Characterization of cardiovascular reflexes evoked by airway stimulation with allylisothiocyanate, capsaicin, and ATP in Sprague-Dawley rats.

Acute inhalation of airborne pollutants alters cardiovascular function and evidence suggests that pollutant-induced activation of airway sensory nerves via the gating of ion channels is critical to these systemic responses. Here, we have investigated the effect of capsaicin [transient receptor potential (TRP) vanilloid 1 (TRPV1) agonist], AITC [TRP ankyrin 1 (TRPA1) agonist], and ATP (P2X2/3 agonist) on bronchopulmonary sensory activity and cardiovascular responses of conscious Sprague-Dawley (SD) rats. Single fiber recordings show that allyl isothiocyanate (AITC) and capsaicin selectively activate C fibers, whereas subpopulations of both A and C fibers are activated by stimulation of P2X2/3 receptors. Inhalation of the agonists by conscious rats caused significant bradycardia, atrioventricular (AV) block, and prolonged PR intervals, although ATP-induced responses were lesser than those evoked by AITC or capsaicin. Responses to AITC were inhibited by the TRP channel blocker ruthenium red and the muscarinic antagonist atropine. AITC inhalation also caused a biphasic blood pressure response: a brief hypertensive phase followed by a hypotensive phase. Atropine accentuated the hypertensive phase, while preventing the hypotension. AITC-evoked bradycardia was not abolished by terazosin, the α1-adrenoceptor inhibitor, which prevented the hypertensive response. Anesthetics had profound effects on AITC-evoked bradycardia and AV block, which was abolished by urethane, ketamine, and isoflurane. Nevertheless, AITC inhalation caused bradycardia and AV block in paralyzed and ventilated rats following precollicular decerebration. In conclusion, we provide evidence that activation of ion channels expressed on nociceptive airway sensory nerves causes significant cardiovascular effects in conscious SD rats via reflex modulation of the autonomic nervous system.

Peripheral chemoreceptors tune inspiratory drive via tonic expiratory neuron hubs in the medullary ventral respiratory column network.

Models of brain stem ventral respiratory column (VRC) circuits typically emphasize populations of neurons, each active during a particular phase of the respiratory cycle. We have proposed that "tonic" pericolumnar expiratory (t-E) neurons tune breathing during baroreceptor-evoked reductions and central chemoreceptor-evoked enhancements of inspiratory (I) drive. The aims of this study were to further characterize the coordinated activity of t-E neurons and test the hypothesis that peripheral chemoreceptors also modulate drive via inhibition of t-E neurons and disinhibition of their inspiratory neuron targets. Spike trains of 828 VRC neurons were acquired by multielectrode arrays along with phrenic nerve signals from 22 decerebrate, vagotomized, neuromuscularly blocked, artificially ventilated adult cats. Forty-eight of 191 t-E neurons fired synchronously with another t-E neuron as indicated by cross-correlogram central peaks; 32 of the 39 synchronous pairs were elements of groups with mutual pairwise correlations. Gravitational clustering identified fluctuations in t-E neuron synchrony. A network model supported the prediction that inhibitory populations with spike synchrony reduce target neuron firing probabilities, resulting in offset or central correlogram troughs. In five animals, stimulation of carotid chemoreceptors evoked changes in the firing rates of 179 of 240 neurons. Thirty-two neuron pairs had correlogram troughs consistent with convergent and divergent t-E inhibition of I cells and disinhibitory enhancement of drive. Four of 10 t-E neurons that responded to sequential stimulation of peripheral and central chemoreceptors triggered 25 cross-correlograms with offset features. The results support the hypothesis that multiple afferent systems dynamically tune inspiratory drive in part via coordinated t-E neurons.

Discharge Identity of Medullary Inspiratory Neurons is Altered during Repetitive Fictive Cough.

