Cholecystokinin induces Fos expression in catecholaminergic neurons of the macaque monkey caudal medulla

Systemic administration of cholecystokinin octapeptide (CCK) slows gastric emptying, inhibits feeding, and stimulates pituitary hormone release in rats and primates. To characterize the central neural circuits that mediate these effects in primates, the present study analyzes the distribution and chemical phenotypes of caudal medullary neurons that are activated in rhesus and cynomolgus macaque monkeys after CCK treatment. Monkeys were injected intravenously with CCK (3 or 15 μg/kg b.wt) or vehicle solution (0.15 M NaCl), then were anesthetized and perfused with fixative 75 min later. Coronal tissue sections through the caudal medulla were processed for immunocytochemical localization of the immediate-early gene product Fos as a marker of stimulus-induced neuronal activation, and were double-labeled for tyrosine hydroxylase to identify catecholaminergic cells. Many neurons in the area postrema, nucleus of the solitary tract, and ventrolateral medulla were activated to express Fos in monkeys after CCK treatment, similar to previous reports in rats. Treatment-activated neurons included substantial proportions of the A1/C1 and A2/C2 catecholaminergic cell groups, whereas neurons in the locus coeruleus (A6 cell group) were not activated. These results indicate that the autonomic, behavioral, and neuroendocrine effects produced by systemic administration of CCK involve hindbrain neural systems whose anatomical and chemical features are comparable in rats and primates.     

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.     

Exogenous cholecystokinin activates cFos expression in medullary but not hypothalamic neurons in neonatal rats

The central distribution of neurons activated to express cFos immunoreactivity in response to peripheral administration of cholecystokinin octapeptide (CCK) was examined in 2-day-old rats. Similar to previous results in adult rats, neurons in specific subregions of the area postrema and nucleus of the solitary tract (NST) expressed cFos after CCK treatment. However, in marked contrast to results in adult rats, CCK treatment in neonates did not stimulate cFos expression in hypothalamic neurons or in other forebrain areas, and did not increase plasma oxytocin levels. These results suggest that vagal sensory activation of intrinsic brainstem circuits may be sufficient for the known inhibitory effects of exogenous CCK on gastric motility and feeding in neonatal rats. The prominent forebrain activation produced by CCK administration in adult rats likely reflects later maturation of direct and relayed ascending neural projections from the NST.     

Tyrosine hydroxylase-immunoreactive projections from the caudal ventrolateral medulla to the subfornical organ in the rat

The subfornical organ (SFO) is known to be innervated by noradrenergic fibers. One possible origin of these fibers, which carry peripheral baroreceptor information to enhance the activity of SFO neurons, is the nucleus tractus solitarius (NTS). To investigate possible sites of origin of the catecholaminergic projections to the SFO, a retrograde tracing method was combined with immunohistochemistry in the rat. Stereotaxical injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase--colloidal gold complex, into the SFO from the dorsal aspect revealed retrogradely labeled neurons in several catecholaminergic cell groups. A substantial number of retrogradely labeled neurons showing tyrosine hydroxylase (TH) immunoreactivity were found in the NTS and ventrolateral medulla (VLM) at levels caudal to the obex and in the locus coeruleus, while retrogradely labeled neurons without TH immunoreactivity were found in the VLM at levels rostral to the obex and in the nucleus prepositus hypoglossi. When the tracer was injected into the structures dorsal to the SFO, including the triangular septal nucleus, the frequency of retrogradely labeled neurons in the NTS and VLM at the caudal level was very low. These findings indicate the existence of catecholaminergic projections from the VLM (probably A1) to the SFO, in addition to the noradrenergic projections from the NTS previously reported.     

