Synaptic organization of a multifunctional interneuron in the central nervous system of Helix pomatia L.

Summary The morphology, axonal arborization and ultrastructure of synaptic connections of the V21 giant neuron in the visceral ganglion of the snail Helix pomatia has been investigated following intracellular labelling with horseradish peroxidase. The V21 neuron establishes several afferent and efferent axo-axonic connections, mainly along the first half of the primary axon. Collaterals of 200–300 m length originate from the primary axon, which shows further arborization, and both afferent and efferent synaptic contacts are formed on these fine axon profiles. Afferent and efferent contacts of the cell occur within very short distances of a few micrometers. On the basis of ultrastructure and vesicle and granule content, several afferent terminals can be distinguished on V21 labelled axon profiles. The majority of these afferent terminals resembles peptidergic-(neurosecretory)-like terminals. This finding supports the possible transmitter role of neuropeptides in the central nervous system of gastropods. Our results are consistent with and provide morphological evidence for recent electrophysiological observations suggesting that, in addition to integrating input, the V21 neuron functions as an interneuron in Helix central nervous system.     

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     

In vitro recalcification of the demineralized shell-repair membrane of the snail,Helix pomatia L.

Summary The role of the amoebocytes in the calcification process of the shell-repair membrane of the snail, Helix pomatia, was investigated in vitro. The shell-repair membranes were demineralized with 0.5 M EDTA at pH 7.4. For the recalcification of the demineralized membranes two substrates were chosen: (i) Tris-buffered Helix pomatia-saline, pH 7.4, and (ii) Helix pomatia-saline supplemented with 5 mM CaCl₂ and 5 mM NaHCO₃. The membranes were incubated in 2 ml substrate at 37° C and examined after 2 h, 24 h, and 3, 5 and 7 days. Calcium deposition and crystal formation were observed within the membrane incubated in the salt-supplemented substrate. The control membranes were either heat-inactivated or deprived of lipids. No calcium precipitation was observed in control membranes. The experiments show that the recalcification of the shell-repair membrane is under strict cellular control and that the granules released from the amoebocytes serve as sites for calcium deposition. The role of phospholipids in the calcification process is discussed.     

Uptake of serotonin, 5-hydroxytryptophan and tryptophan by giant serotonin-containing neurones and other neurones in the central nervous system of the snail (Helix pomatia)

Summary Following exposure to tritiated 5-HTP, silver grains were observed over the perikarya of the GSCs (Giant serotonin cells) of Helix pomatia and other known serotonin-containing neurones in light and electron microscope autoradiograms. There was no indication that the 5-HTP was taken up by nerve endings or by non-nervous structures. The distribution of radioactivity was completely different in autoradiograms of tissue exposed to tritiated serotonin. Silver grains, often in very high concentrations, were observed only over certain fine axon branches and processes thought to be nerve endings. Electron microscope autoradiography showed that these processes contained small dense-cored vesicles, morphologically identical to those thought to sequester serotonin in the perikarya of the GSCs. The accumulation of tritiated tryptophan was less specific; all the neurone perikarya showed an accumulation of radioactivity after exposure to this substance.We are grateful to Professor J. F. Lamb for the use of the Scintillation Spectrometer.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

Ultrastructure of Merkel corpuscles in the tongue of the finch,Lonchura striata

Summary Merkel corpuscles in the lingual mucosa of the finch, Lonchura striata, were examined by means of the argyrophilic reaction and electron microscopy. These corpuscles are composed of 12 to 20 flattened Merkel cells and enclosed nerve terminals. The present study demonstrated for the first time argyrophilia in avian subepithelial Merkel cells with the use of Grimelius silver stain. Electron-microscopically, the Merkel cell was characterized by the presence of numerous densecore granules, approximately 80 to 140 nm in diameter, as well as specialized contacts with nerve terminals. The granules showed a tendency to accumulate in the cytoplasm in close association with both nerve terminals and basal lamina. This study also provided unequivocal evidence for exocytotic discharge of Merkel-cell granules at the plasma membrane facing not only the nerve terminals but also the basal lamina. The exocytotic figures toward the nerve terminals can be regarded as synaptic discharge of Merkel-cell granules, but the possibility also exists that the Merkel-cell granules may exert a trophic effect on the nerve terminals. The exocytotic release of Merkel-cell granules toward the basal lamina with no relation to nerve terminals may suggest an endocrine (paracrine) function for the Merkel cell. The avian subepithelial Merkel cells qualify as paraneurons, but their exact nature and function remain enigmatic as is the case of intraepithelial Merkel cells in other vertebrates.     

