Angiogenesis and biomarkers of cardiovascular risk in adults with metabolic syndrome.

OBJECTIVES: Metabolic syndrome (MetSyn) is associated with an increased risk of atherosclerosis and fatal cardiovascular events. Angiogenesis is thought to contribute to this risk as it might be involved in the progression of atherosclerotic plaques. We investigated the levels of circulating biomarkers of angiogenesis and cardiovascular risk in adults with MetSyn and assessed their association with established metabolic risk factors. DESIGN: The Genetic Park project is a highly inclusive cross-sectional survey (about 80% of residents) conducted in three isolated populations in Southern Italy. A total of 1000 men and women (age range: 18-98 years) were included in the analysis. Anthropometric and blood pressure measurements were recorded. Metabolic and cardiovascular biomarkers included glucose, triglycerides, total cholesterol, HDL, vascular endothelial growth factor, placental growth factor (PlGF), soluble fms-like tyrosine kinase-1, high-sensitivity C-reactive protein, high-sensitivity troponin T (hs-TnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP). RESULT: Subjects with MetSyn had higher levels of PlGF and NT-proBNP after adjustment for age, smoking and body mass index. Circulating levels of PlGF, hs-TnT and NT-proBNP were directly related to the number of criteria of MetSyn, and this association interacted with gender. There was a strong correlation between ageing and cardiovascular risk. CONCLUSIONS: The increase in circulating levels of biomarkers of angiogenesis and cardiac function in subjects with MetSyn mirrors the pathophysiological changes occurring in the cardiovascular system. Over time, these changes might accelerate the formation and progression of atherosclerotic plaques and contribute significantly to cardiovascular morbidity and mortality risk.

Whole-Exome Sequencing in the Isolated Populations of Cilento from South Italy.

The present study describes the genetic architecture of the isolated populations of Cilento, through the analysis of exome sequence data of 245 representative individuals of these populations. By annotating the exome variants and cataloguing them according to their frequency and functional effects, we identified 347,684 variants, 67.4% of which are rare and low frequency variants, and 1% of them (corresponding to 319 variants per person) are classified as high functional impact variants; also, 39,946 (11.5% of the total) are novel variants, for which we determined a significant enrichment for deleterious effects. By comparing the allele frequencies in Cilento with those from the Tuscan population from the 1000 Genomes Project Phase 3, we highlighted an increase in allele frequency in Cilento especially for variants which map to genes involved in extracellular matrix formation and organization. Furthermore, among the variants showing increased frequency we identified several known rare disease-causing variants. By different population genetics analyses, we corroborated the status of the Cilento populations as genetic isolates. Finally, we showed that exome data of Cilento represents a useful local reference panel capable of improving the accuracy of genetic imputation, thus adding power to genetic studies of human traits in these populations.

Glucagon gene regulatory region directs oncoprotein expression to neurons and pancreatic alpha cells.

The regulatory region of the rat preproglucagon gene targets expression of the SV40 large T oncoprotein to two cell types in transgenic mice, the pancreatic alpha cells and a set of neurons localized in the hindbrain, both of which normally produce preproglucagon. Additional neurons in the forebrain and midbrain stain for T antigen but do not express the endogenous glucagon gene. Synthesis of T antigen in endocrine alpha cells results in the heritable development of pancreatic glucagonomas. In brains of transgenic mice from three independent lineages, expression of the hybrid gene begins at embryonic day 12 in neuroblasts of the hindbrain, where it continues throughout adult life, most notably in the medulla. Remarkably, oncoprotein expression in both proliferating neuroblasts and mature neurons has no apparent consequences, either phenotypic or tumorigenic. Expression of the hybrid glucagon gene in both neurons and islet cells supports a possible interrelationship between these cell types.

Albumin antioxidant capacity is modified by methylglyoxal.

