Stimulation of bursting in pre-Bötzinger neurons by Epac through calcium release and modulation of TRPM4 and K-ATP channels

The exchange factor directly activated by cAMP (Epac) can couple cAMP production to the activation of particular membrane and cytoplasmic targets. Using patch-clamp recordings and calcium imaging in organotypic brainstem slices, we examined the role of Epac in pre-B?tzinger complex, an essential part of the respiratory network. The selective agonist 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) sensitized calcium mobilisation from inositol-1,4,5-trisphosphate-sensitive internal stores that stimulated TRPM4 (transient receptor potential cation channel, subfamily M, Melastatin) channels and potentiated the bursts of action potentials. 8-pCPT actions were abolished after inhibition of phospholipase C with U73122 and depletion of calcium stores with thapsigargin. Caffeine-sensitive release channels were not modulated by 8-pCPT. Epac inhibited ATP-sensitive K(+) channels that also led to the enhancement of bursting by 8-pCPT. Bursting activity, spontaneous calcium transients and activity of TRPM4 and ATP-sensitive K(+) channels were potentiated after brief exposures to bradykinin and incubation with wortmannin produced opposite effects that can be explained by changes in phosphatidylinositol 4,5-bisphosphate levels. 8-pCPT stimulated the respiratory motor output in functionally intact preparations and the effects of bradykinin and wortmannin were identical to those observed in organotypic slices. The data thus indicate a novel pathway of controlling bursting activity in pre-B?tzinger complex neurons through Epac that can involved in reinforcement of the respiratory activity by cAMP.     

Dynamics of in-phase and anti-phase bursting in the coupled pre-Bötzinger complex cells

Activity of neurons in the pre-Bötzinger complex within the mammalian brain stem has an important role in the generation of respiratory rhythms. Previous experimental results have shown that the dynamics of sodium and calcium within each cell may be responsible for various bursting mechanisms. In this paper, we study the bursting dynamics of the two-coupled pre-Bötzinger complex neurons. Using a combination of fast-slow decomposition and two-parameter bifurcation analysis, we explore the possible forms of dynamics that the model network can produce as well the transitions of in-phase and anti-phase bursting respectively.     

Effect of thyroliberin on membrane potential and pattern of spontaneous activity of neurons of the respiratory center in rats in vitro

On frontal brainstem slices of rat by means of whole-clamp recordings, we investigated effects of TRH (10(-8) [symbol: see text]) on membrane potential and firing pattern of the neurones in ventrolateral area of the solitary tract nucleus and pre-Botzinger complex. TRH induced a membrane depolarisation and an increase in spontaneous activity of the respiratory centre neurones. After TRH administration, a shortening of time intervals between the beginning of bursts was found in bursting neurones of the pre-Botzinger complex. In some silent neurones, TRH elicited appearance of firing activity, so the silent neurones of the solitary tract nucleus were transformed into tonic while the silent pre-Botzinger complex neurones were transformed into bursting ones. Thus, there is a direct regulatory effect of TRH on the respiratory centre neurones at the level of their membrane.     

Two types of independent bursting mechanisms in inspiratory neurons: an integrative model

The network of coupled neurons in the pre-Bötzinger complex (pBC) of the medulla generates a bursting rhythm, which underlies the inspiratory phase of respiration. In some of these neurons, bursting persists even when synaptic coupling in the network is blocked and respiratory rhythmic discharge stops. Bursting in inspiratory neurons has been extensively studied, and two classes of bursting neurons have been identified, with bursting mechanism depends on either persistent sodium current or changes in intracellular Ca²⁺, respectively. Motivated by experimental evidence from these intrinsically bursting neurons, we present a two-compartment mathematical model of an isolated pBC neuron with two independent bursting mechanisms. Bursting in the somatic compartment is modeled via inactivation of a persistent sodium current, whereas bursting in the dendritic compartment relies on Ca²⁺ oscillations, which are determined by the neuromodulatory tone. The model explains a number of conflicting experimental results and is able to generate a robust bursting rhythm, over a large range of parameters, with a frequency adjusted by neuromodulators.     

