Synaptosomes Poisoning

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Raciborska DA, Charlton MP (1999) Retention of cleaved synaptosome-associated protein of 25 kDa (SNAP-25) in neuromuscular junctions a new hypothesis to explain persistence of botulinum A poisoning. Can J Physiol Pharmacol 77 679-88 Raciborska DA, Trimble WS, Charlton MP (1998) Presynaptic protein interactions in vivo evidence from botulinum A, C, D and E action at frog neuromuscular junction. Eur J Neurosci 10 2617-28... 

Otto, H., Hanson, P.I., Chapman, E.R., Blasi, J., Jahn, R. (1995). Poisoning by hotulinum neurotoxin A does not inhibit formation or disassembly of the synaptosomal fusion complex. Biochem. Biophys. Res. Commun. 212 945-52. 

Fig. 2. Schematic drawing that summarizes the procedure for monitoring the blockade of neurotransmitter release from brain synaptosomes, followed by immu-noblot analysis to detect specific proteolytic activity of clostridial neurotoxins on poisoned synaptosomes...

This fraction (P2) can be used for poisoning and release experiments if high purity of the synaptosomal fraction is not required. In this case the protein concentration should be determined. Then, the sample should be divided into aliquots containing the desired amount of protein (usually 1-3 mg), recentrifuged at 14 000 g for 12 min and stored as pellets on ice until use. 

If you do not detect any inhibition of the transmitter release from the poisoned synaptosomes check the activity of the neurotoxins using one of the following methods ... 

Sellaijevic, L., Kriolica, K., and Boskovic, B., The effect of soman poisoning on phos-phorylating capability and adenylate cyclase activity of isolated synaptosomal membranes. Biochem. Pharmacol, 33, 3714, 1984. 

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