This study investigated the stability of the discharge identity of inspiratory decrementing (I-Dec) and augmenting (I-Aug) neurons in the caudal (cVRC) and rostral (rVRC) ventral respiratory column during repetitive fictive cough in the cat. Inspiratory neurons in the cVRC (n = 23) and rVRC (n = 17) were recorded with microelectrodes. Fictive cough was elicited by mechanical stimulation of the intrathoracic trachea. Approximately 43% (10 of 23) of I-Dec neurons shifted to an augmenting discharge pattern during the first cough cycle (C1). By the second cough cycle (C2), half of these returned to a decrementing pattern. Approximately 94% (16 of 17) of I-Aug neurons retained an augmenting pattern during C1 of a multi-cough response episode. Phrenic burst amplitude and inspiratory duration increased during C1, but decreased with each subsequent cough in a series of repetitive coughs. As a step in evaluating the model-driven hypothesis that VRC I-Dec neurons contribute to the augmentation of inspiratory drive during cough via inhibition of VRC tonic expiratory neurons that inhibit premotor inspiratory neurons, cross-correlation analysis was used to assess relationships of tonic expiratory cells with simultaneously recorded inspiratory neurons. Our results suggest that reconfiguration of inspiratory-related sub-networks of the respiratory pattern generator occurs on a cycle-by-cycle basis during repetitive coughing.

Respiratory and Mayer wave-related discharge patterns of raphé and pontine neurons change with vagotomy.

Previous models have attributed changes in respiratory modulation of pontine neurons after vagotomy to a loss of pulmonary stretch receptor "gating" of an efference copy of inspiratory drive. Recently, our group confirmed that pontine neurons change firing patterns and become more respiratory modulated after vagotomy, although average peak and mean firing rates of the sample did not increase (Dick et al., J Physiol 586: 4265-4282, 2008). Because raphé neurons are also elements of the brain stem respiratory network, we tested the hypotheses that after vagotomy raphé neurons have increased respiratory modulation and that alterations in their firing patterns are similar to those seen for pontine neurons during withheld lung inflation. Raphé and pontine neurons were recorded simultaneously before and after vagotomy in decerebrated cats. Before vagotomy, 14% of 95 raphé neurons had increased activity during single respiratory cycles prolonged by withholding lung inflation; 13% exhibited decreased activity. After vagotomy, the average index of respiratory modulation (eta(2)) increased (0.05 +/- 0.10 to 0.12 +/- 0.18 SD; Student's paired t-test, P < 0.01). Time series and frequency domain analyses identified pontine and raphé neuron firing rate modulations with a 0.1-Hz rhythm coherent with blood pressure Mayer waves. These "Mayer wave-related oscillations" (MWROs) were coupled with central respiratory drive and became synchronized with the central respiratory rhythm after vagotomy (7 of 10 animals). Cross-correlation analysis identified functional connectivity in 52 of 360 pairs of neurons with MWROs. Collectively, the results suggest that a distributed network participates in the generation of MWROs and in the coordination of respiratory and vasomotor rhythms.

Reconfiguration of the pontomedullary respiratory network: a computational modeling study with coordinated in vivo experiments.

A large body of data suggests that the pontine respiratory group (PRG) is involved in respiratory phase-switching and the reconfiguration of the brain stem respiratory network. However, connectivity between the PRG and ventral respiratory column (VRC) in computational models has been largely ad hoc. We developed a network model with PRG-VRC connectivity inferred from coordinated in vivo experiments. Neurons were modeled in the "integrate-and-fire" style; some neurons had pacemaker properties derived from the model of Breen et al. We recapitulated earlier modeling results, including reproduction of activity profiles of different respiratory neurons and motor outputs, and their changes under different conditions (vagotomy, pontine lesions, etc.). The model also reproduced characteristic changes in neuronal and motor patterns observed in vivo during fictive cough and during hypoxia in non-rapid eye movement sleep. Our simulations suggested possible mechanisms for respiratory pattern reorganization during these behaviors. The model predicted that network- and pacemaker-generated rhythms could be co-expressed during the transition from gasping to eupnea, producing a combined "burst-ramp" pattern of phrenic discharges. To test this prediction, phrenic activity and multiple single neuron spike trains were monitored in vagotomized, decerebrate, immobilized, thoracotomized, and artificially ventilated cats during hypoxia and recovery. In most experiments, phrenic discharge patterns during recovery from hypoxia were similar to those predicted by the model. We conclude that under certain conditions, e.g., during recovery from severe brain hypoxia, components of a distributed network activity present during eupnea can be co-expressed with gasp patterns generated by a distinct, functionally "simplified" mechanism.

