Electrophysiological characterization of putative C1 adrenergic neurons in the rat.

Recent studies in the rat have demonstrated that at least two populations of sympathoexcitatory reticulospinal neurons reside in the nucleus reticularis rostroventrolateralis. It appears that only one of these populations consists of C1 adrenergic neurons. The present study used both double-labeling (one retrograde tracer and immunohistochemistry) and triple-labeling (two retrograde tracers and immunohistochemistry) to determine if C1 adrenergic neurons, which are immunoreactive for phenylethanolamine N-methyltransferase, exhibit a projection pattern that is sufficiently unique to permit the electrophysiological discrimination between C1 adrenergic and non-adrenergic neurons in the nucleus reticularis rostroventrolateralis. Double-labeling experiments indicated that 71% (range: 53-80) of phenylethanolamine-N-methyltransferase-immunoreactive neurons in the nucleus reticularis rostroventrolateralis could be retrogradely labeled from the thoracic cord, as were 76% (range: 67-94) following tracer injection in the central tegmental tract at pontine levels. Triple-labeling experiments indicated that 88% (range: 82-93) of nucleus reticularis rostroventrolateralis neurons with projections to both spinal cord and central tegmental tract were phenylethanolamine-N-methyltransferase-immunoreactive. Single-unit recording, in nucleus reticularis rostroventrolateralis, was used to identify antidromic potentials elicted from stimulation sites in the spinal cord and/or central tegmental tract. Since clonidine is known to reduce central adrenaline turnover, sensitivity to this drug was used to identify putative adrenergic neurons. Twenty-six nucleus reticularis rostroventrolateralis neurons with axonal projections to both the ipsilateral spinal cord and the central tegmental tract were recorded in halothane-anesthetized rats. All these cells were barosensitive, pulse-modulated, and 16 of the 16 cells tested exhibited a 66 +/- 8% reduction in activity upon the intravenous administration of clonidine (20 micrograms/kg). Most (13 out of 16) exhibited a strong respiratory modulation. The conduction velocity of their spinal collateral was generally low (0.9 +/- 0.1 m/s) and their firing rate moderate (7.4 +/- 1.2 spikes/s). Forty-three nucleus reticularis rostroventrolateralis cells with axonal projections exclusively to the thoracic cord were studied for comparison. These cells were strongly barosensitive and pulse-synchronous, had a high discharge rate (25 +/- 3 spikes/s) and a moderate conduction velocity (3.4 +/- 0.3 m/s). Only one of the 15 cells tested was inhibited by clonidine and only two to these 15 cells exhibited a detectable respiratory modulation. Thus barosensitive nucleus reticularis rostroventrolateralis neurons with axonal projections to both the spinal cord and the central tegmental tract likely belong to the C1 adrenergic cell group. It is concluded that this subgroup of adrenergic neurons probably subserves a vasomotor function.

Sympathoexcitatory neurons of the rostroventrolateral medulla and the origin of the sympathetic vasomotor tone.

In summary, a substantial portion of the excitatory drive to vasomotor sympathetic preganglionic neurons originates from reticulospinal tonically active cells located in the RVLM. This interpretation does not exclude the possible contribution of other tonically active bulbospinal or propriospinal inputs in generating the vasomotor outflow but under usual anesthetic conditions it seems that these alternative inputs are simply insufficient to bring the vasomotor preganglionic neurons to their firing threshold. Such may not be the case after plastic rearrangements consecutive to complete spinalization or chronic lesions of large portions of the RVLM have occurred (Cochrane and Nathan, 1987; for review see Schramm, 1986). It is also clear at present that the RVLM is not merely a final common pathway consisting of premotoneurons passively driven by tonic synaptic inputs originating elsewhere. Indeed the existence of a population of reticulospinal neurons with intrinsic pacemaker activity indicates that the RVLM contains at least one major intrinsic source of tonic activity. These neurons may release a glutamate-like substance and are not phenotypically adrenergic. They have no documented projections outside the cord and could subserve a tone-generating function specific to the sympathetic outflow, e.g. providing a background excitatory input to a large number of preganglionic neurons with vasoconstrictor of cardioaccelerator function. Strong anatomical evidence backed by weaker electrophysiological evidence also support the notion that C1 adrenergic neurons may have a vasomotor role and contribute an excitatory drive to preganglionic neurons. This could be mediated via alpha 1-adrenergic receptors or by receptors to substance P or neuropeptide Y. There is no evidence yet that C1 cells might have intrinsic pacemaker activity. The origin of the ongoing activity of many of these cells "in vivo" is therefore unclear and could depend on an excitatory drive from outside the RVLM. One might speculate that because these cells appear to have collateral interactions (PNMT-immunoreactive boutons synapse on C1 cells, Milner et al., 1987), they could play a role in synchronizing the sympathetic vasomotor outflow (an unexplained phenomenon observable even in the absence of baroreceptor input). Because of the large variety of peptides which they contain, another speculative view could be that they make rather specific connections with subsets of preganglionic neurons and therefore might be responsible for the differential control of regional blood flows by the rostral medulla (Dampney and McAllen, 1988). C1 cells are inhibited by low systemic doses of clonidine and therefore may be in part responsible for the hypotensive effect of this drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of caudal ventrolateral medulla in reflex and central control of airway caliber.

