Dense-core vesicles and non-synaptic exocytosis in the central body of the crayfish brain

Summary The central body in the median protocerebrum of the brain of the crayfish Cherax destructor is a distinctive area of dense neuropile, the nerve fibres of which contain three main types of vesicles: electronlucent vesicles (diameter 35 nm), dense-core vesicles (diameter 64 nm), and large structured dense-core vesicles (diameter 98 nm, maximum 170 nm). Different vesicle types were found together in the same neurons. Electronlucent vesicles were seen at presynaptic sites and rarely observed in the state of exocytosis. Exocytosis of densecore and structured dense-core vesicles was a regular feature on non-synaptic release sites either close to, or at some distance from pre- and subsynaptic sites. Non-synaptic exocytotic sites are more often observed than chemical synapses. Different forms of exocytosis seen at non-synaptic sites included the release of single densecore vesicles, packets of dense-core vesicles, and rows of dense-core vesicles lined up along cell membranes and around fibre invaginations. Swelling and the enhanced electron density of extracellular non-synaptic spaces may mark the positions of prior exocytotic events. In vitro treatment of the brain with tannic acid buffer solution followed by conventional double fixation resulted in the augmentation of non-synaptic exocytosis. Electron microscopy of proctolin- and serotonin-immunoreactive nerve fibres shows them to contain dense-core and electron-lucent vesicles and to be surrounded by many unlabelled profiles similarly laden with dense-core vesicles and electron-lucent vesicles, indicating the presence of other, not yet identified, neuroactive compounds.     

The fine structure of the central synaptic contacts on an identified crustacean motoneuron

Summary Small nerve terminals in the neuropile of the brain of the crab Scylla serrata make close contact with the secondary, tertiary and higher order central branches of the reflex eye-withdrawal motoneurons. Most contacts have the characteristics of chemically transmitting synapses in that the presynaptic terminals contain agranular vesicles of 25 to 50 nm in diameter and are separated from the motoneuron by a synaptic cleft of about 16 nm. Some terminals contain synaptic ribbons, others contain a mixture of larger (50 to 80 nm) agranular and also dense cored vesicles. In addition large blunt-ended contacts unaccompanied by vesicles, occur between neurons in the neuropile and the motoneuron. It is suggested that the absence of synaptic contacts over the large primary branches of the motoneuron could explain previous physiological findings that little or no resistance changes can be detected in this part of the neuron during excitation or inhibition.We thank Mrs. Joan Goodrum for the preparation of Fig. 1.     

Zur Innervation des glatten Penisretraktormuskels von Helix pomatia: Allgemeine Histologie und Histochemie des monoaminergen Nervensystems

Summary The penis retractor muscle of Helix pomatia is passed by thick nerve trunks, which probably are nerve nets. In the contracted muscle, they are folded meanderlike, in the extended they are pulled smooth. Four types of vesicles different in size and contents can be distinguished in the nerves.Varicose fibres accompany the muscle cells. Frequently the terminals are running in a groove of the muscle fibre. In certain axon types occur presynaptic accumulations of vesicles and thickenings of the terminal membrane. Terminal and muscle cell are separated by a cleft of 300 AE width. The muscle fibre membrane has no subsynaptic infoldings. Beside these axons thin, naked neurites are running through the connective tissue. They are characterized by a high content of neurotubuli.One part of the axons presumably possesses a monoaminergic transmitter. After the glutaraldehyde-dichromate-reaction they contain dense grana, whose diameters are mainly below 1000 AE. The nerve trunks fluoresce after exposure to paraformaldehyde vapour, excited with UV-light, green to green-yellow. The maximum of the excitation was determined at 413 nm and the maximum of emission at 496 respectively at 510 nm. It is concluded, that both, a catecholamine and 5-HT are responsible for the fluorescence. Extraction and paperchromatographic separation lead to the opinion, that the catecholamine is dopamine.

Auszug aus der Dissertation Histologische und histochemische Untersuchungen zur Innervation des glatten Penisretraktormuskels der Weinbergschnecke Helix pomatia L. (Göttingen, 1970). Auf Anregung von Herrn Prof. Dr. Fr.-W. Schlote, mit Unterstützung durch die Deutsche Forschungsgemeinschaft und die Akademie der Wissenschaften.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.     

