Simultaneous imaging of structural plasticity and calcium dynamics in developing dendrites and axons

During nervous system development, the formation of synapses between pre- and postsynaptic neurons is a remarkably specific process. Both structural and functional plasticity are critical for the selection of synaptic partners and for the establishment and maturation of synapses. To unravel the respective contributions of structural and functional mechanisms as well as their interactions during synaptogenesis, it is important to directly observe structural changes and functional signaling simultaneously. Here, we present an imaging approach to simultaneously follow changes in structure and function. Differential labeling of individual cells and the neuronal network with distinct dyes allows the study of structural plasticity and changes in calcium signaling associated with neural activity at the same time and with high resolution. This is achieved by bulk loading of neuronal populations with a calcium-sensitive indicator in combination with electroporation of individual cells with a calcium indicator and an additional noncalcium-sensitive dye with a different excitation spectrum. Recordings of the two differently labeled structures can be acquired simultaneously using confocal microscopy. Thus, structural plasticity and calcium dynamics of the individually labeled neuron and the surrounding network can be related to each other. This combined imaging approach can be applied to virtually all systems of neuronal networks to study structure and function. We provide a comprehensive description of the labeling procedure, the imaging parameters, and the important aspects of analysis for simultaneous recordings of structure and function in individual neurons.