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Coughing and sneezing are protective reflexes that defend the airways against inhaled particles and noxious substances and remove mucus. The central neuronal network mechanisms that process "cough" and "sneeze" receptor inputs and produce the appropriate changes in respiratory muscle activity are poorly understood. Respiratory premotor and motoneurons are concentrated in two regions of the cat medulla. One population is referred to as the dorsal respiratory group (DRG) and corresponds to the ventrolateral nucleus of the solitary tract and the surrounding reticular formation. The second population is called the Botzinger complex/ventral respiratory group (BOT/VRG) and constitutes a longitudinal column of neurons from the rostral to the caudal end of the ventral lateral medulla. Both the DRG and the BOT/VRG also include neurons with only intramedullary projection sites; these cells have various respiratory-modulated discharge patterns and are referred to as propriobulbar.

Since respiration requires muscular activity, its control must ultimately be neural. This neural control has voluntary and involuntary components. The diaphragm is controlled by the phrenic nerve, which originates from C3–C5 vertebrae; the external and internal intercostals are controlled by spinal nerves in the thoracic cord; the abdominal muscles are controlled by spinal nerves in the lumbar cord. Respiratory control goes through centers in the brain stem, the pons and medulla, including the pontine respiratory group and dorsal and ventral respiratory groups of the medulla. Sensory information reaches these groups over the vagus and glossopharyngeal nerves. Sensory information includes stretch, P aCO 2 P aCO 2 , P aO 2 P aO 2 , and pH. Central chemoreceptors in the dorsal medulla also sense P aCO 2 P aCO 2 and pH. The DRG provides inspiratory drive, the VRG both inspiratory and expiratory drives. Peripheral chemoreceptors increase firing with decreased pH, increased P aCO 2 P aCO 2 , and decreased P aO 2 P aO 2 . Increased activity of the peripheral chemoreceptors increases ventilatory drive. Compensation for hypercapnia, hypoxia, and metabolic acidosis is discussed.

[Purpose] The purpose of this study was to examine changes in neuronal expression of c-Fos protein in the medulla oblongata after unilateral phrenicotomy in Wistar rats. [Subjects and Methods] After left side phrenicotomy, rats were maintained for 4 weeks (test experiments), then transcardially perfused with fixatives. Brain sections were prepared, and c-Fos protein induced in neurons in the medulla oblongata was immunohistochemically stained by the ABC method. Control rats were maintained for the same period after sham operation, and the brain sections were similarly processed as in the test experiments. [Results] The number of c-Fos immunoreactive (c-Fos-ir) neurons in the medulla oblongata was counted and their spatial distributions were mapped. In both sides of the ventral respiratory group (VRG) and the left side of dorsal respiratory group (DRG), numbers of c-Fos-ir neurons were significantly increased in phrenicotomy rats compared to the control rats (P

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3–C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Botzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 μm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2–C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Botzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Botzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.

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.

It has been well established that inspiratory neurons of the dorsal respiratory group (DRG) are classified into two types based on whether they receive inputs from slowly-adapting pulmonary stretch receptors (SARs) or not. Inspiratory neurons with SAR inputs are called Iβ and the others are called Iα neurons. In this study, we identified a novel group of inspiratory neurons in Nembutal-anesthetized, paralyzed, and artificially-ventilated rats. (1) These DRG inspiratory neurons were activated characteristically by lung deflation. (2) They were orthodromically activated by electrical stimulation of the vagus nerve at a low intensity just above the threshold for afferents from SARs or rapidly-adapting pulmonary stretch receptors (RARs). (3) The orthodromic latencies (ranged from 1.9 to 2.5 ms) indicated that they receive direct inputs from low-threshold vagal afferents. (4) Unlike Iβ neurons, they hardly responded to lung inflation and never exhibited tonic firing in response to maintained lung inflation. (5) The majority (92%) of them were antidromically activated by electrical stimulation of the cervical spinal cord. These deflation-sensitive inspiratory neurons clearly form a distinct group, and their firing pattern is consistent with the hypothesis that they receive inputs from RAR afferents as well as the central inspiratory drive. The results indicating that DRG inspiratory neurons are classified into at least three groups provide new insights into the organization and role of the DRG.

Using a histochemical technique, we examined distribution of the neurons containing a marker of nitric oxide synthase (NOS), NADPH-diaphorase (NADPH-d), on frontal slices of the medulla and upper cervical spinal segments of 4-day-old rats. It was demonstrated that NADPH-d-positive cells are present within the dorsal and ventral medullary respiratory groups. The highest density of the labeled middle-size multipolar neurons (27.9±2.6 cells per 0.1 mm2 of the slice) was observed in the rostral part of the ventral respiratory group, within the reticular lateral paragigantocellular nucleus. Similar NADPH-d-positive neurons were also observed in other reticular formation structures: rostroventrolateral reticular, gigantocellular, and ventral medullary nuclei, and in the ventral part of the paramedial nucleus. There were no labeled neurons in the lateral reticular nucleus. Single small and medium-size labeled neurons were found at all rostro-caudal levels of thenucl. ambiguous (nuclei retrofacialis, ambiguous, andretroam-biguous). Groups of NADPH-d-positive neurons were also revealed within the dorsal respiratory group, along the whole length of thenucl. tractus solitarii (mostly in its ventrolateral parts). Single labeled neurons were also observed in thenucl. n. hypoglossi, and their groups were observed in the dorsal motor part of thenucl. n. vagus. Involvement of the structures containing NADPH-d-positive neurons in the processes related to generation of the respiratory activity is discussed. Our neuroanatomical experiments prove that in early postnatal mammals NO is actively involved in generation and regulation of the medullary respiratory rhythm.

In order to characterize the brain stem circuitry that produces vocalization, the activities of brain stem respiratory neurons were recorded extracellularly during vocalization induced by electrical stimulation of the periaqueductal gray in decerebrate cats. After the onset of stimulation, the respiratory rhythm ceases, and a preparatory inspiration is induced. Following this initial inspiration, vocalization characterized by increased activities of the intrinsic laryngeal adductor and the major expiratory muscles is induced. During vocalization, most of the dorsal respiratory group inspiratory neurons increase their firing rates in phase with an increase of diaphragm activity. Inspiratory neurons with a continuous discharge pattern in the rostral ventral respiratory group increase their firing rates to augment intrinsic laryngeal abductor motoneurons and bulbospinal inspiratory neurons in the dorsal respiratory group. On the other hand, most of the bulbospinal augmenting expiratory neurons in the Bötzinger complex cease firing just after the onset of periaqueductal gray stimulation for the remainder of the stimulation period. These results indicate that at least some part of the coordinated activations of intrinsic laryngeal and respiratory muscles during vocalization are mediated via the central respiratory neurons that produce breathing.

To clarify the difference between the firing patterns of the dorsal respiratory group (DRG) and ventral respiratory group (VRG) neurons during spontaneous augmented breaths, extracellular single unit recording of 139 respiratory-related neurons (inspiratory: 98/139, expiratory: 41/139) was performed in pentobarbital anesthetized rats. Both the I and E neurons were further classified into six groups: (1) I-augmenting, (2) I-decrementing, (3) I-other cells, (4) E-augmenting, (5) E-decrementing and (6) E-other cells. During the augmented breaths, most inspiratory neurons in the DRG (25/26) and VRG (67/72) show an increase in their discharge frequency irrespective of the cell type, but the discharges after an augmented breath were inhibited. Changes in these inspiratory neurons coincided with those of diaphragm electromyogram activity. With regard to relative changes in the mean firing rate during the inspiratory phase II of augmented breaths, there was a significant difference between the I-augmenting DRG and VRG neurons (353.5 +/- 56.9% vs. 237.5 +/- 17.1%, P0.01), but not in the I-decrementing and I-other neurons. On the other hand, the activities of the expiratory DRG (9/9) and VRG (27/32) neurons decreased during the augmented breath. A significant difference in the relative mean firing rate during the expiratory phase of augmented breaths was observed between the E-decrementing DRG and VRG neurons (27.3 +/- 5.2% vs. 58.0 +/- 6.3%, P0.05), but not between the E-augmenting and E-other neurons. These results suggested that during spontaneous augmented breaths the firing patterns of the DRG neurons were not qualitatively different from those of the VRG neurons.

