Marco Capogna

Botulinum (BoNT/A-G) and tetanus toxins (TeNT) are zinc endopeptidases that cleave proteins associated with presynaptic terminals (SNAP-25, syntaxin, or VAMP/synaptobrevin) and block neurotransmitter release. Treatment of hippocampal slice cultures with BoNT/A, BoNT/C, BoNT/E, or TeNT prevented the occurrence of spontaneous or miniature EPSCs (sEPSCs or mEPSCs) as well as the [Ca2+]o-independent increase in their frequency induced by phorbol ester, 0.5 nM alpha-latrotoxin, or sucrose. [Ca2+]o-independent and -dependent release thus requires that the target proteins of clostridial neurotoxins be uncleaved. In contrast, significant increases in mEPSC frequency were produced in BoNT-treated, but not TeNT-treated, cultures by application of the Ca2+ ionophore ionomycin in the presence of 10 mM [Ca2+]o. The frequency of sEPSCs was increased in BoNT-treated, but not TeNT-treated, cultures by increasing [Ca2+]o from 2.8 to 5-10 mM or by applying 5 mM Sr2+. Large Ca2+ and Sr2+ influxes thus can rescue release after BoNT treatment, albeit less than in control cultures. The nature of the toxin-induced modification of Ca2+-dependent release was assessed by recordings from monosynaptically coupled CA3 cell pairs. The paired-pulse ratio of unitary EPSCs evoked by two presynaptic action potentials in close succession was 0.5 in control cultures, but it was 1.4 and 1.2 in BoNT/A- or BoNT/C-treated cultures when recorded in 10 mM [Ca2+]o. Log-log plots of unitary EPSC amplitude versus [Ca2+]o were shifted toward higher [Ca2+]o in BoNT/A- or BoNT/C-treated cultures, but their slope was unchanged and the maximal EPSC amplitudes were reduced. We conclude that BoNTs reduce the Ca2+ sensitivity of the exocytotic machinery and the number of quanta released.