Influences of superior laryngeal afferent stimulation on expiratory activity in cats

The effects of superior laryngeal nerve (SLN) stimulation on the activity of the expiratory muscles and medullary expiration-related (ER) neurons were investigated in 24 pentobarbital-anesthetized cats. In some experiments the animals were also paralyzed and artificially ventilated. Sustained tetanic stimulation of SLN consistently caused an apneic response associated with the appearance of tonic CO2-dependent activity in the expiratory muscles and in ER neurons located in the caudal ventral respiratory group (VRG) and the B?tzinger complex. Single shocks or brief tetani at the same stimulation intensities failed to evoke excitatory responses in the expiratory muscles and in the vast majority of ER neurons tested. At higher stimulation strengths, single shocks or short tetani elicited excitatory responses in the expiratory muscles (20- to 35-ms latency) and in the majority of ER neurons of the caudal VRG (7.5- to 15.5-ms latency). These responses were obtained only during the expiratory phase and proved to be CO2 independent. On the contrary, only inhibitory responses were evoked in the activity of B?tzinger complex neurons. The observed tonic expiratory activity most likely represents a disinhibition phenomenon due to the suppression of inspiratory activity; activation of expiratory muscles at higher stimulation intensities appears to be a polysynaptic reflex mediated by ER neurons of the caudal VRG but not by B?tzinger complex neurons.     

The role of spiking and bursting pacemakers in the neuronal control of breathing

Breathing is controlled by a distributed network involving areas in the neocortex, cerebellum, pons, medulla, spinal cord, and various other subcortical regions. However, only one area seems to be essential and sufficient for generating the respiratory rhythm: the preBötzinger complex (preBötC). Lesioning this area abolishes breathing and following isolation in a brain slice the preBötC continues to generate different forms of respiratory activities. The use of slice preparations led to a thorough understanding of the cellular mechanisms that underlie the generation of inspiratory activity within this network. Two types of inward currents, the persistent sodium current (I_NaP) and the calcium-activated non-specific cation current (I_CAN), play important roles in respiratory rhythm generation. These currents give rise to autonomous pacemaker activity within respiratory neurons, leading to the generation of intrinsic spiking and bursting activity. These membrane properties amplify as well as activate synaptic mechanisms that are critical for the initiation and maintenance of inspiratory activity. In this review, we describe the dynamic interplay between synaptic and intrinsic membrane properties in the generation of the respiratory rhythm and we relate these mechanisms to rhythm generating networks involved in other behaviors.     

The pre-B?tzinger oscillator in the mouse embryo.

Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-B?tzinger complex (preB?tC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preB?tC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation.     

Parameter estimation for bursting neural models

This paper presents work on parameter estimation methods for bursting neural models. In our approach we use both geometrical features specific to bursting, as well as general features such as periodic orbits and their bifurcations. We use the geometry underlying bursting to introduce defining equations for burst initiation and termination, and restrict the estimation algorithms to the space of bursting periodic orbits when trying to fit periodic burst data. These geometrical ideas are combined with automatic differentiation to accurately compute parameter sensitivities for the burst timing and period. In addition to being of inherent interest, these sensitivities are used in standard gradient-based optimization algorithms to fit model burst duration and period to data. As an application, we fit Butera et al.'s (Journal of Neurophysiology 81, 382-397, 1999) model of preB?tzinger complex neurons to empirical data both in control conditions and when the neuromodulator norepinephrine is added (Viemari and Ramirez, Journal of Neurophysiology 95, 2070-2082, 2006). The results suggest possible modulatory mechanisms in the preB?tzinger complex, including modulation of the persistent sodium current.     

First return maps for the dynamics of synaptically coupled conditional bursters

The pre-Bötzinger complex (preBötc) in the mammalian brainstem has an important role in generating respiratory rhythms. An influential differential equation model for the activity of individual neurons in the preBötc yields transitions from quiescence to bursting to tonic spiking as a parameter is varied. Further, past work has established that bursting dynamics can arise from a pair of tonic model cells coupled with synaptic excitation. In this paper, we analytically derive one- and two-dimensional maps from the differential equations for a self-coupled neuron and a two-neuron network, respectively. Using a combination of analysis and simulations of these maps, we explore the possible forms of dynamics that the model networks can produce as well as which transitions between dynamic regimes are mathematically possible.     

