Synaptic Vesicle Clusters Research Articles

Firing patterns of isolated goldfish retinal ganglion cells and their regulation by a Ba2 +-sensitive K + current

Spontaneous and light-evoked excitatory postsynaptic currents (sEPSCs and leEPSCs) in retinal ganglion cells of the larval tiger salamander were recorded under voltage clamp conditions from living retinal slices. sEPSCs were isolated from the spontaneous inhibitory postsynaptic currents (sIPSCs) by application of 100 M picrotoxin+1 μM strychnine. In addition to the previously reported sEPSCs [K. Matsui, N. Hosoi, M. Tachibana, Excitatory synaptic transmission in the inner retina: pair recordings of bipolar cells and neurons of the ganglion cell layer, J. Neurosci. 18 (1998) 4500–4510; W.R. Taylor, E. Chen, D.R. Copenhagen, Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells, J. Physiol. 486 (1995) 207–221] [which are equivalent to our fast AMPA receptor-mediated sEPSCs (fAMPAsEPSCs)], we found another type of AMPA receptor-mediated sEPSC with slower rise and decay time courses and larger peak amplitudes (sAMPAsEPSCs), and the NMDA receptor-mediated sEPSCs (NMDAsEPSCs) in ON–OFF ganglion cells. The frequency of all three types of sEPSCs is greatly reduced by cobalt (with zero calcium) and increased by hyperosmotic solution, suggesting that these events are mediated by calcium-dependent exocytosis of glutamatergic synaptic vesicles. The amplitude histograms of sEPSCs do not show multiple peaks, suggesting that larger events are not discrete multiples of elementary events, or quanta, of similar neurotransmitter contents, as in the neuromuscular junction [P. Fatt, B. Katz, Spontaneous subthreshold activity at motor nerve endings, J. Physiol. 117 (1952) 109–128]. The average I–V relations of the fAMPAsEPSCs and sAMPAsEPSCs were outward rectified with reversal potentials at −12.2 mV and −10.8 mV, and that of the NMDAsEPSCs was N-shaped with a reversal potential at −5.8 mV. The average conductance increase associated with a single fAMPAsEPSC, a single sAMPAsEPSC, and a single NMDAsEPSC were 163.26±51.02 pS, 233.33±163.64 pS, and 37.5±50.0 pS at −110 mV; 241.67±22.92 pS, 444.90±469.94 pS, and 25.93±70.37 pS at −60 mV; and 440.48±183.33 pS, 1,192.68±651.22 pS, and 517.71±238.24 pS at +30 mV, respectively. The average frequency of the three sEPSCs at +30 mV were 15 Hz, 3.7 Hz and 3.6 Hz, respectively. The rise time (time to peak) of fAMPAsEPSCs was 1.5±1.05 ms and the decay time could be fitted with a single exponential with an average time constant of 3.4±4.1 ms. The rise and decay time course of the sAMPAsEPSCs and NMDAsEPSCs were much slower and sawtooth-shaped, and each `sawtooth' had time course and amplitude similar to those of individual fAMPAsEPSCs. We propose that each fAMPAsEPSC is mediated by single or synchronized multiples of glutamatergic synaptic vesicles from bipolar cells, and each sAMPAsEPSC or NMDAsEPSC is mediated by larger clusters of synaptic vesicles triggered by spontaneous calcium spikes in bipolar cell axon terminals [J. Burrone, L. Lagnado, Electrical resonance and calcium influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina, J. Physiol. 505 (1997) 571–584; D. Zenisek, G. Matthews, Calcium action potentials in retinal bipolar neurons, Vis. Neurosci. 15 (1998) 69–75].

Fluorescence microscopy of calcium and synaptic vesicle dynamics during synapse formation in tissue culture.

The signal transduction process involved in the development of the nerve terminal is an intriguing question in developmental neurobiology. During the formation of the neuromuscular junction, presynaptic development is induced by growth cone's contact with the target muscle cell. Fluorescence microscopy with specific markers has made it possible to follow signalling events during this process. By using fluorescent calcium indicators, such as fura-2 and fluo-3, we found that a rise in intracellular calcium is elicited in the growth cone upon its contact with a target, and this calcium signal can also be elicited by local application of basic fibroblast growth factor. To monitor the clustering of synaptic vesicles in response to target contact, the fluorescent vesicular probe FMl-43 was used. With this probe, we observed that packets of synaptic vesicle are already present along the length of naïve neurite, which has not encountered its synaptic target. The activity-dependent loading of FMl-43 indicates that these packets can undergo exocytosis and endocytosis upon depolarization. Time-lapse recording showed that these packets are quite mobile. Upon target contact, synaptic vesicles become clustered and immobilized at the contact site. The methodology and instrumentation used in these studies are described in this article.