OBJECTIVE: Oxidative stress seems to play a major role in diabetic vascular complication development. Plasma albumin, via its thiol groups, is the main extracellular antioxidant molecule. Methylglyoxal (MG) is a very reactive dicarbonyl compound increased in diabetes which strongly modifies proteins by non-enzymatic glycosylation. The aim of this work was to study if MG could modify albumin antioxidant capacity. METHODS: Bovine serum albumin was incubated with 1 mM MG at 37 degrees C for 7 days (MG-BSA). Albumin physico-chemical changes were evaluated by tryptophan autofluorescence measurement in the presence or in the absence of a quencher (acrylamide). Albumin antioxidant capacity was determined by thiol measurement using Ellman's reagent as well as in a cellular system (HeLa cells stressed by H2O2). RESULTS: MG-BSA exhibited important modifications as shown by conformational changes, decreased tryptophan autofluorescence (30%) and significant thiol loss (40%). MG-BSA led to important modifications resulting in oxidation and loss of albumin antioxidant capacity. MG-BSA modifications were close to the one observed in albumin isolated from diabetic patients. CONCLUSION: Our results suggest that deleterious effects induced by carbonyl stress in diabetes could also originate from a loss of albumin antioxidant capacity by dicarbonyl compound attack. The biological consequences of these findings have now to be investigated.

The C1 area of the brainstem in tonic and reflex control of blood pressure. State of the art lecture.

Recent studies have demonstrated that the neurons of the lower brainstem that are responsible for maintaining normal levels of arterial pressure reside in a specific area of the rostral ventrolateral medulla. In rat, the critical zone corresponds to a small region containing a subpopulation of the adrenergic C1 group, defined immunocytochemically by the presence of the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase. Neurons of this region (the C1 area), possibly including the adrenergic neurons, directly innervate preganglionic neurons in the spinal cord, and are tonically active and sympathoexcitatory. The excitatory transmitter released into the spinal cord is unknown. The discharge of C1 area neurons is locked to the cardiac cycle and, in turn, leads to firing of sympathetic preganglionic neurons. The C1 area neurons are inhibited by baroceptor input and mediate the vascular component of baroceptor reflexes. They also mediate somato-sympathetic pressor responses from skin and muscle and participate in reflex responses to hypoxia. The neurons are directly innervated by local neurons containing gamma-aminobutyric acid, acetylcholine, enkephalin, and substance P, all of which modulate arterial pressure. The C1 area is the site of the hypotensive actions of clonidine. Clonidine appears to act on imidazole receptors in the C1 area to lower arterial pressure. The natural ligand for these receptors may be a newly defined substance in brain, clonidine-displacing substance. Neurons of the C1 area appear to be the critical neuronal group governing the normal resting and reflex control of arterial pressure. They may play a critical role in the maintenance of elevated arterial pressure in hypertension and as a site of action of antihypertensive drugs.

Lesions of epinephrine neurons in the rostral ventrolateral medulla abolish the vasodepressor components of baroreflex and cardiopulmonary reflex.

Epinephrine-containing neurons of the rostral ventrolateral medulla (RVL) (the C1 group of Hökfelt) in the rat are primarily unilaterally innervated by neurons in the nucleus tractus solitarius (NTS) and in turn project to autonomic spinal neurons. In this study, we investigated whether the C1 area of the RVL mediates the vasodepressor responses (VDR) induced by either electrical stimulation of the vagus nerve or carotid sinus stretch. In all experiments, C1 neurons were localized immunocytochemically with antibodies to phenylethanolamine N-methyltransferase (PNMT). Bilateral lesions of the C1 area decreased arterial pressure (AP) and heart rate (HR) to spinal cord transection levels and blocked the VDR induced by vagal stimulation and carotid sinus stretch. Combined lesions of the contralateral NTS and C1 area ipsilateral to the stimulated vagus nerve maintained AP and HR at normal levels, and totally blocked the VDR to vagal stimulation and carotid sinus stretch. Since projections from the vagus nerve to NTS are bilateral and those from NTS to C1 unilateral, the combined contralateral NTS/ipsilateral C1 lesions isolated and interrupted the ipsilateral NTS-C1 pathway and, therefore, blocked the baroreceptor reflex. The results are consistent with the hypothesis that neurons in the NTS synapsing in or projecting through the C1 area mediate the baro- and cardiopulmonary mechanoreceptor reflex.

Brain stem catecholamine mechanisms in tonic and reflex control of blood pressure.

