Modulation of expiratory motor output evoked by chemical activation of pre-Bötzinger complex in vivo

We have previously demonstrated that chemical stimulation of the pre-B?tzinger complex (pre-B?tC) in the anesthetized cat produces either phasic or tonic excitation of phrenic nerve discharge. This region is characterized by a mixture of inspiratory-modulated, expiratory-modulated, and phase-spanning (including pre-inspiratory (pre-I)) neurons; however, its influence on expiratory motor output is unknown. We, therefore, examined the effects of chemical stimulation of the pre-B?tC on expiratory motor output recorded from the caudal iliohypogastric (lumbar, L(2)) nerve. We found that unilateral microinjection of DL-homocysteic acid (DLH; 10 mM; 10-20 nl) into 16 sites in the pre-B?tC enhanced lumbar nerve discharge, including changes in timing and patterning similar to those previously reported for phrenic motor output. Both increased peak amplitude and frequency of phasic lumbar bursts as well as tonic excitation of lumbar motor activity were observed. In some cases, evoked phasic lumbar nerve activity was synchronized in phase with phrenic nerve discharge. These findings demonstrate that chemical stimulation of the pre-B?tC not only excites inspiratory motor activity but also excites expiratory motor output, suggesting a role for the pre-B?tC in generation and modulation of inspiratory and expiratory rhythm and pattern.     

Patterns of phrenic motor output evoked by chemical stimulation of neurons located in the pre-Bötzinger complex in vivo

The pre-B?tzinger complex (pre-B?tC) has been proposed to be essential for respiratory rhythm generation from work in vitro. Much less, however, is known about its role in the generation and modulation of respiratory rhythm in vivo. Therefore we examined whether chemical stimulation of the in vivo pre-B?tC manifests respiratory modulation consistent with a respiratory rhythm generator. In chloralose- or chloralose/urethan-anesthetized, vagotomized cats, we recorded phrenic nerve discharge and arterial blood pressure in response to chemical stimulation of neurons located in the pre-B?tC with DL-homocysteic acid (DLH; 10 mM; 21 nl). In 115 of the 122 sites examined in the pre-B?tC, unilateral microinjection of DLH produced an increase in phrenic nerve discharge that was characterized by one of the following changes in cycle timing and pattern: 1) a rapid series of high-amplitude, rapid rate of rise, short-duration bursts, 2) tonic excitation (with or without respiratory oscillations), 3) an integration of the first two types of responses (i.e., tonic excitation with high-amplitude, short-duration bursts superimposed), or 4) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a high-amplitude, short-duration burst). In 107 of these sites, the phrenic neurogram response was accompanied by an increase or decrease (>/=10 mmHg) in arterial blood pressure. Thus increases in respiratory burst frequency and production of tonic discharge of inspiratory output, both of which have been seen in vitro, as well as modulation of burst pattern can be produced by local perturbations of excitatory amino acid neurotransmission in the pre-B?tC in vivo. These findings are consistent with the proposed role of this region as the locus for respiratory rhythm generation.     

Developmental changes in the spatio-temporal pattern of respiratory neuron activity in the medulla of late fetal rat

We investigated how the spatio-temporal pattern of respiratory neuron network activity in the ventral medulla changes during the late fetal period of rat. Brainstem-spinal cord preparations isolated from rat fetuses on embryonic days 17–21 (E17–E21) were stained with a voltage-sensitive dye for optical image analysis of neuronal activity of the ventral medulla. The spatio-temporal pattern of respiratory neuron activity in the preparation from E20 to E21 was basically identical to that of neonatal rat; pre-inspiratory activity in a limited region of the rostral ventrolateral medulla, the para-facial region, preceded by several hundred milliseconds the onset of inspiratory activity in the more caudal ventrolateral medulla, the pre-Bötzinger complex level. In contrast, in E17–E18 specimens, pre-inspiratory activity could not be detected in the rostral medulla at the level of the facial nucleus. Neuronal activity appeared to begin at the pre-Bötzinger complex level shortly before onset of the inspiratory burst. Strong activity then developed in the facial nucleus and peaked in the post-inspiratory phase. The transition of these patterns of respiratory activity occurred at E19. We conclude that the changes in the spatio-temporal pattern of neuronal activity reflect developmental changes in the cellular elements underlying rhythm generation in the fetal respiratory neuron network. We suggest that the pre-inspiratory neuron network of the para-facial region in the rostral ventrolateral medulla functions as the rhythm generator after E19/20.     