This study tested the hypothesis that the short-latency excitation of the phrenic motor output elicited by superior laryngeal nerve (SLN) stimulation requires non-NMDA receptor-mediated neurotransmission in the region of the dorsal respiratory group (DRG) of the adult cat. Injection of the non-NMDA receptor antagonist NBQX into the DRG severely attenuated or abolished the short-latency excitation, indicating that the short-latency excitation requires non-NMDA receptor-mediated neurotransmission within the DRG.

1. The vestibular system responds to accelerations of the head and produces reflex responses that serve a variety of compensatory functions. The neuronal circuitry that mediates vestibulo-respiratory reflexes is largely unknown. The purpose of the present study was to investigate the possible role of bulbospinal inspiratory neurons located in the para-ambigual region of the ventral respiratory group (VRG) in mediating these reflexes. Experiments were carried out in cats that were decerebrated, paralyzed, and artificially ventilated. 2. Activation of the vestibular nerve by electrical stimulation produced prominent bilateral reflex responses recorded from the phrenic nerve, which supplies the diaphragm. The responses could be complex and consisted of a decrease and/or increase in nerve discharge. 3. Extracellular recordings were made from 35 VRG inspiratory neurons that were antidromically activated from the upper cervical spinal cord. Almost one-half of these neurons (15/35, 43%) responded to vestibular stimulation. The neuronal response patterns were consistent with VRG inspiratory neurons contributing to the vestibular reflex response simultaneously recorded from the phrenic nerve. 4. The present results indicate that approximately one-half of VRG bulbospinal inspiratory neurons contribute to vestibulo-respiratory reflexes. These findings are in contrast to our recent neuroanatomic and electrophysiological studies which revealed a paucity of vestibular inputs to the dorsal respiratory group (DRG) located in the ventrolateral nucleus of the solitary tract. Thus there appears to be a difference between inspiratory neurons in the DRG and VRG in regard to participating in vestibulo-respiratory reflexes.

The interaction between afferent inputs from carotid body chemoreceptors (CCRs) and from slowly adapting pulmonary stretch receptors (PSRs) on the discharge patterns of medullary inspiratory (I) and expiratory (E) neurons was characterized in thiopental sodium-anesthetized, paralyzed, ventilated dogs. A cycle-triggered ventilator was used to produce control and test pulmonary afferent input patterns. The CCRs were stimulated by phase-synchronized bolus injections of CO2-saturated saline into the common carotid arteries. Only those neurons whose discharge time course was altered by both inflation and CCR activation were studied. The dorsal respiratory group (DRG) I inflation-insensitive neurons were also included. Cycle-triggered histograms of unit activity were obtained for the neuronal responses to inflation, CO2 bolus, and their combination, as well as for the spontaneous control condition. Linearity of the interaction was tested by comparing the sum of the net individual responses to the net response of the combined afferent inputs. The results suggest that a linear (additive) interaction between CCR and PSR inputs exists for the DRG I inflation-sensitive neurons, the ventral respiratory group (VRG) I decrementing, and caudal VRG E augmenting neurons, while a nonadditive interaction exists for caudal VRG E decrementing bulbospinal neurons. The implications of these findings are discussed.

There has been controversy whether the dorsal respiratory group (DRG), identified in the cat and several other species as a concentration of mainly inspiratory neurons located in the ventrolateral subnucleus of the solitary tract, also exists in the rat. The aim of this study was to re-examine this question by systematically exploring this region with extracellular microelectrodes, in anesthetized and artificially ventilated rats. One-hundred and forty-two units were recorded which fired in phase with central respiratory cycles (determined by recording from the phrenic nerve) and/or lung inflations. One-hundred and nineteen recordings were thought to be from neuronal cell bodies (confirmed in some cases by excitatory responses to microelectrophoretic administration of dl -homocysteic acid), while the remaining 23 were from lung vagal afferents. Most neurons in the former group (87/119) were inspiratory. Out of 96 neurons tested for spinal projections only 14 (12 inspiratory, 2 expiratory) responded antidromically following stimulation at C3 segment. These results confirm the existence of the DRG in the rat and demonstrate that neurons located in this region have firing patterns generally similar to those previously described in the cat. The main difference is the relative paucity in the rat of neurons projecting spinally below the C2 level, which indicates that most DRG neurons in this species do not project directly to phrenic and intercostal motoneurons, but to other, as yet unidentified, neuronal groups within the brainstem or upper cervical segments.

Vomiting is a nonrespiratory behavior during which the major respiratory muscles contract in a characteristic pattern which generates changes in the intrathoracic and intra abdominal pressures that lead to expulsion of gastric content. During vomiting, the major inspiratory muscles (the diaphragm and the external intercostal) co-contract with the expiratory abdominal muscles in a series of activity bursts.The question arises whether brain stem respiratory neurons are involved in the control of these muscles during vomiting. In the present study, we undertook to determine whether bulbospinal inspiratory neurons in the dorsal respiratory group (DRG) and in the para-ambiguus region of the ventral respiratory group (VRG), together with the upper cervical inspiratory propriospinal neurons, are important for activation of the diaphragm and external intercostal muscles during vomiting. To analyze the behavior of these neurons, we elicited “fictive vomiting” using either emetic agents or electrical stimulation of the supradiaphragmatic vagal nerve afferents in decerebrate paralyzed cats. This fictive vomiting was identified by a characteristic series of bursts from coactivation of the phrenic nerve and some other nerves innervating abdominal muscles, which would be expected to produce an expulsion of gastric contents in nonparalyzed animals.Only a few (

Selective neuronal vulnerability of the motor cortex, basal ganglia, brainstem, medulla, cerebellum, C6 and L6 segments of the spinal cord were studied after 15 min of cardiac arrest followed by 1 h of normoxic or hyperoxic resuscitation using the suppressive Nauta method in dogs. Hyperoxic resuscitation causes characteristic somatodendritic argyrophilia of the interneuronal pool in the spinal cord and lower medulla. Cuneate, lateral reticular, supraspinal, and caudal trigeminal nuclei as well as the dorsal and ventral respiratory neuronal groups were heavily involved. Similarly, the Purkinje cells, neurons in the middle and deep portions of the mesencephalic tectum, perirubral, pretectal, posterior commissure, middle-sized striatal and giant pyramidal (Betz's) neurons in the motor cortex became argyrophilic. Hyperoxic resuscitation versus normoxic resuscitation causes statistically significant somatodendritic argyrophilia of the dorsal respiratory group, cuneate, dorsal lateral geniculate and thalamic reticular nuclei.

The location and axonal projection of a type of respiratory neurones (termed bIE neurones), which show burst firing at the time of phase transition from inspiration to expiration, were studied in Nembutal-anaesthetized, paralysed and artificially ventilated cats. The bIE neurones showed maximum firing at the sharp decline of inspiratory activity. All of the bIE neurones tested projected to medullary respiration-related areas of the ventral respiratory group (VRG), the dorsal respiratory group (DRG), and the Bötzinger complex (BOT). The bIE neurones were distributed in the dorso-medial border of the main assembly of respiratory neurones of the VRG and BOT. The possibility that these bIE neurones participate in inspiratory termination is discussed.

A great number of severed central nervous system (CNS) neurons of the adult rat have the capability to regrow axons into peripheral nerve autografts. In the present experiment, autologous segments of the peroneal nerve were inserted perpendicularly to the dorsal surface of the medulla oblongata more or less laterally within either the ventral respiratory group or the so-called dorsal respiratory group (ventrolateral grafts, n = 5; dorsolateral grafts, n = 5). From 2 to 4.5 months after the graft implantation, spontaneous unitary activities ( n = 197) were recorded within all the grafted nerves: they were found to arise from both central respiratory (R, n = 60) and non-respiratory (NR, n = 137) neurons which were giving off regenerated axons along the nerve grafts. The graft reinnervation by respiratory axons was found to be significantly more abundant within the medullary ventrolateral grafts than within the dorsolateral ones. The low rate of axonal regeneration from respiratory neurons observed within the dorsolateral grafts provides further evidence that the number of the respiratory neurons in the dorsal respiratory group, if present at all, is much smaller than that of the ventral respiratory group in the rat.