Association of cortactin with developing neuromuscular specializations.

During the development of the neuromuscular junction (NMJ), motoneurons grow to the muscle cell and the nerve-muscle contact triggers the development of both presynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of acetylcholine receptors (AChRs). Previous studies have shown that the activation of tyrosine kinases and the local assembly of an actin-based cytoskeletal specialization are involved in the development of both types of specializations. To understand the link between tyrosine phosphorylation and the assembly of the cytoskeleton, we examined the localization of cortactin in relationship to synaptic development. Cortactin is a 80/85 kD F-actin binding protein and is a substrate for tyrosine kinases. It contains a proline-rich motif and an SH3 domain and is localized at sites of active F-actin assembly. Using a monoclonal antibody against cortactin, its localization at developing NMJs in culture was observed. To understand the spatial and temporal relationship between cortactin and developing synaptic structures, cultured muscle cells and spinal neurons from Xenopus embryos were treated with beads coated with heparin-binding growth-associated molecule to induce the formation of AChR clusters and SV clusters and the localization of cortactin was followed by immunofluorescence. In untreated muscle cells, cortactin is often co-localized with spontaneously formed AChR clusters. After cells were treated with beads, cortactin became localized at bead-induced AChR clusters at their earliest appearance (1 h after the addition of beads). This association was most reliably detected at the early stage of the clustering process. On the presynaptic side, cortactin localization could be detected as early as 10 min after the bead-neurite contact was established. Cortactin-enriched contacts later showed concentration of F-actin (at 1 h) and clusters of SVs (at 24 h). These data suggest that cortactin mediates the local assembly of the cytoskeletal specialization triggered by the synaptogenic signal on both nerve and muscle.

Traffic of Dynamin within IndividualDrosophilaSynaptic Boutons Relative to Compartment-Specific Markers

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identified Drosophila neuromuscular synapse that several of these compartments-synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton-labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers and Drosophila shibire(ts1) mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. In shi(ts1) nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show "hot spots" of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.

Synaptic Vesicle Movements Monitored by Fluorescence Recovery after Photobleaching in Nerve Terminals Stained with FM1-43

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Dynamics of Synaptic Vesicles in Cultured Spinal Cord Neurons in Relationship to Synaptogenesis

The dynamics of synaptic vesicles (SVs) during the development of presynaptic specializations in culturedXenopusspinal cord neurons was studied with the fluorescent vesicular probe FM1-43. In naive neurons that have not contacted synaptic targets, packets of SVs are distributed along the entire neurite and are quite mobile. The interaction with the synaptic target, such as a muscle cell or a latex bead coated with basic fibroblast growth factor, results in the localization and immobilization of SV packets at the contact site. Depolarization resulted in exocytosis of SVs in both naive and target-contacted neurites. Okadaic acid, a phosphatase inhibitor, caused a dispersal of SV packets in both naive and target-contacted neurites. Thus, prior to target contact, SVs are already organized into packets capable of release and recycling by a phosphorylation-dependent mechanism. Target interaction then recruits and anchors these functional SV packets into forming the presynaptic nerve terminal. With fluorescent phalloidin as a probe, F-actin was found to colocalize with SV clusters at bead-neurite contacts. Although okadaic acid caused a dispersal of SVs at the beads, F-actin localization there was relatively resistant to this drug treatment. This suggests that SVs become localized at the target by interacting with an actin-based cytoskeletal specialization in a phosphorylation-sensitive manner. The induction of this cytoskeletal specialization by the target may be an early event in presynaptic differentiation.

Structural characterization of peripheral nerve cells and nerve-muscle junctions of the oviduct of stable fly (Diptera:Muscidae).

