title: Four-dimensional multi-site photolysis of caged neurotransmitters.
abstract: 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.
authors.affiliation: Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University Canberra, ACT, Australia.
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Go
authors.affiliation: Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University Canberra, ACT, Australia ; Department of Human Physiology and Centre for Neuroscience, Flinders University Adelaide, SA, Australia.
authors.forename: Minh-Son
authors.initials: MS
authors.lastname: To
authors.affiliation: Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University Canberra, ACT, Australia ; Medical School, College of Medicine, Biology and Environment, The Australian National University Canberra, ACT, Australia.
authors.forename: Christian
authors.initials: C
authors.lastname: Stricker
authors.affiliation: Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University Canberra, ACT, Australia.
authors.forename: Stephen
authors.initials: S
authors.lastname: Redman
authors.affiliation: Department of Quantum Science, Research School of Physics and Engineering, The Australian National University Canberra, ACT, Australia.
authors.forename: Hans-A
authors.initials: HA
authors.lastname: Bachor
authors.affiliation: Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University Canberra, ACT, Australia.
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Stuart
authors.affiliation: Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University Canberra, ACT, Australia.
authors.forename: Vincent R
authors.initials: VR
authors.lastname: Daria
keywords: caged neurotransmitters; holographic projection; synaptic integration; two-photon microscopy; two-photon photolysis