Polarized distribution of γ interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells)

Summary The fine structure of single identified muscle fibers and their nerve terminals in the limb closer muscle of the shore crab Eriphia spinifrons was examined, using a previous classification based on histochemical evidence which recognizes a slow (Type-I) fiber and three fast (Type-II, Type-III, Type-IV) fibers. All four fiber types have a fine structure characteristic of crustacean slow muscle, with 10–12 thin filaments surrounding each thick filament and sarcomere lengths of 6–13 m. Type-IV fibers have sarcomere lengths of 6 m while the other three types have substantially longer sarcomeres (10–13 m). Structural features of nerve terminals revealed excitatory innervation in all four fiber types but inhibitory innervation in Type-I, Type-II, and Type-III fibers only. Thus fibers with longer sarcomeres receive the inhibitor axon but those with shorter sarcomeres do not. Amongst the former, synaptic contact from an inhibitory nerve terminal onto an excitatory one, denoting presynaptic inhibition, was seen in Type-I and Type-II fibers but not in Type-III and Type-IV fibers. Inhibitory innervation of the walking leg closer muscle is therefore highly differentiated: some fibers lack inhibitory nerve terminals, some possess postsynaptic inhibition, and some possess both postsynaptic and presynaptic inhibition.     

Vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibres in the penile cavernous tissue of green monkeys (Cercopithecus aethiops)

Summary The cavernous body of green monkeys contains many unmyelinated and few myelinated axons. The unmyelinated axons form terminals in the adventitia of the arteries, between trabecular muscle cells, in the interstitium, and close to endothelium cells of the sinuses. All terminals displayed predominantly small clear vesicles and very few large granular vesicles; small granular vesicles were not seen. However, in rabbit penises, terminals with many large granular vesicles are prominent. Immunohistochemistry (PAP technique) showed a dense network of VIP- and NPY-reactive fibres around the arteries and around trabecular muscles. The density of nerve fibres was particularly high around the subendothelial cushions of the helicine arteries. Double staining for NPY and VIP revealed that both peptides were colocalized. Immunocytochemistry (preembedding PAP technique) showed VIP- and NPY-reactivity in terminals with small clear vesicles; the reaction product was bound to the cytoplasmic face of different membrane types. Although the intracellular localization of the reaction product is probably due to artefactual displacement during preparation, the uniformity of the terminals questions the view that large and small granular vesicles in all species characterize peptidergic and noradrenergic terminals, respectively. The essential findings can be summarized as (1) a high degree of uniformity of nerve terminals, (2) colocalization of VIP and NPY, (3) heavy innervation of the subendothelial cushions of the helicine arteries, and (4) possible innervation of endothelial cells.     

An immunohistochemical study of synaptogenesis in the electric organ of Torpedo marmorata by use of antisera to vesicular and presynaptic plasma membrane components

Summary Synaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1–2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled -bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.     

Ultrastructural Identification of gastrin-like immunoreactive nerve fibres in the brain of Xenopus laevis by means of colloidal gold or ferritin immunocytochemical methods

Summary By use of an anti-gastrin serum and colloidal gold- or ferritin-labelled sheep anti-rabbit -globulins, nerve fibres and nerve terminals containing a gastrin-like substance were characterized at the ultrastructural level in the median eminence of Xenopus laevis. These immunoreactive fibres contain neurosecretory granules displaying medium to high electron density and a mean diameter of 75 nm. Labelling intensity varies from granule to granule. This is the first demonstration at the ultrastructural level of the precise location of a gastrin-like hormone in the median eminence of a vertebrate.Supported by the D.G.R.S.T., Contrat no 80.7.0242     

The efferent innervation of the genital chamber by an identified serotonergic neuron in the female cricket Acheta domestica

