Distribution Of Synaptic Vesicles Research Articles

Regulation of neurotransmitter release by synapsin III.

Synapsin III is the most recently identified member of the synapsin family, a group of synaptic vesicle proteins that play essential roles in neurotransmitter release and neurite outgrowth. Here, through the generation and analysis of synapsin III knock-out mice, we demonstrate that synapsin III regulates neurotransmitter release in a manner that is distinct from that of synapsin I or synapsin II. In mice lacking synapsin III, the size of the recycling pool of synaptic vesicles was increased, and synaptic depression was reduced. The number of vesicles that fuse per action potential was similar between synapsin III knock-out and wild-type mice, and there was no change in the quantal content of EPSCs; however, IPSCs were greatly reduced in synapsin III-deficient neurons. The density and distribution of synaptic vesicles in presynaptic terminals did not appear to be different in synapsin III knock-out mice in comparison to wild-type littermates. In addition to the changes in neurotransmitter release, we observed a specific delay in axon outgrowth in cultured hippocampal neurons from synapsin III knock-out mice. Our data indicate that synapsin III plays unique roles both in early axon outgrowth and in the regulation of synaptic vesicle trafficking.

Regulation of Neurotransmitter Release by Synapsin III

Synapsin III is the most recently identified member of the synapsin family, a group of synaptic vesicle proteins that play essential roles in neurotransmitter release and neurite outgrowth. Here, through the generation and analysis of synapsin III knock-out mice, we demonstrate that synapsin III regulates neurotransmitter release in a manner that is distinct from that of synapsin I or synapsin II. In mice lacking synapsin III, the size of the recycling pool of synaptic vesicles was increased, and synaptic depression was reduced. The number of vesicles that fuse per action potential was similar between synapsin III knock-out and wild-type mice, and there was no change in the quantal content of EPSCs; however, IPSCs were greatly reduced in synapsin III-deficient neurons. The density and distribution of synaptic vesicles in presynaptic terminals did not appear to be different in synapsin III knock-out mice in comparison to wild-type littermates. In addition to the changes in neurotransmitter release, we observed a specific delay in axon outgrowth in cultured hippocampal neurons from synapsin III knock-out mice. Our data indicate that synapsin III plays unique roles both in early axon outgrowth and in the regulation of synaptic vesicle trafficking.

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals.

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.

Contact-dependent aggregation of functional Ca2+ channels, synaptic vesicles and postsynaptic receptors in active zones of a neuromuscular junction.

To examine whether Ca2+ channels aggregate in a contact-dependent manner, we characterized the distribution of synaptic vesicles and postsynaptic receptors, and compared it to the location of Ca2+ entry sites, in a Xenopus laevis nerve-muscle coculture preparation using a localized Ca2+ detection method. The majority (75%) of Ca2+ entry sites at spontaneously formed nerve-muscle contacts were associated with enhanced immunofluorescence to the synaptic vesicle protein, SV2. In contrast, only 11% of recorded sites without Ca2+ transients exhibited significant SV2 immunofluorescence. When comparing the spatial distribution of synaptic markers with that of Ca2+ entry sites, we found that the majority of Ca2+ entry sites (61%) were associated with both enhanced SV2 immunofluorescence and R-BTX fluorescence, thereby identifying putative neurotransmitter release sites where Ca2+ channels, synaptic vesicles and postsynaptic receptors are colocalized. Using polystyrene beads coated with a heparin binding protein known to mediate in vitro postsynaptic receptor clustering, we show that the location of Ca2+ domains was associated with enhanced SV2 immunofluorescence at neurite-to-bead contacts. We conclude that the localization of functional Ca2+ channels to putative active zones follows a contact-dependent signalling mechanism similar to that known to mediate vesicle aggregation and AChR clustering.

BDNF Enhances Quantal Neurotransmitter Release and Increases the Number of Docked Vesicles at the Active Zones of Hippocampal Excitatory Synapses

Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF-TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory.

