Four-dimensional multi-site photolysis of caged neurotransmitters

• Main Authors: Mary Ann eGo, Minh-Son eTo, Christian eStricker, Stephen eRedman, Hans-A. eBachor, Greg eStuart, Vincent eDaria
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2013-12-01
• Series: Frontiers in Cellular Neuroscience
• Subjects: Synaptic integration; two-photon microscopy; Two-photon photolysis; holographic projection; caged neurotransmitters
• Online Access: http://journal.frontiersin.org/Journal/10.3389/fncel.2013.00231/full

Neurons receive thousands of synaptic inputs that are distributed in space and time. The systematic study of how neurons process these inputs requires a technique to stimulate multiple yet highly targeted points of interest along the neuron's dendritic tree. Three-dimensional multi-focal patterns produced via holographic projection combined with two-photon photolysis of caged compounds can provide for highly localized release of neurotransmitters within each diffraction-limited focus, and in this way emulate simultaneous synaptic inputs to the neuron. However, this technique so far cannot achieve time-dependent stimulation patterns due to fundamental limitations of the hologram-encoding device and other factors that affect the consistency of controlled synaptic stimulation. Here, we report an advanced technique that enables the design and application of arbitrary spatio-temporal photostimulation patterns that resemble physiological synaptic inputs. By combining holographic projection with a programmable high-speed light-switching array, we have overcome temporal limitations with holographic projection, allowing us to mimic distributed activation of synaptic inputs leading to action potential generation. Our experiments uniquely demonstrate multi-site two-photon glutamate uncaging in three dimensions with submillisecond temporal resolution. Implementing this approach opens up new prospects for studying neuronal synaptic integration in four dimensions.