10.1155/2016/3905257 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Marc Chevalier ◽

Rafaël De Sa ◽

Laura Cardoit ◽

Muriel Thoby-Brisson

Keyword(s):

Ph Regulation ◽

Respiratory Rhythm ◽

Network Activity ◽

Pacemaker Neurons ◽

Functional Flexibility ◽

Respiratory Rhythmogenesis ◽

Brainstem Slice ◽

Respiratory Network ◽

The Impact

Breathing is a rhythmic behavior that requires organized contractions of respiratory effector muscles. This behavior must adapt to constantly changing conditions in order to ensure homeostasis, proper body oxygenation, and CO2/pH regulation. Respiratory rhythmogenesis is controlled by neural networks located in the brainstem. One area considered to be essential for generating the inspiratory phase of the respiratory rhythm is the preBötzinger complex (preBötC). Rhythmogenesis emerges from this network through the interplay between the activation of intrinsic cellular properties (pacemaker properties) and intercellular synaptic connections. Respiratory activity continuously changes under the impact of numerous modulatory substances depending on organismal needs and environmental conditions. The preBötC network has been shown to become active during the last third of gestation. But only little is known regarding the modulation of inspiratory rhythmicity at embryonic stages and even less on a possible role of pacemaker neurons in this functional flexibility during the prenatal period. By combining electrophysiology and calcium imaging performed on embryonic brainstem slice preparations, we provide evidence showing that embryonic inspiratory pacemaker neurons are already intrinsically sensitive to neuromodulation and external conditions (i.e., temperature) affecting respiratory network activity, suggesting a potential role of pacemaker neurons in mediating rhythm adaptation to modulatory stimuli in the embryo.

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Norepinephrine Differentially Modulates Different Types of Respiratory Pacemaker and Nonpacemaker Neurons

Journal of Neurophysiology ◽

10.1152/jn.01308.2005 ◽

2006 ◽

Vol 95 (4) ◽

pp. 2070-2082 ◽

Cited By ~ 77

Author(s):

Jean-Charles Viemari ◽

Jan-Marino Ramirez

Keyword(s):

Frequency Modulation ◽

Respiratory Rhythm ◽

Network Activity ◽

Flufenamic Acid ◽

Pacemaker Activity ◽

Rhythm Generation ◽

Burst Frequency ◽

Pacemaker Neurons ◽

Respiratory Network ◽

Different Types

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

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Astrocytes release prostaglandin E2 to modify respiratory network activity

10.1101/150250 ◽

2017 ◽

Author(s):

David Forsberg ◽

Thomas Ringstedt ◽

Eric Herlenius

Keyword(s):

Prostaglandin E2 ◽

Oscillation Frequency ◽

Respiratory Rhythm ◽

Active Role ◽

Time Lapse ◽

Network Activity ◽

Calcium Oscillation ◽

Network Activation ◽

Respiratory Network ◽

Pge2 Release

AbstractProstaglandin E2 (PGE2) released during hypercapnic challenge increases Ca2+ oscillation frequency in the chemosensitive parafacial respiratory group (pFRG/RTN). Here, we demonstrate that pFRG/RTN astrocytes are the PGE2 source. Two distinct astrocyte subtypes were found using transgenic mice expressing GFP and MrgA1 receptors in astrocytes. Although most astrocytes appeared dormant during time-lapse calcium imaging, a subgroup displayed persistent, rhythmic oscillating calcium activity. These active astrocytes formed a subnetwork within the respiratory network distinct from the neuronal network. Activation of exogenous MrgA1 receptors expressed in astrocytes tripled their calcium oscillation frequency activity in both the preBötzinger complex and pFRG/RTN. However, neurons in the preBötC were unaffected, whereas neuronal calcium oscillatory frequency in pFRG/RTN doubled. Notably, astrocyte activation in pFRG/RTN triggered local PGE2 release and blunted the hypercapnic response. Thus, astrocytes play an active role in respiratory rhythm modulation, modifying respiratory-related behaviorthrough PGE2 release in the pFRG/RTN.

