Mechanisms Of Respiratory Rhythm Generation Research Articles

Release your inhibitions: The role of post-inhibitory rebound and synaptic inhibition in the generation of expiratory activity.

Release your inhibitions: The role of post-inhibitory rebound and synaptic inhibition in the generation of expiratory activity.

Analysis of the neuronal network of the medullary respiratory center in transgenic rats expressing archaerhodopsin-3 in Phox2b-expressing cells

Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) comprise one of the respiratory rhythm generators in the medulla of the neonatal rat. A subgroup of pFRG/Pre-I neurons expresses the transcription factor Phox2b. To further analyze detailed neuronal mechanisms of respiratory rhythm generation in the neonatal rat, we developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch). Brainstem-spinal cord preparations were isolated from 0–2-day-old Tg newborn rats and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25–26 °C. Inspiratory fourth cervical ventral root (C4) activity was monitored, and membrane potentials of neurons in the pFRG including Pre-I and inspiratory neurons were recorded. Phox2b-positive cells in the Tg rats were essentially positive for enhanced green fluorescent protein (EGFP) signals (reporter for Arch) in the pFRG. Continuous photo-stimulation of the rostral ventral medulla for up to 90 s by covering the pFRG with green laser light (532 nm) induced a decrease of respiratory rate measured at C4 accompanied by membrane hyperpolarization of Phox2b-positive pFRG/Pre-I neurons. In contrast, Phox2b-negative inspiratory neurons were not hyperpolarized during the photo-stimulation. Our findings showed that Phox2b-expressing pFRG/Pre-I neurons are involved in the maintenance of the basic respiratory rhythm in neonatal rat.

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

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.

The Orchestration of the respiratory rhythm: how to integrate multiple modulators and cellular properties

Event Abstract Back to Event The Orchestration of the respiratory rhythm: how to integrate multiple modulators and cellular properties Jan M. Ramirez1* 1 University of Chicago, Department of Organismal Biology and Anatomy, United States It is well established that the pre-Bötzinger Complex plays a critical role in mammalian respiratory rhythm generation. This relatively small network is located within the ventrolateral medulla and it gives rise to different types of inspiratory activities that contribute to the generation of eupnea, sighs and gasps. The respiratory rhythm emerges through the interaction between various synaptic and intrinsic membrane properties. Non-NMDA, NMDA and metabotropic glutamatergic mechanisms provide an important excitatory synaptic drive, which is counterbalanced by prominent glycinergic and GABAergic inhibitory mechanisms. Synaptic inhibition plays equally important roles in establishing the different phases of respiration and in regulating the relative contribution of intrinsic membrane properties, of which bursting or pacemaker properties received considerable attention. In this study, we review the mechanisms of respiratory rhythm generation, but we will also emonstrate how multi-array recordings from slices containing the pre-Bötzinger Complex reveal important new insights into the dynamics of respiratory ensemble activity. Neurons exhibiting intrinsic bursting and pacemaker properties are characterized by a higher instantaneous discharge frequency during the burst and during baseline activity. On average, these neurons tend to lead an inspiratory cycle. But to our surprise, these neurons showed considerable cycle-bycycle variability. On some cycles, pacemaker neurons discharged before, on other cycles they discharged relatively late in the inspiratory phase. We therefore propose that the respiratory rhythm is assembled in a cycle-by-cycle manner. A given neuron can lead and potentially initiate an inspiration during a given cycle, while being a follower and amplifier of synaptic mechanisms in subsequent cycles. We speculate that the dynamic assembling of respiratory network activity imbues the respiratory network with an enormous flexibility, which is necessary to adjust to continuous changes in metabolic and behavioural conditions. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Symposium 05 – Interactions of synaptic and intrinsic properties in rhythmic motor activities Citation: Ramirez JM (2009). The Orchestration of the respiratory rhythm: how to integrate multiple modulators and cellular properties. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.030 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Nov 2009; Published Online: 19 Nov 2009. * Correspondence: Jan M Ramirez, University of Chicago, Department of Organismal Biology and Anatomy, Chicago, United States, jramire@midway.uchicago.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jan M Ramirez Google Jan M Ramirez Google Scholar Jan M Ramirez PubMed Jan M Ramirez Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Nociceptin/orphanin FQ causes non-quantal slowing of respiratory rhythm in brainstem–spinal cord preparation from newborn rat

Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the N/OFQ peptide receptor, an inhibitory G protein-coupled receptor. N/OFQ acts as a neuromodulator to depress respiratory rhythm in the brainstem. Although the mechanisms of respiratory rhythm generation remain poorly understood, the pre-inspiratory neuron (Pre-I) and the pre-Bötzinger complex (preBötC) inspiratory neuron (Insp) network in the rostral ventrolateral medulla (RVLM) have been proposed to be essential for respiratory rhythm generation. Opioids presumably cause quantal slowing via selective depression of preBötC Insps. However, it is unclear whether N/OFQ depresses respiratory rhythm via the same mechanism. In this study, using in vitro newborn rat en bloc preparations, we examined the slowing pattern of N/OFQ (quantal or non-quantal) and the effects of N/OFQ on the extracellularly recorded discharge of Pre-Is and Insps in the RVLM. N/OFQ caused non-quantal slowing with a synchronous decrease in burst rates of Insps and of C4 discharge whereas the intraburst spike number in Insps remained unchanged. It also caused a significant decrease in burst rates and intraburst spike numbers in Pre-Is, while the 1:1 coupling of Pre-Is bursts to C4 bursts was preserved. When superfusate K(+) was elevated from 6.2 to 11.2mM, Pre-I activity was increasingly uncoupled from C4 bursts. After the application of N/OFQ in a high [K(+)] superfusate, the 1:1 coupling of Pre-Is to C4 bursts was restored. We conclude that N/OFQ suppresses burst and spike generation of Pre-Is, and that suppression of Pre-Is activity with synchronous coupling to the Insps network contributes to N/OFQ-induced non-quantal slowing.

Novel Data Supporting the Two Respiratory Rhythm Oscillator Hypothesis. Focus on “Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats”

Among the most rewarding moments in scientific discovery are when the pieces of a puzzle provided by different laboratories start to fall into place, forming a coherent and new view of the world. Several years ago, a consensus was emerging that respiratory rhythm in mammals arose from a single

Neuronal mechanisms of respiratory rhythm generation

Neuronal mechanisms of respiratory rhythm generation

Development of respiratory rhythm generation in ectothermic vertebrates

Compared with birds and mammals, very little is known about the development and regulation of respiratory rhythm generation in ectothermic vertebrates. The development and regulation of respiratory rhythm generation in ectothermic vertebrates (fish, amphibians and reptiles) should provide insight into the evolution of these mechanisms. One useful model for examining the development of respiratory rhythm generation in ectothermic vertebrates has emerged from studies with the North American bullfrog (Rana catesbeiana). A major advantage of bullfrogs as a comparative model for respiratory rhythm generation is that respiratory output may be measured at all stages of development, both in vivo and in vitro. An emerging view of recent studies in developing bullfrogs is that many of the mechanisms of respiratory rhythm generation are very similar to those seen in birds and mammals. The overall conclusion from these studies is that respiratory rhythm generation during development may be highly conserved during evolution. The development of respiratory rhythm generation in mammals may, therefore, reflect the antecedent mechanisms seen in ectothermic vertebrates. The main focus of this brief review is to discuss recent data on the development of respiratory rhythm generation in ectothermic vertebrates, with particular emphasis on the North American bullfrog (R. catesbeiana) as a model.

Mechanisms of respiratory rhythm generation in vitro. I. Pacemaker neurons and networks in the pre-Bötzinger complex (pre-BötC)

