Synaptic vesicles, preparation

If there is any problem with protein detection, enriched synaptic protein preparations (synaptosomes, presynaptic plasma membrane of synaptic vesicle preparations, depending of the toxin and the substrate) can be better starting material than brain homogenate to check the proteolytic activity of clostridial neurotoxins L chains. 

Kramer and coworkers (1989) investigated the effect of nicotine on the accumulation of dopamine in synaptic vesicles prepared from mouse cerebral cortex or bovine striatum. It was found to be a weak inhibitor of dopamine accumulation. 

Huttner, W., et al. (1983). Synapsin I (Protein I), a Nerve Terminal-specific Phosphoprotein. Ill Its Association with Synaptic Vesicles Studied in a Highly Purified Synaptic Vesicle Preparation, J. Cell Biol. 96 1374-1388. 

Kuo, C.-H., Hata, F., Yoshida, H., Yamatodani, A., and Wada, H., 1979, Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain, Life Sci. 24 911-916. 

To prepare a synapse to respond to another signal, the neurotransmitter must be removed quickly from the synaptic cleft. In some cases, the transmitter is taken up by the presynaptic neuron and repackaged in synaptic vesicles in other cases, it is broken down by extracellular enzymes. Acetylcholine, for example, is hydrolyzed rapidly to choline and acetate by the enzyme acetylcholinesterase. 

The —50 kDa band (48-53 kDa) is identified as dysbindin-1 A in our WESTERNS, because it runs close to the molecular mass of our histidine-tagged recombinant mouse dysbindin-1 A. Its identity is confirmed by the fact that it is recognized by antibodies we have recently generated to amino acid sequences in the CTR of human dysbindin-lA, but not found in dysbindin-lB, -2, or -3. The —50 kDa band is the most consistently observed dysbindin-1 band across tissues. We find it in all tissues examined to date the adrenal gland, heart, kidney, liver, lung, spleen, skeletal muscle, testes, spinal cord, cerebellum, striatum, hippocampus, and cerebral cortex (e.g., Figure 2.2-12a and c). In mouse and human synaptosomes, the —50 kDa isoform is heavily concentrated in the PSD fraction with a much lesser amount in the presynaptic membrane and no detectable amount in the synaptic vesicle fraction (Talbot et al., in preparation). 

The —33 kDa band (32-34 kDa) is close in molecular mass to dysbindin-1C, which lacks the NTR of dysbindin-1 A but is otherwise identical to dysbindin-1 A (see Section 2.2.2.2.1). It is consequently difficult to determine if the —33 kDa band represents (1) dysbindin-lC itself, (2) a degradative product of dysbindin-1 A missing the NTR, or (c) some combination of the first two possibilities. The first possibility seems most likely, however, because the —33 kDa band is absent in some tissues (e.g., cerebellum, kidney, and liver) where the —50 kDa band (i.e., dysbindin-1 A) is present and hence subject to degradation ( Figure 2.2-12c). Moreover, the —33 kDa band is often stronger than the 50 kDa band even in fresh tissue. We thus believe that the —33 kDa band does represent dysbindin-1C. It is seen in the heart, lung, skeletal muscle, striatum, hippocampal formation, and cerebral cortex (e.g., O Figure 2.2-12a and c). In synaptosomes of the mouse and human brain, the —33 kDa isoform is concentrated in synaptic vesicle and PSD fractions with very little, if any, present in the presynaptic membrane fraction (Talbot et al., in preparation). 

Although plasma membrane monoamine transporters are responsible for the reuptake of neurotransmitters from the synapse, vesicular monoamine transporters (VMAT) sequester monoamines into synaptic vesicles in preparation for fusion with the plasma membrane and release into the synapse (Schuldiner et ah, 1995). Vesicular uptake is coupled to a proton gradient across the vesicle membrane rather than the sodium gradient used with the plasma membrane transporters (Schuldiner et ah, 1995). These vesicular transporters are not neurotransmitter-speciflc rather, they transport the monoamines nonselectively (Johnson, Jr., 1988 Henry et ah, 1998). 

Fluorescent styryl dyes such as FMl-43 have been used to approximate neurotransmitter release by measuring rates of ex-ocytosis (16, 72, 73). These dyes reversibly label endosomal membranes and can be taken up into intracellular synaptic vesicles during endocytosis in systems in which vesicle recycling takes place. Typically, tissue is incubated in the fluorescent dye and then stimulated to promote vesicle cycling and therefore uptake of the dye. The preparation then is washed in fresh buffer to remove dye that remained extracellular. Using fluorescent microscopy, vesicle dynamics can be tracked. Neurotransmitter release is estimated from the rate of destaining (because of exocytosis) usually during stimulation. 

Protease-free preparations of TeTx and BoNT/B, D, F and G cleave a membrane protein of small synaptic vesicles (SSV) called VAMP or synaptobrevin (Schiavo ef ai, 1992 a, 1993 a,c, Yamasaki et ai, 1994 a, b). Conversely, BoNT/A, C and E act on proteins associated with the presynaptic membrane BoNT/A and E cut SNAP-25, while serotype C cleaves syntaxin, in addition to SNAP-25 (Blasi efai, 1993 a, b ... 

The procedure for the preparation of synaptosomes and small synaptic vesicles (SSV) from rat brain cortex follows established methods with minor modifications (Schiavo and Montecucco, 1995). 

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. 

Fig. 7. Reversible trapping of synaptic vesicle membrane in the plasma membrane in reticulospinal synapses. (A) Electron micrograph of a lamprey reticulospinal synapse stimulated with action potentials at 20 Hz for 20 min and then incubated for 90 min in Ca " -free solution with 10 mM EGTA. Note the reduction in the number of synaptic vesicles and the presence of large membrane expansions compared to an unstimulated synapse (inset). (B) Activation of clathrin-mediated endocytosis in reticulospinal synapses by addition of Ca +-containing extracellular solution. Spinal cord preparations were stimulated at 20 Hz for 20 min, incubated for 90 min in Ca -free solution, and then incubated in Ca -containing solution (2.6 mM) for 120 s. Electron micrograph of a synapse shows the appearance of coated pits (arrows) lateral to the active zone. Designations as in Fig. 1. Scale bar, 0.2 p.m. Modified from Gad et al. (1998) Neuron 21 601-616, with permission copyright is held by Cell Press.

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