Distribution of serotonin-immunoreactive neurons in the brain of sockeye salmon fry.

The organization of the serotonergic cell groups in the brainstem of fishes and amphibians has received relatively little attention. It has been generally assumed that they are little differentiated and constitute a median cell column throughout the brainstem, and that laterally migrated serotonergic cell groups are largely lacking. In the present study we present evidence to the contrary. By the use of a sensitive immunocytochemical technique for the visualization of serotonin-immunoreactive (5HTir) neurons, we have been able to make a detailed delineation of the putatively serotonergic neuronal groups throughout the brain. In the epithalamus, 5HTir neurons were located in the left habenular nucleus in its dorsal subdivision. 5HTir neural elements, primarily photoreceptor cells, were present throughout the pineal organ and in some cases also in the parapineal organ. In the periventricular zones of the hypothalamus and posterior tuberculum, 5THir cerebrospinal fluid-contacting neurons were located in the paraventricular organ and in the dorsal, ventral and caudal zones of the periventricular hypothalamus. In the dorsal thalamus/synencephalon, 5THir neurons surround the tractus habenulo-interpeduncularis (fasciculus retroflexus). In the brainstem, several groups of 5HTir neurons could be discerned, that for reasons of topological similarity were named according to Lidov and Molliver a raphe pallidus/obscurus-complex (B1 and B2), raphe magnus (part of B3), median raphe (B8) possibly including raphe pontis (B5), raphe dorsalis (B4, B6 and B7), and B9. 5HTir neurons were observed in the central gray of the IVth ventricle, dorsal to the noradrenergic isthmal neurons and lateral to the brachium conjunctivum, in an area topologically equivalent with the dorsal subdivision of the locus coeruleus in mammals. In addition, small numbers of 5HTir neurons were located in the lobi faciales. Thus, the presence of well-differentiated groups of migrated serotonergic neurons is not an advanced trait of amniote brains, but may be a pattern common to all vertebrates.

Substance P-immunoreactivity in the dorsal medial region of the medulla in the cat: effects of nodosectomy.

The distribution of substance P in the vagal system of the cat was studied by immunohistochemistry. Substance P-immunoreactive cell bodies and fibres were observed in the nodose ganglion. Numerous substance P-immunoreactive terminals and fibres were localized in their bulbar projection area, i.e. throughout the caudo-rostral extent of the nucleus of the solitary tract. Four subnuclei, among the nine forming the nucleus of the solitary tract, were strongly labelled: interstitial, gelatinosus, dorsal and commissural. The dorsal motor nucleus of the vagus nerve also exhibited numerous substance P-immunoreactive terminals, sometimes closely apposed on the somata of preganglionic neurons. To determine the substance P component of the vagal afferent system a nodose ganglion was removed on one side. The ablation triggered ipsilaterally a large decrease of substance P immunoreactivity in the four subnuclei strongly labelled on normal cats. These results suggest the involvement of substance P-containing vagal fibres in integrative processes of the central regulation of cardiovascular, digestive and respiratory systems, viscerotopically organized throughout these four subnuclei. The nodose ablation also resulted in a decrease of substance P immunoreactivity in the ipsilateral dorsal motor nucleus of the vagus nerve, suggesting monosynaptic vago-vagal interactions.

Thyrotropin-releasing hormone-like immunoreactive neurons in rabbit medulla oblongata.

Thyrotropin-releasing hormone-like immunoreactive (TRH-LI) neuronal cell bodies and processes were identified by using the peroxidase-antiperoxidase method in the medullar oblongata of rabbits. TRH-LI cell bodies were mainly distributed in the ventral medulla (paraolivary and parapyramidal regions), and caudal raphe nuclei (nucleus raphe obscurus and nucleus raphe pallidus). TRH-LI processes with varicosities were densely distributed in the solitary nucleus, dorsal motor nucleus of the vagus nerve and area postrema. TRH-LI processes appeared to be in contact with unlabelled cell bodies in the dorsal motor nucleus of the vagus nerve.

