Presynaptic Dense Projections Research Articles

C. elegans neurons have functional dendritic spines.

Dendritic spines are specialized postsynaptic structures that transduce presynaptic signals, are regulated by neural activity and correlated with learning and memory. Most studies of spine function have focused on the mammalian nervous system. However, spine-like protrusions have been reported in C. elegans (Philbrook et al., 2018), suggesting that the experimental advantages of smaller model organisms could be exploited to study the biology of dendritic spines. Here, we used super-resolution microscopy, electron microscopy, live-cell imaging and genetics to show that C. elegans motor neurons have functional dendritic spines that: (1) are structurally defined by a dynamic actin cytoskeleton; (2) appose presynaptic dense projections; (3) localize ER and ribosomes; (4) display calcium transients triggered by presynaptic activity and propagated by internal Ca++ stores; (5) respond to activity-dependent signals that regulate spine density. These studies provide a solid foundation for a new experimental paradigm that exploits the power of C. elegans genetics and live-cell imaging for fundamental studies of dendritic spine morphogenesis and function.

Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release.

Active zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens, we isolated a novel Caenorhabditis elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, active zone structure and synapse number. As a result, they have reduced spontaneous vesicle release and increased synaptic depression. cla-1 mutants show defects in vesicle distribution near the presynaptic dense projection, with fewer undocked vesicles contacting the dense projection and more docked vesicles at the plasma membrane. cla-1 encodes three isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The C-termini of all isoforms localize to the active zone. Specific loss of the ~9000 amino acid long isoform results in vesicle clustering defects and increased synaptic depression. Our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, vesicle clustering and release.

The Presynaptic Dense Projection of theCaenorhabiditis elegansCholinergic Neuromuscular Junction Localizes Synaptic Vesicles at the Active Zone through SYD-2/Liprin and UNC-10/RIM-Dependent Interactions

The active zone (AZ) of chemical synapses is a specialized area of the presynaptic bouton in which vesicles fuse with the plasma membrane and release neurotransmitters. Efficient signaling requires synaptic vesicles (SVs) to be recruited, primed, and retained at the AZ, in close proximity to voltage-dependent calcium channels that are activated during presynaptic depolarization. The electron-dense specializations at the AZ might provide a molecular platform for the spatial coordination of these different processes. To investigate this hypothesis, we examined high-resolution three-dimensional models of Caenorhabditis elegans cholinergic neuromuscular junctions generated by electron tomography. First, we found that SVs are interconnected within the bouton by filaments similar to those described in vertebrates. Second, we resolved the three-dimensional structure of the dense projection centered in the AZ. The dense projection is a more complex structure than previously anticipated, with filaments radiating from a core structure that directly contact SVs in the interior of the bouton as well as SVs docked at the plasma membrane. Third, we investigated the functional correlate of these contacts by analyzing mutants disrupting two key AZ proteins: UNC-10/RIM and SYD-2/liprin. In both mutants, the number of contacts between SVs and the dense projection was significantly reduced. Similar to unc-10 mutants, the dependence of SV fusion on extracellular calcium concentration was exacerbated in syd-2 mutants when compared with the wild type. Hence, we propose that the dense projection ensures proper coupling of primed vesicles with calcium signaling by retaining them at the AZ via UNC-10/RIM and SYD-2/liprin-dependent mechanisms.

Protein localization in electron micrographs using fluorescence nanoscopy

A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immuno-electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies, and the inaccessibility of the antigens. Here, we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. Proteins tagged with Citrine or tdEos were expressed in Caenorhabditis elegans, fixed and embedded. Tagged proteins were imaged from ultrathin sections using stimulated emission depletion microscopy (STED) or photoactivated localization microscopy (PALM). Fluorescence was correlated with organelles imaged in electron micrographs from the same sections. These methods were used to successfully localize histones, a mitochondrial protein, and a presynaptic dense projection protein in electron micrographs.