Effects of Staurosporine on Exocytosis and Endocytosis at Frog Motor Nerve Terminals

Observations of the dynamic staining and destaining of FM1-43 in frog motor nerve terminals (Henkel and Betz, 1995) suggested that staurosporine might shorten the interval between exocytosis and endocytosis, inducing a "kiss and run" mode of exocytosis and endocytosis. We tested this hypothesis by using FM1-43 imaging (to measure the time course of FM1-43 endocytosis), intracellular recording of evoked synaptic potentials (to measure acetylcholine release), and electron microscopy (to examine synaptic vesicle distribution). Staurosporine reduced FM1-43 uptake during but not after a tetanus, increased the speed of end plate potential (EPP) amplitude rundown, and greatly slowed the recovery from synaptic depression. Ultrastructural observations showed pronounced vesicle depletion near active zones after tetanic stimulation in staurosporine-treated preparations. These results suggest that staurosporine acted primarily to impair mobilization of synaptic vesicles during tetanic stimulation.

Aberrant synaptic transmission in the hippocampal CA3 region and cognitive deterioration in protein‐repair enzyme‐deficient mice

L-aspartate is the amino-acid residue most susceptible to spontaneous isomerization. This denaturation causes an alteration in the biological activity of the protein and is regarded as an aging process of the protein. Protein L-isoaspartyl methyltransferase (PIMT) repairs this post-translational modification and thus is implicated in retarding the aging process of proteins. PIMT is highly expressed in the brain, and its deficiency results in progressive epilepsy after 4 weeks of age, with a fatal seizure in mice. Here we report the pathophysiological role of this repair system in the hippocampal slice of PIMT-deficient mice. The hippocampal mossy fiber-CA3 synapses of PIMT-deficient mice showed hyperexcitation that was repressed by a gamma-aminobutyric acid (GABA)A receptor agonist muscimol. In addition, the mossy fiber-CA3 synapses failed to show long-term potentiation or paired-pulse facilitation. No abnormality, however, was observed in Schaffer collateral-CA1 synapses or in perforant path-dentate gyrus synapses. Electron microscopic study revealed aberrant distribution of synaptic vesicles in the mossy fiber terminals and vacuolar degeneration at the axon hillock of dentate granule cells in PIMT-deficient mice. Furthermore, the PIMT-deficient mice showed impaired spatial memory in Morris water maze test and exhibited fewer anxiety-related behaviors in the elevated-plus test. These results suggest that the mossy fiber-CA3 system is vulnerable to aspartate isomerization and that the PIMT-mediated repair system is essential for maintenance of normal functions of the hippocampus.

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.

Specialised sympathetic neuroeffector associations in immature rat iris arterioles.

Sympathetic nerve-mediated vasoconstriction in iris arterioles of mature rats occurs via the activation of alpha(1B)-adrenoceptors alone, while in immature rat iris arterioles, vasoconstriction occurs via activation of both alpha1- and alpha2-adrenoceptors. In mature rats the vast majority of sympathetic varicosities form close neuroeffector junctions. Serial section electron microscopy of 14 d iris arterioles has been used to determine whether restriction in physiological receptor types with age may result from the establishment of these close neuroeffector junctions. Ninety varicosities which lay within 4 microm of arteriolar smooth muscle were followed for their entire length. Varicosities rarely contained dense cored vesicles even after treatment with 5-hydroxydopamine. 47 % of varicosities formed close associations with muscle cells and 88 % formed close associations with muscle cells or melanocytes. Varicosities in bundles were as likely as single varicosities to form close associations with vascular smooth muscle cells, although the distribution of synaptic vesicles in single varicosities did not show the asymmetric accumulation towards the smooth muscle cells seen in the varicosities in bundles which were frequently clustered together. We conclude that restriction of physiological receptor types during development does not appear to correlate with the establishment of close neuroeffector junctions, although changes in presynaptic structures may contribute to the refinement of postsynaptic responses.

