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1.  Background Sodium Current Underlying Respiratory Rhythm Regularity 
Rhythm generating neural circuits underlying diverse behaviors such as locomotion, sleep states, digestion and respiration play critical roles in our lives. Irregularities in these rhythmic behaviors characterize disease states - thus, it is essential that we identify ionic and/or cellular mechanisms that are necessary for triggering these rhythmic behaviors on a regular basis. Here, we examine which ionic conductances underlie regular or “stable” respiratory activities, proposed to underlie eupnea, or normal quiet breathing. We used a mouse in vitro medullary slice preparation containing the rhythmogenic respiratory neural circuit, called the preBötzinger Complex (preBötC) that underlies inspiratory respiratory activity. We varied either [K+]o, [Na+]o, or blocked voltage gated calcium channels (VGCC) while recording from synaptically isolated respiratory pacemakers and examined which of these manipulations resulted in their endogenous bursting to become more irregular. Of these, lowering [Na+]o increased the irregularity of endogenous bursting by synaptically isolated pacemakers. Lowering [Na+]o also decreased the regularity of fictive eupneic activity generated by the ventral respiratory group (VRG) population and hypoglossal motor output. Voltage clamp data indicate that lowering [Na+]o, in a range that results in irregular population rhythm generation, decreased persistent sodium currents, but not transient sodium currents underlying action potentials. Our data suggest that background sodium currents play a major role in determining the regularity of the fictive eupneic respiratory rhythm.
doi:10.1111/j.1460-9568.2008.06537.x
PMCID: PMC2815345  PMID: 19032590
2.  Glycinergic pacemaker neurons in preBötzinger Complex of neonatal mouse 
The preBötzinger Complex (preBötC) is essential for normal respiratory rhythm generation in rodents, for which the underlying mechanisms remain unknown. Excitatory preBötC pacemaker neurons are proposed to be necessary for rhythm generation. Here we report the presence of a population of preBötC glycinergic pacemaker neurons. We used rhythmic in vitro transverse slice preparations from transgenic mice where neurons expressing the glycine transporter 2 (GlyT2) gene co-express enhanced green fluorescent protein (EGFP). We combined epifluorescence and whole-cell patch-clamp recording to study preBötC EGFP-labeled, i.e., glycinergic, inspiratory-modulated neurons with pacemaker properties. We defined glycinergic pacemaker neurons as those preBötC EGFP neurons that exhibited: 1) ectopic bursting in rhythmic slices when depolarized during their normally silent period, and; 2) bursting when depolarized in non-rhythmic slices (following AMPA receptor blockade). 42% of EGFP-labeled neurons were inspiratory (n=48 of 115), of which 23% (n=11 of 48 inspiratory; 10% of the total recorded) were pacemakers. We conclude that there is a population of preBötC inspiratory-modulated glycinergic, presumably inhibitory, pacemaker neurons that constitute a substantial fraction of all preBötC pacemaker neurons. These findings challenge contemporary models for respiratory rhythmogenesis that assume the excitatory nature of preBötC pacemaker neurons. Testable and non-trivial predictions of the functional role of excitatory and inhibitory pacemaker neurons need to be proposed and the necessary experiments performed.
doi:10.1523/JNEUROSCI.3040-09.2010
PMCID: PMC2947441  PMID: 20219997
respiratory rhythm generation; breathing; GlyT2-EGFP mice; glycine; inhibition; Pacemaker
3.  Spatial organization and state-dependent mechanisms for respiratory rhythm and pattern generation 
Progress in brain research  2007;165:201-220.
The brainstem respiratory network can operate in multiple functional states engaging different state-dependent neural mechanisms. These mechanisms were studied in the in situ perfused rat brainstem–spinal cord preparation using sequential brainstem transections and administration of riluzole, a pharmacological blocker of persistent sodium current (INaP). Dramatic transformations in the rhythmogenic mechanisms and respiratory motor pattern were observed after removal of the pons and subsequent medullary transactions down to the rostral end of pre-Bötzinger complex (pre-BötC). A computational model of the brainstem respiratory network was developed to reproduce and explain these experimental findings. The model incorporates several interacting neuronal compartments, including the ventral respiratory group (VRG), pre-BötC, Bötzinger complex (BötC), and pons. Simulations mimicking the removal of circuit components following transections closely reproduce the respiratory motor output patterns recorded from the intact and sequentially reduced brainstem preparations. The model suggests that both the operating rhythmogenic mechanism (i.e., network-based or pacemaker-driven) and the respiratory pattern generated (e.g., three-phase, two-phase, or one-phase) depend on the state of the pre-BötC (expression of INaP-dependent intrinsic rhythmogenic mechanisms) and the BötC (providing expiratory inhibition in the network). At the same time, tonic drives from pons and multiple medullary chemoreceptive sites appear to control the state of these compartments and hence the operating rhythmogenic mechanism and motor pattern. Our results suggest that the brainstem respiratory network has a spatial (rostral-to-caudal) organization extending from the rostral pons to the VRG, in which each functional compartment is controlled by more rostral compartments. The model predicts a continuum of respiratory network states relying on different contributions of intrinsic cellular properties versus synaptic interactions for the generation and control of the respiratory rhythm and pattern.
doi:10.1016/S0079-6123(06)65013-9
PMCID: PMC2408750  PMID: 17925248
respiratory CPG; brainstem; medulla; pons; pre-Bötzinger complex; computational modeling; respiratory rhythm generation
4.  The role of spiking and bursting pacemakers in the neuronal control of breathing 
Journal of Biological Physics  2011;37(3):241-261.
