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Volume 17, Number 19,
Issue of October 1, 1997
pp. 7190-7202
Copyright ©1997 Society for Neuroscience
Ca2+ or Sr2+ Partially Rescues
Synaptic Transmission in Hippocampal Cultures Treated with Botulinum
Toxin A and C, But Not Tetanus Toxin
Received April 21, 1997; revised June 16, 1997; accepted July 11, 1997.
Marco Capogna1,
R. Anne McKinney1,
Vincent O'Connor2,
Beat H. Gähwiler1, and
Scott M. Thompson1
1 Brain Research Institute, University of Zurich,
CH-8029 Zurich, Switzerland, and 2 Max Planck Institute for
Brain Research, Department of Neurochemistry, D-60528 Frankfurt,
Germany
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 -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.
Key words:
Ca2+;
clostridial neurotoxins;
exocytosis;
-latrotoxin;
protein kinase;
transmitter release
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