Guided Growth Of Neurons On Micro-Structured Surfaces

Synaptic cell adhesion molecules (SCAMs) are well known to interact across the synaptic cleft of central mammalian synapses. However, their functional role in transsynaptic modulation of the synaptic vesicle cycle (i.e. from the postsynaptic to the presynaptic neuron) is poorly understood. Several families of SCAMs have been characterized at the molecular level. Transsynaptic interactions mediated by SCAMs potentially control initial synapse formation, regulate structural maturation of synapses, modulate basal synaptic function including vesicle endocytosis, and participate in different forms of long-term synaptic plasticity. In order to better separate pre- and postsynaptic effects we grew neurons on microstructured surfaces, functionalized with SCAM proteins. Glass coverslips were patterned with the Neurexin-binding Neuroligin fragment via microcontactprinting by either coating them with silanes to which we covalently coupled Neuroligin with cysteine tag, or with Poly-l-lysine-polyethylenglycol-HaloTag-ligand, covalently linked to Neuroligin via a HaloTag. Both approaches lead to controlled and guided growth of neuronal outgrowths. Formation of presynaptic sites was triggered within one to two days. These sites showed a positive staining with antibodies against the active zone markers RIM1/2 and the synaptic vesicle protein Synaptophysin1. They often opposed the structured Neuroligin patches, as revealed by 4Pi microscopy. Such varicosities contained vesicles that could be stained with FM 1-43 upon electrical stimulation. Release of FM 1-43 by repeated stimulation could be monitored by TIRF microscopy, displaying similar kinetics as control synapses. Formation of synapses on structured surfaces opens up the possibility to study presynapse formation and dynamics under controlled conditions.