Electron microscope investigation of synapses,synapse-like structures,and possible sites of release of neurosecretory material in the buccal ganglia of Helix pomatia (Mollusca)

Summary In the buccal ganglia of Helix pomatia synapses and sites of possible release of neurosecretory material were investigated electron microscopically. There is one chemical synapse and one electrotonic synapse in the neuropile of the ganglion. No synapses could be detected in the buccal nerves, cerebro-buccal connectives, or in the buccal commissure. The synaptic cleft of the chemical synapse is about 25 nm wide and contains electron-dense material whereas the cleft of the electrotonic synapse is only 5 nm wide. The presynaptic fibre of the chemical synapse contains clear vesicles and dense core vesicles. The release sites of neurosecretory material are found at the initial segment of the axons, at perikarya of neurones, and at the perineurium of the ganglion. If the terminals are located at the plasmalemma of a nerve cell, these release sites are called synapse-like structures according to Roubos and Moorer-van Delft (1979). The synapse-like structures show all structural elements of synapses, except the 25 nm cleft containing dense material; the cleft is only 15–20 nm wide here like the normal cleft between neurones and glial cells or between two fibres. If the secretory material is released at the periphery through the perineurium the terminal is called synaptoid according to Scharrer (1970). In all cases, i.e. synapses, synapse-like structures, and synaptoids, clear vesicles were found in the axon terminal. This finding provides further evidence that clear vesicles always accompany the release of substances from axon endings.     

MORPHOLOGICAL CORRELATES OF INCREASED COUPLING RESISTANCE AT AN ELECTROTONIC SYNAPSE

Close appositions between axonal membranes are present in the septum between adjacent axonal segments of the septate or lateral giant axons of the crayfish Procambarus. In sections the closely apposed membranes appear separated by a space or gap. The use of lanthanum indicates that there may be structures connecting the apposed membranes. The apparent gap is actually a network of channels continuous with the extracellular space. Adjacent axonal segments are electrotonically coupled at the septa. The coupling resistance is increased by mechanical injury of an axon, immersion in low Cl^- solutions, and immersion in low Ca⁺⁺ solutions, followed by a return to normal physiological solution. Septa at which coupling resistance had been measured were examined in the electron microscope. The induced increases in coupling resistance are associated with separation of the junctional membranes (with the exception of the moderate increases during immersion in low Ca⁺⁺ solutions). Schwann cell processes are present between the separated axonal membranes. When nerve cords in low Cl^- solutions are returned to normal physiological solution, coupling, i.e., electrotonic synapses. A model of an electrotonic synapse is proposed in which tween axonal membranes are again found. The association between the morphological and physiological findings provides further evidence that the junctions are the sites of electrotonic coupling, i.e., electrotonic, synapses. A model of an electrotonic synapse is proposed in which intercytoplasmic channels not open to the extracellular space are interlaced with a hexagonal network of extracellular channels between the apposed junctional membranes.     

Reliable, responsive pacemaking and pattern generation with minimal cell numbers: the crustacean cardiac ganglion

Investigations of the electrophysiology of crustacean cardiac ganglia over the last half-century are reviewed for their contributions to elucidating the cellular mechanisms and interactions by which a small (as few as nine cells) neuronal network accomplishes extremely reliable, rhythmical, patterned activation of muscular activity-in this case, beating of the neurogenic heart. This ganglion is thus a model for pacemaking and central pattern generation. Favorable anatomy has permitted voltage- and space-clamp analyses of voltage-dependent ionic currents that endow each neuron with the intrinsic ability to respond with rhythmical, patterned impulse activity to nonpatterned stimulation. The crustacean soma and initial axon segment do not support impulse generation but integrate input from stretch-sensitive dendrites and electrotonic and chemically mediated synapses on axonal processes in neuropils. The soma and initial axon produce a depolarization-activated, calcium-mediated, sustained potential, the "driver potential," so-called because it drives a train of impulses at the "trigger zone" of the axon. Extreme reliability results from redundancy and the electrotonic coupling and synaptic interaction among all the neurons. Complex modulation by central nervous system inputs and by neurohormones to adjust heart pumping to physiological demands has long been demonstrated, but much remains to be learned about the cellular and molecular mechanisms of action. The continuing relevance of the crustacean cardiac ganglion as a relatively simple model for pacemaking and central pattern generation is confirmed by the rapidly widening documentation of intrinsic potentials such as plateau potentials in neurons of all major animal groups. The suite of ionic currents (a slowly inactivating calcium current and various potassium currents, with variations) observed for the crustacean cardiac ganglion have been implicated in or proven to underlie a majority of the intrinsic potentials of neurons involved in pattern generation.     

