Fish electroreception as a model for vincristine-induced neuropathies and a possible preventive role for Org 2766 treatment

1. Subcutaneous injection of vincristine (more than 3 μg) in the catfish, Ictalurus nebulosus, produces a black spot on the skin with a grey surround. Doses between 1 and 3 μg give a less pronounced discolouration.2. Two to three days after injection of 5 μg vincristine, repetitive activity is detected in primary afferents of unstimulated electroreceptor organs close to the site of injection.3. Vincristine increases the phase-lag of the modulation of afferent activity in electroreceptor organs during electrical stimulation without a clear effect on the sensitivity of catfish electroreceptor organs.4. The amplitude of the action potentials of the primary afferents begins to decrease after 2 days; after3 days they gradually disappear in the background noise.5. Application of Org 2766, a potentially neurotrophic compound, at 2 days, but not 1 day, before vincristine application prevents vincristine effects on the phase shift.6. Preliminary electron-microscopical studies of the synapse shows a severe depletion of glycogen granules in the afferent nerve fibre after vincristine application,7. It is concluded that electroreceptor organs can be used to study neuropathies caused by vincristine, and that Org 2766 may be useful for preventive treatment of such neuropathies.     

Evidence for transmitter operated electrical synapses (TOES) in ampullary electroreceptor organs

Afferent fibres of ampullary electroreceptor organs in electrosensitive fish fire spontaneously, that is, they fire without external stimulus. In the past it has been postulated that the spontaneous activity originates from a sustained level of neurotransmitter release delivered by the electroreceptor cells. The spontaneous activity can be modulated by electrical stimuli. Blocking of the chemical synapse, however, reduces the susceptibility to electrical stimuli to 2% or less, but the spontaneous activity to 60% only. By evaluating existing experimental evidence it is concluded that spontaneous firing of afferents is based on two processes. (1) A membrane bound oscillator, which does not depend on transmitter release, is almost free of frequency fluctuations, and is described by Hodgkin/Huxley-equations (HH-equations). (2) Release of neurotransmitter, which increases the firing level, adds frequency noise, and raises the susceptibility of the afferent to electrical stimuli. There is evidence that neurotransmitter release acts as a gating process, which makes the generator area of the afferents directly accessible to electrical stimuli from the outside. Apparently, the activated synapse behaves as a transmitter operated electrical synapse (TOES).     

Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.     

End buds: non-ampullary electroreceptors in adult lampreys

Summary Shared anatomical and physiological characters indicate that the low-frequency sensitive electrosensory system of lampreys is homologous with those of non-teleost fishes and amphibians. However, the ampullary electroreceptor organs which characterize all of these gnathostomes are not found in lampreys. Experimental anatomical and physiological studies reported here demonstrate that the epidermal end buds are the electroreceptors of adult lampreys.End buds, consisting of both sensory and supporting cells, are goblet-shaped with the top (25–60 m diameter) at the epidermal surface and the stem directed toward the dermis (Fig. 1A). Short lines or clusters of 2–8 end buds (Fig. 1B) are distributed over both trunk and head. Injections of horseradish peroxidase (HRP) into vitally-stained end buds labeled anterior lateral line afferents terminating in the ipsilateral dorsal nucleus (Fig. 2A) — the primary electrosensory nucleus of the lamprey medulla. Conversely, after HRP injection into the dorsal nucleus HRP-filled fibers and terminals were present on ipsilateral end buds (Fig. 2B).End buds are usually not visible without staining. However, in adult sea lampreys the presence of end buds was histologically confirmed in skin patches containing the receptive fields of electroreceptor fibers recorded in the anterior lateral line nerve. Additionally, in the rare instance of two silver lampreys in which end buds were visible without staining, electrosensory activity indistinguishable from that of the primary electroreceptor afferents was recorded from the end bud surface (Figs. 3, 4).End buds were initially characterized as chemoreceptors (Johnston 1902) but were later correctly advanced as lateralis receptors based on the presence of presynaptic dense bodies in the receptor cells (Whitear and Lane 1981). Unlike all other low-frequency electroreceptors, end buds lack canals. The receptor cells contact the epidermal surface and possess apical microvilli rather than the kinocilium of most gnathostomes with homologous electrosensory systems of the primitive (non-teleost) type.Larval lampreys and newly transformed adults lack end buds although at least the latter are electroreceptive. End buds, therefore, may be the form taken by electroreceptors only in the final portion of a lamprey's life.     