Summary The serotonergic innervation of the genital chamber of the female cricket, Acheta domestica, has been investigated applying anti-serotonin (5-HT) immunocyto-chemistry at both light- and electron-microscopic levels as well as using conventional electron microscopy. Whole mount and pre-embedding chopper techniques of immuno-cytochemistry reveal a dense 5-HT-immunoreactive network of varicose fibers in the musculature of the genital chamber. All of these immunoreactive fibers originate from the efferent serotonergic neuron projecting through the nerve 8v to the genital chamber (Hustert and Topel 1986; Elekes et al. 1987). At the electron-microscopic level, 5-HT-immunoreactive nerve terminals, which contain small (50–60 nm) and large ( 100 nm) agranular vesicles as well as granular vesicles (100nm), contact the muscle fibers or the sarcoplasmic processes without establishing specialized neuromuscular connections. In addition to the 5-HT-immunoreactive axons, two types of immunonegative axons can also be found in the musculature. By use of conventional electron microscopy, three ultrastructurally distinct types of axon processes can be observed, one of which resembles 5-HT-immunoreactive axons. While the majority of the varicosities do not synapse on the muscle fibers, terminals containing small (50–60 nm) agranular vesicles occasionally form specialized neuromuscular contacts. It is suggested that the 5-HTergic innervation plays a non-synaptic modulatory role in the regulation circular musculature in the genital chamber of the cricket, while the musculature as a whole may be influenced by both synaptic and modulatory mechanisms.Fellow of the Alexander von Humboldt-Stiftung     

5-HT nerve terminals in the fourth ventricle of the rat brain: Their identification and distribution studied by fluorescence histochemistry and electron microscopy

Summary The occurrence and distribution of supra-ependymal nerve terminals storing serotonin (5-HT) are described for the fourth ventricle of the rat brain. The nerve terminals were identified as monoaminergic 1) fluorescence-histochemically, by the presence of a varicose, formaldehyde-induced fluorescence (FIF) on the free surface of the ependyma, 2) electron microscopically, by the presence of electron dense (chromaffin) cores in small (50 nm) and large (100 nm) vesicles found within the varicose regions of supra-ependymal nerve fibres, and 3) by the absence of both the FIF and chromaffin dense cores after treatment with reserpine. Moreover, the serotonergic nature of these nerve fibres could be concluded from 1) the yellow colour of the FIF, 2) the increased FIF after treatment with nialamide or reserpine+nialamide, 3) the diminished FIF and absence of chromaffin dense cores after treatment with p-CPA, and finally 4) the persistence of the FIF and chromaffin dense cores after treatment with -MPT.A high density of 5-HT nerve terminals occurred throughout the floor of the fourth ventricle and on the floor and roof of the lateral recess. Few 5-HT nerve terminals occurred only on the roof of the fourth ventricle (velum medullare, lamina epithelialis of the tela chorioidea), and the surface of the choroid plexus epithelia was devoid of such nerves. Virtually all nerve terminals in the fourth ventricle appear to be serotonergic.Results presented in part at the Autumn Meeting of the British and Italian Pharmacological Societies, Bristol, 1974 (Lorez et al., 1974). The skilful assistance of Mr. R. Wybrecht, Mr. R. Reese and Mrs. M. Gschwind is gratefully acknowledged.     

Ultrastructure of epidermal sensory receptors in Amphibolurus barbatus (Lacertilis: Agamidae)

Summary The histological and ultrastructural organisation of the epidermal sensory organs in Amphibolurus barbatus has been described with respect to their position and possible functions. The sensory organs, located at the scale's edge, are most numerous in scales of the dorsal surface of the head. Most other scales of the body surface have two receptors located laterally to the spine or keel of the scale. In the imbricate scales of the ventral body region, the receptors lie just beneath the reinforced scale lip. Scanning electron microscopy has revealed the surface of the organ to be a crater lacking any surface projections. These sensory organs have a dermal papilla consisting of a nerve plexus and loose connective tissue. The nerve fibres arising from the plexus, pass to the epidermal columnar cells, where some form nerve terminals at the base of the cells, while others pass between them to form nerve terminals embedded in a superficial layer of cuboidal cells. The superficial terminals are held against the overlying keratin by masses of tonofilaments. The keratin is thickened to form a collar around the periphery of the organ but is only about 0.5 m thick immediately above it. Mechanical deformation of the scale's spine or reinforced scale lip may initiate stimulation of the nerve terminals described.     