Distribution and neurochemical phenotypes of caudal medullary neurons activated to express cFos following peripheral administration of cholecystokinin

Immunocytochemical localization of the protein product of the proto-oncogene C-fos allows anatomical identification of physiologically activated neurons. The present study examined the subnuclear distribution of cFos protein in the rat caudal medulla following peripheral administration of cholecystokinin octapeptide, which reduces feeding and gastric motility by a vagally mediated mechanism. To begin phenotypic characterization of neurons activated to express cFos following cholecystokinin treatment, double-labeling techniques were used to identify vagal motor neurons and neurons immunoreactive for tyrosine hydroxylase, neuropeptide Y, and neurotensin. Activated cells were most prevalent in the subnucleus medialis of the nucleus of the solitary tract, less prevalent in the subnucleus commissuralis, and virtually absent in the subnuclei centralis and gelatinosus. Many activated cells occupied the caudal area postrema; some of these were catecholaminergic. In contrast, activated cells were sparse within the medial rostral area postrema. Other activated cells occupied the dorso- and ventrolateral medulla and the midline raphe nuclei. Retrograde labeling of vagal motor neurons confirmed that very few were activated. Those that were activated occupied the caudal dorsal motor nucleus. In the dorsomedial medulla, 51% of catecholaminergic neurons and 39% of neurons positive for neuropeptide Y were activated, but no neurotensin-positive neurons were activated. In the ventrolateral medulla, 25% of catecholaminergic neurons and 27% of neuropeptide Y-positive neurons were activated. By characterizing the subnuclear distribution and chemical phenotypes of neurons activated by exogenous cholecystokinin, these data contribute to elucidation of the neural circuits mediating the behavioral, physiological, and neuroendocrine effects produced by this peptide. © 1993 Wiley-Liss, Inc.     

Depletion of catecholaminergic neurons of the rostral ventrolateral medulla in multiple systems atrophy with autonomic failure

The ventrolateral portion of the intermediate reticular formation of the medulla (ventrolateral medulla, VLM), including the C1/A1 groups of catecholaminergic neurons, is thought to be involved in control of sympathetic cardiovascular outflow, cardiorespiratory interactions, and reflex control of vasopressin release. As all these functions are affected in patients with multiple systems atrophy (MSA) with autonomic failure, we sought to test the hypothesis that catecholaminergic (tyrosine hydroxylase [TH]-positive) neurons of the VLM are depleted in these patients. Medullas were obtained at autopsy from 4 patients with MSA with prominent autonomic failure and 5 patients with no neurological disease. Patients with MSA had laboratory evidence of severe adrenergic sudomotor and cardiovagal failure. Tissue was immersion fixed in 2% paraformaldehyde at 4°C for 24 hours and cut into 1-cm blocks in the coronal plane from throughout the medulla. Serial 50-μm sections were collected and one section every 300 μm was stained for TH. There was a pronounced depletion of TH neurons in the rostral VLM in all cases of MSA. There was also significant reduction of TH neurons in the caudal VLM in 3 MSA patients compared with 3 control subjects. In 2 MSA cases and in 2 control subjects, the thoracic spinal cord was available for study. There was also depletion of TH fibers and sympathetic preganglionic neurons (SPNs) in the 2 MSA cases examined. Thus, depletion of catecholaminergic neurons in the VLM may provide a substrate for some of the autonomic and endocrine manifestations of MSA.     

Cholecystokinin induces c-fos expression in hypothalamic oxytocinergic neurons projecting to the dorsal vagal complex.

Systemic administration of cholecystokinin (CCK) decreases gastric motility and stimulates pituitary secretion of oxytocin (OT). Although peripheral OT does not affect gastric function, increasing evidence suggests that central OT secretion acting within the dorsal vagal complex (DVC) can alter gastric motility. To evaluate whether systemically administered CCK is capable of activating oxytocinergic neurons projecting to the DVC, we utilized fluorogold retrograde labeling from the DVC in combination with c-fos and OT immunocytochemical staining to quantitatively analyze paraventricular nucleus (PVN) neurons of rats following injection of CCK at a dose known to cause maximal pituitary OT secretion (100 micrograms/kg i.p.). Our results showed that 2320 +/- 63 PVN neurons were retrogradely labeled from the DVC; 146 +/- 21 (6.3%) of these contained OT, and these cells were predominantly located in the medial parvocellular subdivision of the PVN. Of all retrogradely labeled cells, 671 +/- 112 (28.9%) expressed c-fos after CCK stimulation, and 68 +/- 14 of these (10.1%) contained OT. Approximately 50% of the OT-containing neurons retrogradely labeled from the DVC stained positively for c-fos. Many magnocellular OT neurons in the PVN that were not retrogradely labeled from the DVC also expressed c-fos after CCK stimulation. These results demonstrate that parvocellular OT neurons projecting to the DVC are co-activated along with magnocellular OT neurons projecting to the pituitary following administration of a large dose of CCK, and lend support to a possible functional role for OT as a central neurotransmitter that modulates vagal efferent traffic to the gastrointestinal tract.     

