Hypoxia evokes a sequence of raphe-pontomedullary network operations for inspiratory drive amplification and gasping.

Hypoxia can trigger a sequence of breathing-related behaviors, from tachypnea to apneusis to apnea and gasping, an autoresuscitative behavior that, via large tidal volumes and altered intrathoracic pressure, can enhance coronary perfusion, carotid blood flow, and sympathetic activity, and thereby coordinate cardiac and respiratory functions. We tested the hypothesis that hypoxia-evoked gasps are amplified through a disinhibitory microcircuit within the inspiratory neuron chain and a distributed efference copy mechanism that generates coordinated gasp-like discharges concurrently in other circuits of the raphe-pontomedullary respiratory network. Data were obtained from 6 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated adult cats. Arterial blood pressure, phrenic nerve activity, end-tidal CO2, and other parameters were monitored. Hypoxia was produced by ventilation with a gas mixture of 5% O2 in nitrogen (N2). Neuron spike trains were recorded at multiple pontomedullary sites simultaneously and evaluated for firing rate modulations and short-time scale correlations indicative of functional connectivity. Experimental perturbations evoked reconfiguration of raphe-pontomedullary circuits during tachypnea, apneusis and augmented bursts, apnea, and gasping. The functional connectivity, altered firing rates, efference copy of gasp drive, and coordinated step increments in blood pressure reported here support a distributed brain stem network model for amplification and broadcasting of inspiratory drive during autoresuscitative gasping that begins with a reduction in inhibition by expiratory neurons and an initial loss of inspiratory drive during hypoxic apnea.

Initiating daily acute intermittent hypoxia (dAIH) therapy at 1-week after contusion spinal cord injury (SCI) improves lower urinary tract function in rat.

Spinal cord injury (SCI) above the level of the lumbosacral spinal cord produces lower urinary tract (LUT) dysfunction, resulting in impairment of urine storage and elimination (voiding). While spontaneous functional recovery occurs due to remodeling of spinal reflex micturition pathways, it is incomplete, indicating that additional strategies to further augment neural plasticity following SCI are essential. To this end, acute intermittent hypoxia (AIH) exposure has been proposed as a therapeutic strategy for improving recovery of respiratory and other somatic motor function following SCI; however, the impact of AIH as a therapeutic intervention to improve LUT dysfunction remains to be determined. Therefore, we examined the effects of daily AIH (dAIH) on both spontaneous micturition patterns and reflex micturition event (rME) behaviors in adult female Sprague-Dawley rats with mid-thoracic moderate contusion SCI. For these experiments, dAIH gas exposures (five alternating 3 min 12% O2 and 21% O2 episodes) were delivered for 7 consecutive days beginning at 1-week after SCI, with awake micturition patterns being evaluated weekly for 2-3 sessions before and for 4 weeks after SCI and rME behaviors elicited by continuous infusion of saline into the bladder being evaluated under urethane anesthesia at 4-weeks after SCI; daily normoxia (dNx; 21% O2 episodes) served as a control. At 1-week post-SCI, both an areflexic phenotype (i.e., no effective voiding events) and a functional voiding phenotype (i.e., infrequent voiding events with large volumes) were observed in spontaneous micturition patterns (as expected), and subsequent dAIH, but not dNx, treatment led to recovery of spontaneous void frequency pattern to pre-SCI levels; both dAIH- and dNx-treated rats exhibited slightly increased void volumes. At 4-weeks post-SCI, rME behaviors showed increased effectiveness in voiding in dAIH-treated (compared to dNx-treated) rats that included an increase in both bladder contraction pressure (delta BP; P = 0.014) and dynamic voiding efficiency (P = 0.018). Based on the voiding and non-voiding bladder contraction behaviors (VC and NVC, respectively) observed in the BP records, bladder dysfunction severity was classified into mild, moderate, and severe phenotypes, and while rats in both treatment groups included each severity phenotype, the primary phenotype observed in dAIH-treated rats was mild and that in dNx-treated rats was moderate (P = 0.044). Taken together, these findings suggest that 7-day dAIH treatment produces beneficial improvements in LUT function that include recovery of micturition pattern, more efficient voiding, and decreased NVCs, and extend support to the use of dAIH therapy to treat SCI-induced LUT dysfunction.

