scholarly journals Inspiratory Off-Switch Mediated by Optogenetic Activation of Inhibitory Neurons in the preBötzinger Complex In Vivo

2021 ◽  
Vol 22 (4) ◽  
pp. 2019
Author(s):  
Swen Hülsmann ◽  
Liya Hagos ◽  
Volker Eulenburg ◽  
Johannes Hirrlinger
Keyword(s):  
Respiratory Rate ◽  
Respiratory Rhythm ◽  
Inhibitory Neurons ◽  
Respiratory Neurons ◽  
Ventrolateral Medulla ◽  
Transgenic Mouse Line ◽  
Respiratory Network ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex

The role of inhibitory neurons in the respiratory network is a matter of ongoing debate. Conflicting and contradicting results are manifold and the question whether inhibitory neurons are essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons are required in pulmonary reflexes for adapting the activity of the central respiratory network to the status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch. Over the years, optogenetic tools have been developed that allow for cell-specific activation of subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses Channelrhodopsin 2 in inhibitory neurons. A 200 µm multimode optical fiber ferrule was implanted in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that, in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some cases leading to complete cessation of breathing. However, a lower stimulation frequency (4–5 Hz) could induce a significant increase in the respiratory rate. This phenomenon can be explained by the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated breath and thereby increased the respiratory rate, while stimulation during the expiratory interval reduced the respiratory rate. Taken together, these results support the concept that activation of inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the preBötzinger complex.

scholarly journals Opioid-induced Respiratory Depression Is Only Partially Mediated by the preBötzinger Complex in Young and Adult Rabbits In Vivo

2015 ◽  
Vol 122 (6) ◽  
pp. 1288-1298 ◽  
Author(s):  
Astrid G. Stucke ◽  
Justin R. Miller ◽  
Ivana Prkic ◽  
Edward J. Zuperku ◽  
Francis A. Hopp ◽  
...  
Keyword(s):  
Respiratory Rate ◽  
Respiratory Depression ◽  
Respiratory Rhythm ◽  
Respiratory Drive ◽  
Phase Duration ◽  
Homocysteic Acid ◽  
Opioid Receptor Agonist ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex

Abstract Background: The preBötzinger Complex (preBC) plays an important role in respiratory rhythm generation. This study was designed to determine whether the preBC mediated opioid-induced respiratory rate depression at clinically relevant opioid concentrations in vivo and whether this role was age dependent. Methods: Studies were performed in 22 young and 32 adult New Zealand White rabbits. Animals were anesthetized, mechanically ventilated, and decerebrated. The preBC was identified by the tachypneic response to injection of d,l-homocysteic acid. (1) The μ-opioid receptor agonist [d-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO, 100 μM) was microinjected into the bilateral preBC and reversed with naloxone (1 mM) injection into the preBC. (2) Respiratory depression was achieved with intravenous remifentanil (0.08 to 0.5 μg kg−1 min−1). Naloxone (1 mM) was microinjected into the preBC in an attempt to reverse the respiratory depression. Results: (1) DAMGO injection depressed respiratory rate by 6 ± 8 breaths/min in young and adult rabbits (mean ± SD, P < 0.001). DAMGO shortened the inspiratory and lengthened the expiratory fraction of the respiratory cycle by 0.24 ± 0.2 in adult and young animals (P < 0.001). (2) During intravenous remifentanil infusion, local injection of naloxone into the preBC partially reversed the decrease in inspiratory fraction/increase in expiratory fraction in young and adult animals (0.14 ± 0.14, P < 0.001), but not the depression of respiratory rate (P = 0.19). PreBC injections did not affect respiratory drive. In adult rabbits, the contribution of non-preBC inputs to expiratory phase duration was larger than preBC inputs (3.5 [−5.2 to 1.1], median [25 to 75%], P = 0.04). Conclusions: Systemic opioid effects on respiratory phase timing can be partially reversed in the preBC without reversing the depression of respiratory rate.