Fine structure of both peripheral nerve cells and neuromuscular junctions associated with the oviduct of stable fly. Stomoxys calcitrans (L.), was described. Twelve or more multipolar peripheral neurons were found along major branch nerves that enter the ovipositor. Several were suspended in the haemacoel and others were in close proximity to the surface of the oviduct. Some peripheral neurons contained an abundance of neurosecretory granules that ranged in size from 32 to 180 nm in diameter. No glial elements enveloped the perikarya of such cells. Neurosecretory axons were usually found in the boundary region of large nerves just beneath the stroma. Peripheral nerve cells in close apposition to oviduct muscles were generally non-neurosecretory and were ensheathed in a glial perineurium. Peripheral neurons were surrounded occasionally by an extensive network of extracellular spaces in the glial perineurium. Other smaller neurons were found within large nerve trunks. Nerve-muscle junctions contained large clusters of synaptic vesicles and occasionally included small groups of neuro-secretory granules. Many nerve terminals on the surface of the oviduct contained a complex postsynaptic folding of sarcolemma. Active transmission sites were indicated by increased densities along the neurolemma. Some neurohemal release sites were also evident.

Synaptic Spinules Attendant on Post-tetanic Potentiation in Cat Sympathetic Ganglion.

A synaptic spinule formed on the postsynaptic membrane in the cat superior cervical ganglion (SCG) in vivo was electron microscopically studied. When the preganglionic nerve fiber was tetanically stimulated at 10Hz for more than 10 seconds, a post-tetanic potentiation (PTP) was electrophysiologically detected and maintained up to 30min. Concurrently with PTP, a small process, termed as synaptic spinule, was generated as a small projection standing out from the postsynaptic dendritic membrane with a straightly extended contour. The spinule produced a break at about a middle point of the postsynaptic density (PSD) and divided it into two parts forming a perforated synapse. Then, the spinule increased in size, invaginated into the cytoplasm of the nerve terminal, and subdivided the presynaptic terminal bag into two compartments. Each of the compartments retained a morphology of an active synaptic site with a cluster of synaptic vesicles associated with a presynaptic active zone standing opposite to the one part of the subdivided PSD. The longer the tetanic stimulation in period, the more the synapses with the spinule increased in number. In addition to this, a few spinules (multiple type of spinule) were often formed after the stimulation of 1min and more than two compartments were generated in a single nerve ending. The present observations have suggested that the synaptic spinule is a morphological representation of the “synaptic plasticity” which is electrophysiologically displayed as the PTP in cat SCG in vivo.

Chapter 2 Redistribution of clathrin and synaptophysin at the frog neuromuscular junction triggered by nerve stimulation: immunocytochemical studies of vesicle trafficking

This chapter focuses on the study of the dynamics of synaptic vesicles during nerve stimulation; it uses antibodies against clathrin—the major protein of coated vesicles—and synaptophysin—an integral membrane protein of synaptic vesicles—to monitor the distribution of synaptic vesicles and clathrin. There are currently three models for synaptic vesicle recycling under debate—recycling via clathrin coated vesicles, recycling via endocytotic cistemae, and the kiss and run mechanism that does not postulate the total fusion of the vesicle with the plasma membrane, but rather proposes a short opening of a fusion pore. The major component of coated vesicles is accumulated in small intra-synaptic spots if the terminal is highly stimulated. In a resting terminal, this protein shows a more or less widespread distribution inside the synapse with a trend toward clustering. These clusters resemble synaptic vesicle clusters regarding shape and distribution, but future double-labeling experiments have to be done to show the identity of clathrin and vesicle clusters. It has not yet become clear if the stimulation-dependent clathrin aggregation is directly related to the formation of coated vesicles.

Characterization of synaptic vesicles and related neuronal features in nerve growth factor and ras oncogene differentiated PC12 cells.

PC12 cells can differentiate into neuron-like cells after treatment with either nerve growth factor (NGF) or transduction with a retrovirus which expresses the K-ras oncogene. The concomitant treatment of NGF plus ras differentiates PC12 cells further than either agent alone with respect to neurite outgrowth, acetylcholinesterase levels, and most strikingly, the number of synaptic vesicle (SV) clusters. These SV clusters in PC12 cell neurites closely resemble those in the presynaptic terminals of neurons. Such SV clusters have not been described in cell lines previously. The SV clusters from all three differentiated groups (NGF, ras, and NGF plus ras) were similar in size, shape, and configuration, except that the ones in the doubly treated group occur in higher frequency and have more vesicles. The synaptic nature of these vesicle clusters was demonstrated by their regulated depletion after potassium stimulation. Furthermore, these vesicle clusters stained positively for two SV-associated proteins, synapsin I and synaptophysin, by EM immunocytochemistry (ICC). Such SV clusters in a cell line are very useful for characterizing the regulated release of SVs and the distribution of SV-related antigens in intact cells. Analysis by SDS-gel electrophoresis and immunoblotting indicated that synapsin I levels are higher in all three differentiated groups compared to untreated cells; whereas synaptophysin levels are lower in cells exposed to NGF alone or with NGF and ras double treatment. Possible convergence and/or divergence on the mechanisms of NGF and ras differentiation in PC12 cells are discussed.