Fine structure of nerve-melanophore contacts in the angelfish Pterophyllum scalare

Contacts between small unmyelinated nerve fibres and dermal melanophores of the angelfish, Pterophyllum scalare, exhibit several features characteristic of synapses, including small synaptic vesicles and dense core vesicles, a narrow synaptic cleft, electron-dense material at the postsynaptic membrane (cell membrane of the melanophore) and, occasionally, presynaptic densities. An analysis of serial thin sections shows that the synapses described here represent varicosities of an otherwise more or less straight nerve fibre. A single axon thereby may form several en passant synapses with a single melanophore. It is suggested that the synaptic contacts described here not only represent sites of transmitter release but also play a role as sites of firm attachment between nerves and melanophores which guarantee a stable arrangement of nerve fibres and melanophores.Supported by the Deutsche Forschungsgemeinschaft     

Neurosecretory fibres in the median eyes of the scorpion,Androctonus australis L.

Summary The retina of the median eyes of the North African scorpion, Androctonus australis L., is supplied with numerous neurosecretory nerve fibres which establish synaptoid contacts on the retinula cells. The number of fibres or profiles of varicosities of fibre terminals associated with a retinular unit (five retinula cells with a fused rhabdom) varies between 10 and 20. Electron-opaque vesicles with a diameter of 80–100 nm are abundant within the axonal profiles. The synaptoid junctions are characterized by postsynaptic electron-dense material on the inner leaflet of the retinula cell membrane and, frequently, presynaptic submembranous dense material. Because of these ultrastructural features, the junctions observed here resemble typical interneuronal synaptic contacts. Hence this kind of neurosecretory junction appears to be unique among arthropods.It is suggested that the neurosecretory fibres within the retina represent the efferent pathways for the control of the circadian pigment movements within the retinula cells.Supported by the Deutsche Forschungsgemeinschaft (F1 77/7)     

Demonstration and localization of synapsin I in vestibular receptors in the cat: immunocytochemical study

The presence and localization of synapsin I, a neuron-specific phosphoprotein, was investigated in the cat vestibular epithelium, using a rabbit antisynapsin I anti-serum. The staining was performed by immunofluorescence or by a peroxidase-antiperoxidase (PAP) technique. A strong immunoreactivity was observed with both methods. This immunoreactivity appeared as spherical patches distributed in the lower part of the epithelium. This distribution pattern is very similar to that of the efferent synaptic endings which form axodendritic synapses with the afferent nerve chalice of type I hair cells, or axosomatic synapses with type II hair cells. Some of the nerve chalices were also labelled; in this case, the immunoreactivity was more evident with PAP staining. These results thus suggest the presence of large amounts of synapsin I in the vestibular efferent nerve endings. These endings are known to be filled with numerous synaptic vesicles. This localization of synapsin I is well correlated with previous work that report a close association between synapsin I and small synaptic vesicles. The presence of synapsin I in sensory endings such as the afferent nerve chalices was unexpected and is under investigation.     

Efferent fibers and daily rhabdomal changes in the anteromedial eye of the liphistiid spider,Heptathela kimurai

The presence of efferent fibers in the retina of liphistiid spiders, kept in natural daily cycles of illuminance, was examined by electron microscopy. The efferent fibers were observed to extend their processes through the ocellar nerve to the retina. They contained characteristic large electron-dense granules and branched repeatedly within the retina with varicosities, to provide synaptoid contacts with the receptor cells. They ran mostly among receptor cells and glial cells but sometimes protruded into receptor cells to establish invaginated synaptoid contacts. The synaptoid structures were characterized by spherical clear vesicles located at the presynaptic region, with electron-dense material adhering to the plasma membranes of the receptor cell and the efferent fiber, and a cleft about 10 nm wide formed by the two opposed parallel membranes. The clear vesicles and the electron-dense granules were secreted by exocytosis. The efferent fiber was characteristically presynaptic in relation to the receptor cell. In addition, the rhabdoms differed in size from day to night.     

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.     

