Franco R. Calaresu

Central projections of afferent renal fibers in the rat: an anterograde transport study of horseradish peroxidase.

The projections of afferent renal fibers (ARN) to dorsal root ganglia and into the spinal cord of the rat were studied using the anterograde transport of horseradish peroxidase (HRP). Crystalline HRP was applied to the proximal cut ends of renal nerves or injected as a concentrated solution into the kidney, on either the right or left side. After a survival time of 40-120 h, sections of thoraco-lumbar dorsal root ganglia (DRG) and spinal cord were cut and processed according to the tetramethyl benzidine method. HRP applied either to the ARN or to the kidney on the left labeled neurons in the DRG from T8 to L2. On the other hand, HRP application on the right side resulted in labeling of neurons in DRG T6 to T13. No labeled neurons were found in the contralateral DRG. Labeled neurons in the DRG were of the small (11-20 micron) and medium (30-42 micron) size and were distributed in all portions of the DRG. In the spinal cord the greatest concentration of labeled ARN from the left were found in segments T10-L1, whereas projections from the right ARN were concentrated primarily in segments T7-T10. Labeled fibers entered along the medial aspect of the ipsilateral dorsal horn and projected both rostrally and caudally in the medial portion of Lissauers tract, sending some collaterals into lamina I. The majority of labeled fibers coursed ventrally along the medial aspect of the dorsal horn towards the midline where they terminated in the region of the dorsal gray commissure, just dorsal to the central canal. Additionally, labeled fibers from the medial projection passed into laminae III-V. No labeled fibers or terminals were observed in the contralateral spinal cord. These data show that ARN enter the spinal cord through several DRG and provide the first anatomical demonstration of central sites of termination of ARN. These spinal sites of projection of sensory information from the kidney are likely to be central sites of integration of reno-renal and visceral reflexes.

Horseradish peroxidase study of brain stem projections of carotid sinus and aortic depressor nerves in the cat.

Abstract The connections of carotid sinus (CSN) and aortic depressor (ADN) afferent fibers in the brain stem of the cat were studied using horseradish peroxidase (HRP). Crystalline HRP was applied to the proximal cut end of either the CSN or ADN for 4–10.5 h and after a survival period of 24–120 h the animals were perfused and frozen sections of brain stem, nodose and petrosal ganglia were processed according to the tetramethyl-benzidine method. CSN labeled axons were found to project ipsilaterally to several nuclei of the solitary complex: the dorsal aspect of the medial (Sm), the lateral (Slt), the ventrolateral (Svl), the commissural (Com), the intermediate (Int) and the dorsomedial aspect of the parvocellular (Spc) solitary nuclei. These nuclei, except for the Svl, also received a less intense contralateral projection. Additional terminal labeling was observed on the ipsilateral side along the dorsal border of the dorsal motor nucleus of the vagus (DMV), in the reticular formation ventral to the solitary complex, and in the ventrolateral external cuneate nucleus, and labeled fibers were found in the dorsolateral spinal trigeminal tract. On the other hand, ADN labeled fibers were found to project only to the solitary complex, bilaterally. Terminal labeling was found primarily in the Sm, Slt, Com and dorsal Spc. After HRP application to the CSN few cells were found labeled in the petrosal ganglion; in addition clusters of labelled neurons were found bilaterally in the region of the rostral nucleus ambiguus, retrofacial and facial nuclei, and in the ipsilateral rostral dorsomedial reticular formation. ADN labeled ganglion cells were identified in clusters in the medial aspect of the nodose ganglion primarily near the entry of the superior laryngeal nerve; additional clusters of labeled smaller neurons were found in the ventromedial portion of the ganglion and intermingled with vagal fibers just caudal to the ganglion. No HRP-positive cells were identified in the brain stem after ADN labeling. These data demonstrate that different regions of the solitary complex receive direct inputs from either one or both buffer nerves, suggesting a degree of separation of central pathways carrying CSN and ADN afferent information. Furthermore, the finding of labeled cell bodies in the medulla after CSN labeling suggests a possible route by which the central nervous system may alter activity of receptors in the carotid sinus and body.

Direct pathway from cardiovascular neurons in the ventrolateral medulla to the region of the intermediolateral nucleus of the upper thoracic cord: an anatomical and electrophysiological investigation in the cat.