Production of reflex cough by brainstem respiratory networks.

Delineation of neural mechanisms involved in reflex cough is essential for understanding its many physiological and clinical complexities, and the development of more desirable antitussive agents. Brainstem networks that generate and modulate the breathing pattern are also involved in producing the motor patterns during reflex cough. Neurones of the ventrolateral medulla respiratory pattern generator mutually interact with neural networks in the pons, medulla and cerebellum to form a larger dynamic network. This paper discusses evidence from our laboratory and others supporting the involvement of the nucleus tractus solitarii, midline raphe nuclei and lateral tegmental field in the medulla, and the pontine respiratory group and cerebellum in the production of reflex cough. Gaps in our knowledge are identified to stimulate further research on this complicated issue.

Invited review: Neural network plasticity in respiratory control.

Respiratory network plasticity is a modification in respiratory control that persists longer than the stimuli that evoke it or that changes the behavior produced by the network. Different durations and patterns of hypoxia can induce different types of respiratory memories. Lateral pontine neurons are required for decreases in respiratory frequency that follow brief hypoxia. Changes in synchrony and firing rates of ventrolateral and midline medullary neurons may contribute to the long-term facilitation of breathing after brief intermittent hypoxia. Long-term changes in central respiratory motor control may occur after spinal cord injury, and the brain stem network implicated in the production of the respiratory rhythm could be reconfigured to produce the cough motor pattern. Preliminary analysis suggests that elements of brain stem respiratory neural networks respond differently to hypoxia and hypercapnia and interact with areas involved in cardiovascular control. Plasticity or alterations in these networks may contribute to the chronic upregulation of sympathetic nerve activity and hypertension in sleep apnea syndrome and may also be involved in sudden infant death syndrome.

Medullary respiratory neurones and control of laryngeal motoneurones during fictive eupnoea and cough in the cat.

1. This study addressed the hypothesis that ventrolateral medullary respiratory neurones participate in the control of laryngeal motoneurones during both eupnoea and coughing. 2. Data were obtained from 28 mid-collicular decerebrated, artificially ventilated cats. Cough-like motor patterns (fictive cough) in phrenic, lumbar and recurrent laryngeal nerves were elicited by mechanical stimulation of the intrathoracic trachea. Microelectrode arrays were used to monitor simultaneously several neurones in the ventral respiratory group, including the Bötzinger and pre-Bötzinger complexes. Spike trains were evaluated for responses during fictive cough and evidence of functional connectivity with spike-triggered averages of efferent recurrent laryngeal nerve activity. 3. Primary features were observed in averages triggered by 94 of 332 (28 %) neurones. An offset biphasic wave with a positive time lag was present in the unrectified average for 10 inspiratory and 13 expiratory neurones. These trigger neurones were respectively identified as inspiratory laryngeal motoneurones with augmenting, decrementing, plateau and "other" discharge patterns, and expiratory laryngeal motoneurones with decrementing firing patterns. 4. Rectified averages triggered by inspiratory neurones included 37 offset peaks, 11 central peaks and one offset trough. Averages triggered by expiratory neurones had 12 offset peaks, six central peaks and four offset troughs. Relationships inferred from these features included premotor actions of inspiratory neurones with augmenting, decrementing, plateau and "other" patterns on inspiratory laryngeal motoneurones, and premotor actions of decrementing and "other" expiratory neurones on expiratory laryngeal motoneurones. Corresponding changes in neuronal firing patterns during fictive cough supported these inferences. 5. The data confirm and extend previous results on the control of laryngeal motoneurones during eupnoea and support the hypothesis that the same premotor neurones help to shape motoneurone firing patterns during both eupnoea and coughing.