Neurons of the lower brain stem maintain resting levels of arterial pressure (AP), mediate reflex responses from cardiopulmonary receptors, and are an important site of the hypotensive actions of alpha 2-adrenergic agonists. Details of the pathways and transmitters that mediate tonic and reflex control of AP are emerging. Afferent fibers of cardiopulmonary receptors in the ninth and tenth nerves terminate bilaterally in the nucleus of the tractus solitarius (NTS). Although some neurons contain substance P, the primary neurotransmitter appears to be the excitatory amino acid L-glutamate (L-glu). Neurons in rostral ventrolateral medulla, which most probably comprise the C1 group of epinephrine neurons, are also critical in AP control. C1 neurons project to innervate cholinergic preganglionic sympathetic neurons in the spinal cord. Stimulation of the C1 area electrically or with L-glu increases AP, while lesions or local injection of the inhibitory amino acid gamma-aminobutyric acid (GABA) lowers AP to levels comparable to spinal cord transection. Lesions of C1 neurons or their pathways abolish vasodepressor reflexes from baroreceptors and vagal afferents. In contrast, noradrenergic neurons of the caudal ventrolateral medulla, the A1 group, project rostrally to innervate, in part, vasopressin neurons of the hypothalamus. Stimulation of A1 neurons lowers AP, while lesions or GABA elevates it. We propose that C1 neurons comprise the so-called tonic vasomotor center of the brain stem and also mediate, via a projection from the NTS, the vasodepressor limb of baroreflexes. The NTS-C1 projection may be GABAergic.

Modification of enzymatic antioxidants in retinal microvascular cells by glucose or advanced glycation end products.

Oxidative stress is one possible pathogenic mechanism to explain diabetic microangiopathy. In the present study, we determined the antioxidant enzyme activities in bovine retinal microvessels and cultured retinal microvascular cells: endothelial cells (BREC) and pericytes (BRP). We further investigated the effects of high glucose and advanced glycation end products (AGE) on these enzyme activities in BREC and BRP. Antioxidant enzyme activities in native retinal microvessels and BREC were quite similar but differed markedly from the BRP ones. High glucose decreased Se-GPx activity (about 20%) in BREC compared to mannitol. High concentrations of mannitol or NaCl increased Se-GPx activity (up to 40%) compared to control medium, suggesting that hyperosmolarity could regulate Se-GPx in BREC. No changes in antioxidant enzyme activities were observed when BRP were cultured with glucose or mannitol at high concentrations. AGE-BSA had no effect on enzyme activities in BREC, whereas 20 microM AGE-BSA increased catalase (40%) and superoxide dismutase (60%) activities in BRP. Differences in antioxidant enzyme activities observed between BREC and BRP, cultured with high concentrations of glucose or AGE, might help to explain their different behavior during the pathogenesis of diabetic retinopathy, i.e., early pericyte drop-out and late endothelial cell proliferation.

Projections to the subcortical forebrain from anatomically defined regions of the medial geniculate body in the rat.

Although the auditory cortex is believed to be the principal efferent target of the medial geniculate body (MG), our recent behavioral studies indicate that in rats the conditioned coupling of emotional responses to an acoustic stimulus is mediated by subcortical projections of the MG. In the present study we have therefore used WGA-HRP as an anterograde and retrograde axonal marker to (1) define the full range of subcortical efferent projections of the MG; (2) identify the cells of origin within the MG of each projection; and (3) determine whether the subregions of the MG that project to subcortical areas receive inputs from acoustic relay nuclei of the mid-brain, particularly the inferior colliculus. The rat MG was first parcelled into three major cytoarchitectural areas: the ventral, medial, and dorsal divisions. The suprageniculate nucleus, located within the body of the MG just dorsal to the medial division, was also identified. Efferent projections of the MG were determined by combined anterograde and retrograde tracing methods. Injections of WGA-HRP in the MG produced anterograde transport to cortex and several subcortical areas, including the posterior caudate-putamen and amygdala, the ventromedial nucleus of the hypothalamus, and the subparafascicular thalamic nucleus. The cells of origin of the subcortical projections were then mapped retrogradely after injections in the anterogradely labeled areas. Injections in the caudate-putamen or amygdala retrogradely labeled the medial division of the MG and the suprageniculate nucleus, as well as several adjacent areas of the posterior thalamus surrounding the MG. In contrast, injections in the ventromedial nucleus of the hypothalamus or the subparafascicular thalamic nucleus only produced labeling in the areas surrounding MG. Afferents to MG from the inferior colliculus were then identified. The central nucleus of the inferior colliculus, the main lemniscal acoustic relay nucleus in the midbrain, was found to project to the ventral and medial divisions of the MG. In contrast, the dorsal cortex and external nucleus of the inferior colliculus project to each division of the MG and to several additional nuclei in adjacent areas of the posterior thalamus. These data demonstrate that the medial division of MG, the suprageniculate nucleus, and immediately adjacent areas of the posterior thalamus provide a direct linkage between auditory neurons in the inferior colliculus and subcortical areas of the forebrain and thereby support the view that thalamic sensory nuclei relay afferent signals to subcortical as well as cortical areas.