Localization and properties of respiratory neurons in the rostral pons of the newborn rat

The distribution and discharge pattern of respiratory neurons in the ‘pneumotaxic center’ of the rostral pons in the rat has remained unknown. We performed optical recordings and whole-cell patch clamp recordings to clarify respiratory neuron activity in the rostral pons of a brainstem-spinal cord preparation from a newborn rat. Inspiratory nerve activity was recorded in the 4th cervical nerve and used as a trigger signal for optical recordings. Respiratory neuron activity was detected in the limited region of the rostral-lateral pons. The main active region was presumed to be primarily the Kölliker-Fuse nucleus. The location of respiratory neurons was further confirmed by Lucifer Yellow staining after conducting whole-cell recordings. From a membrane potential analysis of the respiratory neurons in the rostral pons, the respiratory neurons were divided into four types: inspiratory neuron (71.9%), pre-inspiratory neuron (5.3%), post-inspiratory neuron (19.3%), and expiratory neuron (3.5%). A noticeable difference between pontine and medullary respiratory neurons was that post-inspiratory neurons were more frequently encountered in the pons. Application of a μ-opioid agonist, [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin, transformed the burst pattern of post-inspiratory neurons into that of pre-inspiratory neurons. The electrical stimulation of the sensory root of the trigeminal nerve induced three types of responses in 85% of pontine respiratory neurons: inhibitory postsynaptic potentials (42.7%), excitatory postsynaptic potentials (37.7%) and no response (15.1%). Our findings provide the first evidence in the rat for the presence of respiratory neurons in the rostral pons, with localization in the lateral region approximately overlapping with the Kölliker-Fuse nucleus.     

Nasal trigeminal inputs release the A5 inhibition received by the respiratory rhythm generator of the mouse neonate

Experiments were performed on neonatal mice to analyze why, in vitro, the respiratory rhythm generator (RRG) was silent and how it could be activated. We demonstrated that in vitro the RRG in intact brain stems is silenced by a powerful inhibition arising from the pontine A5 neurons through medullary alpha(2) adrenoceptors and that in vivo nasal trigeminal inputs facilitate the RRG as nasal continuous positive airway pressure increases the breathing frequency, whereas nasal occlusion and nasal afferent anesthesia depress it. Because nasal trigeminal afferents project to the A5 nuclei, we applied single trains of negative electric shocks to the trigeminal nerve in inactive ponto-medullary preparations. They induced rhythmic phrenic bursts during the stimulation and for 2-3 min afterward, whereas repetitive trains produced on-going rhythmic activity up to the end of the experiments. Electrolytic lesion or pharmacological inactivation of the ipsilateral A5 neurons altered both the phrenic burst frequency and occurrence after the stimulation. Extracellular unitary recordings and trans-neuronal tracing experiments with the rabies virus show that the medullary lateral reticular area contains respiratory-modulated neurons, not necessary for respiratory rhythmogenesis, but that may provide an excitatory pathway from the trigeminal inputs to the RRG as their electrolytic lesion suppresses any phrenic activity induced by the trigeminal nerve stimulation. The results lead to the hypothesis that the trigeminal afferents in the mouse neonate involve at least two pathways to activate the RRG, one that may act through the medullary lateral reticular area and one that releases the A5 inhibition received by the RRG.     

