Poisoning of Synaptosomes

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins 207 

Follow the protocol above for monitoring neurotransmitter release, starting at step 2 (Section 15.4.4). 

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  

Bioassay. This assay is sensitive, direct and reliable but it requires the sacrifice of many laboratory mice. Various dilutions of the neurotoxin are injected intraperitoneally into mice. The amount of toxin that kills half a population of mice in tour days is calculated (LD50). Toxin can also be injected intravenously. The delay time between injection and death can then be used as a parameter to evaluate the potency of the toxin, allowing calculation of the LD50 (Borott and Fleck, 1966). 

In vitro Assay of L Chain Activity. Since the elucidation of the molecular mechanisms of all clostridial neurotoxins, it has become convenient to test the proteolytic activity of the L chain in vitro. For this purpose, L chain or reduced di-chain toxin is incubated with substrate protein. The cleavage products are then separated by SDS-PAGE and analyzed by immunoblotting or autoradiography. 

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