Inhibition of transmitter release correlates with the proteolytic activity of tetanus toxin and botulinus toxin A in individual cultured synapses of Hirudo medicinalis

J Neurosci. 1997 Mar 15;17(6):1898-910. doi: 10.1523/JNEUROSCI.17-06-01898.1997.


We have studied the effects of tetanus toxin and botulinus toxin A on neurotransmitter release in the Retzius-->P-cell synapse of the leech and exploited the unique properties of this system, which allow for combined physiological and biochemical analyses in single-cell pairs. The sequences of Hirudo medicinalis synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25), deduced by cDNA cloning, are 61 and 55% identical, respectively, to their corresponding mammalian homologs. Whereas Hirudo synaptobrevin is proteolyzed by tetanus toxin, its SNAP-25 isoform is resistant to botulinus toxin A cleavage because of amino acid substitutions within and around the putative cleavage site. In close correlation, microinjection of tetanus toxin into the presynaptic neuron produced a block of transmitter release, whereas botulinus toxin A had no effect on synaptic transmission. Subsequent immunoblotting of single-cell pairs demonstrated directly that the tetanus toxin-mediated block of exocytosis is accompanied by cleavage of synaptobrevin in the injected neuron, resulting in the generation of a detectable C-terminal cleavage product. Immunoblotting also confirmed the resistance of SNAP-25 to botulinus toxin A cleavage in vivo. Using recombinant proteins, we show that the N-terminal fragment of synaptobrevin released by tetanus toxin, but not its C-terminal membrane-anchored cleavage product, participates with syntaxin and SNAP-25 in synaptic SNAP receptor (SNARE) ternary complex formation in Hirudo. Our data demonstrate a direct correlation between the inhibition of transmitter release and the ability of the neurotoxin to proteolyze its target protein and support the view that SNARE ternary complex formation is an important step leading to synaptic vesicle exocytosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Botulinum Toxins / pharmacology*
  • Cells, Cultured / drug effects
  • Leeches
  • Molecular Sequence Data
  • Neurotransmitter Agents / metabolism*
  • Synapses / drug effects*
  • Tetanus Toxin / pharmacology*


  • Neurotransmitter Agents
  • Tetanus Toxin
  • Botulinum Toxins

Associated data

  • GENBANK/U85805
  • GENBANK/U85806
  • GENBANK/U85807