Biphasic effects of substance P on respiratory activity and respiration-related neurones in ventrolateral medulla in the neonatal rat brainstem in vitro

The effects of substance P (SP) on respiratory activity in the brainstem-spinal cord preparation from neonatal rats (0-4 days old) were investigated. The respiratory activity was recorded from C4 ventral roots and intracellularly from three types of respiration-related neurones, i.e. pre-inspiratory (or biphasic E), three subtypes of inspiratory; expiratory and tonic neurones in the ventrolateral medulla (VLM). After the onset of SP bath application (10 nM-1 microM) a dose-dependent decline of burst rate (by 48%) occurred, followed by a weaker dose-dependent increase (by 17.5%) in burst rate. The biphasic effect of SP on inspiratory burst rate was associated with sustained membrane depolarization (in a range of 0.5-13 mV) of respiration-related and tonic neurones. There were no significant changes in membrane resistance in any type of neurones when SP was applied alone or when synaptic transmission was blocked with tetrodotoxin (TTX). The initial depolarization was associated with an increase in inspiratory drive potential (by 25%) as well as in bursting time (by 65%) and membrane excitability in inspiratory and pre-inspiratory neurones, which corresponded to the decrease in burst rate (C4 activity). The spiking frequency of expiratory and tonic neurones was also increased (by 36 and 48%). This activation was followed by restoration of the synaptic drive potential and bursting time in inspiratory and to a less extent in pre-inspiratory neurones, which corresponded to the increase in burst rate. The discharge frequency of expiratory and tonic neurones also decreased to control values. This phase followed the peak membrane depolarization. At the peak depolarization, SP reduced the amplitude of the action potential by 4-8% in all types of neurones. Our results suggest that SP exerts a general excitatory effect on respiration-related neurones and synaptic coupling within the respiratory network in the VLM. The transient changes in neuronal activity in the VLM may underlie the biphasic effect of SP in the brainstem respiration activity recorded in C4 roots. However, the biphasic effect of SP on inspiratory burst rate seems to be also defined by the balance in activity of other SP-sensitive systems and neurones in the respiratory network in the brainstem and spinal cord, which can modify the activity of medullary respiratory rhythm generator.     

Augmentation of muscle nociceptive respiratory reflex facilitation by vagal afferents

Vagal influence on the facilitation of phrenic neural activity during respiratory phase-locked, gastrocnemius muscle nerve nociceptive electrical stimulation was examined in anesthetized, glomectomized, paralyzed, and artificially ventilated cats. (1) In the vagi-intact state, respiratory reflex facilitation was characterized by a sharp rise in peak amplitude, maximum rate of rise or slope, and mean rate of rise of integrated phrenic nerve activity. This was greater during inspiratory phase-locked (T1-locked) muscle nerve electrical stimulation than during expiratory phase-locked (TE-locked) muscle nerve electrical stimulation. "Evoked post-inspiratory phrenic activity" during the early expiratory phase was also observed during TE-locked muscle nerve electrical stimulation. (2) Bilateral vagotomy significantly attenuated the respiratory facilitation during both T1- and TE-locked muscle nerve electrical stimulation. In particular, the "evoked post-inspiratory phrenic activity" during TE-locked muscle nerve electrical stimulation was also attenuated or almost completely abolished. (3) Conditioning electrical stimulation of the vagus nerve revealed facilitatory reflexes which co-exist with inspiratory inhibitory reflexes. (4) The "evoked post-inspiratory phrenic activity" during TE-locked muscle nerve electrical stimulation, which was attenuated or abolished after vagotomy, was restored after vagal T1-locked conditioning stimuli combined with TE-locked muscle nerve electrical stimulation. The results suggest that vagal facilitatory reflexes augment the respiratory reflex facilitation during muscle nociceptive stimulation.     