Catecholaminergic nerves in the embryonic chick ovary: co-localization with β2-adrenoceptor-bearing steroidogenic cells

Summary The present study investigates the innervation of the embryonic chick ovary with regard to (i) development and compartmentalization of catecholaminergic nerves, and (ii) presence of adrenoceptors on steroidogenic target cells of catecholaminergic nerve terminals. Catecholaminergic nerve fibers visualized by glyoxylic acid-induced histofluorescence first appeared at embryonic day (E) 13. From E15 through E21 the density of fluorescent aminergic nerves increased markedly in parallel with the concentration of catecholamines and numbers of nerve bundles and single axons seen at the electron-microscopic level. Catecholaminergic nerves were confined to the ovarian medulla and closely associated with interstitial cells. Nerve terminals approached interstitial cells up to a distance of 20 nm and, in their majority, exhibited uptake of the false adrenergic transmitter 5-hydroxydopamine. Although adrenaline amounted to 14% of the total catecholamine content at E21, adrenaline immunoreactivity was only detected in adrenal chromaffin cells, but not in nerve fibers or cell bodies within the ovary. Interstitial cells structurally matured between E15 and E21 as documented by an increase of smooth endoplasmic reticulum and tubular mitochondria. Monoclonal antibodies mAB 120 and BRK 2 raised against avian ₁ and mammalian ₂-adrenergic receptors revealed the presence of ₂-adrenoceptor-like immunoreactivity on the surface of interstitial cells, but not on any other cell type.The results are consistent with the notion of a dense adrenergic innervation of the embryonic chick ovarian medulla and its steroidogenic interstitial cells, and suggest the chick ovary as an excellent model for elucidating the functional role of a neural input to steroidogenic cells during development.     

Observations on the cellular localization of dopamine in the caudate nucleus of the rat

Summary The cellular localization of dopamine in the caudate nucleus of the rat hat been studied with the highly sensitive and specific fluorescence method of Falck and Hillarp, and by electron microscopy. The histochemical studies provided strong support for the view that the dopamine is concentrated within very fine nerve fibres which have abundant varicosities with an intense fluorescence. The electron microscopical studies revealed the presence of a tightly packed plexus built up i.a. of abundant synaptic nerve terminals, many of which had a diameter below 0.4 . The terminals made synaptic contact mainly with processes that seemed to belong to an extensive dendrite net.The investigation was supported by research grants from the United States Public Health Service (02854-04), The Swedish Medical Research Council and the Knut and Alice Wallenberg Foundation.     

Structure and ultrastructure of the frog motor endplate

Summary The frog motor endplate in its simplest form consists of an elongated, slender nerve ending embedded in a gutter-like depression of the sarcolemma. This nerve terminal contains the usual synaptic organelles. It is covered by a thin coating of Schwann cell cytoplasm which embraces the terminal with thin finger-like processes from both sides, thereby sub-dividing it into 300–1000 regularly spaced compartments. The individual synaptic compartments correspond to the strings of varicosities or grape-like configurations of motor nerve terminals in endplates of other species and in the cerebral neuropil of vertebrates.Each compartment contains one or more bar-like densities of the presynaptic membrane, active zones, which are associated with the attachment sites between synaptic vesicles and plasmalemma. Active zones have a regular transverse arrangement and occur at specific loci opposite the junctional folds. The attachment sites for synaptic vesicles are at the edges of the bars which are bilaterally delineated by a double row of 10 nm particles attached to the A-face. The structural appearance of vesicle attachment sites in freeze-etch replicas corresponds to that of micropinocytosis. The active zones are often fragmented and the frequency of their association with vesicle attachment sites is highly variable.The junctional folds are characterized by specific sites in which intramembranous particle aggregations occur at relatively high packing density (7500/m²). These sites are located opposite the active zones at the juxtaneural lips, a location where one would expect ACh-sensitive receptors on the postsynaptic membrane.This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 38, Projekt N), The Swiss National Foundation for Scientific Research (Grants Nr. 3 82372 and 3 77472) and the Dr. Eric Slack-Gyr Foundation Zürich.     