Cd2+- and K+-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction

Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd(2+)- or K(+)-induced ACH release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd2+ in Ca(2+)-free medium for a period of 3 h, (2) 25 or 40 mM K+ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluorescein-conjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated alpha-bungarotoxin. Unstimulated and K(+)-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd2+ in Ca(2+)-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K(+)-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd2+ stimulation in Ca(2+)-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd2+ stimulation in Ca(2+)-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K+ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.

Presynaptic differentiation induced in cultured neurons by local application of basic fibroblast growth factor

Recent studies have suggested a role for molecules residing at the muscle surface in signaling presynaptic development at the neuromuscular junction (NMJ). Since heparan sulfate-proteoglycan is a major component of the extracellular matrix of skeletal muscle, factors that are bound to this proteoglycan, such as basic fibroblast growth factor (bFGF), are in a strategic position for neuronal signaling. To test this idea, we applied bFGF to cultured Xenopus spinal cord neurons and monitored the change in intracellular Ca2+ level with fura-2 ratio imaging. In one-third of the neurons, local application of bFGF elicited a 30-140% increase in intracellular Ca2+ level. Ca(2+)-free medium or suramin abolished this change. Latex beads coated with bFGF induced clustering of synaptic vesicles at the bead-neurite contacts as evidenced by anti-synaptotagmin antibody labeling and electron microscopy. This response was also blocked by Ca(2+)-free medium and by suramin. Uncoated beads or beads coated with PDGF were ineffective. This induction was also inhibited by a tyrosine kinase inhibitor, tyrphostin RG-50864, suggesting the role of receptor tyrosine kinase activation in this process. In addition, bFGF-coated beads also induced the localization of depolarization-dependent Ca2+ influx to the bead-neurite contacts. In contrast, depolarization caused a distributed Ca2+ elevation in untreated neurites. These results suggest that local presentation of bFGF can mimic the muscle target in signaling the development of both a cytoplasmic and a membranous specialization for excitation-secretion coupling observed at the NMJ.

Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin

Antibodies directed against the lumenal domain of synaptotagmin I conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons. When applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization. The increase of the rate of CY3-Syt1-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min. In axons developing in isolation, CY3-Syt1-Abs, in combination with electron microscopy immunocytochemistry, revealed the presence of synaptic vesicle clusters which move in bulk in anterograde and retrograde direction. Clusters are present both in the axon shaft and in filopodia but not in the filopodia of the growth cone. Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previously shown for synaptic vesicle clusters at the frog neuromuscular junction. These findings indicate that synaptic vesicle aggregation may occur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a given region of the cell surface. Labeling of synaptic vesicles in immature isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic characteristic of synaptic vesicles irrespective of their localization at a synapse.

Synaptic vesicle depletion in reticulospinal axons is reduced by 5-hydroxytryptamine: direct evidence for presynaptic modulation of glutamatergic transmission.

5-hydroxytryptamine (5-HT; serotonin) is known to depress glutamatergic synaptic transmission in the spinal cord of vertebrates. To test directly whether 5-HT inhibits synaptic glutamate release, we examined its effect on the ultrastructure of synaptic vesicle clusters in giant reticulospinal axons in a lower vertebrate (lamprey; Lampetra fluviatilis). The size of these axons makes it possible to selectively expose only a part of the presynaptic element to 5-HT, while another part of the same axon is maintained in control solution. Action potential stimulation at 20 Hz for 20 min caused a marked reduction in the number of synaptic vesicles in active zones maintained in control solution, while in the part exposed to 5-HT (20 microM) the number of synaptic vesicles per active zone was approximately 3-fold higher. In contrast, 5-HT had no effect on the number of vesicles in resting axons. To examine whether 5-HT acts by reducing presynaptic Ca2+ influx, intra-axonal recordings of Ba2+ potentials were performed. No reduction of the axonal Ba2+ potential could be detected after application of 20 or 200 microM 5-HT. The present results show that 5-HT reduces the rate of synaptic exocytosis in reticulospinal axons. The effect appears to be mediated by a mechanism distinct from the presynaptic Ca2+ channels.