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Astrocytes release prostaglandin E2 to modify respiratory network activity

eLife ◽

10.7554/elife.29566 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 13

Author(s):

David Forsberg ◽

Thomas Ringstedt ◽

Eric Herlenius

Keyword(s):

Prostaglandin E2 ◽

Respiratory Rhythm ◽

Active Role ◽

Time Lapse ◽

Calcium Oscillations ◽

Network Activity ◽

Calcium Oscillation ◽

Network Activation ◽

Respiratory Network ◽

Pge2 Release

Previously (Forsberg et al., 2016), we revealed that prostaglandin E2 (PGE2), released during hypercapnic challenge, increases calcium oscillations in the chemosensitive parafacial respiratory group (pFRG/RTN). Here, we demonstrate that pFRG/RTN astrocytes are the PGE2 source. Two distinct astrocyte subtypes were found using transgenic mice expressing GFP and MrgA1 receptors in astrocytes. Although most astrocytes appeared dormant during time-lapse calcium imaging, a subgroup displayed persistent, rhythmic oscillating calcium activity. These active astrocytes formed a subnetwork within the respiratory network distinct from the neuronal network. Activation of exogenous MrgA1Rs expressed in astrocytes tripled astrocytic calcium oscillation frequency in both the preBötzinger complex and pFRG/RTN. However, neurons in the preBötC were unaffected, whereas neuronal calcium oscillatory frequency in pFRG/RTN doubled. Notably, astrocyte activation in pFRG/RTN triggered local PGE2 release and blunted the hypercapnic response. Thus, astrocytes play an active role in respiratory rhythm modulation, modifying respiratory-related behavior through PGE2 release in the pFRG/RTN.

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Reciprocal connectivity of the periaqueductal gray with the ponto-medullary respiratory network in rat

10.1101/2020.09.15.298927 ◽

2020 ◽

Author(s):

Pedro Trevizan-Baú ◽

Werner I. Furuya ◽

Stuart B. Mazzone ◽

Davor Stanić ◽

Rishi R. Dhingra ◽

...

Keyword(s):

Respiratory Rhythm ◽

Upper Airway ◽

Periaqueductal Gray ◽

List Type ◽

Airway Patency ◽

Respiratory Network ◽

Cholera Toxin Subunit B ◽

Motor Activities ◽

Descending Projections ◽

Subunit B

AbstractSynaptic activities of the periaqueductal gray (PAG) can modulate or appropriate the respiratory motor activities in the context of behavior and emotion via descending projections to nucleus retroambiguus. However, alternative anatomical pathways for the mediation of PAG-evoked respiratory modulation via core nuclei of the brainstem respiratory network remains only partially described. We injected the retrograde tracer Cholera toxin subunit B (CT-B) in the pontine Kölliker-Fuse nucleus (KFn, n=5), medullary Bötzinger (BötC, n=3) and pre-Bötzinger complexes (pre-BötC; n=3), and the caudal raphé nuclei (n=3), and quantified the ascending and descending connectivity of the PAG. CT-B injections in the KFn, pre-BötC, and caudal raphé, but not in the BötC, resulted in CT-B-labeled neurons that were predominantly located in the lateral and ventrolateral PAG columns. In turn, CT-B injections into the lateral and ventrolateral PAG columns (n=4) yield the highest numbers of CT-B-labeled neurons in the KFn and far fewer numbers of labeled neurons in the pre-BötC and caudal raphé. Analysis of the relative projection strength revealed that the KFn shares the densest reciprocal connectivity with the PAG (ventrolateral and lateral columns, in particular). Overall, our data imply that the PAG may engage a distributed respiratory rhythm and pattern generating network beyond the nucleus retroambiguus to mediate downstream modulation of breathing. However, the reciprocal connectivity of the KFn and PAG suggests specific roles for synaptic interaction between these two nuclei that are most likely related to the regulation of upper airway patency during vocalization or other volitional orofacial behaviors.HighlightsThe lateral and ventrolateral PAG project to the primary respiratory network.The Kölliker-Fuse nucleus shares the densest reciprocal connectivity with the PAG.The Bötzinger complex appears to have very little connectivity with the PAG.

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