Expansion Microscopy of Synaptic Contacts on the Mauthner Cells of Larval Zebrafish

Because of its genetic tractability and amenability for live imaging, larval zebrafish (Danio rerio) have emerged as a model to study the cellular and synaptic properties underlying behavior. The accessibility of Mauthner cells, a pair of escape-organizing neurons located in the brainstem of teleost fish, along with their associated sensory inputs, enables exploration of the correlation between structural and functional synaptic features. This is the case of the endings of auditory afferents on the lateral dendrite of this cell, known as large myelinated club endings, which provide the excitatory drive for the initiation of auditory-evoked escape responses mediated by the Mauthner cell and its spinal network. Here, we describe the procedures that make it possible to expose the molecular composition of these synapses using protein-retention expansion microscopy (proExM). This method allowed us to generate a map of the distribution of synaptic proteins at these identifiable synapses, which could also be applied to examine the organization of other synaptic contacts in this cell. Key features • This protocol builds upon the method developed by Tillberg et al. [1] • Optimized for the examination of the organization of molecular components at synaptic contacts on the Mauthner cells of larval zebrafish • Requires at least three days to complete and should be preceded by immunostaining. • Results in a linear expansion factor of ~3.9× and an area expansion factor of ~13×.