Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate

Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.     

Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins

The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.     

Characterization of fluo-3 labelling of dense bodies at the hair cell''s presynaptic active zone

Summary The presynaptic active zone is the critical region of a chemical synapse at which Ca²⁺ entry provokes neurotransmitter release by exocytotic fusion of synaptic vesicles. To facilitate investigations of synaptic function, we have identified a group of fluorescent substances that label individual active zones in living hair cells. The Ca²⁺ indicator fluo-3, the compound studied in most detail, binds to the presynaptic dense bodies that are characteristic of active zones in hair cells and other cells that tonically release transmitter. The indicator's binding is reversible, with a dissociation constant of approximately 350 m. Because fluo-3 that is bound to a presynaptic dense body continues to detect Ca²⁺ with an unaltered dissociation constant, the binding of this substance provides a valuable tool for exploration of the Ca²⁺ concentration at the site of vesicle fusion.     

N type Ca2+ channels and RIM scaffold protein covary at the presynaptic transmitter release face but are components of independent protein complexes

Fast neurotransmitter release at presynaptic terminals occurs at specialized transmitter release sites where docked secretory vesicles are triggered to fuse with the membrane by the influx of Ca²⁺ ions that enter through local N type (CaV2.2) calcium channels. Thus, neurosecretion involves two key processes: the docking of vesicles at the transmitter release site, a process that involves the scaffold protein RIM (Rab3A interacting molecule) and its binding partner Munc-13, and the subsequent gating of vesicle fusion by activation of the Ca²⁺ channels. It is not known, however, whether the vesicle fusion complex with its attached Ca²⁺ channels and the vesicle docking complex are parts of a single multifunctional entity. The Ca²⁺ channel itself and RIM were used as markers for these two elements to address this question. We carried out immunostaining at the giant calyx-type synapse of the chick ciliary ganglion to localize the proteins at a native, undisturbed presynaptic nerve terminal. Quantitative immunostaining (intensity correlation analysis/intensity correlation quotient method) was used to test the relationship between these two proteins at the nerve terminal transmitter release face. The staining intensities for CaV2.2 and RIM covary strongly, consistent with the expectation that they are both components of the transmitter release sites. We then used immunoprecipitation to test if these proteins are also parts of a common molecular complex. However, precipitation of CaV2.2 failed to capture either RIM or Munc-13, a RIM binding partner. These findings indicate that although the vesicle fusion and the vesicle docking mechanisms coexist at the transmitter release face they are not parts of a common stable complex.     

Effects of restraint stress on the expression of proteins involved in synaptic vesicle exocytosis in the hippocampus

Chronic restraint stress has been associated with induction of morphological changes in the hippocampus. Postsynaptically, these changes include decreased length and branching of apical dendrites from CA3 pyramidal neurons, while presynaptically, depletion and clustering of synaptic vesicles have been observed. However, the molecular correlates of these changes remain poorly defined; while some studies have identified changes in the levels of some presynaptic proteins, none have assessed the coordinate expression of components of the membrane fusion complex, including synaptobrevin, syntaxin, and synaptosomal-associated protein 25 kDa, and their major regulatory molecules synaptotagmin, synaptophysin, and synapsin. Therefore, we undertook to assess the immunoreactivity of these proteins in hippocampal slices obtained from rats subjected to either acute (one 6 h session) or chronic (21 days at 6 h per day) of restraint stress. Specifically, we observed a significant increase in synaptobrevin immunoreactivity in the inner molecular layer of the dentate gyrus (54.2%; P=0.005), the stratum radiatum in the CA1 subfield (55.5%; P=0.007), and a region including the stratum lucidum and the proximal portion of the stratum radiatum in the CA3 subfield (52.7%; P=0.002); we also observed a trend toward increased synaptophysin levels in the stratum lucidum/radiatum of the CA3 subfield (8.0%; P=0.051) following chronic, but not acute, restraint stress. In that synaptobrevin has been associated with replenishment of the "readily-releasable" pool of synaptic vesicles and the efficiency of neurotransmitter release, the present results suggest that stress-induced changes in synaptobrevin may at least in part underlie the previously observed changes in synaptic and neuronal morphology.     