Central and primary visceral afferents to nucleus tractus solitarii may generate nitric oxide as a membrane-permeant neuronal messenger.

An anatomical basis was sought for the postulated roles of nitric oxide (NO) as a labile transcellular messenger in the dorsal vagal complex (NTS-X). The diaphorase activity of NO synthase was used as a marker of neurons in NTS-X that are presumed to convert L-arginine to L-citrulline and NO. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) staining patterns in the nucleus tractus solitarii (NTS) were spatially related to terminal sites of primary visceral afferents from 1) orosensory receptors (e.g., rostral-central nucleus); 2) soft palate, pharynx, larynx, and tracheobronchial tree (e.g., dorsal, intermediate, and interstitial nuclei); 3) esophagus (nucleus centralis); 4) stomach (nucleus gelatinosus); 5) hepatic and coeliac nerves (nucleus subpostrema); and 6) carotid body and baroreceptors (medial commissural and dorsal-lateral nuclei). Primary visceral afferents were identified as sources of NADPHd-stained fiber plexuses in the NTS-X based on three findings: 1) the presence of NADPHd in nodose ganglion cells with morphological features of first-order sensory relay neurons; 2) retrograde transport of Fluoro-Gold (FG) or cholera toxin B (CT-B) from NTS-X to NADPHd-positive nodose ganglion neurons; and 3) striking reductions of NADPHd-stained processes within primary vagal projection fields ipsilateral to unilateral nodose ganglionectomy. A central origin of NADPHd-stained processes in NTS-X was identified in the medial parvicellular subdivision of the paraventricular hypothalamic nucleus. We conclude that NO of peripheral and central origin may modulate viscerosensory signal processing in the NTS-X and autonomic reflex function.

Medullary visceral reflex circuits: local afferents to nucleus tractus solitarii synthesize catecholamines and project to thoracic spinal cord.

Visceral feedback circuits in lower brainstem were elucidated with retrograde tracers by mapping neurons that issue local projections to the general visceral afferent division of the nucleus tractus solitarii (NTS) and dorsomotor vagal nucleus (DMX) in adult male rats. In study 1, spinal and intramedullary afferents to the visceral-sensorimotor complex (NTS-X) were traced to contiguous populations of cell bodies arranged in cylindrical segmental organization. NTS-X afferents derive from curvilinear arrays of neurons that parallel the efferent radiations of the solitariotegmental tract. Newly discovered afferents arise from circumscribed cell groups in the dorsal reticular formation and periventricular zone. Another source was traced to a paraambigual cell column in the apex of the rostral ventrolateral reticular nucleus (n.RVL). In study 2, catecholaminergic afferents were initially defined with combined retrograde transport-immunocytochemical methods. Deposits of retrograde tracers into NTS-X transported to neurons containing tyrosine hydroxylase (TH) in the A1, C1, and C3 areas or phenylethanolamine N-methyltransferase (PNMT) in the C1 area of the n.RVL and C3 area. In study 3, it was revealed that NTS-X afferents arise, in part, as collaterals of thoracic reticulospinal neurons. Deposits of the retrograde fluorescent tracer Fluorogold into the upper thoracic cord and rhodamine-labeled microbeads into NTS-X transported to the same neurons within a subambigual locus in n.RVL and parts of nucleus raphe magnus. In study 4, dual retrograde tracer-immunocytochemical analysis demonstrated that catecholamines are synthesized by a subset of neurons in the n.RVL that issue collaterals to the NTS-X and thoracic cord. Double retrogradely labeled TH- or PNMT-immunoreactive cell bodies were restricted to the C1 area within a 450-microns column bordered rostrally by the facial nucleus and ventrally by the medullary subpial surface. We conclude that visceral reflex arcs are reciprocally organized. Targets of NTS projection are also sources of local NTS-X afferent innervation. Catecholaminergic and other local afferents from reticular formation, periventricular, and spinal gray may, via collaterals, simultaneously modulate visceral reflex excitability at the level of NTS and the outflow of autonomic and respiratory motoneurons.

