Combining Membrane Potential Imaging with l-Glutamate or GABA Photorelease

Abstract

Combining membrane potential imaging using voltage sensitive dyes with photolysis of l-glutamate or GABA allows the monitoring of electrical activity elicited by the neurotransmitter at different sub-cellular sites. Here we describe a simple system and some basic experimental protocols to achieve these measurements. We show how to apply the neurotransmitter and how to vary the dimension of the area of photolysis. We assess the localisation of photolysis and of the recorded membrane potential changes by depolarising the dendrites of cerebellar Purkinje neurons with l-glutamate photorelease using different experimental protocols. We further show in the apical dendrites of CA1 hippocampal pyramidal neurons how l-glutamate photorelease can be used to calibrate fluorescence changes from voltage sensitive dyes in terms of membrane potential changes (in mV) and how GABA photorelease can be used to investigate the phenomenon of shunting inhibition. We also show how GABA photorelease can be used to measure chloride-mediated changes of membrane potential under physiological conditions originating from different regions of a neuron, providing important information on the local intracellular chloride concentrations. The method and the proof of principle reported here open the gateway to a variety of important applications where the advantages of this approach are necessary.

Spatially Selective Holographic Photoactivation and Functional Fluorescence Imaging in Freely Behaving Mice with a Fiberscope

“…This has been achieved in brain slices and in anesthetized animals by combining imaging of Ca 2+ -sensitive probes or voltage-sensitive dye (VSD) with glutamate uncaging, either with 1P excitation (Anselmi et al, 2011;Vogt et al, 2011;Zahid et al, 2010) or 2P excitation (Bloodgood and Sabatini, 2007;Losonczy and Magee, 2006;Losonczy et al, 2008;Nikolenko et al, 2007;Noguchi et al, 2011) or by combining VSD imaging with 1P ChR2 photoactivation (Lim et al, 2012;Tsuda et al, 2013;Willadt et al, 2014). All these approaches benefit from a reduced overlap between the absorption spectra of the functional reporter and the photosensitive actuator.…”

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

“…Optical stimulation has been paired with voltage imaging 11–14 . However, robust and crosstalk-free genetically targeted all-optical electrophysiology has not been achieved due to limitations on the speed and sensitivity of genetically encoded voltage indicators (GEVIs), and spectral overlap between existing GEVIs and optogenetic actuators.…”

Using simultaneous voltage and calcium imaging to study fast Ca2+channels

“…The JPW1114-ΔF∕F 0 signal was calibrated in terms of V m change using a previously demonstrated protocol based on wide-field photorelease of L-glutamate from the caged compound 4-methoxy-7-nitroindolinyl-caged-L-glutamate (MNI-glutamate). 17 Briefly, a calibration of the JPW1114-ΔF∕F 0 signal can be done if an electrical signal of known amplitude is available at all recording sites. In many neuronal types, activation of a large portion of ionotropic glutamate receptors makes them the dominant conductance and the resulting V m will be 0 mV in all the illuminated area.…”