The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei.

Silver enhancement of small gold particles can be used with pre-embedding immunocytochemistry to analyze the distribution of label over cell organelles. We have developed a method that improves tissue morphology, has good penetration of reagents, and allows greater control of silver enhancement of 1.4-nm gold. In this study we analyzed the distribution of glutamic acid decarboxylase (GAD), a synthetic enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), in the cerebellar nuclei of the mouse. Pre-embedding immunocytochemistry was carried out on brain sections fixed with high concentrations of glutaraldehyde and sodium metabisulfite. After incubations with a monoclonal antibody to GAD and a 1.4-nm NanoGold-labeled secondary antibody, sections were silver-enhanced with N-propyl gallate as a reducing agent and MES as a new buffer system. In the cerebellar nuclei, GAD label was specifically localized in axon terminals over clusters of synaptic vesicles. These terminals formed axosomatic and axodendritic contacts. The majority of GAD-labeled terminals had cytological characteristics indicating their origin from Purkinje cells, which are known to contain GAD.

Peripheral connections of FMRFamide-like immunoreactive neurons in the snail, helix pomatia: an immunogold electron microscopic study.

Using a postembedding immunogold electron microscopic method, the ultrastructure and synaptic connections of FMRFamide-like immunoreactive varicosities were investigated in different peripheral organs of the snail Helix pomatia, including the heart (auricle), intestine, hepatopancreas, upper tentacle and salivary gland. The FMRFamide-like immunoreactive varicosities contained granules and vesicles as described in a previous study of the CNS of this species, and additionally, based on their granule content, two novel types of varicosities were found in the auricle. A selective accumulation of gold particles over the granules could be demonstrated. The FMRFamide-like immunoreactive varicosities formed unspecialized contacts with postsynaptic target cells in all peripheral organs investigated, with the exception of the tentacle retractor muscle. Both the neuro-muscular and the neuro-glandular contacts were characterized by either unspecialized close apposition of the 'pre- and postsynaptic' membranes or the immunoreactive elements faced the target cell(s) across a relatively wide extracellular space. In the tentacle retractor muscle some of the neuromuscular contacts showed appositions of electron dense material along the presynaptic membrane, clustering of agranular synaptic vesicles and intersynaptic cleft material. The present observations support previous electrophysiological findings and suggest a versatile modulatory role of FMRFamide and related substances in the Helix PNS.

Extrasynaptic localization of taurine-like immunoreactivity in the lamprey spinal cord.

Taurine is an endogenous amino acid that can occur in nerve terminals in the central nervous system and that can produce inhibitory neuronal responses. It is unclear, however, whether this amino acid can function as a synaptic transmitter. To examine the distribution of taurine at high anatomical resolution in a vertebrate, light and electron microscopic immunocytochemical postembedding techniques were applied to the lamprey spinal cord (Ichtyomyzon unicuspis and Lampetra fluviatilis), which contains many large, unmyelinated axons. The most intense immunolabeling occurred in a population of liquor-contacting cells (tanycytes), located around the central canal, which extended processes to the dorsal, lateral, and ventral margins of the spinal cord. In addition, a proportion of the taurine-immunoreactive cells contained gamma-aminobutyric acid (GABA)-like immunoreactivity. A moderate level of taurine immunoreactivity was also present in ependymal cells, located around the central canal, as well as in astrocytes throughout all regions of the spinal cord. At the ultrastructural level, the taurine immunoreactivity showed an even distribution in the cytoplasm of the labeled cells. In contrast to the glial labeling, neuronal cell bodies and axons exhibited very low levels of taurine labeling, which were similar to the level of background labeling. The synaptic vesicle clusters within the axons did not show any clear accumulation of taurine immunoreactivity. These results suggest that taurine may have metabolic roles in the lamprey spinal cord, and, as in other systems, it may take part in osmoregulation. However, the lack of immunolabeling in presynaptic elements is not consistent with a role of taurine as a synaptic transmitter.