Immunohistochemical study of neuropeptides in vagal and glossopharyngeal afferent neurons in the rat.

The presence and distribution of multiple neuropeptides in vagal and glossopharyngeal afferent ganglia of the rat were studied using immunohistochemistry. Substance P-, calcitonin-gene related peptide-, cholecystokinin-, neurokinin A-, vasoactive intestinal polypeptide-, and somatostatin-immunoreactive neurons were detected in each visceral afferent ganglion. Neurotensin-immunoreactive cells were not observed. In the nodose ganglion (inferior ganglion of the vagus nerve) occasional immunoreactive cells were scattered throughout the main (caudal) portion of the ganglion with small clusters of cells seen in the rostral portion. The pattern of distribution of the various peptides in the nodose ganglion was similar, with the exception of vasoactive intestinal polypeptide-immunoreactive neurons which exhibited a more caudal distribution. The relative numbers of immunoreactive cells varied, with the greatest numbers being immunoreactive for substance P or vasoactive intestinal polypeptide, and the lowest numbers being immunoreactive for neurokinin A and somatostatin. A build-up of immunoreactivity for each of the peptides, except somatostatin and neurotensin, was detected in vagal nerve fibers of colchicine-injected ganglia. Numerous peptide-immunoreactive cells were also found in the petrosal (inferior ganglion of the glossopharyngeal nerve) and jugular (superior ganglion of the vagus nerve) ganglia. No specific intraganglionic distribution was noted although the relative numbers of cells which were immunoreactive for the different peptides varied considerably. Substance P and calcitonin-gene related peptide were found in large numbers of cells, cholecystokinin was seen in moderate numbers of cells, and neurokinin A, vasoactive intestinal polypeptide and somatostatin were seen in fewer cells. These data provide evidence for the presence and non-uniform distribution of multiple peptide neurotransmitters in vagal and glossopharyngeal afferent neurons. In general, relatively greater numbers of immunoreactive cells were located in the rostral compared with caudal nodose ganglion, and in the petrosal and jugular ganglia compared with the nodose ganglion. Thus, multiple neuropeptides may be involved as afferent neurotransmitters in the reflexes mediated by vagal and glossopharyngeal sensory nerves.

Central distribution of subdiaphragmatic vagal branches in the rat.

In the rat, the subdiaphragmatic vagus nerves (SDX) have five major branches--the right gastric, the left gastric, the coeliac, the accessory coeliac, and the hepatic. Although these branches innervate more than the organs after which they are named, some mediate specific behavioral functions. In addition to the SDX trunk, the central stump of each of these branches was incubated in horseradish peroxidase (HRP) for 6 hours in anesthetized rats. After processing the vagal ganglia, pons, medulla, and upper cervical spinal cord of each preparation, the sections were examined for both retrogradely and anterogradely transported HRP reaction product. When only one nerve had been incubated, retrogradely labeled neurons were confined primarily to the ipsilateral ganglion, medulla, and spinal cord. Within the brain, a few labeled neurons occurred within the nucleus ambiguus (NA) and the reticular formation caudal to the NA, but the vast majority appeared in the dorsal motor nucleus of the vagus (DMX). The axons of most labeled neurons in the NA distributed in the gastric branches; those from cells caudal to the NA, probably distributed in the coeliac branch. Most labeled DMX cells also distributed with the gastric branches. Those on the lateral tip of the right DMX, however, had axons in the coeliac branch; those on the left DMX tip, in the accessory coeliac. After incubation of the SDX trunk, anterograde HRP reaction product occurred in the caudomedial nucleus of the solitary tract (NST) just rostral and subjacent to the area postrema (AP). Unlike the retrograde label, anterograde reaction product was bilateral, but always weaker contralaterally. Within the SDX distribution, the afferent axons from the gastric branches exhibited one pattern of termination; those from the coeliac, accessory coeliac, and hepatic branches, another. The gastric branch distributions began dorsolaterally in the SDX termination zone and continued caudally beneath the AP. Immediately subjacent to the AP, gastric branch terminals were never dense and the entire distribution faded at the level of the obex. The coeliac and accessory coeliac distributions began dorsomedially within the SDX termination zone and intensified caudally in a thin band immediately subjacent to the AP. The densest label was associated with the caudal half of the AP, but the distribution thinned rapidly caudal to the obex. The hepatic distribution was similar to that of the coeliac branches but never achieved similar density. Physiological and behavioral data correlate with the anatomical picture in that the efferent functions appear to be more densely localized than the afferent functions.