Interactions of endogenous lectin from theHelix pomatia reproductive system (HPA) with the surface of CNS neurons of the snail

The surface of unidentified cultured neurons and electrophysiologically identified units RPa1, RPa2, and LPa3 of the snailHelix pomatia was studied using a colloidal gold-labelled endogenous gonad lectin of this molluse (Helix pomatia agglutinin -HPA), which is specific to -anomer N-acetyl-D-galactosamine, and some other lectins. It was found that only two populations of cultured neurons with a specific ultrastructure typical of peptidergic cells and differing from the ultrastructure of other peptidergic and non-peptidergic neurons possessHPA receptors. These neurons average about 1% of cultured nerve cells. Using a variety of plant lectins withHPA-like specificity (LBA, VVA, SBA) showed that the surface of many peptidergic neurons contains -anomer-like structures, yet it does not bindHPA. These data were supported in the experiments on identified snall neurons with the use ofSBA. The results suggest a putative role of endogenous lectins in regulation of activity of neurons participating in processes of secretion in the gonads.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 17–29, January–February, 1996.     

Light- and electron-microscopic immunocytochemistry of a molluscan insulin-related peptide in the central nervous system of Planorbarius corneus

Summary Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIP_B-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIP_B- and anti-MIP_C-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurohormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes.     

On the occurrence of merkel cells in the epidermis of teleost fishes

Summary The ultrastructure of a differentiated cell type in the epidermis of two species of teleost fish, Ictalurus melas and Phoxinus phoxinus, is described. This cell type has a synaptic association with nerve fibres, microvillus-like peripheral processes, and membrane-bounded inclusions, which together are the diagnostic features of the Merkel cells of tetrapod vertebrates. Other cytoplasmic features are shared with the epithelial cells. The appearance of the membrane-bounded granules depends on the fixative used; after fixation with glutaraldehyde the granules are of a size and electron-density comparable to that found in tetrapod Merkel cells, but after fixing in osmium tetroxide the granules are inconspicuous.Our thanks are due to Mr. A.C. Wheeler of the British Museum (Natural History) for help with the identification of the species of Ictalurus, and to Mr. E. Perry for technical assistance. One author (EBL) was supported by a SRC research studentship     

Isolation of a galactogen utilizing strain of Arthrobacter

The land snail, Helix pomatia, is known to deposit eggs that contain the galactose homopolymer, galactogen. Selective enrichment for galactogen utilizing bacteria in a Helix pomatia habitat resulted in the isolation of a new strain of Arthrobacter. The strain's ability to metabolize galactogen was confirmed by the release of ¹⁴CO₂ from (¹C)-galactogen. The new isolate was able to utilize galactogen, galactose and glucose but not glycogen as sole carbon sources. The type strain A. globiformis ATCC 8010 utilized glucose and galactose, but not galactogen, as carbon sources.     

Secretory morphology of the intermediate lobe of the rat pituitary incubated in vitro

Summary The ultrastructure of the incubated intermediate lobe of the rat pituitary and the morphological effect of isoproterenol stimulation on its cells were studied under in vitro conditions. The general structure of isolated neurointermediate lobes maintained for 2–3 h in vitro was well preserved, and the presence of intact nerve terminals establishing synaptic contacts with the glandular cells of the intermediate lobe was confirmed. Removal of the intermediate lobe from central inhibition leads to increased hormonal secretion, which was reflected by large Golgi areas and the appearance of secretory images. However, no obvious degranulation or peripheral migration of the secretory granules after 2–3 h in vitro was seen. The secretory granules varied in electron density; totally electron-lucent granules were regularly observed and exocytotic phenomena were shown. In addition, more extensive invaginations suggesting secretion by compound exocytosis were seen. A three-fold increase in the -endorphin secretion during a 4-min stimulation with 10^-6 M isoproterenol did not induce any morphometrically detectable changes in the incubated cells. This indicates that only a minor fraction of the total granule content is mobilized during an acute increase in secretory activity.     