Formation of the active zone at developing neuromuscular junctions in larval and adult bullfrogs

Summary Development of the presynaptic active zone was studied at neuromuscular junctions with freeze-fracture electron microscopy in larval and adult bullfrogs. In rudimentary larval neuromuscular junctions, clusters of active zone particles were scattered over the P-face of the presynaptic membrane. Vesicle openings were observed at these terminals even though active zone particles lacked the mature pattern of two double rows. Gradually, active zone particles became organized into rows, but they were still randomly located and oriented. Once junctional folds were observed in replicas, developing active zones were located opposite to the folds, as in mature terminals. Multiple terminals occupying the same junctional gutters were also observed. At the end of metamorphosis, most active zones were still immature in appearance and had only grown to one third of their mature length. After metamorphosis, the number of active zone segments aligned at the same junctional fold increased. These discontinuous short active zones then elongated, joined together, and finally formed the mature active zones. Signs suggesting synapse elimination such as disorganization of active zones, absence of intramembrane particles in varicosities, and exposed muscle membranes with patches of acetylcholine receptor aggregates were observed. In some multiply innervated junctions, one terminal had mature active zones with vesicle openings, the other in the same gutter displayed disorganized active zones without vesicle openings, although both terminals showed similar sizes and distributions of background particles. This study suggests that developing active zones, as is the case for regenerating active zones in the adult, are functional before the mature organization is formed. The sequence of development of active zones is also similar to that of regeneration except for the random location and orientation of early active zones in tadpoles. The comparison between regeneration and development further indicates that the process of active zone formation is related to junctional folds and/or associated structures. It is also suggested that synapse elimination may involve degenerative changes in presynaptic membranes, although direct evidence remains to be provided.     

Prenatal stress alters presynaptic marker proteins in the hippocampus of rat pups

Exposure to stress during critical periods of an organism's maturation can result in permanent behavioral changes and induced hyper-responsive to aversive stimuli as adult. Hippocampus is a plastic and vulnerable brain structure that is susceptible to damage during aging and repeated stress. The present study examines the effect of maternal restraint stress on the level of GAP-43, pGAP-43 and synaptophysin in the hippocampus of rat pups. Prenatal stress (PS) causes a significant increase of GAP-43 and pGAP-43 (p ≤ 0.001) in the pup's hippocampus during postnatal days 7 and 14, but not at later ages. Up-regulation of GAP-43 and pGAP-43 may alter the pattern of axonal growth and synapses’ formation in the pup's brain since the first two postnatal weeks are correlated with peak period of synaptogenesis in the rat brain. We also examined the level of synaptophysin, a synaptic vesicle membrane protein, in the pup's brain. Our finding revealed that, PS causes a significant decrease of synaptophysin in the pup's hippocampus as compared to control (p ≤ 0.001). These changes are due to the direct effects of maternal stress hormone since repeated injection with corticosterone (CORT, 40 mg/kg) to pregnant rat during gestation days (GDs) 14–21 also gave the same results. Abnormal axonal sprouting and reorganization together with the alterations in synaptic vesicle membrane protein during the critical period of synaptogenesis may lead to a defect in synapse formation and axonal pruning in the hippocampus. These changes may be associated with stress-induced impairment of hippocampal function that occurs in later life of the offspring.     

Synapsin III is a key component of α‐synuclein fibrils in Lewy bodies of PD brains

Lewy bodies (LB) and Lewy neurites (LN), which are primarily composed of α‐synuclein (α‐syn), are neuropathological hallmarks of Parkinson''s disease (PD) and dementia with Lewy bodies (DLB). We recently found that the neuronal phosphoprotein synapsin III (syn III) controls dopamine release via cooperation with α‐syn and modulates α‐syn aggregation. Here, we observed that LB and LN, in the substantia nigra of PD patients and hippocampus of one subject with DLB, displayed a marked immunopositivity for syn III. The in situ proximity ligation assay revealed the accumulation of numerous proteinase K‐resistant neuropathological inclusions that contained both α‐syn and syn III in tight association in the brain of affected subjects. Most strikingly, syn III was identified as a component of α‐syn‐positive fibrils in LB‐enriched protein extracts from PD brains. Finally, a positive correlation between syn III and α‐syn levels was detected in the caudate putamen of PD subjects. Collectively, these findings indicate that syn III is a crucial α‐syn interactant and a key component of LB fibrils in the brain of patients affected by PD.     

Expression of the 3-fucosyl N-acetyllactosamine (CD 15) antigen in normal, metaplastic, dysplastic, and neoplastic squamous epithelia

The 3-fucosyl N-acetyllactosamine residue is the antigen recognized by the monoclonal antibody MC2. Using MC2, we demonstrated the distribution of this antigen in a variety of squamous epithelia. The antigen is expressed to a variable degree on supra-basal cells in most normal non-keratinizing squamous mucosae, with a similar distribution in metaplastic squamous epithelia; antibody-labelled latex microspheres and immunogold electron microscopy show the antigen to form part of the glycocalyx. In dysplastic and neoplastic squamous lesions, expression is reduced or absent except in cells around areas of differentiation. Prior neuraminidase treatment of sections had little effect on the amount or distribution of demonstrable antigen. Expression of this antigen by cells in non-keratinizing squamous epithelia gives an indication of cell maturity and may provide a histological marker for the grading of dysplastic and malignant squamous mucosal lesions. A possible role for these carbohydrate residues in squamous mucosal defence is discussed.     