Presynaptic to postsynaptic relationships of the neuromuscular junction are held constant across age and muscle fiber type

The neuromuscular junction (NMJ) displays considerable morphological plasticity as a result of differences in activity level, as well as aging. This is true of both presynaptic and postsynaptic components of the NMJ. Yet, despite these variations in NMJ structure, proper presynaptic to postsynaptic coupling must be maintained in order for effective cell‐to‐cell communication to occur. Here, we examined the NMJs of muscles with different activity profiles (soleus and EDL), on both slow‐ and fast‐twitch fibers in those muscles, and among young adult and aged animals. We used immunofluorescent techniques to stain nerve terminal branching, presynaptic vesicles, postsynaptic receptors, as well as fast/slow myosin heavy chain. Confocal microscopy was used to capture images of NMJs for later quantitative analysis. Data were subjected to a two‐way ANOVA (main effects for myofiber type and age), and in the event of a significant (p < 0.05) F ratio, a post hoc analysis was performed to identify pairwise differences. Results showed that the NMJs of different myofiber types routinely displayed differences in presynaptic and postsynaptic morphology (although the effect on NMJ size was reversed in the soleus and the EDL), but presynaptic to postsynaptic relationships were tightly maintained. Moreover, the ratio of presynaptic vesicles relative to nerve terminal branch length also was similar despite differences in muscles, their fiber type, and age. Thus, in the face of considerable overall structural differences of the NMJ, presynaptic to postsynaptic coupling remains constant, as does the relationship between presynaptic vesicles and the nerve terminal branches that support them. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 744–753, 2013     

Ultrastructural localization of monoamines in the central nervous system of Lumbricus terrestris (L.) with remarks on neurosecretory vesicles

Summary The cerebral ganglion and the ventral nerve cord of Lumbricus terrestris have been studied with the electron microscope. The results are as follows: In the neuropile small granular vesicles (300 to 500 Å) occur in some varicose nerve fibres after fixation with potassium permanganate. This indicates the presence of noradrenaline. Sometimes only a few of the vesicles produce a positive reaction. After incubation with -methyl-noradrenaline the numbers of nerve terminals with small granular vesicles greatly increase, indicating the presence of dopamine and/or 5-hydroxytryptamine. In this case the reaction is now complete. The number of small granular vesicles is largest in the terminal swellings.These findings are consistent with histofluorescence, chemical, and microspectrofluorometric analyses, which have demonstrated noradrenaline, dopamine, and 5-hydroxytryptamine in neurones in the central nervous system.Large granular vesicles (600 to 900 Å) are to be found in some perikarya, not identical with neurosecretory cell bodies. In this case the granular vesicles in the axon are smaller and fewer. This indicates a simultaneous proximo-distal transport and gradual decrease in size of the granular vesicles. The intraneuronal distribution of the vesicles is in agreement with the distribution of the fluorophores in the fluorescent neurones.Neurosecretory neurones are found most likely not to contain monoamines.This work was supported by grants from the Helge Axison Johnson Foundation, the Magn. Bergvall Foundation, and the Roy. Physiographic Society at Lund.I am greatly indebted to Mrs. Lena Eriksson, Miss Rita Jönsson, Miss Inger Norling, Mrs. Lena Svenre, and Mr. Henryk Keff for their excellent technical assistance.     

Evidence for the existence of serotonin-, dopamine-, and noradrenaline-containing neurons in the gut of Lampetra fluviatilis

Summary Monoamine-containing neurons in the gut of Lampetra fluviatilis are characterized by histochemical, electron microscopical and biochemical methods. Strongly yellow fluorescent, probably serotonin-containing intrinsic neurons are found along the entire length of the intestine. Their processes aggregate to form large bundles of mainly non-terminal axons, constituting a subepithelial fibre plexus. This subepithelial, ganglion cell comprising plexus is connected to a wide-meshed subserosal plexus which has ganglion cells of different size and few varicose, single axons. Intermingled with both plexus there occur — in the anterior and middle but not in the preanal portion of the lamprey intestine — scattered green fluorescent intrinsic perikarya, emanating faintly green fluorescent, poorly varicosed axons.The formaldehyde-induced neuronal fluorophores conform to serotonin (yellow fluorescent compound), noradrenaline, and dopamine (green fluorescent substance), as revealed in microspectrofluorimetric recordings. The electron microscopical analysis of the yellow fluorescent intrinsic neurons in the terminal hindgut shows nerve cell pericarya and axons equipped with a typical population of occasional small granular and many large granular vesicles (750–1600 Å). The number and opacity of cores of the small and the osmiophilia of the cores of the large granular vesicles are significantly increased following short-term treatment with 5,6-dihydroxytryptamine. Long-term treatment with 5,6- or 5,7-dihydroxytryptamine provokes severe signs of ultrastructure impairment and eventual degeneration in the supposed serotonin-containing axons, besides indications of piling-up of organelles in the non-terminal axons due to arrest of axonal transport.Chromatography of acid extracts from the lamprey intestine, gills and kidney reveals the presence of serotonin (besides another unidentified indoleamine) and dopamine and noradrenaline in the gut, but only dopamine in the brain. The detection of serotonin, noradrenaline and dopamine in the lamprey gut is confirmed by chemical determinations.The occurrence of intrinsic serotonin-, noradrenaline- and dopamine-containing neurons in the gut of Lampetra fluviatilis deviates from the established pattern of innervation of the vertebrate intestine and is considered to be a remnant of an autonomic innervation principle common in invertebrates.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by grants from the Swedish Medical Research Council (No. 14X-712 and 14X-56.The authors are indebted to Lilan Bengtsson, Gertrude Stridsberg, Eva Svensson and Rolf Frank for skilful technical assistance.     