Methods for patch clamp capacitance recordings from the calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.     

Ultrastructural observations on rapid formation of neuro-muscular junctions in vitro

Summary The development of neuro-muscular junctions (mouse, rat) from the time of first contact between neurons and myotubes in culture and the changes which lead to the formation of functional synaptic contacts have been investigated using light microscopy and ultrastructural techniques.An extensive basal lamina was present when the neuronal cell population was added to the developing myotubes in culture. The nerve cells were initially strongly attracted to each other and nerve cell aggregates formed rapidly. It was only when nerve fibres began to grow out of these aggregates to contact developing myotubes that changes within the cytoplasm of the two adjacent cells were observed. These developments included accumulations of filaments, membrane densities, mitochondria and large clear vesicles within both cells in the region of contact. In addition, collections of glycogen granules and an extensive membrane reticular complex were found within myotubes, and an extensive granular material filled many of the nerve processes. The basal lamina within the intercellular space appeared more electron-dense than elsewhere and was traversed by strands linking the two cell membranes. These features all appeared to be stages in the initial formation of neuro-muscular junctions. It was only after these events had occurred that presynaptic vesicles gradually appeared within the future nerve terminal. The results of this paper therefore support the view that synaptic transmission at developing mammalian neuromuscular junctions is not necessarily dependent on the presence of presynaptic vesicles.     

Quantitative characteristics of ultrastructural changes in axo-dendritic synapses under the influence of tetanus toxin

Changes in synaptic ultrastructure of the external geniculate body (EGB) were investigated in rats when a generator of pathologically intensified excitation (GPIE) was produced in this nucleus under the influence of tetanus toxin (TT). At the period of pronounced convulsive activity (24 h after TT injection), synaptic changes were estimated electronmicroscopically and with quantitative comparison of the materials from three groups. The first group included EGB synapses where TT was injected, the second group included contralateral EGB synapses and the third included EGB from the rats injected with inactivated toxin. By means of electron optic computer "Klassimat" average amount of round, flat, anomalous and adjacent to the presynaptic membrane vesicles was measured, average relative length of the active zone, average area of the presynaptic terminal, average relative section areas of pre- and postsynaptic cytoplasm condensation were estimated. In the area of GPIE formation, under the influence of TT, the increased amount of the vesicles related to the presynaptic membrane and that of flat vesicles were statistically significant. At the same time, the synaptic terminals, by the number of vesicles, have bimodal, while the control groups have unimodal distribution.     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

CHANGES IN THE FINE STRUCTURE OF THE NEUROMUSCULAR JUNCTION OF THE FROG CAUSED BY BLACK WIDOW SPIDER VENOM

Application of black widow spider venom to the neuromuscular junction of the frog causes an increase in the frequency of miniature end-plate potentials (min.e.p.p.) and a reduction in the number of synaptic vesicles in the nerve terminal. Shortly after the increase in min.e.p.p. frequency, the presynaptic membrane of the nerve terminal has either infolded or "lifted." Examination of these infoldings or lifts reveals synaptic vesicles in various stages of fusion with the presynaptic membrane. After the supply of synaptic vesicles has been exhausted, the presynaptic membrane returns to its original position directly opposite the end-plate membrane. The terminal contains all of its usual components with the exception of the synaptic vesicles. The only other alteration of the structures making up the neuromuscular junction occurs in the axon leading to the terminal. Instead of completely filling out its Schwann sheath, the axon has pulled away and its axoplasm appears to be denser than the control. The relation of these events to the vesicle hypothesis is discussed.     

Phosphorylation of VAMP/Synaptobrevin in Synaptic Vesicles by Endogenous Protein Kinases

Abstract: VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G and is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.     