scholarly journals Maturation of the Mammalian Respiratory System

1999 ◽  
Vol 79 (2) ◽  
pp. 325-360 ◽  
Author(s):  
Gérard Hilaire ◽  
Bernard Duron
Keyword(s):  
Respiratory Activity ◽  
Respiratory Rhythm ◽  
Ventrolateral Medulla ◽  
Pacemaker Neurons ◽  
Inhibitory Processes ◽  
Respiratory Rhythmogenesis ◽  
Respiratory Network ◽  
Network Functions

In this review, the maturational changes occurring in the mammalian respiratory network from fetal to adult ages are analyzed. Most of the data presented were obtained on rodents using in vitro approaches. In gestational day 18 (E18) fetuses, this network functions but is not yet able to sustain a stable respiratory activity, and most of the neonatal modulatory processes are not yet efficient. Respiratory motoneurons undergo relatively little cell death, and even if not yet fully mature at E18, they are capable of firing sustained bursts of potentials. Endogenous serotonin exerts a potent facilitation on the network and appears to be necessary for the respiratory rhythm to be expressed. In E20 fetuses and neonates, the respiratory activity has become quite stable. Inhibitory processes are not yet necessary for respiratory rhythmogenesis, and the rostral ventrolateral medulla (RVLM) contains inspiratory bursting pacemaker neurons that seem to constitute the kernel of the network. The activity of the network depends on CO2 and pH levels, via cholinergic relays, as well as being modulated at both the RVLM and motoneuronal levels by endogenous serotonin, substance P, and catecholamine mechanisms. In adults, the inhibitory processes become more important, but the RVLM is still a crucial area. The neonatal modulatory processes are likely to continue during adulthood, but they are difficult to investigate in vivo. In conclusion, 1) serotonin, which greatly facilitates the activity of the respiratory network at all developmental ages, may at least partly define its maturation; 2) the RVLM bursting pacemaker neurons may be the kernel of the network from E20 to adulthood, but their existence and their role in vivo need to be further confirmed in both neonatal and adult mammals.

Vagal Feedback Obscures the Effects of Systemic Opioids on Respiratory Rate in the preBötzinger Complex in Young Rabbits in vivo

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Astrid G. Stucke ◽  
Barbara Palkovic ◽  
Jennifer J. Callison ◽  
Vitaliy Marchenko ◽  
Eckehard A.E. Stuth ◽  
...  
Keyword(s):  
Respiratory Rate ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex

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

2007 ◽  
Vol 292 (1) ◽  
pp. C508-C516 ◽  
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.

scholarly journals PreBötzinger complex neurons drive respiratory modulation of blood pressure and heart rate

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Clément Menuet ◽  
Angela A Connelly ◽  
Jaspreet K Bassi ◽  
Mariana R Melo ◽  
Sheng Le ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Heart Rate ◽  
Respiratory Neurons ◽  
Neuron Activity ◽  
Neuronal Tracing ◽  
Cardiovascular Neurons ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex

Heart rate and blood pressure oscillate in phase with respiratory activity. A component of these oscillations is generated centrally, with respiratory neurons entraining the activity of pre-sympathetic and parasympathetic cardiovascular neurons. Using a combination of optogenetic inhibition and excitation in vivo and in situ in rats, as well as neuronal tracing, we demonstrate that preBötzinger Complex (preBötC) neurons, which form the kernel for inspiratory rhythm generation, directly modulate cardiovascular activity. Specifically, inhibitory preBötC neurons modulate cardiac parasympathetic neuron activity whilst excitatory preBötC neurons modulate sympathetic vasomotor neuron activity, generating heart rate and blood pressure oscillations in phase with respiration. Our data reveal yet more functions entrained to the activity of the preBötC, with a role in generating cardiorespiratory oscillations. The findings have implications for cardiovascular pathologies, such as hypertension and heart failure, where respiratory entrainment of heart rate is diminished and respiratory entrainment of blood pressure exaggerated.

