pre-Bötzinger Complex Research Articles

Variability in respiratory rhythm generation: in vitro and in silico models

The variability inherent in rhythmic physiological patterns may arise from both stochastic and deterministic sources. Though variability is disruptive when in excess, it may also be functional in evolved systems. Here we focus on the neural control of respiration which is critical for survival in many animals. The sources of respiratory variability, as well as its possible function, are unknown. A fundamental component of respiratory pattern generation is the preBotzinger complex (preBotC) which is part of the ventral respiratory column located in the brainstem. Here we use in vitro and in silico models of the preBotC to study the variability of respiratory rate. The in vitro model exhibits a bounded range of variability in which the upper and lower limits are functions of the respiratory rate. The in silico model consists of two types of neurons: intrinsically bursting “pacemaker” cells and tonically spiking cells that relay chemosensory and mechanosensory feedback. When fitted to the experimental data, the in silico model shows the existence of both stochastic and deterministic sources for variability in the respiratory rate. The upper and lower limits of variability seen experimentally are reproduced in silico using a range of network connection strengths. Simulations demonstrate that stochastic spiking in sensory relay neurons may be utilized to stabilize changes in variability when the respiratory rate changes due to physiological demand.

Evolutionary algorithm search for connectivity patterns conducive to bursting in respiratory neural networks

The respiratory neural network in the pre-Botzinger complex of the ventrolateral medulla controls and flexibly maintains the breathing rhythm, coordinating network-wide bursting to signal the inspiratory phase of the breath. Interestingly, however, the connectivity of the network by which it achieves this coordination is still a subject of active research. We search for connectivity patterns which yield high performance in a measure of network bursting, using an evolutionary algorithm (EA)-based approach, in networks of leaky integrate-and-fire (LIF) and conductance-based model (CBM) neurons. In LIF networks, the connectivity patterns of bursting networks are characterized by regularities in several network statistics: closeness centrality(CC), betweenness centrality (BC) and out-degree (OD) distributions. We find additionally that intentionally selecting for these statistical regularities in CC, BC and OD leads to steady improvement in the network bursting measure. We examine the extent to which these trends in network statistics are present in well-adapted networks of CBM neurons without intrinsically bursting neurons, finding that the regularities in BC and OD are also visible. We also present initial results on the statistical regularities in networks of CBM neurons with intrinsic bursters. Figure 1 illustrates the regularities in closeness centrality (CC), betweenness centality (BC) and out-degree (OD) distributions in well-adapted networks of LIF neurons. In well-adapted networks with (a) 2, (b) 3, (c) 4 and (d) 5 tonically spiking neurons present ...

Ascending projections of the nucleus retroambiguus target key areas of the ponto‐medullary respiratory network (714.1)

Purpose: We aim to elucidate the role and contribution of the nucleus retroambiguus (NRA) in the generation of respiratory rhythm. We previously identified a putative rhythmogenic area 1.5 mm caudal to obex, destruction of which abolished respiratory rhythm in the in‐situ perfused brainstem‐spinal cord preparation of rat. Histological analysis indicated that the putative rhythmogenic cell population resides at the planar level of the pyramidal decussation, therefore likely to overlap with the NRA. We investigated possible involvement of the NRA in rhythm generation by mapping its neuronal connectivity in rat. Methods: Anterograde neuronal tracer, biotinylated dextran amine, was injected into the NRA. Respiratory areas in which labelled terminal fields were seen were injected with retrograde tracer Fast Blue in subsequent experiments. Results: Anterograde tracing from the NRA revealed dense clusters of labelled nerve terminals within important respiratory nuclei including the pre‐Bötzinger complex (PBC), parafacial respiratory group (pFRG) and Kölliker Fuse nucleus (KF). Injection of retrograde tracer into PBC, KF and pFRG show clusters of retrogradely labelled cell bodies in the NRA. Conclusion: Our data provides the anatomical framework for a rhythmogenic role of the NRA within the respiratory network.Grant Funding Source: ARC

Implementation of rapid prototyping to produce a 3‐dimensional physical model of the anatomical distribution of functionally identified cat pre‐Bötzinger complex microinjection sites (714.4)