Neurotransmitter release: the dark side of the vacuolar-H+ATPase

Vacuolar-H⁺ATPase (V-ATPase) is a complex enzyme with numerous subunits organized in two domains. The membrane domain V0 contains a proteolipid hexameric ring that translocates protons when ATP is hydrolysed by the catalytic cytoplasmic sector (V1). In nerve terminals, V-ATPase generates an electrochemical proton gradient that is acid and positive inside synaptic vesicles. It is used by specific neurotransmitter-proton antiporters to accumulate neurotransmitters inside their storage organelles. During synaptic activity, neurotransmitters are released from synaptic vesicles docked at specialized portions of the presynaptic plasma membrane, the active zones. A fusion pore opens that allows the neurotransmitter to be released from the synaptic vesicle lumen into the synaptic cleft. We briefly review experimental data suggesting that the membrane domain of V-ATPase could be such a fusion pore.We also discuss the functional implications for quantal neurotransmitter release of the sequential use of the same V-ATPase membrane domain in two different events, neurotransmitter accumulation in synaptic vesicles first, and then release from these organelles during synaptic activity.     

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.     

Some features of the submicroscopic morphology of synapses in frog and earthworm

Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.     

Are there efferent synapses in fish taste buds?

In fish, nerve fibers of taste buds are organized within the bud's nerve fiber plexus. It is located between the sensory epithelium consisting of light and dark elongated cells and the basal cells. It comprises the basal parts and processes of light and dark cells that intermingle with nerve fibers, which are the dendritic endings of the taste sensory neurons belonging to the cranial nerves VII, IX or X. Most of the synapses at the plexus are afferent; they have synaptic vesicles on the light (or dark) cells side, which is presynaptic. In contrast, the presumed efferent synapses may be rich in synaptic vesicles on the nerve fibers (presynaptic) side, whereas the cells (postsynaptic) side may contain a subsynaptic cistern; a flat compartment of the smooth endoplasmic reticulum. This structure is regarded as a prerequisite of a typical efferent synapse, as occurring in cochlear and vestibular hair cells. In fish taste buds, efferent synapses are rare and were found only in a few species that belong to different taxa. The significance of efferent synapses in fish taste buds is not well understood, because efferent connections between the gustatory nuclei of the medulla with taste buds are not yet proved.     

Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products

Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC+FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC+FA. Here, we show that LysoPC+FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC+FA lipid mixture as a novel and effective agonist of synaptic vesicle release.     

SOME FEATURES OF THE SUBMICROSCOPIC MORPHOLOGY OF SYNAPSES IN FROG AND EARTHWORM

Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.     

How do presynaptic PLA2 neurotoxins block nerve terminals?

Snake presynaptic neurotoxins with phospholipase A2 activity block nerve terminals in an unknown way. Here, we propose that they enter the lumen of synaptic vesicles following endocytosis and hydrolyse phospholipids of the inner leaflet of the membrane. The transmembrane pH gradient drives the translocation of fatty acids to the cytosolic monolayer, leaving lysophospholipids on the lumenal layer. Such vesicles are highly fusogenic and release neurotransmitter upon fusion with the presynaptic membrane, but cannot be retrieved because of the high local concentration of fatty acids and lysophospholipids, which prevents vesicle neck closure.     

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone

Black widow spider venom (BWSV) was applied to frog nerve-muscle preparations bathed in Ca2+-containing, or Ca2+-free, solutions and the neuromuscular junctions were studied by the freeze-fracture technique. When BWSV was applied for short periods (10-15 min) in the presence of Ca2+, numerous dimples (P face) or protuberances (E face) appeared on the presynaptive membrane and approximately 86% were located immediately adjacent to the double rows of large intramembrane particles that line the active zones. When BWSV was applied for 1 h in the presence of Ca2+, the nerve terminals were depleted of vesicles, few dimples or protuberances were seen, and the active zones were almost completely disorganized. The P face of the presynaptic membrane still contained large intramembrane particles. When muscles were soaked for 2-3 h in Ca2+-free solutions, the active zones became disorganized, and isolated remnants of the double rows of particles were found scattered over the P face of the presynaptic membrane. When BWSV was applied to these preparations, dimples or protuberances occurred almost exclusively alongside disorganized active zones or alongside dispersed fragments of the active zones. The loss of synaptic vesicles from terminals treated with BWSV probably occurs because BWSV interferes with the endocytosis of vesicle membrane. Therefore, we assume that the dimples or protuberances seen on these terminals identify the sites of exocytosis, and we conclude that exocytosis can occur mostly in the immediate vicinity of the large intramembrane particles. Extracellular Ca2+ seems to be required to maintain the grouping of the large particles into double rows at the active zones, but is not required for these particles to specify the sites of exocytosis.