Morphology and central synaptic connections of the efferent neurons innervating the crayfish hindgut

Summary The distribution, morphology and synaptic connections of the hindgut efferent neurons in the last (sixth) abdominal ganglion of the crayfish, Orconectes limosus, have been investigated using light and electron microscopy in conjunction with retrograde cobalt/nickel and HRP labeling through the intestinal nerve. The hindgut efferent neurons occur singly and in clusters, and are unipolar. Their axonal projections are uniform and consist of a thick primary neurite with typical lateral projections and limited arborization of varicose fibers in the ganglionic neuropil. They also send lower order axon processes to the ganglionic neural sheath, where they arborize profusely, forming a network of varicose fibers. The majority of the efferent neurons project to the anterior part of the hindgut. HRP-labeled axon profiles are found in both pre- and postsynaptic position in the neuropil of the ganglion. HRP-labeled axon profiles also establish pre- and postsynaptic contacts in the intestinal nerve root. All hindgut efferent terminals contain similar synaptic vesicle populations: ovoid agranular vesicles (50–60 nm) and a few large granular vesicles (100–200 nm). It is suggested that the hindgut efferent neurons in the last abdominal ganglion are involved in: (1) innervation of the hindgut; (2) central integrative processes; (3) en route synaptic modification of efferent and afferent signals in the intestinal nerve; (4) neurohumoral modulation of peripheral physiological processes.Fellow of the Alexander von Humboldt Stiftung     

The connection between spinal cord and notochord in Amphioxus (Branchiostoma lanceolatum)

Summary Silver impregnation of nerves, histochemical reactions for acetylcholinesterase, and electron microscopy reveal an efferent innervation of the notochord in amphioxus. Extensions of the notochordal lamellae end in groups (the notochordal horns) just below the ventro-lateral surface of the spinal cord where they are opposed to large nerve terminals originating as short collaterals of axons running longitudinally in the nerve cord. This neurochordal junction resembles an ordinary neuro-muscular junction in several respects and is interpreted as a part of the muscular system found in the notochord itself. Acknowledgement. The author is indebted to the staffs at the Marine laboratory in Plymouth and the Biological station at Helgoland for supply of material. The expert advices and criticism of Q. Bone, Ph. D., Plymouth, and Dr. U. Welsch, Kiel, are also greatly appreciated.     

Substance P-like immunoreactivity in the parietal eye visual system of the lizard Uta stansburiana

Summary We examined the parietal eye visual system of the iguanid lizard Uta stansburiana for the presence of substance P-like immunoreactivity by use of both immunofluorescence and peroxidase-antiperoxidase techniques. In the parietal eye no substance P-containing somata were found; however, its plexiform layer contained small (ca. 1 m diam) immunoreactive fibers. These fibers apparently originate outside the parietal eye. Immunoreactive fibers also were found in the parietal nerve, the dorsal sac, and the leptomeninx of the pineal gland. No labeled somata were observed in any of these regions in either normal or colchicine treated animals. Previously we demonstrated that a system of centrifugal fibers to the parietal eye originates from neurons in the dorsal sac (Engbretson et al. 1981). The apparent absence of substance P-containing neurons in the dorsal sac suggests that the substance P-containing fibers in the parietal eye are not the previously observed centrifugal fibers. The source of the substance P-containing fibers in the parietal eye is unknown. The pars dorsolateralis of the left medial habenular nucleus receives a dense substance P-positive projection. No such projection was seen in the right habenula. Simultaneous visualization of the terminals of ganglion cells of the parietal eye (labeled with orthograde intraaxonally transported horseradish peroxidase) and substance P-like immunofluorescence showed that the locus of habenular immunoreactivity is distinct from the projection field of the parietal eye. Thus the substance P-positive terminals in the habenula do not originate in the parietal eye. Transection of the parietal nerve confirmed this conclusion.     