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 (INaP) and the calcium-activated non-specific cation current (ICAN), 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.
doi:10.1007/s10867-011-9214-z
PMCID: PMC3101325  PMID: 22654176
Pacemaker; Respiration; Persistent sodium current; Calcium-activated non-specific cation current; preBötzinger complex; Rhythm generation; Bursting
5.  Substance P modulation of TRPC3/7 channels improves respiratory rhythm regularity and ICAN-dependent pacemaker activity 
The European journal of neuroscience  2010;31(7):1219-1232.
Neuromodulators, such as Substance P (SubP) play an important role in modulating many rhythmic activities driven by central pattern generators (e.g., locomotion, respiration). However, the mechanism by which SubP enhances breathing regularity has not been determined. Here, we used mouse brainstem slices containing the pre-Bötzinger Complex (Pre-BötC) to demonstrate, for the first time, that SubP activates transient receptor protein canonical (TRPC) channels to enhance respiratory rhythm regularity. Moreover, SubP enhancement of network regularity is accomplished via selective enhancement of ICAN-dependent intrinsic bursting properties. In contrast to INaP-dependant pacemakers, ICAN-dependant pacemaker bursting activity is TRPC dependent. Western Blots reveal TRPC3 and TRPC7 channels are expressed in rhythmically active ventral respiratory group (VRG) island preparations. Taken together, these data suggest that SubP-mediated activation of TRPC3/7 channels underlies rhythmic ICAN-dependent pacemaker activity and enhances the regularity of respiratory rhythm activity.
doi:10.1111/j.1460-9568.2010.07156.x
PMCID: PMC3036165  PMID: 20345918
6.  Spatial and Functional Architecture of the Mammalian Brain Stem Respiratory Network: A Hierarchy of Three Oscillatory Mechanisms 
Journal of neurophysiology  2007;98(6):3370-3387.
Mammalian central pattern generators (CPGs) producing rhythmic movements exhibit extremely robust and flexible behavior. Network architectures that enable these features are not well understood. Here we studied organization of the brain stem respiratory CPG. By sequential rostral to caudal transections through the pontine-medullary respiratory network within an in situ perfused rat brain stem–spinal cord preparation, we showed that network dynamics reorganized and new rhythmogenic mechanisms emerged. The normal three-phase respiratory rhythm transformed to a two-phase and then to a one-phase rhythm as the network was reduced. Expression of the three-phase rhythm required the presence of the pons, generation of the two-phase rhythm depended on the integrity of Bötzinger and pre-Bötzinger complexes and interactions between them, and the one-phase rhythm was generated within the pre-Bötzinger complex. Transformation from the three-phase to a two-phase pattern also occurred in intact preparations when chloride-mediated synaptic inhibition was reduced. In contrast to the three-phase and two-phase rhythms, the one-phase rhythm was abolished by blockade of persistent sodium current (INaP). A model of the respiratory network was developed to reproduce and explain these observations. The model incorporated interacting populations of respiratory neurons within spatially organized brain stem compartments. Our simulations reproduced the respiratory patterns recorded from intact and sequentially reduced preparations. Our results suggest that the three-phase and two-phase rhythms involve inhibitory network interactions, whereas the one-phase rhythm depends on INaP. We conclude that the respiratory network has rhythmogenic capabilities at multiple levels of network organization, allowing expression of motor patterns specific for various physiological and pathophysiological respiratory behaviors.
doi:10.1152/jn.00985.2007
PMCID: PMC2225347  PMID: 17913982
7.  Rhythmic Bursting in the Pre-Bötzinger Complex: Mechanisms and Models 
Progress in brain research  2014;209:1-23.
The pre-Bötzinger complex (pre-BötC), a neural structure involved in respiratory rhythm generation, can generate rhythmic bursting activity in vitro that persists after blockade of synaptic inhibition. Experimental studies have identified two mechanisms potentially involved in this activity: one based on the persistent sodium current (INaP) and the other involving calcium (ICa) and/or calcium-activated nonspecific cation (ICAN) currents. In this modeling study, we investigated bursting generated in single neurons and excitatory neural populations with randomly distributed conductances of INaP and ICa. We analyzed the possible roles of these currents, the Na+/K+ pump, synaptic mechanisms, and network interactions in rhythmic bursting generated under different conditions. We show that a population of synaptically coupled excitatory neurons with randomly distributed INaP- and/or ICAN-mediated burst generating mechanisms can operate in different oscillatory regimes with bursting dependent on either current or independent of both. The existence of multiple oscillatory regimes and their state dependence may explain rhythmic activities observed in the pre-BötC under different conditions.
doi:10.1016/B978-0-444-63274-6.00001-1
PMCID: PMC4059190  PMID: 24746040
neural oscillations; respiration; persistent sodium current; calcium-activated nonspecific cation current; sodium–potassium pump
8.  Identification of the pre‐Bötzinger complex inspiratory center in calibrated “sandwich” slices from newborn mice with fluorescent Dbx1 interneurons 
Physiological Reports  2014;2(8):e12111.
