Immunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum

Summary The localization of serotonin (5-HT)-immunoreactive nerve fibers in the cerebellum of the rat and cat was investigated by means of the peroxidase-anti-peroxidase (PAP) method using highly specific antibodies to 5-HT.Serotonin-containing nerve fibers were distributed throughout the entire cerebellum including the deep cerebellar nuclei, while 5-HT-positive neuronal somata were not detected in the cerebellum of either species. A different pattern of 5-HT innervation was found among the three layers of the cerebellar cortex. There were also interspecific differences in the pattern of distribution of 5-HT. In the rat, the pool of 5-HT nerve fibers mainly consisted of tangential elements, which were predominant in the molecular layer, while in the cat only a few 5-HT fibers were found in the molecular layer of the cerebellar cortex; dense networks of 5-HT nerve fibers were present in the granular layer. Some differences are evident in the pattern of distribution of 5-HT fibers in cerebellar regions classified on an anatomical and functional basis.This work was supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the hypothalamus of the monkey,Macaca fuscata

Summary The distributional pattern of serotonin-containing nerve fibers in the hypothalamus of the monkey (Macaca fuscata) was analyzed with the use of the peroxidaseantiperoxidase method in conjunction with a highly sensitive and specific anti-serotonin serum. The highest concentrations of serotonin-immunoreactive varicose fibers were found in the nucleus praeopticus medialis, nucleus ventromedialis hypothalami, and the complex of mammillary nuclei (nucleus praemamillaris, supramamillaris, mamillaris medialis et lateralis). However, the nucleus suprachiasmaticus, where numerous serotoninergic fibers have been reported to occur in the rat, appeared to be almost devoid of these fibers. The infundibular stalk, and the intermediate and posterior lobes of the pituitary contained considerable numbers of immunoreactive fibers. The present study provides a morphological basis for possible clarification of the influence of serotoninergic projections on various neuroendocrine mechanisms in primates. Furthermore, an attempt was made to clarify the differences and similarities concerning the distributional patterns of serotoninergic nerve fibers within the monkey hypothalamus in contrast to the rat hypothalamus.Supported by grants (No. 56440022, 57214028) from the Ministry of Education, Science and Culture, Japan     

Immunohistochemical demonstration of serotonin nerve fibers in the cat neurohypophysis

Summary Distribution of the serotonin nerve fibers in the neurohypophysis of adult cats was demonstrated using the peroxidase-antiperoxidase method. The serotonin nerve fibers were distributed in the internal and external layers of the infundibulum, terminating on the wall of the capillary loops and the outer surface of the external layer. A small number of the fibers scattered about in the posterior lobe penetrated the intercellular spaces in the intermediate lobe.Supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, JapanTo whom offprint requests should be sent     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag1 fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag2 fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag1 fibers.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Summary Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag₁ fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag₂ fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag₁ fibers.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Immunohistochemical demonstration of serotonergic and peptidergic nerve fibers in the subcommissural organ of the dog

Summary The distributional patterns of serotonin-, luteinizing hormone-releasing hormone (LHRH)-, oxytocin (OXT)- and vasopressin (VP)-immunoreactive nerve fibers were studied in the subcommissural organ (SCO) of the dog by use of the peroxidase-antiperoxidase technique.Abundant serotonergic and moderate numbers of peptidergic nerve fibers running toward the ventricular surface were observed among the cylindrical ependymal cells in the SCO of the dog. Concerning the distributional density of the peptidergic nerve fibers, VP-immunoreactive fibers displayed the highest and LHRH-immunoreactive fibers the lowest values. Most serotonergic and peptidergic fibers returned to the basal portion of the SCO after forming loops immediately beneath the ventricular surface of the ependymal layer. Serotonin-immunoreactive fibers often established a perivascular plexus around the blood vessels in the SCO.At the electron-microscopic level, after use of antiserum to serotonin dark immunoprecipitate was observed in large granular vesicles and the matrix surrounding small and large, clear vesicles and mitochondria; VP immunoreactivity was localized in the large granular vesicles.Serotonergic nerve fibers could be detected in the SCO of the newborn dog. Although the distributional density was in principle not different from that in the adult animal, individual fibers showed immature features such as growth cones and insufficiently swollen varicosities. After penetrating into the ventricle, in the newborn dog, a few serotonin-immunoreactive fibers ran for a relatively long distance on the ependymal surface.     