Electrotonic coupling between cercal afferents and giant interneurons in the American cockroach

Stimulation of the cercal nerve of the female American cockroach evokes a short-latency action potential in one giant axon in the ipsilateral connective of the ventral nerve cord. Neither procion yellow nor cobalt passes from the nerve cord into the cercal nerve, and the short-latency response disappears several weeks after removal of the cercus. Therefore, the short-latency spike is not due to a branch of the giant interneuron extending into the cercal nerve, but is presumably due to electrotonic coupling of cercal afferents to the giant. Responses of the presumed electrotonic junction to drugs, varied ionic concentrations and tonicity, and to cold are described. These responses and the impermeability of the junction to procion yellow suggest that the coupling is not by means of a gap junction. There is evidence for electrotonic coupling to another giant axon in the female, but this junction does not ordinarily transmit a spike. Electrotonic coupling is rare in males. In some females action potentials in giant interneurons excite cercal afferents electrically, and the afferents then re-excite the giants chemically. Electrotonic coupling may reduce fatigue and habituation of chemical synapses by depolarizing presynaptic terminals whenever the giants are active.     

Axonal branching pattern and coupling mechanisms of the cerebral giant neurones in the snail,lymnaea stagnalis

The axonal branching pattern of the two cerebral giant neurones (CGCs) of Lymnaea stagnalis was studied with intrasomatically applied horseradish peroxidase. The cells are symmetrical. Each CGC projects to the ipsilateral n. labialis medius and n. arteriae labialis, the subcerebral commissure, and to all ipsi- and contralateral buccal nerves. The contralateral buccal nerves are reached via the ipsilateral cerebro-buccal connective and the buccal commissure. The CGC fire action potentials 1:1 in a driver-follower relationship. Each cell is capable of both driving and following. The relationship depends on the membrane potentials of the somata. In driving CGC spikes are initiated in a cerebral spike trigger zone located near the soma. In following cells spikes are initiated in a distal zone located in the buccal ganglia. The buccal zone is only affected by the partner CGC. CGC are synchronized by three coupling mechanisms: mutual excitatory chemical synapses, electrotonic coupling, and common input. The chemical and electrotonic connections are located in the buccal ganglia. All spikes are relayed to the partner cell via the chemical synapses. The electrotonic coupling improves the efficiency of the chemical synapses. The dual connection selectively synchronizes the CGC-axonal spikes from each side of the buccal mass. Common excitatory input affects the cerebral spike trigger zones and can initiate simultaneous spikes in both cells. This results in bilateral synchrony of spikes in the CGC-axons in both the buccal and the lip nerves.     