Effects of calcium (Ca(2+)) extrusion mechanisms on electrophysiological properties in a hypoglossal motoneuron: insight from a mathematical model.

Spike-frequency dynamics and spike shape can provide insight into the types of ion channels present in any given neuron and give a sense for the precise response any neuron may have to a given input stimulus. Motoneuron firing frequency over time is especially important due to its direct effect on motor output. Of particular interest is intracellular Ca(2+), which exerts a powerful influence on both firing properties over time and spike shape. In order to better understand the cellular mechanisms for the regulation of intracellular Ca(2+) and their effect on spiking behavior, we have modified a computational model of an HM to include a variety of Ca(2+) handling processes. For the current study, a series of HM models that include Ca(2+) pumps, Na(+)/Ca(2+) exchangers, and a generic exponential decay of excess Ca(2+) were generated. Simulations from these models indicate that although each extrusion mechanism exerts a similar effect on voltage, the firing properties change distinctly with the inclusion of additional Ca(2+)-related mechanisms: BK channels, Ca(2+) buffering, and diffusion of [Ca(2+)]i modeled via a linear diffusion partial differential equation. While an exponential decay of Ca(2+) seems to adequately capture short-term changes in firing frequency seen in biological data, internal diffusion of Ca(2+) appears to be necessary for capturing longer term frequency changes.

Using a computational model to analyze the effects of firing frequency on synchrony of a network of gap junction-coupled hypoglossal motoneurons.

Hypoglossal motoneurons (HMs) are located in the brainstem and play an important role in the maintenance of upper airway patency. HMs are known to be coupled to one another via gap junctions and exhibit synchronous firing behavior when driven by premotor inputs. In the current study, we used a computational model to analyze the influence of firing frequency on synchronous firing behavior of a network of gap junction-coupled HMs. As there are many factors that can influence excitability and firing frequency of HMs, our simulations were focused on the effects of modulation of SK channel conductance and the magnitude of the input currents as a means of modifying firing frequency. Our simulations revealed that regardless of the mechanism by which firing frequency was modulated, increasing the firing frequency disrupted the synchrony in gap junction-coupled HM networks with low, medium, and high levels of gap junction coupling.

Intermittent hypoxia-induced respiratory long-term facilitation is dominated by enhanced burst frequency, not amplitude, in spontaneously breathing urethane-anesthetized neonatal rats.

Acute intermittent hypoxia (AIH) triggers a form of respiratory plasticity known as long-term facilitation (LTF), which is manifested as a progressive increase in respiratory motor activity that lasts for minutes to hours after the hypoxic stimulus is removed. Respiratory LTF has been reported in numerous animal models, but it appears to be influenced by a variety of factors (e.g., species, age, and gender). While most studies focusing on respiratory LTF have been conducted in adult (including young adult) rat preparations, little is known about the influence of postnatal maturation on AIH-induced respiratory LTF. To begin to address this issue, we examined diaphragm EMG activity in response to and at 5-min intervals for 60 min following three 5-min episodes of hypoxia (8% O2) in urethane-anesthetized spontaneously breathing P14-P15 neonatal rats (n=15). For these experiments, the hypoxic episodes were separated by hyperoxia (40% O2), and all rats were continuously supplied with ~4% CO2. During the AIH trials, burst frequency was increased by ~20-90% above baseline in each of the rats examined while changes in burst amplitude were highly variable. Following the AIH episodes, respiratory LTF was characterized by predominantly an increase in burst frequency (fLTF) ranging from ~10% to 55%, with most rats exhibiting a 20-40% increase. In seven rats, however, an increase in amplitude (ampLTF) (~10%, n=3; ~20%, n=3; ~30%, n=1) was also noted. These data suggest that in contrast to observations in anesthetized ventilated adult rats, in anesthetized spontaneously breathing P14-P15 neonatal rats, respiratory LTF is dominated by fLTF, not ampLTF.