Abdominal expiratory activity in the rat brainstem–spinal cord in situ: patterns, origins and implications for respiratory rhythm generation

We studied respiratory neural activity generated during expiration. Motoneuronal activity was recorded simultaneously from abdominal (AbN), phrenic (PN), hypoglossal (HN) and central vagus nerves from neonatal and juvenile rats in situ . During eupnoeic activity, low-amplitude post-inspiratory (post-I) discharge was only present in AbN motor outflow. Expression of AbN late-expiratory (late-E) activity, preceding PN bursts, occurred during hypercapnia. Biphasic expiratory (biphasic-E) activity with pre-inspiratory (pre-I) and post-I discharges occurred only during eucapnic anoxia or hypercapnic anoxia. Late-E activity generated during hypercapnia (7–10% CO₂) was abolished with pontine transections or chemical suppression of retrotrapezoid nucleus/ventrolateral parafacial (RTN/vlPF). AbN late-E activity during hypercapnia is coupled with augmented pre-I discharge in HN, truncated PN burst, and was quiescent during inspiration. Our data suggest that the pons provides a necessary excitatory drive to an additional neural oscillatory mechanism that is only activated under conditions of high respiratory drive to generate late-E activity destined for AbN motoneurones. This mechanism may arise from neurons located in the RTN/vlPF or the latter may relay late-E activity generated elsewhere. We hypothesize that this oscillatory mechanism is not a necessary component of the respiratory central pattern generator but constitutes a defensive mechanism activated under critical metabolic conditions to provide forced expiration and reduced upper airway resistance simultaneously. Possible interactions of this oscillator with components of the brainstem respiratory network are discussed.     

Episodic phrenic-inhibitory vagus nerve stimulation paradoxically induces phrenic long-term facilitation in rats

All respiratory long-term facilitation (LTF) is induced by inspiratory-excitatory stimulation, suggesting that LTF needs inspiratory augmentation and is the result of a Hebbian mechanism (coincident pre- and post-synaptic activity strengthens synapses). The present study examined the long-term effects of episodic inspiratory-inhibitory vagus nerve stimulation (VNS) on phrenic nerve activity. We hypothesized that episodic VNS would induce phrenic long-term depression. The results are compared with those obtained following serotonin receptor antagonism or episodic carotid sinus nerve stimulation (CSNS). Integrated phrenic neurograms were measured before, during and after three episodes of 5 min VNS (50 Hz, 0.1 ms), each separated by a 5 min interval, at a low (˜50 μA), medium (˜200 μA) or high (˜500 μA) stimulus intensity in anaesthetized, vagotomized, neuromuscularly blocked and artificially ventilated rats. Medium- and high-intensity VNS eliminated rhythmic phrenic activity during VNS, while low-intensity VNS only reduced phrenic burst frequency. At 60 min post-VNS, phrenic amplitude was higher than baseline (35 ± 5 % above baseline, mean ± S.E.M., P < 0.05) in the high-intensity group but not in the low- (-4 ± 4 %) or medium-intensity groups (-10 ± 15 %), or in the high-intensity with methysergide group (4 mg kg⁻¹, I.P.) (-11 ± 5 %). These data, which are inconsistent with our hypothesis, indicate that phrenic-inhibitory VNS induces a serotonin-dependent phrenic LTF similar to that induced by phrenic-excitatory CSNS (33 ± 7 %) and may require activation of high-threshold afferent fibres. These data also suggest that the synapses on phrenic motoneurons do not use the Hebbian mechanism in this LTF, as these motoneurons were suppressed during VNS.     

Rhythmic phrenic,intercostal and sympathetic activity in relation to limb and trunk motor activity in spinal cats

During L-DOPA-induced fictive spinal locomotion rhythmic activities in nerves to internal intercostal and external oblique abdominal muscles and in phrenic and sympathetic nerves were observed which were always coordinated with locomotor activity in forelimb and hindlimb muscle nerves. A periodicity with longer lasting tonic phases could be induced by cutaneous nerve stimulation or asphyxia. This activity was observed in limb motor nerves as well as in respiratory motor and sympathetic nerves. A slow independent activity of the phrenic and intercostal nerves or the sympathetic nerves, which could be related to a normal respiratory rhythm or independent sympathetic rhythms was not observed. The findings indicate that during fictive spinal locomotion the activity of spinal rhythm generators for locomotion also projects onto respiratory and sympathetic spinal neurones.     