UNC-13 and UNC-10/Rim Localize Synaptic Vesicles to Specific Membrane Domains

Synaptic vesicles undergo a maturation step, termed priming, in which they become competent to fuse with the plasma membrane. To morphologically define the site of vesicle priming and identify fusion-competent synaptic vesicles, we combined a rapid physical-fixation technique with immunogold staining and high-resolution morphometric analysis at Caenorhabditis elegans neuromuscular junctions. In these presynaptic terminals, a subset of synaptic vesicles contact the plasma membrane within approximately 100 nm of a presynaptic dense projection. UNC-13, a protein required for vesicle priming, localizes to this same region of the plasma membrane. In an unc-13 null mutant, few synaptic vesicles contact the plasma membrane, suggesting that membrane-contacting synaptic vesicles represent the morphological correlates of primed vesicles. Interestingly, a subpopulation of membrane-contacting vesicles, located within 30 nm of a dense projection, are unperturbed in unc-13 mutants. We show that UNC-10/Rim, a protein implicated in presynaptic plasticity, localizes to dense projections and that loss of UNC-10/Rim causes an UNC-13-independent reduction in membrane-contacting synaptic vesicles within 30 nm of the dense projections. Our data together identify a discrete domain for vesicle priming within 100 nm of dense projections and further suggest that UNC-10/Rim and UNC-13 separately contribute to the membrane localization of synaptic vesicles within this domain.

An ultrastructural study into the effect of global transient cerebral ischaemia on the synaptic population of the cerebellar cortex in rats

The density of synapses, the shape and size of presynaptic dense projections (PDP), and the curvature of synaptic appositions in the molecular layer of the cerebellum cortex of rat at 10 min ischaemia and after 90 min, 1, 3, 7, 30 days of re-circulation were examined using quantitative ultra-structural techniques. The numerical density of mature junctions decreased significantly (44.0%) after 1 and 3 days of re-circulation, and was increased to 149.8% of the value in the control animals after 7 days of re-circulation. The restoration of the population of mature synaptic junctions was accompanied by a considerable increase of the number of immature junctions.We found a close association between the synaptic curvature and the size of PDP. The curvature of the larger junctions was consistently associated with a reduced height of PDP and a rounder shape. Synaptic curvature increased from 0.0885 (control) to 0.2041 (3 days of re-circulation) and to 0.2128 (7 days re-circulation). The maximum reduction in synaptic numerical density and larger junction curvature was found in zones of irreversibley damaged Purkinje cells. Our results revealed that the synaptic curvature and the height of the pre-synaptic dense projections undergo reciprocal changes after global transient cerebral ischemia. It is tempting to hypothesize that the positive synaptic curvature occurs as a result of changes in morphological conditions for the PDP filaments and in the shape and size of PDP and depends on the level of Ca 2+ in synaptic appositions.

Hundredth Anniversary of the “Synapse”: II. Study of the Cholinergic Synapse

Major contributions to the research on the cholinergic synapse in the last five decades was described. The original notion of active zone, composed of a presynaptic dense projection and associated synaptic vesicles, was analyzed in order to underline its functional meaning in synaptic transmission. An overview was done on the findings made on acetylcholine release (ultrastructural aspect, cytochemistry, non-vesicular hypothesis and mediatophore), acetylcholinesterase (ultrastructural localization, discovery of molecular polymorphism) and acetylcholine receptor (characterization, purification). The structural base of the smallest functional unit in a synapse was attributed to a single synaptic vesicle of active zone and corresponding area of synaptic cleft and postsynaptic membrane interacting with the neurotransmitter released from the vesicle.

Structural plasticity of optic synapses in the rat suprachiasmatic nucleus: adaptation to long-term influence of light and darkness.