Application of a "staggered walk" algorithm for generating large-scale morphological neuronal networks.

Large-scale models of neuronal structures are needed to explore emergent properties of mammalian brains. Because these models have trillions of synapses, a major problem in their creation is synapse placement. Here we present a novel method for exploiting consistent fiber orientation in a neural tissue to perform a highly efficient modified plane-sweep algorithm, which identifies all regions of 3D overlaps between dendritic and axonal projection fields. The first step in placing synapses in physiological models is neurite-overlap detection, at large scales a computationally intensive task. We have developed an efficient "Staggered Walk" algorithm that can find all 3D overlaps of neurites where trillions of synapses connect billions of neurons.

Chronic serotonin-norepinephrine reuptake transporter inhibition modifies basal respiratory output in adult mouse in vitro and in vivo.

Respiratory disturbances are a common feature of panic disorder and present as breathing irregularity, hyperventilation, and increased sensitivity to carbon dioxide. Common therapeutic interventions, such as tricyclic (TCA) and selective serotonin reuptake inhibitor (SSRI) antidepressants, have been shown to ameliorate not only the psychological components of panic disorder but also the respiratory disturbances. These drugs are also prescribed for generalized anxiety and depressive disorders, neither of which are characterized by respiratory disturbances, and previous studies have demonstrated that TCAs and SSRIs exert effects on basal respiratory activity in animal models without panic disorder symptoms. Whether serotonin-norepinephrine reuptake inhibitors (SNRIs) have similar effects on respiratory activity remains to be determined. Therefore, the current study was designed to investigate the effects of chronic administration of the SNRI antidepressant venlafaxine (VHCL) on basal respiratory output. For these experiments, we recorded phrenic nerve discharge in an in vitro arterially-perfused adult mouse preparation and diaphragm electromyogram (EMG) activity in an in vivo urethane-anesthetized adult mouse preparation. We found that following 28-d VHCL administration, basal respiratory burst frequency was markedly reduced due to an increase in expiratory duration (T(E)), and the inspiratory duty cycle (T(I)/T(tot)) was significantly shortened. In addition, post-inspiratory and spurious expiratory discharges were seen in vitro. Based on our observations, we suggest that drugs capable of simultaneously blocking both 5-HT and NE reuptake transporters have the potential to influence the respiratory control network in patients using SNRI therapy.

Emergent central pattern generator behavior in gap-junction-coupled Hodgkin-Huxley style neuron model.

Most models of central pattern generators (CPGs) involve two distinct nuclei mutually inhibiting one another via synapses. Here, we present a single-nucleus model of biologically realistic Hodgkin-Huxley neurons with random gap junction coupling. Despite no explicit division of neurons into two groups, we observe a spontaneous division of neurons into two distinct firing groups. In addition, we also demonstrate this phenomenon in a simplified version of the model, highlighting the importance of afterhyperpolarization currents (I(AHP)) to CPGs utilizing gap junction coupling. The properties of these CPGs also appear sensitive to gap junction conductance, probability of gap junction coupling between cells, topology of gap junction coupling, and, to a lesser extent, input current into our simulated nucleus.

Analyzing the effects of gap junction blockade on neural synchrony via a motoneuron network computational model.

In specific regions of the central nervous system (CNS), gap junctions have been shown to participate in neuronal synchrony. Amongst the CNS regions identified, some populations of brainstem motoneurons are known to be coupled by gap junctions. The application of various gap junction blockers to these motoneuron populations, however, has led to mixed results regarding their synchronous firing behavior, with some studies reporting a decrease in synchrony while others surprisingly find an increase in synchrony. To address this discrepancy, we employ a neuronal network model of Hodgkin-Huxley-style motoneurons connected by gap junctions. Using this model, we implement a series of simulations and rigorously analyze their outcome, including the calculation of a measure of neuronal synchrony. Our simulations demonstrate that under specific conditions, uncoupling of gap junctions is capable of producing either a decrease or an increase in neuronal synchrony. Subsequently, these simulations provide mechanistic insight into these different outcomes.

