Gap junctions (GJs) provide a common form of intercellular communication in most animal cells and tissues, from Hydra to human, including electrical synaptic signalling. Cell coupling via GJs has an important role in development in general, and in neural network development in particular. However, quantitative studies monitoring GJ proteins throughout nervous system development are few. Direct investigations demonstrating a role for GJ proteins by way of experimental manipulation of their expression are also rare. In the current work we focused on the role of invertebrate GJ proteins (innexins) in the in vitro development of neural network functional topology, using two-dimensional neural culture preparations derived from the frontal ganglion of the desert locust, Schistocerca gregaria. Immunocytochemistry and quantitative real-time PCR revealed a dynamic expression pattern of the innexins during development of the cultured networks. Changes were observed both in the levels and in the localization of expression. Down-regulating the expression of innexins, by using double-strand RNA for the first time in locust neural cultures, induced clear changes in network morphology, as well as inhibition of synaptogenesis, thus suggesting a role for GJs during the development of the functional topology of neuronal networks.