We investigated connections between the rostral ventrolateral medulla (rVLM) and the two main inspiration-related medullary areas, i.e., the dorsal respiratory group (DRG) and the rostral ventral respiratory group (rVRG) in the cat. Non respiration-related tonically firing units encountered in the rVLM displayed either antidromic or orthodromic responses to DRG or rVRG microstimulation. Some units responded to the stimulation of both regions. We suggest that at least part of rVLM neurons are components of medullary loops operating in the control of breathing.

Axonal projections of second order neurons activated by vagal afferent fibers originating from pulmonary rapidly adapting receptors (RARs) were studied electrophysiologically in Nembutal-anesthetized, paralyzed and artificially ventilated cats. Extracellular recordings from these neurons (referred to as 'RAR-cells') were made in the commissural subnucleus (COM) of the nucleus of the solitary tract (NTS). They were identified by a combination of stimuli including stimulation of the vagus nerve(s), hyperinflation and deflation of the lungs, and a brief period of ammonia vapor inhalation. A total of 80 RAR-cells were tested for axonal projections to a respiration-related area in the brain-stem, either the dorsolateral rostral pons or the dorsal respiratory group (DRG) or the ventral respiratory group (VRG) or the Bötzinger complex (BOT) and/or the spinal cord. Twenty-two of the 47 (47%) RAR-cells tested for ipsilateral pontine projection could be antidromically activated, and in 8 cases evidence for axonal arborization was obtained. Only 1 of the 11 RAR-cells tested for DRG projection, and 1 of the 10 RAR-cells tested for VRG projection, were antidromically activated. No RAR-cells were activated from the BOT (n = 8) or from the C3-C4 segments of the spinal cord (n = 11). Bilateral lesions of the COM abolished the reflex responses induced by ammonia inhalation or hyperinflation of the lungs, but not the Hering-Breuer reflex. These results indicate that a pathway from the COM to the rostral pons forms part of the reflex arc originating from RARs.

To determine distribution, discharge patterns and the spinal projections of medullary respiratory neurons (RNs), a systematic mapping of 806 RNs was made in the medulla of anesthetized rabbits. In disagreement with previous reports that there are no discrete medullary respiratory neuronal groups in rabbits, two neuronal groups were identified: (1) dorsal respiratory group (DRG), associated with the nucleus tractus solitarius; and (2) ventral respiratory group (VRG), associated with the nucleus ambiguus compact formation. The density of RNs in the DRG was much lower than that in the VRG. In the VRG, 3 subdivisions of RN populations were found: predominantly expiratory neurons in the caudal and the rostral parts, and mainly inspiratory neurons in the intermediate region. Nine distint types of RNs were classified on the basis of firing patterns. Nearly all types were found in both the DRG and each VRG subdivision. Antidromic mapping of 64 VRG neurons revealed that 67% projected to the spinal cord. Expiratory bulbospinal neurons in the rostral subdivision of the VRG projected only to the cervical cord (mainly ipsilaterally). Most neurons of the intermediate and caudal subdivisions of the VRG (74%) appeared to project either contralaterally or ipsilaterally below T1. The axonal conduction velocity was 40–50 m/s by two-point determinations. We conclude that respiratory neuronal groups in the medulla of the rabbit are generally similar to those of the cat. Nearly equal proportions of bulbospinal RNs projected to the ipsilateral vs contralateral spinal cord.

To determine whether bulbar respiratory neurons are active in a manner similar to respiratory motor nerves, in which diverse activity patterns have been observed in dogs, the discharge of 112 bulbospinal and 155 propriobulbar respiratory neurons was observed during retching and vomiting evoked by stimulation of the vagal nerve in decerebrate, paralyzed dogs. During retching, the respiratory activities of all respiratory neurons changed. The discharge patterns during retching were classified into seven types in accordance with the discharge phase in the retching cycle and the discharge frequency. Inspiratory bullbospinal neurons (39) in the ventral respiratory group (VRG) exhibited discharge synchronous with retches at higher (14/39) or lower (4/39) frequencies than those of their respiratory discharge, discharge between retches at higher (3/39) or lower (14/39) frequencies and discharge depressed throughout the retching period (4/39). Inspiratory bulbospinal neurons (30) in the dorsal respiratory group (DRG) and expiratory bulbospinal neurons (23) in the VRG also fired with some of the seven types of discharge. No significant difference was recognized between the ratio of the numbers of inspiratory bulbospinal neurons firing with the seven types of discharge and that of expiratory bulbospinal neurons. On the other hand, the respiratory propriobulbar neurons (73) in the VRG and those (45) in the DRG seemed to be differently activated; that is, 44% of the propriobulbar neurons in the VRG fired synchronously with retches at a higher frequency than their frequency during breathing, while only 22% of those in the DRG fired at such a high frequency with retches. However, the ratio of whole inspiratory neurons (174) exhibiting the seven types of discharge was almost the same as that of whole expiratory neurons (93). During vomiting (fictive expulsion), three of five expiratory neurons fired, but most (14/16) of inspiratory neurons did not. In conclusion, all of the activity patterns which have been observed during retching in motor fibers innervating respiratory muscles are found in both bulbospinal and propriobulbar respiratory neurons in dogs. Furthermore, since the firing patterns of inspiratory bulbospinal neurons during retching in the dogs in this study are apparently different from those in cats (Miller et al., 1990), inspiratory neurons and muscles seem to be differently organized during retching in cats and dogs.

The purpose of this study was to examine whether treadmill running exercise after phrenicotomy resulted in change in neuronal expression of c-Fos protein in the medulla oblongata in Wistar rats. All rats were subjected to left-sided unilateral phrenicotomy. In test experiments, rats were trained with treadmill running for 4 weeks, while in control experiments rats were not provided with the opportunity to exercise. c-Fos protein, a transient transcription factor, was immunohistochemically stained by the ABC method, and surveyed in the medulla oblongata. Numbers of c-Fos-immunoreactive (c-Fos-ir) neurons were counted microscopically, and their distribution in the medulla oblongata was mapped. In the dorsal respiratory group (DRG), numbers of c-Fos-ir neurons were found to be significantly increased in the test rats compared with control rats, while in the ventral respiratory group (VRG) no significant differences were found in numbers of c-Fos-ir neurons between test and control rats. These findings suggest that DRG neurons may play more important roles than VRG neurons in recovery of respiration in treadmill exercise after unilateral phrenicotomy in Wistar rats.

The effect of enflurane on the firing activity (spikes/sec) of the inspiratory neurons of the dorsal respiratory group (DRG) of the medulla oblongata was studied in decerebrate, paralyzed, mechanically ventilated cats before and after bilateral cervical vagotomy. Inspiratory neuronal activity, phrenic neurogram, arterial blood pressure, tracheal pressure, and end tidal CO2 concentration were recorded. Cells whose firing activity was in phase with that of the phrenic nerve were considered inspiratory neurons. Administration of 1 and 2% enflurane in oxygen produced gradual, significant, and dose-dependent depression of the cell activity with cervical vagi either intact or severed. Recovery of the cell activity occurred after termination of enflurane administration. In cats with intact vagi, 10 min after introduction of 1 and 2% enflurane, the cell activity (mean +/- SE) expressed as percentage of the control was 70 +/- 6% (P less than 0.05) and 48 +/- 5% (P less than 0.01), respectively. Bilateral cervical vagotomy did not affect the degree of cell depression due to enflurane. Hypercarbia induced by inhalation of 5% CO2 increased cell activity, but it did not block enflurane-induced cell depression, although it reduced it. It may be concluded that enflurane depresses the activity of the inspiratory neurons of the DRG. The results also suggest that the respiratory depressant effect of enflurane has a central component and that the DRG region may serve as a site to mediate the enflurane-induced respiratory depression.