FMRFamide-like and allatostatin-like immunoreativity in the lateral heart nerve of Periplaneta americana: Colocalization at the electron-microscopic level

Both allatostatin immunoreactivity (AS-IR) and FMRFamide immunoreactivity (FMRFa-IR) have been demonstrated light-microscopically in the lateral heart nerve of Periplaneta americana. The identifical labeling of some fibers suggests the coexistence of the two antigens. Electron-microscopically, six granule types in the peripheral part of the lateral heart nerve can be distinguished according to their size and density (types 1–6). These granule types can be subdivided immunocytochemically by means of a new mirror-section technique. Granules of types 4 and 5 always exclusively show FMRFa-IR. In the populations of fibers containing granules of types 1 and 6, axon profiles can be found that contain granules colocalizing FMRFa-IR and AS-IR. Other axon profiles of these populations only contain immunonegative granules of the same ultrastructure. Granules of type 2 can be differentiated immunocytochemically in three forms in the same section: In some fibers, they are nonreactive; in other fibers of the same section, they show FMRFa-IR, whereas in a third fiber type, granules show AS-IR. Finally, granules of type 3 can be observed with FMRFA-IR. In other fibers, they occur with the same ultrastructure but exhibit no immunoreactivity. Two soma types occur in the lateral heart nerve. Soma type I is characterized by the production of electron-dense granules that show FMRFa-IR. Type II is in close contact with various fibers, forming different types of axosomatic synapses, hitherto unknown in Insecta.     

Nonlinear dynamics and information processing in pacemaker neurons

The paper treats some nonlinear dynamic phenomena in oscillatory activity of a single nerve cell. Based on experiments with CNS bursting pacemaker neurons ofHelix pomatia snail, a mathematical model was studied. The model demonstrates the majority of experimentally observable phenomena and allows one to investigate the role of its separate components. The phenomena demonstrated by model neuron (chaotic behavior, bistability, and sensitivity to parameter variations, initial conditions, and stimuli) may be relevant to information processing in nerve cells. The complexity of [Ca²⁺] _in −V phase diagrams of initial conditions depends on parameters. Transient synaptic impulse produces stable parameter-independent changes in activity of model neuron. These results indicate that a single bursting neuron can work in the neuronal ensemble as a dynamic switch. The sensitivity of this switch is regulated by a neurotransmitter.     

The axosomatic contacts on the bursting neuron of the snailHelix pomatia. I. ultrastructural features of the axosomatic contacts

The analysis of serial ultrathin sections of the RPAI bursting neuron of the snail Helix pomatia reveals the presence of axosomatic contacts on its surface membrane. These contacts have a number of specific features: the presynaptic axon contains synaptic vesicles and electron-dense granules, typical of peptidergic terminals; the terminal part of the axon forms many finger-like processes which invaginate the neuronal soma; the width of the cleft (80 nm) in the area of the contact is larger than that in usual synaptic contacts; and there is a system of lacoons in the region of the axosomatic contact; this system is formed by protrusions of the soma and it accompanies the contact along its extent. It is suggested that the system of lacoons which communicates with the space between the terminal and the soma may serve as a ramified synaptic cleft into which the secretion from the terminal is released. This system may contribute to a considerable prolongation of the time of action of the secretory product on the membrane of the RPAI neuron.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

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.     

Electron Microscopy of Peripheral Nerves and Neuromuscular Junctions in the Wasp Leg

The peripheral nerve branch innervating the femoral muscles of the common yellow jacket (Vespula carolina) has been found to possess a thick lemnoblast basement membrane and a complex mesaxon. The term "tunicated nerve" is proposed to designate the type of peripheral nerve in which one or several axons are loosely mantled by meandering, cytoplasm-enclosing membranes of the lemnoblast. The peripheral axon courses longitudinally in a groove in the muscle fiber between the plasma membrane of the muscle fiber and a cap formed by lemnoblast and tracheoblast. The junction is characterized by apposition of plasma membranes of axon and muscle fiber, abundant mitochondria, and synaptic vesicles in the axon, and aggregates of "aposynaptic granules" plus mitochondria and endoplasmic reticulum on the muscle side of the synapse. Unlike the vertebrate striated muscle fiber, no complex infolding of the synapsing plasma membrane of the muscle fiber occurs. The "connecting tissue" of the insect is formed by tracheoblasts, their basement membranes, and the basement membranes of other cells. Further mechanical support is given by the ramifying tracheoles. The physiologic roles of the specialized structures are considered.     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

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.