Immunohistochemical localization of serotonin- and substance P-containing fibers around respiratory muscle motoneurons in the nucleus ambiguus of the cat.

Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to determine if the putative neurotransmitters, serotonin and substance P, are present around posterior cricoarytenoid muscle motoneurons. Fast Blue was injected into the posterior cricoarytenoid muscle of the larynx. Following a 14-21 day survival time to allow for transport of the dye, the animals were perfusion fixed and the brainstem was removed for analysis under the fluorescence microscope. Retrogradely labeled cell bodies containing Fast Blue were found within the nucleus ambiguus from 0.5 to 3.0 mm rostral to obex. These motoneurons ranged in size from 23 to 38 micron. The same tissue sections containing labeled posterior cricoarytenoid muscle motoneurons were then used to determine the distribution of serotonin and substance P around these motoneurons using the indirect immunofluorescence technique. A dense network of serotonin-containing immunoreactive fibers was found around posterior cricoarytenoid muscle motoneurons. The fibers contained varicosities which were in close proximity, actually appearing to surround these motoneurons. Substance P immunoreactive fibers and varicosities were also found around posterior cricoarytenoid muscle motoneurons. The density and pattern of distribution of the substance P immunoreactivity was similar to that of the serotonin immunoreactivity. These results suggest that these putative neurotransmitters may be involved in influencing the activity of posterior cricoarytenoid muscle motoneurons. Serotonin and substance P are also present around other respiratory motoneurons such as phrenic motoneurons. Therefore, these two neurotransmitters may have a more general role in influencing respiratory motor outflow.

Catecholaminergic parasympathetic efferents within the dorsal motor nucleus of the vagus in the rat: a quantitative analysis.

Catecholaminergic vagal motor neurones were identified within the dorsal motor nucleus of the vagus, by retrograde tracing of True blue from the stomach followed by immunocytochemistry using antibodies directed against tyrosine hydroxylase. Presumed dopaminergic efferents were largely confined to caudal regions, where they averaged as much as 30% of the labelled efferents. Most but not all of these were also identified on the basis of acetylcholinesterase histochemistry.

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers.

Calcium efflux was measured in desheathed rabbit vagus nerves loaded with 45Ca2+. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of 45Ca2+ efflux profiles. The 45Ca2+ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min-1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min-1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm-2 sec-1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.

Occurrence of an insulin-like peptide in extracts of human nervous tissue.

Peripheral nerves of the cat such as the vagal and the sciatic nerves have been shown to contain a peptide with insulin-like properties. The aim of the present study was to investigate whether insulin-like immunoreactivity (ILI) can be demonstrated in human nervous tissue collected from autopsy material. Biopsies were taken in connection with autopsy from various peripheral nerves and their content of ILI was investigated. ILI in amounts up to about 12 ng g-1 was found in about 30% of all biopsies taken from peripheral nerves. The ILI coeluted with a bovine insulin standard in an HPLC system indicating that it corresponds to a peptide identical with or similar to pancreatic insulin. Autopsy specimens taken from the sciatic nerve of individuals with diabetes type II or from individuals without established diabetes contained similar amounts of ILI.

Differences in neuronal lipid composition between superior cervical ganglia and nodose ganglia of the rat.