Brainstem projections to the phrenic nucleus were studied in rabbits using horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) as a retrograde and anterograde neuronal tracer. Injections of 1% WGA-HRP were centered in the phrenic nucleus in the C4-C5 ventral horn in 4 rabbits to identify pontomedullary nuclear groups that contain neurons projecting to the midcervical spinal cord. Regions of the rabbit brainstem that are homologous to the ventral respiratory group (VRG), dorsal respiratory group (DRG), Bötzinger Complex (BötC) and Kölliker-Fuse nucleus in the cat and rat were shown to provide the major pontomedullary projections to the phrenic nucleus. Injections of WGA-HRP into physiologically identified locations within DRG, VRG and BötC anterogradely labelled bulbospinal axons of these groups. These injections produced presumptive terminal labelling in the C4-C5 ventral horn in the region containing the phrenic cell column and the transverse phrenic motoneuron dendrite bundles as defined by WGA-HRP labelling of phrenic motoneurons. These results indicate: 1) The presumptive excitatory (DRG, VRG) and inhibitory (BötC) bulbospinal control of phrenic motoneurons arise from the same medullary respiratory groups in the rabbit as in the cat and rat. 2) The bulbospinal control of phrenic motoneurons is primarily via direct projections to the phrenic motor nucleus, and not through segmental propriospinal interneurons. 3) As in the rat, the bulbospinal contribution of the DRG is less pronounced in the rabbit than in the cat. 4) The rabbit and rat have a slight ipsilateral predominance in their bulbospinal projections to phrenic nucleus; whereas these projections have a contralateral predominance in the cat.

In decerebrate, paralyzed and ventilated cats, we monitored the intracellular responses of 30 inspiratory neurons of the dorsal respiratory group (DRG) to stimulation of vagal and expiratory muscle (internal intercostal and abdominal) afferents. We hypothesized that the inhibitory effects of stimulation of expiratory muscle afferents, previously reported, would block the excitatory responses of inspiratory neurons of the DRG to vagal stimulation. Although prolonged stimulus trains to expiratory muscle afferents caused respiratory phase-switching, single shocks or short trains elicited no responses in 17 bulbospinal neurons, excitatory responses in 6, and inhibitory responses in 2. Of the 4 propriobulbar neurons tested, 2 had inhibitory responses and 2 did not respond. In only 2 neurons, both bulbospinal, did conditioning stimuli to expiratory muscle afferents block or reduce the excitatory effects of vagal stimulation. These results suggest that interaction of vagal and expiratory muscle afferents, which might account for the absence of a change in inspiratory duration despite increased vagal afferent feedback at elevated end-expiratory lung volumes, does not occur within the DRG.

Previous transneuronal tracing studies conducted in the ferret revealed that a large population of neurons that provides inputs to diaphragm and abdominal motoneurons is located in the ventral magnocellular portion of the medial medullary reticular formation. These observations raise the possibility that the neural substrate underlying respiratory rhythmogenesis may be different in the ferret than in other species in which this circuitry has been explored. In the present study, systematic tracking was conducted through the ferret medulla to map the locations of neurons with activity related to the contractions of respiratory muscles. As in the cat, rat, and rabbit, neurons with respiratory-related discharges were distributed either lateral or ventrolateral to the solitary nucleus (dorsal respiratory group) or in the vicinity of nucleus retroambigualis, nucleus ambiguus and the retrofacial nucleus (ventral respiratory group). Although the general organization of respiratory group neurons appeared to be similar in the ferret to that in other mammals, a difference was that few expiratory neurons were located rostrally in the ventral respiratory group. These data suggest that the ventral magnocellular medullary reticular formation is not essential for respiratory rhythm generation, at least during quiet breathing, but may participate in regulating the excitability of respiratory motoneurons or in coordinating the contractions of respiratory muscles during nonrespiratory responses (e.g. coughing or emesis).

We examined the role of dorsal respiratory group (DRG) inspiratory neurons as transmitters of respiratory drive to phrenic and intercostal motoneurons and as relays of afferent information to ventral respiratory group (VRG) bulbospinal, inspiratory neurons. Attempts to antidromically activate 76 DRG neurons from the spinal cord at the C7 segment resulted in only 4 (5.3%) successes (3 contralateral, 1 ipsilateral). Cross-correlating DRG neuron discharge with that of the ipsilateral (56) and contralateral (20) phrenic nerve detected common activation peaks in 2 and 3 cases respectively, with no evidence for monosynaptic connections. Cross-correlating DRG neuron discharge with that of bulbospinal, inspiratory VRG neurons found some evidence for interaction. Peaks in 7 of 73 (10%) cross-correlation histograms were attributed to a monosynaptic excitation of DRG neurons by VRG neurons, although a common activation cannot be ruled out; troughs, some with an accompanying peak, in 9 (12.3%) histograms were interpreted as a combined excitation of the DRG neuron and delayed inhibition of the VRG neuron. In addition, 2 cross-correlation histograms showed peaks with latencies and half-amplitude widths consistent with a disynaptic excitation of a DRG neuron by a bulbospinal inspiratory VRG neuron. Cross-correlating the discharge of 57 pairs of DRG inspiratory neurons (6 contralateral) detected common activation peaks in 7 (12.3%) cases (none contralateral) and one case interpreted as evidence for a disynaptic excitation. These findings suggest that the role of the DRG inspiratory neurons in rats differs from that in cats, primarily because they do not act to transmit respiratory rhythmic drive directly to phrenic and intercostal motoneurons. The results offer some support for an excitation of DRG neurons by VRG inspiratory neurons, but no support for a role of DRG inspiratory neurons as mediators of afferent information transfer to VRG bulbospinal inspiratory neurons.

Extracellular recordings were made in the dorsal respiratory group (DRG) and adjacent reticular formation following single-shock stimulation of the anterior ethmoidal nerve (AEN) and during sneeze evoked by repetitive stimulation of the AEN in nembutal-anaesthetized, curarized and ventilated cats. These neurones were characterised according to (i) their activity during the respiratory cycle (as inspiratory augmenting or decrementing (I Aug or I Dec), expiratory augmenting or decrementing (E Aug or E Dec), silent or tonic), and (ii) their axonal projection (bulbospinal or non-bulbospinal-non-vagal (BS or NBS-NV)). Following single-shock stimulation of the AEN, most of the inspiratory neurones were transiently inhibited, whereas E Aug neurones were activated and E Dec neurones were activated and then inhibited. Silent neurones responded with a multispike or a paucispike pattern. Following repetitive stimulation of the AEN and during the resulting sneeze reflex, I Aug neurones increased their activity in parallel with the phrenic activity, I Dec neurones fired at the onset and at the end of the inspiration, E Dec and some silent neurones fired either during the compressive phase or after the expulsive phase, whereas E Aug and some silent neurones fired during the expulsive phase. We conclude that sneeze involves a reconfiguration of the central respiratory drive which uses, at least partly, the respiratory network to trigger a non-ventilatory defensive motor act.

Membrane potential changes and/or discharges from 36 inspiratory neurons were recorded intracellularly in the dorsal respiratory group (DRG; i.e., the ventrolateral subdivision of the nucleus tractus solitarii) in decerebrate, paralyzed, and ventilated cats. Electrical activities were recorded from both somata (n = 10) and axons (n = 26). Activities during quiet breathing were compared with those observed during fictive coughing and swallowing evoked by repetitive electrical stimulation of afferent fibers of the superior laryngeal nerve (SLN). These nonrespiratory behaviors were evident in paralyzed animals as characteristic discharge patterns of the phrenic, abdominal, and hypoglossal nerves. Twenty-six neurons exhibiting antidromic action potentials in response to electrical stimuli applied to the cervical (C3-5) spinal cord were classified as inspiratory bulbospinal neurons (IBSNs). These neurons were considered as premotoneurons. The remaining 10 inspiratory neurons (I-NAA) were not antidromically activated by electrical stimuli applied to either cervical spinal cord or ipsilateral cervical vagus. These neurons are thought to be propriobulbar neurons. We recorded the activity of 31 DRG inspiratory neurons (24 IBSNs and 7 I-NAA) during coughing. All but one (a late-recruited IBSN) discharged a burst of action potentials during the coughing-related phrenic nerve activity. Typically, ramp-like membrane depolarization trajectories and discharge frequencies during coughing were similar to those observed during inspiration. We recorded the activity of 33 DRG inspiratory neurons (23 IBSNs and 10 I-NAA) during swallowing. Most (28/33) neurons were briefly activated, i.e., discharged a burst of action potentials during swallowing, but peak discharge frequency decreased compared with that measured during inspiration. The membrane potentials of nine somata exhibited a brief bell-shaped depolarization during swallowing, the amplitude of which was similar to that observed during inspiration. These results suggest that some inspiratory premotoneurons and propriobulbar neurons of the DRG might be involved in nonrespiratory motor activities, even if clearly antagonistic to breathing (e.g., swallowing). We postulate the existence in the medulla oblongata of adult mammals of neurons exhibiting a "functional flexibility".

