Bulbospinal Neurons Research Articles

Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters

Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters

Abdominal vagal inputs to catecholamine neurons in the ventrolateral medulla.

Stimulation of the abdominal vagus nerve increases arterial pressure and causes secretion of vasopressin. We have shown in rabbits that the arterial pressure increases are mediated by activation of barosensitive bulbospinal neurons in the C1 region of the rostral medulla. Secretion of vasopressin involves activation of neurons in the A1 region of the caudal ventrolateral medulla. Neuroanatomical studies with Fos indicate that approximately 50% of the C1 area cells are C1 neurons. Approximately 70% of the A1 area cells are A1 neurons. These neurons may mediate physiological responses occurring during abdominal malaise resulting from ingestion of toxins. Our discovery of a major excitatory input to A1 and C1 neurons from the abdominal vagus should prove useful in studies of the function of these neurons.

Central nervous mechanisms responsible for cardio-respiratory homeostasis.

There is a body of data that indicates that the generation of respiratory activity involves the interplay of a limited number of distinct classes of respiratory neurones in the ventrolateral medulla [1]. Similarly the cardiovascular system is regulated by the activity of premotor and preganglionic neurones localised in an equivalent region of the CNS [2]. Cardio-respiratory homeostasis depends on the integration of the activity of the two systems and it could be envisaged that this was achieved by the action of two independent control systems that are loosely dependent on one another. Studies over the last decade have drawn attention, however, to the fact that the two systems are tightly coupled being sensitive to the same reflex inputs and participating in the expression of behavioral activities in a totally co-ordinated manner [3]. This has led to a recognition that rather than operating through independent control systems the CNS acts to maintain cardio-respiratory homeostasis by means of a single group of‘ cardio-respiratory’ neurones located in the ventrolateral medulla which regulate the activity of the two systems. This is achieved by generating complementary patterns of discharge of bulbospinal neurones that control respiratory motoneuronal and sympathetic preganglionic neuronal firing and through brainstem interneurones influencing the activity of vagal motoneurones that supply the heart, lungs and airways (and subsidiary muscles of respiration). This notion is not novel being firmly based in the 19th century literature but the experimental justification is only now emerging [4]. This results from an extensive electrophysiological analysis of the properties of the various motoneurones and interneurones in these networks, the nature of the reflex inputs impinging on them and the action of regions of the forebrain in generating changes in their activity.

Respiratory-related discharge patterns of caudal raphe neurones projecting to the upper thoracic spinal cord in the rat

Sympathetic activity is modulated by central resptiratory drive. Bulbospinal neurones arising in the ventrolateral medulla and A5 region probably contribute to this modulation. In the present investigation the involvement of caudal raphe-spinal neurones in relaying respiratory-related inputs to sympathetic preganglionic neurones was investigated. Experiments were carried out on anaesthetized, vagotomized, paralysed and artificially ventilated rats. Extracellular recordings were made from the cell bodies of 53 caudal raphe neurones activated antidromically stimulating the spinal cord between T1 and T3. Their axonal conduction velocities ranged from 0.7–9.1 m/s (median = 3.8 m/s). Thirty-six of 53 neurones (consisting of neurones with on-going activity and quiescent neurones activated with glutamate) were held long enough for detailed analysis. Of those recorded 26 were in the region of raphe obscurus, nine in raphe pallidus and one in raphe magnus. Twenty-five of 36 neurones had firing patterns related to phrenic nerve discharge. Of the four firing patterns defined: seven neurones had the highest probability of firing during inspiration (inspiratory-related), 10 neurones had the highest probability of firing during expiration (expiratory-related), 3 had the highest probability of firing during post-inspiration (post-inspiratory-related) and 5 had lowest levels of firing during early- and post-inspiratory phases (early and post-inspiratory depressed). Of 27 neurones with axonal projections through or to the region of the intermediolateral cell column in the upper thoracic cord 19 had a respiratory-related discharge pattern. For respiratory-modulated neurones with on-going activity the median of the modal inter-spike intervals was 0.08 s. None of the neurones had an ECG-related firing pattern. The findings of this study also indicate a species difference between rats and cats regarding the physiological properties of some raphe-spinal neurones; i.e., an absence of ECG-related activity in the rats. The characteristics of the neurones recorded in this study are not those of ‘typical’ 5-HT-containing neurones with reference to axonal conduction velocities and discharge characteristics.