Changes in cat medullary neurone firing rates and synchrony following induction of respiratory long-term facilitation.

1. Long-term facilitation is a respiratory memory expressed as an increase in motor output lasting more than an hour. This change is induced by repeated hypoxia, stimulation of carotid chemoreceptors, or electrical stimulation of the carotid sinus nerve or brainstem mid-line. The present work addressed the hypothesis that persistent changes in medullary respiratory neural networks contribute to long-term facilitation. 2. Carotid chemoreceptors were stimulated by close arterial injection of CO(2)-saturated saline solution. Phrenic nerve efferent activity and up to 30 single medullary neurones were recorded simultaneously in nucleus tractus solitarii (NTS) including the dorsal respiratory group (DRG), Botzinger-ventral respiratory group (Böt-VRG), and nucleus raphe obscurus of nine adult cats, anaesthetized, injected with a neuromuscular blocking agent, vagotomized and artificially ventilated. 3. The firing rates of 87 of 105 neurones (83 %) changed following induction of long-term facilitation. Nine of eleven DRG and Böt-VRG putative premotor inspiratory neurones had increased firing rates with long-term facilitation. Fourteen of twenty-one raphe obscurus neurones with control firing rates less than 4 Hz had significant long-term increases in activity. 4. Cross-correlogram analysis suggested that there were changes in effective connectivity of neuron pairs with long-term facilitation. Joint peristimulus time histograms and pattern detection methods used with 'gravity' analysis also detected changes in short time scale correlations associated with long-term facilitation. 5. The results suggest that changes in firing rates and synchrony of VRG and DRG premotor neurones and altered effective connectivity among other functionally antecedent elements of the medullary respiratory network contribute to the expression of long-term facilitation.

Repeated sequences of interspike intervals in baroresponsive respiratory related neuronal assemblies of the cat brain stem.

Many neurons exhibit spontaneous activity in the absence of any specific experimental perturbation. Patterns of distributed synchrony embedded in such activity have been detected in the brain stem, suggesting that it represents more than "baseline" firing rates subject only to being regulated up or down. This work tested the hypothesis that nonrandom sequences of impulses recur in baroresponsive respiratory-related brain stem neurons that are elements of correlational neuronal assemblies. In 15 Dial-urethan anesthetized vagotomized adult cats, neuronal impulses were monitored with microelectrode arrays in the ventral respiratory group, nucleus tractus solitarius, and medullary raphe nuclei. Efferent phrenic nerve activity was recorded. Spike trains were analyzed with cycle-triggered histograms and tested for respiratory-modulated firing rates. Baroreceptors were stimulated by unilateral pressure changes in the carotid sinus or occlusion of the descending aorta; changes in firing rates were assessed with peristimulus time and cumulative sum histograms. Cross-correlation analysis was used to test for nonrandom temporal relationships between spike trains. Favored patterns of interspike interval sequences were detected in 31 of 58 single spike trains; 18 of the neurons with significant sequences also had short-time scale correlations with other simultaneously recorded cells. The number of distributed patterns exceeded that expected under the null hypothesis in 12 of 14 data sets composed of 4-11 simultaneously recorded spike trains. The data support the hypothesis that baroresponsive brain stem neurons operate in transiently configured coordinated assemblies and suggest that single neuron patterns may be fragments of distributed impulse sequences. The results further encourage the search for coding functions of spike patterns in the respiratory network.

Respiratory neuronal assemblies.