Immunohistochemical demonstration of serotonin nerve fibers in the olfactory bulb of the rat,cat and monkey

Summary The distribution of serotonin (5-HT) positive fibers in the olfactory bulb of the rat, cat and monkey was studied using the peroxidase-anti-peroxidase (PAP) immunohistochemical method and highly specific antibodies to 5-HT. In general, 5-HT fibers were present throughout all layers in the olfactory bulb of these species except for the olfactory nerve layer and different as well as restricted laminar patterns of 5-HT distribution were observed. There were also species-related differences in the pattern of 5-HT distribution, in each layer. The most notable species difference was apparent in the glomerular layer of the main olfactory bulb. In case of the rat and cat, a very dense plexus of 5-HT fibers was observed to be diffuse in the glomerulus, while in the monkey, the distribution of 5-HT fibers was scanty and partial, as was seen in the accessory olfactory bulb of the rat.This work was supported by grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

Immunohistochemical demonstration of corticotropin releasing factor containing nerve fibers in the median eminence of the rat and monkey

Summary By means of the peroxidase-antiperoxidase technique, in conjunction with a specific anti-CRF serum, it was shown that a large number of immunoreactive nerve fibers were demonstrable on the capillary loops of the hypophysial portal vessels in the external layer of the median eminence of the rat and monkey, particularly in its medial part. This result confirms the radioimmunological determination of CRF immunoreactivity in the median eminence.Supported by a grant (No. 56440022) from the Ministry of Education, Science, and Culture, Japan     

Immunohistochemical demonstration of calbindin-containing nerve endings in the rat esophagus

Immunoreactivity for calbindin was found in nerve endings with irregular laminar shapes in the rat esophagus. In the myenteric ganglia, laminar endings of a range of sizes formed a complex network and appeared to lie at the surface of the ganglion. The myenteric ganglia that contained nerve endings were most abundant in the upper portion of the eosphagus, their number decreasing orally to anally. Calbindin-immunoreactive nerve cell bodies were scattered throughout the esophagus. Laminar terminals were found in the connective tissue of the lamina propria immediately beneath the epithelium and in the muscularis mucosae. Occasional nerve branches formed a network of aborizing endings that surrounded part of the submucosal arterioles. Immunoreactive nerve endings in the mucosa and submucosa were present only in the upper part of the cervical esophagus. Unilateral vagotomy caused a remarkable decrease in the number of the myenteric ganglia containing the calbindin-immunoreactive laminar endings after 15 days or survival; in some of ganglia, the laminar structures disappeared and nerve endings showing weak immunoreactivity had an indistinct appearance, so that the outline of the ganglia became obscure. In operated rats at 24 days, the number of innervated ganglia was about half that in normal rats. However, there was no change in the morphology and the occurrence of the immunoreactive laminar structures in the mucosa and submucosa after denervation. The results show that many of the laminar endings that are immunoreactive for calbindin in the myenteric ganglia are derived from the vagus nerve. Thus, the calbindin-immunoreactive nerve endings with laminar expansions that are found in the rat eosphageal wall could be sensory receptors.     

Myelin movements in mature mammalian peripheral nerve fibers

Mature mouse and cat peripheral nerve fibers have been examined in vitro by time-lapse photography. Some Schmidt-Lanterman clefts which were open at the start closed later; other were seen to open and then to close, some of them more than once. The implications of these movements are considered, especially in regard to the question of the passage of materials from the endoneurial connective tissue spaces to the axon. Myelin movements other than those occurring at the Schmidt-Lanterman clefts consisted primarily of the development and frequent regression of indentations of the myelin sheath. A single evagination was seen to develop and then to recede. These myelin movements suggest that previously described invaginations and evaginations of the myelin sheath, including flaps of “redundant myelin”, are not static but rather that they are in a state of movement, forming and regressing at intervals. The possible functional significance of the development and regression of myelin sheath indentations in relationship to axoplasmic flow is discussed.     

