Glutamate-dependent stabilization of presynaptic terminals

Dissecting the mechanisms underlying synapse formation and elimination is fundamental to understand how the nervous system is constructed and subsequently modified. Two studies by Tashiro et al. and by Hashimoto and Kano in this issue of Neuron provide new insights into the roles of neurotransmitter glutamate release in regulating the motility of hippocampal mossy fiber filopodia and synaptic competition among climbing fibers.     

Ultrastructural organization of presynaptic terminals

In response to calcium influx, synaptic vesicles fuse very rapidly with the plasma membrane to release their neurotransmitter content. An important mechanism for sustained release includes the formation of new vesicles by local endocytosis. How synaptic vesicles are trafficked from the sites of endocytosis to the sites of release and how they are maintained at the release sites remain poorly understood. Recent studies using fast freezing immobilization and electron tomography have led to insights on the ultrastructural organization of presynaptic boutons and how these structural elements may maintain synaptic vesicles and organize their exocytosis at particular areas of the plasma membrane.     

Immunohistochemical localization of cholinergic nerve terminals

Summary Most of the published light-microscopic methods for the localization of cholinergic nerve pathways present various difficulties of interpretation. The production and characterization of an antiserum that binds specifically to cholinergic terminals is described. The antiserum was raised to small synaptosomes prepared from the purely cholinergic electric organ of Torpedo marmorata. It was shown to lyse cholinergic synaptosomes in a mixed population derived from guinea-pig cortex. After partial purification by adsorption onto nonspecific antigens, it was used to label nerve endings in several tissues of Torpedo, rats and guinea pigs using indirect immunofluorescence histochemistry. The antiserum appears to provide a highly specific means of localizing cholinergic nerve endings in these tissues.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Dense-core vesicles in motor nerve terminals

Summary Motor nerve terminals on white and intermediate muscle fibers of the Atlantic hagfish (Myxine glutinosa, L.) contain translucent synpatic vesicles and about 1–2% dense-core vesicles. Terminals on red muscle fibers contain up to 40% dense-core vesicles with diameter 800–1100 Å. Examinations for formaldehyde-induced fluorescence indicate yellow fluorescence (5-HT ?) apparently corresponding with terminal axons on red muscle fibers in craniovelar muscles. Possibly red muscle fibers of Myxine receive monoaminergic innervation.The author is indebted to Dr. Finn Walvig, Biological station, University of Oslo, Drøbak, for supply of hagfishes.     

Heterogeneity of reducing terminals of urinary chondroitin sulfates

Human urinary chondroitin sulfates were isolated by precipitation with cetylpyridinium chloride of the non-dialyzable fraction of pooled urine, followed by ethanol fractionation and successive enzymic digestions with neuraminidase and mucopolysaccharides. Further purification was achieved by Dowex-1 chromatography with stepwise elution by increasing the concentration of NaCl at intervals of 0.25 M from 0.75 M to 1.5 M. The chondroitin sulfates thus obtained were characterized by the analysis and quantification on of carbohydrate, amino acid and sulfate, and by electrophoresis on cellulose acetate membrane. Then reducing terminals were identified by gas liquid chromatographic analyses of the acetyl and butaneboronate derivatives of hydrolysates, after reduction of the reducing terminals with sodium borohydride. About 22.8% of the urinary chondroitin sulfate in the 1.5 M fraction was peptide-bound, and the remainder was peptide-free, with xylose (29.8%), galactose (23.6%) and glucuronic acid (18.7%) at the reducing terminal. The amount of peptide-free chondroitin sulfate with xylose and galactose at its reducing terminals in the 0.75 M-, 1.0 M-, 1.25 M- and 1.5 M-fractions increased in the order described in parallel with the increase of sulfation and the decrease of peptide content. It was thus suggested that the endo-types of beta-xylosidase, beta-galactosidase and beta-glucuronidase acted on the carbohydrate-peptide linkage region of proteo-chondroitin sulfate in the tissues and produced various types of urinary chondroitin sulfate with heterogeneity at reducing terminals.     

Structural plasticity of axon terminals in the adult

There is now conclusive evidence for widespread ongoing structural plasticity of presynaptic boutons and axon side-branches in the adult brain. The plasticity complements that of postsynaptic spines, but axonal plasticity samples larger volumes of neuropil, and has a larger impact on circuit remodeling. Axons from distinct neurons exhibit unique ratios of stable (t1/2>9 months) and dynamic (t1/2 5-20 days) boutons, which persist as spatially intermingled subgroups along terminal arbors. In addition, phases of side-branch dynamics mediate larger scale remodeling guided by synaptogenesis. The plasticity is most pronounced during critical periods; its patterns and outcome are controlled by Hebbian mechanisms and intrinsic neuronal factors. Novel experience, skill learning, life-style, and age can persistently modify local circuit structure through axonal structural plasticity.     

Isolation of synaptic terminals from Alzheimer's disease cortex

Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aβ, but the Aβ and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aβ-positive synaptosomes with the goal of understanding the nature of Aβ and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aβ were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aβ-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aβ oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aβ and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.     

Stoichiometry of the sodium-calcium exchanger in nerve terminals

The stoichiometry of the Na+-Ca2+ exchanger from synaptic plasma membranes was studied in both native and reconstituted preparations. In kinetic experiments performed with the native preparation, initial rates of Na+ gradient-dependent Ca2+ influx were compared to Ca2+-dependent Na+ efflux. These experiments showed that 4.82 Na+ ions are exchanged for each Ca2+ ion. A thermodynamic approach in which equilibrium measurements were made with the reconstituted preparation resulted in a similar (4.76) stoichiometry. The effects of membrane potential generated by valinomycin-induced K+ fluxes could be demonstrated in the reconstituted preparation. In addition, the direct contribution of the Na+-Ca2+ exchanger to the membrane potential across the reconstituted vesicle membrane could be demonstrated by using the lipophilic cation tetraphenylphosphonium.