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 >or=10(-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.