This review describes results from in vivo experiments on brain stem network mechanisms that control breathing. Multi-array recording technology and computational methods were used to test predictions derived from simulations of respiratory network models. This highly efficient approach has the advantage that many simultaneously recorded neurons are subject to shared stimulus, history, and state-dependent conditions. Our results have provided evidence for concurrent or parallel network interactions in the generation and modulation of the respiratory motor pattern. Recent data suggest that baroreceptors, chemoreceptors, nociceptors, and airway cough receptors shape the respiratory motor pattern, at least in part, through a system of shared coordinated 'multifunctional' neurons distributed in the brain stem. The 'gravity method' for the analysis and representation of multi-neuron data has demonstrated respiratory phase-dependent impulse synchrony among neurons with no respiratory modulation of their individual firing rates. The detection of this emergent property motivated the development of pattern detection methods that subsequently identified repeated transient configurations of these 'correlational assemblies'. These results support the view that information can be 'coded' in the nervous system by spike timing relationships, in addition to firing rate changes that traditionally have been measured by neurophysiologists.

Respiratory neural activity during long-term facilitation.

Intermittent hypoxia results in a long-term facilitation (LTF) of respiratory efferent activity. The studies reviewed here presented data from both anesthetized and decerebrate, paralyzed, vagotomized, artificially ventilated adult cats. Multiple arrays of tungsten microelectrodes were used to record the concurrent responses of brain stem neurons that contribute to respiratory motor pattern generation. Spike trains were analyzed with firing rate histograms, peristimulus time histograms, cycle triggered histograms, spike triggered averages with multiunit phrenic efferent activity, cross correlation histograms, joint peristimulus time histograms and the gravity method. These studies addressed several hypotheses. (1) There is parallel processing of input from carotid chemoreceptors to the brain stem. (2) Respiratory related midline neurons are involved in the induction and maintenance of LTF. (3) There is a change in effective connectivity of brain stem neurons with LTF. (4) Neural networks involved in the induction and maintenance of LTF have patterns of synchrony that recur with a frequency greater than expected by chance.

Transient configurations of baroresponsive respiratory-related brainstem neuronal assemblies in the cat.

The regulation of gas exchange requires coordination of the respiratory and cardiovascular systems. Previous work suggested that medullary raphe neurones transform and transmit information from baroreceptors to neurones in the ventral respiratory group. This study tested the hypothesis that distributed brainstem neuronal assemblies are transiently reconfigured during the respiratory cycle and baroreceptor stimulation. Blood pressure was perturbed by intravenous injection of an alpha1-adrenergic receptor agonist, unilateral pressure changes in the carotid sinus, or occlusion of the descending aorta in 14 Dial-urethane anaesthetized, vagotomized, paralysed, artificially ventilated cats. Neurones were monitored simultaneously with microelectrode arrays in two or more of the following sites: n. raphe obscurus, n. raphe magnus, rostral and caudal ventrolateral medulla, and the nucleus tractus solitarii. Transient configurations of baroresponsive assemblies were detected with joint pericycle-triggered histograms, the gravitational representation, and related pattern detection methods. Data were also analysed with cycle-triggered histograms, peristimulus-time and cumulative sum histograms, cross-correlograms, spike-triggered averages of efferent phrenic activity, and joint impulse configuration scatter diagrams (snowflakes). Five to nine simultaneously recorded spike trains from control expiratory phases were compared with data from interleaved equal-duration time blocks from control inspiratory phases. In each of seven animals, significant impulse synchrony detected by gravity analysis was confined to one phase of the respiratory cycle. Repeated patterns of distributed synchrony confined to periods of altered baroreceptor activity were detected and involved neurones that individually did not change firing rates during stimulation. Snowflakes and logical cross-correlation analysis provided evidence for the cooperative actions of impulses in concurrently active parallel channels. In 12 of 17 pairs of neurones with at least one baroresponsive cell, joint pericycle-triggered histograms detected synchrony indicative of shared inputs or functional excitatory interactions that varied as a function of time in the respiratory cycle. Neurones in four of the pairs had no respiratory modulation of their individual firing rates. Data from eight other pairs were indicative of fluctuations in inhibition during the respiratory cycle. The results demonstrate repeated transient configurations of baroresponsive neuronal assemblies during the respiratory cycle, without concomitant firing rate changes in the constituent neurones, and suggest distributed network mechanisms for the modulation of baroreceptor-mediated reflexes.

Functional connectivity among ventrolateral medullary respiratory neurones and responses during fictive cough in the cat.