Abstract
Inspiratory active pre‐Bötzinger complex (preBötC) networks produce the neural rhythm that initiates and controls breathing movements. We previously identified the preBötC in the newborn rat brainstem and established anatomically defined transverse slices in which the preBötC remains active when exposed at one surface. This follow‐up study uses a neonatal mouse model in which the preBötC as well as a genetically defined class of respiratory interneurons can be identified and selectively targeted for physiological recordings. The population of glutamatergic interneurons whose precursors express the transcription factor Dbx1 putatively comprises the core respiratory rhythmogenic circuit. Here, we used intersectional mouse genetics to identify the brainstem distribution of Dbx1‐derived neurons in the context of observable respiratory marker structures. This reference brainstem atlas enabled online histology for generating calibrated sandwich slices to identify the preBötC location, which was heretofore unspecified for perinatal mice. Sensitivity to opioids ensured that slice rhythms originated from preBötC neurons and not parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) cells because opioids depress preBötC, but not pFRG/RTN rhythms. We found that the preBötC is centered ~0.4 mm caudal to the facial motor nucleus in this Cre/lox reporter mouse during postnatal days 0–4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imaging‐based studies, as well as the application of other Cre‐dependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratory‐related oscillations of preBötC Dbx1‐derived neurons and thus breathing.
Breathing movements emanate from Dbx1‐derived interneurons of the brainstem pre‐Bötzinger complex (preBötC). We generated a histology atlas of the medulla in newborn Dbx1 Cre/lox reporter mice and performed physiological tests to pinpoint the preBötC location and map the Dbx1 neuron distribution, which will facilitate neurobiological studies of respiratory rhythm generation.
doi:10.14814/phy2.12111
PMCID: PMC4246597  PMID: 25138790
Breathing; central pattern generator; respiration
9.  Differential Modulation of Neural Network and Pacemaker Activity Underlying Eupnea and Sigh-Breathing Activities 
Journal of neurophysiology  2008;99(5):10.1152/jn.01192.2007.
Many networks generate distinct rhythms with multiple frequency and amplitude characteristics. The respiratory network in the pre-Bötzinger complex (pre-Böt) generates both the low-frequency, large-amplitude sigh rhythm and a faster, smaller-amplitude eupneic rhythm. Could the same set of pacemakers generate both rhythms? Here we used an in vitro respiratory brainslice preparation. We describe a subset of synaptically isolated pacemakers that spontaneously generate two distinct bursting patterns. These two patterns resemble network activity including sigh-like bursts that occur at low frequencies and have large amplitudes and eupneic-like bursts with higher frequency and smaller amplitudes. Cholinergic neuromodulation altered the network and pacemaker bursting: fictive sigh frequency is increased dramatically, whereas fictive eupneic frequency is drastically lowered. The data suggest that timing and amplitude characteristics of fictive eupneic and sigh rhythms are set by the same set of pacemakers that are tuned by changes in the neuromodulatory state.
doi:10.1152/jn.01192.2007
PMCID: PMC3860370  PMID: 18287547
10.  Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre-Bötzinger complex: a computational modelling study 
The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre-Bötzinger complex (pre-BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre-BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na+ current (INaP), and the other involving the voltage-gated Ca2+ current (ICa) and the Ca2+-activated nonspecific cation current (ICAN), activated by intracellular Ca2+ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na+-dependent and Ca2+-dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with INaP and ICa randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca2+ release, and the Na+/K+ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on INaP and/or ICAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre-BötC and other brainstem/spinal cord circuits under different experimental conditions.
doi:10.1111/ejn.12042
PMCID: PMC3659238  PMID: 23121313
Ca2+-activated nonspecific cation current; Na+/K+ pump; neural oscillations; persistent Na+ current; respiration
11.  ROLE OF INHIBITION IN RESPIRATORY PATTERN GENERATION 
Postsynaptic inhibition is a key element of neural circuits underlying behavior, with 20-50% of all mammalian (non-granule) neurons considered inhibitory. For rhythmic movements in mammals, e.g., walking, swimming, suckling, chewing, breathing, inhibition is often hypothesized to play an essential rhythmogenic role. Here we study the role of fast synaptic inhibitory neurotransmission in the generation of breathing pattern by blocking GABAA and glycine receptors in the preBötzinger Complex (preBötC), a site essential for generation of normal breathing pattern, and in the neighboring Bötzinger Complex (BötC). The breathing rhythm continued following this blockade, but the lung inflation-induced Breuer-Hering inspiratory-inhibitory reflex was suppressed. The antagonists were efficacious, as this blockade abolished the profound effects of the exogenously applied GABAA receptor agonist muscimol or glycine, either of which under control conditions stopped breathing in vagus-intact or vagotomized, anesthetized, spontaneously breathing adult rats. In vagotomized rats, GABAAergic and glycinergic antagonists had little, if any, effect on rhythm. The effect in vagus intact rats was to slow the rhythm to a pace equivalent to that seen after suppression of the aforementioned Breuer-Hering inflation reflex. We conclude that postsynaptic inhibition within the preBötC and BötC is not essential for generation of normal respiratory rhythm in intact mammals. We suggest the primary role of inhibition is in shaping the pattern of respiratory motor output, assuring its stability, and in mediating reflex or volitional apnea, but not in the generation of rhythm per se.