1. This study addressed the hypothesis that there is a parallel processing of input from carotid chemoreceptors to brainstem neurones involved in inspiratory phase timing and control of inspiratory motor output amplitude. Data were from fifteen anaesthetized, bilaterally vagotomized, paralysed, artificially ventilated cats. Carotid chemoreceptors were stimulated by close arterial injection of 200 microliters of CO2-saturated saline solution. 2. Planar arrays of tungsten microelectrodes were used to monitor simultaneously up to twenty-two neurones in the nucleus tractus solitarii (NTS) and ventral respiratory group (VRG). Spike trains were analysed with two statistical tests of respiratory modulation, cycle-triggered histograms, peristimulus-time histograms, cumulative sum histograms and cross-correlograms. 3. In NTS, 16 of 26 neurones with respiratory and 12 of 27 without respiratory modulation changed firing rate during carotid chemoreceptor stimulation. In the VRG 72 of 112 respiratory and 14 of 48 non-respiratory neurones changed firing rate during stimulation. 4. The spike trains of 85 of 1276 pairs (6.7%) of cells exhibited short time scale correlations indicative of paucisynaptic interactions. Ten pairs of neurones were each composed of a rostral VRG phasic inspiratory neurone that responded to carotid chemoreceptor stimulation with a decline in firing rate and a caudal VRG phasic inspiratory neurone that increased its firing rate. Cross-correlograms from two of the pairs had features consistent with excitation of the caudal neurones by the rostral cells. A decrease in the duration of activity of the rostral VRG neurones was paralleled by the decrease in inspiratory time of phrenic nerve activity. Caudal VRG inspiratory neurones increased their activity as phrenic amplitude increased. Spike-triggered averages of all four neurones indicated post-spike facilitation of phrenic motoneurones. 5. The results support the hypothesis that unilateral stimulation of carotid chemoreceptors results in parallel actions. (a) Inhibition of rostral VRG I-Driver neurones decreases inspiratory duration. (b) Concurrent excitation of premotor VRG and dorsal respiratory group inspiratory neurones increases inspiratory drive to phrenic motoneurones. Other data suggest that responsive ipsilateral neurones act to regulate contralateral neurones.

Exposure of anesthetized paralyzed vagotomized peripherally chemodenervated cats to hypoxia results in initial depression and subsequent loss of the phrenic neurogram. To determine whether hypoxic respiratory depression results from the inhibition of respiratory premotor neurons by bulbospinal neurons of the Botzinger complex (Bot-E neurons), extracellular recordings were made of dorsal and ventral respiratory group bulbospinal inspiratory neurons and Bot-E neurons during acute hypoxic hypoxia. All neurons recorded decreased firing rate during hypoxia. Bot-E neurons became silent before the loss of phasic phrenic activity during hypoxia and commenced firing before or coincident with the return of the phrenic neurogram during reoxygenation. Inspiratory neurons ceased firing coincident with phrenic silence. Dorsal respiratory group and ventral respiratory group neurons that had a late onset of firing with respect to the phrenic neurogram during normoxia fired progressively earlier in inspiration during hypoxia, an effect that was reversed during reoxygenation. These data are consistent with inhibition and/or disfacilitation as the mechanism of hypoxic respiratory depression but suggest that Bot-E neurons are not the source of this inhibition.

Ipsilateral and contralateral pairs of augmenting expiratory neurons were recorded simultaneously from the Bötzinger complex using glass-coated tungsten microelectrodes in pentobarbitone-anaesthetized cats. The neurons were identified both by firing pattern and by antidromic activation from the contralateral site of the dorsal respiratory group. Cross-correlation histograms of the extracellularly recorded action potentials were calculated in order to detect short time-scale synchronizations of firing indicative of synaptic connections between the neurons. The cross-correlation histograms for 40 ipsilateral pairs of neurons less than 1 mm apart showed eight (20%) narrow troughs (mean half-amplitude width +/- SD, 1.1 +/- 0.37 ms) at short latencies (mean latency +/- SD, 1.0 +/- 0.35 ms) suggestive of monosynaptic inhibition. These included two cross-correlation histograms which showed troughs on both sides of time zero, indicating a mutual inhibition. For another four pairs of neurons (10%), a central broad peak suggestive of common activation due to either excitation or release from inhibition was evident. Contralateral pairs of expiratory neurons of the Bötzinger complex were examined in a similar manner. The cross-correlation histograms for 43 pairs of neurons showed five (12%) narrow troughs (mean half-amplitude width +/- SD, 1.2 +/- 0.67 ms) at short latencies (mean latency +/- SD, 2.7 +/- 1.47 ms) suggestive of monosynaptic inhibition. These included one cross-correlation histogram which showed troughs (one not statistically significant) on both sides of time zero, indicating a mutual inhibition. For another two pairs of neurons (4.6%) a central, broad peak suggestive of common activation due to either excitation or release from inhibition was evident. We conclude that inhibitory interconnections exist between augmenting expiratory neurons of the Bötzinger complex ipsilaterally and contralaterally. These connections may synchronize the expiratory burst of activity within this population and assist in the patterning of the burst.

The location and firing patterns of medullary respiratory neurons have been described in a small number of species. The cat has been the most widely studied species, but some potentially important differences have recently been noted in others. A more complete survey of species is required to determine the significance of these differences. We describe the location and firing patterns of respiratory neurons in the medulla of anesthetized, paralyzed and mechanically ventilated adult guinea pigs. Extracellular single-unit recordings were made from the medulla, their phase relationship with phrenic nerve activity used to define them as respiratory and their location marked with fast green. Respiratory units were concentrated ventrolateral to the nucleus tractus solitarius (NTS) and within and surrounding the nucleus ambiguus (NA), corresponding to the dorsal respiratory group (DRG) and ventral respiratory group (VRG) of the cat, respectively. Most DRG respiratory units were inspiratory, while the VRG contained equal numbers of inspiratory and expiratory units. The DRG and VRG both contained early, late and constant-frequency inspiratory and expiratory units. In general, these findings are similar to those in other mammalian species examined, consistent with these basic aspects of the respiratory network being highly conserved.

Synaptic inputs from afferents in the superior laryngeal nerve (SLN) to medullary respiratory neurons (n=154) in the dorsal respiratory group (DRG), ventral respiratory group (VRG) and the region of the Bo¨tzinger complex (BOT) were studied in anesthetized cats. Single pulse stimulation of the SLN-evoked monosynaptic EPSPs in most inspiratory bulbospinal (I-BS) neurons in the DRG, and disynaptic or oligosynaptic chloride-dependent IPSPs in other I-BS neurons in the DRG and VRG. Stimulation of laryngeal afferents also inhibited oligosynaptically expiratory bulbospinal neurons in the VRG, and all types of respiratory neurons recorded in the BOT region. Oligosynaptic potentials (usually EPSPs) were recorded in inspiratory and expiratory laryngeal motoneurons. These results provide evidence of a processing of SLN-evoked synaptic responses by all tested groups of medullary respiratory neurons. The pathways mediating these synaptic responses are discussed.

Experiments were performed on anaesthetized, vagotomized rabbits. Respiratory movement and phrenic rhythmical discharge were reversibly abolished by the symmetrical injection of 1% procaine into the medial area of the nucleus retrofacialis (mNRF). Blocking other areas of the medulla had no obvious effect on respiratory rhythm, with the exception of the rostral portion of the ventral respiratory group (VRG), which overlaps with the mNRF. When the mNRF was blocked, most inspiratory and expiratory neurons recorded in the VRG and DRG (dorsal respiratory group) gradually started to fire continuously, and no longer exhibited respiratory rhythm. A minority of respiratory neurons was inactivated during apnea. Stimulation of the caudal portion of the DRG and VRG evoked only a short cluster of phrenic discharges instead of rhythmical firing, indicating that the respiratory neurons situated in these areas cannot generate rhythmic activity by themselves. This suggests that the mNRF plays an important role in the genesis and maintenance of basic respiratory rhythm.