We have proposed that the inspiratory rhythm-generating kernel in the pre-BotC consists of a heterogeneous network of glutamatergic neurons with voltage-dependent pacemaker-like bursting properties. This model is based on our optical imaging [1], electrophysiological [2], and mathematical modeling [3] studies of inspiratory (I) neurons within the pre-BotC of rhythmically active in vitro transverse slice preparations from neonatal rats. For electrophysiological and imaging studies, we have developed novel methods to optically identify I neurons with Ca2+-sensitive dyes combined with IR-DIC imaging for whole-cell current camp (CC) and voltage clamp (VC) analyses of neuronal membrane conductance and synaptic mechanisms. In computational modeling studies we have developed models of single pacemaker neurons and synaptically-coupled networks of these neurons (50–500 cells) with heterogeneous distributions of cellular/network parameters. Model predictions have been tested experimentally from single-cell electrophysiological measurements and from macroscopic recordings of neuron population activity within the pre-BotC [2]. Electrophysiological and optical measurements demonstrate a sub-population of pre-BotC I neurons that exhibit voltage-dependent rhythmic bursting under CC after blockade of non-NMDA gluta-matergic synaptic transmission or after blocking synaptic transmission with Ca2+ channel blockers. The intrinsic bursting frequency of these pacemaker-type cells was a monotonic function of baseline membrane potential, spanning a frequency range of over an order of magnitude (~0.05–1 Hz); the voltage-dependence and frequency range varied for different cells indicating heterogeneity. Under VC with synaptic transmission intact, these pacemaker neurons exhibited glutamatergic synaptic drive currents. Optical imaging and cross-correlation of rhythmic Ca2+ activities of multiple cells demonstrate that the glutamatergic synaptic interactions synchronize bursting within the heterogeneous population, but with a temporal dispersion in neuronal spiking including pre-I spiking patterns [2,3]. Measurements of pre-BotC population activity show network frequency control by changing pre-BotC excitability, like predictions from our network models [3]. We have obtained evidence that a persistent Na+ current (INaP) is the main subthreshold-activating cationic conductance underlying the voltage-dependent pacemaker bursting. We measured Na+ currents in bursting pacemaker and non-bursting pre-BotC I cells. In all cells tested under VC, voltage ramp commands at rates of <100 mV/s inactivated the fast Na+ current and yielded N-shaped IV curves with a negative slope region between -60 and -35 mV. TTX (1 μM) blocked this inward current. The slowest ramp speed tested (3.3 mV/s) failed to fully inactivate the negative slope generating current. The current is therefore a TTX-sensitive INaP. The peak amplitude of INaP ranged from -50 to -100 pA at Em = -20 mV (peak conductance = ~1–2 nS). Boltzmann plots gave half-maximal activation voltage of ~-40 mV and a slope factor of ~5, very similar to values used for INaP in our pacemaker neuron models [3]. In non-pacemaker Icells, the ratio of INaP/Ileak was insufficient to support intrinsic bursting as predicted by our models. Further tests of the role of INaP have been performed using the dynamic clamp to artificially add INaP to non-pacemaker neurons. INaP transformed these neurons into rhythmic bursters with voltage-dependent behavior quantitatively similar to our models and experimental observations. These results generally support our conceptual and computational models for the rhythm-generation kernel as a heterogeneous pacemaker network.

Optical recording of the neuronal activity in the brainstem-spinal cord: application of a voltage-sensitive dye.

Although there are several limitations, optical recording techniques are superior to multi-electrode mapping methods, as it is possible to record at large number of points in a small area without destroying the tissue and possible to know relative changes of membrane potentials. Optical recording techniques using voltage-sensitive dyes will be more importantly applied in the study of central respiratory control (e.g., mechanisms of respiratory rhythm generation) in the near future.

Newborn rat brainstem preparation with the trigeminal nerve attached for pain study

