Date Published: October 11, 2011
Publisher: Public Library of Science
Author(s): Kaspar E. Vogt, Stephan Gerharz, Jeremy Graham, Marco Canepari, Olivier Jacques Manzoni. http://doi.org/10.1371/journal.pone.0024911
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.
Partial Text: In physiological research, optical techniques offer the possibility to stimulate and record from multiple sub-cellular sites, potentially with high spatial resolution. The advantages of optical methods are greater when optical stimulation is combined with optical recording. Chemical stimulation can be delivered optically by photorelease of a molecule from a caged compound . Because most available caged compounds do not photorelease above a certain wavelength, uncaging stimulation can be optimally combined with fluorescence recording if the light used to excite the fluorescent molecule is inert to the caged compound. A notable example is the recording of Ca2+ signals associated with photorelease of inositol triphosphate, obtained initially at broad spatial resolution ,  and later with a resolution in the order of a micron . Similarly, Ca2+ signals mediated by synaptic glutamate receptors were recorded from neuronal dendrites after large-field l-glutamate photorelease , , or recorded from dendritic spines after two-photon l-glutamate photorelease .
In this report we describe how to perform simultaneous voltage imaging and photolytic release of l-glutamate or GABA. These measurements were based on the resolution of several technical problems, including the modification of the microscope for dual illumination and the development and assessment of simple protocols for local perfusion of caged compounds and the regulation of the dimension of the photolysis spot. The caged compounds used in this report are stable, pharmacologically inert, efficient and kinetically fast , . Thus, the approach described here can be used for a variety of applications. The optimal size of the area of photolysis will depend on the particular application. For some applications, the optimal diameter of the illumination spot is 1–5 µm. This can be achieved either by using a UV laser , ,  or by two photon uncaging , –. UV lasers can be coupled to our microscope through the epifluorescence pathway replacing the UV LED that was used in the present configuration. For other applications a less localised photolysis or the uncaging over the whole field of view are more advantageous. This can be done, as described here, using UV LED illumination. In this report we have shown how to use simultaneous voltage imaging and photolysis as tools to calibrate the fluorescence signals in terms of absolute membrane potential changes. This is a notable result because it allows the possibility to quantitatively compare signals from different sites, a requirement necessary to analyse the spatial distribution of membrane potential changes. The method described here is valid in the cell types where the glutamate receptor conductance becomes dominant over the background conductance and can be possibly extended to other cell types using different caged compounds. We also showed how to record Cl− mediated membrane potential changes under physiological conditions and how to investigate local mechanisms of signal integration such as shunting inhibition. The most salient finding from these experiments is the extreme focal restriction of the shunting effect and the resulting rapid loss in action potential backpropagation. This despite the fact that the GABA uncaging occurred over a relatively large area – considerably larger than what would be expected for a synaptic signal. These applications were used to validate the method, thus opening the gate to novel physiological investigations. The apparatus to achieve these measurements is based on commercially available equipment that can be adapted to conventional microscopes.