Effect of leu-enkephalin on potassium currents in neurons of the respiratory center of rats in vitro

Action of opioid peptide: leu-enkephalin (10 nM - 1 microM), on K+A-current and inward rectifier in neurons of two divisions of the respiratory center: ventrolateral area of the solitary tract nucleus and the pre-Botzinger complex, was investigated in brainstem slices of Wistar rats by whole-cell voltage-clamp recordings. A-current and inward rectifier were found in all the neurons under study. A-current did not change after the application of leu-enkephalin to the bath solution while the amplitude of inward rectifier did reversibly increase. The data obtained suggest that the inhibitory effect of leu-enkephalin on neurons of the respiratory center, at least in part, can be based on its ability to modulate inward rectifier.     

Intrinsic bursting activity in the pre-Bötzinger Complex: Role of persistent sodium and potassium currents

Computational models of single pacemaker neuron and neural population in the pre-Bötzinger Complex (pBC) were developed based on the previous models by Butera et al. (1999a,b). Our modeling study focused on the conditions that could define endogenous bursting vs. tonic activity in single pacemaker neurons and population bursting vs. asynchronous firing in populations of pacemaker neurons. We show that both bursting activity in single pacemaker neurons and population bursting activity may be released or suppressed depending on the expression of persistent sodium (I_NaP) and delayed-rectifier potassium (I_K) currents. Specifically, a transition from asynchronous firing to population bursting could be induced by a reduction of I_K via a direct suppression of the potassium conductance or through an elevation of extracellular potassium concentration. Similar population bursting activity could be triggered by an augmentation of I_NaP. These findings are discussed in the context of the possible role of population bursting activity in the pBC in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping.     

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.     

Cooperation of intrinsic bursting and calcium oscillations underlying activity patterns of model pre-Bötzinger complex neurons

Activity of neurons in the pre-Bötzinger complex (pre-BötC) within the mammalian brainstem drives the inspiratory phase of the respiratory rhythm. Experimental results have suggested that multiple bursting mechanisms based on a calcium-activated nonspecific cationic (CAN) current, a persistent sodium (NaP) current, and calcium dynamics may be incorporated within the pre-BötC. Previous modeling works have incorporated representations of some or all of these mechanisms. In this study, we consider a single-compartment model of a pre-BötC inspiratory neuron that encompasses particular aspects of all of these features. We present a novel mathematical analysis of the interaction of the corresponding rhythmic mechanisms arising in the model, including square-wave bursting and autonomous calcium oscillations, which requires treatment of a system of differential equations incorporating three slow variables.     

Background sodium current stabilizes bursting in respiratory pacemaker neurons

Endogenous pacemaker properties have been proposed to generate rhythmic activity underlying many behaviors including respiration. For pacemakers to generate regenerative bursting, background currents maintain their membrane potential (Vm) within a range where bi-stable properties are expressed, thereby stabilizing rhythmogenesis. We previously found that the baseline Vm of respiratory pacemakers is stabilized against hyperpolarizing shifts in their Vm. In response to prolonged hyperpolarizing current injection synaptically isolated respiratory pacemakers steadily depolarize and resume bursting, suggesting a stabilizing background current is involved. What is the ionic basis of this background current in respiratory pacemakers? Here we demonstrate that in low-[Na(+)](o) ACSF, synaptically isolated respiratory pacemakers hyperpolarized and remained outside the bursting window, but could burst upon depolarizing current injection. These data suggest that pacemakers possess a background sodium current that is necessary to bring their Vm into a bursting range. Low-[Na(+)](o) ACSF also abolished the depolarizing shift evoked during prolonged hyperpolarizing current injection, and bursting did not resume. This depolarizing shift persisted in the presence of I(h)-current blockers, but was abolished in tetrodotoxin. Although, under control conditions, the Vm of synaptically isolated respiratory pacemaker neurons was not significantly affected when [K(+)](o) was changed from 3 to 8 mM, the Vm is altered when [K(+)](o) was raised in low-[Na(+)](o) ACSF. Thus, current-clamp studies suggest that respiratory pacemaker neurons possess a background sodium current that maintains their membrane potential within a range where they express bursting, thereby stabilizing rhythmogenesis.     

Distribution and colocalization of neurotransmitters and receptors in the pre-B?tzinger complex of rats.