Effects of ouabain and electrical stimulation on the fine structure of nerve endings in the electric organ of Torpedo marmorata

Summary The cycle of synaptic vesicles was studied in isolated nerve terminals and in the electric tissue of Torpedo marmorata. The synaptosomes, as used in this investigation, were a pure cholinergic subcellular fraction that captured dextran particles as an extracellular marker. This endocytotic phenomenon was enhanced by potassium depolarization. Field electrical stimulation (1 Hz and 10 Hz) of the electric organ induced the appearance of membrane foldings into presynaptic terminals. Morphometric studies showed that the number of synaptic vesicles did not decline until after at least 30 min. On the other hand, at 10 Hz these changes were accompanied by an increase in length of the membrane of the terminal. At 15 min of recovery after prolonged stimulation, there was a great increase in density of synaptic vesicles with a large number of vesicles of small diameter. This increase was accompanied by a decrease of membrane length, suggesting that reformation of vesicles is related to retrieval of membrane. Pharmacological stimulation with ouabain produced changes similar to those of long-term electrical stimulation. These changes in membrane were accompanied by a decrease of the population of synaptic vesicles and a wide variation in their diameters. It is concluded that structural changes reported here could not be correlated with kinetics of the transmitter release.We are grateful to Dr. E. Cañadas, Prof. Dr. D. Ribas and Dr. J. Tomás for valuable help and encouragement. We are indebted to Dr. P. Arté and to the staff of the Acuario de Barcelona del Instituto de Investigaciones Pesqueras for providing specimens of Torpedo marmorata. This investigation was supported by a grant Formación Personal Investigador del Ministerio de Universidades e Investigación     

Synaptic organization in the lateral geniculate nucleus of the monkey

Summary The synaptic organization in the lateral geniculate nucleus of the monkey has been studied by electron microscopy.The axon terminals in the lateral geniculate nucleus can be identified by the synaptic vesicles that they contain and by the specialized contacts that they make with adjacent neural processes. Two types of axon terminal have been recognized. The first type is relatively large (from 3–20 ) and contains relatively pale mitochondria, a great many vesicles and, in normal material, a small bundle of neurofilaments. These terminals have been called LP terminals. The second type is smaller (1–3 ), contains darker mitochondria, synaptic vesicles, and no neurofilaments. These have been called SD terminals.Both types of terminal make specialized axo-somatic and axo-dendritic synaptic contacts, but the axo-somatic contacts are relatively rare. In addition the LP terminals frequently make specialized contacts with the SD terminals, that is, axo-axonal contacts, and at these contacts the asymmetry of the membranes is such that the LP terminal must be regarded as pre-synaptic to the SD terminal.The majority of the synaptic contacts are identical to those that have been described previously (Gray, 1959 and 1963a) but, in addition, a new type of contact has been found. This is characterized by neurofilaments that lie close to the post-synaptic membrane, and by an irregular post-synaptic thickening. Such filamentous contacts have been found only where an LP terminal contacts a dendrite or a soma.The degeneration that follows removal of one eye demonstrates that the LP terminals are terminals of optic nerve fibres. The origin of the SD terminals is not known.The glial cells often form thin lamellae around the neural processes and tend to isolate synaptic complexes. These lamellae occasionally show a complex concentric organization similar to that of myelin.It is a pleasure to thank Prof. J. Z. Young for advice and encouragement and Dr. E. G. Gray for the considerable help he has given us. Dr. J. L. de C. Downer gave us much help with the care of the animals and with the operations. We also wish to thank Mr. K. Watkins for technical assistance and Mr. S. Waterman for the photography.     