We investigated the role played by the caudal ventrolateral (CVL) medulla in the reflex and central neural control of airway caliber in chloralose-anesthetized dogs. Changes in total lung resistance were evoked by four different stimuli. These changes were compared before and after bilateral injection of either ibotenic acid (75 nl; 100 mM) or cobalt chloride (75 nl; 50 mM) into the CVL medulla. The four stimuli used to change lung resistance were static muscular contraction, electrical stimulation of thin fiber afferents in the sciatic nerve, electrical stimulation of the posterior diencephalon, and hypoxia. The first three stimuli have been shown to decrease total lung resistance, whereas the latter stimulus has been shown to increase resistance. We found that injection of both ibotenic acid, which destroys cell bodies but not fibers of passage, and cobalt, which prevents synaptic transmission, either abolished or greatly attenuated the decrease in total lung resistance evoked by static contraction, by sciatic nerve stimulation, and by posterior diencephalic stimulation. We also found that injection of ibotenic acid and cobalt attenuated the reflex increase in lung resistance evoked by hypoxia. In control experiments, we found that bilateral injection of ibotenic acid into the dorsal medulla had no effect on the changes in total lung resistance evoked by these four stimuli. We conclude that the CVL medulla plays an important role in the reflex and central control of airway caliber.

Vagal neuroeffector mechanisms affecting transpulmonary pressure in the intact rat.

Experiments were conducted with chloralose-urethan anesthetized rats to assess the effects of 1) bilateral stimulation of the cervical vagus nerves and 2) parasympathomimetic and sympathomimetic agents. Transpulmonary pressure (Ptp) was used as an index of airway smooth muscle tone, and peak inspiratory Ptp (Ptppeak) values were used for a comparison of responses. In untreated animals, vagal stimulation elicited an increase in Ptppeak of 155%. Cooling of the vagus nerves to 15 degrees C abolished the response of Ptppeak to vagal stimulation. Although isoproterenol (1-10 micrograms/kg i.v.) did not alter resting Ptppeak, it did prevent vagal stimulation from evoking an increase in Ptppeak. Nadolol (1.5 mg/kg i.v.) augmented the increase in Ptppeak elicited by vagal stimulation. Vagal stimulation did not evoke any change in Ptppeak after the administration of both nadolol and atropine or after combined administration of nadolol, atropine, and either serotonin aerosol or prostaglandin F2 alpha. In rats pretreated with capsaicin 1 wk before the experiment, vagal stimulation evoked an increase in Ptppeak that was not statistically different from that of untreated control animals. Therefore, nonadrenergic noncholinergic systems did not appear to play an independent role in the response of the airways to the activation of the vagus nerves.

Activation of neurons in the rostral ventrolateral medulla increases bronchomotor tone in dogs.

Previous work from this laboratory has demonstrated that the chemical activation of cell bodies in the caudal ventrolateral medulla of chloralose-anesthetized dogs decreased bronchomotor tone by withdrawing cholinergic input to airway smooth muscle. In the present study we determined the bronchomotor responses to microinjection of DL-homocysteic acid (100 mM; 25-50 nl) into the rostral ventrolateral (RVL) medulla of chloralose-anesthetized dogs. Total lung resistance was used as a functional index of bronchomotor tone. Microinjection of DL-homocysteic acid into the 20 sites located in the lateral aspect of the RVL medulla increased both total lung resistance [from 6.5 +/- 0.4 to 9.1 +/- 0.8 (SE) cmH2O.l-1.s; P less than 0.05] and mean arterial pressure (from 125 +/- 5 to 148 +/- 8 mmHg; P less than 0.05). Microinjection of this amino acid into nine sites located in the medial aspect of the RVL medulla increased mean arterial pressure (from 130 +/- 6 to 153 +/- 6 mmHg; P less than 0.05) but had no effect on total lung resistance. We confirmed in three sites that the increase in total lung resistance evoked by microinjection of DL-homocysteic acid was accompanied by an increase in tracheal smooth muscle tension. The increase in total lung resistance evoked by DL-homocysteic acid was not affected by beta-adrenergic blockade but was abolished by muscarinic blockade.

