Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals.

The location of neurons generating the rhythm of breathing in mammals is unknown. By microsection of the neonatal rat brainstem in vitro, a limited region of the ventral medulla (the pre-Bötzinger Complex) that contains neurons essential for rhythmogenesis was identified. Rhythm generation was eliminated by removal of only this region. Medullary slices containing the pre-Bötzinger Complex generated respiratory-related oscillations similar to those generated by the whole brainstem in vitro, and neurons with voltage-dependent pacemaker-like properties were identified in this region. Thus, the respiratory rhythm in the mammalian neonatal nervous system may result from a population of conditional bursting pacemaker neurons in the pre-Bötzinger Complex.

Distribution of bulbospinal gamma-aminobutyric acid-synthesizing neurons of the ventral respiratory group of the rat.

Spinal respiratory motoneuron activity is controlled primarily by excitatory and inhibitory neurons in the medulla oblongata. To identify bulbospinal inhibitory neurons, immunohistochemistry for glutamic acid decarboxylase (GAD) was combined with retrograde labeling of projections to the C(4) ventral horn with Fluoro-Gold. GAD-immunoreactive bulbospinal neurons were located in the ventrolateral portion of the intermediate reticular nucleus, the ventral portion of the medial reticular nuclei, and the raphe and spinal vestibular nuclei. Small numbers of bulbospinal ventral respiratory group neurons were GAD immunoreactive. These neurons were distributed throughout the rostral ventral respiratory group and the Bötzinger complex. Surprisingly, low numbers of Bötzinger neurons, a population thought to be exclusively inhibitory, were GAD immunoreactive. These results suggest that the rostral ventral respiratory group and the Bötzinger complex both contain heterogeneous bulbospinal neuron populations, only some of which have gamma-aminobutyric acid (GABA)-mediated inhibitory control over phrenic motoneurons. Furthermore, the ventral respiratory group contained many GABAergic neurons that lacked bulbospinal projections.

Monosynaptic transmission of respiratory drive to phrenic motoneurons from brainstem bulbospinal neurons in rats.

The termination patterns in the rat phrenic nucleus of neurons within two respiratory cell groups of the ventrolateral medulla (Bötzinger Complex and the rostral ventral respiratory group) were determined. The plant lectin, Phaseolus vulgaris leuco-agglutinin, was used as an anterograde tracer to label presynaptic processes of bulbospinal neurons, and horseradish peroxidase was used simultaneously to label phrenic motoneurons. Labeled bulbospinal axons ended with dense terminal arborizations within the phrenic cell column and on radial phrenic motoneuron dendrite bundles, which represented the exclusive site of termination of Bötzinger Complex and rostral ventral respiratory group neurons in the lower cervical spinal cord. Terminals of these descending axons formed presumptive synaptic contacts within longitudinal and radial dendrite bundles, and on the cell somata of phrenic motoneurons.

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.

Subnuclear organization of the lateral tegmental field of the rat. II: Catecholamine neurons and ventral respiratory group.

Bulbospinal and propriobulbar respiratory neurons of the ventral respiratory group and catecholamine neurons of the A1 and C1 cell groups were simultaneously labelled in the rat medulla by a combination of retrograde tracing and immunohistochemical identification. The ventral respiratory group and catecholamine cell groups form adjacent, parallel cell columns in the lateral tegmental field of the ventrolateral medulla. The ventral respiratory group is located immediately dorsal to the A1 and C1 groups, although some A1 neurons are intermingled with neurons of the rostral ventral respiratory group, and some C1 neurons are intermingled with those of the Bötzinger complex. The proximate populations of respiratory, catecholamine, and (presumptive) cardiovascular neurons identified in this study provide further support to the hypothesis that this region of the lateral tegmental field of the ventrolateral medulla is a site of cardiorespiratory coordination.

Ventral respiratory group projections to phrenic motoneurons: electron microscopic evidence for monosynaptic connections.