Synapses of optic afferents (optic synapses) in the suprachiasmatic nucleus of hooded rats were morphometrically evaluated after exposing the animals to 12 h, 14 days, 2 months, and 8 months of constant light (light rats) and darkness (dark rats). Compared with dark rats, optic synapses from light rats have larger boutons with larger mitochondria, more clear vesicles, fewer dense-core vesicles and front-line vesicles, smaller presynaptic dense projections, a smaller amount of postsynaptic density material, a smaller relative number of Gray-type I (asymmetric) junctions, a greater relative number of Gray-type II (symmetric) junctions, as well as more and larger mitochondria in the postsynaptic dendrites. Junctions of optic synapses are mostly straight, but the small number of positively curved contacts are more flattened in light rats than in dark rats. An age-related increase in the size of presynaptic dense projections was also observed. There are no changes in the sizes of clear and dense-core vesicles, in the size of synaptic junctions and their numerical density in area, and in the unspecific contact area between pre- and postsynaptic elements. The changes in optic boutons are characteristic for activated and relatively disused synapses with a slow, tonic firing rate. It appears that (1) the amount of postsynaptic density material is proportional to the strength of Gray-type I synapses, and that (2) some excitatory optic synapses become inhibitory after long-term activity, whereas some inhibitory synapses turn into excitatory contacts after long-term disuse.

Morphology of the release site of inhibitory synapses on the soma and dendrite of an identified neuron

Synapses are complex arrangements of pre- and postsynaptic differentiations involved in neural communication. A key element in this synaptic transmission is the presynaptic active zone where the release of neurotransmitter occurs. Active zones can be visualized and analyzed after staining with ethanolic phosphotungstic acid (EPTA) on semithin (0.5 micron) sections. This staining has been used in association with postembedding immunogold labeling for the neurotransmitters glycine or GABA, to investigate the organization of chemically defined inhibitory active zones, viewed in their full extent, on different regions of the goldfish Mauthner (M-) cell. With this approach, a marked variability in size and shape was observed for the release sites contacting the different parts of the postsynaptic neuron. In the axon cap and on the soma, glycinergic afferent terminals have small presynaptic grids (0.066 +/- 0.029 micron2, n = 30 and 0.076 +/- 0.037 micron2, n = 46, respectively). These grids are quite circular and they include 12 to 13 presynaptic dense projections (PDPs). The situation is different on the lateral dendrite, where glycinergic and GABAergic active zones display a greater variability in their surface areas (mean = 0.147 +/- 0.100 micron2, n = 115 and 0.139 +/- 0.080 micron2, n = 125, respectively), and their number of PDPs (mean = 19 +/- 9) per individual grid. Similarly, the shape of the release sites over the dendrite is more complex (annular, horseshoe-shaped) when compared to those on the soma. These differences of dendritic versus somatic release sites could represent a structural basis to maximize the shunting effect of glycinergic and GABAergic inhibitory junctions, i.e., close to excitatory inputs. We also observed that the proportion of endings containing 1 or more active zones also varies. More precisely, 96% and 82% of glycinergic terminals in the axon cap and on the soma, respectively, display only one active zone. On the dendrite, their proportion falls to 65.5% for both glycine- and GABA-containing boutons. The remaining inhibitory terminals contain 2 (30%) and 3 to 4 (4.5%) presynaptic grids. These results reveal a greater variability of morphology and organization of the inhibitory release sites at dendritic versus somatic locations. The functional significance of this observation for the synaptic transmission is discussed.

Membrane structure of parallel-fibre synaptic terminals in the cerebellum of the jaundiced Gunn rat: freeze-fracture and E-PTA study

Parallel-fibre synaptic membranes were examined by freeze-fracture and ethanolic-phosphotungstic acid methods in the cerebellum of homozygous (j/j) Gunn rats with hereditary jaundice. Parallel-fibre synapses with dendritic spines of Purkinje cell were severely affected since many Purkinje cells degenerated during the early postnatal period. Some parallel-fibre synaptic terminals lacked their postsynaptic partners and faced astrocytic processes from 18 days of age to the adult stage. These parallel-fibre terminals contained clusters of synaptic vesicles adjacent to synaptic membranes, and synaptic membranes and synaptic cleft materials were identical to those of parallel fibres with postsynaptic partners, as visualized by conventional electron microscopy with osmium tetroxide postfixation and staining of sections with uranyl acetate and lead citrate. In freeze-fractured specimens, the presynaptic membrane of parallel fibres had diffusely distributed large particles and tiny pits on the P-face and protuberances on the E-face, together representing synaptic vesicle attachment sites. Such vesicle attachment sites were present on the presynaptic membranes of parallel fibres without postsynaptic partners from day 18 to the adult stage. After ethanolic-phosphotungstic acid staining, parallel-fibre terminals displayed presynaptic dense projections, intercleft materials and postsynaptic thickening, but some parallel fibres lacked postsynaptic thickening. These observations suggest that the presynaptic membrane structure of the parallel fibre is preserved, even in the absence of a postsynaptic partner, in j/j cerebella. A mechanism for persistence of presynaptic membrane structures without postsynaptic partners in j/j cerebella is discussed.