We analyzed the membrane potential of 161 respiratory neurons in the medulla of decerebrate rats which were paralyzed and ventilated. Three types of inspiratory (I) neurons were observed: those displaying progressive depolarization in inspiration (augmenting I neurons), those which gradually repolarized after maximal depolarization at the onset of inspiration (decrementing I neurons) and those exhibiting a plateau or bell-shaped membrane potential trajectory throughout inspiration (I-all neurons). Three types of expiratory (E) neurons were also encountered: those in which the membrane potential progressively depolarized (augmenting E neurons), those in which the membrane potential repolarized during the interval between phrenic bursts (decrementing E or post-I neurons) and those exhibiting a plateau or bell-shaped membrane potential trajectory throughout expiration (E-all neurons). Axonal projections of these medullary neurons were identified in the cranial nerves (n = 34), or in the spinal cord (n = 19) as revealed by antidromic stimulation and/or by reconstruction following horseradish peroxidase (HRP) labeling. The other 108 neurons were not antidromically activated (NAA) by the stimulations tested, or had their axons terminating inside the medulla as revealed by HRP labeling. All these respiratory neurons, except for 3 which were hypoglossal motoneurons, had their somata within the ventrolateral medulla, in the region of the nucleus ambiguus, homologous to the ventral respiratory group (VRG) of the cat. No dorsal respiratory group (DRG) was detected within the medulla of the rats. Due to this absence of a DRG, it is concluded that the neural organization of respiratory centers is quite different in cats and rats.

The nucleus tractus solitarii (NTS) relays information from primary visceral receptors to the central nervous system and is critically involved in the reflex control of autonomic functions. In the respiratory system, it is a part of so-called dorsal respiratory group (DRG) and plays an important role in the regulation of respiration. Although people have noticed that the NTS in general is not a simple relay nucleus but a place of information processing, such concept has not necessarily been realized in the respiratory system. Based on our recent data on NTS neurons, we now show some aspects of information processing taking place at the NTS. Inflation or deflation of the lungs evokes various respiratory reflexes by activating mainly two types of pulmonary stretch receptors. Slowly adapting receptors (SARs) are activated solely by inflation of the lungs and firmly involved in the Hering-Breuer reflex; on the other hand, rapidly adapting receptors (RARs) are activated by both inflation and deflation of the lungs but their functional role is still controversial. We aimed to determine the input-output properties of their second-order relay neurons (P-cells and RAR-cells, respectively) in the NTS. P-cells are key neurons of SAR-induced reflexes and have long been regarded as simple relay neurons; however, our experiments in the rat clearly showed that firing of P-cells (and RAR-cells) was modulated with central (not via afferents) respiratory rhythm, indicating that these second-order relay neurons are under influence of the respiratory center. In other words, the respiratory center gates the afferent inputs at the NTS before such inputs act on respiratory neurons that underlie the elaboration of various respiratory reflexes. Now P-cells and RAR-cells have been revealed to receive complex synaptic inputs involving glycinergic and GABAergic inhibitions and non-NMDA and NMDA glutamate receptor-mediated excitations [1]. Therefore, these "relay neurons" are crucially organized into the medullary respiratory network and can exert a phasic influence on the central rhythm generating mechanisms even without receiving afferent inputs from SARs and RARs. RAR-cells respond characteristically to lung deflation but do not necessarily respond to lung inflation. This is somewhat peculiar since RAR afferents respond to both inflation and deflation of the lungs. This suggests the possibility that lung inflation that activates RARs on one hand suppresses RAR-cell firing on the other hand. Recently we found evidence that this suppression is caused by synaptic inhibition of RAR-cells from P-cells (P-R linkage) [2]. This implies that some P-cells are inhibitory and that the SAR and RAR systems are not independent but work cooperatively to evoke respiratory reflexes. On the assumption that RAR pathways mediate inspiratory facilitation, this P-R linkage seems to explain the neuronal mechanisms underlying the Hering-Breuer inflation and deflation reflexes. That is, inflation of the lungs activates preferentially SAR pathways by inhibiting RAR pathways and deflation of the lungs activates RAR pathways. In the NTS area, ie in the DRG, we identified a novel group of inspiratory neurons, which receive monosynaptic inputs from low-threshold vagal afferents and respond to lung deflation [3]. It is quite possible that RAR afferents innervate these deflation-sensitive inspiratory neurons (tentatively termed Iγ neurons). Until now we have no suitable answer to the question why two types of inspiratory (Iα and Iβ) neurons exist in the DRG. The fact that DRG inspiratory neurons are classified into at least three groups, Iα, Iβ and Iγ neurons, has made the situation more complex but provides new insight into the organization and role of the NTS, which integrates afferent inputs and the central respiratory rhythm.

In the cat the dorsal respiratory group comprising the nucleus tractus solitarius (NTS) is regarded as an important respiratory centre. However the relevance of this nucleus in the control of respiration in the rat is still unclear. The present work aimed to identify and localize the various neurons involved in respiration in the nucleus tractus solitarius region of the rat medulla. Further, we intended to study the responses of these identified respiratory neurons to central and peripheral stimulation, in particular: 1)effect of stimulation of midbrain periaqueductal gray (PAG) matter and, 2)effect of stimulation of the vagus (vagal feedback). Nembutal-anaesthetised (80 mg/Kg), spontaneously-breathing rats (n = 60, 300–400 g) were used for the study. Neurons firing in relation to diaphragm electromyogram (dEMG) were located between 0.6–1.5 mm lateral to the midline, extending 0.5 mm caudal to 1.8 mm rostral to the posterior end of the area postrema (AP). Four different populations of respiratory-related neurons were recorded. They have been classified (with reference to dEMG) as: expiratory-inspiratory phase-spanning cells, early inspiratory neurons (eI-cells), cells that fire only in phase with diaphragm (I-all cells), and expiratory cells. The phase-spanning and eI-cell populations were located both in the ventrolateral and ventromedial NTS rostral to the area postrema, while the other cells were found predominantly in the more lateral regions. eI-cells were observed to start firing up to 20 ms prior to dEMG and ceased firing about one third of the way through diaphragm activity. They also showed a decrementing discharge pattern. Excitatory amino acid (DLH, 15–60 nl of 0.2 M) stimulation of PAG along its dorso-ventral axis produced dose-dependent changes in the cardiorespiratory parameters: increased respiratory frequency, decreased inspiratory and expiratory durations, increased blood pressure (BP) and heart rate. PAG stimulation elicited increases in activity in both phase-spanning and eI-neuronal populations in the NTS. The PAG sites evoking an increase in the phase-spanning cell activity were located more laterally than the PAG sites that led to increased eI-cell activity. The increase in the responses of the phase-spanning neurons was not accompanied by any changes in the respiratory frequency, whereas the PAG stimulation giving rise to an increase in eI-neuronal activity also elicited simultaneous increases in dEMG and the respiratory frequency. The central end of the cut vagus was electrically stimulated to activate pulmonary stretch-receptor afferents. Vagal stimulation caused depression of inspiratory cell activity along with bradycardia and a fall in BP. Both eI-neurons and I-all cells were inhibited. These cells exhibited a 'vagal escape' phenomenon ie, resuming activity whilst vagal stimulation continued. Phase-spanning cells and expiratory cells are yet to be tested for responses to vagal stimulation. The behaviours of these respiratory-related cells of the NTS, their temporal relationships with the dEMG, firing patterns, their responses to central and peripheral stimuli are discussed and a possible role for each in the brainstem control of respiration is proposed. Similar studies have also been carried out to elucidate the roles of ventrolateral column (VLM)/ventral respiratory group (VRG) neurons in the control of respiration in the rat. The findings of these studies are presented in an accompanying abstract.