Barosensory neurons in the ventrolateral medulla in rabbits and their responses to various afferent inputs from peripheral and central sources

In 55 anesthetized and paralyzed adult rabbits, 161 spontaneously active neurons which responded to electrical stimulation of A-fibers of the aortic nerve were found within the ventrolateral medulla (VLM). They were termed barosensory VLM neurons, since the aortic nerve A-fibers were considered to consist exclusively of afferents from arterial baroreceptors. Forty percent of barosensory VLM neurons tested (49/123) were activated antidromically by stimulation of the dorsolateral funiculus indicating that they send descending bulbospinal projections. Spontaneous discharges of barosensory VLM neurons were invariably inhibited by stimulation of aortic nerve A-fibers. Ninety-three percent of 80 neurons tested also responded to stimulation of aortic nerve C-fibers, a mixture of barosensory and nonbarosensory afferents. Natural stimulation of carotid sinus baroreceptors by an intravenous injection of phenylephrine in 19 vagotomized rabbits with aortic nerves disrupted inhibited spontaneous activity of all the 50 barosensory VLM neurons tested. By contrast, pharmacological stimulation of right or left carotid body chemoreceptors by close arterial injection of NaCN into the carotid sinus augmented activity of 93% of barosensory VLM neurons tested (41/44). The neuronal response was always greater to stimulation of chemoreceptors in the contralateral carotid sinus. Seven out of 8 barosensory VLM neurons tested (88%) were orthodromically excited by stimulation of the posterior hypothalamic area. In 74% of the 97 neurons examined in 29 vagotomized animals, a distinct respiratory-related rhythm, locked to that of phrenic nerve activity, was discerned. Thus, spontaneous activity of barosensory VLM neurons is inhibited by afferent inputs from aortic and carotid sinus baroreceptors, but is excited by incoming signals from carotid body chemoreceptors and the posterior hypothalamic area. It is also subject to the influence of the central mechanism generating the respiratory rhythm.     

Role of the ventrolateral region of the nucleus of the tractus solitarius in processing respiratory afferent input from vagus and superior laryngeal nerves

Summary The role of respiratory neurons located within and adjacent to the region of the ventrolateral nucleus of the tractus solitarius (vlNTS) in processing respiratory related afferent input from the vagus and superior laryngeal nerves was examined. Responses in phrenic neural discharge to electrical stimulation of the cervical vagus or superior laryngeal nerve afferents were determined before and after lesioning the vlNTS region. Studies were conducted on anesthetized, vagotomized, paralyzed and artificially ventilated cats. Arrays of 2 to 4 tungsten microelectrodes were used to record neuronal activity and for lesioning. Constant current lesions were made in the vlNTS region where respiratory neuronal discharges were recorded. The region of the vlNTS was probed with the microelectrodes and lesions made until no further respiratory related neuronal discharge could be recorded. The size and placement of lesions was determined in subsequent microscopic examination of 50 m thick sections. Prior to making lesions, electrical stimulation of the superior laryngeal nerve (4–100 A, 10 Hz, 0.1 ms pulse duration) elicited a short latency increase in discharge of phrenic motoneurons, primarily contralateral to the stimulated nerve. This was followed by a bilateral decrease in phrenic nerve discharge and, at higher currents, a longer latency increase in discharge. Stimulation of the vagus nerve at intensities chosen to selectively activate pulmonary stretch receptor afferent fibers produced a stimulus (current) dependent shortening of inspiratory duration. Responses were compared between measurements made immediately before and immediately after each lesion so that changes in response efficacy due to lesions per se could be distinguished from other factors, such as slight changes in the level of anesthesia over the several hours necessary in some cases to complete the lesions. Neither uni- nor bi-lateral lesions altered the efficacy with which stimulation of the vagus nerve shortened inspiratory duration. The short latency excitation of the phrenic motoneurons due to stimulation of the superior laryngeal nerve was severely attenuated by unilateral lesions of the vlNTS region ipsilateral to the stimulated nerve. Neither the bilateral inhibition nor the longer latency excitation due to superior laryngeal nerve stimulation was reduced by uni- or bi-lateral lesions of the vlNTS region. These results demonstrate that extensive destruction of the region of the vlNTS: a) does not markedly affect the inspiratory terminating reflex associated with electrical stimulation of the vagus nerve in a current range selective for activation of pulmonary stretch receptor afferents, and b) abolishes the short-latency increase, but not the bilateral decrease or longer latency increase in phrenic motoneuronal discharge which follows stimulation of the superior laryngeal nerve. We conclude that respiratory neurons in the region of the vlNTS do not play an obligatory role in the respiratory phase transitions in this experimental preparation. Neurons in the vlNTS region may participate in other reflexes, such as the generation of augmented phrenic motoneuronal discharge in response to activation of certain superior laryngeal or vagus nerve afferents.     

