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.

Fine structure and distribution of axon terminals from the cochlear nucleus on neurons in the medial superior olivary nucleus of the cat.

The morphology and distribution of axon terminals on central column and marginal neurons of the cat medial superior olivary nucleus (MSO) were analyzed by electron microscopy. Individual neurons or groups of cells oriented such that substantial lengths of their dendrites were within a 5-7 mu thich section were selected for detailed study. Thin sections were cut from remounted thick sections. Boutons with spherical vesicles arise directly from myelinated axons; more than one synaptic region of an axon, each separated by a myelinated segment, may contact a given dendrite. Boutons with flattened and occasionally dense core vesicles arise from both myelinated and unmyelinated portions of axons; these axons may also have more than one synaptic region. Both kinds of synaptic profiles are found on the somata and dendrites of all MSO neurons. To determine which nerve endings are from the cochlear nucleus (CN) lesions were made to produce orthograde degeneration. Following unilateral CN lesions degenerating spherical vesicle terminals were observed on the lateral dendrites and somata of ipsilateral central column cells and the medial dendrites and somata of contralateral neurons. Degenerating terminals were rarely seen on the opposite dendrite (three of 48 cells). In six of seven instances where medial and lateral dendrites of two cells overlapped degeneration was limited to one oriented toward the lesion. Marginal cells examined received virtually all spherical vesicle terminals from only one CN. Terminals with flattened vesicles persisted on the somata and dendrites of all neurons studied including cells from cats with bilateral lesions.

Respiratory pattern changes produced by intercostal muscle/rib vibration.

Large-amplitude vibration of the intercostal muscles/ribs has an inhibitory effect on inspiratory motor output. This effect has been attributed, in part, to the stimulation of intercostal muscle tendon organs. Intercostal muscle/rib vibration can also produce a decrease or increase in respiratory frequency. Studies were conducted 1) to determine whether, in addition to intercostal tendon organs, costovertebral joint mechanoreceptors (CVJR's) contribute to the inspiratory inhibitory effect of intercostal muscle/rib vibration (IMV) and 2) to explain the different respiratory frequency responses to IMV previously reported. Phrenic (C5) activity was monitored in paralyzed thoracotomized, artificially ventilated cats. Vibration (125 Hz) at amplitudes greater than 1,200 micron of one T6 intercostal space in decerebrated vagotomized rats reduced phrenic activity. This response was still present but weaker in some animals after denervation of the T6 intercostal muscles. Subsequent denervation of the T6 CVJR's by dorsal root sections eliminated this effect. Respiratory frequency decreased during simultaneous vibration (greater than 1,200 micron) of the T5 and T7 intercostal spaces in vagotomized cats. Respiratory frequency increased during IMV of two intercostal spaces (greater than 1,300 micron) in vagal intact cats. The use of different anesthetics (pentobarbital, allobarbital) did not alter these results. We conclude that CVJR's may contribute to the inhibitory effect of IMV on medullary inspiratory activity. The presence or absence of pulmonary vagal afferents can account for the different respiratory frequency responses to IMV, and different anesthetics did not influence these results.

Medullary neurons mediating the inhibition of inspiration by intercostal muscle tendon organs?

Studies were conducted to test the hypothesis that nonrespiratory-modulated units are last-order interneurons mediating the effects of intercostal muscle tendon organs on medullary inspiratory neuron activity. Vagotomized, anesthetized, or decerebrate cats were used. Results show the following. 1) Afferents from different receptor types (i.e., intercostal tendon organs and chest wall cutaneous receptors) that inhibit medullary inspiratory neuron activities evoke the same units. 2) Gastrocnemius muscle group I afferent fibers evoke some of the same units as intercostal afferents but do not alter respiratory activity. 3) The "pneumotaxic center" and laryngeal nerve afferents, which inhibit medullary inspiratory activity, evoke different medullary units than intercostal afferents. 4) Evoked units are not active in spontaneously breathing cats. Additional results suggest that a few respiratory neurons near the retrofacial nucleus may be involved in the mediation of the inspiratory inhibitory effects of intercostal tendon organs. These results do not establish the mechanism by which intercostal muscle tendon organs reduces medullary inspiratory activity.

Medullary inspiratory activity: influence of intercostal tendon organs and muscle spindle endings.

