The distribution of and interactions between GABA-immunoreactive and non-immunoreactive processes presynaptic to afferents from campaniform sensilla on the trochanter of the locust leg

Summary Campaniform sensilla on the trochanter of the mesothoracic legs of the locust were backfilled with cobalt salts or horseradish peroxidase for light and electron microscopy. The distribution of the terminal branches of afferent neurones in the thoracic ganglia were described from wholemount preparations and from thick slices through the ganglia. Ultrathin sections of identified branches were processed with GABA antibodies using a post-embedding immunogold technique and examined in the electron microscope. Input synapses were observed on fine varicose branches in all regions of the terminal arborisations close to the sites of afferent output. The major branches neither make nor receive synapses. Seventy-two percent of the input synapses are made by processes immunoreactive for GABA. Immunoreactive and non-immunoreactive processes synapse onto afferent terminals in close proximity. In some instances GABA-immunoreactive processes presynaptic to an afferent are also presynaptic to a non-immunoreactive presynaptic processes strongly suggesting that different presynaptic influences can interact directly with each other.     

GABA and glutamate-like immunoreactivity at synapses received by dorsal unpaired median neurones in the abdominal nerve cord of the locust

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.     

Types, numbers and distribution of synapses on the dendritic tree of an identified visual interneuron in the brain of the locust

The descending contralateral movement detector (DCMD), an identified descending interneuron in the brain of the locust Schistocerca gregaria has been investigated by using light and electron microscopy. We describe the fine structure, distribution and numbers of synapses that it receives from another identified brain neuron, the lobular giant movement detector (LGMD), and from unidentified neurons. The DCMD dendrites emerging from the integrative segment vary in form and number between individuals and sexes but always form a flattened dendritic domain. The arborizations and the integrative segment appear to be exclusively postsynaptic. Two types of synaptic contacts (Type 1 and 2) onto the DCMD can be discerned as having either round (Type 1) or pleiomorphic synaptic vesicles (Type 2) and by large (Type 1) or small (Type 2) subsynaptic appositions. Contact zones of Type 1 synapses are smaller than those of Type 2. LGMD-synapses are of Type 1 and occur intermingled with presynaptic sites of unidentified units. Some branches of the DCMD receiving input from unidentified units are devoid of contacting LGMD processes. Synapses of both types are randomly distributed over the DCMD integrative segment and at fibres with similar sizes.Type 1 synapses are much more frequent than Type 2 synapses and their number is negatively correlated with fibre diameter. For a whole DCMD dendritic arborization, a total of 8500 active zones of chemical synapses has been calculated, including a minimum of 2250 LGMD-synapses and about 1000 Type 2 synapses. The DCMD may thus receive a considerable amount of input from as yet unidentified neurons.     

Spatial distribution of synapses onto thoracic motor neurones in locusts

Summary Aggregates of synaptic vesicles, stained black by the zinc iodide-osmium procedure, can be visualised with the light microscope in 1 m plastic sections. This allows the main branches of a neurone to be reconstructed relatively rapidly and the associated vesicle aggregates to be plotted. By resectioning, the identity of the vesicle aggregates has been confirmed with the electron microscope. Two flight motor neurones in the mesothoracic ganglion of the locust have been examined. One is identified as a dorsal longitudinal muscle motor neurone (muscle 112) and the other is probably a subalar neurone (muscle 99). Both have a large density of vesicle aggregates on the neuropilar segment, the widest part of the main neuronal axis, but few on the neurite within 250 m of the cell body. The larger branches arising from the neuropilar segment tend to have a lower density of aggregates than fine branches, which suggests that synapses to the branches may occur mainly on the distal twigs. These results are an important preliminary step in determining the integrative functions of such neurones and have immediate implications in the interpretation of microelectrode recordings.JSA is supported by grant KU 240/3 from the Deutsche Forschungsgemeinschaft to Dr. W. Kutsch. We thank Mrs. Christine Davies for valuable assistance with the resectioning technique.     

Arrested exocytosis of synaptic vesicles at a glutamatergic synapse,the insect fast excitatory neuromuscular junction

New ultrastructural evidence supporting the vesicular theory of neurotransmitter release has been obtained using a tannic acid incubation technique. In tannic acid-treated muscles of the locust (Schistocerca gregaria), sites of arrested synaptic vesicle fusion are present at synapses of the fast excitatory neuromuscular junction (NMJ). Tannic acid treatment, prior to aldehyde fixation, permits a degree of normal cell function to be maintained so that exocytosis can continue. This results in large numbers of fused vesicles, found merging fully with the presynaptic membrane throughout the synapses. A possible mechanism of membrane retrieval has also been identified, involving large invaginations from the presynaptic membrane.     