Brain stem afferents to the fastigial nucleus in the cat demonstrated by transport of horseradish peroxidase.

Although retrograde and anterograde degeneration studies have provided important information concerning brain stem afferents to the fastigal nucleus (FN), these data may be incomplete and should be confirmed by axonal transport methods. Attempts were made to inject horseradish peroxidase (HRP) unilaterally into the FN in a series of adult cats. Animals were perfused with dextran and a fixative solution of paraformaldehyde and glutaraldehyde in 0.1 M phospate buffer. Representative sections were treated by the Graham and Karnovsky ('66) method. Selective HRP injections in one FN resulted in retrograde transport of the marker to Purkinje cells of the ipsilateral vermis and distinctive appendages of the contralateral medial accessory olivary (MAO) nucleus (nucleus beta and the dorso-medial cell column). Retrograde transport of the label was found bilaterally in cells of the medial (MVN) and inferior (IVN) vestibular nuclei, in cell group x and in the nucleus prepositus (PP). Labeled vestibular neurons, most numerous in MVN, were identified in dorsal, caudal and lateral regions, with a slight ipsilateral preponderance. Only a few neurons in caudal, dorsal and lateral regions of the IVN were labeled and none of these included cells of group f. Labeled cells in the caudal third of PP were greatest ipsilaterally. Rostral and caudal injections of FN labeled smaller numbers of cells in MVN, IVN, cell group x and PP. HRP injections of FN and portions of lobules VIII and IX resulted in bilateral retrograde labeling of larger numbers of cells in MVN, IVN and cell group x, and ipsilateral labeling of cells in group y and the interstitial nucleus of the vestibular nerve. Injections of HRP into basal folia of lobules V and VI resulted in retrograde transport of the marker to cells of the medial and dorsal accessory olivary nuclei contralaterally, and to cells of the ipsilateral accessory cuneate nucleus. Transport of label injected into portions of the pyramis was detected in parts of the contralateral MAO and bilaterally in parts of the pontine and reticulotegmental nuclei. This study suggests that the principal afferents of the fastigial nucleus arise from: (1) Purkinje cells of the ipsilateral vermis, (2) restricted portions of the contralateral MAO (nucleus beta and dorsomedial cell column), (3) portions of the MVN and IVN (bilaterally) and (4) caudal parts of the PP. Secondary vestibular inputs to the fastigial nucleus probably are relayed mainly by Purkinje cells in the cerebellar cortex.

Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla.

Projections from the nucleus tractus solitarii (NTS) to autonomic control regions of the ventrolateral medulla, particularly the nucleus reticularis rostroventrolateralis (RVL), which serves as a tonic vasomotor center, were analyzed in rat by anterograde, retrograde, and combined axonal transport techniques. Autonomic portions of the NTS, including its commissural, dorsal, intermediate, interstitial, ventral, and ventrolateral subnuclei directly project to RVL as well as to other regions of the ventrolateral medulla. The projections are organized topographically. Rostrally, a small cluster of neurons in the intermediate third of NTS, the subnucleus centralis, and neurons in proximity to the solitary tract selectively innervate neurons in the retrofacial nucleus and nucleus ambiguus. Neurons generally located in more caudal and lateral sites in the NTS innervate the caudal ventrolateral medulla (CVL). The RVL, CVL, and nucleus retroambiguus are interconnected. A combined retrograde and anterograde transport technique was developed so as to prove that projections from the NTS to the ventrolateral medulla specifically innervate the region of RVL containing neurons projecting to the thoracic spinal cord or the region of the nucleus containing vagal preganglionic neurons. When the retrograde tracer, fast blue, was injected into the thoracic spinal cord, and wheat germ agglutinin-conjugate horseradish peroxidase (HRP) was injected into the NTS, anterogradely labeled terminals from the NTS surrounded the retrogradely labeled neurons in the RVL and in the nucleus retroambiguus in the caudal medulla. Among the bulbospinal neurons in the RVL innervated by the NTS were adrenaline-synthesizing neurons of the C1 group. When fast blue was applied to the cervical vagus, and HRP was injected into the NTS, anterogradely labeled terminals from the NTS surrounded retrogradely labeled neurons in the rostral dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, the retrofacial nucleus, and the dorsal portion of the RVL, a region previously shown to contain cardiac vagal preganglionic neurons. This combined anterograde and retrograde transport technique provides a useful method for tracing disynaptic connections in the brain. These data suggest that the RVL is part of a complex of visceral output regions in the ventrolateral medulla, all of which receive afferent projections from autonomic portions of the NTS. Bulbospinal neurons in the RVL, in particular the C1 adrenaline neurons, may provide a portion of the anatomic substrate of the baroreceptor and other visceral reflexes.