Dose-dependent Effects of Halothane on the Carbon Dioxide Responses of Expiratory and Inspiratory Bulbospinal Neurons and the Phrenic Nerve Activities in Dogs

Expiratory bulbospinal and inspiratory bulbospinal neurons in the ventral respiratory group provide drive for thoracoabdominal expiratory and phrenic and thoracic inspiratory motor neurons. Potent inhalational agents such as halothane may have differential effects on inspiratory and expiratory neurons, but detailed studies comparing neurons at a homologous level are lacking. The dose-dependent effects of anesthesia with 1.0-2.5 minimum alveolar concentration halothane on the CO2 responses of single expiratory and inspiratory bulbospinal neurons of the ventral respiratory group and on phrenic neural activities were studied in nonpremedicated, anesthetized, paralyzed, vagotomized dogs. Hyperventilation with O2 and the addition of CO2-O2 mixtures were used to produce low, medium, and high steady-state levels of central CO2 drive. Peak neuron discharge frequency decreased progressively with increasing halothane dose at all levels of CO2 drive for both types of neurons. The sensitivities of inspiratory and expiratory bulbospinal neuronal activities to halothane were not significantly different from one another, whereas the sensitivity to halothane of the peak phrenic activity was markedly greater than those of the neurons. Increasing halothane dose caused a downward, predominantly parallel shift of the CO2 response curves. Phrenic nerve activity also showed a decrease in slope of the CO2 response. The activities of respiratory premotor neurons are less depressed by increasing doses of halothane than is phrenic nerve activity. The greater depression of phrenic activity may result from additional anesthetic actions on the efferent motor pathways, resulting in decreased descending synaptic inputs to phrenic motor neurons.

Role of bulbospinal respiratory neurons in the generation of the breathing rhythm.

Role of bulbospinal respiratory neurons in the generation of the breathing rhythm.

CENTRAL NEURONS AND NEUROTRANSMITTERS IN THE CONTROL OF BLOOD PRESSURE

1. In this paper we review recent work from our laboratory on two major pathways important in the central control of blood pressure. 2. We report experiments on the sympatho-excitatory bulbospinal pathway from the rostral ventral medulla. Here we focus particularly on the role of excitatory amino acids. 3. We review studies on the short inhibitory or depressor pathway ascending from the caudal to the rostral ventral medulla, which is thought to use gamma-aminobutyric acid (GABA) as its neurotransmitter. We report on experiments with the immediate early gene, c-fos, demonstrating that its expression in the bulbospinal pressor neurons is increased by stimuli that activate these nerves, and that this expression can be blocked in vivo by treatment with an antisense oligonucleotide. We also show that basal and stimulated expression of the c-fos gene is important in the central control of blood pressure.

Brainstem network controlling descending drive to phrenic motoneurons in rat.

Contraction of the diaphragm is controlled by phrenic motoneurons that receive input from sources that are not fully established. Bulbospinal (second-order) neurons projecting to phrenic motoneurons and propriobulbar (third-order) neurons projecting to these bulbspinal neurons were investigated in rat by transsynaptic transport of the neuroinvasive pseudorabies virus. Bulbospinal neurons were located predominantly in the medullary lateral tegmental field in two functionally described regions, the ventral respiratory group and Bötzinger complex. An intervening region, the pre-Bötzinger complex, contained essentially no phrenic premotoneurons. Bulbospinal neurons were also located in ventral, interstitial, and ventrolateral subnuclei of the solitary tract, and gigantocellular, Kölliker-Fuse, parabrachial, and medullary raphe nuclei. A monosynaptic pathway to phrenic motoneurons from the nucleus of the solitary tract was confirmed; monosynaptic pathways from upper cervical spinal cord, spinal trigeminal nucleus, medical and lateral vestibular nuclei, and medial pontine tegmentum were not verified. Locations of third-order neurons were consistent with described projections to the ventral respiratory group, from contralateral ventral respiratory group, Bötzinger complex, A5 noradrenergic cell group, and the following nuclei; solitary, raphe, Kölliker-Fuse, parabrachial, retrotrapezoid, and paragigantocellular. Novel findings included a projection from locus coeruleus to respiratory premotoneurons and the lack of previously described pathways from area postrema and spinal trigeminal nucleus. These second- and third-order neurons from the output network for diphragm motor control which includes numerous behaviors (e.g., respiration, phonation, defecation). Of the premotoneurons, the rostral ventral respiratory group is the primary population controlling phrenic motoneurons.