Spatial distribution of synaptic vesicles in presynaptic terminals: Computer simulation

A procedure for computer simulation is proposed, which allows one to quantitatively characterize the spatial distribution of synaptic vesicles in presynaptic terminals (PST) using ultrathin sections of such terminals. The procedure includes three stages: simulation, topographical analysis, and comparison. At the first stage, the spatial distribution of vesicles within a PST and the process of random sectioning of it are simulated using the corresponding mathematical model. At the second stage, the topographical distribution of vesicle profiles within the plane of PST section is estimated; three respective approaches have been used: (i) nearest neighbor distance distribution; (ii) minimal spanning tree; and (iii) Voronoi paving. At the third stage, the simulated parameters are compared with the parameters of native terminal sections; when the coincidence of these two parameter groups is satisfactory, we believe that the simulated spatial distribution agrees with the real distribution. The software for the procedure is written in C++ programing langage. The results of a pilot study on ultrathin sections of cultured rat hippocampal neurons showed that the method offers broad possibilities for spatial interpretation and quantitative characterization of distributions of synaptic vesicles.

A Neuronal Sec1 Homolog Regulates Neurotransmitter Release at the Squid Giant Synapse

Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the squid giant synapse. Microinjection of s-Sec1 into the presynaptic terminal of the giant synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro, completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites ("active zones"), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin-at a stage blocked by the peptide-is necessary for docked vesicles to fuse.

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.

A functional role for GTP-binding proteins in synaptic vesicle cycling.

The squid giant synapse was used to test the hypothesis that guanosine-5'-triphosphate (GTP)-binding proteins regulate the local distribution of synaptic vesicles within nerve terminals. Presynaptic injection of the nonhydrolyzable GTP analog GTP gamma S irreversibly inhibited neurotransmitter release without changing either the size of the calcium signals produced by presynaptic action potentials or the number of synaptic vesicles docked at presynaptic active zones. Neurotransmitter release was also inhibited by injection of the nonhydrolyzable guanosine diphosphate (GDP) analog GDP beta S but not by injection of AIF4-. These results suggest that a small molecular weight GTP-binding protein directs the docking of synaptic vesicles that occurs before calcium-dependent neurotransmitter release. Depletion of undocked synaptic vesicles by GTP gamma S indicates that additional GTP-binding proteins function in the terminal at other steps responsible for synaptic vesicle replenishment.

Monoaminergic innervation of the rat kidney: a quantitative study

The sympathetic innervation of the renal tubules and vasculature was characterized by measuring the overlap of accumulations of autoradiographic grains (AAGs) on these structures in autoradiograms of kidney sections from rats injected with tritiated norepinephrine. AAG overlap was used as an indirect measure of the innervation of those structures. The renal vasculature showed x 4.5 more AAG overlap than observed on renal tubules. The greatest amount of AAG overlap occurred on afferent arterioles, followed by efferent arterioles, interlobular arteries, cortical capillaries, arcuate arteries, and renal veins. High concentration of AAGs occurred along the vascular bundles of the outer stripe. In the tubular nephron the proximal tubule had the greatest amount of AAG overlap, followed by the cortical thick ascending limb of Henle, the connecting tubule, the distal convoluted tubule, and the collecting duct. It was found that afferent arterioles had significantly higher mean density of AAG overlap than efferent arterioles for the superficial, midcortical, and juxtamedullary (vascular bundles excluded) renal cortex. There was consistently more AAG perimeter facing the interstitium than overlapping the vasculature. These observations, together with the ultrastructural distribution of synaptic vesicles in varicosities, suggest that the interstitium might be an additional pathway of neurotransmitter access to the effector structures.

Proliferation in non-Hodgkin's lymphoma: a comparison of Ki-67 staining on fine needle aspiration and cryostat sections.

Assessment of the growth fraction of non-Hodgkin's lymphomas may provide useful additional prognostic information to that obtained with conventional histological criteria. The monoclonal antibody Ki-67 has been reported to provide such information immunocytochemically in tissue biopsy specimens from lymphoma as well as other tumours. This study was undertaken to assess whether this approach could be extended to fine needle aspiration (FNA) biopsy specimens which are becoming increasingly important in the diagnosis of lymphoma. In 21 cases of non-Hodgkin's lymphoma the rate of tumour proliferation estimated by Ki-67 immunostaining of FNA material, obtained from surgically removed specimens, was compared with that obtained on tissue biopsy. The correlation between both preparations was excellent, indicating that FNA biopsy material is suitable for the immunocytochemical assessment of the growth fraction of non-Hodgkin's lymphoma.