Previous work from our laboratory has used histological analyses to confirm functionally identified sites in the cat pre‐Botzinger complex (pre‐BotC). These sites have been represented in a 2‐D fashion based on our histological sections in conjunction with plates from Berman's stereotactic atlas (1968). To gain further insight into the anatomical distribution of these sites, we have used rapid prototyping technology to generate a 3‐D physical model representing the relevant regions/nuclei of the cat brainstem (based on Berman’s atlas) and the histological/stereotaxic coordinates associated with our microinjection sites. To accomplish this, plates from Berman’s atlas were scanned and compiled into a 3‐D configuration slide box, the stereotaxic coordinates of 348 pre‐BötC microinjection sites were entered into an x/y/z matrix based on histologically confirmed coordinates with the origin (0, 0, 0) referenced to the caudal pole of the retrofacial nucleus, midline, and dorsal surface of the medulla, and a 3‐D digital representation was generated and printed. We believe that this approach compliments both the 2‐D representations (previously used) and the digital 3‐D representation, and provides additional insight into the anatomical specificity of our functionally identified and histologically confirmed microinjection sites.Grant Funding Source: Supported by HL 63175

CrossTalk proposal: The preBötzinger complex is essential for the respiratory depression following systemic administration of opioid analgesics

Drugs acting on μ-opioid receptors (MOR) are widely used as analgesics but present serious side-effects such as addiction and respiratory depression. The latter is critical considering its potential lethality and the current absence of treatments to prevent it. The development of therapies to reduce respiratory depression is limited because the critical neural sites and mechanisms of action of opioids in causing respiratory depression are unclear. Here we discuss evidence highlighting the importance of the preBotzinger complex (preBotC), a critical site in the medulla for respiratory rhythm generation, in mediating respiratory rate depression by MOR drugs. This is of significance given that the isolated preBotC in vitro is widely utilized to develop and test new therapies to prevent respiratory depression (Manzke et al. 2003; Ren et al. 2006).

Breathing variability and brainstem serotonergic loss in a genetic model of multiple system atrophy

Breathing disorders like sleep apnea, stridor, and dysrythmic breathing are frequent in patients with multiple system atrophy (MSA). These observations have been related to neurodegeneration in several pontomedullary respiratory nuclei and may explain the occurrence of sudden death. In this study, we sought to determine whether these functional and neuropathological characteristics could be replicated in a transgenic model of MSA. Mice expressing human wild-type α-synuclein under the control of the proteolipid promoter (PLP-αSYN) were compared with age-matched controls. Using whole-body, unrestrained plethysmography, the following breathing parameters were measured: inspiratory and expiratory times, tidal volume, expiratory volume, peak inspiratory and expiratory flows, and respiratory frequency. For each category, the mean, coefficient of variation, and irregularity score were analyzed. Brains were then processed for stereological cell counts of pontomedullary respiratory nuclei. A significant increase in the coefficient of variation and irregularity score was observed for inspiratory time, tidal volume, and expiratory volume in PLP-αSYN mice (P < 0.05). Glial cytoplasmic inclusions were found in the medullary raphe of PLP-αSYN mice, together with a loss of serotonergic immunoreactivity in the raphe obscurus (P < 0.001) and pallidus (P < 0.01). There was a negative correlation between α-synuclein burden and raphe pallidus cell counts (P < 0.05). There was no significant neuronal loss in the pre-Botzinger complex. The PLP-αSYN mouse model replicates the breathing variability and part of the neuronal depletion in pontomedullary respiratory nuclei observed in patients with MSA. Our findings support the use of this model for future candidate drugs in the breathing disorders observed in MSA.

Opioid-Induced Central Sleep Apnea

Patients on chronic opioids have significant central sleep apnea (CSA) and complex sleep apnea. The mechanism of opioid-induced CSA most probably has to do with suppression of inspiration generated by the pre-Botzinger complex. The best approach for treatment of opioid-induced CSA is a programmed withdrawal of opioids. Another option is treatment with bilevel devices with backup rate, or the new adaptive servoventilation devices, which are effective in eliminating sleep-related breathing disorders. However, randomized clinical trials are needed to determine if treatment with positive airway pressure devices has any effect on quality of life or excess mortality.