Horseradish peroxidase (HRP) and single unit recording experiments were done in cats to identify neurons in the ventrolateral medulla (VLM) projecting directly to the intermediolateral nucleus (IML) of the thoracic cord and relaying cardiovascular afferent information from the buffer nerves and hypothalamus. In the first series, HRP was allowed to diffuse from a micropipette into the region of the IML at the level of T2. After a survival period of 30-138 h, transverse and horizontal sections of the brainstem were processed according to the tetramethyl benzidine method. Labeled neurons were found in the VLM 1-5 mm rostral to the obex, bilaterally, but with an ipsilateral predominance. The majority were observed in sections 2-4 mm rostral to the obex, clustered in an area lateral to the inferior olivary nucleus around the intramedullary rootlets of the hypoglossal nerve. Additional labeled neurons were found scattered along the ventral surface of the medulla; most of these neurons were oval in shape, 15-30 micron in diameter, and had dendritic processes which lay parallel to the ventral surface. In the second series, the region of the VLM shown to contain labeled neurons was systematically explored for single units antidromically activated by electrical stimulation of the IML in chloralosed, paralyzed and artificially ventilated animals. These antidromically identified units were then tested for their responses to electrical stimulation of the carotid sinus (CSN) and aortic depressor (ADN) nerves, and the paraventricular nucleus (PVH). Ninety-four single units in the VLM were antidromically activated with latencies corresponding to a mean conduction velocity of 19.1 +/- 1.5 m/s. Of these units 52% (49/94) were orthodromically excited by stimulation of buffer nerves; 12 by stimulation of the CSN only (mean latency, 16.0 +/- 3.6 ms), 5 by stimulation of the ADN only (mean latency, 9.5 +/- 2.0 ms), 7 by both buffer nerves, and the remaining 25 units responded to at least one of the buffer nerves and to PVH. Stimulation of PVH excited orthodromically 42 of the 94 units (45%), of which 17 responded only to stimulation of PVH (mean latency, 17.9 +/- 3.5 ms). These experiments provide anatomical and electrophysiological evidence for the existence of a direct cardiovascular pathway from the VLM to the region of the IML and suggest that neurons in the VLM are involved in the integration of cardiovascular afferent inputs from buffer nerves and the hypothalamus to provide an excitatory input to vasoconstrictor neurons in the IML.

Renal afferent nerves affect discharge rate of medullary and hypothalamic single units in the cat

Abstract Electrical activity of spontaneously firing single units in the medulla and hypothalamus of 22 cats anesthetized with chloralose was monitored for changes in firing frequency during electrical stimulation of afferent renal (ARN) and carotid sinus (CSN) nerves. Stimulation of ARN altered the firing frequency of 214 out of 540 units studied in the ipsi- and contralateral medulla; the majority of the responses were excitatory but a few units (8%) were inhibited by stimulation. Of the units responding to ARN stimulation, 57% were found to respond in the same manner to stimulation of the CSN. Responsive units were found primarily in 3 regions: the lateral tegmental field, the area of the paramedian reticular nucleus and the region of the dorsal vagal complex around the obex. In the hypothalamus stimulation of ARN affected the activity of 197 of the 407 units studied ipsi- and contralaterally; the majority of the units were excited but 8% were found to be inhibited. Of the units responding to ARN 75% also responded to stimulation of the CSN. Responsive units were found in most areas but were concentrated in 3 anterior regions: lateral preoptic area, lateral hypothalamic area and the region of the paraventricular nucleus. This is the first demonstration that stimulation of afferent renal nerves can influence the electrical activity of medullary and hypothalamic neurons bilaterally. Because of the demonstrated physiological role of the structures where these responsive units were found these results suggest that sensory receptors in the kidney convey important information to central sites involved in physiological responses related to cardiovascular adjustments and fluid balance. Furthermore it has been demonstrated that the majority of medullary and hypothalamic neurons responding to stimulation of ARN also receive an input from the CSN suggesting that certain regions of both medulla and hypothalamus can integrate peripheral information from the kidney and from cardiovascular receptors to bring about appropriate homeostatic responses.