Different kinds of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells. II. Synaptic junctions

Four different types of axon terminals form symmetric synapses with the cell bodies and initial axon segments of pyramidal cells in layer II/III of rat visual cortex. One type belongs to chandelier cells, and the other three kinds of terminals have origins that have not been established yet. These latter are referred to as large, medium-sized and dense terminals. The purpose of the present study was to examine the synaptic junctions formed by all four types of terminal. The synapses formed by the chandelier cell terminals are readily recognized in thin sections because of the characteristics features of both the terminals and the initial axon segments, which are the neuronal elements postsynaptic to them. In en face views of these axo-axonal synapses the junctions can be seen to have presynaptic dense projections that form a grid in which they are triagonally spaced, and have an average centre-to-centre spacing of 84 nm. As an ensemble the projections form the presynaptic grid, which usually has an oval or round outline, but may be notched on one side where projections are absent. The synaptic junctions of the large, medium-sized and dense terminals were examined by making reconstructions of the terminals from serial thin sections. It was found that at the interfaces between the axon terminals and the cell bodies of pyramidal cells, several separate synaptic junctions may be present, in addition to a number of puncta adhaerentia. Thus, there may be as many as five separate synaptic junctions and as few as one.(ABSTRACT TRUNCATED AT 250 WORDS)

Freeze‐fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze-fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P-face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P-face of the afferent postsynaptic membrane at a density of 3,000/microns 2. In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased. Dense populations of IMPs of about 9 nm in diameter were observed on the P-face of the efferent postsynaptic membrane at a density of 4,000/microns 2. A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group. In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.

Structural aspects, potassium stimulation and calcium dependence of nonsynaptic neuropeptide release by the egg laying controlling caudodorsal cells of Lymnaea stagnalis

The cerebral peptidergic caudodorsal cells of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying behaviour by releasing peptides into (1) the haemolymph, from neurohaemal axon terminals in the periphery of the cerebral commissure and (2) the intercellular space of the central nervous system, from collaterals in the inner compartment of this commissure. Recently, it was shown that collateral release occurs from nonsynaptic release sites, which lack the morphological specializations that are characteristic of classical synapses. Probably, these sites enable the caudodorsal cells to communicate with central neurons in a nonsynaptic (“paracrine”, “diffuse”, “hormone-like”) fashion. The structural and ionic bases of nonsynaptic release were studied using the tannic acid-Ringer incubation-method for the detection of exocytotic release of secretory granule contents in vitro. Elevation of the extracellular potassium concentration strongly stimulates exocytotic activity in the collaterals. No stimulation was found in the absence of extracellular calcium ions. Similar results have been obtained for the neurohaemal axon terminals. Electron-dense material occurs apposed at the cytoplasmic side of the axolemma of collaterals (ethanolic phosphotungstic acid method). This material appears homologous with the presynaptic dense projections forming the “vesicular grid” in classical synapses. Such projections are also present in the neurohaemal axon terminals. It is concluded that secretion from nonsynaptic release sites in caudodorsal cell collaterals shares fundamental characteristics with secretion from conventional neuronal release sites (neurohaemal axon terminals and classical synapses); release occurs by exocytosis of secretory granules, is associated with a vesicular grid, is stimulated by membrane depolarization, and depends on the presence of extracellular calcium ions. The results underline the importance of nonsynaptic release sites for interneuronal communication within the central nervous system.

Structural components in the synaptic cleft captured by freeze-substitution and deep etching of directly frozen cerebellar cortex.