Distinct roles of inotropic and metabotropic glutamate receptors in rhythmic activity generated by pre-Botzinger complex

To keep up with oxygen demands, the central nervous system regulates frequency of respiratory rhythm. The inspiratory phase of the respiratory rhythm originates in the pre-Botzinger complex (preBotC), a region of the ventrolateral medulla. preBotC neurons generate stable rhythmic output when isolated from the rest of respiratory column. The frequency of the respiratory rhythm is modulated via release of excitatory neuromodulators by the Raphe nucleus. The respiratory rhythm observed in the preBotC depends on excitatory neurotransmission between neurons in the population, since blocking excitatory neurotransmission eliminates a coordinated rhythm at the level of the preBotC. In this work we extend previously developed model of pre-BotC neuron (Figure ​(Figure1)1) to determine the role of ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors on generation of the respiratory rhythm at different levels of neuromodulatory tone. Using distribution of persistent sodium and voltage-gated calcium conductances, we generated the heterogeneous population of neurons connected through both, ionotropic and metabotropic glutamate receptors. Our simulations demonstrate that mGluR and iGluR has distinct roles, determined by neuormodulatory tone. In low neuromodulatory tone, the rhythmic activity of the preBotC network relies on activation of iGluR. In high neuromodulatory tone, the rhythmic activity of the network relies on activation of second messenger cascade by mGluR. Figure 1 Schematic representation of electrical activity in single preBotC neuron

Coarse-graining of the dynamics seen in neural networks

The pre-Botzinger complex (pre-BotC) is a population of neurons, located in the brainstem of mammals within a central pattern generator (CPG) that produces the neural oscillation underlying the breathing rhythm. Breathing is achieved when the diaphragm (the respiratory muscle) is activated and contracts, leading to movement of air into the lungs followed by a relaxation of the diaphragm and a movement of air out of the lungs. Control of breathing is needed to maintain adequate oxygen and carbon dioxide levels in the blood and support metabolism as well as for the purpose of vocalization and is accomplished by feedback signals to the brainstem networks that affect the pre-BotC activity. Studying the integrated behavior of the respiratory system is a major challenge both computationally and conceptually, aggravated by the complex dynamics at the level of the pre-BotC population. However, the exact details of the neural dynamics may not be important when studying the integrated behavior of the respiratory system as the diaphragm contracts when excited by spiking neurons whether the spiking is chaotic or not, provided the spikes are dense enough. One would therefore like to represent the pre-BotC population with a simplified mathematical model that captures the important coarse-grained features of its dynamics. Motivated by the example above, we have developed a numerical method that enables us to estimate the dynamics a simplified model should retain. The method maps between the variables of a bursting neural network (for which the equations are known) and the variables of a simplified model (for which the equations are unknown). By moving backward and forward between the variables of the detailed neural network and the variables of the simplified system using restriction and lifting operators, and simulating the detailed neural network for short periods of time, one can calculate the steady states of the simplified model and their stabilities and create bifurcation diagrams for the simplified model even though the equations of the simplified model are unknown. Using this approach we created bifurcation diagrams for 2D simplified models representing networks of 1 to 50 cells and compared them with bifurcation diagrams of the detailed networks (see Figure ​Figure11 for an example). Our calculations show that a 2D model can capture the dynamical behavior of the neural system roughly for certain parameter values and identify the domain of parameters for which the 2D simplification is invalid. Figure 1 Coarse-graining of the dynamics seen in a 2-cells neural network (described by 8 differential equations). A is a measure of the network activity and μ is a parameter related to tonic drive. The green dots represent stable equilibrium of a 2D model ...

Medroxy‐progesterone acetate (MPA) treatment increases pentobarbital resistance in neurons within the pre‐Botzinger complex (PBC), consistent with increased epsilon subunit expression in GABAA receptors (GABAARs)

PBC neurons are critical for inspiratory rhythm generation and naturally increase epsilon subunit expression in GABAARs during pregnancy. Since epsilon subunits confer resistance to positive allosteric modulators (allopregnanolone and pentobarbital) that enhance chloride ion influx, epsilon expression may protect breathing during pregnancy when central allopregnanolone levels increase 3‐fold. We hypothesize that increased epsilon subunit expression increases when central neurosteroid concentrations are increased. Thus, we tested whether MPA treatment (progesterone analogue, 10 mg/kg, SQ, 7 days) increases epsilon subunit expression in the PBC. Multichannel electrodes were used to record spontaneous action potentials of PBC‐region neurons in medullary slices from control (n=7) and MPA‐treated (n=4) P30 rats before bath‐application of 300 μM pentobarbital. In neurons (n=68 cells) from control rats, the mean neuronal firing rate decreased to 44 ± 5% of baseline; 13% of cells were pentobarbital resistant. In neurons (n=31) from MPA‐treated rats, the mean neuronal firing rate was 91 ± 12% of baseline; 52% of cells were pentobarbital resistant. These data suggest that increased central neurosteroid levels are sufficient to increase epsilon expression in PBC‐region neurons, and thereby protect inspiratory motor output from excessive inhibition. Supported by NIH T32 HL07654.