Studies were conducted to determine the effects of intercostal muscle spindle endings (MSEs) and tendon organs (TOs) on medullary inspiratory activity in decerebrate and allobarbital-anesthetized cats. Impeded muscle contractions, elicited by electrical stimulation of the peripheral cut end of the T6 ventral root, were used to stimulate external and internal intercostal TOs without MSEs. Impeded contractions of either the external or internal intercostal muscles reduced phrenic and medullary inspiratory neuronal activities. Vibration was used to selectively stimulate external or internal intercostal MSEs (90 and 40 micron amplitude, respectively). Selective stimulation of either external or internal intercostal MSEs did not change phrenic or medullary inspiratory neuronal activities. It is concluded that both external and internal intercostal TOs have a generalized inhibitory effect on medullary inspiratory activity and intercostal MSEs have no effect on medullary inspiratory activity.

Medullary expiratory activity: influence of intercostal tendon organs and muscle spindle endings.

Studies were conducted to determine the effects of intercostal muscle spindle endings (MSEs) and tendon organs (TOs) on medullary expiratory activity in decerebrate cats. Impeded intercostal muscle contractions, elicited by electrical stimulation of the peripheral cut end of the T6 ventral root, were used to stimulate intercostal TOs without MSEs. Impeded contractions of the intercostal muscles augmented expiratory laryngeal motoneuron activity, and either had no effect on or reduced the activity of bulbospinal expiratory neurons. Vibration was used to stimulate intercostal MSEs. Intercostal MSEs had no effect on medullary expiratory neuron activity. It is concluded that both external and internal intercostal TOs have an excitatory effect on expiratory laryngeal motoneuron activity and an inhibitory effect on a subpopulation of expiratory neurons driving intercostal and/or abdominal muscles, and intercostal MSEs have no direct influence on medullary expiratory activity.

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.

Cerebral cortical evoked potentials elicited by cat intercostal muscle mechanoreceptors.

Intercostal muscle afferents discharge in response to changes in intercostal muscle mechanics and have spinal and brain stem projections. It was hypothesized that intercostal muscle mechanoreceptors also project to the sensorimotor cortex. In cats, the proximal muscle branch of an intercostal nerve was used for electrical stimulation. The mechanical stimulation was stretch of an isolated intercostal space. The sensorimotor cortex was mapped with a surface ball electrode. Primary cortical evoked potentials (CEP) were found in area 3a of the sensorimotor cortex with mechanical and electrical stimulation. The CEP was elicited with the smallest stretch amplitude used, 50 microns. The CEP response showed little increase beyond 300-microns stretch. The CEP elicited by 50-microns stretch suggests an initial cortical activation by intercostal muscle spindles. The minimal increase in CEP amplitude with stretch > 300 microns suggests that the CEP response is primarily due to muscle spindle recruitment. The increase in amplitude beyond this stretch may be due to recruitment of tendon organs. These results demonstrate a short-latency projection of intercostal muscle mechanoreceptors to the sensorimotor region of the cerebral cortex. This cortical activation may be involved in respiratory sensations and/or transcortical reflex responses to changes in respiratory muscle mechanics.

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.

Suppression of proprioceptor--motor neuron interactions by proprioceptors in crayfish claw.

Crayfish claw proprioceptors and slow closer exciter and opener inhibitor motor neurons were monitored simultaneously during imposed claw displacements. With increasing displacement velocity and decreasing joint angle, the activity of closing sensitive receptors increased, while dynamic-static opening sensitive receptor activity decreased during claw closing. Motor neuron activity evoked by claw opening varied inversely as a function of preceding closing velocity, and directly with preceding pause duration at the closed position. This dependence on closing history cannot be accounted for by changes in opening sensitive receptor activity. Data demonstrate that closing sensitive receptors can suppress excitatory interactions between claw proprioceptors and motor neurons.

Gravitational representation of simultaneously recorded brainstem respiratory neuron spike trains.

Experiments designed to study concurrent processes in neural networks have been hampered by limitations of available analytical methods. A recently described gravitational representation of spike train data was used to evaluate groups of simultaneously monitored medullary respiratory related neurons in anesthetized, vagotomized cats. The results establish that the method can detect and define functional associations among elements of such groups after as few as 20 respiratory cycles.

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.

Proprioceptive fields of crayfish claw motor neurons.

