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.

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

2007 ◽  
Vol 97 (3) ◽  
pp. 2283-2292 ◽  
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.

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

2001 ◽  
Vol 86 (1) ◽  
pp. 104-112 ◽  
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.

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.

Hyperthermia Modulates Respiratory Pacemaker Bursting Properties

2004 ◽  
Vol 92 (5) ◽  
pp. 2844-2852 ◽  
Author(s):  
Andrew K. Tryba ◽  
Jan-Marino Ramirez
Keyword(s):  
Network Effects ◽  
Elevated Temperatures ◽  
Heat Stroke ◽  
Network Activity ◽  
Respiratory Neurons ◽  
Population Activity ◽  
Normal Body Temperature ◽  
Respiratory Network ◽  
Hyperthermic Response

Most mammals modulate respiratory frequency (RF) to dissipate heat (e.g., panting) and avoid heat stroke during hyperthermic conditions. Respiratory neural network activity recorded in an isolated brain stem-slice preparation of mice exhibits a similar RF modulation in response to hyperthermia; fictive eupneic frequency increases while inspiratory network activity amplitude and duration are significantly reduced. Here, we study the effects of hyperthermia on the activity of synaptically isolated respiratory pacemakers to examine the possibility that these changes may account for the hyperthermic RF modulation of the respiratory network. During heating, modulation of the bursting frequency of synaptically isolated pacemakers paralleled that of population bursting recorded from the intact network, whereas nonpacemaker neurons were unaffected, suggesting that pacemaker bursting may account for the temperature-enhanced RF observed at the network level. Some respiratory neurons that were tonically active at hypothermic conditions exhibited pacemaker properties at approximately the normal body temperature of eutherian mammals (36.81 ± 1.17°C; mean ± SD) and continued to burst at 40°C. At elevated temperatures (40°C), there was an enhancement of the depolarizing drive potential in synaptically isolated pacemakers, while the amplitude of integrated population activity declined. Isolated pacemaker bursting ceased at 41–42°C ( n = 5), which corresponds to temperatures at which hyperthermic-apnea typically occurs in vivo. We conclude that pacemaker properties may play an important role in the hyperthermic frequency modulation and apnea, while network effects may play important roles in generating other aspects of the hyperthermic response, such as the decreased amplitude of ventral respiratory group activity during hyperthermia.

scholarly journals Assessing the Impact of Single-Cell Stimulation on Local Networks in Rat Barrel Cortex—A Feasibility Study

2019 ◽  
Vol 20 (10) ◽  
pp. 2604
Author(s):  
Beate Knauer ◽  
Maik C. Stüttgen
Keyword(s):  
Single Cell ◽  
Statistical Power ◽  
Single Cells ◽  
Network Activity ◽  
Local Network ◽  
Brain State ◽  
Population Activity ◽  
Local Networks ◽  
Cell Stimulation ◽  
The Impact

In contrast to the long-standing notion that the role of individual neurons in population activity is vanishingly small, recent studies have shown that electrical activation of only a single cortical neuron can have measurable effects on global brain state, movement, and perception. Although highly important for understanding how neuronal activity in cortex is orchestrated, the cellular and network mechanisms underlying this phenomenon are unresolved. Here, we first briefly review the current state of knowledge regarding the phenomenon of single-cell induced network modulation and discuss possible underpinnings. Secondly, we show proof of principle for an experimental approach to elucidate the mechanisms of single-cell induced changes in cortical activity. The setup allows simultaneous recordings of the spiking activity of multiple neurons across all layers of the cortex using a multi-electrode array, while manipulating the activity of one individual neuron in close proximity to the array. We demonstrate that single cells can be recorded and stimulated reliably for hundreds of trials, conferring high statistical power even for expectedly small effects of single-neuron spiking on network activity. Preliminary results suggest that single-cell stimulation on average decreases the firing rate of local network units. We expect that characterization of the spatiotemporal spread of single-cell evoked activity across layers and columns will yield novel insights into intracortical processing.

scholarly journals Cycle-by-cycle assembly of respiratory network activity is dynamic and stochastic

2013 ◽  
Vol 109 (2) ◽  
pp. 296-305 ◽  
Author(s):  
Michael S. Carroll ◽  
Jan-Marino Ramirez
Keyword(s):  
Computational Models ◽  
De Novo ◽  
Activity Patterns ◽  
Respiratory Rhythm ◽  
Network Models ◽  
Network Activity ◽  
Respiratory Neurons ◽  
Intrinsic Activity ◽  
Pacemaker Neurons ◽  
Network Output

Rhythmically active networks are typically composed of neurons that can be classified as silent, tonic spiking, or rhythmic bursting based on their intrinsic activity patterns. Within these networks, neurons are thought to discharge in distinct phase relationships with their overall network output, and it has been hypothesized that bursting pacemaker neurons may lead and potentially trigger cycle onsets. We used multielectrode recording from 72 experiments to test these ideas in rhythmically active slices containing the pre-Bötzinger complex, a region critical for breathing. Following synaptic blockade, respiratory neurons exhibited a gradient of intrinsic spiking to rhythmic bursting activities and thus defied an easy classification into bursting pacemaker and nonbursting categories. Features of their firing activity within the functional network were analyzed for correlation with subsequent rhythmic bursting in synaptic isolation. Higher firing rates through all phases of fictive respiration statistically predicted bursting pacemaker behavior. However, a cycle-by-cycle analysis indicated that respiratory neurons were stochastically activated with each burst. Intrinsically bursting pacemakers led some population bursts and followed others. This variability was not reproduced in traditional fully interconnected computational models, while sparsely connected network models reproduced these results both qualitatively and quantitatively. We hypothesize that pacemaker neurons do not act as clock-like drivers of the respiratory rhythm but rather play a flexible and dynamic role in the initiation and stabilization of each burst. Thus, at the behavioral level, each breath can be thought of as de novo assembly of a stochastic collaboration of network topology and intrinsic properties.

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 Epileptogenesis due to glia-mediated synaptic scaling

2008 ◽  
Vol 6 (37) ◽  
pp. 655-668 ◽  
Author(s):  
Cristina Savin ◽  
Jochen Triesch ◽  
Michael Meyer-Hermann
Keyword(s):  
Immune System ◽  
Neuronal Activity ◽  
Activity Patterns ◽  
Brain Inflammation ◽  
Network Activity ◽  
Homeostatic Regulation ◽  
Synaptic Scaling ◽  
Tnf Α ◽  
Factor Α ◽  
Potential Interactions

Homeostatic regulation of neuronal activity is fundamental for the stable functioning of the cerebral cortex. One form of homeostatic synaptic scaling has been recently shown to be mediated by glial cells that interact with neurons through the diffusible messenger tumour necrosis factor-α (TNF-α). Interestingly, TNF-α is also used by the immune system as a pro-inflammatory messenger, suggesting potential interactions between immune system signalling and the homeostatic regulation of neuronal activity. We present the first computational model of neuron–glia interaction in TNF-α-mediated synaptic scaling. The model shows how under normal conditions the homeostatic mechanism is effective in balancing network activity. After chronic immune activation or TNF-α overexpression by glia, however, the network develops seizure-like activity patterns. This may explain why under certain conditions brain inflammation increases the risk of seizures. Additionally, the model shows that TNF-α diffusion may be responsible for epileptogenesis after localized brain lesions.

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.

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.

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