The hypothesis that excitatory drive is transmitted monosynaptically from bulbospinal medullary respiratory neurons to spinal respiratory motoneurons was tested by an ultrastructural analysis of the phrenic motoneuronal pool in the rat. Combined anterograde labeling of the principal inspiratory bulbospinal neuron population (ventral respiratory group) and retrograde labeling of the phrenic motoneuron pool demonstrated the presence of labeled synaptic profiles, indicating that at least some bulbospinal inspiratory neurons make monosynaptic contacts with phrenic motoneurons. The synaptic boutons of ventral respiratory group neurons that were labeled in the phrenic nucleus had asymmetrical membrane densities at sites of synaptic contact with labeled phrenic somal or dendritic profiles, supporting the notion that this bulbospinal pathway has excitatory contacts with phrenic motoneurons. The morphological types of labeled boutons included three of the eight previously identified bouton types in the phrenic nucleus (Goshgarian and Rafols: Journal of Neurocytology 13:85-109, 1984), including the "S"-terminal, the "NFs"-terminal, and the "F"-terminal. There was no conclusive evidence of labeled double synapses, indicating that this type of synaptic contact is not common in the intact bulbospinal pathway.

Brainstem projections to the major respiratory neuron populations in the medulla of the cat.

Efferent and afferent connections of the dorsal and ventral respiratory groups in the medulla of the cat were mapped by axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase. Injections of wheat germ agglutinin-horseradish peroxidase into the dorsal respiratory group and the three principal subdivisions of the ventral respiratory group (caudal, rostral, and Bötzinger Complex) revealed extensive interconnections between these regions and with a limited number of other brainstem neuron populations. Major neuron populations with efferent projections to the regions of the dorsal and ventral respiratory groups include the parabrachial nuclear complex (medial parabrachial, lateral parabrachial, and Kölliker-Fuse nuclei), subregions of the lateral paragigantocellular reticular nucleus, subregions of the lateral and magnocellular tegmental fields, inferior central and postpyramidal nuclei of the raphe, and sensory trigeminal nuclei. A previously unidentified neuron population with extensive efferent projections to the dorsal and ventral respiratory groups was found near the ventral surface of the rostral medulla; we refer to this group as the retrotrapezoid nucleus. The results suggest that the dorsal and ventral respiratory groups form an extensively interconnected neuronal system receiving convergent inputs from the same brainstem nuclear groups, consistent with the hypothesis that the dorsal and ventral groups are primarily sites for integration of sensory and premotor respiratory drive inputs. Neuron populations in the rostral ventrolateral medulla with projections to both the dorsal and ventral respiratory groups, particularly the retrotrapezoid nucleus and neighboring subregions of the lateral paragigantocellular reticular nucleus, are candidate sites for participation in respiratory rhythmogenesis or other critical functions of the brainstem respiratory control system such as intracranial chemoreception.

Monoaminergic and GABAergic terminations in phrenic nucleus of rat identified by immunohistochemical labeling.

The termination patterns of axons in the phrenic nucleus immunoreactive to synthetic enzymes for catecholamines and for serotonin and GABA were studied in rats. Spinal cord tissue in which phrenic motoneurons were retrogradely labeled with horseradish peroxidase was incubated with antisera against dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, 5-hydroxytryptamine, and GABA to identify presumptive terminations of monoaminergic and GABAergic neurons onto identified phrenic motoneurons. In the C3 to C5 spinal cord, 5-hydroxytryptamine-, dopamine beta-hydroxylase- and GABA-like positive terminals with varicosities formed a dense network, with presumptive synaptic contacts on dendrites and somas of phrenic motoneurons. A similar pattern of terminations was also observed in adjacent (non-respiratory muscle) motoneuron pools. There were fewer phenylethanolamine-N-methyltransferase-positive terminal arborizations in the cervical spinal cord compared to thoracic spinal cord; phenylethanolamine-N-methyltransferase terminals were not seen in the vicinity of phrenic motoneurons. These results suggest that phrenic motoneuronal activity is influenced by multiple supraspinal inputs utilizing different neurotransmitters. These transmitters also mediate inputs to other (nearby) spinal motoneurons and thus are not unique for signal transmission to phrenic motoneurons.