This study tested predictions from a network model of ventrolateral medullary respiratory neurone interactions for the generation of the cough motor pattern observed in inspiratory and expiratory pump muscles. Data were from 34 mid-collicularly decerebrated, paralysed, artificially ventilated cats. Cough-like patterns (fictive cough) in efferent phrenic and lumbar nerve activities were elicited by mechanical stimulation of the intrathoracic trachea. Neurones in the ventral respiratory group, including the Botzinger and pre-Botzinger complexes, were monitored simultaneously with microelectrode arrays. Spike trains were analysed for evidence of functional connectivity and responses during fictive cough with cycle-triggered histograms, autocorrelograms, cross-correlograms, and spike-triggered averages of phrenic and recurrent laryngeal nerve activities. Significant cross-correlogram features were detected in 151 of 1988 pairs of respiratory modulated neurones. There were 59 central peaks, 5 central troughs, 11 offset peaks and 2 offset troughs among inspiratory neurone pairs. Among expiratory neurones there were 23 central peaks, 8 offset peaks and 4 offset troughs. Correlations between inspiratory and expiratory neurones included 20 central peaks, 10 central troughs and 9 offset troughs. Spike-triggered averages of phrenic motoneurone activity had 51 offset peaks and 5 offset troughs. The concurrent responses and multiple short time scale correlations support parallel and serial network interactions proposed in our model for the generation of the cough motor pattern in the respiratory pump muscles. Inferred associations included the following. (a) Excitation of augmenting inspiratory (I-Aug) neurones and phrenic motoneurones by I-Aug neurones. (b) Inhibition of augmenting expiratory (E-Aug) neurones by decrementing inspiratory (I-Dec) neurones. (c) Inhibition of I-Aug, I-Dec and E-Aug neurones by E-Dec neurones. (d) Inhibition of I-Aug and I-Dec neurones and phrenic motoneurones by E-Aug neurones. The data also confirm previous results and support hypotheses in current network models for the generation of the eupnoeic pattern.

Responses of simultaneously recorded respiratory-related medullary neurons to stimulation of multiple sensory modalities.

This study addresses the hypothesis that multiple afferent systems share elements of a distributed brain stem network that modulates the respiratory motor pattern. Data were collected from 18 decerebrate, bilaterally vagotomized, paralyzed, artificially ventilated cats. Up to 28 neurons distributed in the rostral and caudal ventral respiratory group, nucleus tractus solitarius, and raphe obscurus were recorded simultaneously with microelectrode arrays. Phases of the respiratory cycle and inspiratory drive were assessed from integrated efferent phrenic nerve activity. Carotid chemoreceptors were stimulated by injection of CO2-saturated saline solution via the external carotid artery. Baroreceptors were stimulated by increased blood pressure secondary to inflation of an embolectomy catheter in the descending aorta. Cutaneous nociceptors were stimulated by pinching a footpad. Four hundred seventy-four neurons were tested for respiratory modulated firing rates and responses; 403 neurons were tested with stimulation of all 3 modalities. Chemoreceptor stimulation and pinch, perturbations that tend to increase respiratory drive, caused similar responses in 52 neurons; 28 responded oppositely. Chemoreceptor and baroreceptor stimulation resulted in similar primary responses in 45 neurons; 48 responded oppositely. Similar responses to baroreceptor stimulation and pinch were recorded for 38 neurons; opposite effects were measured in 26 neurons. Among simultaneously recorded neurons, distinct combinations of firing rate changes were evoked in response to stimulation of the different modalities. The results show a functional convergence of information from carotid chemoreceptors, baroreceptors, and cutaneous nociceptors on respiratory-modulated neurons distributed in the medulla. The data are consistent with the hypothesis that brain stem neurons have overlapping memberships in multifunctional groups that influence the respiratory motor pattern.

Multimodal medullary neurons and correlational linkages of the respiratory network.