内皮素—1基因内含子4Taq I片段多态性的研究

根据EDN1第4内含子设计相应引物,建立检测EDN1第4内含子TaqI多态性PCR＋RFLP技术。利用该技术,扩增到具有特异性的PCR产物,长为358bp。经限制性核酸内切酶TaqI酶酶切检测中国汉族人群该片段多态性状况。实验显示中国汉人群中存在该位点的多态性。基因T1的频率为0．664;T2基因型频率为0．336;T1T1基因型频率为0．418;T1T2基因型频率为0．492;T2T2基因型频率为0．090。该位点可作为遗传标记探讨中国人群中与EDN1相关遗传病的关系。     

Revealing the molecular layer of the primate dorsal cochlear nucleus

In nonprimate mammals, the dorsal cochlear nucleus (DCN) is thought to play a role in the orientation of the head toward sounds of interest by integrating acoustic and somatosensory information. Humans and higher primates might not use this system because of reported phylogenetic changes in DCN cytoarchitecture [Moskowitz N (1969) Comparative aspects of some features of the central auditory system of primates. Ann N Y Acad Sci 167:357-369; Moore JK, Osen KK (1979) The cochlear nuclei in man. Am J Anat 154:393-418; Moore JK (1980) The primate cochlear nuclei: loss of lamination as a phylogenetic process. J Comp Neurol 193:609-629]. In this study, we re-evaluated this question from a comparative perspective and examined the rhesus monkey (cercopithecoid primate) using more sensitive probes and higher resolution imaging methods. We used electron microscopy to identify parallel fibers and their synapses, and molecular markers to determine that primates exhibit the main components of excitatory neurotransmission as other mammals. We observed that characteristics of the monkey molecular layer resembled what has been reported for nonprimates: (1) immunohistochemistry revealed many unmyelinated, thin axons and en passant glutamatergic synapses on dendritic spines; (2) immunohistochemistry for phosphodiesterase (PDE10A) showed the nuclei of granule cells distributed in the external molecular layer and the deep layers in the DCN; (3) antibodies for the inositol trisphosphate receptor (IP3r) and calbindin immunostained cartwheel cells; (4) postembedding immunogold labeling revealed synaptic expression of AMPA and delta glutamate receptor subunits on spines in parallel fiber endings; and (5) parallel fibers use vesicular glutamate transporter 1 (VGLUT1) to package glutamate into the synaptic vesicles and to mediate glutamate transport. These observations are consistent with the argument that the rhesus monkey DCN has neuronal features similar to those of other nonprimate mammals.     

Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the nucleus accumbens of cocaine-treated rats

There is significant pharmacological and behavioral evidence that group I metabotropic glutamate receptors (mGluR1a and mGluR5) in the nucleus accumbens play an important role in the neurochemical and pathophysiological mechanisms that underlie addiction to psychostimulants. To further address this issue, we undertook a detailed ultrastructural analysis to characterize changes in the subcellular and subsynaptic localization of mGluR1a and mGluR5 in the core and shell of nucleus accumbens following acute or chronic cocaine administration in rats. After a single cocaine injection (30 mg/kg) and 45 min withdrawal, there was a significant decrease in the proportion of plasma membrane-bound mGluR1a in accumbens shell dendrites. Similarly, the proportion of plasma membrane-bound mGluR1a was decreased in large dendrites of accumbens core neurons following chronic cocaine exposure (i.e. 1-week treatment followed by 3-week withdrawal). However, neither acute nor chronic cocaine treatments induced significant change in the localization of mGluR5 in accumbens core and shell, which is in contrast with the significant reduction of plasma membrane-bound mGluR1a and mGluR5 induced by local intra-accumbens administration of the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG). In conclusion, these findings demonstrate that cocaine-induced glutamate imbalance has modest effects on the trafficking of group I mGluRs in the nucleus accumbens. These results provide valuable information on the neuroadaptive mechanisms of accumbens group I mGluRs in response to cocaine administration.     

Comparative Study of Two Immunocytochemical Techniques in the Electron Microscopical Detection of Alpha-1-Antitrypsin in Routinely Processed Liver Biopsies

Two indirect immunocytochemical techniques using different markers, namely peroxidase and gold, were applied to ultrathin sections of liver biopsies to detect alpha-1-antitrypsin. The blocks used were taken from the routine electron microscopy files and had been processed optimally for maximum ultrastructural preservation. The immunogold technique provided the best method for localizing alpha-1 -antitrypsin and was associated with ultrastructural preservation equivalent to that seen in routinely processed liver biopsies. The procedure may be a useful adjunct to understanding the pathogenesis of alpha-1-antitrypsin deficiency and in recognizing further variants of this disorder.