doi:10.1523/JNEUROSCI.1595-12.2013
PMCID: PMC3724454  PMID: 23536061
breathing; respiratory rhythm; inhibition; preBötzinger Complex; Bötzinger Complex; Breuer-Hering reflex
12.  Dual oscillator model of the respiratory neuronal network generating quantal slowing of respiratory rhythm 
We developed a dual oscillator model to facilitate the understanding of dynamic interactions between the parafacial respiratory group (pFRG) and the preBötzinger complex (preBötC) neurons in the respiratory rhythm generation. Both neuronal groups were modeled as groups of 81 interconnected pacemaker neurons; the bursting cell model described by Butera and others [model 1 in Butera et al. (J Neurophysiol 81:382–397, 1999a)] were used to model the pacemaker neurons. We assumed (1) both pFRG and preBötC networks are rhythm generators, (2) preBötC receives excitatory inputs from pFRG, and pFRG receives inhibitory inputs from preBötC, and (3) persistent Na+ current conductance and synaptic current conductances are randomly distributed within each population. Our model could reproduce 1:1 coupling of bursting rhythms between pFRG and preBötC with the characteristic biphasic firing pattern of pFRG neurons, i.e., firings during pre-inspiratory and post-inspiratory phases. Compatible with experimental results, the model predicted the changes in firing pattern of pFRG neurons from biphasic expiratory to monophasic inspiratory, synchronous with preBötC neurons. Quantal slowing, a phenomena of prolonged respiratory period that jumps non-deterministically to integer multiples of the control period, was observed when the excitability of preBötC network decreased while strengths of synaptic connections between the two groups remained unchanged, suggesting that, in contrast to the earlier suggestions (Mellen et al., Neuron 37:821–826, 2003; Wittmeier et al., Proc Natl Acad Sci USA 105(46):18000–18005, 2008), quantal slowing could occur without suppressed or stochastic excitatory synaptic transmission. With a reduced excitability of preBötC network, the breakdown of synchronous bursting of preBötC neurons was predicted by simulation. We suggest that quantal slowing could result from a breakdown of synchronized bursting within the preBötC.
doi:10.1007/s10827-010-0249-0
PMCID: PMC3058346  PMID: 20544264
Rhythm coupling; Neuronal networks; Respiratory rhythm generation; Quantal slowing; Numerical simulation
13.  A Gradient in Endogenous Rhythmicity and Oscillatory Drive Matches Recruitment Order in an Axial Motor Pool 
The rhythmic firing behavior of spinal motoneurons is a function of their electrical properties and synaptic inputs. However, the relative contribution of endogenous versus network-based rhythmogenic mechanisms to locomotion is unclear. To address this issue, we have recorded from identified motoneurons and compared their current-evoked firing patterns to network-driven ones in the larval zebrafish (Danio rerio). Zebrafish axial motoneurons are recruited topographically from the bottom of the spinal cord up. Here, we have explored differences in the morphology of axial motoneurons, their electrical properties and their synaptic drive, to reveal how they match the topographic pattern of recruitment. More ventrally located ‘secondary’ motoneurons generate bursts of action potentials in response to constant current steps, demonstrating a strong inherent rhythmogenesis. The membrane potential oscillations underlying bursting behavior occur in the normal frequency range of swimming. In contrast, more dorsal secondaries chatter in response to current, while the most dorsally distributed ‘primary’ motoneurons all fire tonically. We find that systematic variations in excitability and endogenous rhythmicity are inversely related to the level of oscillatory synaptic drive within the entire axial motor pool. Specifically, bursting cells exhibit the least amount of drive while tonic cells exhibit the most. Our data suggest that increases in swimming frequency are accomplished by the recruitment of axial motoneurons that progressively rely on instructive synaptic drive to shape their oscillatory activity appropriately. Thus, within the zebrafish spinal cord there are differences in the relative contribution of endogenous versus network-based rhythms to locomotion and these vary predictably according to order of recruitment.
doi:10.1523/JNEUROSCI.1809-12.2012
PMCID: PMC3428065  PMID: 22875927
14.  Identifying Crucial Parameter Correlations Maintaining Bursting Activity 
PLoS Computational Biology  2014;10(6):e1003678.
Recent experimental and computational studies suggest that linearly correlated sets of parameters (intrinsic and synaptic properties of neurons) allow central pattern-generating networks to produce and maintain their rhythmic activity regardless of changing internal and external conditions. To determine the role of correlated conductances in the robust maintenance of functional bursting activity, we used our existing database of half-center oscillator (HCO) model instances of the leech heartbeat CPG. From the database, we identified functional activity groups of burster (isolated neuron) and half-center oscillator model instances and realistic subgroups of each that showed burst characteristics (principally period and spike frequency) similar to the animal. To find linear correlations among the conductance parameters maintaining functional leech bursting activity, we applied Principal Component Analysis (PCA) to each of these four groups. PCA identified a set of three maximal conductances (leak current, Leak; a persistent K current, K2; and of a persistent Na+ current, P) that correlate linearly for the two groups of burster instances but not for the HCO groups. Visualizations of HCO instances in a reduced space suggested that there might be non-linear relationships between these parameters for these instances. Experimental studies have shown that period is a key attribute influenced by modulatory inputs and temperature variations in heart interneurons. Thus, we explored the sensitivity of period to changes in maximal conductances of Leak, K2, and P, and we found that for our realistic bursters the effect of these parameters on period could not be assessed because when varied individually bursting activity was not maintained.