Pyroglutamyl peptidase II activity is not in the processes of bulbospinal TRHergic neurons

Pyroglutamyl peptidase II (PPII) is a neuronal ectoenzyme involved in released thyrotropin-releasing hormone (TRH) inactivation. In an attempt to define if it is present in the pre or postsynaptic membrane, we induced neuronal degeneration of serotonin-TRHergic cells that project from raphe nuclei to the spinal cord. 2–4 weeks after intracisternal injection of 5,7-dihydroxytryptamine, TRH levels decreased over 70% in the cervical, thoracic or lumbar regions of spinal cord. In contrast, no change of PPII activity was observed. Longer times after injection (6–8 weeks), a 59–66% increase in activity was detected in the lumbar region. These data suggest that PPII is not localized in these TRHergic neurons but probably in the target cells.

Different effects of respiratory and metabolic acidosis on preganglionic sympathetic nerve activity

We studied sympathetic nerve activity (SNA) responses, recorded in multifiber preparations of left third thoracic white ramus, to respiratory or isocapnic metabolic acidosis or to CO2 enhancement at constant pH in chloralose-anesthetized paralyzed artificially ventilated cats. Cardiopulmonary, baro-, and peripheral chemoreceptors were denervated by bilaterally cutting vagus and carotid sinus nerves. Acidosis was induced by either decreasing artificial ventilation or infusing HCl (0.5 M i.v.). Both respiratory and isocapnic metabolic acidosis induced a decrease in local extracellular pH, measured directly with pH-sensitive microelectrodes within medulla region containing sympathoexcitatory bulbospinal neurons. The magnitude of changes in medullary pH was independent of the way systemic acidosis was generated. Despite uniformity of changes in local medullary extracellular pH due to systemic respiratory or isocapnic metabolic acidosis, different responses were observed in preganglionic SNA. Isocapnic metabolic acidosis resulted in a slight increase in SNA, averaging 6.4% per 0.05 systemic pH unit decrease. In contrast, respiratory acidosis induced by decreasing artificial ventilation produced a more pronounced increase of SNA, reaching peak changes of approximately 70% compared with control level with normal blood gases, an average increase of 13% per 0.05 systemic pH unit decrease. We conclude that systemic CO2 and H+ concentrations represent different stimuli to sympathetic nervous system. Despite similar changes of local extracellular pH within rostral ventrolateral medulla during systemic acidosis, different responses of SNA suggest other sites or as yet unknown additional effects of CO2 as being responsible for excitation of sympathetic activity.

Homeodynamics versus homeostasis: periodicities superimposed on non-linear dynamic sympathetic tone generated in ventral medulla

Homeodynamics based on theories of complexity and chaos and its impact on mechanisms generating sympathetic activity are presented. Activity in rats cervical, lumbar and renal sympathetic nerves was analyzed. In time domain glutamate stimulation of neurons within medullary periambigual area (PAA) disturbed temporal pattern of respiratory-sympathetic synchronization. Divalent calcium antagonists, Co2+ and Mg2+, blockers of synaptic transmission, uncoupled respiratory oscillator and sympathetic activity. PAA neurons act as an interphase between different subsets of respiratory neurons and bulbospinal sympathoexcitatory neurons in rostral ventrolateral medulla (RVLM). In frequency domain sympathetic activity analyzed by FFT algorithm and power density spectra (PDS) exhibited periodicities at the range from 0.4 Hz to 7.5 Hz. Blockers of synaptic transmission microinjected bilaterally into RVLM reduced total power exhibited in PDS to low level of magnitude generated in spinal cord and increased total, yet non-synchronized sympathetic activity and arterial blood pressure. A two component hybrid model of generation of sympathetic activity was proposed: a tone-generating system confined mainly to intrinsic activity of RVLM pacemaker neurons responsible for chaos-like discharges and a second component - ­neuronal circuits superimposed on tone-generating neurons and shaping the pattern of PDS. Contribution of spinal cord oscillatory mechanism to overall power of sympathetic periodicities was discussed.

