Pacemaker Network Research Articles

Mechanisms of Intrinsic Beating Variability in Cardiac Cell Cultures and Model Pacemaker Networks

Heart rate variability (HRV) exhibits fluctuations characterized by a power law behavior of its power spectrum. The interpretation of this nonlinear HRV behavior, resulting from interactions between extracardiac regulatory mechanisms, could be clinically useful. However, the involvement of intrinsic variations of pacemaker rate in HRV has scarcely been investigated. We examined beating variability in spontaneously active incubating cultures of neonatal rat ventricular myocytes using microelectrode arrays. In networks of mathematical model pacemaker cells, we evaluated the variability induced by the stochastic gating of transmembrane currents and of calcium release channels and by the dynamic turnover of ion channels. In the cultures, spontaneous activity originated from a mobile focus. Both the beat-to-beat movement of the focus and beat rate variability exhibited a power law behavior. In the model networks, stochastic fluctuations in transmembrane currents and stochastic gating of calcium release channels did not reproduce the spatiotemporal patterns observed in vitro. In contrast, long-term correlations produced by the turnover of ion channels induced variability patterns with a power law behavior similar to those observed experimentally. Therefore, phenomena leading to long-term correlated variations in pacemaker cellular function may, in conjunction with extracardiac regulatory mechanisms, contribute to the nonlinear characteristics of HRV.

Mechanisms of Clock Output in the Drosophila Circadian Pacemaker System

Molecular oscillations that underlie the circadian clock are coupled to different output signals by which daily rhythms in downstream events are evoked and/or synchronized. Here the authors review the literature that describes circadian output mechanisms in Drosophila. They begin at the most proximal level, within oscillator cells themselves, by surveying studies of rhythmic gene expression within Drosophila heads. Next the authors describe the several neuron groups that compose the circadian pacemaker network underlying rhythmic locomotor activity, and they detail current models of how that network is organized and coordinated. The authors outline the body of evidence that describes a role for the neuropeptide pigment dispersing factor (PDF) as a circadian transmitter in the fly brain. Finally, in the context of PDF, they consider studies that address mechanisms of signaling from the circadian pacemaker network to downstream neurons and nonneuronal cells that directly control rhythmic outputs.

Pigment-Dispersing Factor and GABA Synchronize Cells of the Isolated Circadian Clock of the CockroachLeucophaea maderae

Pigment-dispersing factor-immunoreactive circadian pacemaker cells, which arborize in the accessory medulla, control circadian locomotor activity rhythms in Drosophila as well as in the cockroach Leucophaea maderae via unknown mechanisms. Here, we show that circadian pacemaker candidates of the accessory medulla of the cockroach produce regular interspike intervals. Therefore, the membrane potential of the cells oscillates with ultradian periods. Most or all oscillating cells within the accessory medulla are coupled via synaptic and nonsynaptic mechanisms, forming different assemblies. The cells within an assembly share the same ultradian period (interspike interval) and the same phase (timing of spikes), whereas cells between assemblies differ in phase. Apparently, the majority of these assemblies are formed by inhibitory GABAergic synaptic interactions. Application of pigment-dispersing factor phase locked and thereby synchronized different assemblies. The data suggest that pigment-dispersing factor inhibits GABAergic interneurons, resulting in disinhibition and phase locking of their postsynaptic cells, which previously belonged to different assemblies. Our data suggest that phase control of action potential oscillations in the ultradian range is a main task of the circadian pacemaker network. We hypothesize that neuropeptide-dependent phase control is used to gate circadian outputs to locomotor control centers.