Expression of c-fos protein in rat brain after electrical stimulation of the aortic depressor nerve

To reveal central nervous system (CNS) structures involved in the baroreceptor reflex we studied the distribution of Fos protein-like immunoreactivity in the rat brain after one hour of electrical stimulation of the aortic depressor nerve (ADN). In 13 male Wistar rats under urethane the ADN was cut on both sides and the central ends were placed on stimulating electrodes. Intermittent (11 s on, 6 s off) electrical stimulation at parameters set to elicit a drop in mean arterial pressure of 15-30 mmHg was applied to one, both or neither ADNs for 1 h. CNS sections were incubated for 48 h in anti-Fos antibody and prepared for visualization of the reaction product using the ABC immunoperoxidase technique. Label was found in several discrete brain nuclei primarily on the side ipsilateral to the side of stimulation. In the medulla labelled nuclei were found in the nucleus tractus solitarius, area postrema, rostral and caudal ventrolateral medulla, nucleus ambiguus and medullary reticular formation. In the pons labelled neurons were found in the lateral and ventrolateral parabrachial nucleus, locus coeruleus, pontine reticular field and A5 region. In the forebrain labelled nuclei were observed in the peri- and paraventricular hypothalamus, supraoptic nucleus, subfornical organ, preoptic area, central nucleus of the amygdala, median preoptic area, horizontal limb of the diagonal band, bed nucleus of the stria terminalis and islands of Calleja. In control animals moderate amounts of label were present in the supraoptic nucleus and periventricular hypothalamus bilaterally. These results define central pathways involved in mediating the baroreceptor reflex.

Glossopharyngeal and vagal afferent projections to the brain stem of the cat: A horseradish peroxidase study

Brain stem projections of the glossopharyngeal and vagus nerves in the cat were studied using the anterograde transport of horseradish peroxidase (HRP). Crystalline HRP was applied to the proximal cut ends of the nerves for a period of 4-10.5 h, and after a survival time of 24-120 h, transverse and horizontal sections of the brain stem were processed according to the tetramethylbenzidine method. Labeled fibers from both nerves were found to project bilaterally to the solitary complex, and ipsilaterally to the ventral region of the external cuneate nucleus and to the medial region of the nucleus praepositus hypoglossi, just dorsolateral to the medial longitudinal fasciculus. Within the solitary complex terminal labeling was found in the parvocellular, ventrolateral, lateral, medial and commissural solitary nuclei. Exclusive glossopharyngeal nerve projections were found ipsilaterally in the rostral dorsal motor nucleus of the vagus, the ventrolateral portion of the medial cuneate nucleus, the dorsal part of the nuclei caudalis and interpolaris of the trigeminal complex, the nuclei insulae cuneati lateralis, and the dorsolateral aspect of the nucleus medullae oblongata centralis. Finally, in the area postrema a bilateral projection of vagal and an ipsilateral projection of glossopharyngeal fibers were found. These findings demonstrate that the glossopharyngeal nerve has more widely distributed brain stem projections that the vagus nerve and provide essential information on projection sites of visceral and taste inputs to the central nervous system.

Monosynaptic connection from caudal to rostral ventrolateral medulla in the baroreceptor reflex pathway

Experiments were done to test the hypothesis that caudal ventrolateral medulla (CVLM) neurons excited by activation of arterial baroreceptors and by stimulation of depressor sites in the nucleus tractus solitarii (NTS) project monosynaptically to the rostral ventrolateral medulla (RVLM). In urethan anaesthetized and artificially ventilated rats we recorded extracellular activity from 46 spontaneously firing units in the CVLM. Twenty of these units were excited by baroreceptor activation (1-3 micrograms phenylephrine i.v.) and of these 6 were excited (mean latency of 9.8 +/- 2.3 ms) by single pulses (0.1 ms, 30 +/- 8.3 microA) delivered once per second to a depressor site in the ipsilateral NTS. These 6 units were also antidromically activated with a latency of 4.1 +/- 0.12 ms by stimulation of a pressor region in the ipsilateral RVLM. These results provide evidence for the existence of an excitatory projection from the NTS to the CVLM which, in turn, projects monosynaptically to sympathoexcitatory neurons in the RVLM.

