• growth balls;
  • growth cones;
  • Lymnaea stagnalis;
  • neurodevelopment;
  • synapse formation;
  • synapse specificity


All brain functions, ranging from motor behaviour to cognition, depend on precise developmental patterns of synapse formation between the growth cones of both pre- and postsynaptic neurons. While the molecular evidence for the presence of ‘pre-assembled’ elements of synaptic machinery prior to physical contact is beginning to emerge, the precise timing of functional synaptogenesis between the growth cones has not yet been defined. Moreover, it is unclear whether an initial assembly of various synaptic molecules located at the extrasomal regions (e.g. growth cones) can indeed result in fully mature and consolidated synapses in the absence of somata signalling. Such evidence is difficult to obtain both in vivo and in vitro because the extrasomal sites are often challenging, if not impossible, to access for electrophysiological analysis. Here we demonstrate a novel approach to precisely define various steps underlying synapse formation between the isolated growth cones of individually identifiable pre- and postsynaptic neurons from the mollusc Lymnaea stagnalis. We show for the first time that isolated growth cones transformed into ‘growth balls’ have an innate propensity to develop specific and multiple synapses within minutes of physical contact. We also demonstrate that a prior ‘synaptic history’ primes the presynaptic growth ball to form synapses quicker with subsequent partners. This is the first demonstration that isolated Lymnaea growth cones have the necessary machinery to form functional synapses.