Bronchomotor vagal preganglionic cell bodies in the dog: an anatomic and functional study.

A previous study in our laboratory demonstrated that the stimulation with microinjection of DL-homocysteic acid of cell bodies in the rostral portion of the external formation of the nucleus ambiguus (Aext) increased total lung resistance in dogs. In the present study anatomic experiments were conducted in dogs to determine if the rostral Aext contains vagal preganglionic cell bodies that give rise to axons in the pulmonary branches of the vagus nerve. The application of horseradish peroxidase (HRP) to either the pulmonary branches or the vagus at a point between the pulmonary branches and the cardiac branches resulted in retrograde labeling of cell bodies in both rostral Aext and the dorsal motor nucleus of the vagus (DMN). On the other hand, application of HRP to the vagus at a point below the pulmonary branches did not result in any retrogradely labeled cell bodies in rostral Aext but did result in labeled cell bodies in DMN. In another series of experiments DL-homocysteic acid (2.5 nmol in 25 nl) was microinjected at sites in rostral Aext and DMN. As we previously reported the injection of DL-homocysteic acid in rostral Aext increased total lung resistance. In contrast, in the same animals, the injection of DL-homocysteic acid in DMN did not change total lung resistance. We conclude that bronchomotor vagal preganglionic cell bodies are located in rostral Aext but not in DMN. The functional significance of vagal preganglionic cell bodies in DMN whose axons contribute to the pulmonary branches of the vagus nerve remains to be determined.

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.

Anatomical and functional connections of neurons of the rostral medullary raphe of the rabbit.

Single cell recordings were made from neurons in the rostral medullary raphe (RMR) of the rabbit. The recording sites were ones that had been shown to yield pressor responses from electrical stimulation and by pressure injections of glutamate. Electrical stimulation of the intermediolateral (IML) region of the spinal cord led to antidromic activation of 12 of the 100 cells studied. Eleven of these cells were located in raphe pallidus or raphe magnus, and one cell was located in raphe obscurus. These findings were consistent with the results of horseradish peroxidase (HRP) histochemistry experiments. Injections of HRP into the IML led to heavy cell body labeling in raphe pallidus and raphe magnus, but sparse labeling in raphe obscurus. Cells in the RMR could be orthodromically activated by electrical stimulation of the putative defense area of the periaqueductal (PAG) but not by stimulation of putative defense areas in the hypothalamus. Most of these cells were located in raphe pallidus or raphe magnus. Similarly, HRP injections into raphe pallidus and raphe magnus led to heavy cell body labeling in the PAG but not the hypothalamus; no cell body labeling was found in the PAG when injections were made into raphe obscurus.

Cardiovascular responses elicited by electrical and chemical stimulation of the rostral medullary raphe of the rabbit.

Electrical stimulation of the rostral medullary raphe (RMR) of the rabbit elicited pressor responses that were accompanied by tachycardia or bradycardia. Stimulation of dorsal sites (the dorsal raphe obscurus) evoked a pressor/tachycardia response and stimulation of ventral sites (the ventral raphe obscurus, raphe magnus and raphe pallidus) produced a pressor/bradycardia response. Electrical stimulation of the RMR after sinoaortic denervation led to an increase in the magnitude of the pressor response elicited from all stimulation sites, a decrease in the magnitude of the bradycardia produced by stimulation at the ventral sites, but had no effect upon the magnitude of the tachycardia observed from stimulation of the dorsal sites. These findings suggest that electrical stimulation of the dorsal sites leads to inhibition of the cardiomotor component of the baroreceptor reflex. The results of vagal blockade experiments demonstrated that baroreceptor attenuation of the pressor responses at ventral sites was mediated primarily by parasympathetic input to the heart. Chemical stimulation of the RMR with L-glutamate also led to a pressor/tachycardia response at the dorsal sites and a pressor/brachycardia response at the ventral sites. This finding provides evidence that neuronal cell bodies, not axon of passage, mediated the responses elicited by electrical stimulation.

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.

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.

Neuronal cell bodies in paraventricular nucleus affect renal hemodynamics and excretion via the renal nerves.

