Respiratory neurons in the medulla of the rabbit: distribution, discharge patterns and spinal projections

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 distinct 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 T. 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.     

Distribution of medullary respiratory neurons in the rat

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 B?tzinger 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.     

Types and locations of respiratory-related neurons in lateral tegmental field of cat medulla oblongata

Extracellular microelectrode recordings were made from a total of 868 neurons in the medullas of cats in regions known to contain high densities of respiratory-related neurons (solitary tract complex, nucleus ambiguus/retroambigualis, lateral tegmental field). Both the discharge patterns and the locations of units were noted and correlated with a recently described substructure of the tegmental field of the cat medulla in which neuronal cell bodies are found associated with sheets of blood vessels supplying the brainstem. The majority of cells were phasically firing (59%) with activity confined to either the inspiratory or the expiratory phase, 21% were tonically firing cells with no discernible respiratory modulation and 20% were silent neurons, responsive to electrical stimulation of the vagus nerves or the dorsolateral pons in the vicinity of nucleus parabrachialis, but not to various respiratory stimuli. Within the solitary tract complex inspiratory discharge patterns were predominant (94%), while in nucleus ambiguus/retroambigualis 26% of the neurons had expiratory patterns with the rest being inspiratory (68%) or tonic (6%). Within the lateral tegmental field, the percentages of inspiratory, expiratory and tonic patterns were 51, 9 and 40%. Thus, inspiratory type patterns were found throughout the medulla, but expiratory patterns were most common in the ambiguus/retroambigualis nuclei. Found within all 3 major regions, but primarily within the lateral tegmental field of the rostral medulla were neurons that discharged with a brief burst at the inspiratory to expiratory phase transition. These cells had properties consistent with the off-switch mechanism: extreme late-inspiratory onset of discharge with the onset time being delayed by lung inflation, peak discharge at or slightly after the peak activity of the diaphragmatic EMG and a discharge rate which was insensitive to lung inflation. Within the lateral tegmental field, where longitudinal sheets of blood vessels running radially with respect to the IVth ventricle have been described, it was found that 85% of the tonically active units and 93% of the respiratory modulated cells were located less than 200 microns from the planes of these sheets. In addition, 87% of the neurons that could be antidromically or synaptically activated from the dorsolateral rostral pons were similarly located.     

Discharge patterns of dorsal and ventral respiratory group neurons during spontaneous augmented breaths observed in pentobarbital anesthetized rats

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: , expiratory: ) 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 () and VRG () 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%, P < 0.01), but not in the I-decrementing and I-other neurons. On the other hand, the activities of the expiratory DRG () and VRG () 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%, P < 0.05), 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.     

Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat

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 ofdl-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.     

Patterns of membrane potentials and distributions of the medullary respiratory neurons in the decerebrate rat

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.     

Synaptic inputs to medullary respiratory neurons from superior laryngeal afferents in the cat.

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 B?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.     

Localization of respiratory bulbospinal and propriobulbar neurons in the region of the nucleus ambiguus of the rat

The location of neurons within the ventral respiratory group (VRG) of rat was mapped following injections of 3 different fluorochrome tracers into different sites known to receive projections from VRG neurons. Injection sites included muscles innervated by the vagus (X) and glossopharyngeal (IX) nerves, and the sites of expiratory activity in the caudal medulla and of inspiratory activity in the spinal cord at the C4 level. Labeling of vagal motoneurons resulting from fluorochrome injections into muscles innervated by X and IX nerves was always ipsilateral to the site of injection. Both propriobulbar and bulbospinal neurons had primarily ipsilateral projections. No double-labeled cell bodies were observed. The cell bodies of the 3 types of neurons, propriobulbar, bulbospinal and vagal/glossopharyngeal, were unevenly distributed along the rostrocaudal axis of the VRG, suggesting a complex mosaic of neurons which regulate respiratory-related functions such as swallowing and vocalization.     

Projections to B?tzinger expiratory neurons by dorsal and ventral respiratory group neurons.