Innervation of chromatophore muscle fibres in the octopus Eledone cirrhosa

Summary Cephalopod chromatophores are made of a central pigment cell surrounded by 10 to 20 radially arranged muscle fibres under direct nervous control. Innervation of these muscle fibres was studied with anterograde cobalt fills of peripheral nerve bundles and light and electron microscopy. Individual axons branch repeatedly to innervate the muscles of chromatophores scattered over several millimeters. Axons contained in several dermal nerves converge to innervate the same chromatophores. Among the chromaophores, axons were found running either singly or in small bundles, often accompanied by sheath cells. Single chromatophore muscles were innervated by at least one axon running across or along its length. Since nerves terminating on chromatophore muscles are very rare, neuromuscular contact seems to be made en passant. Varicosities of the axons apposed to the muscles are thought to be presynaptic sites. However, morphological differentiations of the pre-or post-synaptic membranes were not visible. Two types of innervating processes were found containing either electron-clear or a mixture of electron-clear and dark-core synaptic vesicles.Supported by a postgraduate award from the University of Aberdeen (GB)     

CORRELATION OF FINE STRUCTURE AND PHYSIOLOGY OF THE INNERVATION OF SMOOTH MUSCLE IN THE GUINEA PIG VAS DEFERENS

An electron microscope study of the innervation of smooth muscle of the guinea pig vas deferens was undertaken in order to find a structural basis for recent electrophysiological observations. The external longitudinal muscle coat was examined in transverse section. Large areas of the surfaces of adjacent muscle cells were 500 to 800 A apart. Closer contacts were rare. A special type of close contact suggested cytoplasmic transfer between neighbouring cells. Groups of non-myelinated axons from ganglia at the distal end of the hypogastric nerve ramified throughout the muscle. Some small axon bundles and single axons lay in narrow fissures within closely packed muscle masses. Many axons contained "synaptic vesicles." About 25 per cent of the muscle fibres in the plane of section were within 0.25 µ of a partly naked axon; of these 15 per cent were within 500 A of the axon, and about 1 per cent made close contact (200 A) with a naked axon. It is unlikely that every muscle fibre is in close contact with an axon, and it is not possible for every fibre to have many such contacts. Muscle fibres are probably activated by both diffusion of transmitter from naked portions of axons a fraction of a micron distant, and electrotonic spread of activity from neighbouring cells.     

Electrotonic transmission in the mammalian central nervous system (author's transl]

The aim of this review is to present the electrophysiological data, obtained in the mammalian central nervous system, which show that depolarisations recorded intracellularly, under certain experimental conditions can be interpreted in terms of electrotonic coupling. The results were obtained from very different structures: primary sensory nuclei, sensori-motor integration centres and motor nuclei. The association of the phenomenon of electrotonic transmission with a known ultrastructural substrate--the "gap junction"--has been defined by the term electrotonic coupling. In the cases where it has not been possible to link depolarisations with the presence of gap junctions, other possible morphological correlates have been envisaged. The functional significance of electrotonic interactions are discussed on the basis of information obtained from different experimental approaches.     

Multisegmental cobalt filling of the dorsal giant fibers in the nervous system of the earthworm,Lumbricus terrestris

Summary The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.Abbreviations CL collateral of lateral giant fiber - CM collateral of medial giant fiber - GIN giant interneuron - LGF lateral giant fiber - MGF medial giant fiber - SN segmental nerve     

Drugs that block calmodulin activity inhibit cell-to-cell coupling in the epidermis oftenebrio molitor

Summary In many cell systems, the permeability of membrane junctions is modulated by the cytoplasmic level of free Ca⁺⁺. To examine whether the calcium-dependent regulatory protein calmodulin is involved in this process, the ability of anticalmodulin drugs to influence the cell-to-cell passage of injected current and an organic tracer was tested using standard intracellular glass microelectrode techniques. Several antipsychotics and local anesthetics were found to block junctional communication in the epidermis of the beetleTenebrio molitor. Treatment of the epidermis with chlorpromazine (0.25 mM) raised intercellular resistance two- to threefold within 20 to 25 min; cell-to-cell passage of electrical current was abolished within 41±5 min. Loss of electrotonic coupling was accompanied by a block in the cell-to-cell movement of the organic tracer carboxyfluorescein. The reaction is fully reversible, with normal electrotonic coupling being restored within 2 to 4 hr. Other antipsychotics and local anesthetics had similar effects on cell coupling. The order of potency found was: trifluoperazine>thioridazine> d-butaclamol>chlorprothixine=chlorpromazine> l-butaclamol> dibucaine>tetracaine. The relative uncoupling potencies of these drugs correlate well with their known ability to inhibit calmodulin-dependent phosphodiesterase activity. Other anesthetic compounds, procaine and pentobarbital, did not block cell-to-cell communication. Altering the extracellular Ca⁺⁺ concentration did not affect the rate of uncoupling by antipsychotics, while chelation of extracellular Ca⁺⁺ with EGTA raised electrotonic coupling. The effect of three metabolic inhibitors on coupling was also examined. Iodoacetate uncoupled the epidermal cells while DNP and cyanide did not. These results are discussed in terms of possible mechanisms by which calmodulin may control junctional communication in this tissue.     