Ultrastructure and distribution of somatostatin-like immunoreactive neurons and nerve fibres in the coeliac ganglion of cats

Summary Somatostatin-like immunoreactivity was localized in nerve cell bodies and nerve terminals in the cat coeliac ganglion. Two types of somatostatin-immunoreactive cell bodies were revealed, the first being large (diameter 35 m), numerous and weakly labelled, where—as the second was considerably smaller (diameter 10.4 m), sparsely distributed and heavily stained. The immunoreactive nerve terminals were in synaptic contact with many immunonegative large neurons and dendrites. However, in a few cases, somatostatin-immunoreactive nerve terminals could also be observed on the surface of lightly stained neurons. Transection of vagal or mesenteric nerve failed to affect the distribution or density of somatostatin-like immunoreactive nerve terminals. These results demonstrate the existence of a synaptic input to the principal neurons of the coeliac ganglion of the cat by somatostatin-containing nerve terminals and suggest that this peptide may act as a neuromodulator or neurotransmitter. It is proposed that somatostatin-positive neurons provide intrinsic projections to other somatostatin-positive and to somatostatin-negative neurons throughout the coeliac ganglion, thereby creating a complex interneuronal system.     

Intracisternal neurotoxins and monoamine neurons innervating the spinal cord: Acute and chronic effects on cell and axon counts and nerve terminal densities

Summary The total number of catecholamine (CA) and 5-hydroxy-trydroxy-tryptamine (5-HT) containing nerve cell bodies in groups A1-7 and B1-3 were counted using Falck-Hillarp fluorescence histochemistry and found to be about 5000 and about 6300 respectively. The total number of CA axons in the white matter of the spinal cord was found to be about 4300 at the cervical level and about 3300 at the lumbar level as revealed in cryostat sections. Together with previous studies our quantitative results suggest that individual cell bodies in locus coeruleus send axon collaterals to the spinal cord, cortex cerebri, cortex cerebelli and probably also other areas of the brain.The decrease in number of CA axons in spinal cord occurred approximately at the level where the CA nerve terminal innervation of the sympathetic lateral column ends. Single CA axons were observed to give off multiple branches at right angles, suggesting that single CA axons innervate several different levels of the grey matter.A transient loss of visible CA cell bodies in groups A1-7 was observed after intracisternal injection of 25–50 g 6-hydroxydopamine (6-OH-DA). A permanent loss of visible 5-HT cell bodies in groups B1-3 was observed after 25 g 5,6-dihydroxy-tryptamine (5,6-HT) given intracisternally, a transient decrease was obtained when the same dose of 5,6-HT was given intraventricularly.CA nerve terminals regenerated in a cranio-caudal direction along the motoneuron columns after lesioning with 6-OH-DA given intracisternally. The reappearing CA nerve terminals seemed to outline the longitudinally oriented dendritic bundles of the motoneurons and was exclusively found in these areas of the ventral horn. Thus, no substantial CA nerve terminal reinnervation of zona intermedia or the dorsal horn was observed. About 10% of the CA axons running in white matter were left after intracisternal 6-OH-DA and found at all levels of the cord. The number of remaining CA axons did not increase with time.After 10–50 g 5,6-HT given intracisternally a return of 5-HT nerve terminals was only observed in cervical segments. There was a 60–90% decrease in number of axons that accumulated 5-HT after an acute transverse lesion and no recovery of the number of such accumulations was observed during the following six months.It was concluded that regeneration of CA nerve terminals in the spinal cord can occur after lesioning with 6-OH-DA given intracisternally. Regeneration of 5-HT nerve terminals following intracisternal 5,6-HT is very restricted probably because of the permanent loss of 5-HT cell bodies obtained by the intracisternal route of administration as opposed to the intraventricular route. It was suggested that the reappearing CA nerve terminals were derived from locus coeruleus and that they were specifically guided by the motoneuron columns.     

