Ca2+or Sr2+Partially Rescues Synaptic Transmission in Hippocampal Cultures Treated with Botulinum Toxin A and C, But Not Tetanus Toxin

Abstract

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 nmα-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⁺]_ofrom 2.8 to 5–10 mmor by applying 5 mmSr²⁺. 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⁺]_owere shifted toward higher [Ca²⁺]_oin 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.