Several lines of evidence support the existence of an oligosynaptic projection from the paraventricular nucleus of the hypothalamus (PVN) to the kidney in the rat. We sought to provide evidence that this neural pathway is capable of influencing renal function in rats. Bilateral microinjections of bicuculline (Bic; 1 nmol) into the PVN decreased glomerular filtration rate (59%), effective renal plasma flow (71%), urine flow (UV; 57%), and urinary sodium excretion (UNaV; 54%), accompanied by increased mean arterial pressure (17%) and heart rate (17%). These results were not obtained when Bic was injected outside the PVN or when vehicle (0.9% saline) was injected into the PVN. Bilateral renal denervation (5-7 days before the experiments) significantly reduced the renal vasoconstriction, attenuated the antidiuresis, and abolished the antinatriuresis evoked by PVN stimulation. On the other hand, both the antidiuresis and antinatriuresis evoked by PVN stimulation were undiminished after treatment with either of two vasopressin receptor antagonists ([beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,O-Et-Tyr2, Val4,Arg8]vasopressin, a vasopressin V1 receptor antagonist, or [adamantaneacetyl1,O-Et-D-Tyr2,Val4,aminobutyryl6,Arg8, 9]-vasopressin, a V2 receptor antagonist). In renal-denervated rats treated with the same V2 receptor antagonist, PVN stimulation produced highly variable increases in both UV and UNaV, which overall were not statistically different than zero. We conclude that the activation of neurons in PVN evokes 1) renal vasoconstriction accompanied by antinatriuresis, both of which are attributable to the renal nerves, and 2) decreased water excretion, which is mediated by the renal nerves and vasopressin V2 receptors.

Ascending collaterals of medullary barosensitive neurons and C1 cells in rats.

Triple-label experiments were conducted in rats to determine whether reticulospinal C1 adrenergic neurons of rostral ventrolateral medulla (RVLM) also innervate dorsolateral pons (LPBN/KF), mesencephalic central gray area (CG), and hypothalamus (LH). Ten percent of RVLM neurons that project to LPBN/KF were immunoreactive for tyrosine hydroxylase (TH), and 50% of those projecting to either pariaqueductal gray (PAG) or LH were TH immunoreactive. A large proportion of RVLM TH-immunoreactive neurons that were retrogradely labeled from either LPBN/KF or PAG (65.5 and 28.5%, respectively) were retrogradely labeled from the spinal cord as well. A smaller fraction of RVLM TH-immunoreactive neurons that were labeled from LH were also labeled from the spinal cord (13.1%). The vast majority of the cohaterized RVLM neurons were TH immunoreactive (LPBN/KF 50%; PAG 82%; LH 84%), and the remaining ones may be nonadrenergic neurons. Antidromic (AD) mapping techniques were also used to determine if RVLM reticulospinal barosensitive neurons (putative vasomotor sympathoexcitatory cells, total sample of 106) send axonal collaterals to the supramedullary areas investigated anatomically. Twenty percent of the cells sampled (n = 21) were also AD activated from 1 of 20 supramedullary sites. The combined approaches support the following conclusions: 1) RVLM barosensitive neurons include phenotypically adrenergic cells, and 2) RVLM barosensitive cells do not represent an output pathway solely dedicated to providing an excitatory drive to sympathetic preganglionic neurons.

Central respiratory modulation of medullary sympathoexcitatory neurons in rat.

The central respiratory generator exerts a modulatory influence on sympathetic nerve discharge. In cats the sympathoexcitatory neurons of the rostroventrolateral medulla (RVL) exhibit central respiratory modulation as well. Because RVL sympathoexcitatory neurons are largely responsible for the maintenance of sympathetic vasomotor tone, it is likely that the modulation of these neurons accounts for the central respiratory modulation of sympathetic discharge. In the present study experiments were performed to characterize the pattern of respiratory modulation of lumbar sympathetic nerve discharge (LSND) in the halothane-anesthetized rat. Phrenic-triggered averaging of LSND exhibited a small depression coincident with the onset of the phrenic burst followed by a large peak that was coincident with the cessation of the phrenic burst. Phrenic-triggered histograms of the activity of RVL sympathoexcitatory neurons exhibited three patterns of central respiratory modulation: inspiratory depression (I), inspiratory peak (II), and early inspiratory depression followed by a postinspiratory peak (III), a pattern that was very similar to that seen in LSND. Both nerve recording and single-unit recording experiments were performed in vagotomized rats with or without intact barosensory afferents. A comparison of the results suggested that, in the rat, the baroreflex does not modify or contribute to the central respiratory modulation of sympathetic output. Finally, a comparison was made between presumed nonadrenergic pacemaker-like neurons and putative C1 adrenergic neurons in the RVL. No differences were found in the patterns of central respiratory modulation.