Immunohistochemical Changes in 5 Respiratory Nuclei after Bilateral Carotid Body Denervation (CBD) in Sprague Dawley Rats

Bilateral CBD in rat has no effect on CO2 sensitivity but elicits hypoventilation 1–2 d post‐CBD followed by a return to baseline values 7–8 d post‐CBD. To investigate the neural mechanisms driving the recovery of breathing after CBD, we compared NK1 receptor staining intensity on neuronal membranes from 5 different respiratory nuclei (solitary tract, NTS; dorsal motor, DMV; hypoglossal, nXII; nucleus ambiguous, NA; pre‐botzinger complex, PBC). Brain tissue was obtained from sham (n=1) and CBD animals sacrificed 4–5 (n=1), and &gt;;21 d (n=2) post‐CBD. Optical density (OD) measurements of NK1 in NTS, DMV, and nXII in the sham rat (0.91, 0.96, 0.80 OD, respectively) were similar to rats sacrificed after 21 d post CBD (0.84, 0.96, 0.74 OD, respectively). The rat sacrificed 4–5 d post‐CBD had less intense NK1 staining in the NTS (0.68 OD), DMV (0.77 OD), and nXII (0.65 OD) relative to the other group of rats. The sham rat had lower OD values in the NA (0.54 OD) and PBC (0.70 OD) than rats sacrificed after 21 d post‐CBD (0.93 and 0.93 OD, respectively). These preliminary data suggest that CBD causes a down‐regulation, and recovery from CBD due to up‐regulation of the NK1 receptor. Remaining tissue sections from the same animals will be used to compare AMPA type 2, NMDA type 1, the serotonin transporter, and 5HT2A receptor staining intensities in the same respiratory nuclei. Supported by NIH HL097033

I think ICAN: modulation of TRPM4 channels may contribute not only to the emergence of rhythm, but robust output and metabolic sensitivity of the preBötzinger Complex inspiratory network

Understanding the mechanisms by which central rhythm generators (CRGs) produce the neural rhythms that drive behaviours such as locomotion, chewing and breathing is a longstanding, fundamental problem that also has great clinical relevance. A topic of current debate is whether the basis of CRG function resides in the intrinsic auto-rhythmic properties of pacemaker neurons or emergent properties of the network (Del Negro & Hayes, 2008). Strong support for the latter comes from work in the preBotzinger complex (preBotC, a region critical for rhythm generation) of the inspiratory network that shows powerful functional links among AMPA receptors, Ca2+ channels, group I metabotropic glutamate receptors (mGluRs) and the TRPM4-mediated (transient receptor potential melastatin 4 subtype), burst-generating, non-specific cation current, ICAN (Mironov 2008; Pace & Del Negro, 2008). In this model, referred to as the group pacemaker hypothesis, synaptically released glutamate periodically activates AMPA and group I mGluRs. AMPA receptor-mediated depolarization evokes Ca2+ current via voltage-gated Ca2+ channels. Inositol 1,4,5-trisphosphate (IP3) receptors bind this Ca2+ as well as IP3 synthesized as a result of mGluR activation to cause regenerative intracellular Ca2+ release. Ca2+ from Ca2+ channels and intracellular stores evokes ICAN, which provides the bulk of the inward current underlying the inspiratory drive potential. Thus, the mechanism of rhythm generation in vitro emerges from the properties of a network in which latent burst-generating, intrinsic conductances are recruited by excitatory synaptic interactions among preBotC inspiratory neurons (Feldman & Del Negro, 2006; Pace & Del Negro, 2008).

Physiological and morphological properties of Dbx1-derived respiratory neurons in the pre-Botzinger complex of neonatal mice.