Respiratory responses to ionotropic glutamate receptor antagonists in the ventral respiratory group of the rabbit

Selective excitatory amino acid receptor antagonists acting on either N-methyl-D-aspartic acid (NMDA) or non-NMDA receptors were microinjected (30-50 nl) bilaterally into different subregions of the ventral respiratory group (VRG) of alpha-chloralose-urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits. Blockade of NMDA receptors by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 or 10 mM) within the inspiratory portion of the VRG (iVRG) dose-dependently decreased the peak amplitude and rate of rise of phrenic nerve activity, without significant changes in respiratory timing. Decreases in respiratory frequency and peak phrenic amplitude up to apnoea were evoked by 20 mM D-AP5; phrenic nerve activity was restored transiently by hypoxic or hypercapnic stimulation during D-AP5-induced apnoea. Microinjections of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1, 10 or 20 mM) into the iVRG provoked less intense depressant respiratory effects. No significant respiratory responses were evoked by microinjections of these antagonists into more caudal VRG subregions. The results suggest that ionotropic glutamate receptors within the iVRG are involved mainly in the control of the intensity of inspiratory activity, with a major role played by NMDA receptors. Glutamate receptor antagonism in the iVRG does not seem to impair the basic mechanisms underlying respiratory rhythm generation.     

The influence of pulmonary receptors on respiratory drive in a rabbit model of pulmonary emphysema

We have observed that pulmonary rapidly adapting receptor activity is greater in emphysematous rats than in controls. Pulmonary receptor activity, if modified by lung disease, may produce an inappropriate drive to breathe which may be perceived as dyspnoea. To investigate the efferent (drive) component of this hypothesis respiratory drive (phrenic nerve activity) was recorded in a rabbit model of emphysema. Drive was measured as slope and peak height of phrenic nerve activity. Slope and peak height were greater in emphysematous rabbits than controls, by 28% and 34%, respectively. Block of slowly adapting pulmonary stretch receptors by inhaled sulphur dioxide (which left only rapidly adapting and C-fibre receptors active) decreased drive in control (slope: 38.89+/-2.29 to 24.09+/-1.26, P<0.01) but not emphysematous rabbits (slope: 49.92+/-4.11 to 54.51+/-5.28, NS). Subsequent vagotomy decreased drive in emphysematous rabbits (slope: 54.51+/-5.28 to 41.41+/-3.90, P<0.05) but not controls (24.09+/-1.26 to 23.07+/-1.84, NS). Increased rapidly adapting receptor activity may, in part, increase respiratory drive in emphysema. This vagal component is only part of the total increased drive which may be perceived as dyspnoea in man.     