B?tzinger complex (BOT) augmenting expiratory neuron efferent connection are well established, but little is known concerning the afferent neural projections to BOT. The dorsal (DRG) and ventral (VRG) respiratory groups were extensively searched in 17 pentobarbital anaesthetized cats for inspiratory neurons that were anti-dromically activated from BOT. Only 1 of the 60 VRG inspiratory neurons with confirmed spinal projection was antidromically activated from BOT. Another 3 VRG inspiratory neurons and 4 of the 30 DRG inspiratory neurons were activated from BOT, but none of these neurons had confirmed spinal cord projections. All 15 early burst neurons were antidromically activated from BOT. Neural projections to BOT from DRG and VRG inspiratory neurons are rare, but neural projections from early burst neurons are common.     

Respiratory rhythmicity after extensive lesions of the dorsal and ventral respiratory groups in the decerebrate cat

It was previously demonstrated that extensive destruction of the regions of the dorsal (DRG) and rostral portions of the ventral respiratory groups (VRG) in the medulla does not disrupt respiratory rhythmicity in the anesthetized cat. The present experiments examined if either higher CNS structures or the caudal expiratory VRG might have been responsible for preserving rhythm in those studies. Results indicate that the DRG and VRG are not required for respiratory rhythmicity in the midcollicularly decerebrated cat.     

Morphological study of long axonal projections of ventral medullary inspiratory neurons in the rat

The aim of this study was to examine medullary and spinal axonal projections of inspiratory bulbospinal neurons of the rostral ventral respiratory group (VRG) in the rat. A direct visualization of long (9.8–33 mm) axonal branches, including those projecting to the contralateral side of the medulla oblongata and the spinal cord, was possible due to intracellular labeling with neurobiotin and long survival times (up to 22 h) after injections. Seven of the nine labeled neurons had bilateral descending axons, which were located in discrete regions of the spinal white matter; ipsilateral axons in the lateral and dorsolateral funiculus, contralateral in the ventral and ventromedial funiculus. The collaterals issued by these axons at the mid-cervical level formed close appositions with dendrites of phrenic motoneurons, which had also been labeled with neurobiotin. None of these collaterals crossed the midline. The significance of this finding is discussed in relation to the crossed-phrenic phenomenon. Additional spinal collaterals were identified in the C₁ and T₁ segments. Within the medulla, collaterals with multiple varicosities were identified in the lateral tegmental field and in the dorsomedial medulla (in the hypoglossal nucleus and in the nucleus of the solitary tract). These results demonstrate that inspiratory VRG neurons in the rat have some features which have not been previously described in the cat, including frequent bilateral spinal projection and projection to the nucleus of the solitary tract. In addition, this study shows that intracellular labeling with neurobiotin offers an effective way of tracing long axonal projections, supplementing results previously obtainable only with antidromic mapping, and providing morphological details which could not be observed in previous studies using labeling with horseradish peroxidase.     

The identification of brainstem neurones projecting to thoracic respiratory motoneurones in the cat as demonstrated by retrograde transport of HRP

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 ion-tophoretic 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].     

Influence of trigeminal nasal afferents on bulbar respiratory neuronal activity

This study examined the influence of nasal trigeminal afferents, the anterior ethmoidal nerve (AEN) and posterior nasal nerves (PNN) on the spike discharges of respiratory-related neurons recorded in the ventral respiratory group (VRG) (2.6-3.5 mm lateral to the midline, from 1 mm rostral to 3 mm caudal to the obex and at depth of 2-4 mm below the dorsal surface). Electrical stimulations to the AEN and PNN were administered to 10 pentobarbital anaesthetized cats and to 8 ketamine anaesthetized, vagotomized, curarized and ventilated cats. Single shock stimulations of either nerve evoked transient and total inhibition of inspiratory activities. Expiratory-related neurons of the VRG presented three patterns of activity in response to stimulation:excitation, inhibition or inhibition followed by excitation. More generally, expiratory units are activated with a short latency. In the course of repetitive stimulation of the AEN and PNN we observed a prolongation of the spontaneous inspiratory discharge which presented transient, short inhibition in response to each shock. Most expiratory units presented a short activation which was synchronous with the transient inhibition of inspiratory activities. When repetitive stimulation provoked a sneeze-like response, we observed a progressive increase in the duration of transient inspiratory inhibition first, associated with a progressive reinforcement of transient expiratory activation. Secondarily, just before the expiratory thrust, we noted a stronger inhibition of the inspiratory activity which preceded a high-frequency (400 Hz) expiratory discharge. Nasal afferents exert a forceful excitatory effect on bulbospinal (BS) and non-bulbospinal-non-vagal (NBS-NV) expiratory cells of the VRG. The effects due to vagotomy and curarization are discussed.     