Breathing in mammals depends on an inspiratory-related rhythm that is generated by glutamatergic neurons in the pre-Bötzinger complex (preBötC) of the lower brainstem. A substantial subset of putative rhythm-generating preBötC neurons derive from a single genetic line that expresses the transcription factor Dbx1, but the cellular mechanisms of rhythmogenesis remain incompletely understood. To elucidate these mechanisms, we carried out a comparative analysis of Dbx1-expressing neurons (Dbx1(+)) and non-Dbx1-derived (Dbx1(-)) neurons in the preBötC. Whole-cell recordings in rhythmically active newborn mouse slice preparations showed that Dbx1(+) neurons activate earlier in the respiratory cycle and discharge greater magnitude inspiratory bursts compared with Dbx1(-) neurons. Furthermore, Dbx1(+) neurons required less input current to discharge spikes (rheobase) in the context of network activity. The expression of intrinsic membrane properties indicative of A-current (IA) and hyperpolarization-activated current (Ih) tended to be mutually exclusive in Dbx1(+) neurons. In contrast, there was no such relationship in the expression of currents IA and Ih in Dbx1(-) neurons. Confocal imaging and digital morphological reconstruction of recorded neurons revealed dendritic spines on Dbx1(-) neurons, but Dbx1(+) neurons were spineless. The morphology of Dbx1(+) neurons was largely confined to the transverse plane, whereas Dbx1(-) neurons projected dendrites to a greater extent in the parasagittal plane. The putative rhythmogenic nature of Dbx1(+) neurons may be attributable, in part, to a higher level of intrinsic excitability in the context of network synaptic activity. Furthermore, Dbx1(+) neuronal morphology may facilitate temporal summation and integration of local synaptic inputs from other Dbx1(+) neurons, taking place largely in the dendrites, which could be important for initiating and maintaining bursts and synchronizing activity during the inspiratory phase.

THE EFFECTS OF GASTRIN-RELEASING PEPTIDE MICROINJECTIONS INTO THE PRE-BOTZINGER COMPLEX ON THE RESPIRATORY PARAMETERS OF RATS

In the article, the results of investigation of the respiratory effects of gastrinreleasing peptide microjections into the pre-Botzinger complex are presented. It was found that microinjections of neuropeptide induced an increase in respiratory rate, tidal volume, amplitude of diaphragm and external intercostal muscles bursting activity. The statistically significant respiratory responses observed when 10^-5 M of GRP was used, while 10^-8 M of GRP appeared to be sub-threshold and didn’t alter the breathing pattern and activity of inspiratory muscles. The suggested mechanisms of action of GRP at the level of pre-Botzinger complex are discussed.

The H3K27 Demethylase JMJD3 Is Required for Maintenance of the Embryonic Respiratory Neuronal Network, Neonatal Breathing, and Survival

JMJD3 (KDM6B) antagonizes Polycomb silencing by demethylating lysine 27 on histone H3. The interplay of methyltransferases and demethylases at this residue is thought to underlie critical cell fate transitions, and the dynamics of H3K27me3 during neurogenesis posited for JMJD3 a critical role in the acquisition of neural fate. Despite evidence of its involvement in early neural commitment, however, its role in the emergence and maturation of the mammalian CNS remains unknown. Here, we inactivated Jmjd3 in the mouse and found that its loss causes perinatal lethality with the complete and selective disruption of the pre-Bötzinger complex (PBC), the pacemaker of the respiratory rhythm generator. Through genetic and electrophysiological approaches, we show that the enzymatic activity of JMJD3 is selectively required for the maintenance of the PBC and controls critical regulators of PBC activity, uncovering an unanticipated role of this enzyme in the late structuring and function of neuronal networks.

COMPARATIVE ANALYSIS OF RESPIRATORY REACTIONS TO MICROINJECTION OF GABA AND PENICILLIN INTO THE BOTZINGER COMPLEX AND THE PRE-BOTZINGER COMPLEX IN RATS

In acute experiments on anesthetized rats, the respiratory responses to local administration of GABA (10^{-5}М) and noncompetitive GABAA antagonist penicillin (10^{-7} М) into B¨otzinger complex (BC) and pre-B¨otzinger complex (PBC) were studied. The differences in the responses of respiratory frequency, inspiratory and expiratory duration after injection of GABA into the BC and PBC were found. The effects were more pronounced when GABA administered into the PBC. It is shown that the response of tidal volume, in contrast, was more frequent when both GABA and penicillin injected into the BC. The question of the role of GABA receptors of BC and PBC in the realization of the inhibitory action of GABA on the temporal and volumetric parameters of breathing pattern is discussed.