Collateral axonal projections from ventrolateral medullary non-catecholaminergic neurons to central nucleus of the amygdala

Retrograde tract-tracing techniques were used to investigate whether catecholaminergic neurons in the ventrolateral medulla (VLM) send collateral axonal projections to both central nuclei of the amygdala (ACe) in the rat. Rhodamine-labelled latex microspheres or fluorogold (2%) were microinjected into the region of either the right or left ACe. After a survival period of 10–12 days, the rats were sacrificed and transverse sections of the brainstem were processed immunohistochemically for the identification of cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) or phenylethanolamine-N-methyltransferase (PNMT). Neuronal perikarya containing the retrogradely transported tracers were observed throughout the rostrocaudal extent of VLM, bilaterally. Approximately 10% of the retrogradely labelled neurons were observed to contain both retrograde tracers. The majority (79 ± 6.8%) of these double labelled neurons were located within the caudal VLM and their number decreased rostrally. In addition, the proportion of double labelled neurons to single labelled neurons in VLM decreased rostrally; approximately 11% in the caudal VLM and 6% in the rostral VLM. Furthermore, approximately 21% of all VLM neurons that projected to ACe were found to be catecholaminergic: 75% of these were immunoreactive to TH and 25% to PNMT. However, no neurons were found in VLM that contained both retrograde tracers and immunoreactivity to TH or PNMT. These data demonstrate that axons originating from non-catecholaminergic neurons in VLM bifurcate to innervate ACe bilaterally. Although the function of these VLM neurons that project to both ACe is not known, they may be the anatomical substrate by which VLM neurons relay simultaneously autonomic and/or visceral sensory information to influence the activity of ACe.     

Organization of immune-responsive medullary projections to the bed nucleus of the stria terminalis, central amygdala, and paraventricular nucleus of the hypothalamus: evidence for parallel viscerosensory pathways in the rat brain

Immune-responsive neurons in the brainstem, primarily in the nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), contribute to a significant drive on forebrain nuclei responsible for brain-mediated host defense responses. The current study investigated the relative contribution of brainstem-derived ascending pathways to forebrain immune-responsive nuclei in the rat by means of retrograde tract tracing and c-Fos immunohistochemistry. Fluorogold was iontophoresed into the bed nucleus of stria terminalis (BST), central nucleus of the amygdala (CEA), paraventricular nucleus of the hypothalamus (PVN), and the pontine lateral parabrachial nucleus (PBL; an important component of ascending viscerosensensory pathways) followed 2 weeks later by intraperitoneal injection of lipopolysaccharide (LPS, 0.1 mg/kg) or saline. The NTS and VLM provide immune-responsive input to all four regions, via direct, predominantly catecholaminergic, projections to the PVN, the lateral BST, and the CEA, and mostly non-catecholaminergic projections to the PBL. The PBL provides a major LPS-activated input to the BST and CEA. The pattern of LPS-activated catecholaminergic projections from the VLM and NTS to the forebrain is characterized by a strong predominance of VLM input to the PVN, whereas the NTS provides a greater contribution to the BST. These findings indicate that direct and indirect pathways originate in the caudal brainstem that propagate immune-related information from the periphery with multiple levels of processing en route to the forebrain nuclei, which may allow for integration of brain responses to infection.     