Dendro-dendritic and reciprocal synapses in the primate motor cortex.

Dendro-dendritic synapses have been observed infrequently in the deep layers of the motor cortex. The presynaptic dendrites are of a varicose type and themselves receive a considerable density of synapses both of the asymmetric and symmetrical type. The ultrastructure of the dendro-dendritic synapse itself shows the typical arrangement of presynaptic and postsynaptic membrane densities, often with presynaptic dense projections, and the membrane specialization is of the symmetrical type. There is the usual cleft containing electron-dense material between the presynaptic and postsynaptic profiles. The synaptic vesicles occur in a small cluster confined to a region close to the presynaptic membrane specialization; some of the vesicles are flattened and were shown by tilt analysis to be of the discoid type. Two examples were found of reciprocal dendro-dendritic synapses, both components being of the symmetrical type. A single axon terminal may make a synapse on to both dendrites involved in a dendro-dendritic synapse.     

The monosynaptic connection: the modulating effect of opioid peptides on the plasticity of presynaptic neurons and identified synapses]

Study of opioid peptides (leucine-enkephalin and methionine-enkephalin) action on plastic properties of the system of monosynaptically connected neurones LPa7--LPa3, PPa3 and LPa8--LPa3, PPa3 was conducted in the snail brain. It has been shown that all three links in the system studied (presynaptic neurone, postsynaptic neurone and synapse) manifest one and the same type of plasticity--habituation to rhythmic stimulation. Enkephalins have a modulating action on plastic properties of the presynaptic neurone and synapse: they retard the habituation of the presynaptic neurone to intracellular stimulation and retard the development of habituation at synaptic level. However, changes in the character of postsynaptic response in the presence of enkephalins are not a direct consequence of their influence on plastic properties of the presynaptic neurone. Besides, enkephalines reduce the effectiveness of synaptic transmission in the given system: they reduce EPSP duration in the postsynaptic neurone.     

Presynaptic depolarization rate controls transmission at an invertebrate synapse

Second-order neurons L1-3 of the locust ocellar pathway make inhibitory synapses with each other. Although the synapses transmit graded potentials, transmission depresses rapidly and completely so that a synapse only transmits when the presynaptic terminal depolarizes rapidly. The rate at which a presynaptic neuron depolarizes determines the rate at which a postsynaptic neuron hyperpolarizes, and neurotransmitter is only released during a fixed 2 ms long period. Consequently, the amplitude of a postsynaptic potential depends on the rate rather than the amplitude of a presynaptic depolarization. Following a postsynaptic potential, a synapse recovers from depression over about a second. The synapse recovers from depression even if the presynaptic terminal is held depolarized.     

The monosynaptic connection: identified synapses in the CNS of the edible snail

By electrophysiological and microanatomical methods of analysis of snail CNS small neurones it was shown that a number of neurones form a monosynaptic connection (MSC) with the gigantic polyfunctional neurone LPa3. By using cobalt and nickel staining, the structure of MSC cells LPa7--LPa3 was studied. Six identified synapses in two LPa3 processes zones were found. Physiological analysis showed that the revealed MSC was plastic. The described MSC with identified synapses is convenient for studying synaptic transmission mechanisms.     

Preparation-dependent distribution of intramembranous particles in freeze-fracture replicas of the neuromuscular junction of the locust Schistocerca gregaria

Summary Freeze-fracture replicas of the neuromuscular junction were prepared from untreated retractor unguis muscles of the locust Schistocerca gregaria that were rapidly frozen by contact with a copper block cooled by liquid helium. These replicas were compared with others prepared from tissue following fixation with glutaraldehyde and cryoprotection in glycerol. Freeze-fracture of rapidly frozen tissue produced replicas of high quality with little evidence of tissue damage by ice crystals in the superficial layers. The gross fracturing characteristics of the neuromuscular junction were consistent with replicas from fixed and cryoprotected tissue; all of the membrane specializations were recognisable but with some alterations in infrastructure. In tissue replicas prepared using either method intramembranous particles in the presynaptic membrane were arranged in a bar-like array. The intramembranous particles of this presynaptic bar array of the rapidly frozen material were large and found on the E-face of the cleaved membrane. This contrasts with the P-face distribution of the comparable particles in muscles fixed in glutaraldehyde and cryoprotected in glycerol, in which they are also smaller and more numerous. This difference in partitioning between rapidly frozen, and fixed, cryoprotected nerve terminals is not found at cholinergic synapses and thus may reflect functional differences between the two types of junction.Indentations of the nerve-terminal membrane occur in replicas from rapidly frozen muscle as well as fixed and cryoprotected muscle suggesting they are not fixation or glycerol-induced artifacts. It is suggested from their position and size that these indentations are more likely to be part of a membrane retrieval system than exocytotic figures.This work was supported by an S.E.R.C. project grant to I.R.D.     