Neonatal deafferentation prevents normal expression of synaptic vesicle antigens in the developing rat superior cervical ganglion.

The effect of neonatal deafferentation of the rat superior cervical ganglion (SCG) on the expression of two synaptic vesicle proteins was studied to assess the role of transsynaptic influences in the regulation of these neural antigens in the SCG. The two proteins studied were a 65 kilodalton integral membrane protein of synaptic vesicles (SV), and synapsin-1 (S-1), a synaptic vesicle phosphoprotein. Antigen levels were quantified by radioimmunoassay using antibodies directed against the proteins. Distribution of SV in control, deafferented and reinnervated ganglia from 30-day-old rats was visualized by immunohistochemical labeling. Levels of both antigens were reduced following deafferentation of the SCG on postnatal days 1-3. The reduction in S-1 levels at 30 days was less than that observed for SV. The amount of S-1 remaining in deafferented ganglia was consistent with estimates of the postsynaptic pool in the SCG reported previously. SV levels, in contrast, were reduced to 24% of control levels, suggesting that SV synthesis in principal ganglionic neurons might be affected. The time course of postnatal development of S-1 in the SCG differed from previous studies of SV expression, with significant increases occurring after the second week after birth. The differences in response to deafferentation may reflect functional differences of the two vesicle-associated proteins. These studies demonstrate that transsynaptic regulation of antigens other than those directly associated with neurotransmitters occurs in the SCG.

Distribution of synaptic specializations along isolated motor units formed in Xenopus nerve-muscle cultures

The capacity of individual muscle cells and neurons to establish synaptic specializations along the entire extent of their neurite-muscle contacts was assessed in culture. Spinal cord neurons derived from embryos of Xenopus laevis were plated at low density in cultures of Xenopus myotomal muscle cells in order to obtain isolated motor units whose neuron and muscle cells were not contacted by any other neuron. These isolated motor units were examined for localization of acetylcholine receptors (AChRs) after staining with fluorescent alpha-bungarotoxin and, in some cases, for localization of a synaptic vesicle antigen by immunofluorescence. The neurite-muscle contacts formed by competent neurons exhibited discontinuous sites of AChR localization occupying about 25% of the contact length as compared with 2% for incompetent neurons. Competent neurons, unlike incompetent ones, established these neurite-associated receptor patches (NARPs) on virtually all the muscle cells they contacted and functionally innervated them. These and other observations on the distribution of NARPs throughout the isolated motor units indicate that the capacity of competent neurons to establish NARPs extends to the limits of growth of most if not all of their neurites, that this capacity is least in the most proximal portions of initial neuritic segments, and that the overall capacity of muscle cells to generate NARPs can be saturated by long lengths of neurite-muscle contact. The results also suggest that even in the absence of competitive interactions between neurons there are spatial discontinuities in neuritic action and/or muscle response during the establishment of NARPs. Synaptic vesicle antigen patches (SVAPs) occurred along the neuritic arbor of all neurons, but their distribution in competent neurons (those which established NARPs) and in incompetent ones differed. For competent neurons the percentage of neurite length occupied by SVAPs was 4.8-fold greater on muscle cells than off, whereas the corresponding value for incompetent neurons was only 1.5-fold. This large preferential localization of SVAPs along neurite-muscle contacts of competent neurons was further associated with a colocalization of SVAPs and NARPs that was greater than predicted by chance. These results suggest that muscle cells are much more effective in influencing the distribution of synaptic vesicles along the neuritic arbor of competent neurons than along the arbor of incompetent neurons and that this influence is greatest at sites of postsynaptic differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptic vesicle diameters and synaptic cleft widths at the mouse diaphragm in Neonates and Adults

