Confocal Imaging and 3D Reconstruction to Determine How Genetic Perturbations Impact Presynaptic Morphology at the Mouse Calyx of Held.

Neurons communicate via synapses-specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit. Key features Confocal imaging and 3D reconstruction of presynaptic terminals. Used with a virus-mediated expression of mEGFP to achieve efficient, cell-type specific labeling of the presynaptic compartment. Protocol was developed with the calyx of Held but is suitable for pre- and postsynaptic compartments of various neurons across multiple mammalian and invertebrate species.