Interaction between chemoreceptor and stretch receptor inputs at medullary respiratory neurons

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.

Disinhibition of the rostral ventral medulla increases blood pressure and Fos expression in bulbospinal neurons

The GABA agonist muscimol, injected into the depressor area of the caudal ventrolateral medulla, increased blood pressure and increased the expression of the immediate early gene c-fos in the rostral ventral medulla (RVM) of the rat. The number of Fos-immunoreactive (Fos-IR) neurons seen in the RVM was increased 3-fold after muscimol compared to Fos-IR after vehicle treatment. In the rostral aspect of the RVM approximately half of the Fos-IR neurons were identified as spinally projecting after the injection of the retrograde tracer cholera toxin B subunit into the upper thoracic spinal cord. These bulbospinal Fos-IR neurons were identified in the lateral aspects of the RVM, in the area where baroreceptor-sensitive neurons have been identified in electrophysiological studies, and alos in more medial areas of the RVM. Fos-IR neurons were also identified in the intermediolateral cell column of the thoracic spinal cord after muscimol injection, but were rarely observed in this area after vehicle treatment. This study demonstrates the functional connectivity of the caudal and rostral areas of the medulla oblongata and the spinal cord, supporting the view that the caudal ventrolateral medulla contains neurons that provide a tonic inhibitory control over neurons in the RVM and that, in turn, the spinally projecting neurons in the RVM provide an excitatory input to the spinal cord sympathetic preganglionic neurons. The results show that the withdrawal of an inhibitory input is a sufficient stimulus to increase the expression of c-fos and increase neuronal activity in the RVM. c-fos expression also increases in the neurons that receive an excitatory input from these RVM neurons.

Bulbospinal neuropeptide Y-immunoreactive neurons in the rat: comparison with adrenaline-synthesising neurons.

Immunohistochemistry and retrograde tracing using cholera toxin B subunit colloidal gold (CTB-gold) has been used to identify neurons in the medulla that contain neuropeptide Y and project to the area of the intermediolateral cell column in either the upper (T2-T4) or the lower (T8-T9) thoracic spinal cord. The rostrocaudal distributions of neuropeptide Y neurons and neuropeptide Y/CTB-gold neurons have been compared with the distributions of adrenaline-synthesising, phenylethanolamine N-methyltransferase-containing neurons and phenylethanolamine N-methyltransferase/CTB-gold neurons visualised in adjacent sections. In particular areas of the rostral medulla similarities in the numbers and distributions of neuropeptide Y neurons and phenylethanolamine N-methyltransferase neurons suggested a coexistence of the peptide within the catecholamine neurons. However, at the most rostral levels of the rostral ventral medulla, the large numbers of phenylethanolamine N-methyltransferase neurons were not matched by similar numbers of neuropeptide Y neurons, so that the phenylethanolamine N-methyltransferase neurons in this area could not all contain neuropeptide Y. In the rostral ventral medulla fewer neuropeptide Y/CTB-gold neurons than phenylethanolamine N-methyltransferase/CTB-gold neurons were observed, so that these bulbospinal peptide neurons might define a subset of the phenylethanolamine N-methyltransferase/CTB-gold neurons, accounting for 25% of the total phenylethanolamine N-methyltransferase bulbospinal projection from the rostral ventral medulla. Other neuropeptide Y/CTB-gold neurons in the dorsal medulla are also likely to contain phenylethanolamine N-methyltransferase. Finally, a population of neuropeptide Y/CTB-gold neurons was identified in the caudal ventral medulla, these neurons appear not to contain catecholamine synthesising enzymes.