Immunohistochemical localization of a synaptic-vesicle antigen in a cholinergic neuron under conditions of stimulation and rest

Summary An antiserum against a specific component (a glycosamino glycan) of the cholinergic synaptic-vesicle of Torpedo marmorata has been used to investigate the localization of the component in the cell body, its movement within the electromotor axon and its fate within the nerve terminal upon electrical stimulation. After immunofluorescent staining, spots are observed throughout the cytoplasm of the lobe perikarya, although they are concentrated in the region of the axon hillock. Ligation of the electromotor nerves leading from the lobe to electric organ produces a proximal build-up of material which stains readily with the antivesicle antiserum, indicating that the vesicle antigen is transported from the cell body to the nerve terminal. A marked increase in indirect immunofluorescent staining of the electric organ is observed in the nerve ending upon electrical stimulation. We interpret this result as fusion of the vesicles with the presynaptic plasma membrane and exteriorization of the vesicle antigen to the extracellular space, thereby facilitating its staining. After recovery of the system the fluorescence declines, a result that is consistent with the reinternalization of the vesicle antigen into the core of reformed vesicles. The results support a mechanism whereby vesicles recycle within the nerve terminal and transmitter is released by exocytosis.     

Occurrence of innervation in labral glands of Daphnia obtusa (crustacea,cladocera)

Electron microscopy discloses nerve endings in contact with gland cells situated in the labrum of Daphnia. Swellings of nerve fibers are in close contact with gland cell membranes, either on the cell surface or inserted into infoldings of plasma membrane. The axonal processes are single or double and lack glial wrappings. Inside the nerve fibers are vesicles of different sizes and electron density. These include granular vesicles, which often are dense-cored, and also clearer vesicles. Some presynaptic differentiations lie along the contact line of the axonal process with the gland cell membrane. The significance of the vesicles is discussed in terms of their possible content of biogenic amines, as described in other invertebrates.     

Identification of the endophypte ofDryas andRubus (Rosaceae)

Summary Root nodules ofDryas drummondii are of the coralloid type (Alnus type). The endophyte is present in the middle cortical cells of the root-nodule tissue. Transmission electron micrographs revealed an actinorhizal endophyte with septate hyphae and non-septate spherical or ovoid vesicles. Vesicles always possess at the base a septum; septa formation in the endophyte is always associated with the presence of mesosomes. Branching of the endophyte is not necessarily correlated with septum formation. Hyphal structures are more prominent in the apical part of the root nodule and vesicles are more numerous in a broad zone below this. In the middle and towards the base of the root nodule the endophytic structures appear in a stage of disintegration. Vesicles appear in a broad region near the periphery of the host cell and regularly show no strict orientation towards the host-cell wall. In the center of the host cells only hyphae occur. In the intercellular spaces between the host cells theFrankia endophyte produces spore-like structures although the outline of the sporangia is often faint.The coralloid root ofRubus ellipticus shows characteristically a basal rootlet initiated below the dichotomous branching of the nodular lobes, but extending beyond the root nodule. The endophyte is only present in the outer cortex of the root nodule in a 1–2 cell wide layer. This endophytic layer is bounded, internally as well as externally, with a 4–5 cell wide layer of tannin-filled host cells. The implications of this situation are discussed. Tannin-filled cells occur regularly inRubus species and their arrangement has been used for taxonomic purposes within the genus. TheRubus endophyte is aFrankia species with septate hyphae and distinctly septate spherical vesicles. The ultrastructure of the vesicles of theRubus endophyte is very similar to that of theAlnus endophyte.     