Pattern variability in a computational model of respiratory rhythm generation

Variability is inherent to rhythmic physiological patterns. Fluctuations in neural patterns are often regarded as noise. However it has been suggested that neurally generated rhythms result from systems that are not homeostatic but instead are homeodynamic [1][2]. In this view, excessive variability may indicate an instability while insufficient variability could result from an inability to adapt to environmental stressors. Understanding the inherent mechanisms of variability in the nervous system may enable us to use statistical measures to evaluate stability and robustness. Studies have addressed the presence of variability of the neural control of respiration in both the clinical context [3] as well as the experimental context [4]. Experimental studies have primarily focused on in vivo models that incorporate chemosensory and mechanosensory feedback which are likely to be necessary mechanisms of respiratory variability. A fundamental source of respiratory pattern generation is the preBotzinger complex (preBotC) which is part of the ventral respiratory column located in the brainstem. Here we use a computational model of the preBotC to analyze the variability of behavioral patterns at both the population and single-cell levels. Our model is based on previous computational studies of the in vitro respiratory slice [5][6]. It utilizes Hodgking-Huxley style neurons [7] that are divided into four cell types: hypoglossal motoneurons, premotoneurons, and preBotC cells that are configured for bursting by means of a persistent Na+ current. The architecture consists a feedforward hierarchy such that motoneurons are driven by premotoneurons and premotoneurons are driven by preBotC cells. The preBotC cells receive inputs from each other and also receive random tonic drive in the form of excitatory and inhibitory postsynaptic currents. This tonic drive represents chemosensory and mechanosensory feedback. Hierarchical levels are randomly connected with a fixed connection probability or density. Additionally the model includes extracellular K+ diffusion/buffering and a cellular Na+/ K+ exchange pump. We analyzed the burst patterns of individual cells as well as the spike histogram of the hypoglossal motoneuron population which represents the whole-nerve activity recorded from cranial nerve XII in experiments. Variability was measured for both the single-cell bursts and motoneuron population bursts. Burst properties that were analyzed included respiratory cycle length, inspiratory phase length, and peak burst amplitude for the population spike histogram. Three variability measures were used: the linear measure of coefficient of variation and two nonlinear measures: mutual information and sample entropy. We focused on two network areas and their influence on burst pattern variability. The first was the tonic drive to preBotC cells. We varied the quantity of tonic inputs and connection densities. We found that an increase in the connection density to preBotC cells produced a decrease in overall linear and nonlinear variability. This is due to the increase in postsynaptic event correlation that results from the combined common inputs. The second area that was studied was the interconnection density between the preBotC cells themselves. Again an increase in connection density produced a decrease in overall linear and nonlinear variability. However, this effect was not as pronounced as that of the tonic drive.

Modeling Na+- and Ca2+-dependent mechanisms of rhythmic bursting in excitatory neural networks

The mechanisms generating neural oscillations in the mammalian brainstem, particularly in the pre-Botzinger complex (pre-BotC) involved in control of respiration, and the spinal cord (e.g. circuits controlling locomotion) that persist after blockade of synaptic inhibition, remain poorly understood. Experimental studies in medullary slices from neonatal rodents containing the pre-BotC identified two mechanisms that could potentially contribute to generation of rhythmic bursting in the pre-BotC: one based on the persistent sodium current (INaP) [1,2], and the other involving the voltage-gated calcium (ICa) [3] and/or the calcium-activated nonspecific cation current (ICAN), activated by intracellular Ca2+ accumulated from extra- and/or intracellular sources [4]. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modeling study we investigated Na+- and Ca2+-dependent bursting generated in single cells and in a heterogeneous population of synaptically interconnected excitatory neurons with INaP, and ICa randomly distributed within the population. We analyzed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca2+ release, and the Na+/K+ pump in rhythmic bursting activity generated under different conditions. We show that the heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on INaP and/or ICAN, or independent of both (Fig. ​(Fig.1).1). The oscillatory regime and operating bursting mechanism may depend on neuronal excitability, synaptic interactions and relative expression of particular ionic currents. Figure 1 A map of bursting activities derived from multiple simulations of the excitatory neuron population and presented in a 2D parameter space (gtonic, ), where gtonic is the external excitatory drive to all neurons, N is the number of neurons in the population, ... The existence of multiple oscillatory regimes and their state-dependency may provide explanations for different rhythmic activities observed in the brainstem and spinal cord under different experimental conditions.