The organization of synaptic vesicles at tonically transmitting connections of locust visual interneurons

Large, second‐order neurons of locust ocelli, or L‐neurons, make some output connections that transmit small changes in membrane potential and can sustain transmission tonically. The synaptic connections are made from the axons of L‐neurons in the lateral ocellar tracts, and are characterized by bar‐shaped presynaptic densities and densely packed clouds of vesicles near to the cell membrane. A cloud of vesicles can extend much of the length of this synaptic zone, and there is no border between the vesicles that are associated with neighboring presynaptic densities. In some axons, presynaptic densities are associated with discrete small clusters of vesicles. Up to 6% of the volume of a length of axon in a synaptic zone can be occupied with a vesicle cloud, packed with 4.5 to 5.5 thousand vesicles per μm³. Presynaptic densities vary in length, from less than 70 nm to 1.5 μm, with shorter presynaptic densities being most frequent. The distribution of vesicles around short presynaptic densities was indistinguishable from that around long presynaptic densities, and vesicles were distributed in a similar way right along the length of a presynaptic density. Within the cytoplasm, vesicles are homogeneously distributed within a cloud. We found no differences in the distribution of vesicles in clouds between locusts that had been dark‐adapted and locusts that had been light‐adapted before fixation. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 93–105, 2002; DOI 10.1002/neu.10018     

Effect of hypoxia on the structure of the presynaptic grid of the interneuronal contacts of the rat neocortex

Hypoxic effect (a 6 minute asphyxia) on the presynaptic grid structure and on the amount of synapses in the neocortex has been studied in white rats using the method of selective staining of synapses with phosphoric tungsten acid. Neurofilamentous formations of the presynaptic grid appeared to be most labile structures. Dense projections of the presynaptic grid are most sensitive to hypoxia: their height, distinctness of the frame, and the intensity degree of phosphoric tungsten acid staining decrease. The content of intermediate form contacts with low indistict dense projections increases with the increase in the number of light type changed synapses. The principle organization of the presynaptic grid does not change in the posthypoxic period: hexagonal division of dense projections and places of vesicular attachment are kept. The hypertrophy of the presynaptic grid is also kept during that process.     

The innervation of the closer muscle of the mesothoracic spiracle of the locust.

The closer muscle of the mesothoracic spiracle of the locust, Schistocerca gregaria is innervated by two excitatory motoneurones and also by processes of a peripherally located neurosecretory cell. Within the muscle, ultrastructural studies show the presence of two types of excitatory nerve terminal which differ in the content of dense cored vesicles and in their distribution. The ventral segment of the muscle is innervated predominantly by terminals with small clear vesicles and only an occasional dense-cored vesicle. The central part of the muscle is innervated predominantly by terminals with small clear vesicles and larger numbers of dense-cored vesicles. The dorsal segment of the muscle is innervated exclusively by a neurosecretory type innervation. The small neurohaemal organ of the median nerve close to the spiracle muscle is immunoreactive to an antibody raised against bovine pancreatic polypeptide but no immunoreactive processes enter the muscle itself. The muscle possesses specific octopaminergic receptors that increase cyclic AMP levels and the possibility that the neurosecretory input to the muscle is provided by either a central or peripheral octopamine containing neurone is discussed.     

Peculiarities of ultrastructural organization of the metathoracic ganglion in the locustLocusta migratoria larva

In electron microscopic study of structural organization of the thoracic ganglion of the locust larva of the 1st age (1–2 days after hatching), the data on the structure of motoneurons of the 1st nerve, basal and motor neuropil of the larva were obtained. The effector elements of the larval locust CNS are formed rather early and have the structural plan similar to that in adult insects. However, in the larval motoneurons innervating the flight muscles (longitudinal dorsal muscles, wing depressors) the clearly seen features of immaturity of these nervous elements are revealed. Study of the larval ganglion neuropil has shown that the basal neuropil is morphologically formed sufficiently completely as early as in larvae of the first days after hatching. There are shown longitudinal contacts between axons of the ventral neuropil zone, the presence of axons forming theen-passant contacts as well as the synapses with a heterogeneous set of vesicles in the presynaptic area. The presence of the great number of granular vesicles in the basal neuropil of the locust larva may indicate an important role of catecholamines in the early development of the nervous system in the locust larva.     