Miniature excitatory postsynaptic currents in bipolar cells of the tiger salamander retina

The synapse between photoreceptor and bipolar cell is important for at least three reasons: (1) it is the first synapse in the visual pathway; (2) it is the best-known tonic chemical synapse; (3) it has perhaps the most complex and highly organized synaptic morphology in the entire brain. Yet little is known about how neurotransmitter is released from this synapse. We present in this report evidence which suggests that the release of photoreceptor neurotransmitter, presumably glutamate, is probably mediated by clusters of synaptic vesicles which give rise to discrete miniature excitatory postsynaptic currents (MEPSCs) in bipolar cells. The MEPSCs are Ca 2+-, osmotic- and CNQX-sensitive, and they share the same reversal potential (near − 3mV) as the glutamate-induced postsynaptic current. The frequency of MEPSCs increases upon presynaptic depolarization, and the mean peak conductance is about 54 pS. MEPSCs exhibit wide variations of amplitudes and durations, probably resulting from random variations of number of synaptic vesicles and the degree of synchronization in individual release clusters.

Synaptic targeting domains of synapsin I revealed by transgenic expression in photoreceptor cells.

Synapsins are abundant nerve terminal proteins present at all synapses except for ribbon synapses, e.g. photoreceptor cell synapses. Multiple functions have been proposed for synapsins, including clustering of synaptic vesicles and regulation of synaptic vesicle exocytosis. To investigate the physiological functions of synapsin and to ascertain which domains of synapsin are involved in synaptic targeting in vivo, we expressed synapsin Ib and its N- and C-terminal domains in the photoreceptor cells of transgenic mice. In these cells synapsin Ib is targeted efficiently to synaptic vesicles but has no significant effect on the development, structure or physiology of the synapses. This suggests that synapsin I does not have dominant physiological or morphoregulatory functions at these synapses. Full-length synapsin Ib and the N-terminal domains of synapsin Ib but not its C-terminal domains are transported to synapses, revealing that the molecular apparatus for synaptic targeting of synapsins is also present in cells which form ribbon synapses that normally lack synapsins. This apparatus appears to utilize the conserved N-terminal domains that are shared between all synapsins.

Extraordinary synapses of the unipolar brush cell: An electron microscopic study in the rat cerebellum

A neglected type of neuron, termed the unipolar brush cell, was recently characterized in the granular layer of the mammalian cerebellar cortex with several procedures, including light and electron microscopic immunocytochemistry utilizing antibodies to calretinin and neurofilament proteins. Although certain features of the unipolar brush cells were highlighted in these studies, the internal fine structure was partially obfuscated by immunoreaction product. In this study, rat cerebella were prepared for electron microscopy after perfusion fixation and Araldite embedding, and folia of the vestibulo-cerebellum, where unipolar brush cells are known to be enriched, were studied by light microscopy in semithin (0.5-1 micron) sections and by electron microscopy in ultrathin sections. Unipolar brush cells were easily identified in semithin sections immunostained with antibodies to GABA and/or glycine, and counterstained with toluidine blue. The unipolar brush cells have a pale cytoplasm and are GABA and glycine negative, while Golgi cells are darker and appear positive for GABA and, for the most part, also for glycine. Sets of identification criteria to differentiate unipolar brush cells from granule and Golgi cells in standard electron micrographs are presented. The unipolar brush cells possess many distinctive features that make them easily distinguishable from other cerebellar neurons and form unusually conspicuous and elaborate synapses with mossy rosettes. The unipolar brush cell has a deeply indented nucleus containing little condensed chromatin. The Golgi apparatus is large and the cytoplasm is rich in neurofilaments, microtubules, mitochondria, and large dense core vesicles, but contains few cisterns of granular endoplasmic reticulum. In addition, unipolar brush cells contain an unusual inclusion, which invariably lacks a limiting membrane and is made up of peculiar ringlet subunits. The cell body usually emits a thin axon and is provided with a single, large dendritic trunk that terminates with a paintbrush-like bunch of branchlets. Numerous nonsynaptic appendages emanate from the cell body, the dendritic stem, and the branchlets. The appendages contain rare organelles and lack neurofilaments. The branchlets contain numerous mitochondria, neurofilaments, large dense core vesicles, and clusters of clear, small, and round synaptic vesicles. They form extensive asymmetric synaptic junctions with one or two mossy fibers, which indicates minimal convergence of excitatory inputs. Under the postsynaptic densities, the branchlet cytoplasm displays a microfilamentous web. Besides their contact with mossy rosettes, the branchlets form symmetric and asymmetric synaptic junctions with presumed Golgi cell boutons that contain pleomorphic synaptic vesicles, indicating that the unipolar brush cells receive an inhibitory modulation. Some of these junctions are unusually extensive.(ABSTRACT TRUNCATED AT 400 WORDS)