1. Action potentials of crayfish claw motor neurons were recorded during both imposed constant-velocity displacements and imposed alternating sequences of opening and closing step movements of the dactyl. 2. Peristimulus time (PST) histograms show that the firing probabilities of two neurons, the opener inhibitor (OI) and the slow closer excitor (CE) consistently increased during opening ramp movements and declined during closing ramp movements. Hyperpolarizing synaptic potentials were observed in both cells during closing movements. 3. The proprioceptive field organizations of OI and CE were analyzed with response planes and contour planes. Each PST histogram in a plane displays the firing probability of the neuron as a function of time following step displacements at a given position. A relatively uniform early primary response followed each successive opening step. The probability of occurrence of later activity, when present, usually became more pronounced as the joint angle increased. Often both cells were silent during closing steps; when the cells were active, their firing probabilities were highest at the more open joint angles. 4. When both OI and CE were active, their spike trains were usually temporally correlated. 5. The other claw efferents did not respond to imposed movements in a consistent manner. When CE was active it was most likely to respond to closing movements near the closed position. 6. It is concluded that OI and CE are strongly and similarly influenced by proprioceptive reflexes. The responses of the two cells to imposed dactyl movements change as a function of joint angle, time after movement, and direction of movement.

Interactions among an ensemble of chordotonal organ receptors and motor neurons of the crayfish claw.

1. Action potentials of crayfish propodite-dactyl (PD) chordotonal organ receptors and two claw motor neurons, the opener inhibitor (OI) and slow closer excitor CE) were simultaneously monitored during imposed step and ramp movements of the dactyl or while the dactyl was held at various positions. 2. The activities of the cells during imposed displacements were analyzed using peristimulus time histograms and response and contour planes. The proprioceptive fields (PFs) of individual receptors resemble components of the more complex motor neuron PFs. Some receptors are briefly active after each successive opening step, while others do not respond to steps near the closed position but respond as the joint angle increases, becoming active when the claw is held open. Another type of receptor responds to closing movements. 3. Interactions among the various types of receptors and the two motor neurons were detected and analyzed by various statistical methods and intracellular recording techniques. The results indicate that receptors activated during opening movements and when the dactyl is held at open positions excite OI and CE via divergent functional connections. The efficacies of the connections made by a receptor may differ. Receptors activated by closing movements produce hyperpolarizing synaptic potentials in both efferents, possible directly or via interneurons. 4. It is concluded that several types of chordotonal organ receptors form an ensemble of parallel input channels, which modulates the activities of OI and CE and contributes to the generation of the spatial-temporal nonuniformities of their proprioceptive reflex responses.

Intercostal and abdominal muscle afferent influence on pneumotaxic center respiratory neurons.

The experiments were performed on mid-collicular decerebrated, vagotomized, paralyzed, artificially ventilated cats. Phrenic (PA) and pneumotaxic center (PC) respiratory neuron (extracellular) activities were recorded during electrical stimulation of intercostal nerve proprioceptor afferents (external, internal or lateral intercostal nerves). Intercostal nerve stimulation (INS) of sufficient intensity to reduce PA also reduced the activity of phasic PC I-neurons and the I-modulated portion of tonic firing I-neurons. The stimulus-response latency for the reduction in PA was always shorter than the latency for the reduction in I-neuron activity. Baseline tonic activity (during E-phase) was unaffected by INS in most tonic I-neurons. The predominant response of PC IE- and E-neurons to INS was augmentation of their activity. Stimulus-response latency studies showed that the increase in IE- and E-neuron activity occurred after the decrease in PA. It is concluded that: (1) the reduced PC I-neuron activity following INS is due primarily to disfacilitation resulting probably from decreased activity in medullary I-neurons that drive the PC I-neurons, (2) PC IE- and E-neurons are not the primary neurons mediating the inspiratory inhibitory effects of intercostal and abdominal muscle proprioceptors on medullary I drive, (3) the changes in PC IE- and E-neuron activity is not due secondarily to changes in DRG and VRG IE- or E-neuron activities, and (4) the reflex effects are due to stimulation of low threshold Group I afferent fibers.

Expiratory neurons in the region of the retrofacial nucleus: inhibitory effects of intercostal tendon organs.

Intercostal muscle tendon organs have an inhibitory effect on the medullary neurons driving all inspiratory muscles, and on a subpopulation of medullary bulbospinal expiratory neurons. Results from this study showed that the activity of expiratory related neurons in the region of the retrofacial nucleus (Botzinger complex) decreases during tendon organ afferent stimulation; the response is similar to that of caudal bulbospinal expiratory neurons. We conclude that these expiratory neurons do not mediate the tendon organ inhibition of medullary inspiratory neuronal activity.