Studies using a brainstem–spinal cord preparation isolated from newborn rats have provided substantial information on neuro-physiology, -pharmacology and -anatomy of the respiratory center, such as mechanisms of respiratory rhythm generation, development of a respiratory center or respiratory reflex [T. Murakoshi, T. Suzue, S. Tamai, A pharmacological study on respiratory rhythm in the isolated brainstem–spinal cord preparation from newborn rat, Br. J. Pharmac. 86 (1985) 95–104 [5]; H. Onimaru, A. Arata, I. Homma, Primary respiratory rhythm generator in the medulla of brainstem–spinal cord preparations from newborn rats, Brain Res. 445 (1988) 314–324 [6]; H. Onimaru, I. Homma, Whole cell recordings from respiratory neurons in the medulla of brainstem–spinal cord preparations isolated from newborn rats, Pflügers Arch. 420 (1992) 399–406 [7]; J.C. Smith, K. Ballanyi, D.W. Richter, Whole-cell patch clamp recordings from respiratory neurons in neonatal rat brainstem in vitro, Neurosci. Lett. 314 (1992) 153–156 [10]; T. Suzue, Respiratory rhythm generation in the in vitro brainstem–spinal cord preparation of the neonatal rat, J. Physiol. (London) 354 (1984) 173–183 [11], reviewed in H. Onimaru, A. Arata, I. Homma, Neuronal mechanisms of respiratory rhythm generation: An approach using in vitro preparation, Jpn. J. Physiol. 47 (1997) 385–403 [8]]. Recently, the dissecting method of the preparation was modified to introduce a brainstem preparation with the trigeminal primary afferents attached for pain studies [M. Hamba, Repetitive stimulation potentiated the stimulus-evoked firing in the trigeminal caudalis—in vitro study. Neurosci. Res. 20 (1996) s163 [2]; M. Hamba, Stimulation-induced responses of the trigeminal caudal neurons in the brainstem preparation isolated from newborn rats, Brain Res. 785 (1998) 66–74 [3]]. As reported previously [3], the activity-dependent change in the excitability of pain-processing neurons, wind-up phenomenon, was studied in the trigeminal caudalis by stimulating the mandibular nerve, using a modified brainstem preparation isolated from newborn rats. The caudalis, the medulla dorsal horn, is known as the center for processing pain and sensory information from the cranio-facial area. The results indicated that the brainstem preparation is applicable for studies on the neuroplasticity at the pain-processing synapses. Here, we describe the method for isolation of a brainstem preparation with the trigeminal mandibular nerve attached and for recording the synaptic response evoked in the caudal neurons, using a whole-cell patch clamp technique. In the present study, we demonstrated repetitive stimulation-induced responses of caudal neurons at postnatal day 1 as an example showing the feasibility of the preparation for pain studies. Themes: Sensory systems Topics: Pain modulation: anatomy and physiology

Involvement of glycine receptors in coordination between inspiratory and expiratory motor activities in the newborn rat in vitro

Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the N/OFQ peptide receptor, an inhibitory G protein-coupled receptor. N/OFQ acts as a neuromodulator to depress respiratory rhythm in the brainstem. Although the mechanisms of respiratory rhythm generation remain poorly understood, the pre-inspiratory neuron (Pre-I) and the pre-Bötzinger complex (preBötC) inspiratory neuron (Insp) network in the rostral ventrolateral medulla (RVLM) have been proposed to be essential for respiratory rhythm generation. Opioids presumably cause quantal slowing via selective depression of preBötC Insps. However, it is unclear whether N/OFQ depresses respiratory rhythm via the same mechanism. In this study, using in vitro newborn rat en bloc preparations, we examined the slowing pattern of N/OFQ (quantal or non-quantal) and the effects of N/OFQ on the extracellularly recorded discharge of Pre-Is and Insps in the RVLM. N/OFQ caused non-quantal slowing with a synchronous decrease in burst rates of Insps and of C4 discharge whereas the intraburst spike number in Insps remained unchanged. It also caused a significant decrease in burst rates and intraburst spike numbers in Pre-Is, while the 1:1 coupling of Pre-Is bursts to C4 bursts was preserved. When superfusate K+ was elevated from 6.2 to 11.2 mM, Pre-I activity was increasingly uncoupled from C4 bursts. After the application of N/OFQ in a high [K+] superfusate, the 1:1 coupling of Pre-Is to C4 bursts was restored. We conclude that N/OFQ suppresses burst and spike generation of Pre-Is, and that suppression of Pre-Is activity with synchronous coupling to the Insps network contributes to N/OFQ-induced non-quantal slowing.

