scholarly journals Organotypic slice cultures containing the preBötzinger complex generate respiratory-like rhythms

2016 ◽  
Vol 115 (2) ◽  
pp. 1063-1070 ◽  
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.

Serotonin elicits long-lasting enhancement of rhythmic respiratory activity in turtle brain stems in vitro

2001 ◽  
Vol 91 (6) ◽  
pp. 2703-2712 ◽  
Author(s):  
Stephen M. Johnson ◽  
Julia E. R. Wilkerson ◽  
Daniel R. Henderson ◽  
Michael R. Wenninger ◽  
Gordon S. Mitchell
Keyword(s):  
Brain Stem ◽  
Respiratory Activity ◽  
Dependent Manner ◽  
Frequency Increase ◽  
Burst Frequency ◽  
Bath Application ◽  
Burst Amplitude ◽  
Dose Dependent ◽  
The Brain

Brain stem preparations from adult turtles were used to determine how bath-applied serotonin (5-HT) alters respiration-related hypoglossal activity in a mature vertebrate. 5-HT (5–20 μM) reversibly decreased integrated burst amplitude by ∼45% ( P < 0.05); burst frequency decreased in a dose-dependent manner with 20 μM abolishing bursts in 9 of 13 preparations ( P < 0.05). These 5-HT-dependent effects were mimicked by application of a 5-HT1A agonist, but not a 5-HT1B agonist, and were abolished by the broad-spectrum 5-HT antagonist, methiothepin. During 5-HT (20 μM) washout, frequency rebounded to levels above the original baseline for 40 min ( P < 0.05) and remained above baseline for 2 h. A 5-HT3 antagonist (tropesitron) blocked the post-5-HT rebound and persistent frequency increase. A 5-HT3 agonist (phenylbiguanide) increased frequency during and after bath application ( P < 0.05). When phenylbiguanide was applied to the brain stem of brain stem/spinal cord preparations, there was a persistent frequency increase ( P < 0.05), but neither spinal-expiratory nor -inspiratory burst amplitude were altered. The 5-HT3receptor-dependent persistent frequency increase represents a unique model of plasticity in vertebrate rhythm generation.

cAMP‐dependent modulation of I h underlies the P2Y 1 receptor‐mediated excitation of the preBötzinger Complex inspiratory network in vitro

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Yong Zhang ◽  
Vivian Biancardi ◽  
Ana Miranda Tapia ◽  
Toka Abu Jaib ◽  
Alexander Gourine ◽  
...  
Keyword(s):  
Prebotzinger Complex ◽  
Prebötzinger Complex

scholarly journals Neuronal Mediators of the Frequency Increase Evoked by Activation of P2Y 1 Receptors in the preBötzinger Complex Inspiratory Rhythm Generating Network in Vitro

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Ana Miranda Tapia ◽  
Vivian Biancardi ◽  
Robert Reklow ◽  
Wei Zhang ◽  
Vladimir Rancic ◽  
...  
Keyword(s):  
Frequency Increase ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex

scholarly journals Microglia attenuate the opioid‐induced depression of preBötzinger Complex (preBötC) inspiratory rhythm in vitro via a TLR4‐independent pathway

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Jennifer D Zwicker ◽  
Yong Zhang ◽  
Mark R Hutchinson ◽  
Kenner C Rice ◽  
Linda R Watkins ◽  
...  
Keyword(s):  
Prebotzinger Complex ◽  
Prebötzinger Complex

Serotonergic modulation of respiratory motor output during tadpole development

2002 ◽  
Vol 93 (3) ◽  
pp. 936-946 ◽  
Author(s):  
Richard Kinkead ◽  
Olivier Belzile ◽  
Roumiana Gulemetova
Keyword(s):  
Brain Stem ◽  
Lung Ventilation ◽  
Receptor Activation ◽  
Motor Output ◽  
Burst Frequency ◽  
Bath Application ◽  
Age Dependent ◽  
Dose Dependent ◽  
Serotonergic Modulation

To test the hypothesis that serotonin (5-hydroxytryptamine; 5-HT)-receptor activation elicits age-dependent changes in respiratory motor output, we compared the effects of 5-HT bath application (5-HT concentration = 0.5–25 μM) onto in vitro brain stem preparations from pre- and postmetamorphic bullfrog tadpoles. Recording of motor output related to gill and lung ventilation showed that 5-HT elicits a dose-dependent depression of gill burst frequency in both groups. In contrast, the lung burst frequency response was stage dependent; an increase in lung burst frequency at low 5-HT concentration (≤0.5 μM) was observed only in the postmetamorphic group. Higher 5-HT concentrations decreased lung burst frequency in all preparations. Gill burst frequency attenuation is mediated (at least in part) by 5-HT1A-receptor activation in an age-dependent fashion. We conclude that serotonergic modulation of respiratory motor output 1) changes during tadpole development and 2) is distinct for gill and lung ventilation.

scholarly journals Gender influence on in vitro rhythmogenesis from the preBotzinger complex

2011 ◽  
Vol 25 (S1) ◽  
Author(s):  
Naama Rotem‐Kohavi ◽  
Alfredo J. Garcia ◽  
Sebastien Zanella ◽  
Jan‐Marino Ramirez
Keyword(s):  
Gender Influence ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex

scholarly journals Purinergic signaling mediates neuroglial interactions to generate sighs

2021 ◽  
Author(s):  
Jan Marino Ramirez ◽  
Liza Severs ◽  
Nicholas Bush ◽  
Lely Quina ◽  
Nicholas Burgraff ◽  
...  
Keyword(s):  
Experimental Evidence ◽  
Purinergic Signaling ◽  
Emergent Property ◽  
Calcium Oscillations ◽  
Computational Simulations ◽  
Hypoxic Conditions ◽  
Neuroglial Interactions ◽  
Rhythmic Behaviors ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex

Abstract Sighs prevent the collapse of alveoli in the lungs, initiate arousal under hypoxic conditions, and even express sadness and relief. Sighs are periodically superimposed on normal breaths, known as eupnea. Implicated in the generation of these rhythmic behaviors is the preBötzinger complex (preBötC)1. Yet how this small microcircuit can produce two rhythms with strikingly different periodicities remains unresolved. Our computational simulations predict that sighs are generated by the coincidence of two temporally distinct calcium oscillations and are in agreement with experimental evidence suggesting that astrocytes drive sigh behavior through slower, extrinsically driven calcium oscillations that link the eupnea and sigh rhythms. We found that purinergic signaling is necessary to generate spontaneous and hypoxia- induced sighs, and photo-activation of preBötC astrocytes is sufficient to elicit sigh activity. We conclude that sighs are an emergent property of the preBötC network generated by neuroglial interactions, where the distinct modulatory responses of neurons and glia allow for both rhythms to be independently regulated.

scholarly journals Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides

2021 ◽  
Vol 22 (6) ◽  
pp. 2820
Author(s):  
Stephan Altmann ◽  
Jürgen Mut ◽  
Natalia Wolf ◽  
Jutta Meißner-Weigl ◽  
Maximilian Rudert ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Adipogenic Differentiation ◽  
Culture Conditions ◽  
Surface Proteins ◽  
Differentiation Potential ◽  
Or Gene ◽  
Mannose Derivatives ◽  
Metabolic Glycoengineering ◽  
Scientific Questions

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4ManNAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.

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