The Dynamic Range of Bursting in a Model Respiratory Pacemaker Network

A network of excitatory neurons within the pre-Botzinger complex (pre-BotC) of the mammalian brain stem has been found experimentally to generate robust, synchronized population bursts of activity. An experimentally calibrated model for pre-BotC cells yields typical square-wave bursting behavior in the absence of coupling, over a certain parameter range, with quiescence or tonic spiking outside of this range. Previous simulations of this model showed that the introduction of synaptic coupling extends the bursting parameter range significantly and induces complex effects on burst characteristics. In this paper, we use geometric dynamical systems techniques, predominantly a fast/slow decomposition and bifurcation analysis approach, to explain these effects in a two-cell model network. Our analysis yields the novel finding that, over a broad range of synaptic coupling strengths, the network can support two qualitatively distinct forms of synchronized bursting, which we call symmetric and asymmetric bursting, as well as both symmetric and asymmetric tonic spiking. By elucidating the dynamical mechanisms underlying the transitions between these states, we also gain insight into how relevant parameters influence burst duration and interburst intervals. We find that, in the two-cell network with synaptic coupling, the stable family of periodic orbits for the fast subsystem features spike asynchrony within otherwise synchronized bursts and terminates in a saddle-node bifurcation, rather than in a homoclinic bifurcation, over a wide parameter range. As a result, square-wave bursting is replaced by what we call top hat bursting (also known as fold/fold cycle bursting), at least for a broad range of parameter values. Further, spike asynchrony is a key ingredient in shaping the dynamic range of bursting, leading to a significant enhancement in the parameter range over which bursting occurs and an abrupt increase in burst duration as an appropriate parameter is varied.

Follower neurons in lobster (Panulirus interruptus) pyloric network regulate pacemaker period in complementary ways.

Distributed neural networks (ones characterized by high levels of interconnectivity among network neurons) are not well understood. Increased insight into these systems can be obtained by perturbing network activity so as to study the functions of specific neurons not only in the network's "baseline" activity but across a range of network activities. We applied this technique to study cycle period control in the rhythmic pyloric network of the lobster, Panulirus interruptus. Pyloric rhythmicity is driven by an endogenous oscillator, the Anterior Burster (AB) neuron. Two network neurons feed back onto the pacemaker, the Lateral Pyloric (LP) neuron by inhibition and the Ventricular Dilator (VD) neuron by electrical coupling. LP and VD neuron effects on pyloric cycle period can be studied across a range of periods by altering period by injecting current into the AB neuron and functionally removing (by hyperpolarization) the LP and VD neurons from the network at each period. Within a range of pacemaker periods, the LP and VD neurons regulate period in complementary ways. LP neuron removal speeds the network and VD neuron removal slows it. Outside this range, network activity is disrupted because the LP neuron cannot follow slow periods, and the VD neuron cannot follow fast periods. These neurons thus also limit, in complementary ways, normal pyloric activity to a certain period range. These data show that follower neurons in pacemaker networks can play central roles in controlling pacemaker period and suggest that in some cases specific functions can be assigned to individual network neurons.

Why do cubomedusae have only four swim pacemakers?

The classic view of swimming control in scyphozoan and cubozoan jellyfish involves a diffuse motor nerve net activated by multiple pacemaker sites that interact in a simple resetting hierarchy. Earlier modeling studies of jellyfish swimming, utilizing resetting linkages of multiple pacemakers, indicated that increases in pacemaker number were correlated with increases in the rate and regularity of network activity. We conducted a similar study using the cubozoan jellyfish Carybdea marsupialis, concentrating not only on the adaptive features of multiple pacemaker networks but also on the mechanism of pacemaker interaction. The best fit for our experimental data is a model in which pacemakers express a degree of independence. Thus, our results challenge the idea that pacemaker interactions in scyphozoan and cubozoan medusae are based on a strict resetting hierarchy. Furthermore, our data suggest that the combination of semi-independent linkage of pacemakers with the small pacemaker number characteristic of cubomedusae is important in (i) maintaining a biphasic modulatory capability in the swimming system, and (ii) allowing behaviorally appropriate directional responses to asymmetrical sensory inputs in the radially arranged jellyfish nervous system.

Stationary organotypic culture of the pre-Bötzinger complex from the newborn rat.