Structural components in the synaptic cleft were examined in cerebellar excitatory synapses by conventional electron microscopy and by rapid freezing followed by freeze-substitution or deep etching. Two transverse components and one parallel element were identified in the clefts of rapidly frozen and freeze-substituted synapses: (i) bridging fibrils, 4-6 nm in diameter, that span the cleft; (ii) columnar pegs, 4-6 nm wide and 8-15 nm high, projecting from the postsynaptic surface; and (iii) intervening fine fibrils running parallel to the apposed synaptic membranes. These were more clearly visible in deep-etched synapses, although the postsynaptic pegs were difficult to distinguish from intramembrane particles in the cross-fractured postsynaptic membranes. Deep etching also revealed other fibrils on the cytoplasmic surface of the postsynaptic membrane. These appear to contact the membrane surface or the intramembrane particles. Freeze-substituted materials also displayed the fibrillar components in the postsynaptic dense fuzz, but failed to display the presynaptic dense projections typically observed in thin sections or deep-etched replicas of the conventionally fixed materials. The bridging fibrils are likely to play a mechanical role in holding the synapse together, while the short pegs may be integral parts of the receptor molecules.

Feline ‘C’-type terminals possess synaptic sites associated with a hypolemmal cistern and Nissl body

Previous findings suggest that axosomatic ‘C’-type terminals on rat spinal motoneurones possess ‘active’ synaptic sites associated with the characteristic subsynaptic cistern and postsynaptic Nissl body, but ‘C’-terminals in cat do not. A re-examination of feline ‘C’-terminals undertook to compare the synaptic ultrastructure in aldehyde-fixed material stained by either osmium or ethanolic phosphotungstic acid (E-PTA). In agreement with previous findings osmicated cat ‘C’-terminals failed to reveal synaptic complexes in regions possessing the subsynaptic cistern (‘cisternal regions’). In contrast, cisternal regions of E-PTA stained ‘C’-terminals exhibited a linear array of presynaptic dense projection all opposed by a single extended postsynaptic density which abutted directly onto both postsynaptic membrane and the cistern. This close topographical relationship is suggested to be functionally significant in the trans-synaptic trophism previously demonstrated for feline ‘C’-terminals.

Plasticity of the synaptic contact zone following loss of synapses in the cerebral cortex of aging humans

Quantitative ultrastructural analyses of ethanolic phosphotungstic acid-stained human layer 1 precentral mortor cortex (Brodmann's area 4) and layer 1 postcentral somatosensory cortex (Brodmann's area 3) were undertaken to determine the nature of synaptic changes occurring over a series of ages (45–84 years) of a normal aging human population. In the precentral cortex, a significant decrease in the number of synapses was accompanied by an increase in mean length of the postsynaptic contact zone and a decrease in the mean width of the presynaptic paramembranous density. The frequency of mature type A and immature type E synaptic profiles decreased with age. There were no changes in the width of the postsynaptic paramembranous density, cleft width or the number of presynaptic dense projections per synapse. In the postcentral cortex there were no significant changes in synaptic number or in any of the synaptic parameters measured. The present study demonstrates that age-related synapse loss in the human cerebral cortex may be confined to specific cortical regions. The data suggest that in the precentral cortex the plasticity of the synaptic contact zone may be a compensatory response by the remaining synapses to age-related synapse loss.

Synaptic structural changes during development and aging

Although a great deal is known about the development of synaptic number, comparatively little is known about the effects of development, and particularly aging, on the structure of the synapse. The present study examined synaptic structure in the molecular layer of the motor-sensory neocortex during early development (postnatal days (P) 1, 3, 5, 7, 10, 15, 20, 30), adulthood (P60, 90), and old age (28 months). Tissue was stained with osmium tetroxide (osmium) or ethanol phosphotungstic acid and the following synaptic characteristics were quantified: (1) presynaptic element length, area, thickness, maximal projection height and smoothness, and number and size of vesicles adjacent to the presynaptic element; (2) postsynaptic element length, area, and thickness; and (3) cleft width. There is an early developmental increase in synaptic element length, followed by an increase in thickness into adulthood. During development the height and width of the presynaptic dense projections increase, after which they remain stable. While the number of adjacent synaptic vesicles increases throughout the lifespan, there is a parallel decrease in their size. During the period of rapid synaptogenesis in this brain region there are no decreases in any of the synaptic structural parameters examined, indicating that newly generated synapses are either formed the same size as the existing mature synapses, or are extremely plastic and grow very rapidly. Unlike age-associated changes in synaptic number, no changes were found in synaptic structure during aging.