Visceral noxious stimulation induced expression of Fos protein in medullary catecholaminergic neurons projecting to nucleus accumbens in the rat: a study with triple labeling method of HRP tracing combined with Fos and TH immunohistochemistry

Horseradish peroxidase (HRP) was stereotaxically injected into the nucleus accumbens (Acb), and visceral noxious stimulation given by injecting formalin into the stomach. Sections of the medulla were subjected to HRP reaction combined with immunohistochemical reactions for Fos protein (ABC method) and tyrosine hydroxylase (TH, PAP method). The catecholaminergic neurons of the medulla (including vagal complex, ventrolateral medulla and reticular formation between them) which expressed Fos protein and projected to Acb were studied. The results showed that HRP retrogradely labeled cells were seen in the medulla bilaterally with apparent ipsilateral predominance and TH-LI and Fos-LI single labeled cells were bilaterally distributed; HRP/TH,TH/Fos double labeled neurons were more numerous than HRP/Fos double-labeled neurons. HRP/TH/Fos triple-labeled neurons were small in number and were mainly distributed in the nucleus tractus solitarii (nTS) and ventrolateral medulla (VLM), but only a few labeled cells were located in RF between nTS and VLM. It is concluded that TH-LI neurons in the medulla projected to Acb and some of them expressed Fos protein after noxious stimulation of the stomach.     

Catecholaminergic neurons in the ventrolateral medulla and nucleus of the solitary tract in the human

Catecholaminergic neurons in the ventrolateral medulla (VLM) and nucleus of the solitary tract (NTS) are important because of their presumed roles in autonomic regulation, including the tonic and reflex control of arterial pressure, neuroendocrine functions, and the chemosensitivity associated with the ventral medullary surface. However, little is known about the connections of these neurons in the human brain. As a first step in analyzing the functional biochemical anatomy of catecholamine neurons in the human, we used antisera against tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) to localize medullary catecholamine-containing neurons and processes in the VLM and the NTS. Cells staining for TH were located throughout the VLM. Most cells staining for TH and PNMT, which are therefore adrenergic, occurred in an area of the VLM probably corresponding to the rostroventrolateral reticular nucleus. Axons of TH-immunoreactive neurons in the VLM projected (1) dorsally, in a series of parallel transtegmental trajectories, toward the dorsomedial reticular formation, the NTS, and vagal motor nucleus, (2) longitudinally, through the central tegmental field, as fascicles running parallel to the neuraxis, (3) ventrolaterally toward the ventral surface (VS) of the rostral VLM where they appeared to terminate, and (4) medially into the raphe, where they arborized. Similar systems of fibers were labeled for PNMT; the longitudinal bundles of PNMT-labeled axons were limited to the principal tegmental bundle and concentrated dorsally. Fibers containing PNMT were also identified in the medullary raphe, on the medullary ventral surface, and contacting intraparenchymal blood vessels. In the NTS, neurons exhibited immunoreactivity to both TH and PNMT: Four principal subgroups of TH-immunoreactive neurons were seen: a ventral, an intermediate, a medial, and a dorsal group. Perikarya containing PNMT were restricted to the dorsolateral aspect of the NTS. Processes containing TH and PNMT immunoreactivity were identified in the medial and dorsolateral NTS; others appeared to project between the NTS and the VLM and within the solitary tract. The presence of catecholaminergic fibers of the VLM interconnecting with the NTS, raphe, intraparenchymal microvessels, VS, and possibly the spinal cord suggests that the autonomic and chemoreceptor functions attributed to these neurons also may apply to the human.     

Activation of gastric afferents increases noradrenaline release in the paraventricular nucleus and plasma oxytocin level

Effects of electrical stimulation of the gastric vagal nerves on plasma levels of oxytocin (OXT) and arginine vasopressin (AVP) were examined in rats anesthetized with urethane. Electrical stimulation of the gastric vagal nerves increased the plasma levels of OXT, but not AVP. The concentrations of extracellular noradrenaline (NA) in the paraventricular nucleus (PVN) were measured by in vivo microdialysis in rats anesthetized with urethane. Electrical stimulation of the gastric vagal nerves evoked an increase followed by a slight decrease in the concentrations of NA. The responses of spontaneous firing magnocellular neurosecretory neurons in the PVN to both electrical stimulation of the gastric vagal nerves and intravenous (i.v.) administration of CCK-8 were examined. Most of the putative OXT-secreting cells recorded were excited by both electrical stimulation of gastric vagal nerves and i.v. administration of CCK-8. These results suggest that gastric vagal afferents activate the central noradrenergic system from the brainstem to the PVN and secretion of OXT.     

