Subcellular synaptic vesicles

The action of catecholamines released at the synapse is modulated by diffusion and reuptake into presynaptic nerve terminals. Catecholamines diffuse from the site of release, interact with receptors and are transported back into the nerve terminal. Some of the catecholamine molecules may be catabolized by MAO and COMT. The cate-cholamine-reuptake process was originally described by Axelrod [18]. He observed that, when radioactive norepinephrine was injected intravenously, it accumulated in tissues in direct proportion to the density of the sympathetic innervation in the tissue. The amine taken up into the tissues was protected from catabolic degradation, and studies of the subcellular distribution of catecholamines showed that they were localized to synaptic vesicles. Ablation of the sympathetic input to organs abolished the ability of vesicles to accumulate and store radioactive norepinephrine. Subsequent studies demonstrated that this Na+- and Cl -dependent uptake process is a characteristic feature of catecholamine-containing neurons in both the periphery and the brain (Table 12-2). 

Synaptobrevin, SNAP-25, and syntaxin are all highly abundant membrane proteins in the mammalian CNS. Thus, a sufficiently enriched membrane preparation can be obtained by crude subfractionation techniques. While this approach is convenient, particularly for laboratories with no expertise in molecular techniques, it has a number of shortcomings due to the inherent property of the proteins to form toxin-resistant complexes (Hayashi ef.al., 1994). A procedure based on a crude tissue extract is given below. Better results can be achieved when using more highly purified subcellular fractions, e.g., synaptic vesicle fractions, for the assay of synaptobrevin-cleaving toxins. 

Cu is normally found at relatively high levels in the brain (100-150 xM) with substantial variations at the cellular and subcellular level [55-57]. Ionic Cu is compartmentalized into a post-synaptic vesicle and released upon activation of the NMDA-R but not AMPA/kainate-type glutamate receptors [58]. The Menkes Cu7aATPase is the vesicular membrane Cu transporter, and upon NMDA-R activation, it traffics rapidly and reversibly to neuronal processes, independent of the intracellular Cu concentration [58]. Cu ions function to suppress NMDA activation and prevent excitotoxicity by catalyzing S-nitrosylation of specific cysteine residues on the extracellular domain of the NRl and NR2A subunits of the NMDA receptor [58]. The concentrations of Cu in the synaptic cleft can reach approximately 15 xM. Subsequently, Cu is cleared by uptake mechanisms from the synaptic cleft. Several studies have shown that Cu levels increase with age in the brains of mice [22-24]. 

Synaptic vesicles, isolated from rat brain cortex, and cholinergic vesicles, isolated from the electric organ of Torpedo nobiliana, were broken down by a phospholipase A from cobra venom. The breakdown was accompanied by a release of acetylcholine. Morphological analysis revealed membrane fragments about one-third the size of the vesicle circumference. Subcellular fractions enriched in nerve ending membranes showed some phospholipase A activity. On the basis of these findings models of transmitter release, involving specific alterations of lipid components in the vesicular membrane, are discussed. 

SYNAPTIC VESICLES. SPECIFIC GRANULES, AUTOPHARMACOLOGY S.2.6.3. Cellular and Subcellular Localization of the Biosynthetic Enzymes... 

All of the known neurotransmitters synthesised in mammalian neurons share certain chemical properties all are low-molecular-weight, water-soluble compounds which are ionised at the pH of body fluids. All are synthesised primarily in the parts of the neuron (the synaptic bulbs, or nerve terminals) which form synaptic contacts with the cells to which the neuron transmits signals (the post-synaptic cells), and all are stored within subcellular organelles (synaptic vesicles) or are attached to cytoplasmic proteins, prior to being released into a synapse (Fig. 1). Release occurs when the neuron that synthesised the neurotransmitter is depolarised. Thereafter, some of... 

The subcellular localization of the enzyme is interesting. In contrast to tyrosine hydroxylase, which is localized to the cytoplasmic compartment, dopamine hydroxylase is largely contained within the aminergic storage vesicles. Thus, it appears that dopamine, which is synthesized in the cytosol, must be taken up into these vesicles in order to be converted to norepinephrine. Approximately 50% of the enzyme is associated with the membranous portion of the vesicles, whereas the other 50% is in a soluble form within the vesicle. During synaptic transmission noradrenergic neurons release both norepinephrine and its biosynthetic enzyme by an exocytotic mechanism. While most of the released norepinephrine is taken back up by the terminal, the enzyme is believed to diffuse of the synaptic cleft into the extracellular fluid and eventually into the serum. There are relatively large amounts of dopamine p-hydroxylase in human serum that are believed to arise from release from sympathetic neurons. 

When an action potential traveling down the axon of a motoneuron reaches the myoneural endplate, a process occurs that releases acetylcholine into the synaptic cleft and consequently depolarizes the postsynaptic membrane. A similar process probably occurs at cholinergic synapses in the central nervous system. In 1950 Fatt and Katz discovered a spontaneous subthreshold activity (MEPP) of motor nerve endings and were thereby led to the concept that acetylcholine is released in definite units (quanta) of 10 to 10 molecules. Electron microscopy subsequently revealed characteristic vesicles about 40 nm in diameter, clustered near presynaptic membranes. Subcellular fractionation procedures were devised by Whittaker and de Robertis for the isolation of these vesicles from brain homogenates in sucrose density gradients, and it was soon demonstrated that they were indeed concentrated reservoirs of acetylcholine. The hypothesis that the vesicles discharge the quanta of transmitter became irresistible. 

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