This study addresses the hypothesis that multiple sensory systems, each capable of reflexly altering breathing, jointly influence neurons of the brain stem respiratory network. Carotid chemoreceptors, baroreceptors, and foot pad nociceptors were stimulated sequentially in 33 Dial-urethan-anesthetized or decerebrate vagotomized adult cats. Neuronal impulses were monitored with microelectrode arrays in the rostral and caudal ventral respiratory group (VRG), nucleus tractus solitarius (NTS), and n. raphe obscurus. Efferent phrenic nerve activity was recorded. Spike trains of 889 neurons were analyzed with cycle-triggered histograms and tested for respiratory-modulated firing rates. Responses to stimulus protocols were assessed with peristimulus time and cumulative sum histograms. Cross-correlation analysis was used to test for nonrandom temporal relationships between spike trains. Spike-triggered averages of efferent phrenic activity and antidromic stimulation methods provided evidence for functional associations of bulbar neurons with phrenic motoneurons. Spike train cross-correlograms were calculated for 6,471 pairs of neurons. Significant correlogram features were detected for 425 pairs, including 189 primary central peaks or troughs, 156 offset peaks or troughs, and 80 pairs with multiple peaks and troughs. The results provide evidence that correlational medullary assemblies include neurons with overlapping memberships in groups responsive to different sets of sensory modalities. The data suggest and support several hypotheses concerning cooperative relationships that modulate the respiratory motor pattern. 1) Neurons responsive to a single tested modality promote or limit changes in firing rate of multimodal target neurons. 2) Multimodal neurons contribute to changes in firing rate of neurons responsive to a single tested modality. 3) Multimodal neurons may promote responses during stimulation of one modality and "limit" changes in firing rates during stimulation of another sensory modality. 4) Caudal VRG inspiratory neurons have inhibitory connections that provide negative feedback regulation of inspiratory drive and phase duration.

Analysis of diffusion coefficient distributions in humic and fulvic acids by means of diffusion ordered NMR spectroscopy.

The use of the computer program CONTIN to analyze pulsed-field gradient NMR (PFG-NMR) data for several standard humic and fulvic acids is described. An advantage of PFG-NMR analysis is that integration of different spectral regions provides a picture of how the diffusion coefficients vary with functional group composition for a given sample. Using prior knowledge of the sample and the principle of parsimony, CONTIN approximates a solution to the inverse Laplace transform applied to the decay of peak intensity with gradient area in the PFG-NMR experiment. Thus, a continuous distribution of diffusion coefficients is resolved for the polydisperse humic and fulvic acids. The results of the CONTIN analyses are in the form of a distribution function and a two-dimensional DOSY plot. The 2D DOSY spectrum displays chemical shifts along one axis and diffusion coefficients along the other, while a number-average diffusion coefficient, D(N), a weight-average diffusion coefficient, D(W), and a most probable diffusion coefficient, D(P), are realized from the diffusion coefficient distribution. For all spectral regions of each humic sample, D(W) was greater than D(N), which in turn was greater than or equal to the D(P), suggesting that the diffusion coefficient distribution is weighted toward smaller, more rapidly diffusing molecules. Polydispersities, estimated from the ratio D(W)/D(N), were less than the reported M(W)/M(N) values for similar humic substances. Thus, the D(W)/D(N) ratio obtained by CONTIN analysis of PFG-NMR data can be at least a qualitative, and at best a semiquantitative, indication of the polydispersity of the humic sample, but should not be used as a quantitative measure of polydispersity.

Medullary raphe neurones and baroreceptor modulation of the respiratory motor pattern in the cat.