Cytoarchitectonic Analysis of Fos-immunoreactivity in Brainstem Neurones Following Visceral Stimuli in Conscious Rats

Visceral inputs to the brain make their initial synapses within the nucleus of the solitary tract (NTS), where information is relayed to other brain regions. These inputs relate to markedly different physiological functions and provide a tool for investigating the topography of visceral processing in brainstem nuclei. Therefore, Fos immunoreactivity was used to determine whether a gastric stimulus affects neurones within different or similar parts of the NTS, ventrolateral medulla (VLM) and parabrachial nucleus (PBN), compared to a baroreceptive stimulus. The contribution of catecholaminergic neurones in these areas was studied by combining Fos and tyrosine hydroxylase (TH) immunoreactivity. Conscious male rats received either cholecystokinin (CCK) intraperitoneally to activate gastrointestinal afferents, or were made hypertensive by intravenous infusion of phenylephrine (PE) to activate baroreceptors. Tissue sections were processed immunocytochemically for Fos and/or TH. Phenylephrine infusion and CCK injection elicited Fos expression in distinct and in overlapping regions of the NTS and the VLM. Cholecystokinin injections increased the number of Fos-immunoreactive neurones in the area postrema (AP) and throughout the rostral-caudal extent of the NTS, including commissural neurones and the medial subnuclei. Some reactive neurones in NTS were also positive for TH, but most were not, and most of the TH-positive NTS neurones were not Fos-positive. In contrast, PE infusion produced a more restricted distribution of Fos-positive neurones in the NTS, with most neurones confined to a dorsolateral strip containing few TH-positive neurones. The medial NTS at the level of the AP and the AP itself were largely unresponsive, but rostral to the AP the medial NTS was labelled, including some TH-positive neurones. Both treatments produced labelling in the caudal and mid-VLM, but PE infusion had a stronger effect in the rostral VLM. In the PBN, CCK elevated Fos expression in several subregions, whereas PE infusion failed to specifically alter any subdivision. The results suggest that stimulation of baroreceptor and gastric afferents evoke both overlapping and cytoarchitectonically distinct pathways in the brainstem.     

Regional specificity of the long-term regulation of tyrosine hydroxylase in some catecholaminergic rat brainstem areas. I. Influence of long-term hypoxia

The present study was carried out to investigate the influence of long-term hypoxia on tyrosine hydroxylase (TH) protein quantity in some catecholaminergic rat brainstem areas such as the dorsomedial medulla (DMM), the ventrolateral medulla (VLM) and the locus coeruleus (LC). TH protein quantity was also measured in a dopaminergic structure, the substantia nigra (SN). Male Sprague-Dawley rats were exposed to normobaric hypoxia (10% O₂/90% N₂) for 3, 7, 14 or 22 days. Controls were kept in normoxia for the same period. This study demonstrates that: (1) 3 days of hypoxia produced a 50% and a 26% increase in the quantity of TH protein in the rostral and caudal LC, respectively; (2) 14 days of hypoxia produced a 44% increase of TH protein content exclusively in the caudal part of DMM and a 31% increase in the VLM area; and (3) the stimulus failed to alter the TH protein quantity in the SN. After 14 and 22 days of hypoxia respectively, the TH protein content in the LC and DMM returned to the level of controls. To determine whether the increase in TH protein quantity could be related to a change in norepinephrine (NE) content, the rate constant of disappearance (k) of NE was measured in the catecholaminergic areas of intact or chemodenervated rats submitted to long-term hypoxia. Our results show that hypoxia causes an increase of TH protein quantity within the subpopulations of catecholaminergic areas additionally with an elevation in the NE content. These data suggest a selective response of the TH regulation to long-term hypoxia within the caudal DMM catecholaminergic area which receives chemosensory inputs.     

Projection from the ventrolateral medullary neurons containing tyrosine hydroxylase to the central amygdaloid nucleus in the rat.