Geometrical analysis of bursting pacemaker neurons generated by computational models: comparison to in vitro pre-Bötzinger complex bursting neurons.

Despite an incredible amount of progress toward understanding respiratory rhythm generation through the use of reduced mathematical models, controversy exists concerning the role of various ionic conductances in generating bursting behavior. Moreover, the dynamical behavior of these model neurons has not been examined. Here, we have used two well described pre-Bötzinger complex (pre-BötC) pacemaker neuron models to investigate their dynamical features. Based on our observations, we identify one of the models to better represent the general characteristics of the bursting dynamics and propose additional modifications to better capture the dynamics of our own experimental recordings.

Influence of 5-HT2A receptor blockade on phrenic nerve discharge at three levels of extracellular K+ in arterially-perfused adult rat.

Recent observations from in vitro rodent preparations suggest an important role for the serotonin-2A (5-HT(2A)) receptor in eupneic (basal) and gasping respiratory activities, although the precise role appears to be different in different preparations. Since these in vitro preparations are typically supplied with elevated (and different) levels of K(+) to increase neuronal excitability, the role of endogenous activation of 5-HT(2A) receptors in these respiratory behaviors under "normal" levels of extracellular K(+) ([K(+)](o)) requires clarification. The current study sought to evaluate the influence of [K(+)](o) on the 5-HT(2A) receptor-mediated effects on basal respiratory activity and the phases of the hypoxic ventilatory response (HVR), including ischemia-induced gasping in an arterially-perfused adult rat preparation. Our data demonstrate that at each level of [K(+)](o) examined, blockade of 5-HT(2A) receptors increases basal phrenic burst frequency, decreases basal phrenic burst amplitude, alters basal phrenic burst pattern, and eliminates the phases of the HVR. These data support an important role for 5-HT(2A) receptors in respiratory control, and indicate the their role is not dependent on the level of [K(+)](o).

Upper airway and abdominal motor output during sneezing: is the in vivo decererate rat an adequate model?

While numerous studies have focused on identifying and characterizing the neural mechanisms mediating upper airway defense reflexes in the anesthetized or decerebrate adult cat, little is known about these behaviors in in vivo rodent models. The current study was undertaken to investigate whether the in vivo decelerate adult rat might serve as an acceptable model for studying these behaviors. To begin to address this possibility, we examined multiple respiratory motor activities in response to mechanical stimulation of the anterior nasal cavity (sufficient to elicit fictive sneezing) in in vivo decerebrate adult rats. We found that the neural activities observed during nasal stimulation were consistent with those previously reported during fictive sneezing in the adult cat model. We suggest that the in vivo decerebrate rat is an acceptable model for studying the sneezing reflex.

Influence of extracellular [K+]o on inspiratory network complexity of phrenic and hypoglossal nerve discharge in arterially-perfused adult rat.

Many in vitro mammalian preparations are used to study multiple aspects of central respiratory control. In these preparations, recordings of respiratory-related outputs that range from individual and population neuronal activities to hypoglossal (XII) nerve output to phrenic (PHR) nerve discharge commonly are used. These reduced preparations typically are supplied with an artificial cerebral spinal fluid (aCSF) containing an extracellular potassium level ([K(+)](o)) elevated above physiological levels in order to increase excitability and maintain a stable respiratory output. To begin to investigate the effects of [K(+)](o) on the relationship between PHR and XII phase components, as well as the complexity underlying their respiratory-related network components, we examined the effects of various [K(+)](o) levels on simultaneously recorded PHR and XII nerve activities in an arterially-perfused adult rat preparation.

Pontine-ventral respiratory column interactions through raphe circuits detected using multi-array spike train recordings.