Parvocellular neurons of the paraventricular nucleus are involved in the reduction in renal nerve discharge during isotonic volume expansion.

The parvocellular neurons of the paraventricular nucleus (PVN) of the hypothalamus are known to be involved in the control of central autonomic outflow. While the PVN is also known to be involved in the control of fluid-balance, most of the studies examining this nucleus have emphasized the magnocellular neurons, which are involved in the humoral control of fluid-balance and related hemodynamics. The present study took advantage of the differential sensitivity of these two cell types to kainic acid as a means of investigating the role of the parvocellular neurons in the reflex reduction of renal sympathetic nerve discharge (RSND) during acute isotonic volume expansion. Kainic acid (18 pmol), which destroys parvocellular but not magnocellular neurons in the PVN, was microinjected (20 nl) bilaterally at sites in and adjacent to the PVN 3-4 days prior to acute isotonic volume expansion. In anesthetized rats RSND decreased by 59% at the completion of acute isotonic volume expansion (10% of body weight) in the vehicle-injected control group; on the other hand, it decreased by 33% in the kainic acid-treated group. The effect of destruction of the parvocellular neurons on the baroreceptor reflex was also examined. Neither the renal nerve component (delta %RSND/delta AP), nor the heart rate component (delta HR/delta AP), of the baroreceptor reflex were different in the kainic acid-treated group (3.1 +/- 0.4, and 1.1 +/- 0.1, respectively) than in the vehicle-injected control group (2.9 +/- 0.7, and 0.8 +/- 0.1, respectively). We conclude that the parvocellular neurons of the PVN are an important synaptic relay site in the reflex are that is activated during isotonic volume expansion, but not in the baroreceptor reflex.

Ischemia potentiates the reflex bronchodilation evoked by static muscular contraction in dogs.

In eleven anesthetized dogs, we found that static contraction of hindlimb muscles that were freely perfused decreased total lung resistance by 0.7 +/- 0.1 cm H2O.L-1.sec, whereas static contraction of the same muscles rendered ischemic decreased total lung resistance by 1.5 +/- 0.4 cm H2O.L-1.sec (P less than 0.025). In ten other dogs, we found that static contraction of freely perfused hindlimb muscles decreased total lung resistance by 0.9 +/- 0.2 cm H2O.L-1.sec, whereas dynamic contraction of the same freely perfused muscles decreased total lung resistance by 1.1 +/- 0.3 cm H2O.L-1.sec. The difference in the magnitudes of the bronchodilator responses to the two modes of contraction was not significant (P greater than 0.05). We conclude that a mismatch between blood supply and demand in working skeletal muscle increases the reflex bronchodilator response to static contraction. We also conclude that dynamic contraction evokes a reflex bronchodilation equivalent to that evoked by static contraction provided that the tension produced by the two modes of contraction are equal.

The location of chronotropic cardioinhibitory vagal motoneurons in the medulla of the rabbit.

Vagal preganglionic motoneurons originating in nucleus ambiguus (NA) and dorsal vagal nucleus (DVN) were identified via retrograde labeling with horseradish peroxidase (HRP). DVN and NA were then explored for cardiovascular responsive sites using microstimulation. Stimulation within DVN from slightly caudal to obex to 3.00 mm rostral to obex produced a primary bradycardia (n = 15, X = -123 bpm). Stimulation within NA from slightly rostral to obex to 1.5 mm caudal to obex produced a similar primary bradycardia (n = 15, X = -127 bpm). Extracellular recordings were made from 7 cells in DVN and 10 cells in NA in regions producing maximal bradycardia to electrical stimulation. These cells were antidromically activated by cervical vagus nerve (VN) stimulation, increased their firing rates to systemic injection of phenylephrine (PE), revealed an expiratory rhythm, showed an increase in firing rate coinciding with spontaneous and elicited decreases in heart rate, had conduction velocities in the A-delta and B-fiber range, and produced bradycardia upon stimulation through the recording electrode with thresholds as low as 4 microA. The data indicate that in rabbits, chronotropic cardioinhibitory vagal motoneurons are discretely localized on the lateral, caudal portions of DVN and NA between 0.5 mm caudal and 1.5 mm rostral to obex.