Author Summary
Central pattern-generating networks (CPGs) must be remarkably robust, maintaining functional rhythmic activity despite fluctuations in internal and external conditions. Recent experimental evidence suggests that this robustness is achieved by the coordinated regulation of many membrane and synaptic current parameters. Experimental and computational studies showed that linearly correlated sets of such parameters allow CPG neurons to produce and maintain their rhythmic activity. However, the mechanisms that allow multiple parameters to interact, thereby producing and maintaining rhythmic single cell and network activity, are not clear. Here, we use a half-center oscillator (HCO) model that replicates the electrical activity (rhythmic alternating bursting of mutually inhibitory interneurons) of the leech heartbeat CPG to investigate potential relationships between parameters that maintain functional bursting activity in the HCOs and the isolated component neurons (bursters). We found a linearly correlated set of three maximal conductances that maintains functional bursting activity similar to the animal in burster model instances, therefore increasing robustness of bursting activity. In addition, we found that bursting activity was very sensitive to individual variation of these parameters; only correlated changes could maintain the activity type.
doi:10.1371/journal.pcbi.1003678
PMCID: PMC4063674  PMID: 24945358
15.  General Principles of Rhythmogenesis in Central Pattern Networks 
Progress in brain research  2010;187:213-222.
The cellular and ionic mechanisms that generate the rhythm in central pattern generator (CPG) networks for simple movements are not well understood. Using vertebrate locomotion, respiration and mastication as exemplars, I describe four main principles of rhythmogenesis: (1) rhythmogenic ionic currents underlie all CPG networks, regardless of whether they are driven by a network pacemaker or an endogenous pacemaker neuron kernel; (2) fast synaptic transmission often evokes slow currents that can affect cycle frequency; (3) there are likely to be multiple and redundant mechanisms for rhythmogenesis in any essential CPG network; and (4) glial cells may participate in CPG network function.
The neural basis for rhythmogenesis in simple behaviors has been studied for almost 100 years, yet we cannot identify with certainty the detailed mechanisms by which rhythmic behaviors are generated in any vertebrate system. Early studies focused on whether locomotor rhythms were generated by a chain of coupled reflexes that require sensory feedback, or by a central neural network. By now there is general agreement that for the major rhythmic behaviors (including locomotion, respiration, and mastication, the subjects of this book), there exist CPG networks within the central nervous system that are able to drive the basic rhythmic behavior in the complete absence of sensory feedback. This of course does not eliminate an important role for sensory feedback, which certainly affects cycle frequency and for some behaviors determines the timing of one phase of the behavior. Given the existence of CPGs, the question of rhythmogenesis can be rephrased to ask how these networks determine the timing of the rhythmic behavior. In this chapter, I focus on cellular and molecular mechanisms that could underlie rhythmogenesis in CPG networks, especially those that drive locomotion, respiration, and mastication.
doi:10.1016/B978-0-444-53613-6.00014-9
PMCID: PMC3005202  PMID: 21111210
Central Pattern Generator; rhythmogenesis; bursting; modulation; ion channel; receptor; calcium
16.  Growth Dynamics Explain the Development of Spatiotemporal Burst Activity of Young Cultured Neuronal Networks in Detail 
PLoS ONE  2012;7(9):e43352.
A typical property of isolated cultured neuronal networks of dissociated rat cortical cells is synchronized spiking, called bursting, starting about one week after plating, when the dissociated cells have sufficiently sent out their neurites and formed enough synaptic connections. This paper is the third in a series of three on simulation models of cultured networks. Our two previous studies [26], [27] have shown that random recurrent network activity models generate intra- and inter-bursting patterns similar to experimental data. The networks were noise or pacemaker-driven and had Izhikevich-neuronal elements with only short-term plastic (STP) synapses (so, no long-term potentiation, LTP, or depression, LTD, was included). However, elevated pre-phases (burst leaders) and after-phases of burst main shapes, that usually arise during the development of the network, were not yet simulated in sufficient detail. This lack of detail may be due to the fact that the random models completely missed network topology .and a growth model. Therefore, the present paper adds, for the first time, a growth model to the activity model, to give the network a time dependent topology and to explain burst shapes in more detail. Again, without LTP or LTD mechanisms. The integrated growth-activity model yielded realistic bursting patterns. The automatic adjustment of various mutually interdependent network parameters is one of the major advantages of our current approach. Spatio-temporal bursting activity was validated against experiment. Depending on network size, wave reverberation mechanisms were seen along the network boundaries, which may explain the generation of phases of elevated firing before and after the main phase of the burst shape.In summary, the results show that adding topology and growth explain burst shapes in great detail and suggest that young networks still lack/do not need LTP or LTD mechanisms.
doi:10.1371/journal.pone.0043352
PMCID: PMC3447003  PMID: 23028450
17.  Computational Models and Emergent Properties of Respiratory Neural Networks 
Comprehensive Physiology  2012;2(3):1619-1670.