Reevaluation of projections from the mesencephalic trigeminal nucleus to the medulla and spinal cord: new projections. a combined retrograde and anterograde horseradish peroxidase study.

Microinjection of horseradish peroxidase (HRP) into the medullary parvocellular reticular formation (NPvc) resulted in retrograde labeling of neurons throughout the mesencephalic trigeminal nucleus (Mes V). Labeled cells were large and ovoid and were distributed primarily in the expanded pontine part of the nucleus. However, none of the small neurons in Mes V were labeled. Injections of HRP made into adjacent brainstem structures including the nucleus gigantocellularis, ventrolateral reticular formation, vestibular complex, and the spinal trigeminal nucleus failed to label neurons in Mes V. Injections made into the medullary raphe and into regions reported to receive inputs from Mes V--spinal cord, nucleus tractus solitarius, hypoglossal nucleus, and facial nucleus--were also not followed by transport to Mes V. Anterograde axonal transport of HRP from the region of reticular formation innervated by Mes V also labeled axons projecting to Mes V and to visceral and somatic sensorimotor nuclei in the lower brainstem. Recent reports of afferents from the amygdala to Mes V suggest that reflexes involving the mesencephalic trigeminal nucleus might be modulated by signals from limbic and autonomic as well as somatic centers in the brain.

Distribution of catecholamine neurons in the hypothalamus and preoptic region of mouse.

The distribution and morphology of cells containing tyrosine hydroxylase (TH) were mapped by using the immunoperoxidase technique in the hypothalamus and preoptic area in two strains of mouse, CBA/J and BALB/cJ. On the basis of rostral-caudal contiguities between cell aggregates, hypothalamic preoptic neurons were subdivided into three arbitrary groups: (1) dorsal, (2) intermediate, and (3) ventral. New or more prominent collections of TH cells were observed, and in some regions, cells were more complexly organized than originally described. In the dorsal group, a rostral collection of small ovoid cells, previously not described, were located in the anterior preoptic nucleus (APN) of Loo ('31) and extended rostrally and ventrally into the preoptic periventricular gray. The next constituent occupied the paraventricular nucleus (PVN), and was composed of two classes of cells: (1) a small ovoid cell within anterior and medial parvocellular PVN in contiguity rostrally with a similar cell in APN and (2) a larger, angular cell within and adjacent to the lateral PVN in contiguity caudally with cells in the zona incerta (ZI). Further caudally, a larger and more pleomorphic collection of TH neurons was localized in the medial ZI, particularly at midtuberal levels. These cells were not scattered, as previously reported, but were differentiated into two clear-cut densities, a larger medial island and a more elongated lateral island. Cells of ZI, both large and small, extended caudally into the dorsal hypothalamic and subparafascicular nuclei and periventricular gray. In contrast to previous descriptions, no cells were seen in the nucleus reuniens. In the intermediate group, the most rostral constituent occupied the preoptic periventricular gray, extended as far as the lamina terminalis, and merged dorsocaudally with cells in APN. While the variably shaped cells of the hypothalamic periventricular gray (PVG) were still present in the retrochiasmatic region, a striking absence of these cells was noted at midtuberal levels between the dorsomedial and arcuate hypothalamic nuclei. At this level, a new group of small-round TH cells, resembling those of the arcuate nucleus, was identified in the dorsomedial hypothalamic nucleus (DMN). At caudal tuberal levels, similar neurons were found in the posterior hypothalamic nucleus (PH). These neurons overflowed medially into the PVG and caudoventrally into the arcuate nucleus. In the ventral group, the most rostral constituent, composed of both small and ovoid cells in the retrochiasmatic area, appeared to represent the rostral commissural portion of the arcuate nucleus (Arc).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Anatomical substrates of cholinergic-autonomic regulation in the rat.