Role of potassium in the phosphate efflux from mammalian nerve fibers

Summary The efflux of phosphate was measured in rabbit vagus nerve loaded with radiophosphate. The efflux was found to depend on the K concentration of the bathing solutions; increasing the K from 5.6 up to 150mm produced a maximal lowering of 28%; K-free solution produced a transient increase whose peak was 86% above the normal efflux. In the presence of Na, the K-free effect could be repeated; in Na-free solution, it was found only for the first application of the K-free solution. The phosphate efflux was not altered when K was replaced by Rb; replacement with Cs showed that this ion only partially mimics the effect of K.The results suggest that the transient increase in phosphate efflux is due to release of label from a K-dependent saturable binding site, which is distinct from the main intracellular pool. The binding site appears to be labeled from the inside by the Na-dependent phosphate efflux previously described. It may correspond to the phosphorylation of membrane phospholipids. A mathematical model of this system is developed and curves simulated by an analog computer are compared to the experimental results.Measurements of the membrane potential and the internal inorganic phosphate showed that the effect of K on the phosphate efflux could not be explained by changes in the membrane potential or in the internal phosphate pool.     

Immunohistochemical study of neuropeptide Y-containing nerve fibers in the human clitoris and penis

The occurrence and distribution of neuropeptide Y in the human clitoris and penis was investigated by light immunohistochemistry. Neuropeptide Y-containing nerve fibers were detected in the tunicae of arteries and veins as well as among trabecular smooth muscle. The distribution pattern of the peptide was similar in both organs although a higher density of immunoreactive nerve fibers was detected in the penis. The immunolocalization of neuropeptide Y was also compared with that of neuron-specific enolase, a neuronal marker which labels the entire nerve network. It is suggested that neuropeptide Y is involved in the physiology of the penis and the clitoris, affecting vascular and nonvascular smooth muscle activity.     

Cytochemical demonstration of nucleoside phosphatase activity in myelinated nerve fibers of the rat

Summary The histochemical and cytochemical localization of end product from the Wachstein-Meisel reaction was examined in transverse frozen sections of rat ischiatic nerve and spinal nerve roots. A variety of fixatives, substrates, and inhibitors were used at varying pH. Following glutaraldehyde fixation, nucleoside phosphatase activity was noted in the axon-Schwann cell interface of both unmyelinated and myelinated nerve fibers. The presence of this enzymatic activity at this strategic location in the nerve fiber complex attests to the importance of this space as a metabolically active site.Additional deposits of end product occurred on neurofilaments of myelinated and unmyelinated axons and were observed as diffuse axonal stains by light microscopy. These precipitates had a predilection for tissue fixed in formalin or hydroxyadipaldehyde and were relatively more prominent following acidic incubations. The resemblance between these deposits and spurious axonal acid phosphatase precipitates was discussed.This study was supported by Grant NB 04161 of the National Institute of Neurological Disease and Blindness.     

Involvement of intracellular calcium in the phosphate efflux from mammalian nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from desheathed rabbit vagus nerves after loading with radiophosphate. The effects of strategies designed to increase intracellular calcium were investigated. At the same time, the exchangeable calcium content was measured using⁴⁵Ca. Application of calcium ionophore A23187 increased phosphate efflux in the presence of external calcium in parallel with an increase in calcium content. In the absence of external calcium, there was only a late, small increase in phosphate efflux. For nerves already treated with the calcium ionophore, the phosphate efflux was sensitive to small changes in external calcium, in the range 0.2 to 2mm calcium, whereas similar increases in calcium in absence of ionophore gave much smaller increases in phosphate efflux. Removal of external sodium (choline substitution) produced an initial increase in phosphate efflux followed by a fall. The initial increase in phosphate efflux was much larger in the presence of calcium, than in its absence. The difference was again paralleled by an increase in calcium content of the preparation, thought to be due to inhibition of Na/Ca exchange by removal of external sodium. Measurements of ATP content and ATP, ADP, phosphate and creatine phosphate ratios did not indicate significant metabolic changes when the calcium content was increased. Stimulation of phosphate efflux by an increase in intracellular calcium may be due to stimulation of phospholipid metabolism. Alternatively, it is suggested that stimulation of phosphate efflux is associated with the stimulation of calcium efflux, possibly by cotransport of calcium and phosphate.     

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers

Summary Calcium efflux was measured in desheathed rabbit vagus nerves loaded with⁴⁵Ca²⁺. 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 of⁴⁵Ca²⁺ efflux profiles. The⁴⁵Ca²⁺ 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.Deceased 18 April 1988