Multiple putative neuromessenger inputs to the phrenic nucleus in rat.

Immunohistochemical reactions for 12 putative neuromessengers combined with retrograde labeling of phrenic motoneurons identified seven neuromessengers (5-hydroxytryptamine, substance P, thyrotropin-releasing hormone, methionine enkephalin, cholecystokinin, galanin, neuropeptide Y) located within terminal varicosities in the phrenic nucleus. The degree of terminal labeling in the phrenic nucleus varied depending on the peptide. Substance P, thyrotropin-releasing hormone and methionine enkephalin were each tested for colocalization with 5-hydroxytryptamine within terminal varicosities in the phrenic nucleus, and the coincidence of double-labeling varied for each peptide. These results indicate that phrenic motoneurons are subject to modulation by many peptide neuromessengers that may alter their responsiveness to primary excitatory and inhibitory inputs.

Origins of excitatory drive within the respiratory network: anatomical localization.

This study identified several sources of excitatory drive within the rat brain stem respiratory network. Excitatory neuronal projections to the ventral respiratory group (the largest brain stem respiratory neuron population in the rat) were identified by selective retrograde labeling with tritiated aspartate. Neurons were labeled within portions of the nucleus of the tractus solitarius, the region of the ventral respiratory group, superficial to the facial nucleus, the medullary raphe nuclei and the parabrachial/Kölliker-Fuse nuclei. These findings are the first anatomical demonstration of sources of excitatory drive within the respiratory network, and are consistent with electrophysiological data localizing a source of excitatory drive within the pre-Bötzinger complex subdivision of the ventral respiratory group.

Brainstem connections of the rostral ventral respiratory group of the rat.

The pontomedullary connections of the rostral division of the ventral respiratory group (rVRG), the largest medullary population of inspiratory bulbospinal and propriobulbar neurons, were identified in the rat by retrograde and anterograde tracing techniques. These experiments revealed that: (i) the Kölliker-Fuse nucleus, portions of the medial and lateral parabrachial nuclei, and all levels of the ipsilateral and contralateral VRG complex have dense reciprocal connections with the rVRG; (ii) the lateral paragigantocellular nucleus has reciprocal but less dense connections with rVRG; (iii) portions of the nucleus of the solitary tract have prominent projections to, but weaker inputs from rVRG; (iv) the raphe, magnocellular tegmental field and spinal trigeminal nuclei have minor projections to rVRG and receive only sparse inputs from rVRG, and; (v) the retrotrapezoid nucleus, pontine lateral tegmental field and area postrema each have only efferent projections to rVRG. These findings are consistent with previous studies of pontomedullary connections of rVRG in the cat, and further document the extensive reciprocal connections between principal respiratory groups. These connections are likely to be important for generation of the respiratory pattern, for the coordination of effector responses of the cranial and spinal respiratory motor neurons to afferent stimuli, and coordination of the central respiratory and cardiovascular control systems.

Bulbospinal respiratory neurons are a source of double synapses onto phrenic motoneurons following cervical spinal cord hemisection in adult rats.

The purpose of this study was to determine if the medullary neurons that provide the primary excitatory drive to phrenic motoneurons (i.e., rostral ventral respiratory group, rVRG) are a source of double synapse formation in the phrenic nucleus after spinal cord hemisection. The axons of rVRG neurons either ipsilateral or contralateral to the hemisection were labeled by injection of a mixture of HRP and WGA-HRP into the rostral ventral respiratory group. Phrenic motoneurons ipsilateral and caudal to the hemisection were labeled by the retrograde transport of HRP. The ultrastructural results indicated that after hemisection, rVRG neurons from both sides of the medulla formed labelled double synapses in the phrenic nucleus.

The intermediolateral nucleus: an 'open' or 'closed' nucleus?