Unilateral focal cold blocks in the region of the nucleus tractus solitarius and the dorsal respiratory group of neurons, DRG, of anaesthetized cats consistently caused apneustic-type breathing. There was no concomitant change in the initial rate of rise of inspiratory activity. The apneustic prolongation of inspiratory duration, TI, was most pronounced in, but was not confined to, the DRG. The apneustic effects were more marked after vagotomy. In cats with intact vagus nerves being given artificial ventilation, focal cooling at certain sites of the DRG region could produce 'unlocking' of the respiratory rhythm from that of the respiratory pump. At other sites in this region, focal cooling could selectively block the effects of the inspiration-facilitating reflex induced by deflation without blocking the inspiration-inhibiting Hering-Breuer reflex. Unilateral focal cold blocks in the region of the intermediate part of the ventral respiratory group of neurons, VRG, generally caused depression of the rate of rise of inspiratory activity, but almost never apneustic effects. All effects of unilateral focal cooling both in the DRG and VRG were bilaterally symmetrical. No systematic differences between the effects on phrenic and external intercostal inspiratory activity were found in response to focal cooling either of the DRG or VRG suggesting that differential control of phrenic and external intercostal motoneurons is not exerted mainly at the level of these medullary structures. The results suggest that the DRG and VRG areas exert somewhat different effects on the respiratory pattern: DRG appears to be more concerned with integration of vagal and other inputs contributing to the inspiratory off-switch mechanisms which, however, are not confined only to the DRG. The VRG inspiratory mechanisms, on the other hand, appear to be more involved in the gain control of the inspiratory output intensity.

Brainstem neurones which project to the immediate vicinity of the spinal motoneurones which supply the intercostal and abdominal respiratory muscles were identified by means of the retrograde transport of horseradish peroxidase (HRP). A combined electrophysiological and histological technique was used in which recording of phasic inspiratory or expiratory motoneurone activity within upper (T3-T4) or lower (T8-T9) thoracic segments was followed by the iontophoretic injection of HRP at these recording sites. HRP labelled cells were concentrated in those brainstem regions known to contain phasic respiratory neurones, namely the ventrolateral nucleus of the solitary tract (vl-NTS) or dorsal respiratory group (DRG), the ambiguus complex or ventral respiratory group (VRG) and the parabrachial pontine (PB) nuclei. In 18 cats, 248 cells were labelled in these three respiratory regions of the brainstem while 668 were much more diffusely distributed in other regions of the medulla and pons. The ipsilateral and contralateral contributions within the respiratory regions were respectively; 23%:77% (DRG), 33%:67% (VRG), 95%:5% (PB). These results are considered in the general context of previous electrophysiological and histological findings, but also with particular reference to a related study of the projections from brainstem neurones to the phrenic nucleus [32].

The effects of intravenous administration of fentanyl (50 and 100 micrograms/kg) on the discharge activity of the medullary inspiratory neurons and of the phrenic nerve were studied following vagotomy in nine decerebrate, paralyzed mechanically ventilated cats. In six cats, the inspiratory neurons explored were in the dorsal respiratory group (DRG) associated with the nucleus of the tractus solitarius (NTS), while in the remaining three, they were in the ventral respiratory group (VRG). In the former group, the rhythmic discharge of the inspiratory neurons was disrupted by fentanyl and replaced by a continuous discharge superimposed with irregularly occurring bursts. These changes were also reflected by the phrenic nerve discharge. Inspiratory neuronal activity increased significantly (P less than 0.05) at 1 and 5 min after completion of fentanyl injection. Disruption of the rhythmic activity of the inspiratory neurons and its replacement by a continuous and irregular discharge may lead to sustained contraction of inspiratory muscles and cessation of respiration. In the VRG, the activity of the inspiratory neurons was totally abolished by fentanyl. Thus, it appears that different groups of medullary inspiratory neurons have differential sensitivity to fentanyl. Nalbuphine, an opiate agonist-antagonist, restored the normal pattern and magnitude of the activity of the inspiratory neurons.

In Nembutal-anesthetized and spontaneously breathing rats, a total of 226 respiratory neurons were recorded in the medulla extending from the caudal end of the facial nucleus to 1 mm caudal to the obex. They were classified into inspiratory (I) and expiratory (E) neurons by their temporal relationships to diaphragm EMGs. One hundred and seventeen I and 108 E neurons were identified. I and E neurons were further classified into augmenting, decrementing, and other types based on their firing patterns. Almost all the respiratory neurons recorded were located around the nucleus ambiguus and the nucleus retroambigualis, corresponding to the ventral respiratory group (VRG) of the cat. On the other hand, only a few respiratory neurons were identified around the ventrolateral nucleus of the solitary tract, corresponding to the dorsal respiratory group of the cat. In the VRG, 3 subgroups were distinguished rostrocaudally. One group of E neurons was located ventrally to the rostral part of the nucleus ambiguus, presumably corresponding to the Bötzinger complex defined in the cat. Another group of E neurons extended caudally beyond the obex, from the caudal portion of the nucleus ambiguus through the nucleus retroambigualis. Between these two groups of E neurons, an assembly of predominantly I neurons existed in the vicinity of the nucleus ambiguus. These characteristics of distributions were basically similar to those of the VRG of the cat.

Intrabulbar connections of respiratory nuclei and the medullary reticular formation and also descending pathways from these structures in the spinal cord were studied by the retrograde horseradish peroxidase axonal transport method in cats. Neurons of the nucleus ambiguus and nucleus retroambigualis (ventral respiratory group) and of the ventrolateral part of the nucleus of the tractus solitarius (dorsal respiratory group) were shown to form direct two-way connections with each other and with the medial region of the medulla. Neurons of the pneumotaxic center send uncrossed axons to the nucleus ambiguus and to the medial medullary reticular formation. Neurons of the contralateral homonymous nucleus and neurons of the nucleus of the tractus solitarius are sources of projections of the locus coeruleus. A well developed system of direct connections was found between neurons of respiratory nuclei of the two halves of the brain. The possible role of these nuclear formations in genesis of the respiratory rhythm and regulation of the respiratory and other motor functions of the reticular formation is discussed.

Extracellular activity of 200 dorsal respiratory group (DRG) neurons of the ventrolateral nucleus of the tractus solitarius was recorded in anesthetized, paralyzed, artificially ventilated cats. The effects on neural activity of electrically stimulating the contralateral cervical spinal cord, the superior laryngeal nerves (SLN) bilaterally, and inflating the lungs to various levels during the expiratory pause were tested. 1. (1) Of the 200 cells, 157 were inspiratory cells; 32 cells (termed P cells), fired synchronously with lung inflation but did not have inspiratory rhythm of central origin; and 11 were non-inspiratory respiratory cells. Eighty-eight of the inspiratory cells which were inhibited with inhibition of phrenic inspiratory discharge following lung inflation were classified as α-type. However, most also showed short duration activation (gasp reflex) at the onset of lung hyperinflation. Thirty-five inspiratory cells which were excited by lung inflation in addition to showing the gasp reflex were classified as β-type neurons. The discharge of these β-type cells adapted during lung inflation; their excitation threshold was higher and sensitivity lower than that of the Hering-Breuer expiratory facilitatory reflex. I conclude that they are not the interneurons responsible for that reflex, but could possibly receive excitatory inputs from pulmonary irritant receptors. 2. (2) Approximately 80% of both α- and β-type neurons had axonal projections to the cord. A spinal cord collision test error and relative error were calculated for 93 projecting DRG neurons. Only two neurons had a collison error of zero, the rest had positive values. I conclude that these results imply only spinal axonal projections. 3. (3) Fifty-seven (58%) of the inspiratory cells tested were evoked from the ipsilateral SLN, with a mean latency of 3.9 msec ± 0.3 S.E.. All cells that were evoked with short latency followed stimuli delivered at 100 Hz. No cell was evoked from the contralateral SLN. I suggest that some DRG neurons are part of a disynaptic neural circuit involving SLN afferents, ipsilateral DRG neurons, and contralateral phrenic motoneurons.