An ultrastructural study of the polyp and strobila of Atorella japonica (Cnidaria,Coronatae) with respect to muscles and nerves

Atorella japonica were observed by TEM to examine the nerve plexus in the capitulum of the polyp and the cross-striated muscle cells of the strobila. The nerve plexus included a number of neuromuscular junctions and many interneural synapses. Neuromuscular junctions contained two types of synaptic vesicle: clear and small (ca 75 nm diam.), and dense cored and large (ca 120 nm diam.). The first type of vesicle always appeared near the presynaptic membrane and the second type was distributed behind the former. In interneural synapses, two types of vesicle which were similar to neuromuscular synaptic vesicles were recognized. They were distributed in a pattern similar to that of the neuromuscular synaptic vesicles, but these vesicles were found on both sides of the two synaptic membranes.     

Innervation des cellules du pancréas endocrine du Poisson Téléostéen Xiphophorus helleri H.

Summary The pancreatic islet tissue of the bony fish Xiphophorus helleri H. is innervated by amyelinic fibers. Neuroglandular junctions on the islet cells show a structure similar to that of the synapses of the central nervous system. Presynaptic projections, intrasynaptic lines, and postsynaptic bands are to be observed. In the nerve endings two kinds of vesicles occur: large granular vesicles (diameter:850–1150 Å) and small clear vesicles (diameter: 500–650 Å). Synaptic vesicles are gathered around the presynaptic dense projections. This precise organization suggests the existence of a direct nervous control involved in the metabolism of the pancreatic hormones.

Avec la collaboration technique de M. D. Streicher.     

Diurnal changes in the fine structure of photoreceptors in an abalone,Nordotis discus

Summary The ultrastructure of the synapses in the brain of the monogenean Gastrocotyle trachuri (Platyhelminthes) is described. The synapses consist of one presynaptic terminal separated by a uniformly wide synaptic cleft, from one or more postsynaptic elements. The presynaptic terminals are characterized by the presence of paramembranous dense projections and associated synaptic vesicles. The postsynaptic elements while possessing membrane densities, are usually devoid of vesicles.The structure of the synapses in the brain of Gastrocotyle is compared to synapses from other platyhelminths.     

Ultrastructural localization of monoamines in the epidermis of Lumbricus terrestris (L.)

Summary Receptor cells in the epithelium and the basiepithelial nerve net of the prostomium of Lumbricus terrestris were investigated with electron microscope with special regard to the presence of monoamines. The receptor cells are found in groups of about 40 intermingled with supportive cells. After pretreatment with -methyl-noradrenaline and fixation with potassium permanganate a few receptor cells in each group and some nerve fibres in the basiepithelial nerve net contain small granular vesicles (about400 Å) characteristic for monoaminergic neurons. The distribution and relative number of these receptor cells and nerve fibres coincide well with previous reports on fluorescent receptor cells and varicose fibres. That the monoamine-storing small granular vesicles not are visualized until pretreatment with -methyl-noradrenaline is in accordance with recent microspectrofluorometric analysis, which shows that dopamine is the only primary monoamine present in the epithelium.In the epithelium there are occasional receptor cells and nerve fibres containing large vesicles (1000–1800 Å) which resemble the neurosecretory vesicles in the central nervous system. Photoreceptor cells having an intracellular cavity with microvilli and cilia have infrequently been observed at the base of the epithelium.No synapses on the mucous cells have been noticed. Nor have any synaptic specializations been observed in the basiepithelial nerve net. The morphological conditions necessary for the existence of possible axo-axonal synapses are briefly discussed.This work was supported by grants from the Helge Ax: son Johnson Foundation and the Magn. Bergvall Foundation.     

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus)

Summary Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibres containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which form distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P_{CO 2} and so leading to modulation of activity in afferent nerve fibres (type containing agranular vesicles). The presence of NEBs solely with an adrenergic (efferent) innervation poses a problem with this interpretation.