Brain stem projections by axonal collaterals to the rostral and caudal ventral respiratory group in the rat

The location of neurons projecting by axonal collaterals to the rostral and caudal ventral respiratory group (VRG) regions was determined after discrete injections of Fast blue and Diamidino yellow into the physiologically identified rostral inspiratory VRG and the caudal expiratory VRG areas, respectively. In contrast with single fluorochrome labeled neurons found throughout the rostro-caudal extent of the medulla and pons (in a variety of areas known to have cardiorespiratory function), double-labeled neurons were located in discrete pontomedullary regions. The largest number of the double-labeled neurons was counted within the peripheral facial area, lateral paragigantocellular nucleus, and the VRG region, ipsi- and contralaterally to the injected side. Only a few double-labeled neurons were found within the ventrolateral and intermediate subnuclei of the solitary tract, medial parabrachial, and Kölliker-Fuse nuclei. The possible physiological implications of this neuronal network have also been emphasized.     

The pre-Bötzinger complex and phase-spanning neurons in the adult rat

To characterise respiratory neurons in the pre-Bötzinger complex of adult rats, extracellular recordings were made from 302 respiratory neurons in the ventral respiratory group of sodium pentobarbitone anaesthetised adult rats. Neurons were located 0 to 1.6 mm caudal to the facial nucleus, and ventral to the nucleus ambiguus. The pre-Bötzinger complex comprised expiratory neurons (22%, 22/100), inspiratory neurons (37%, 37/100) and phase-spanning neurons (41%, 41/100). In contrast, 80% (125/157) of Bötzinger neurons were expiratory, and 80% (36/45) of rostral ventral respiratory group neurons were inspiratory. Rostrocaudally, the pre-Bötzinger complex extended about 400 μm, starting at the caudal pole of the nucleus ambiguus compact formation. The pre-Bötzinger complex was also characterised by a predominance of propriobulbar neurons (81%, 13/16). Furthermore, 68% (33/48) of expiratory–inspiratory neurons found were located within the pre-Bötzinger complex. The variety of neuronal subtypes in the pre-Bötzinger complex, including many firing during the expiratory–inspiratory transition is consistent with the hypothesis that this nucleus plays a key role in respiratory rhythm generation in the adult rat.     

Distribution and connections of inspiratory premotor neurons in the brainstem of the pigeon (Columba livia)

We have recorded extracellular, inspiratory-related (IR) unit activity in the medulla at locations corresponding to those of neurons retrogradely labeled by injections of retrograde tracers in the lower brachial and upper thoracic spinal cord, injections that covered cell bodies and dendrites of motoneurons innervating inspiratory muscles. Bulbospinal neurons were distributed throughout the dorsomedial and ventrolateral medulla, from the spinomedullary junction through about 0.8 mm rostral to the obex. Almost all of the 104 IR units recorded were located in corresponding parts of the ventrolateral medulla, rostral to nucleus retroambigualis, where expiratory related units are found. Injections of biotinylated dextran amine at the recording sites labeled projections both to the spinal cord and to the brainstem. In the lower brachial and upper thoracic spinal cord, bulbospinal axons traveled predominantly in the contralateral dorsolateral funiculus and terminated in close relation to the dendrites of inspiratory motoneurons retrogradely labeled with cholera toxin B-chain. In the brainstem, there were predominantly ipsilateral projections to the nucleus retroambigualis, tracheosyringeal motor nucleus (XIIts), ventrolateral nucleus of the rostral medulla, infraolivary superior nucleus, ventrolateral parabrachial nucleus, and dorsomedial nucleus of the intercollicular complex. In all these nuclei, except XIIts, retrogradely labeled neurons were also found, indicating reciprocity of the connections. These results suggest the possibility of monosynaptic connections between inspiratory premotor neurons and inspiratory motoneurons, which, together with connections of IR neurons with other brainstem respiratory-vocal nuclei, seem likely to mediate the close coordination that exists in birds between the vocal and respiratory systems. The distribution of IR neurons in birds is similar to that of the rostral ventral respiratory group (rVRG) in mammals. J. Comp. Neurol. 379:347–362, 1997. © 1997 Wiley-Liss, Inc.     