Transient Neuronal Populations Are Required to Guide Callosal Axons: A Role for Semaphorin 3C

The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.     

Impulse Propagation at the Septal and Commissural Junctions of Crayfish Lateral Giant Axons

Transmission across the septal junctions of the segmented giant axons of crayfish is accounted for quantitatively by a simple equivalent circuit. The septal membranes are passive, resistive components and transmission is ephaptic, by the electrotonic spread of the action current of the pre-septal spike. The electrotonic spread appears as a septal potential, considerably smaller than the pre-septal spike, but usually still large enough to initiate a new spike in the post-septal segments. The septal membranes do not exhibit rectification, at least over a range of ± 25 mv polarization and this accounts for their capacity for bidirectional transmission. The commissural branches, which are put forth by each lateral axon, make functional connections between the two axons. Transmission across these junctions can also be bidirectional and is probably also ephaptic. Under various conditions, the ladder-like network of cross-connections formed by the commissural junctions can give rise to circus propagation of impulses from one axon to the other. This can give rise to reverberatory activity of both axons at frequencies as high as 400/sec.     

GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria

Summary Neurones in the suboesophageal ganglion of the locust Schistocerca gregaria were stained with an antiserum raised against gamma amino butyric acid (GABA). This ganglion consists of the fused mandibular, maxillary and labial neuromeres. Immunoreactive cell bodies of similar size and distribution occur in the lateral, ventral and middorsal regions of all three neuromeres. Approximately 200 cell bodies stain in both the mandibular and maxillary neuromeres and 270 in the labial neuromere. A few distinctly larger cells occur in the ventral groups and one large pair occurs in the lateral group of the maxillary neuromere. Dorsal commissures DCIV and DCV are composed mainly of stained fibres, while DCI–DCIII are largely unstained. A ventral commissure also stains in the maxillary neuromere. All longitudinal tracts contain both stained and unstained fibres. Many processes within the neuropil are also immunoreactive. A stained axon is found in the posterior tritocerebral commissure which enters the anterior dorsal region of the mandibular neuromere. The salivary branch of the 7th nerve contains one stained axon and two axons stain in nerve 8 which innervates neck muscles.     

The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.     

EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure

The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor‐mediated signaling contribute to ACp and CC axon guidance. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 405–420, 2016     

Identification of a secretomotor centre in the brain of Locusta migratoria,controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum

Summary After retrograde filling of axons terminating in the glandular lobe of the corpus cardiacum (CC) of Locusta migratoria with cobalt chloride, a paired group of about 15 cobalt containing cells was demonstrated in the lateral area of the protocerebrum. The axons of these cells run via the NCC II into the glandular lobe of the CC. These small neurons have the characteristics of secretory cells; they contain secretory granules of about 1000 Å in diameter. The axon terminals in the glandular lobe, making synaptic contacts with the glandular cells, contain secretory granules of the same size. It is therefore concluded that the cell groups in the protocerebrum control the activity of the glandular cells which produce an adipokinetic hormone. Arborizations of fibers of the lateral secretomotor cells are present in the dorsal neuropile of the protocerebrum, ventral of the mushroom bodies and along the tracts of the NCC I within the brain. It is proposed that these arborizations are sites of synaptic input. It is discussed that the axons of these cells might receive additional synaptic input in the storage lobe of the CC.The localization of cell bodies, the axons of which enter the storage part of the CC is described. The course of the axon tracts of the various cell groups in the protocerebrum and their connections with the NCC I and NCC II are demonstrated.Supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The electron microscopical investigations were performed at the EM-unit of the Faculty of Biology, State University of Utrecht (Director: Prof. Dr. J.C. van de Kamer)The author is greatly indebted to Dr. A.M.Th. Beenakkers and Dr. H.H. Boer for their active interest and helpful advise. Thanks are also due to Mr. H. van Kooten and his staff for making the macro- and microphotographs, to Mr. L.W. van Veenendaal for preparing the electron micrographs and final assistance in the preparation of the photo pages and to Mr. D. Smit, who made the drawings     

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus

Summary The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160–350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160–350 nm in diameter. Synaptoid configurations with 20–50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.This work was done while JTB was supported by USPHS Training Grant HD-0266 from NICHDI wish to express my thanks to Dr. Richard A. Cloney for sharing his expertise in electron microscopy     

Distribution of soil fractions and location of soil bacteria in a vertisol under cultivation and perennial grass

Effects of soil management on soil characteristics were investigated on the rhizosphere (RPP) and the nonrhizosphere (NRPP) soil of a re-grass vertisol underDigitaria decumbens and in the soil under continuous cultivation (CC). A low energy technique allowed to separate eight size and density fractions, including macro- and micro-aggregates while preserving soil bacteria. Organic C and N, microbial biomass C and the number of total bacteria (AODC) and ofAzospirillum brasilense and their distribution were determined in soil fractions isolated from the CC, NRPP and RPP soils. Soil macroaggregates (>2000 m) were similarly predominant in the NRPP and RPP soils when the dispersible clay size fraction (<2 m) respresented more than 25% of the CC soil mass. The main increase of C content in RPP originated from the macroaggregates (> 2000 m) and from the root fraction, not from the finer separates. The proportion of organic C as microbial biomass C revealed the low turnover of microbial C in the PP situations, especially in the clay size fraction of the NRPP soil. A common shift of AODC toward the finer separates from planted soils (CC and RPP) revealed the influence of living plants on the distribution of soil bacteria. The relative abundance ofA. brasilense showed the presence of the active roots ofDigitaria in the macroaggregates and their contact with the dispersible clay size fraction of the rhizosphere soil.     

Recombinant inbreeding in mice reveals thresholds in embryonic corpus callosum development

The inbred strains BALB/cWah1 and 129P1/ReJ both show incomplete penetrance for absent corpus callosum (CC); about 14% of adult mice have no CC at all. Their F(1) hybrid offspring are normal, which proves that the strains differ at two or more loci pertinent to absent CC. Twenty-three recombinant inbred lines were bred from the F(2) cross of BALB/c and 129, and several of these expressed a novel and severe phenotype after only three or four generations of inbreeding - total absence of the CC and severe reduction of the hippocampal commissure (HC) in every adult animal. As inbreeding progressed, intermediate sizes of the CC and the HC remained quite rare. This striking phenotypic distribution in adults arose from developmental thresholds in the embryo. CC axons normally cross to the opposite hemisphere via a tissue bridge in the septal region at midline, where the HC forms before CC axons arrive. The primary defect in callosal agenesis in the BALB/c and 129 strains is severe retardation of fusion of the hemispheres in the septal region, and failure to form a CC is secondary to this defect. The putative CC axons arrive at midline at the correct time and place in all groups, but in certain genotypes, the bridge is not yet present. The relative timing of axon growth and delay of the septal bridge create a narrow critical period for forming a normal brain.