The pre-B?tzinger complex (PBC), thought to be the center of respiratory rhythm generation, is a cell column ventrolateral to the nucleus ambiguus. The present study analyzed its cellular and neurochemical composition in adult rats. PBC neurons were mainly oval, fusiform, or multipolar in shape and small to medium in size. Neurokinin-1 receptor, a marker of the PBC, was present in the plasma membrane of mostly medium and small neurons and their associated processes and boutons. Among neurons immunoreactive for different neurotransmitter or receptor candidates, various numbers were colocalized with neurokinin-1 receptor. The highest ratio was with nitric oxide synthase (52.72%), and the lowest was with glycine receptors (31.93%). Glutamic acid decarboxylase- and glycine transporter 2-immunoreactive boutons, as well as GABA(A) receptor-immunoreactive plasma membrane processes and boutons, were also identified in the PBC. PBC neurons exhibited different levels of cytochrome oxidase activity, indicating their various energy demands. Our results suggest that synaptic interactions within the PBC of adult rats involve a variety of neurotransmitter and receptor types and that nitric oxide may play an important role in addition to glutamate, GABA, glycine, and neurokinin.     

Respiratory changes induced by kainic acid lesions in rostral ventral respiratory group of rabbits

The role played by the B?tzinger complex (B?tC), the pre-B?tzinger complex (pre-B?tC), and the more rostral extent of the inspiratory portion of the ventral respiratory group (iVRG) in the genesis of the eupneic pattern of breathing was investigated in anesthetized, vagotomized, paralyzed, and artificially ventilated rabbits by means of kainic acid (KA, 4.7 mM) microinjections (20-30 nl). Unilateral KA microinjections into all of the investigated VRG subregions caused increases in respiratory frequency associated with moderate decreases in peak phrenic amplitude in the B?tC and pre-B?tC regions. Bilateral KA microinjections into either the B?tC or pre-B?tC transiently eliminated respiratory rhythmicity and caused the appearance of tonic phrenic activity ("tonic apnea"), whereas injections into the rostral iVRG completely suppressed inspiratory activity. Rhythmic activity resumed as low-amplitude, high-frequency oscillations and displayed a progressive, although incomplete, recovery. Combined bilateral KA microinjections (B?tC and pre-B?tC) caused persistent (>3 h) tonic apnea. Results show that all of the investigated VRG subregions exert a potent control on both the intensity and frequency of inspiratory activity, thus suggesting that these areas play a major role in the genesis of the eupneic pattern of breathing.     

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 μm² ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 μm² ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.     

Ventrolateral medullary respiratory network participation in the expiration reflex in the cat.

The expiration reflex is a distinct airway defensive response characterized by a brief, intense expiratory effort and coordinated adduction and abduction of the laryngeal folds. This study addressed the hypothesis that the ventrolateral medullary respiratory network participates in the reflex. Extracellular neuron activity was recorded with microelectrode arrays in decerebrated, neuromuscular-blocked, ventilated cats. In 32 recordings (17 cats), 232 neurons were monitored in the rostral (including B?tzinger and pre-B?tzinger complexes) and caudal ventral respiratory group. Neurons were classified by firing pattern, evaluated for spinal projections, functional associations with recurrent laryngeal and lumbar nerves, and firing rate changes during brief, large increases in lumbar motor nerve discharge (fictive expiration reflex, FER) elicited during mechanical stimulation of the vocal folds. Two hundred eight neurons were respiratory modulated, and 24 were nonrespiratory; 104 of the respiratory and 6 of the nonrespiratory-modulated neurons had altered peak firing rates during the FER. Increased firing rates of bulbospinal neurons and expiratory laryngeal premotor and motoneurons during the expiratory burst of FER were accompanied by changes in the firing patterns of putative propriobulbar neurons proposed to participate in the eupneic respiratory network. The results support the hypothesis that elements of the rostral and caudal ventral respiratory groups participate in generating and shaping the motor output of the FER. A model is proposed for the participation of the respiratory network in the expiration reflex.     

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

The pre-B?tzinger 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 Ca(2+) 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 x20 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.     

Unraveling the mechanism for respiratory rhythm generation

Breathing is generated by a neuronal network located within the caudal brainstem. One area of particular significance for respiratory rhythm generation is the pre-B?tzinger (preBotC) complex in the ventrolateral medulla. An important step towards understanding the cellular and network basis by which neurons within this region generate the respiratory rhythm was made in a recent study by Koshiya and Smith.(1) Using simultaneous image analysis and electrophysiological techniques these authors identified a discrete population of synaptically-coupled pacemaker neurons within the preBotC. They postulated that these neurons constitute the minimal essential network component (kernel) for generating the respiratory rhythm. BioEssays 22:6-9, 2000.