Computational models of the neural control system for breathing in mammals provide a theoretical and computational framework bringing together experimental data obtained from different animal preparations under various experimental conditions. Many of these models were developed in parallel and iteratively with experimental studies and provided predictions guiding new experiments. This data-driven modeling approach has advanced our understanding of respiratory network architecture and neural mechanisms underlying generation of the respiratory rhythm and pattern, including their functional reorganization under different physiological conditions. Models reviewed here vary in neurobiological details and computational complexity and span multiple spatiotemporal scales of respiratory control mechanisms. Recent models describe interacting populations of respiratory neurons spatially distributed within the Bötzinger and pre-Bötzinger complexes and rostral ventrolateral medulla that contain core circuits of the respiratory central pattern generator (CPG). Network interactions within these circuits along with intrinsic rhythmogenic properties of neurons form a hierarchy of multiple rhythm generation mechanisms. The functional expression of these mechanisms is controlled by input drives from other brainstem components, including the retrotrapezoid nucleus and pons, which regulate the dynamic behavior of the core circuitry. The emerging view is that the brainstem respiratory network has rhythmogenic capabilities at multiple levels of circuit organization. This allows flexible, state-dependent expression of different neural pattern-generation mechanisms under various physiological conditions, enabling a wide repertoire of respiratory behaviors. Some models consider control of the respiratory CPG by pulmonary feedback and network reconfiguration during defensive behaviors such as cough. Future directions in modeling of the respiratory CPG are considered.
doi:10.1002/cphy.c110016
PMCID: PMC3656479  PMID: 23687564
18.  Precise Temperature Compensation of Phase in a Rhythmic Motor Pattern 
PLoS Biology  2010;8(8):e1000469.
Computational modeling and experimentation in a model system for network dynamics reveal how network phase relationships are temperature-compensated in terms of their underlying synaptic and intrinsic membrane currents.
Most animal species are cold-blooded, and their neuronal circuits must maintain function despite environmental temperature fluctuations. The central pattern generating circuits that produce rhythmic motor patterns depend on the orderly activation of circuit neurons. We describe the effects of temperature on the pyloric rhythm of the stomatogastric ganglion of the crab, Cancer borealis. The pyloric rhythm is a triphasic motor pattern in which the Pyloric Dilator (PD), Lateral Pyloric (LP), and Pyloric (PY) neurons fire in a repeating sequence. While the frequency of the pyloric rhythm increased about 4-fold (Q10∼2.3) as the temperature was shifted from 7°C to 23°C, the phase relationships of the PD, LP, and PY neurons showed almost perfect temperature compensation. The Q10's of the input conductance, synaptic currents, transient outward current (IA), and the hyperpolarization-activated inward current (Ih), all of which help determine the phase of LP neuron activity, ranged from 1.8 to 4. We studied the effects of temperature in >1,000 computational models (with different sets of maximal conductances) of a bursting neuron and the LP neuron. Many bursting models failed to monotonically increase in frequency as temperature increased. Temperature compensation of LP neuron phase was facilitated when model neurons' currents had Q10's close to 2. Together, these data indicate that although diverse sets of maximal conductances may be found in identified neurons across animals, there may be strong evolutionary pressure to restrict the Q10's of the processes that contribute to temperature compensation of neuronal circuits.
Author Summary
The neural circuits that produce behaviors such as walking, chewing, and swimming must be both robust and flexible to changing internal and environmental demands. How then do cold-blooded animals cope with temperature fluctuations when the underlying processes that give rise to circuit performance are themselves temperature-dependent? We exploit the crab stomatogastric ganglion to understand the extent to which circuit features are robust to temperature perturbations. We subjected these circuits to temperature ranges they normally encounter in the wild. Interestingly, while the frequency of activity in the network increased 4-fold over these temperature ranges, the relative timing between neurons in the network—termed phase relationships—remained constant. To understand how temperature compensation of phase might occur, we characterized the temperature dependence (Q10's) of synapses and membrane currents. We used computational models to show that the experimentally measured Q10's can promote phase maintenance. We also showed that many model bursting neurons fail to burst over the entire temperature range and that phase maintenance is promoted by closely restricting the model neurons' Q10's. These results imply that although ion channel numbers can vary between individuals, there may be strong evolutionary pressure that restricts the temperature dependence of the processes that contribute to temperature compensation of neuronal circuits.
doi:10.1371/journal.pbio.1000469
PMCID: PMC2930868  PMID: 20824168
19.  Structural and functional architecture of respiratory networks in the mammalian brainstem 
Neural circuits controlling breathing in mammals are organized within serially arrayed and functionally interacting brainstem compartments extending from the pons to the lower medulla. The core circuit components that constitute the neural machinery for generating respiratory rhythm and shaping inspiratory and expiratory motor patterns are distributed among three adjacent structural compartments in the ventrolateral medulla: the Bötzinger complex (BötC), pre-Bötzinger complex (pre-BötC) and rostral ventral respiratory group (rVRG). The respiratory rhythm and inspiratory–expiratory patterns emerge from dynamic interactions between: (i) excitatory neuron populations in the pre-BötC and rVRG active during inspiration that form inspiratory motor output; (ii) inhibitory neuron populations in the pre-BötC that provide inspiratory inhibition within the network; and (iii) inhibitory populations in the BötC active during expiration that generate expiratory inhibition. Network interactions within these compartments along with intrinsic rhythmogenic properties of pre-BötC neurons form a hierarchy of multiple oscillatory mechanisms. The functional expression of these mechanisms is controlled by multiple drives from more rostral brainstem components, including the retrotrapezoid nucleus and pons, which regulate the dynamic behaviour of the core circuitry. The emerging view is that the brainstem respiratory network has rhythmogenic capabilities at multiple hierarchical levels, which allows flexible, state-dependent expression of different rhythmogenic mechanisms under different physiological and metabolic conditions and enables a wide repertoire of respiratory behaviours.
