Search

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neurons in the rostral ventromedial medulla (RVM) are thought to modulate nociceptive transmission via projections to spinal and trigeminal dorsal horns. The cellular substrate for this descending modulation has been studied with regard to projections to spinal dorsal horn, but studies of the projections to trigeminal dorsal horn have been less complete. In this study, we combined anterograde tracing from RVM with immunocytochemical detection of the GABAergic synthetic enzyme, GAD67, to determine if the RVM sends inhibitory projections to trigeminal dorsal horn. We also examined the neuronal targets of this projection using immunocytochemical detection of NeuN. Finally, we used electron microscopy to verify cellular targets. We compared projections to both trigeminal and spinal dorsal horns. We found that RVM projections to both trigeminal and spinal dorsal horn were directed to postsynaptic profiles in the dorsal horn, including somata and dendrites, and not to primary afferent terminals. We found that RVM projections to spinal dorsal horn were more likely to contact neuronal somata and were more likely to contain GAD67 than projections from RVM to trigeminal dorsal horn. These findings suggest that RVM neurons send predominantly GABAergic projections to spinal dorsal horn and provide direct input to postsynaptic neurons such as interneurons or ascending projection neurons. The RVM projection to trigeminal dorsal horn is more heavily targeted to dendrites and is only modestly GABAergic in nature. These anatomical features may underlie differences between trigeminal and spinal dorsal horns with regard to the degree of inhibition or facilitation evoked by RVM stimulation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This review focuses on presympathetic neurons in the medulla oblongata including the adrenergic cell groups C1-C3 in the rostral ventrolateral medulla and the serotonergic, GABAergic and glycinergic neurons in the ventromedial medulla. The phenotypes of these neurons including colocalized neuropeptides (e.g., neuropeptide Y, enkephalin, thyrotropin-releasing hormone, substance P) as well as their relative anatomical location are considered in relation to predicting their function in control of sympathetic outflow, in particular the sympathetic outflows controlling blood pressure and thermoregulation. Several explanations are considered for how the neuroeffectors coexisting in these neurons might be functioning, although their exact purpose remains unknown. Although there is abundant data on potential neurotransmitters and neuropeptides contained in the presympathetic neurons, we are still unable to predict function and physiology based solely on the phenotype of these neurons.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.

Alpha-neoendorphin (alpha-NE) is an opiate decapeptide derived from the prodynorphin protein. Its anatomical distribution in the brain of mammals other than the rat, particularly in carnivores, is less well known than for other opiate peptides. In the present work, we have charted the distribution of alpha-NE immunoreactive fibers and perikarya in the diencephalon and the brainstem of the dog. The highest densities of labeled fibers were found in the substantia nigra and in patches within the nucleus of the solitary tract. Moderate densities appeared in the arcuate nucleus (Ar), median eminence, entopeduncular nucleus, ventral tegmental area, retrorubral area, periaqueductal central gray, interpeduncular nucleus and lateral parabrachial nucleus. Groups of numerous labeled perikarya were localized in the magnocellular hypothalamic nuclei, Ar and in the central superior and incertus nuclei in the metencephalon. Moreover, less densely packed fibers and cells appeared widely distributed throughout many nuclei in the region studied. These results are discussed with regard to the pattern described in other species. In addition, the present results were compared with the distribution of met-enkephalin immunoreactivity in the diencephalon and the brainstem of the dog that we have recently described. Although the distributions of these two peptides overlap in many areas, the existence of numerous differences suggest that they form separate opiate systems in the dog.

To reveal neurones in the cat medulla oblongata involved in carotid baroreceptor/chemoreceptor reflexes, the distribution of c-Fos oncoprotein immunoreactivity was studied following electrical stimulation of the right carotid sinus nerve. The neurochemistry of the activated neurones was investigated using antisera to tyrosine hydroxylase, neuropeptide Y, somatostatin, and glutamate. Nitric oxide containing neurones were identified using antiserum to nitric oxide synthase (NOS) and by the histochemical localization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Following sinus nerve stimulation numerous c-Fos-IR cells were detected both ipsilaterally and contralaterally in the nucleus tractus solitarii, the area postrema and throughout the ventrolateral medulla. Dual labelling studies revealed that 3.3% of c-Fos-immunoreactive cells in the nucleus tractus solitarii were also immunoreactive for tyrosine hydroxylase. The double labelled cells were scattered within the medial and ventrolateral subnuclei, predominantly rostral to obex. A higher proportion (10.3%) of c-Fos-IR cells in the ventrolateral medulla also showed tyrosine hydroxylase immunoreactivity. Caudal to obex, these were scattered in the reticular formation between the spinal trigeminal nucleus and the lateral reticular nucleus, while more rostrally they were found within the lateral reticular nucleus, the nucleus ambiguus and the lateral tegmental field. Cells expressing c-fos and reactive for glutamate, neuropeptide Y or NADPH-diaphorase (or NOS) were only rarely seen, and co-localization of c-Fos and somatostatin immunoreactivities was not seen. These results suggest that of the neurones forming pathways within the medulla activated on carotid sinus nerve stimulation, presumably mediating baro- and chemoreceptor reflexes, relatively few utilize catecholamines, glutamate, neuropeptide Y or nitric oxide as their transmitter substance.