Miniature endplate potential (MEPP) amplitude distributions from adult mouse diaphragm junctions show two classes of MEPPs. The larger MEPPs form a bell-shaped distribution (bell-MEPPs) with a variance of 20–30%. The smaller MEPPs form a right-hand skew-distribution composing 1–10% of the MEPPs with a mode 1/10th (s-MEPP class) that of the bell-MEPPs. Junctions of 1-day-old neonates generate mainly s-MEPPs. The MEPP distribution gradually changes to mainly bell-MEPPs during the first 3 weeks of postnatal development. The purpose of this study was to compare the synaptic vesicle diameters and synaptic clefts of 1- to 3-day-old neonatal junctions to those of adults and reinnervated adults. We found no differences between neonates and adults in either synaptic vesicle diameter (56 nm o.d.) or in synaptic cleft widths. Synaptic vesicle diameters were the same for ·touching vesicles’. Junctions of 1-day-old neonates showed no or shallow postsynaptic folds. Postsynaptic folds were essentially developed by the third day although one-quarter of the MEPPs were of the skew-class. Assuming that vesicles are simple containers, we would expect a class of 30-nm o.d. vesicles which would generate the s-MEPP class. Since we found a smooth distribution of synaptic vesicles and no vesicles of 30 nm diameter we conclude that within the constraints of the vesicle hypothesis of transmitter release that s- and bell-MEPPs are from vesicles sized the same and that vesicle volume does not determine the amount of acetylcholine released.

The effect of nerve activity on the distribution of synaptic vesicles.

Certain gymnotid fish (apteronotids) continuously emit a high-frequency electric-organ discharge and thus continuously drive their electroreceptor afferents at high rates. Electroreceptor afferents terminate in one lamina of the electrosensory lateral line lobe (ELL) and can be readily sampled. Normally these terminals have many small vesicles clustered adjacent to the presynaptic membrane. When afferent activity is blocked for 24 hr by an injection of tetrodotoxin (TTX) into the electroreceptor nerve, the density of vesicles adjacent to the synaptic membrane declines; the volume of the remaining vesicles increases. If the nerve of a TTX-treated fish is stimulated proximal to the injection site, these changes can be reversed. These results imply that the migration of vesicles toward the presynaptic membrane is influenced by the level of activity in the nerve.

Structure of the piriform cortex of the opossum. III. Ultrastructural characterization of synaptic terminals of association and olfactory bulb afferent fibers.

Terminals of olfactory bulb afferent (OB) and association (ASSN) fibers within the piriform cortex were characterized ultrastructurally. Identification was by electron microscopic (EM) autoradiography following injections of tritiated amino acids into the olfactory bulb and anterior piriform cortex. The results show that terminals of both fiber systems contain round vesicles and make asymmetrical synaptic contacts predominantly onto dendritic spines. Profiles with pleomorphic vesicles do not appear to be labeled from either site. Since there is strong evidence that both fiber systems generate excitatory postsynaptic potentials (EPSPs) in pyramidal cells, these results provide additional examples in the mammalian CNS of terminals with round vesicles and asymmetrical contacts that mediate an excitatory effect. Percentage density analysis and quantitative study of a large number of heavily labeled terminals revealed that while OB and ASSN terminals are similar in terms of vesicle shape and contact type, they differ in many morphological details including pre- and postsynaptic profile size, the packing density and distribution of synaptic vesicles, synaptic contact shape, and the presence of overlying neuroglial lamellae. However, large variations in appearance of different terminals of the same type are also present so that a small percentage of OB and ASSN terminals are indistinguishable morphologically in the absence of label. An important finding of the quantitative analysis is that spines contacted by lateral olfactory tract (LOT) terminals appear to be of two types based on a bimodal distribution in size and differences in morphology, while spines contacted by ASSN terminals appear to be of a single type. Comparison of these data with results from Golgi analysis indicates that ASSN terminals predominantly contact pyramidal cell spines while OB terminals contact both pyramidal and semilunar cell spines. Quantitative analysis of synaptic vesicles revealed that histograms of vesicle size for OB and ASSN terminals are virtually identical in shape, but peaks are slightly displaced (ASSN vesicles are 5% larger; significant with P less than .002). An analysis of the laminar distribution of OB and ASSN synaptic terminals revealed that while most OB terminals are segregated in layer Ia and most ASSN terminals in layer Ib, occasional OB terminals are observed up to approximately 50 micro deep to the Ia-Ib boundary and occasional ASSN terminals up to approximately 50 micro superficial to this boundary.