Ventilatory responses to cooling the ventrolateral medullary surface of awake and anesthetized goats

1995 ◽  
Vol 78 (1) ◽  
pp. 247-257 ◽  
Author(s):  
P. J. Ohtake ◽  
H. V. Forster ◽  
L. G. Pan ◽  
T. F. Lowry ◽  
M. J. Korducki ◽  
...  
Keyword(s):  
Pulmonary Ventilation ◽  
Respiratory Rhythm ◽  
Respiratory Neurons ◽  
Ventrolateral Medulla ◽  
Arterial Pco2 ◽  
Respiratory Rhythmogenesis ◽  
Ventilatory Responses ◽  
Anesthesia Breathing ◽  
A Site ◽  
Caudal Area

The ventrolateral medulla (VLM) has been reported to be important as a source of tonic facilitation of dorsal respiratory neurons and as a site critical for respiratory rhythmogenesis. We investigated these theories in awake and anesthetized goats (n = 13) by using chronically implanted thermodes to create reversible neuronal dysfunction at superficial VLM sites between the first hypoglossal rootlet and the pontomedullary junction (area M (rostral) and area S). During halothane anesthesia (arterial PCO2 = 57.4 +/- 4.5 Torr), bilateral cooling (thermode temperature = 20 degrees C) of 60–100% of areas M and S for 30 s produced a sustained apnea (46 +/- 4 s) that lasted beyond the period of cooling. While the animals were awake (arterial PCO2 = 36.0 +/- 1.9 Torr), cooling the identical region in the same goats resulted in a decrease (approximately 50%) in pulmonary ventilation, with a brief apnea seen only in one goat. Reductions in both tidal volume and frequency were observed. Qualitatively similar responses were obtained when cooling caudal area M-rostral area S and rostral area M, but the responses were less pronounced. Minimal effects were seen in response to cooling caudal area S. During anesthesia, breathing is critically dependent on superficial VLM neurons, whereas in the awake state these neurons are not essential for the maintenance of respiratory rhythm. Our data are consistent with these superficial VLM neuronal regions providing tonic facilitation to more dorsal respiratory neurons in both the anesthetized and awake states.

scholarly journals Reconfiguration of the Pontomedullary Respiratory Network: A Computational Modeling Study With Coordinated In Vivo Experiments

2008 ◽  
Vol 100 (4) ◽  
pp. 1770-1799 ◽  
Author(s):  
I. A. Rybak ◽  
R. O'Connor ◽  
A. Ross ◽  
N. A. Shevtsova ◽  
S. C. Nuding ◽  
...  
Keyword(s):  
Computational Models ◽  
Ad Hoc ◽  
Large Body ◽  
Network Activity ◽  
Respiratory Pattern ◽  
Respiratory Neurons ◽  
Phase Switching ◽  
In Vivo Experiments ◽  
Respiratory Network

A large body of data suggests that the pontine respiratory group (PRG) is involved in respiratory phase-switching and the reconfiguration of the brain stem respiratory network. However, connectivity between the PRG and ventral respiratory column (VRC) in computational models has been largely ad hoc. We developed a network model with PRG-VRC connectivity inferred from coordinated in vivo experiments. Neurons were modeled in the “integrate-and-fire” style; some neurons had pacemaker properties derived from the model of Breen et al. We recapitulated earlier modeling results, including reproduction of activity profiles of different respiratory neurons and motor outputs, and their changes under different conditions (vagotomy, pontine lesions, etc.). The model also reproduced characteristic changes in neuronal and motor patterns observed in vivo during fictive cough and during hypoxia in non-rapid eye movement sleep. Our simulations suggested possible mechanisms for respiratory pattern reorganization during these behaviors. The model predicted that network- and pacemaker-generated rhythms could be co-expressed during the transition from gasping to eupnea, producing a combined “burst-ramp” pattern of phrenic discharges. To test this prediction, phrenic activity and multiple single neuron spike trains were monitored in vagotomized, decerebrate, immobilized, thoracotomized, and artificially ventilated cats during hypoxia and recovery. In most experiments, phrenic discharge patterns during recovery from hypoxia were similar to those predicted by the model. We conclude that under certain conditions, e.g., during recovery from severe brain hypoxia, components of a distributed network activity present during eupnea can be co-expressed with gasp patterns generated by a distinct, functionally “simplified” mechanism.

Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids

1993 ◽  
Vol 70 (4) ◽  
pp. 1497-1515 ◽  
Author(s):  
G. D. Funk ◽  
J. C. Smith ◽  
J. L. Feldman
Keyword(s):  
Nmda Receptors ◽  
Respiratory Neurons ◽  
Ventrolateral Medulla ◽  
Motor Nucleus ◽  
Burst Frequency ◽  
Respiratory Network ◽  
Bath Application ◽  
Burst Amplitude ◽  
Main Column

1. The involvement of excitatory amino acid (EAA) receptors in the generation of respiratory rhythm and transmission of inspiratory drive to hypoglossal (XII) motoneurons was examined in an in vitro neonatal rat medullary slice preparation. Slices generated rhythmic inspiratory activity in XII nerves. The role of EAAs in rhythm generation was determined by analyzing perturbations of respiratory network activity after bath application of EAA receptor antagonists or local microinjection of antagonists into the main column of respiratory neurons in the ventrolateral medulla (ventral respiratory group), particularly in the pre-Botzinger complex (pre-BotC). The involvement of EAAs in drive transmission to XII motoneurons was examined by recording perturbations in XII nerve discharge or motoneuron synaptic inputs after microinjection of EAA receptor antagonists into either the XII motor nuclei or sites in the ventrolateral medulla containing interneurons of the drive transmission circuit. 2. Block of non-N-methyl-D-aspartate (non-NMDA) receptors by bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reversibly reduced XII nerve burst frequency and amplitude in a concentration-dependent manner, completely blocking respiratory motor output at concentrations > 4 microM. Activation of 2-amino-4-phosphonobutyric acid (AP-4)-sensitive receptors with D,L AP-4 reduced XII nerve burst amplitude by 30% but did not alter burst frequency. Block of NMDA receptor channels by bath application of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate (MK-801) did not perturb the frequency or amplitude of motor output. Inhibition of EAA uptake in the slices by bath application of dihydrokainic acid reversibly increased the frequency and amplitude of XII motor discharge. 3. Block of non-NMDA receptors at multiple sites along the main column of respiratory neurons in the ventrolateral medulla, including the pre-BotC, by unilateral microinjection of CNQX produced a dose-dependent, bilateral reduction in XII nerve burst amplitude without substantial perturbations of the frequency of respiratory oscillations. Block of non-NMDA receptors within the pre-BotC at sites ventral to amplitude altering sites produced a reduction in frequency and ultimately bilateral block of respiratory network oscillations. 4. Non-NMDA receptor block within the XII motor nucleus by unilateral microinjection of CNQX produced a dose-dependent reduction in ipsilateral XII nerve discharge amplitude without perturbing the frequency of respiratory oscillations. Perturbations of contralateral XII nerve burst amplitude were significantly smaller. NMDA channel block within the XII motor nucleus did not affect inspiratory burst amplitude, whereas activation of AP-4 receptors caused a 30% reduction in amplitude.

scholarly journals Multi-Level Regulation of Opioid-Induced Respiratory Depression

Physiology ◽  
2020 ◽  
Vol 35 (6) ◽  
pp. 391-404
Author(s):  
Barbara Palkovic ◽  
Vitaliy Marchenko ◽  
Edward J. Zuperku ◽  
Eckehard A. E. Stuth ◽  
Astrid G. Stucke
Keyword(s):  
Respiratory Rate ◽  
Respiratory Depression ◽  
Minute Ventilation ◽  
Direct Effects ◽  
Respiratory Phase ◽  
Multi Level ◽  
Prebotzinger Complex ◽  
Prebötzinger Complex ◽  
Excitatory Drive

Opioids depress minute ventilation primarily by reducing respiratory rate. This results from direct effects on the preBötzinger Complex as well as from depression of the Parabrachial/Kölliker-Fuse Complex, which provides excitatory drive to preBötzinger Complex neurons mediating respiratory phase-switch. Opioids also depress awake drive from the forebrain and chemodrive.

scholarly journals Defining preBötzinger Complex Rhythm- and Pattern-Generating Neural Microcircuits In Vivo

Neuron ◽  
2016 ◽  
Vol 91 (3) ◽  
pp. 602-614 ◽  
Author(s):  
Yan Cui ◽  
Kaiwen Kam ◽  
David Sherman ◽  
Wiktor A. Janczewski ◽  
Yu Zheng ◽  
...  
Keyword(s):  
Prebotzinger Complex ◽  
Prebötzinger Complex
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