Innervation of muscle in the cestode Hymenolepis microstoma

The innervation of muscle in the cestode Hymenolepis microstoma was investigated by analysis of serial thin sections in the electron microscope. The myocytons, containing the nucleus and cell organelles, formed sarcoplasmic processes that contained the myofibers. Junctional complexes resembling gap junctions were found between sarcoplasmic processes. Sarcoplasmic extensions from the myofibers passed to the segmental ring commissures and formed sarconeural junctions. The presynaptic component of the sarconeural junctions was characterized by an axon terminal, or axon en passant, containing electron-lucent and occasional electron-dense synaptic vesicles, occasional mitochondria, and presynaptic dense projections. The presynaptic terminal was isolated from the postsynaptic terminal by a synaptic cleft of ~20 nm. The postsynaptic sarcoplasmic process, which often was wrapped around the nerve terminal, displayed a postsynaptic thickening. Single axon terminals were observed to form sarconeural junctions with up to four sarcoplasmic processes. Sarcoplasmic processes formed synaptic contacts with more than one axon, and sarcoplasmic processes formed more than one synaptic contact with a single axon, suggesting a polyneuronal multiterminal pattern of innervation. Serial synapses were found in conjunction with sarconeural junctions in the suckers. The functional significance of this type and pattern of innervation of muscle in H. microstoma is discussed.

Vesicle-containing dendrites in the nucleus raphe dorsalis of the cat. A serial section electron microscopic analysis.

The nucleus raphe dorsalis of the cat was examined by serial section electron microscopy and the presence of vesicle-containing dendrites is reported. Such dendrites were divided into two classes according to their synaptic contact. Dendrites containing round and/or pleomorphic vesicles associated with a clear synaptic specialization which was generally intermediate between a Gray's type I or II were classified as presynaptic dendrites. These presynaptic dendrites were presynaptic to conventional dendrites and dendritic spines. In addition, some profiles containing a sparse population of vesicles which may be dendritic in nature were observed involved in serial synaptic arrangements. A second class of dendrites were characterized by the presence of vesicles which were never found associated with any synaptic membrane specialization. Commonly, the vesicles were densely packed and associated with unusual densities. Serial section analysis of these densities showed that they were not presynaptic dense projections. We suggest that the existence of vesicle-containing dendrites in the nucleus raphe dorsalis of the cat constitute the morphological support for the dendritic release of neurotransmitters.

New observations on the substructure of the active zone of brain synapses and motor endplates.

This study offers a new concept on the origin and function of the hitherto enigmatic presynaptic dense projections (dps) of neurons and motor endplates. After a deuterium oxide-albumin pretreatment (da), brain tissue and motor endplate of rat and frog reveal an intricate association of smooth endoplasmic reticulum (ser), microtubules (mts) and synaptic vesicles (sv) at the presynaptic grid-active zone of synapses. The ser entwines the mts, which are clothed in svs, and impinges directly onto the presynaptic membrane as sacs or 'tubular-fibrillar' extensions. Since no dps are seen in these sections, whereas they do occur in conventionally processed material (i.e. without da pretreatment), it is suggested that the dps of conventional material may, in part, originate from improperly fixed ser at these points. Thus for the first time we demonstrate an in vivo system of ser which, because its 'finger' processes come into intimate contact with the presynaptic membrane, may be implicated in Ca2+ ion translocation, presumably out of the presynaptic bulb. Since no such tubular ser has been demonstrated in what are claimed to be sophisticated techniques (i.e. high-speed slam-freezing-freeze substitution) the actual sophistication of such methods is questioned.