UNLABELLED: Acetylcholine (ACh) plays a major role in central autonomic regulation, including the control of arterial blood pressure (AP). Previously unknown neuroanatomic substrates of cholinergic-autonomic control were mapped in this study. Cholinergic perikarya and bouton-like varicosities were localized by an immunocytochemical method employing a monoclonal antiserum against choline acetyltransferase (ChAT), the enzyme synthesizing ACh. In the forebrain, bouton-like varicosities and/or perikarya were detected in the septum, bed nucleus of the stria terminalis, amygdala (in particular, autonomic projection areas AP1 and AP2 bordering the central subnucleus), hypothalamus (rostrolateral/innominata transitional area, perifornical, dorsal, incertal, caudolateral, posterior [PHN], subparafascicular, supramammillary and mammillary nuclei). Few or no punctate varicosities were labeled in the paraventricular (PVN) or supraoptic (SON) hypothalamic nuclei. In the mid- and hindbrain, immunoreactive cells and processes were present in the nucleus of Edinger-Westphal, periaqueductal gray, parabrachial complex (PBC), a periceruleal zone avoiding the locus ceruleus (LC), pontine micturition field, pontomedullary raphe, paramedian reticular formation and periventricular gray, A5 area, lateral tegmental field, nucleus tractus solitarii (NTS), nucleus commissuralis, nucleus reticularis rostroventrolateralis (RVL), and the ventral medullary surface (VMS). In the PBC, immunoreactive varicosities identified areas previously unexplored for cholinergic autonomic responsivity (superior, internal, dorsal, and central divisions of the lateral subnucleus, nucleus of Koelliker-Fuse and the medial subnucleus). In the NTS, previously undescribed ChAT-immunolabeled cells and processes were concentrated at intermediate and subpostremal levels and distributed viscerotopically in areas receiving primary cardiopulmonary afferents. In the nucleus RVL, cholinergic perikarya were in proximity to the VMS and medial to adrenergic cell bodies of the C1 area. Punctate varicosities of unknown origin and dendrites extending ventrally from the nucleus ambiguus overlapped the C1 area and immediate surround of RVL. IN CONCLUSION: 1) Cholinergic perikarya and putative terminal fields, overlap structures that are rich in cholinoreceptors and express autonomic, neuroendocrine, or behavioral responsivity to central cholinergic stimulation (PHN, NTS, RVL). The role of ACh in most immunolabeled areas, however, has yet to be determined. Overall, these data support the concept that cholinergic agents act at multiple sites in the CNS and with topographic specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

A role of insular cortex in cardiovascular function.

We sought to determine whether the insular cortex contributes to the regulation of arterial blood pressure (AP). Responses to electrical and chemical stimulation of the cortex were studied in the anesthetized, paralyzed, and artificially ventilated Sprague-Dawley rat. The insular cortex was initially defined, anatomically, by the distributions of retrogradely labeled perikarya following injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the nucleus tractus solitarii (NTS). Injections of WGA-HRP into the insular cortex anterogradely labeled terminals in cardiopulmonary and other divisions of the NTS and confirmed projections revealed by retrograde tracing experiments. Electrical stimulation of the insular cortex elicited elevations of AP (less than or equal to 50 mm Hg) and cardioacceleration (less than or equal to 40 bpm). The locations of the most active pressor sites corresponded closely to the locations of retrogradely labeled cells in layer V of granular and posterior agranular areas of the insular cortex (areas 14 and 13) and the extreme capsule. Maximal pressor responses were obtained at a stimulus intensity of three to five times threshold current of 20-30 microA. Responses elicited mostly with higher-threshold currents were also mapped in areas 2a and 5lb and the claustrum and within the corpus callosum. Unilateral injections into the insular pressor area of the excitatory amino acid monosodium glutamate (L-Glu; 0.05 nmol to 10 nmol) or the rigid structural analogue of L-Glu, kainic acid (KA) (0.4 nmol) (which specifically excite perikarya), caused topographically specific elevations in AP and tachycardia. During the course of the anatomical transport studies, new findings were obtained on the organization and characteristics of the cortical innervation of the NTS and the nucleus reticularis parvocellularis. Topographic relationships between the cortex and the NTS were organized in a more complex manner than previously thought. Cells projecting to caudal cardiopulmonary segments of the NTS were fewer and generally located ventrally and caudally and in a more restricted area than cells projecting rostrally or to the parvicellular reticular formation. Anterograde transport data revealed new presumptive terminal fields in dorsolateral, ventral, periventricular, and commissural regions of the NTS, including an area overlapping the terminal field of the aortic baroreceptor nerve. We conclude that neurons within an area of the insular cortex projecting to multiple brainstem autonomic nuclei, including a region of the NTS innervated by baroreceptor afferents, increase arterial blood pressure and heart rate.(ABSTRACT TRUNCATED AT 400 WORDS)