Medullary projection of nonaugmenting inspiratory neurons of the ventrolateral medulla in the cat.

In Nembutal-anesthetized, immobilized, and artificially ventilated cats, we studied the morphological characteristics of inspiratory neurons with nonaugmenting firing patterns. HRP was injected intracellularly into a total of 22 neurons of the B?tzinger complex (BOT) and the ventral respiratory group (VRG). In 20 cases somata with their axonal trajectories were stained, and in two cases only axons were stained. None of the neurons stained could be antidromically activated by stimulation of the cervical cord. The somata of 20 neurons were located in the vicinity of the nucleus ambiguus or the retrofacial nucleus (RFN) between 600 microns and 2,800 microns caudal to the rostral end of the RFN. Their axons could be traced for a distance of several millimeters on the side of the somata, and showed various projection patterns. According to these projection patterns, the 20 neurons were tentatively classified into four groups: A (8/20), B (4/20), C (6/20), and motoneurons (2/20). Group A neurons gave off extensive axon collaterals that arborized and distributed boutons predominantly in the BOT and the VRG areas. Group B neurons had less extensive axon collaterals with various projection patterns, projecting rarely to the BOT or the VRG area. Group C neurons sent their stem axons, without issuing any axonal collaterals, to the contralateral side in five cases and to the ipsilateral pons in one case. The two motoneurons had axons leaving the brainstem without any intramedullary collaterals. Thus, the nonaugmenting inspiratory neurons showed morphological variations, which may play different roles in neural control of respiration.     

Subnuclear organization of the lateral tegmental field of the rat. I: Nucleus ambiguus and ventral respiratory group

Three classes of neurons within the lateral tegmental field of the rat medulla having different target projections were identified by retrograde labelling with three different fluorescent tracers. Labelled bulbospinal premotor and propriobulbar interneurons of the ventral respiratory group and vagal motoneurons of nucleus ambiguus formed partially intermingled longitudinal columns encompassed within a common region of the lateral tegmental field. Labelled neurons of each class were organized in a nonuniform distribution within these columns forming subdivisions distinguished by neuron morphology, orientation, and target projection. The three major rostrocaudal divisions of the ventral respiratory group (VRG) previously identified in the cat and rabbit were identified here in the rat, suggesting a common pattern of VRG organization among these species.     

Possible inspiratory off-switch neurones in the ventrolateral medulla of the cat.

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 B?tzinger 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.     

家兔面神经核腹内侧区呼吸神经元的放电模式

通过细胞外记录的方法 ,探测成年家兔面神经核腹内侧区 ( vm NF)呼吸神经元的放电模式。实验采用健康成年家兔 46只 ,氨基甲酸乙酯麻醉 ,记录膈神经放电作为呼吸指标。在 vm NF内记录到 2 57个呼吸神经元单位放电 ,根据神经元放电模式及与膈神经放电的时相关系将记录到的呼吸神经元分为六类 :呼气递增神经元、前吸气神经元、吸气递增神经元、晚吸气神经元、后吸气神经元及吸 -呼跨时相神经元。其中呼气递增神经元和吸 -呼跨时相神经元占总数的 75.9%,这些神经元与 vm NF的“吸气切断”作用有密切关系。