Recently, Segers et al. identified functional connectivity between the ventrolateral respiratory column (VRC) and the pontine respiratory group (PRG). The apparent sparseness of detected paucisynaptic interactions motivated consideration of other potential functional pathways between these two regions. We report here evidence for "indirect" serial functional linkages between the PRG and VRC via intermediary brain stem midline raphé neurons. Arrays of microelectrodes were used to record sets of spike trains from a total of 145 PRG, 282 VRC, and 340 midline neurons in 11 decerebrate, vagotomized, neuromuscularly blocked, ventilated cats. Spike trains of 13,843 pairs of neurons that included at least one raphé cell were screened for respiratory modulation and short-time scale correlations. Significant correlogram features were detected in 7.2% of raphé-raphé (291/4,021), 4.3% of VRC-raphé (292/6,755), and 4.0% of the PRG-raphé (124/3,067) neuron pairs. Central peaks indicative of shared influences were the most common feature in correlations between pairs of raphé neurons, whereas correlated raphé-PRG and raphé-VRC neuron pairs displayed predominantly offset peaks and troughs, features suggesting a paucisynaptic influence of one neuron on the other. Overall, offset correlogram features provided evidence for 33 VRC-to-raphé-to-PRG and 45 PRG-to-raphé-to-VRC correlational linkage chains with one or two intermediate raphé neurons. The results support a respiratory network architecture with parallel VRC-to-PRG and PRG-to-VRC links operating through intervening midline circuits, and suggest that raphé neurons contribute to the respiratory modulation of PRG neurons and shape the respiratory motor pattern through coordinated divergent actions on both the PRG and VRC.

Effects of aerobic and anaerobic metabolic inhibitors on avian intrapulmonary chemoreceptors.

Birds have rapidly responding respiratory chemoreceptors [intrapulmonary chemoreceptors (IPC)] that provide vagal sensory feedback about breathing pattern. IPC are exquisitely sensitive to CO(2) but are unaffected by hypoxia. IPC continue to respond to CO(2) during hypoxic and even anoxic conditions, suggesting that they may generate ATP needed for signal transduction anaerobically. To assess IPC energy metabolism, single-cell action potential discharge and acid-base status were recorded from 26 pentobarbital-anesthetized Anas platyrhynchos before and after intravenous infusion of the glycolytic blocker iodoacetate (10-70 mg/kg), mitochondrial blocker rotenone (2 mg/kg), and/or mitochondrial uncoupler 2,4-dinitrophenol (5-15 mg/kg). After 5 min exposure at the highest dosages, iodoacetate inhibited IPC discharge 65% (15.9 +/- 0.3 s(-1) to 5.5 +/- 0.3 s(-1), P < 0.05), rotenone inhibited discharge 80% (12.9 +/- 0.5 s(-1) to 2.6 +/- 0.6 s(-1), P < 0.05), and 2,4-dinitrophenol inhibited discharge 19% (14.0 +/- 0.3 s(-1) to 11.3 +/- 0.3 s(-1), P < 0.05). These results suggest that IPC utilize glucose, require an intact glycolytic pathway, and metabolize the products of glycolysis to CO(2) and H(2)O by mitochondrial respiration. The small but significant effect of 2,4-dinitrophenol suggests that ATP production by glycolysis may be sufficient to meet IPC energy demands if NADH can be oxidized to NAD experimentally by uncoupling mitochondria, or physiologically by transient lactate production. A model for IPC spike frequency adaptation is proposed, whereby the rapid onset of phasic IPC discharge requires ATP from anaerobic glycolysis, using lactate as the electron acceptor, and the roll-off in IPC discharge reflects transient acidosis due to intracellular lactic acid accumulation.

Automatic selection of the threshold value R for approximate entropy.

Calculation of approximate entropy (ApEn) requires a priori determination of two unknown parameters, m and r. While the recommended values of r, in the range of 0.1-0.2 times the standard deviation of the signal, have been shown to be applicable for a wide variety of signals, in certain cases, r values within this prescribed range can lead to an incorrect assessment of the complexity of a given signal. To circumvent this limitation, we recently advocated finding the maximum ApEn value by assessing all values of r from 0 to 1, and found that maximum ApEn does not always occur within the prescribed range of r values. Our results indicate that finding the maximum ApEn leads to the correct interpretation of a signal's complexity. One major limitation, however, is that the calculation of all choices of r values is often impractical due to the computational burden. Our new method, based on a heuristic stochastic model, overcomes this computational burden, and leads to the automatic selection of the maximum ApEn value for any given signal. Based on Monte Carlo simulations, we derive general equations that can be used to estimate the maximum ApEn with high accuracy for a given value of m. Application to both synthetic and experimental data confirmed the advantages claimed with the proposed approach.