The sympathetic preganglionic neurons located in the intermediolateral nucleus (IML) that project to the superior cervical ganglion of the rabbit were observed to have two major dendritic orientations after retrograde labeling with horseradish peroxidase. One projection extends longitudinally within IML. The second projection courses medially and presents a triangular shape in horizontal sections. The labeled processes that project medially arise from cells in IML and project through the intercalated nucleus towards the central autonomic area and follow the contour of the central canal. Medially oriented dendrites intruding into other areas of the intermediate grey matter show that IML is an 'open' rather than a 'closed' nucleus as has been recently suggested. The location and distribution of the sympathetic preganglionic neurons projecting to the superior cervical ganglion in the rabbit are compared with those reported for other species.

Nucleus reticularis lateralis involvement in the pressor component of the cerebral ischemic response.

Lesions of the ventromedial portion of the nucleus reticularis lateralis (NRL) of the medulla oblongata in the rabbit significantly and markedly reduced the pressor component of the cerebral ischemic response (CIR) without altering vasomotor tone. Use of horseradish peroxidase as an anterograde and retrograde tracer indicated that the ventromedial NRL projects to the intermediolateral cell column (IML) of the upper thoracic spinal cord. Injections of glutamate into ventromedial NRL elevated blood pressure (BP), indicating that neuronal cell bodies in this area are capable of eliciting BP elevations when stimulated. These findings suggest that cell bodies in the ventromedial NRL are capable of eliciting pressor responses during the CIR via direct projections to sympathetic preganglionic cell bodies.

Are there serotonergic projections from raphe and retrotrapezoid nuclei to the ventral respiratory group in the rat?

The relationship of serotonergic neurons in raphe and ventrolateral medullary regions to neurons projecting to the rostral ventral respiratory group in the rat were investigated using combined immunohistochemical and retrograde labeling techniques. Serotonergic and non-serotonergic neurons retrogradely labeled with rhodamine beads were found intermingled in the larger population of serotonin-immunoreactive neurons in raphe pallidus, obscurus and magnus. In addition, a cell group analogous to the retrotrapezoid nucleus in the cat was identified in the rat ventrolateral medulla. Retrotrapezoid neurons, which exhibited exclusively ipsilateral projections to the ventral respiratory group, were located lateral to clusters of serotonergic cells near the ventral surface, but were not serotonin immunoreactive.

Nucleus ambiguus and bulbospinal ventral respiratory group neurons in the neonatal rat.

The in vitro brainstem-spinal cord preparation of the neonatal rat is an important model system for studies of the respiratory control system, yet there have not been studies to anatomically characterize respiratory neuron populations in the neonate. Fluorescent retrograde tracers were used to identify bulbospinal neurons of the ventral respiratory group and motoneurons of nucleus ambiguus in neonatal rats. Fluoro-Gold injections into the C4 ventral horn labeled bulbospinal neurons within a densely packed column within the ventrolateral intermediate reticular nucleus from the level of the pyramidal decussation to the facial nucleus. This cell column corresponded closely to the location of the ventral respiratory group of the adult rat. In particular, neurons were labeled in regions corresponding to the rostral ventral respiratory group and the Bötzinger complex. Unlike adult rats, the preBötzinger complex also contained many bulbospinal neurons. Fluoro-Gold-labeled neurons were also located in the medial reticular nuclei, raphe pallidus, and obscurus and spinal vestibular nucleus. As in adult rats, bulbospinal ventral respiratory group neurons overlapped with cervical vagal motoneurons in the external formation, and partially with those in the loose formation, but not with those in the semicompact or compact formation of nucleus ambiguus. These results indicate that the distribution of bulbospinal ventral respiratory group neurons corresponds with that observed in physiological studies of neonatal rats.

Brainstem projections to the phrenic nucleus: an anterograde and retrograde HRP study in the rabbit.

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.

Sulfated cholecystokinin octapeptide in the rat: pontomedullary distribution and modulation of the respiratory pattern.

