In vivo single-cell excitability probing of neuronal ensembles in the intact and awake developing Xenopus brain.

Sensory experience can elicit long-lasting plasticity of both single neurons and ensemble neural circuit response properties during embryonic development. To investigate their relationship, one must image functional responses of large neuronal populations simultaneously with single-cell resolution. In this protocol, we describe a noninvasive approach to assay functional plasticity of individual neurons and neuronal populations in vivo using targeted infusion of calcium-sensitive dyes, two-photon microscopy and synchronized visual stimuli presentations. This technique allows visualization of approximately 200 neurons while probing visual responses in the optic tectum of awake, immobilized Xenopus laevis tadpoles. The protocol includes visual training paradigms that elicit long-lasting potentiation or depression of functional responses, allowing investigations of population and single-neuron plasticity induced by natural sensory stimuli in the awake, intact, developing brain. Setup time for this protocol, including dye injection and chamber preparation, is approximately 2 h. Excitability probing experiments can then be performed for at least 3 h.