Functional connectivity in the pontomedullary respiratory network.

Current models propose that a neuronal network in the ventrolateral medulla generates the basic respiratory rhythm and that this ventrolateral respiratory column (VRC) is profoundly influenced by the neurons of the pontine respiratory group (PRG). However, functional connectivity among PRG and VRC neurons is poorly understood. This study addressed four model-based hypotheses: 1) the respiratory modulation of PRG neuron populations reflects paucisynaptic actions of multiple VRC populations; 2) functional connections among PRG neurons shape and coordinate their respiratory-modulated activities; 3) the PRG acts on multiple VRC populations, contributing to phase-switching; and 4) neurons with no respiratory modulation located in close proximity to the VRC and PRG have widely distributed actions on respiratory-modulated cells. Two arrays of microelectrodes with individual depth adjustment were used to record sets of spike trains from a total of 145 PRG and 282 VRC neurons in 10 decerebrate, vagotomized, neuromuscularly blocked, ventilated cats. Data were evaluated for respiratory modulation with respect to efferent phrenic motoneuron activity and short-timescale correlations indicative of paucisynaptic functional connectivity using cross-correlation analysis and the "gravity" method. Correlogram features were found for 109 (3%) of the 3,218 pairs composed of a PRG and a VRC neuron, 126 (12%) of the 1,043 PRG-PRG pairs, and 319 (7%) of the 4,340 VRC-VRC neuron pairs evaluated. Correlation linkage maps generated for the data support our four motivating hypotheses and suggest network mechanisms for proposed modulatory functions of the PRG.

A mathematical model of pH(i) regulation in central CO2- chemoreception.

In CO2 chemosensitive neurons, an increase in CO2 (hypercapnia) leads to a maintained reduction in intracellular pH (pH(i)) while in non-chemosensitive neurons pH(i) recovery is observed. The precise mechanisms for the differential regulation of pH(i) recovery between these cell populations remain to be identified; however, studies have begun to explore the role of Na+/H+ exchange (NHE). Here, we compare the results of two different formulations of a mathematical model to begin to explore pH(i) regulation in central CO2 chemoreception.

Fast oscillatory rhythms in inspiratory motor discharge: a mathematical model.

In this paper, a mathematic model is applied to characterize spectral activity associated with fast oscillatory rhythms inherent in inspiratory discharges. Based on the estimated parameters, features are extracted to allow the model to discriminate between changes in the location, magnitude, and shape of spectral activities under basal conditions and during pharmacological blockade of gap junctions.

Respiratory network complexity in neonatal rat in vivo and in vitro.

Numerous experimental preparations from neonatal rodents have been developed to study mechanisms responsible for respiratory rhythm generation. Amongst them, the in vivo anesthetized neonatal rat preparation and the in vitro medullary slice preparation from neonatal rat are commonly used. These two preparations not only contain a different extent of the neuroanatomical axis associated with central respiratory control, but they are also studied under markedly different conditions, all of which may affect the complex dynamics underlying the central inspiratory neural network. Here, we evaluated the approximate entropy (ApEn) underlying inspiratory motor bursts as an index of inspiratory neural network complexity from each preparation to address this possibility. Our findings suggest that the central inspiratory neural network of the in vivo anesthetized neonatal rat exhibits lower complexity (i.e., more order) than that observed in the in vitro transverse medullary slice preparation, both of which are substantially lower than that observed in more intact in vitro (e.g., arterially-perfused rat) and mature in vivo (e.g., anesthetized rat, piglet, cat) preparations. We suggest that additional studies be conducted to identify the precise mechanisms responsible for the differences in central inspiratory neural network complexity between these two neonatal rat preparations.