We performed anatomical and physiological studies to determine the site and actions of sulfated cholecystokinin octapeptide (CCK8-S) on breathing. Peptide locations were determined by combined immunodetection of CCK8-S- containing synaptic varicosities and retrograde labeling of medullary neurons projecting to the ventral respiratory group. Retrogradely labeled neurons and CCK8-S immunolabeled varicosities overlapped within the nuclei of the solitary tract, ventral respiratory group, and the Kolliker-Fuse nucleus. Additional CCK8-S immunoreactive terminals were located in the rostroventrolateral medullary reticular nucleus, lateral paragigantocellular reticular nucleus, and the caudal pontine reticular nucleus. The respiratory effects of CCK8-S, which binds to CCK(A) and CCK(B) receptors, were examined by intravenous injection in adult rats and by bath application in the in vitro neonatal rat brainstem - spinal cord preparation. CCK8-S produced an increase in the mean amplitude of diaphragmatic electromyogram (EMG) of 28 +/- 35% (SD) and a decrease in mean respiratory interval of 13 +/- 4% in vivo. In vitro, CCK8-S significantly increased inspiratory duration and decreased respiratory interval, primarily by shortening expiratory duration. CCK8-unsulfated, a specific agonist for CCK(B) receptors, did not produce these effects. CCK8-S effects in the in vitro preparation were partially blocked by the CCK receptor antagonist lorglumide (final bath concentration 600 nM). These results suggest that CCK8-S modulates the respiratory rhythm via CCK(A) receptors within one or more medullary or pontine respiratory groups in both neonatal and adult rats.

Decussation of bulbospinal respiratory axons at the level of the phrenic nuclei in adult rats: a possible substrate for the crossed phrenic phenomenon.

The axonal trajectories of inspiratory bulbospinal neurons were examined after deposition of the anterograde neuronal tracer phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral ventral respiratory group in rats. At the level of the phrenic nucleus, PHA-L-labeled bulbospinal axons crossed the midline of the spinal cord in both the anterior gray and the anterior white commissure. These spinally decussating neurons provide a possible anatomical substrate for the respiratory reflex known as the crossed phrenic phenomenon.

Respiratory activity in retrotrapezoid nucleus in cat.

An anatomic projection from the retrotrapezoid nucleus to the ventral respiratory group in cat was previously reported by our laboratory (J. C. Smith, D. E. Morrison, H. H. Ellenberger, M. R. Otto, and J. L. Feldman. J. Comp. Neurol. 281: 69-96, 1989). We now report on the properties of neurons in the retrotrapezoid nucleus, investigated with extracellular recording techniques in 10 chloralose-urethane anesthetized, paralyzed, mechanically ventilated cats. A ventral exposure of the medulla facilitated recording from neurons in the retrotrapezoid nucleus, located ventral to the facial nucleus near the medullary surface. Respiratory-modulated, as well as irregularly discharging, spontaneous unit activity was recorded within the retrotrapezoid nucleus. Twelve respiratory-modulated units in the retrotrapezoid nucleus exhibited inspiratory (8 units), expiratory (3 units), or multimodal (1 unit) discharge patterns. Chemical activation of an inspiratory unit in the retrotrapezoid nucleus by pressure ejection of DL-homocysteic acid (less than 0.5 nl of 10 mM solution) demonstrated that the respiratory-modulated activity originated from the cell soma and not fibers of passage coursing through the retrotrapezoid nucleus region. Electrical microstimulation (20-40 microA, approximately 70-microseconds duration, biphasic pulse) within the ipsilateral ventral respiratory group elicited antidromic activation of 7 units in the retrotrapezoid nucleus, three of which were not spontaneously active. Electrical stimulation (5-80 microA, 70-microseconds pulse width, 100 Hz, 400- to 500-ms trains) at sites within retrotrapezoid nucleus affected the respiratory motor output when delivered during late expiration, eliciting premature onset of inspiration. These results suggest that retrotrapezoid nucleus projections to the ventral respiratory group (and dorsal respiratory group) may influence respiratory timing and pattern, perhaps by conveying signals originating in the rostral ventrolateral medulla that result from ventral surface perturbations.