The adrenergic phenotype was analysed in the rat's rostral dorsomedial medulla under normal conditions and 3 days after a single intraperitoneal injection of an eburnamine derivative, RU 24722, which increases tyrosine hydroxylase protein expression in the rostral portion of the nucleus tractus solitarius. This approach was investigated by a double immunofluorescence labelling of tyrosine hydroxylase and phenylethanolamine N-methyltransferase proteins. Under normal conditions, most adrenergic cell bodies are anatomically distributed in the dorsal and rostral medulla oblongata between the rostral part of the dorsal motor nucleus of the vagus nerve and the medial longitudinal fasciculus. Adrenergic neurons detected in this medullar region were distributed between both cell groups. Three days after the pharmacological RU 24722 treatment, an upregulation in tyrosine hydroxylase and phenylethanolamine N-methyltransferase protein expression was detected in both cell groups characterized by a highly increased number of tyrosine hydroxylase- and phenylethanolamine N-methyltransferase-containing cell bodies. The number of TH-mRNA containing neurons was also increased, indicating the transcriptional level of this regulation. These results demonstrated a particular neuronal plasticity of adrenergic phenotype in the medullary cell groups of adult rat.

We have performed a mapping of basic fibroblast growth factor (bFGF) immunoreactive (ir) glial and nerve cell populations in the male rat brain using a rabbit antibody raised against a synthetic peptide of bovine bFGF. Regional morphometric and microdensitometric analysis of the bFGF ir neuronal profiles in coronal brain sections was carried out by means of an automatic image analyser. The density and intensity of the bFGF ir glial profiles were subjectively evaluated. The bFGF immunoreactivity (IR) was detected within the cytoplasm of neurons, except within the pyramidal neurons of hippocampal CA2 region, the fasciola cinerea and the indusium griseum, where bFGF IR was present in the nucleus. In contrast, in glial cells bFGF IR was always found in the nucleus. Neuronal and glial IR was no longer observed after absorption of the bFGF antiserum with recombinant bFGF. Basic FGF IR was found in neuronal and glial cell populations throughout the brain as well as in the choroid plexus and in the ependymal cells lining the ventricles. Basic FGF ir nerve cells were found in all layers of both the neocortex and allocortex. Within the caudate putamen and the nucleus accumbens a low density of weak bFGF ir neuronal profiles was detected. The majority of the thalamic nuclei showed medium to high densities of moderate to strong bFGF ir neuronal profiles. All the hypothalamic nuclei, with the exception of the anterior and lateral hypothalamic area and of the ventral hypothalamic nucleus, contained a high density of bFGF ir profiles. The pons and the medulla oblongata were characterized by the presence of a large number of nuclei containing moderate to high densities of strong bFGF ir profiles. The Purkinje cell layer of the cerebellar cortex contained a high density of moderately bFGF ir profiles. A moderate density of strong bFGF ir nerve cell profiles was observed within all the laminae of the spinal cord, except within the II and III laminae where a high density of strongly ir profiles was found. Histogram analysis of total immunoreactivity showed that the distribution of bFGF ir profiles within the telencephalon and mesencephalon tend to be similar with regard to the central tendency and spread. Using Kendall's tau, a significant correlation between intensity and density values was obtained only in the diencephalon. The cytoplasmic bFGF IR found in distinct nerve cell populations all over the rat brain and spinal cord may represent forms of bFGF which can be released from the nerve cells via non-exocytotic mechanisms in view of the absence of an intracellular signal peptide in bFGF. The presence of nuclear bFGF IR within the glial cells all over the central nervous system (CNS) suggests an intracellular function of bFGF, such as the promotion of mitogenesis and/or participation in the transcriptional regulation of various genes.

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within non-dopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltransferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the noradrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.