The organization of synaptic vesicles at tonically transmitting connections of locust visual interneurons.

Large, second-order neurons of locust ocelli, or L-neurons, make some output connections that transmit small changes in membrane potential and can sustain transmission tonically. The synaptic connections are made from the axons of L-neurons in the lateral ocellar tracts, and are characterized by bar-shaped presynaptic densities and densely packed clouds of vesicles near to the cell membrane. A cloud of vesicles can extend much of the length of this synaptic zone, and there is no border between the vesicles that are associated with neighboring presynaptic densities. In some axons, presynaptic densities are associated with discrete small clusters of vesicles. Up to 6% of the volume of a length of axon in a synaptic zone can be occupied with a vesicle cloud, packed with 4.5 to 5.5 thousand vesicles per microm(3). Presynaptic densities vary in length, from less than 70 nm to 1.5 microm, with shorter presynaptic densities being most frequent. The distribution of vesicles around short presynaptic densities was indistinguishable from that around long presynaptic densities, and vesicles were distributed in a similar way right along the length of a presynaptic density. Within the cytoplasm, vesicles are homogeneously distributed within a cloud. We found no differences in the distribution of vesicles in clouds between locusts that had been dark-adapted and locusts that had been light-adapted before fixation.     

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.     

Serial synapses in Aplysia

Serial synapses occur between small profiles in the neuropil of Aplysia abdominal ganglion. Material was fixed in phosphate buffered OsO₄, embedded in epon, and sections were stained with uranyl acetate and lead citrate. A class of synapses had the following characteristics: (1) synaptic vesicles clustered against the presynaptic membrane, (2) a widened extracellular space of about 20 nm containing electron-dense material, (3) straightening of the pre- and postsynaptic membranes, and (4) no postsynaptic membrane specialization. Some density between the presynaptic membrane and the adjacent synaptic vesicles was occasionally observed. Synapses occurred between small profiles in the neuropil (typical profile diameters were 1–3 m?m). In this sample of approximately 100 synapses, four serial synapses were identified. The serial synaptic profiles were all small. In addition to the finding of serial synapses, 40% of the postsynaptic profiles contained vesicles similar to the synaptic vesicles seen in presynaptic profile. Serial synapses may be the anatomical substrate of presynaptic inhibition and facilitation and of dishabituation.     

Localization of smg p25A/rab3A p25, a small GTP-binding protein, at the active zone of the rat neuromuscular junction.

smg p25A is a small G protein which has been suggested to regulate neurotransmitter release from the synapses. We investigated here the ultrastructural localization of this small G protein in the rat neuromuscular junction by an immunoperoxidase method. The results showed that smg p25A was distributed non-uniformly on the presynaptic plasma membrane and among the synaptic vesicles with the focal accumulation on the discrete presynaptic sites which corresponded to the active zones, the regions of the presynaptic plasma membrane specialized for the exocytosis of the synaptic vesicles. This unique distribution of smg p25A suggests that it plays an important role in the attachment and fusion of the synaptic vesicles with the active zones.     

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling.     

Ultrastructure of the lateral-line sense organs of the ratfish,Chimaera monstrosa

Summary The ultrastructure of the lateral-line neuromasts in the ratfish, Chimaera monstrosa is described. The neuromasts rest at the bottom of open grooves and consist of sensory, supporting, basal and mantle cells. Each sensory cell is equipped with sensory hairs consisting of a single kinocilium and several stereocilia. There are several types of sensory hair arrangement, and cells with a particular arrangement form patches within the neuromast. There are two types of afferent synapse. The most common afferent synapse has a presynaptic body and is typically associated with an extensive system of anastomosing tubules on the presynaptic side. When the tubules are absent, vesicles surround the presynaptic body. These synapses are often associated into synaptic fields, containing up to 35 synaptic sites. The second type of afferent synapse does not have a presynaptic body and is not associated with the tubular system. The afferent synapses of the second type do not form synaptic fields and are uncommon. The efferent synapses are either associated with a postsynaptic sac or more commonly with a strongly osmiophilic postsynaptic membrane. The accessory cells are similar to those in the acoustico-lateralis organs of other aquatic vertebrates. A possibility of movement of the presynaptic bodies and of involvement of the tubular system in the turnover of the transmitter is discussed. A comparison of the hair tuft types in the neuromasts of Ch. monstrosa with those in the labyrinth of the goldfish and of the frog is attempted.