The site of the antiemetic action of tachykinin NK 1 receptor antagonists may exist in the medullar area adjacent to the semicompact part of the nucleus ambiguus

Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the N/OFQ peptide receptor, an inhibitory G protein-coupled receptor. N/OFQ acts as a neuromodulator to depress respiratory rhythm in the brainstem. Although the mechanisms of respiratory rhythm generation remain poorly understood, the pre-inspiratory neuron (Pre-I) and the pre-Bötzinger complex (preBötC) inspiratory neuron (Insp) network in the rostral ventrolateral medulla (RVLM) have been proposed to be essential for respiratory rhythm generation. Opioids presumably cause quantal slowing via selective depression of preBötC Insps. However, it is unclear whether N/OFQ depresses respiratory rhythm via the same mechanism. In this study, using in vitro newborn rat en bloc preparations, we examined the slowing pattern of N/OFQ (quantal or non-quantal) and the effects of N/OFQ on the extracellularly recorded discharge of Pre-Is and Insps in the RVLM. N/OFQ caused non-quantal slowing with a synchronous decrease in burst rates of Insps and of C4 discharge whereas the intraburst spike number in Insps remained unchanged. It also caused a significant decrease in burst rates and intraburst spike numbers in Pre-Is, while the 1:1 coupling of Pre-Is bursts to C4 bursts was preserved. When superfusate K+ was elevated from 6.2 to 11.2 mM, Pre-I activity was increasingly uncoupled from C4 bursts. After the application of N/OFQ in a high [K+] superfusate, the 1:1 coupling of Pre-Is to C4 bursts was restored. We conclude that N/OFQ suppresses burst and spike generation of Pre-Is, and that suppression of Pre-Is activity with synchronous coupling to the Insps network contributes to N/OFQ-induced non-quantal slowing.

The neuronal mechanisms of respiratory rhythm generation

New, improved in vivo and in vitro approaches have led to a better understanding of the mechanisms that generate respiratory rhythm, which depends on a complex interaction between network and intrinsic membrane properties. The pre-Bötzinger complex in the ventrolateral medulla is particularly important for respiratory rhythm generation. This complex can be studied in isolation, and it contains all the known types of respiratory neurons that are now amenable to detailed cellular and molecular analyses.

Studies of the respiratory center using isolated brainstem-spinal cord preparations

It has been ten years since a brainstem-spinal cord preparation isolated from a newborn rat was introduced for study of the mammalian respiratory center. Here, I briefly summarize first, these studies, which include the tissue condition of in vitro preparations, respiratory reflexes, pharmacology, rhythm generation,- respiratory chemoreception, phrenic motoneurons, regulation from pons, and development of a respiratory center. In the latter half of this paper, I focus on the neural mechanisms of respiratory rhythm generation. A current hypothesis for the central pattern generator of respiration proposed by the author's group is that the respiratory rhythm generator, composed of pre-inspiratory neurons in the rostral ventrolateral medulla, produces the primary rhythm of respiration and triggers an inspiratory pattern generator composed of inspiratory neurons in the rostral and the caudal ventrolateral medulla.

Mechanisms of respiratory rhythm generation

In mammals, a three-phasic respiratory rhythm is generated by a network of various types of neurons in the lower brainstem. The cellular mechanisms of rhythmogenesis involve cooperative interactions between synaptic processes and specific membrane properties. The network seems to be driven by extrinsic sources in mature animals, whereas in the immature network pacemaker neurons might be involved.

Mechanisms of respiratory rhythm generation

Mechanisms of respiratory rhythm generation

Neural network implementation of a three-phase model of respiratory rhythm generation

A mathematical model of the central neural mechanisms of respiratory rhythm generation is developed. This model assumes that the respiratory cycle consists of three phases: inspiration, post-inspiration, and expiration. Five respiratory neuronal groups are included: inspiratory, late-inspiratory, post-inspiratory, expiratory, and early-inspiratory neurons. Proposed interconnections among these groups are based substantially on previous physiological findings. The model produces a stable limit cycle and generally reproduces the features of the firing patterns of the 5 neuronal groups. When simulated feedback from pulmonary stretch receptors is made to excite late-inspiratory neurons and inhibit early-inspiratory neurons, the model quantitatively reproduces previous observations of the expiratory-prolonging effects of pulses and steps of vagal afferent activity presented in expiration. In addition the model reproduces expected respiratory cycle timing and amplitude responses to change of chemical drive both in the absence and in the presence of simulated stretch receptor feedback. These results demonstrate the feasibility of generating the respiratory rhythm with a simple neural network based on observed respiratory neuronal groups. Other neuronal groups not included in the model may be more important for shaping the waveforms than for generating the basic oscillation.