Electrical stimulation of the rabbit pulmonary artery increases respiratory output

This study was conducted to test the hypothesis that the pulmonary artery is involved in neural respiratory control and to identify the involved topographical region, if any. Six adult rabbits were anesthetized, artificially ventilated, and the chest was opened. The outer surface of the extra-pulmonary portion of the pulmonary artery was electrically stimulated by monitoring phrenic nerve activity. Phrenic nerve activity increased in three of the six rabbits when the proximal dorsal surface of the pulmonary trunk was stimulated. This positive response was abolished after bilateral vagotomy. In histological examinations we found densely grouped cells, i.e. pulmonary glomic tissue, with a fine nerve bundle in the tissue adjacent to the dorsal surface of the pulmonary trunk where electrical stimulation elicited respiratory augmentation. We suggest that there is a neural substrate which is involved in respiratory control inside the wall of or in the region adjacent to the proximal dorsal surface of the pulmonary trunk. Further studies to anatomically identify the neural substrate and clarify its physiological role in respiratory control are necessary.     

Pre-Bötzinger complex functions as a central hypoxia chemosensor for respiration in vivo

Recently, we identified a region located in the pre-B?tzinger complex (pre-B?tC; the proposed locus of respiratory rhythm generation) in which activation of ionotropic excitatory amino acid receptors using DL-homocysteic acid (DLH) elicits a variety of excitatory responses in the phrenic neurogram, ranging from tonic firing to a rapid series of high-amplitude, rapid rate of rise, short-duration inspiratory bursts that are indistinguishable from gasps produced by severe systemic hypoxia. Therefore we hypothesized that this unique region is chemosensitive to hypoxia. To test this hypothesis, we examined the response to unilateral microinjection of sodium cyanide (NaCN) into the pre-B?tC in chloralose- or chloralose/urethan-anesthetized vagotomized, paralyzed, mechanically ventilated cats. In all experiments, sites in the pre-B?tC were functionally identified using DLH (10 mM, 21 nl) as we have previously described. All sites were histologically confirmed to be in the pre-B?tC after completion of the experiment. Unilateral microinjection of NaCN (1 mM, 21 nl) into the pre-B?tC produced excitation of phrenic nerve discharge in 49 of the 81 sites examined. This augmentation of inspiratory output exhibited one of the following changes in cycle timing and/or pattern: 1) a series of high-amplitude, short-duration bursts in the phrenic neurogram (a discharge similar to a gasp), 2) a tonic excitation of phrenic neurogram output, 3) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a gasplike burst), or 4) an increase in frequency of phrenic bursts accompanied by small increases or decreases in the amplitude of integrated phrenic nerve discharge. Our findings identify a locus in the brain stem in which focal hypoxia augments respiratory output. We propose that the respiratory rhythm generator in the pre-B?tC has intrinsic hypoxic chemosensitivity that may play a role in hypoxia-induced gasping.     

Activity of Brainstem Respiratory Neurones just before the Expiration-Inspiration Transition in the Rat

Inspiratory activity of the hypoglossal nerve (XIIn) often precedes that of the phrenic nerve (PHRn). By manipulating artificial respiration, this preceding activity (pre-I XIIn activity) can be lengthened or isolated prematurely (decoupled XIIn activity) without developing into overt PHRn-associated inspiratory bursts. We hypothesized that these pre-I and decoupled XIIn activities, collectively termed 'XIIn-w/o-PHRn activity', reflect certain internal states of the respiratory centre at the period just prior to the transition from the expiratory phase to the inspiratory phase. In decerebrate, neuromuscularly blocked and artificially ventilated rats, the firing properties of medullary respiratory neurones were examined during the period of the XIIn-w/o-PHRn activity. The majority of the inspiratory neurones examined could be classified into two types: one was active (XIIn-type) and the other was inactive (PHRn-type) during the XIIn-w/o-PHRn period. On the other hand, augmenting expiratory (E-AUG) neurones of the Bötzinger complex (BOT) and the caudal ventral respiratory group (VRG) fired intensively during this period. Their firing stopped at the onset of the overt inspiratory bursts in the XIIn and PHRn, suggesting that BOT E-AUG neurones inhibit PHRn-type, but not XIIn-type, inspiratory neurones. We hypothesize that XIIn-type inspiratory activity facilitates the phase change from expiration to inspiration, through activation of certain inspiratory neurones that inhibit the firing of BOT E-AUG neurones and generation of the overt inspiratory bursts in XIIn-type and PHRn-type inspiratory neurones.     