By using a double-labeling produce of retrograde horseradish peroxidase (HRP) tracing and the immunocytochemical localization of tyrosine hydroxylase (TH), the present study ascertained that the axonal fibers of catecholaminergic neurons in the caudal ventrolateral medulla projected to the central amygdaloid nucleus in the rat. The majority of double-labeled cells were observed primarily around the level of the obex. 92% of HRP retrogradely labeled cells contained TH-like immunoreactive (TH-IR), but only 31% of TH-IR cells was labeled with HRP.     

Reduction of CCK-8 binding in the nucleus of the solitary tract in unilaterally nodosectomized rats

Cholecystokinin octapeptide (CCK-8) binding sites were examined in the caudal hindbrain of unilaterally nodosectomized and intact rats by in vitro autoradiography with 125I-CCK-8. Unilateral nodosectomy caused a significant reduction in CCK-8 binding in the caudal medial nucleus of the solitary tract (NST) on the side ipsilateral to the nodose ganglionectomy. Examination of other caudal hindbrain regions exhibiting CCK-8 binding did not reveal changes in binding in nodosectomized animals compared to controls. These findings suggest that the CCK receptors in this brain region may be located on terminals of vagal afferent neurons.     

Evidence of γ-aminobutyric acidergic control over the catecholaminergic projection from the medulla oblongata to the central nucleus of the amygdala

It is known that the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM) project to the central nucleus of the amygdala (Ce), conveying visceral information. Conversely, the Ce sends projections to the NTS and the VLM. To understand better the role of catecholamine and γ-aminobutyric acid (GABA) in these reciprocal connections, experiments were performed by combining lectin-conjugated horseradish peroxidase (WGA-HRP) anterograde and retrograde transport with preembedding immunocytochemistry to detect tyrosine hydroxylase (TH), and postembedding immunocytochemistry to detect GABA. The light microscopic study suggested that the majority of neurons in the NTS and the VLM projecting to the Ce were TH immunoreactive (TH-IR). Most of them were located at the level of the obex. Under the electron microscope, the GABAergic and non-GABAergic terminals were found to form synaptic contacts with the TH-(IR) or Ce-projecting or TH-IR/Ce-projecting double-labelled neurons of the NTS and VLM. The GABAergic terminals mostly formed symmetrical synaptic contacts with the postsynaptic structure in which perikarya (14-19%), dendrites (79-84%), and spines (2%) were observed. Approximately 94% of the axon terminals in the NTS and 90% of those in the VLM arising from the Ce were GABAergic and appeared not to form synaptic contacts with the TH-IR or Ce-projecting neurons in these regions. The present results demonstrated that the catecholaminergic neurons of the NTS and VLM projecting to the Ce receive an extensive GABAergic innervation and that the amygdala projection to the medulla is mostly GABAergic. J. Comp. Neurol. 381:262-281, 1997. © 1997 Wiley-Liss, Inc.     

Fos expression by glutamatergic neurons of the solitary tract nucleus after phenylephrine-induced hypertension in rats

The baroreflex pathway might include a glutamatergic connection between the nucleus of the solitary tract (NTS) and a segment of the ventrolateral medulla (VLM) called the caudal ventrolateral medulla. The main goal of this study was to seek direct evidence for such a connection. Awake rats were subjected to phenylephrine- (PE-) induced hypertension (N=5) or received saline (N=5). Neuronal activation was gauged by the presence of Fos-immunoreactive (Fos-ir) nuclei. Fos-ir neurons that contained vesicular glutamate transporter 2 mRNA (glutamatergic neurons) or glutamic acid decarboxylase mRNA (GABAergic neurons) were mapped throughout the medulla oblongata. Saline-treated rats had very few Fos-ir neurons. In PE-treated rats, Fos-ir neurons were detected in both NTS and VLM. In NTS, 72% of Fos-ir neurons were glutamatergic and 26% were GABAergic. In the VLM, 41% of Fos-ir neurons were glutamatergic and 56% were GABAergic. In VLM, Fos-ir glutamatergic neurons were evenly distributed and were often catecholaminergic, whereas Fos-ir GABAergic cells were clustered around Bregma -13.0 mm. This region of the VLM was injected with Fluoro-Gold (FG) in eight rats, four of which received PE and the rest saline. Fos-ir NTS neurons retrogradely labeled with FG were detected only in PE-treated rats. These cells were exclusively glutamatergic and were concentrated within the NTS subnuclei that receive the densest inputs from arterial baroreceptors. In conclusion, PE, presumably via baroreceptor stimulation, induces Fos in glutamatergic and GABAergic neurons in both NTS and VLM. At least 29% of the Fos-ir glutamatergic neurons of NTS project to the vicinity of the VLM GABAergic interneurons that are presumed to mediate the sympathetic baroreflex.     