Whole-cell patch-clamp recordings from visualized bulbospinal neurons in the brainstem slices

The purpose of this study was to develop a method for electrophysiological characterization of retrogradely labeled bulbospinal neurons in the specific cytoarchitectonic regions in the brainstem slices. Several days after the spinal cord was injected with the carbocyanine dye, DiI, retrogradely labeled bulbospinal neurons were visualized by epifluorescence optics in the brainstem slices with the aid of a silicon intensifier tube (SIT) camera. Labeled somata were routinely seen in the caudal raphe nuclei, rostroventral medial and lateral portions of the medulla, the A5 group and in other medullary sites known to project to the spinal cord. Electrophysiological properties of the DiI-labeled neurons were assessed by whole-cell recordings using micropipettes filled with biocytin. The slices were subsequently processed for dual visualization of biocytin and serotinin or a marker for noradrenergic neurons, tyrosine hydroxylase (TH). The electrophysiological properties of bulbospinal neurons were correlated with their morphology and neurochemical content. This technique may be useful in other areas of CNS for studying morphology, neurochemical content and physiology of retrogradely labeled neurons.

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 C 1 and T 1 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.

Alpha 2A-adrenergic receptors are present in lower brainstem catecholaminergic and serotonergic neurons innervating spinal cord

A subtype-specific polyclonal antibody was used for the immunohistochemical detection of alpha 2A-adrenergic receptors (α 2A-ARs) in the rat lower brainstem (medulla and pons). Using dual-label fluorescence histochemistry, punctate α 2A-AR-like immunoreactivity (α 2A-AR-LIR) was identified in noradrenergic, adrenergic, and serotonergic neurons of the pontomedullary region. Confocal microscopic examination of material simulataneously labeled for TH-LIR and α 2A-LIR revealed that the clusters of α 2A-LIR were located intracellularly. Lower medullary neurons with spinal protections to segment T 3 were retrogradely labeled using FITC-conjugated microbeads and the material was processed for simulatneous detection of α 2A-LIR and either TH-LIR or 5-HT-LIR. Using this triple-label approach, we found that virtually all medullary serotonergic cells (raphe pallidus, raphe obscurus and parapyramidal area) including those with identified spinal projections contain α 2A-AR-LIR. In contrast, fewer than 10% of dorsal raphe serotonergic cells examined for comparison were immunoreactive. The triple labeling approach also indicated that more than 95% of the TH-immunoreactive cells of the dorsal and ventrolateral medulla, including those with demonstrable spinal projections (A5 noradrenergic and C1/C3 adrenergic) had detectable amounts of α 2A-AR-LIR. The presence of α 2A-ARs in a large fraction of bulbospinal pre-sympathetic neurons (noradrenergic A5, adrenergic C1 and C3 and serotonergic raphe cells) could explain the powerful and relatively selective effect of clonidine and other centrally acting α 2A-AR agonists on sympathetic efferent activity and hypertension.

Chemical activation of caudal medullary expiratory neurones alters the pattern of breathing in the cat.