Respiratory effects on high cervical cord cold blockade on efferent vagal and phrenic discharges in the rabbit

A technique of reversible cold blockade was applied in decerebrate and vagotomized rabbits that were immobilized and artificially ventilated to study the modulation of spontaneous respiratory rhythms. Respiratory discharges were recorded from vagal and phrenic efferents before and during cold blockade at the second cervical segment (C2) with a coolant-circulated thermode (–15° C). Measurement of the cooling profile demonstrated that there was significant hypothermia in the regions of the phrenic nucleus (+25° C) and obex of the medulla (+26° C). Arterial pressure was maintained by continual norepinephrine infusion, end-tidal carbon dioxide tension was held at hypercapnic levels, and rectal temperature was regulated near 38°C. The cold blockade of descending respiratory drives to the cervical phrenic nucleus inhibited the spontaneous activity in the phrenic nerve for more than 90 min. Phrenic activity could be induced by the intravenous injection of strychnine, but not doxapram, although this was not of respiratory quality. These results show that in the absence of descending and pharmacologic drives, but in the presence of phrenic hypothermia, spinalized rabbits are incapable of generating rhythmic patterns of discharge. C2 cold blockade also significantly slowed the spontaneous central respiratory rhythm with no change in integrated vagal amplitude, presumably due to a direct cooling effect on brainstem oscillators for breathing.     

Role of NMDA- and non-NMDA Subtypes of Glutamate Receptors in A5 Neuronal Structures in the Regulation of Respiration and Circulation during Thermal Nociceptive Stimulation in Rats

In narcotized albino rats, thermal nociceptive stimulation elevated systemic blood pressure and increased the frequency of respiratory rhythm generation. Unilateral microinjection of ketamine hydrochloride, a selective blocker for NMDA receptors, into A5 region did not change the baseline parameters of multineuronal activity in the phrenic nerve and systemic blood pressure. Under conditions of NMDA-receptor blockade, thermal nociceptive stimulation evoked more pronounced respiratory response (in comparison to that observed before ketamine treatment), but induced smaller blood pressure rise. Unilateral microinjection of GAMS, a selective blocker for non-NMDA receptors, into A5 region did not modify the examined baseline parameters and the nociceptive response. It is concluded that during thermal nociceptive stimulation, activity of the respiratory center and blood pressure in rats are controlled by neuronal structures in A5 region via NMDA subtype of glutamate receptors. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 11, pp. 484-487, November, 2008     

Responses of ventral respiratory neurones in the rat to vagus stimulation and the functional division of expiration.

In anaesthetized rats, extracellular and intracellular recordings were taken from 106 respiratory neurones in the intermediate region of the nucleus ambiguus. We observed unprovoked shortening of expiratory time accompanied, in all classes of respiratory neurone, by the elimination of the changes in membrane potential that were characteristic of stage II expiration. The demonstration of the elimination of stage II expiration in both the rat and cat strongly supports the functional division of expiration into stage I expiration (post-inspiration) and stage II expiration. In order to identify the neurones in the rat that receive inputs from vagal afferents and modulate the central respiratory rhythm, we examined whether any respiratory neurones responded to stimulation of the vagus nerve. Some post-inspiratory and stage II expiratory neurones responded. The short latency (< 2 ms) of four of the responses indicates that some vagal afferents act on post-inspiratory neurones via a disynaptic pathway. While repetitive stimulation of the vagus nerve could inhibit the phrenic rhythm, it appears that most inspiratory neurones in the intermediate region of the nucleus ambiguous complex are not directly involved in integrating the information from vagal afferents with the central respiratory rhythm.