doi:10.1098/rstb.2009.0081
PMCID: PMC2865112  PMID: 19651658
breathing; brainstem; respiratory rhythm and pattern generation; pons; pre-Bötzinger complex; ventral respiratory column
20.  Membrane resonance in bursting pacemaker neurons of an oscillatory network is correlated with network frequency 
Network oscillations typically span a limited range of frequency. In pacemaker-driven networks, including many Central Pattern Generators (CPGs), this frequency range is determined by the properties of bursting pacemaker neurons and their synaptic connections; thus, factors that affect the burst frequency of pacemaker neurons should play a role in determining the network frequency. We examine the role of membrane resonance of pacemaker neurons on the network frequency in the crab pyloric CPG. The pyloric oscillations (freq ~1 Hz) are generated by a group of pacemaker neurons: the Anterior Burster (AB) and the Pyloric Dilator (PD). We examine the impedance profiles of the AB and PD neurons in response to sinusoidal current injections with varying frequency and find that both neuron types exhibit membrane resonance, i.e. demonstrate maximal impedance at a given preferred frequency. The membrane resonance frequencies of the AB and PD neurons fall within the range of the pyloric network oscillation frequency. Experiments with pharmacological blockers and computational modeling show that both calcium currents ICa and the hyperpolarization-activated inward current Ih, are important in producing the membrane resonance in these neurons. We then demonstrate that both the membrane resonance frequency of the PD neuron and its supra-threshold bursting frequency can be shifted in the same direction by either DC current injection or by using the dynamic clamp technique to inject artificial conductances for Ih or ICa. Together, these results suggest that membrane resonance of pacemaker neurons can be strongly correlated with the CPG oscillation frequency.
doi:10.1523/JNEUROSCI.0545-09.2009
PMCID: PMC2716082  PMID: 19458214
Oscillation; central pattern generator; resonance; stomatogastric; model; Ih
21.  Spontaneous rhythmogenic capabilities of sympathetic neuronal assemblies in the rat spinal cord slice 
Neuroscience  2010;170(3-2):827-838.
Neuronal networks generating rhythmic activity as an emergent property are common throughout the nervous system. Some are responsible for rhythmic behaviours, as is the case for the spinal cord locomotor networks; however, for others the function is more subtle and usually involves information processing and/or transfer. An example of the latter is sympathetic nerve activity, which is synchronized into rhythmic bursts in vivo. This arrangement is postulated to offer improved control of target organ responses compared to tonic nerve activity. Traditionally, oscillogenic circuits in the brainstem are credited with generating these rhythms, despite evidence for the persistence of some frequencies in spinalized preparations. Here, we show that rhythmic population activity can be recorded from the intermediolateral cell column (IML) of thoracic spinal cord slices. Recorded in slices from 10- to 12-day-old rats, this activity was manifest as 8–22 Hz oscillations in the field potential and was spatially restricted to the IML. Oscillations often occurred spontaneously, but could also be induced by application of 5-HT, α-methyl 5-HT or MK212. These agents also significantly increased the strength of spontaneous oscillations. Rhythmic activity was abolished by TTX and attenuated by application of gap junction blockers or by antagonists of GABAA receptors. Together these data indicate that this rhythm is an emergent feature of a population of spinal neurons coupled by gap junctions. This work questions the assumption that sympathetic rhythms are dependent on supraspinal pacemaker circuits, by highlighting a surprisingly strong rhythmogenic capability of the reduced sympathetic networks of the spinal cord slice.
doi:10.1016/j.neuroscience.2010.07.007
PMCID: PMC2989444  PMID: 20650307
oscillation; sympathetic nervous system; serotonin; spontaneous activity; gap junctions; GABA; aCSF, artificial cerebrospinal fluid; Cx36, connexin 36; GABA, gamma amino butyric acid; Hb9, homeobox gene 9; HERG, human Ether-a-go-go Related Gene; mRNA, messenger RNA; IML, intermediolateral cell column; IQ, interquartile; SEM, standard error of the mean; SPNs, sympathetic preganglionic neurons
22.  Distinct inspiratory rhythm and pattern generating mechanisms in the preBötzinger Complex 
In the mammalian respiratory central pattern generator, the preBötzinger Complex (preBötC) produces rhythmic bursts that drive inspiratory motor output. Cellular mechanisms initiated by each burst are hypothesized to be necessary to determine the timing of the subsequent burst, playing a critical role in rhythmogenesis. To explore mechanisms relating inspiratory burst generation to rhythmogenesis, we compared preBötC and hypoglossal (XII) nerve motor activity in medullary slices from neonatal mice in conditions where periods between successive inspiratory XII bursts were highly variable and distributed multimodally. This pattern resulted from rhythmic preBötC neural population activity that consisted of bursts, concurrent with XII bursts, intermingled with significantly smaller “burstlets”. Burstlets occurred at regular intervals during significantly longer XII interburst intervals, at times when a XII burst was expected. When a preBötC burst occurred, its high amplitude inspiratory component (I-burst) was preceded by a preinspiratory component that closely resembled the rising phase of burstlets. Cadmium (8 μM) eliminated preBötC and XII bursts, but rhythmic preBötC burstlets persisted. Burstlets and preinspiratory activity were observed in ~90% of preBötC neurons that were active during I-bursts. When preBötC excitability was raised significantly, burstlets could leak through to motor output in medullary slices and in vivo in adult anesthetized rats. Thus, rhythmic bursting, a fundamental mode of nervous system activity and an essential element of breathing, can be deconstructed into a rhythmogenic process producing low amplitude burstlets and preinspiratory activity that determine timing, and a pattern-generating process producing suprathreshold I-bursts essential for motor output.
doi:10.1523/JNEUROSCI.4143-12.2013
PMCID: PMC3737080  PMID: 23719793
23.  Emergence of Noise-Induced Oscillations in the Central Circadian Pacemaker 
PLoS Biology  2010;8(10):e1000513.