1. Perturbations of arterial blood pressure change medullary raphe neurone activity and the respiratory motor pattern. This study sought evidence for actions of baroresponsive raphe neurones on the medullary respiratory network. 2. Blood pressure was perturbed by intravenous injection of an alpha1-adrenergic receptor agonist, unilateral pressure changes in the carotid sinus, or occlusion of the descending aorta in thirty-six Dial-urethane-anaesthetized, vagotomized, paralysed, artificially ventilated cats. Neurones were monitored with microelectrode arrays in two or three of the following domains: nucleus raphe obscurus-nucleus raphe pallidus, nucleus raphe magnus, and rostral and caudal ventrolateral medulla. Data were analysed with cycle-triggered histograms, peristimulus time and cumulative sum histograms, cross-correlograms and spike-triggered averages of efferent phrenic nerve activity. 3. Prolongation of the expiratory phase and decreased peak integrated phrenic amplitude were most frequently observed. Of 707 neurones studied, 310 had altered firing rates during stimulation; changes in opposite directions were monitored simultaneously in fifty-six of eighty-seven data sets with at least two baroresponsive neurones. 4. Short time scale correlations were detected between neurones in 347 of 3388 pairs. Seventeen pairs of baroresponsive raphe neurones exhibited significant offset correlogram features indicative of paucisynaptic interactions. In correlated raphe-ventrolateral medullary neurone pairs with at least one baroresponsive neurone, six of seven ventrolateral medullary decrementing expiratory (E-Decr) neurones increased their firing rate during baroreceptor stimulation. Thirteen of fifteen ventrolateral medullary inspiratory neurones correlated with raphe cells decreased their firing rate during baroreceptor stimulation. 5. The results support the hypothesis that raphe neuronal assemblies transform and transmit information from baroreceptors to neurones in the ventral respiratory group. The inferred actions both limit and promote responses to sensory perturbations and match predictions from simulations of the respiratory network.

Ventrolateral medullary respiratory network and a model of cough motor pattern generation.

The primary hypothesis of this study was that the cough motor pattern is produced, at least in part, by the medullary respiratory neuronal network in response to inputs from "cough" and pulmonary stretch receptor relay neurons in the nucleus tractus solitarii. Computer simulations of a distributed network model with proposed connections from the nucleus tractus solitarii to ventrolateral medullary respiratory neurons produced coughlike inspiratory and expiratory motor patterns. Predicted responses of various "types" of neurons (I-DRIVER, I-AUG, I-DEC, E-AUG, and E-DEC) derived from the simulations were tested in vivo. Parallel and sequential responses of functionally characterized respiratory-modulated neurons were monitored during fictive cough in decerebrate, paralyzed, ventilated cats. Coughlike patterns in phrenic and lumbar nerves were elicited by mechanical stimulation of the intrathoracic trachea. Altered discharge patterns were measured in most types of respiratory neurons during fictive cough. The results supported many of the specific predictions of our cough generation model and suggested several revisions. The two main conclusions were as follows: 1) The Bötzinger/rostral ventral respiratory group neurons implicated in the generation of the eupneic pattern of breathing also participate in the configuration of the cough motor pattern. 2) This altered activity of Bötzinger/rostral ventral respiratory group neurons is transmitted to phrenic, intercostal, and abdominal motoneurons via the same bulbospinal neurons that provide descending drive during eupnea.

Repeated patterns of distributed synchrony in neuronal assemblies.

Models of brain function predict that the recurrence of a process or state will be reflected in repeated patterns of correlated activity. Previous work on medullary raphe assembly dynamics revealed transient changes in impulse synchrony. This study tested the hypothesis that these variations in synchrony include distributed nonrandom patterns of association. Spike trains were recorded simultaneously in the ventrolateral medulla, n. raphe obscurus, and n. raphe magnus of four anesthetized (Dial), vagotomized, paralyzed, and artificially ventilated adult cats. The "gravitational" representation of spike trains was used to detect moments of impulse synchrony in neuronal assemblies visualized as variations in the aggregation velocities of particles corresponding to each neuron. Template matching algorithms were developed to identify excessively repeating patterns of particle condensation rates. Repeating patterns were detected in each animal. The reiterated patterns represented an emergent property not apparent in either corresponding firing rate histograms or conventional gravity representations. Overlapping subsets of neurons represented in different patterns were unmasked when the template resolution was changed. The results demonstrate repeated transient network configurations defined by the tightness and duration of synchrony in different combinations of neurons and suggest that multiple information streams are conveyed concurrently by fluctuations in the synchrony of on-going activity.