Electrophysiological properties of neurons in the caudal ventrolateral medulla projecting to the paraventricular nucleus of the hypothalamus in rats

Extracellular recordings were made from neurons in the caudal ventrolateral medulla in urethane-chloralose-anesthetized rats. Stimulation of the paraventricular nucleus (PVN) in the hypothalamus evoked antidromic action potentials in 71 neurons. On the basis of antidromic spike latencies, these neurons could be divided into fast- (24 neurons) and slow-conducting cell groups (47 neurons). Slow-conducting cells showed irregular and slow spontaneous discharges, while a majority of the fast-conducting cells did not show spontaneous discharges. The spontaneous activity of slow-conducting cells was suppressed by i.v. clonidine administration. The effects of clonidine could be consistently reversed by administration of the alpha 2-adrenergic antagonist, yohimbine. The responses by clonidine and yohimbine remained unimpaired in baroreceptor-denervated rats. Vagus nerve stimulation produced an excitation in 80% of slow-conducting cells tested. Baroreceptor activation induced by i.v. administration of phenylephrine inhibited about half of slow-conducting cells tested. Similar elevation of blood pressure in baroreceptor-denervated rats did not show any effect. These physiological and pharmacological properties of slow-conducting cells were similar to those previously reported for catecholaminergic cells in other parts of the brain. The results show the existence of two different populations among neurons in the caudal ventrolateral medulla which project directly to the PVN, and suggest that the presumed A1 catecholaminergic cells are involved in the afferent pathway from cardiovascular baroreceptors and the vagus nerve to the PVN.     

Distribution of dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes

The immunocytochemical localization of the biosynthetic enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)--was used to determine the cytological features and precise neuroanatomical location of catecholaminergic neurons in the medulla oblongata of rat. Perikarya labeled with TH were detected in two bilaterally symmetrical columns located in the ventrolateral and dorsomedial medulla. The distribution and the number of neuronal perikarya containing TH were the same as those containing DBH, except in the dorsal motor nucleus of the vagus at the level of the area postrema where the number of neurons immunocytochemically labeled for TH was considerably greater than those labeled for DBH. The detection of perikarya which show immunoreactivity for TH, used in the biosynthesis of dopamine, noradrenaline, and adrenaline, but not DBH, which converts dopamine to noradrenaline, suggests the existence of dopamine-synthesizing neurons in the medulla. Perikarya labeled with PNMT, used in the biosynthesis of adrenaline, were localized in more restricted regions corresponding to rostral subsets of the dorsal and ventral groups labeled for TH and DBH. Counts of neurons immunocytochemically labeled for TH or PNMT were obtained in order to determine the relative ratio of neurons which contain the enzymes necessary for the synthesis of dopamine, noradrenaline, or adrenaline at various levels of the medulla. At the most caudal levels no PNMT labeled neurons were detected. Further rostral, PNMT-labeled neurons were first detected in the ventrolateral medulla. At the level of the area postrema, the number of PNMT-labeled neurons in the ventrolateral medulla was approximately half of the number of cells showing immunoreactivity for TH. In contrast, few PNMT-labeled cells were detected in the dorsomedial medulla at the level of the area postrema compared to many neurons labeled for TH. At rostral medullary levels, in both the ventrolateral and the dorsomedial regions, the number of neurons labeled for TH and PNMT was essentially the same. Thus most, if not all, of the catecholaminergic neurons in the rostral medulla have PNMT, necessary for the synthesis of adrenaline.