The lipid content and composition of rat superior cervical ganglia containing sympathetic motor neurons and nodose ganglia containing parasympathetic sensory neurons were studied for the first time to elucidate the mechanism of the different effects of exogenous gangliosides on these neurons in the culture medium. The ganglioside content of the superior cervical ganglia was almost 3-times that of the nodose ganglia. Although both ganglia contained GM3, GD3, GD1b and GT1b as major gangliosides, the nodose ganglia additionally contained a significant amount of sialosyllactoneotetraosylceramide LM1 (10% of total sialic acids). Contrasting with nodose ganglia, vagus fiber and dorsal root ganglia of rats, superior cervical ganglia had a higher content of sulfatide than galactosylceramide. The phospholipid content was lower in superior cervical ganglia than in nodose ganglia. Superior cervical ganglia contained less ethanolamine plasmalogen and more phosphatidylcholine than nodose ganglia. Sphingomyelin in superior cervical ganglia contained mainly medium-chain fatty acids, while that in nodose ganglia contained mainly longer-chain fatty acids. Differences in the fatty acid composition of glycerophospholipids were also observed. The results indicate that the properties of neuronal cell membranes from superior cervical ganglia and nodose ganglia are quite different, and that the differences may reflect the physiological roles of these ganglia.

Distribution of muscarinic cholinergic receptors in the dorsal vagal complex and other selected nuclei in the human medulla.

Muscarinic cholinergic receptors were localized in human brainstem by quantitative autoradiography, using the radioligand [3H]quinuclidinyl benzilate. Receptor densities were highest in the hypoglossal nucleus. The second highest density was found in the medial region of the nucleus of the solitary tract (NTS). Moderately high numbers of receptors were present in the dorsal motor nucleus of the vagus, the dorsal NTS, subpostremal NTS, lateral NTS and ventral NTS. Intermediate densities were present in the dorsal and medial accessory nuclei of the inferior olive and the spinal trigeminal nucleus pars interpolaris. Low densities were found in the area postrema, principle nucleus of the inferior olive, gracile nucleus, cuneate nucleus and the tractus of the NTS. Muscarinic cholinergic receptors in the dorsal vagal complex are an important component of the neural substrate governing visceral function. These receptors may be the central site of action of anticholinergic medications in suppressing emesis.

The brain stem localization of vagal preganglionic neurones in the ferret, Mustela putorius furo.

Horseradish peroxidase-wheatgerm agglutinin was injected into the cervical vagus nerve of adult ferrets. Efferent fibres coursing in the vagus nerve were shown to originate in the dorsal vagal motor nucleus (DmnX), the nucleus ambiguus (nA), a region ventral to the nucleus ambiguus (VnA) and the reticular formation between DmnX and nA. The distribution of vagal preganglionic neurones in the ferret is compared with data already obtained in similar studies conducted in other mammals.

Angiotensin receptors and the vagal system.

Angiotensin II (Ang II) is known to attenuate the vagal component of the baroreflex at both central and peripheral cardiac sites. Ang II receptor binding sites occur in both the nucleus of the solitary tract (NTS), where they are associated with vagal afferent terminals, and in the dorsal motor nucleus of vagus. In this study we have examined the distribution of Ang II binding sites in the cell bodies of vagal afferents in the nodose ganglion, and investigated whether these receptors are transported in the vagus nerve. Dense Ang II receptor binding was observed over neuronal cell bodies in the nodose ganglion and, in streaks, in the vagus nerve. Vagal ligation distal to the nodose ganglion resulted in a marked accumulation of receptor binding sites, proximal to the ligature, with a moderate increase on the distal side. These results demonstrate that Ang II receptor binding sites occur in the nodose ganglion and are transported centrally in the vagus to be located on presynaptic terminals in the NTS and also peripherally where they may occur on terminals of the vagus.

Electrophysiological evidence for oxytocin receptors on neurones located in the dorsal motor nucleus of the vagus nerve in the rat brainstem.