1. The purpose of this work was to ascertain whether the activation of caudal expiratory neurones located in the caudal part of the ventral respiratory group (VRG) may affect the pattern of breathing via medullary axon collaterals. 2. We used microinjections of DL-homocysteic acid (DLH) to activate this population of neurones in pentobarbitone-anaesthetized, vagotomized, paralysed and artificially ventilated cats. Both phrenic and abdominal nerve activities were monitored; extracellular recordings from medullary and upper cervical cord respiratory neurones were performed. 3. DLH (160 mM) microinjected (10-30 nl for a total of 1.6-4.8 nmol) into the caudal VRG, into sites where expiratory activity was encountered, provoked an intense and sustained activation of the expiratory motor output associated with a corresponding period of silence in phrenic nerve activity. During the progressive decline of the activation of abdominal motoneurones, rhythmic inspiratory activity resumed, displaying a decrease in frequency and a marked reduction or the complete suppression of postinspiratory activity as its most consistent features. 4. Medullary and upper cervical cord inspiratory neurones exhibited inhibitory responses consistent with those observed in phrenic nerve activity, while expiratory neurones in the caudal VRG on the side contralateral to the injection showed excitation patterns similar to those of abdominal motoneurones. On the other hand, in correspondence to expiratory motor output activation, expiratory neurones of the Bötzinger complex displayed tonic discharges whose intensity was markedly lower than the peak level of control breaths. 5. Bilateral lignocaine blockades of neural transmission at C2-C3 affecting the expiratory and, to a varying extent, the inspiratory bulbospinal pathways as well as spinal cord transections at C2-C3 or C1-C2, did not suppress the inhibitory effect on inspiratory neurones of either the ipsi- or contralateral VRG in response to DLH microinjections into the caudal VRG. 6. The results show that neurones within the column of caudal VRG expiratory neurones promote inhibitory effects on phrenic nerve activity and resetting of the respiratory rhythm. We suggest that these effects are mediated by medullary bulbospinal expiratory neurones, which may, therefore, have a function in the control of breathing through medullary axon collaterals.

Role of serotonin and catecholamines in sympathetic responses evoked by stimulation of rostral medulla

In halothane-anesthetized, paralyzed rats, single-pulse stimulation of nucleus raphe pallidus/obscurus (NR) evoked an inhibition followed by a single wave of excitation in 1) the mass discharge of lumbar and splanchnic sympathetic nerves (modal onset latencies: LSND 210 +/- 14 ms, SSND 161 +/- 5 ms) and 2) the unit activity of lumbar sympathetic vasoconstrictor neurons (LSVNs). Stimulation of the rostral ventrolateral medulla (RVL) produced two excitatory responses in LSND, SSND, and LSVN units (modal onset latencies: RVL peak I 98 +/- 8 ms for LSND and 62 +/- 2 ms for SSND; RVL peak II 210 +/- 15 ms for LSND and 160 +/- 6 ms for SSND). Most LSVNs received excitatory inputs from both raphe and RVL. NR peak was selectively attenuated (76%) by 5,7-dihydroxytryptamine [intracisternally (ic), 2-wk] and acute intrathecal (it) methiothepine (10 micrograms, 69%) or methysergide (40 micrograms, 72%). 6-Hydroxydopamine (2-wk ic) produced no effect. Phentolamine (20 micrograms it) or prazosin (20 micrograms it) reduced RVL peak II by > 94% and attenuated NR peak (phentolamine 67%, prazosin 46%). Intrathecal kynurenic acid produced proportionately larger reduction of RVL peak I than raphe peak or RVL peak II. Our interpretations are 1) LSVNs receive convergent excitatory inputs from midline raphe, parphyramidal area, and RVL, 2) bulbospinal serotonergic neurons mediate most of the sympathoexcitation evoked from NR and a portion of RVL peak II, 3) a catecholamine probably released by C1 cells mediates most of RVL peak II and a portion of the NR response, and 4) RVL peak I is predominantly mediated by an excitatory amino acid.

Bulbospinal neurons of the cat that co-contain serotonin and methionine enkephalin.

The present study utilizes a combined retrograde transport of Fast Blue (or rhodamine-labeled latex microspheres) and simultaneous immunofluorescence technique to demonstrate directly the coexistence of serotonin and methionine enkephalin in bulbospinal neurons of the cat. The bulbospinal neurons that immunostained for both serotonin and enkephalin were observed, without any distinct somatotopic organization, in the nuclei raphe pallidus, obscurus and magnus. They were also observed in the nucleus reticularis magnocellularis and the ventrolateral medulla (cell group B1/3). Among the bulbospinal neurons encountered within individual 5-HT-rich medullary nuclei, high proportions of these neurons co-containing serotonin and methionine enkephalin were evidenced in the nucleus raphe obscurus (64%) and nucleus raphe pallidus (56%), less so in cell group B1/3 (41%), nucleus raphe magnus (39%), and the nucleus reticularis magnocellularis (29%). Physiological significance of such a morphological substrate is discussed.