Dynamics of neuromodulatory feedback determines frequency modulation in respiratory network

Neuromodulators, such as amines and neuropeptides, alter the activity of neurons and neuronal networks. In this work, we investigate how neuromodulators which activate Gq-protein second messenger systems can modulate the frequency of bursting neurons in a critical portion of the respiratory neural network, the pre-Botzinger complex (pBC). These neurons are a vital part of the ponto-medullary neuronal network, which generates a stable respiratory rhythm, whose frequency is modulated by neuromodulator release from nearby Raphe nucleus. Using a simulated 50-cell network of excitatory connected pBC neurons with a heterogeneous distribution of persistent sodium conductance and ER Ca2+, we determined conditions for frequency modulation in such network by simulating interaction between Raphe and pBC nuclei. We found that the positive feedback between the Raphe excitability and pBC activity induces frequency modulation in the pBC neurons. In addition, the frequency of the respiratory rhythm can be modulated via phasic release of excitatory neuromodulators from the Raphe nucleus. We further predict that the application of a Gq antagonist will eliminate this frequency modulation by Raphe and keep the network frequency constant and low. In contrast, application of a Gq agonist will result in a high frequency for all levels of Raphe stimulation. Our modeling results also suggest that high [K+] requirement in respiratory brain slice experiments may serve as a compensatory mechanism for low neuromodulatory tone.

Analysis of excitatory and inhibitory interactions at high temporal resolution in core circuits of the respiratory CPG

The pre-Botzinger complex (pre-BotC) is the essential core component of the brainstem respiratory central pattern generator (rCPG) in mammals. Phasic excitatory and inhibitory synaptic inputs during the respiratory cycle are thought to dynamically shape membrane potential trajectories and spiking patterns of pre-BotC neurons. These synaptic inputs reflect the functionally interacting neuron populations involved in rhythm and inspiratory-expiratory pattern generation. The dynamic patterns of these synaptic inputs have not been systematically studied and characterized experimentally. Accurate temporal reconstruction of the synaptic input patterns is important for testing current models of the rCPG that incorporate specific excitatory and inhibitory circuit mechanisms for rhythm and pattern generation. A recent computational model of the rCPG generating a normal “three-phase” respiratory pattern [1] specified patterns of synaptic excitation and inhibition in the preBotC during the respiratory cycle required to produce the behaviorally distinct phases of inspiration (I) and two phases of expiration (E1, or post-inspiration, and E2). This model has not been rigorously tested by experimental data on dynamic changes in phasic postsynaptic excitatory and inhibitory conductances in different populations of pre-BotC inspiratory and expiratory neurons. We developed analytical techniques that allow quantitative reconstruction of the patterns of synaptic conductances at high temporal resolution from intracellular recordings of membrane potential trajectories. Our approach extends previously proposed methods for extracting dynamic patterns of synaptic input conductances [2,3] and provides nearly continuous readouts of inhibitory and excitatory synaptic conductances in electrophysiologically different cell types throughout the respiratory cycle. The membrane potential trajectories of pre-BotC respiratory neurons were obtained by sharp microelectrode current-clamp recording within in situ perfused brainstem-spinal cord preparations of mature rats, which generate a three-phase respiratory pattern and provide mechanically stable conditions for intracellular recordings in functionally intact brainstem circuits. We distinguished different types of inspiratory and expiratory pre-BotC neurons and analyzed the dynamical changes of excitatory (Ge )a nd inhibitory (Gi) conductances. Inspiratory neurons exhibited strong excitatory inputs (large dynamic increases in Ge) during their spiking phase followed by a wave(s) of inhibitory inputs during the expiratory period with large increases in Gi and temporal patterns consistent with two populations of inhibitory neurons providing inputs that coordinate inspiratory to expiratory and expiratory to inspiratory phase transitions. Expiratory neurons exhibited strong inhibition during the inspiratory phase, consistent with the rhythmic alternation of inspiration and expiration, and changes in Ge indicating reciprocal interactions between two major populations of inhibitory neurons coordinating the generation of two (E1 and E2) phases of expiration. The reconstructed patterns of Ge and Gi are generally consistent with previously proposed circuit architecture [1] and also suggest its extension. The techniques that we have developed for high temporal resolution of postsynaptic conductance changes are likely to have broad application for analysis of synaptic interactions in circuit components whenever intracellular recordings of membrane potentials can be