Projections from the nucleus tractus solitarii to the spinal cord.

Projections from the nucleus tractus solitarii (NTS) to the spinal cord were demonstrated in the male Sprague-Dawley rat. In retrograde transport studies, a horseradish peroxidase conjugate or a fluorescent dye, FluoroGold, were injected into midcervical or upper thoracic spinal segments. Most solitariospinal neurons were multipolar or bipolar and located between the obex and spinomedullary junction. Solitariospinal neurons were concentrated in proximity to the ventral border of the solitary tract and extended dorsally into the intermediate division and ventrolaterally into the intermediate reticular zone (IRt) of the lateral tegmental field. This subgroup predominantly projects to midcervical spinal segments. A subset of small neurons was retrogradely labeled from cervical or thoracic spinal segments in the medial commissural nucleus and contiguous with a periventricular group surrounding the central canal. In anterograde transport studies, iontophoretic deposits of Phaseolus vulgaris leucoagglutinin were centered stereotaxically on sites in NTS identified by retrograde transport data. The lectin was incorporated by neurons of the solitary complex and transported bilaterally by axons that emerged from the nucleus and entered the reticular formation. The solitario-reticular (transtegmental) pathway irradiated diagonally across the IRt and extended caudally into the cervical lateral funiculus and spinal gray. A small periventricular-spinal pathway also descended longitudinally to the neuraxis. Solitariospinal neurons project to superficial lamina of the dorsal horn, laminae VII and X and ventral horn. The projections are predominantly contralateral to phrenic and intercostal motor nuclei and ipsilateral to the intermediolateral cell column. The solitariospinal projection represents the shortest route in the central nervous system, other than the local intraspinal reflex, through which first order visceral afferents signal cardiorespiratory and alimentary motor nuclei.

Topographic organization of convergent projections to the thalamus from the inferior colliculus and spinal cord in the rat.

The purpose of this study was to identify thalamic areas receiving convergent sensory inputs from acoustic and spinal projection systems in the rat. The topographic distribution of afferents to the thalamus from the inferior colliculus and spinal cord was examined by using WGA-HRP as an anterograde axonal tracer. Following injections in the inferior colliculus, terminal labeling was present in ventral, medial, and dorsal divisions of the medial genicuate body (MGB) and in adjacent areas of the posterior thalamus, including the posterior limitans nucleus, the posterior intralaminar nucleus, the marginal zone, the peripeduncular region, the lateral or parvicellular part of the subparafascicular nucleus, and a region intercalated between the posterior limitans nucleus and the suprageniculate nucleus. In the caudal thalamus spinal projections remained in the reticular formation medial to the collicular terminal field. At intermediate levels of the MG, however, the spinal projection began to overlap the collicular field, terminating in the medial division of the MG and in the posterior intralaminar nucleus, the marginal zone, the lateral subparafascicular nucleus, and the area between the suprageniculate and posterior limitans nuclei. More rostrally, the convergent field expanded to include aspects of the dorsal MG division. The extent to which afferent projections to the thalamus from the inferior colliculus and spinal cord converge is thus graded in the caudorostral plane, with the greatest overlap occurring at the level of the rostral third of the MGB. These observations identify potential areas of acoustic and somesthetic integration and may account for observations of neuronal plasticity in the thalamus in response to the pairing of acoustic and somesthetic inputs.