Primary cell culture for evaluation of botulinum neurotoxin antagonists

The actions of botulinum neurotoxin (BoNT) were studied on evoked release of the neurotransmitter glycine in primary mouse spinal cord cells. (3)[H]-glycine was taken up by cells in physiological solution and released by depolarization with 56 mM K+ in the presence of 2 mM Ca2+. Release of (3)[H]-glycine was found to be inhibited by BoNT serotypes A, B and E with similar potency ratios to those observed in the acutely isolated mouse diaphragm muscle. When spinal cord cultures were exposed to BoNT/A for 24 h, inhibition of (3)[H]-glycine release was detected at toxin concentrations as low as 10(-14) M, and complete inhibition was observed at concentration greater than or equal to10(-12) M. Preincubation of BoNT/A with polyclonal equine antiserum led to antagonism of toxin-induced inhibition of (3)[H]-glycine release in spinal cord cells and to protection of mice from the lethal effects of BoNT/A. It is concluded that spinal cord neurons are a useful model for studying botulinum intoxication and for evaluating BoNT antagonists. Published by Elsevier Ltd.