Responses of phrenic motoneurones to stimulation of the three medullary raphe nuclei (raphe magnus (r. magnus), raphe obscurus (r. obscurus) and raphe pallidus (r. pallidus] were recorded in anaesthetized and decerebrated cats. Stimulation of r. magnus or r. obscurus depressed phrenic motoneurones. Stimulation at 100 Hz reduced action potential frequency within each inspiratory burst, without appreciable changes in inspiratory duration, or number of inspiratory bursts per unit time. The depression was proportional to the stimulus intensity (40-160 microA) and frequency (12-100 Hz) and lasted throughout the period of stimulation. Intracellular recording revealed concomitant depression of central respiratory drive potentials (c.r.d.p.s) and increased membrane input resistance during r. obscurus or r. magnus stimulation. In motoneurones which discharged action potentials during expiratory as well as inspiratory phases following intracellular chloride injection, stimulation of r. magnus or r. obscurus depressed cell firing during both phases. Both c.r.d.p.s and reversed inhibitory post-synaptic potentials (i.p.s.p.s) were depressed. These findings indicate that the depression is not related to post-synaptic inhibition of phrenic motoneurones. Stimulation (100 Hz) of r. pallidus produced discharges of action potentials in phrenic motoneurones. Stimulation lengthened the duration of each inspiratory discharge in proportion to stimulus intensity. Continuous firing occurred throughout the period of stimulation with maximal intensities. Intracellular recordings revealed sustained depolarization and reduction in membrane input resistance during the discharge. Responses were recorded extracellularly from medullary inspiratory neurones of the dorsal respiratory group (d.r.g.) and ventral respiratory group (v.r.g.) and from vagal axons which fired in phase with phrenic nerve activity. Responses to raphe stimulation were similar to those recorded from phrenic motoneurones. Evidence is presented that the responses are not related to stimulation of decussating bulbo-spinal axons from d.r.g. or v.r.g. neurones. It is suggested that medullary respiratory neurones receive inhibitory and excitatory synaptic inputs from medullary raphe neurones. Hypercapnia (5% CO2 in O2) or hypoxia (15% O2 in N2) reduced markedly the inhibition produced during stimulation of r. obscurus or r. magnus, and restored expiratory-linked silent periods during stimulation of r. pallidus. Activation of Hering-Breuer or baroreceptor reflexes did not alter responses to r. pallidus stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injections were made at the same sites within the midbrain periaqueductal grey region (PAG) of the cat at which microinjections of excitatory amino acids had previously elicited the set of autonomic and somatic reactions (i.e. pupil dilatation, piloerection, retraction of the ears, arching of the back, hissing, howling and growling) known as the ‘defence reaction’. The WGA-HRP injections revealed that this PAG region has an extensive set of afferent and efferent connections with solitary tract nuclei (NTS). Within the NTS the majority of labelled neurons were distributed, in approximately equal numbers, in the ipsilateral medial solitary nucleus (SM) and the ipsilateral and the contralateral ventrolateral solitary nuclei (SVL). The densest anterograde labelling was found in the ipsilateral SM, with lighter anterograde labelling in the contralateral SM and bilaterally in the commissural solitary nucleus and SVL. The described connections between the defence region of the PAG and the NTS not only provide a new anatomical basis for cardiovascular and respiratory components of the reaction, but also add to the evidence that, in addition to the hypothalamus, the PAG is an important integrating center for the autonomic and somatic elements of the defence reaction.

1. An in vitro brain stem slice preparation from adult guinea pigs was used to determine the properties of neurons located in the ventral part of the nucleus tractus solitarius (NTS), an area associated with the dorsal respiratory group. Based upon their morphology and their repetitive firing properties, three classes of ventral NTS neurons, termed types I, II, and III, were observed. 2. Type I neurons were multipolar with pyramidal-shaped cell bodies. These neurons responded to prolonged depolarizations from a resting level of -50 mV with a discrete, high-frequency burst of spikes, which rapidly adapted to a low steady-state level. When depolarized from levels more negative than -65 mV, the initial burst was diminished. 3. Type II neurons were multipolar with fusiform-shaped cell bodies. Type II neurons responded to depolarizations from -50 mV with an initial high spike frequency, which gradually adapted to a steady-state level. When depolarized from levels more negative than -60 mV, these neurons displayed a delay between the onset of the stimulus and the first spike. This delay has been termed “delayed excitation.” The expression of delayed excitation was modulated by both the size and duration of hyperpolarizing prepulses that preceded depolarization. 4. Type III neurons were multipolar with spherical shaped-cell bodies. In response to depolarizations from -50 mV, these neurons displayed high-frequency firing with little adaptation. The repetitive firing properties of type III neurons were not modulated by hyperpolarization. 5. Bulbospinal neurons in the ventral NTS were identified using retrograde transport of rhodamine-labeled latex beads injected into the region of the phrenic motor nucleus at spinal cord levels C4 through C6. Only type I and type II neurons were labeled in the ventral NTS (0.2-1.0 mm rostral to the obex). Both contralateral and ipsilateral projections were observed. Contralaterally, type I and II neurons were evenly distributed. Ipsilaterally, however, type II neurons accounted for two-thirds of the labeled neurons. 6. Type I and II neurons had similar input resistances and time constants: 97.0 +/- 17.6 M omega and 14.4 +/- 2.2 ms (n = 5) for type I and 107.0 +/- 11.2 M omega and 13.7 +/- 1.6 ms for type II (n = 5).(ABSTRACT TRUNCATED AT 400 WORDS)

In cats anaesthetized with pentobarbital, medullary respiratory neurones of both dorsal and ventral populations were recorded intracellularly with 1 mol.l-1 KCl-electrodes. The neurones were classified according to the projection of their axons to the spinal cord (bulbospinal neurones) or to the vagal nerves (vagal neurones). Those neurones which could not be activated antidromically (NAA-neurones) by either procedure were subdivided into (inspiratory) R beta-neurones, which were monosynaptically excited by lung stretch receptor afferents, and into inspiratory and expiratory NAA-neurones, which did not receive a direct synaptic input, from these afferents. All types of neurone investigated revealed postsynaptic activity during both inspiration and expiration. The periods when synaptic activity was minimal were the periods of transition between respiratory phases. The input resistance of most respiratory neurones varied in parallel with the respiratory cycle. A drastic fall of the input resistance during expiration was observed in R beta-neurones and in some inspiratory vagal neurones. This was not seen in inspiratory bulbospinal neurones. In stable intracellular recordings, periodic postsynaptic inhibition was demonstrated in 52 of 53 respiratory neurones by IPSP reversal following chloride injection. Maximal membrane potential then was generally reached during one of the periods of respiratory phase transition. Reasons for the failure of others to demonstrate these IPSPs are presented and discrepancies between other findings and these are discussed. It is concluded that reciprocal inhibition between bulbar respiratory neurones does exist and is a general phenomenon. It is argued that reciprocal inhibition is the fundamental mechanism underlying respiratory gating of afferent inputs. The probable existence of recurrent inhibition is inferred from the changes in the pattern of membrane depolarization during the active period of neurones.

In Nembutal-anesthetized and artificially ventilated cats, we studied the connectivity of burst inspiratory (I) neurons in the Bötzinger complex and the ventral respiratory group (VRG) with spike-triggered averaging methods. Burst I neurons exhibited tonic (I-TON) or decrementing (I-DEC) firing patterns. Spikes of I-TON neurons induced monosynaptic EPSPs in intracellularly recorded I neurons of both the VRG and the dorsal respiratory group (DRG). Spikes of I-DEC neurons induced monosynaptic inhibitory postsynaptic potentials (IPSPs) in both VRG and DRG I neurons.

Activities were recorded from single respiratory neurons in the area of the retrofacial nucleus. Stimuli were delivered in the dorsal (DRN) and ventral (VRN) medullary respiratory nuclei, the rostral pons, the cervical spinal cord and the vagus nerve to elicit antidromic potentials for these neurons and, hence, establish their axonal projections. The great majority of the antidromically activated retrofacial inspiratory neurons sent their axons to the contralateral VRN. A majority of expiratory neurons had their axons in the contralateral DRN or in both the DRN and spinal cord. For the phase-spanning neurons, the projections were found mainly in the VRN, spinal cord or pons.