The distribution of nitric oxide producing neurones in the medulla oblongata of the cat was investigated using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, and nitric oxide synthase (NOS) immunohistochemistry. The pattern of staining obtained with both methods was found to be similar. Strongly diaphorase and NOS reactive neurones were present in the paramedian and lateral tegmental fields, including the regions occupied by the A1/C1 catecholamine cell groups, the nucleus ambiguus and lateral reticular nucleus, and in a number of sensory nuclei including the nucleus of the tractus solitarius and the dorsal column nuclei. The extent of co-localization of NADPH-diaphorase with a number of neuropeptides and neurotransmitters was investigated by combining NADPH-diaphorase histochemistry with immunocytochemistry for neuropeptide Y, somatostatin, glutamate, cholecystokinin and tyrosine hydroxylase. NADPH-diaphorase reaction product was observed in neurones immunoreactive for glutamate and somatostatin. These double-labelled cells were found in the paramedian region, lateral reticular field, the nucleus prepositus hypoglossi and in the rostral nucleus of the tractus solitarius. In the rostral ventrolateral medulla NADPH-diaphorase/somatostatin immunoreactive cells were found in the paragigantocellular nucleus. NADPH-diaphorase/glutamate immunoreactive cells overlapped the nucleus ambiguus, the lateral reticular nucleus and the A1/C1 catecholaminergic cell groups. In addition, a few NADPH-diaphorase/glutamate immunoreactive cells were found in the paraolivary area and gigantocellular tegmental field, in the external cuneate and infratrigeminal nuclei. The functional implications of the co-localization of nitric oxide with these neurotransmitters in areas of the medulla concerned with cardiovascular regulation is discussed.

Secretoneurin is a peptide of 33 amino acids generated in the brain by proteolytic processing of secretogranin II which is a member of the chromogranin/secretogranin family. The distribution of this newly characterized peptide was investigated by immunocytochemistry in the human brain stem. The staining pattern of secretoneurin-like immunoreactivity was compared with that of substance P in adjacent sections. Secretoneurin-like immunoreactivity appeared mainly in dot- and fiber-like structures with densities varying from low to very high. Only a low number of secretoneurin-immunoreactive perikarya was found. Pericellular staining of both secretoneurin-immunopositive and immunonegative cells was frequently observed in the area of the central gray, in the reticular formation and in the solitary nuclear complex. The medial part of the substantia nigra pars reticulata, the nucleus interpeduncularis, the area of the central gray, the raphe complex and the inferior olive displayed a high density of secretoneurin-like immunoreactivity. Furthermore, a very prominent staining was found in the medial, dorsal and gelatinous subnuclei of the solitary tract and the dorsal motor nucleus of vagus. The substantia gelatinosa of the caudal trigeminal nucleus and spinal cord were also very strongly secretoneurin-immunopositive. The staining patterns of secretoneurin- and substance P-like immunoreactivities were to a certain extent overlapping in several areas. The highest degree of coincidence was found in the substantia gelatinosa. This study demonstrated that secretoneurin is distinctly distributed in the human brain stem. Its distributional pattern indicates a role particularly in the modulation of afferent pain transmission and in the regulation of autonomic functions.

Our study investigates the distribution of neurophysins (Nph), proteins that are part of the precursors for vasopressin and oxytocin, and calcitonin gene-related peptide (CGRP) in the human brainstem by immunohistochemistry. Both peptides were found in discrete regions of the human hindbrain. Nph could be demonstrated exclusively in fibers and punctate perineural varicosities that were travelling within the mesencephalic central gray, substantia nigra, as well as locus coeruleus, medial longitudinal fascicle, raphe, nucleus of the solitary tract, lateral reticular nucleus and area postrema. A few varicosities were seen in the substantia gelatinosa of the spinal trigeminal tract and its continuation into the dorsal horn of the cervical spinal cord. In contrast to these observations. CGRP-immunoreactive fibers were found to be densest in the spinal tract of the trigeminal nerve and the dorsal horn of the spinal cord. In addition, fibers and varicosities could be demonstrated in numerous distinct brain regions, such as locus coeruleus and subcoeruleus, solitary tract, cuneate nucleus, raphe and periaqueductal gray. CGRP-immunoreactivity was also present in perikarya in the ventral horn of the spinal cord, as well as motor nuclei of cranial nerves, i.e., hypoglossal nucleus, ambiguous nucleus. Our results suggest that Nph-immunoreactivity in the human brainstem may be present predominantly within long fiber projections from hypothalamic neurosecretory nuclei, in analogy to data obtained from rodents, whereas CGRP may play a role in the branchiomotor system as well as in intrinsic or extrinsic projections involved in autonomic regulation and integration of sensory information.