The release of adenosine at the electric organ ofTorpedo. A study using a continuous chemiluminescent method

Acetylcholine and ATP are costored and coreleased during synaptic activity at the electric organ ofTorpedo. It has been suggested that released ATP is converted to adenosine at the synaptic cleft, and in turn this nucleoside would depress the evoked release of acetylcholine. In the present communication we have used a chemiluminescent reaction that let us to monitor continuously the presence of adenosine in this preparation. The chemiluminescent reaction is based on the conversion of adenosine into uric acid and H₂O₂ by adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzymes. The hydrogen peroxide has been detected by peroxidase-luminol mixture. The reaction has a sensitivity on the picomol range and discerned between Adenosine, AMP, ADP, and ATP. We have developed this technique in the hope of understanding whether adenosine is released during synaptic activity or it comes from the released ATP. We have studied the release or formation of adenosine in fragments of the electric organ and in isolated cholinergic nerve terminals obtained from it. In both conditions we have followed the effect of potassium stimulation upon the detection of adenosine. Potassium stimulation increased the extracellular adenosine either in slices or the synaptosomal fraction ofTorpedo electric organ. The presence of , -methylene ADP, an inhibitor of 5-nucleotidase, inhibits the detection of adenosine, suggesting that extracellular adenosine is a consequence of ectocellular dephosphorylation of released ATP.     

Do vasoactive intestinal peptide (VIP)-and nitric oxide synthase-immunoreactive terminals synapse exclusively with VIP cell bodies in the submucous plexus of the guinea-pig ileum?

In the submucous plexus of the guinea-pig ileum, previous light-microscopic studies have revealed that vasoactive intestinal peptide (VIP)-immunoreactive and nitric oxide synthase (NOS)-immunoreactive terminals are found predominantly in association with VIP-immunoreactive nerve cell bodies. In this study, double-label immunohistochemistry at the light-microscopic level demonstrated co-localization of NOS-immunoreactivity and VIP-immunoreactivity in axon terminals in submucous ganglia. About 90% of nerve fibres with NOS-immunoreactivity or VIP-immunoreactivity were immunoreactive for both antigens; only about 10% of labelled varicosities contained only NOS-immunoreactivity or VIP-immunoreactivity. The VIP/NOS varicosities were more often seen in the central parts of the ganglia, close to the VIP-immunoreactive cell bodies. Ultrastructural immunocytochemistry with antibodies to VIP was used to determine if NOS/VIP terminals synapse exclusively with VIP-immunoreactive nerve cell bodies. We examined the targets of VIP-immunoreactive boutons in two submucous ganglia from different animals. Serial ultrathin sections were taken through the ganglia after they had been processed for VIP immunocytochemistry. For each cell body, the number of VIP inputs (synapses and close contacts) was determined. The number of VIP-immunoreactive synapses received by the cell bodies of submucous neurons varied from 0–4 and the number of VIP-immunoreactive close contacts varied from 3–10. There was no significant difference between VIP-immunoreactive nerve cell bodies and non-VIP nerve cell bodies in the number of VIP-immunoreactive synapses and close contacts they received. Thus, the implication from light microscopy that NOS/VIP terminals end predominantly on VIP nerve cells was not vindicated by electron microscopy.Abbreviations CCK Cholecystokinin - cGMP guanosine-3, 5-cyclic monophosphate - CGRP calcitonin gene-related peptide - ChAT choline acetyltransferase - DYN dynorphin - GAL galanin - GTP guanosine triphosphate - IR immunoreactive(ivity) - NO nitric oxide - NOS nitric oxide synthase - NMU neuromedin U - NPY neuropeptide Y - SOM somatostatin - SP substance P - VIP vasoactive intestinal peptide     

Effects of stimulation and rest on the ultrastructure of the excitatory neuromuscular junctions of Locusta migratoria L.

Summary The terminals of the fast axon on extensor tibiae muscle fibres of Locusta were examined in untreated nerve-muscle preparations and in preparations stimulated electrically at frequencies varying from 0.5 to 100 Hz. The ultrastructure of the terminals in preparations stimulated at the lower range of these frequencies, which induce twitch contractions of the muscles, is similar to that of the controls. Stimulation at the higher frequencies induced tetanic muscle responses and rapid fatigue of the muscles after which they would not respond again to high frequency stimulation for about 1 h. This loss and recovery of the responses of the muscles is correlated with changes in the ultrastructural appearance of the terminals, in particular in the number and shape of the synaptic vesicles. The ultrastructure of these recovering axon terminals closely resembles that of the controls.R.P. Botham gratefully acknowledges the SRC for financial assistance