In the early 1990s, it was suggested that the central neuronal network responsible for breathing relies on pacemaker cells to promote its rhythmic activity.1,2,3These pacemaker neurons were shown to be concentrated in the region of the pre-Bötzinger complex and are critical components of the network generating respiratory rhythm.3,4Previous studies of this neuronal population have been done primarily in fresh tissue slices in vitro which have a limited survival (less than 48 hours) and, due to thickness of the slice, offer poor accessibility to neurons of interest. Tissue culture of the isolated pre-Bötzinger complex may offer some advantages in this respect, but it is not known whether the isolated pre-Bötzinger network would maintain its functional properties. Therefore, we evaluated the viability of the stationary explant of the cultured pre-Bötzinger region (“island”). We hypothesized that the pacemaker network would maintain functional activity for extended periods in culture due to the network robust functional properties.KeywordsORGANOTYPIC CulturePacemaker CellRespiratory RhythmColor RepresentationPacemaker NeuronThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.

Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker–network model

We review a new unified model of respiratory rhythm generation — the hybrid pacemaker–network model. This model represents a comprehensive synthesis of cellular and network mechanisms that can theoretically account for rhythm generation in different functional states, from the most reduced states in the neonatal nervous system in vitro to the intact adult system in vivo. The model incorporates a critical neuronal kernel consisting of a network of excitatory neurons with state-dependent, oscillatory bursting or pacemaker properties. This kernel, located in the pre-Bötzinger complex of the ventrolateral medulla, provides a rudimentary pacemaker network mechanism for generating an inspiratory rhythm, revealed predominately in functionally reduced states in vitro. In vivo the kernel is embedded in a larger network that interacts with the kernel via inhibitory synaptic connections that provide the dynamic control required for the evolution of the complete pattern of inspiratory and expiratory network activity. The resulting hybrid of cellular pacemaker and network properties functionally endows the system with multiple mechanisms of rhythm generation. New biophysically realistic mathematical models of the hybrid pacemaker–network have been developed that illustrate these concepts and provide a computational framework for investigating interactions of cellular and network processes that must be analyzed to understand rhythm generation.

Gap junction effects on precision and frequency of a model pacemaker network.

We investigated the precision of spike timing in a model of gap junction-coupled oscillatory neurons. The model incorporated the known physiology, morphology, and connectivity of the weakly electric fish's high-frequency and extremely precise pacemaker nucleus (Pn). Two neuron classes, pacemaker and relay cells, were each modeled with two compartments containing Hodgkin-Huxley sodium and potassium currents. Isolated pacemaker cells fired periodically, due to a constant current injection; relay cells were silent but slightly depolarized at rest. When coupled by gap junctions to other neurons, a model neuron, like its biological correlate, spiked at frequencies and amplitudes that were largely independent of current injections. The phase distribution in the network was labile to intracellular current injections and to gap junction conductance changes. The model predicts a biologically plausible gap junction conductance of 4-5 nS (200-250 MOmega). This results in a coupling coefficient of approximately 0.02, as observed in vitro. Network parameters were varied to test which could improve the temporal precision of oscillations. Increased gap junction conductances and larger numbers of cells (holding total junctional conductance per cell constant) both substantially reduced the coefficient of variation (CV = standard deviation/mean) of relay cell spike times by 74-85% and more, and did so with lower gap junction conductance when cells were contacted axonically compared with somatically. Pacemaker cell CV was only reduced when the probability of contact was increased, and then only moderately: a fivefold increase in the probability of contact reduced CV by 35%. We conclude that gap junctions facilitate synchronization, can reduce CV, are most effective between axons, and that pacemaker cells must have low intrinsic CV to account for the low CV of cells in the biological network.

Heterogeneity in electrical activity of the canine ileal circular muscle: interaction of two pacemakers.