Intracellular recordings were obtained from vagal neurones and their response to oxytocin was investigated in slices from the rat brainstem. Following recording, Lucifer Yellow was injected into the cells in order to verify their localization within the dorsal motor nucleus of the vagus nerve. Virtually all neurones throughout the rostro-caudal extent of the nucleus increased their rate of firing in the presence of 10-1000 nM oxytocin and their membrane depolarized in a reversible, concentration-dependent manner. This excitation was probably exerted directly on the impaled cells rather than being synaptically mediated, since it persisted in a low calcium-high magnesium medium or in the presence of tetrodotoxin. These data provide evidence for a direct membrane effect of oxytocin on a defined population of neurones in the rat brain.

Substance P-like immunoreactive fibers in the trigeminal sensory nuclei of the pit viper, Trimeresurus flavoviridis.

Substance P-like immunoreactive nerve fibres were located in the trigeminal sensory system of the infrared-sensitive snake, Trimeresurus flavoviridis, using the immunohistochemical method. There are two trigeminal sensory systems in the medulla of this animal: the descending nucleus and the lateral descending nucleus. The descending nucleus is equivalent to the trigeminal spinal nucleus in other vertebrates, and the lateral descending nucleus is a special trigeminal sensory nucleus belonging to the infrared sensory system. In the present study we determined that the lateral descending nucleus is completely ensheathed by large numbers of substance P-like immunoreactive fibers. The distribution of these fibers seems to be similar to that of the thin vagal unmyelinated fibers, rather than to that of the thick trigeminal myelinated fibers. More substance P-like immunoreactive nerve fibers were observed in the lateral descending tract than in the descending tract. Almost no dense substance P-like immunoreactive fibers were found in these tracts rostral to the lateral descending nucleus or rostral to the subnucleus caudalis of the descending nucleus. The substance P-like immunoreactive fibers in the lateral descending tract extended to those of Lissauer's tract of the spinal cord, and the substance P-like immunoreactive fibers surrounding the Lissauer's tract were similar in appearance to those of the lateral descending nucleus. This nucleus seems to have developed from the elements existing in Lissauer's tract, and also to have a similar modulating function. The primary nucleus of the infrared sensory system is the most substance P-like immunoreactive nucleus in the trigeminal sensory system of this animal. Even in the trigeminal sensory system, substance P-like immunoreactive fibers seem not to be related solely to the nociceptive sensation.

Cholinergic neurons in the rat nodose ganglia.

Presence of acetylcholine (ACh) in the vagal afferent fibres of the rat was investigated. In the nodose ganglion, which contains the cell bodies of this sensitive contingent, a choline acetyltransferase (ChAT) activity, a choline (Ch) uptake and an endogenous content of acetylcholine were detected. These data were confirmed by ChAT immunohistological visualization.

Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurones of the vagal motor nucleus in the adult rat.

Catecholaminergic nerve cell bodies have been recently identified in the rat spinal cord. They lie in the rostral cervical segments and at the lumbosacral junction. Among them, many are located in parasympathetic areas. This finding led us to investigate the interactions between these catecholaminergic neurones and the cholinergic ones. To address this question, we performed sequential immunocytochemical detection of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in the same sections. We could then identify the co-expression of both TH and ChAT-like immunoreactivities (LI) in some perikarya of the cervical spinal cord and medulla oblongata. Such cells are located in the caudal extension of the dorsal motor nucleus of the vagus nerve (DMNX) as well as in the caudal part of the medullary DMNX itself. Such a co-expression of TH-LI and ChAT-LI could not be found in the lumbosacral region, another parasympathetic territory where cell bodies displaying TH-LI were intermingled with those containing ChAT-LI. This is one of the first demonstrations of the co-existence of catecholaminergic and cholinergic phenotypes in some neurones of the adult mammalian nervous system. These observations also support the presence of catecholaminergic efferents within the vagus nerve.

Substance P and somatostatin content and transport in vagus and sciatic nerves of the streptozocin-induced diabetic rat.