Neurally mediated cardiovascular responses to stimulation of cell bodies in the hypothalamus of the rat

To study the cardiovascular responses to selective activation of neuronal cell bodies in the hypothalamus, DL-homocysteate (5-50 nl of a 0.15-M solution, pH 7.4) was injected into 417 histologically verified sites in the hypothalamus of 46 urethan-anesthetized, paralyzed and artificially ventilated rats. Injections resulted in depressor responses (-5 to -32 mm Hg) in 271 sites, in pressor responses (5-47 mm Hg) in 77 sites and 69 sites were not responsive. Depressor effects had a shorter latency (85% started within 5 s) than pressor effects (42% started within 5 s). Control injections of 0.15 M NaCl into 126 of the responsive sites were ineffective. Arterial pressure (AP) responses showed a positive correlation (r = 0.61, P less than 0.001) with changes in heart rate (HR). Analysis of the anatomical distribution of responsive sites showed that in all hypothalamic subdivisions depressor responses predominated except in the paraventricular nucleus, where mainly pressor effects and tachycardia were elicited. These results demonstrate that excitation of cell bodies in most hypothalamic regions elicits neurally mediated changes in AP and HR and that the traditional functional division of the hypothalamus into a rostral depressor and a caudal pressor area is probably based on the combined excitation of fibers of passage and cell bodies.

Medullary origin of vagal preganglionic axons to the heart of the cat

It is apparent from the literature that a controversy exists concerning the site of origin of cardiac vagal preganglionic axons. Physiological studies have suggested that the location of these neurons may be different in different species and there has been disagreement between physiological and anatomical findings in the same species. We now present anatomical and neurophysiological studies suggesting that in the cat cardiac vagal preganglionic neurons are located in two medullary regions: the areas of the dorsal motor nucleus of the vagus (DMV) and of the nucleus ambiguus (AMB). This suggestion is based on the following observations. Firstly, after application of horseradish peroxidase to the right cardiac branches of the vagus nerve, labeled neurons were found primarily in the regions of te DMV and AMB. Additional scattered neurons were found in the reticular formation between these two nuclei. Secondly, following injections of tritiated amino acids into either the DMV or AMB, labeled vagal fibers were found in the atrial myocardium. Finally, electrical stimulation of the right cardiac branches of the vagus nerve antidromically activated DMV or AMB, labeled vagal fibers were found in the atrial myocardium. Finally, neurons in the DMV and AMB regions with latencies corresponding to conduction velocities of B-fibers. In addition, these neurons were orthodromically excited by electrical stimulation of the carotid sinus and aortic depressor nerves.

Projections from buffer nerves to the nucleus of the solitary tract: an anatomical and electrophysiological study in the cat

The projections of aorta depressor (ADN) and carotid sinus (CSN) afferent fibers to the region of the nucleus of the solitary tract were studied in the cat with the anterograde transport of horseradish peroxidase (HRP) technique and by recording single unit activity during electrical stimulation of these nerves. In the first series of experiments, after application of crystalline HRP to the proximal cut end of either buffer nerve and a postoperative survival period of 24-120 h, brain stem sections were processed according to the tetramethyl benzidine method. ADN and CSN labeling were found bilaterally, with a predominant ipsilateral labeling, in the medial (Sm), lateral (Slt), commissural (Com) and dorsomedial aspect of the parvocellular solitary nuclei. Additional CSN labeling was found in the ventrolateral and intermediate (Int) solitary nuclei, in the reticular formation ventrolateral to the solitary complex and along the dorsal border of the dorsal motor nucleus of the vagus. In the second series of experiments these areas receiving primary afferent fibers were explored for single units responding to stimulation for the buffer nerves in chloralosed cats, paralyzed and artificially ventilated. Of 177 responsive units, 80 responded only to stimulation of the CSN, 44 only to the ADN and 53 to both nerves. Responsive units were found throughout the rostrocaudal extent of the solitary complex and areas adjacent to the solitary complex. However, most of the units were found in 3 regions: the Sm, Slt and adjacent areas. Units in the Slt and Com were found to respond to only one input, either the CSN or the ADN alone. On the other hand, units in the Int responded only to both buffer nerves and not selectively to one nerve. These results demonstrate that the CSN has a wider distribution in the solitary complex than the ADN and that second order neurons in the solitary complex receive inputs from either one or both buffer nerves, suggesting a degree of separation of central pathways carrying cardiovascular afferent information.