The EU-funded project 'Manipulating neuronal outgrowth using a novel pneumatic micro gene gun' (MANIPULATING TILING) worked on identifying factors that direct and govern neuronal growth. Researchers aimed to develop the ability to study and manipulate neurons at the single cell level, both in vitro and in the intact animal. The nervous system of the medicinal leech was used for in vivo and in vitro studies. These are ideal for study as the leech neurons' activity and morphology are well characterised. Study focused on the mechanosensory neurons that are sensitive to light and pressure touch. Moreover, they can regenerate their branches and synaptic connections. The receptive fields are organised as an array of transducers that cover the skin, similar to how tiles cover a floor ('tiling'). According to previous work, cell-cell interactions are critical for sensory tiling. For the in vivo experiments, researchers used traditional microinjection set-up and a novel technology developed by project members called the pneumatic capillary gene gun. They delivered reagents to a well-defined region in the skin and the nervous system of intact embryos and adults to knock-in and knock-down genes. Neuronal cell culture experiments were performed on isolated leech neurons. Researchers were able to direct neuronal growth and align the neuronal processes to patterned substrates. They studied the role of the axon guidance cue netrin, as a candidate for directing mechanosensory tiling. Extraction of mRNA from leech embryos and analysis of the complementary DNA led to isolation of the netrin receptors — UNC-5 and DCC. Importantly, the DCC receptor of netrin was identified and sequenced for the first time. Results of the project were presented in several publications. Project activities led to a new developmental scheme for sensory neurons and approaches to promote reinnervation of transplanted skin post-trauma. The use of the new generation gene gun will facilitate the development of novel methodologies for gene manipulation and drug delivery.
Neurons, axons, dendrites, networks, leech, mechanosensory, microinjection, gene gun, receptor, netrin