Substance P (SP) and somatostatin (SS) are two widely distributed neuropeptides that within the vagus and sciatic nerves are localized predominantly in sensory fibers. The effect of diabetes mellitus on their content or transport in sensory nerves is unknown. With the nerve ligation technique, the peripheral orthograde 24-h transport of both peptides was quantified in the vagus nerve 3 days or 1 mo after induction of streptozocin (STZ) diabetes and in both the vagus and sciatic nerves after diabetes of 3 mo duration. In acute (3-day) diabetics, neuropeptide transport in the vagus was unaltered. After 1 mo, SP transport was significantly increased; content in unligated contralateral nerve was unaltered. Transport of SS was unchanged, and content in contralateral nerve was too low to reliably quantitate. After diabetes of 3-mo duration, transport of both peptides in the vagus nerve was increased in STZ-induced diabetic (STZ-D) rats versus both weight- and age-matched controls: SP 474 +/- 17 (N = 10) vs. 358 +/- 32 (N = 13) pg/24 h, STZ-D rats vs. controls, mean +/- SE, P less than .03; SS 29 +/- 4 vs. 20 +/- 3 pg/24 h, STZ-D rats vs. controls, P less than .02. In the sciatic nerve, SP transport and content were unaltered. SS content was significantly reduced: 17 +/- 3 vs. 30 +/- 3 pg/3-mm nerve segment, STZ-D rats vs. controls, P less than .01. SS transport in the sciatic nerve of diabetic rats was variably reduced (P less than .07), and transport rates were increased (1.41 +/- 0.13 vs. 0.96 +/- 0.10 mm/h.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative distribution and cardiovascular actions of substance P and substance K within the nucleus tractus solitarius of rats.

An investigation has been made into the comparative distribution and cardiovascular actions of Substance P (SP) and Substance K (SK) in the nucleus tractus solitarius (NTS) of rats. The two tachykinins were similarly distributed in the NTS region, as determined by radioimmunoassay of microdissected brain nuclei, although SP was present in an approximately 10 fold higher concentration than SK. Unilateral nodose ganglionectomy resulted in a non-significant reduction of tachykinin immunoreactivity in the ipsilateral NTS. Microinjection of synthetic SP into the NTS of urethane anaesthetised rats did not cause any significant changes in mean arterial pressure or heart rate. In contrast, SK (18 pmol) elicited a significant bradycardia which was maximal at 2 min after the injection. The bradycardic response was blocked in rats pre-treated with atropine. These results extend previous studies in which contrasting functional activities of SP and SK were demonstrated in peripheral systems, to indicate that tachykinins may exhibit different functional roles in the brain. SK, unlike SP, may be involved in central cardiovascular control via an action within the NTS.

A subset of Schwann cells in peripheral nerves contain a 50-kDa protein antigenically related to astrocyte intermediate filaments.

Antisera raised to the astrocyte intermediate filament structural protein stained elements in the peripheral nerves of several species. These elements were not associated with myelinated nerve fibers, were more common in splenic and vagus nerves than in the sciatic nerve, and persisted after nerve transection. In teased nerve preparations antigen-positive cells appeared to be the Schwann cells that surround small diameter, unmyelinated axons. Absorption of the antiserum with purified rat spinal cord 50-kDa protein or with bovine splenic nerve cytoskeletal extract blocked the reaction with CNS astrocyte processes or with PNS nerve fibers. Immunoblots of cytoskeletal preparations of bovine splenic nerve or rat sciatic nerve showed that the antigen from peripheral nerves comigrated at 50 kDa with antigen from bovine or rat spinal cord or cultured rat astrocytes. The CNS and PNS 50-kDa proteins from bovine tissues were subjected to limited digestion with Staphylococcus aureus protease V8. After separation on SDS-gels, antigenic peptides were detected by immunoblotting. The pattern of antigenic peptides for the CNS and PNS proteins were identical. We conclude that Schwann cells associated with nonmyelinated axons contain a cytoskeletal protein that is the same size and has the same peptide map as the major structural protein of astrocyte intermediate filaments.