Combining multiphoton excitation microscopy with fast microiontophoresis to investigate neuronal signaling

Abstract

Multiphoton excitation (MPE) microscopy allows subcellular structural and functional imaging of neurons and can be combined with techniques for activating postsynaptic receptors at spatial and temporal scales that mimic normal synaptic transmission. Here, we describe procedures for combining MPE imaging of dye-filled neurons with fast microiontophoresis, by which neurotransmitter agonists can be applied from high-resistance micropipettes with subcellular resolution. With adequate compensation of the pipette capacitance, the effective time constant of the pipette is reduced, and this permits application of very brief pulses of receptor agonist (≤1 ms). The consequent high temporal and spatial resolution leads to the high specificity required for single-synapse investigations. This chapter includes detailed procedures for electrophysiological whole-cell recording, structural and functional (Ca2+) MPE imaging of dye-filled neurons, targeting a microiontophoresis pipette to a specific subcellular compartment of a dye-filled neuron under visual control, and capacitance compensation of the microiontophoresis pipette, as well as examples of experimental results that can be obtained.