Computational modeling and experimentation explain how intercellular coupling and intracellular noise can generate oscillations in a mammalian neuronal network even in the absence of cell-autonomous oscillators.
Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN) of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of noise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization.
Author Summary
The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals that controls and coordinates physiological processes in a daily manner. The SCN is composed of a network of cells, with each cell acting as an autonomous oscillator. In isolated individual cells, timekeeping is not precise because of the inherent randomness in the biochemical reactions within each cell, involving its core clock components. However, in the SCN network, precise rhythms can emerge because of intercellular coupling. In this article, we study a loss-of-function mutation of BMAL1, a core clock component, which eliminates timekeeping in isolated cells. Surprisingly, in both experiments and mathematical simulations, we find that noisy rhythms emerge from the SCN network even in the presence of this BMAL1 mutation. This random yet coordinated timekeeping has not been observed in previous modeling and experimental work and indicates that a network of cells can utilize noise to help compensate for loss of a physiological function. In normal function, the SCN network mitigates any variability observed in individual cellular rhythms and produces a precise and rhythmic network timekeeping signal. When the individual cells are no longer rhythmic, the coupling pathways within the SCN network can propagate stochastic rhythms that are a reflection of both feed-forward coupling mechanisms and intracellular noise. Thus, in a manner analogous to central pattern generators in neural circuits, rhythmicity can arise as an emergent property of the network in the absence of component pacemaker or oscillator cells.
doi:10.1371/journal.pbio.1000513
PMCID: PMC2953532  PMID: 20967239
24.  A Sodium Leak Current Regulates Pacemaker Activity of Adult Central Pattern Generator Neurons in Lymnaea Stagnalis 
PLoS ONE  2011;6(4):e18745.
The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na+ (ILeak-Na). The ILeak-Na contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na+ leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.
doi:10.1371/journal.pone.0018745
PMCID: PMC3079709  PMID: 21526173
25.  A Multiscale Model to Investigate Circadian Rhythmicity of Pacemaker Neurons in the Suprachiasmatic Nucleus 
PLoS Computational Biology  2010;6(3):e1000706.
The suprachiasmatic nucleus (SCN) of the hypothalamus is a multicellular system that drives daily rhythms in mammalian behavior and physiology. Although the gene regulatory network that produces daily oscillations within individual neurons is well characterized, less is known about the electrophysiology of the SCN cells and how firing rate correlates with circadian gene expression. We developed a firing rate code model to incorporate known electrophysiological properties of SCN pacemaker cells, including circadian dependent changes in membrane voltage and ion conductances. Calcium dynamics were included in the model as the putative link between electrical firing and gene expression. Individual ion currents exhibited oscillatory patterns matching experimental data both in current levels and phase relationships. VIP and GABA neurotransmitters, which encode synaptic signals across the SCN, were found to play critical roles in daily oscillations of membrane excitability and gene expression. Blocking various mechanisms of intracellular calcium accumulation by simulated pharmacological agents (nimodipine, IP3- and ryanodine-blockers) reproduced experimentally observed trends in firing rate dynamics and core-clock gene transcription. The intracellular calcium concentration was shown to regulate diverse circadian processes such as firing frequency, gene expression and system periodicity. The model predicted a direct relationship between firing frequency and gene expression amplitudes, demonstrated the importance of intracellular pathways for single cell behavior and provided a novel multiscale framework which captured characteristics of the SCN at both the electrophysiological and gene regulatory levels.
Author Summary
Circadian rhythms are ∼24 hour cycles in biochemical, physiological and behavioral processes observed in a diverse range of organisms including Cyanobacteria, Neurospora, Drosophila, mice and humans. In mammals, the dominant circadian pacemaker that drives daily rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is composed of a highly connected network of ∼20,000 neurons. Within each individual SCN neuron core clock genes and proteins interact through intertwined regulatory loops to generate circadian oscillations on the molecular level. These neurons express daily rhythmicity in their firing frequency and other electrophysiological properties. The mechanisms by which the core clock produces synchronized rhythms in neural firing and gene expression are postulated to involve intracellular calcium, a second messenger that regulates many cellular processes. The interaction between the various clock components however remains unknown. In this paper, we present a single cell model that incorporates the circadian gene regulatory pathway, cellular electrophysiological properties, and cytosolic calcium dynamics. Our results suggest a possible system architecture that accounts for the robustness of the circadian clock at the single cell level. Our simulations predict a dual role for intracellular pathways instigated by intracellular calcium and VIP: maintaining the periodicity and amplitude of the core clock genes as well as the firing frequency oscillations.
doi:10.1371/journal.pcbi.1000706
PMCID: PMC2837390  PMID: 20300645

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