In cross-sectioned slabs from the muscularis externa or in the isolated circular muscle devoid of longitudinal muscle and myenteric plexus (MyP) of ileum, a slow wave, sigmoidal or triangular in shape, was recorded from microelectrode impalements near the deep muscular plexus (DMP) region in the whole-thickness preparation. From the MyP region of whole-thickness preparations, a slow wave which oscillated at a similar frequency (9-10 cycles min-1) was characterized by a fast upstroke and a square shape. Slow waves of mumixed pattern were recorded in the outer circular muscle (OCM) while triangular slow waves were present near the submucous plexus (SMP). In this preparation but not in isolated circular muscle, the inhibitory junction potentials (IJPs) produced by supramaximal electrical field stimulation triggered slow waves. The amplitudes (15-25 mV) of spontaneous and triggered slow waves (TSWs) were greatest in the MyP region, significantly so compared to those of DMP and SMP regions and to those in all regions of isolated circular muscle. Frequencies of slow waves recorded from the MyP and DMP were slightly but significantly higher than those recorded from either the OCM or the SMP or from all regions of isolated circular muscle. A 10-15 mV gradient in resting membrane potential (more hyperpolarized near MyP) existed across the intact (but not the isolated) circular muscle layer. Both types of slow waves, TSWs and IJPs were unaffected by muscarinic, adrenergic or tachykinergic blockade. We suggest that a MyP pacemaker network generated a plateau-type slow wave while a DMP one induced a triangular slow wave. Each source can function independently but the MyP network may dominate and entrain DMP slow waves.

Oscillating Networks: Control of Burst Duration by Electrically Coupled Neurons.

The pyloric network of the stomatogastric ganglion in crustacea is a central pattern generator that can produce the same basic rhythm over a wide frequency range. Three electrically coupled neurons, the anterior burster (AB) neuron and two pyloric dilator (PD) neurons, act as a pacemaker unit for the pyloric network. The functional characteristics of the pacemaker network are the result of electrical coupling between neurons with quite different intrinsic properties, each contributing a basic feature to the complete circuit. The AB neuron, a conditional oscillator, plays a dominant role in rhythm generation. In the work described here, we manipulate the frequency of the AB neuron both isolated and electrically coupled to the PD neurons. Physiological and modeling studies indicate that the PD neurons play an important role in regulating the duration of the bursts produced by the pacemaker unit.

Monosynaptic entrainment of an endogenous pacemaker network: A cellular mechanism for von Holst's magnet effect

1. The pyloric rhythm of the lobster (Panulirus interruptus, Palinurus vulgaris) stomatogastric ganglion is generated by a set of 3 electrically coupled endogenously bursting neurons. The phasic coordinating effects of monosynaptic excitatory (EPSP) and inhibitory (IPSP) inputs to these neurons were examined in isolated nervous systems. 2. Periodic stimulation of both inputs at frequencies near the endogenous frequency of the pacemakers can produce large cycle by cycle variations in the burst period of the system (Fig. 4). Similar variation in PD burst period is also observed in intact freely behaving animals (Fig. 2). 3. The effect of both inputs depends on the phase at which they occur in the endogenous pacemaker cycle. Both EPSP's and IPSP's can advance or delay subsequent bursts. The two inputs exhibit qualitatively different characteristic phase response curves (Figs. 5, 6). 4. Both EPSP's and IPSP's can entrain the endogenous rhythm (Figs. 7, 8). The coordinating effects of EPSP's are stronger when their repetition frequency is slightly higher than the endogenous pacemaker frequency, while those produced by IPSP's are stronger when the repetition frequency of the input is slightly slower than the repetition frequency of the oscillator. 5. The phase relationships of the discharge of the pacemakers in the stimulus cycle are qualitatively different when the cyclic stimulus is slower or faster than the endogenous rhythm for both types of inputs (Fig. 9). The entrainment of the oscillator by EPSP inputs which repeat at a frequency which is greater than the endogenous frequency can be attributed to the input triggering the burst of spikes at a fixed latency (Fig. 10).

Simulation of slow-wave electrical activity of small intestine.

Simulation of slow-wave electrical activity of small intestine.

Nature of the intestinal slow-wave frequency gradient.

Nature of the intestinal slow-wave frequency gradient.