Combining voltage and Ca2+ imaging allows the correlation of electrical and chemical activity at sub-cellular level. Here we describe a novel apparatus designed to obtain simultaneous voltage and Ca 2+ measurements with single-trial resolution from sites as small as a few microns. These measurements can be obtained with negligible optical cross-talk between the two signals and negligible photo-damage of the preparation. The capability of the technique was assessed recording either from individual neurons in brain slices or from networks of cultured neurons.The present achievements open the gate to many novel physiological investigations requiring simultaneous measurement of voltage and Ca 2+ signals.
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.
We reinvestigated the solvatochromism of 8-hydroxypyrene-1,3,6-trisulfonate (pyranine) in conjunction with that of 8-methoxypyrene-1,3,6-trisulfonate and of 1-hydroxypyrene (pyrenol) by use of 25 different solvents. Conclusions for the prediction of ESPT behaviour of synthetic dyes were drawn by comparison with the solvatochromism of p-hydroxystyryl Bodipy dyes. Solvents were chosen according to their Kamlet-Taft parameters alpha and beta for elucidating the acidicity of the dyes and the basicity of their conjugated bases in the ground and excited state. Comparison of the spectra of pyranine and pyrenol in solvents with varying beta-values revealed that the acidity of both dyes is similar therein. The well-known ESPT behaviour of pyranine in water is assigned to a change of the electronic state at alpha-values approximately 0.7 to 0.8. The high acidity of this excited state also appears in the vanishing solvatochromism of the photoproduct fluorescence. However, prediction of an ESPT tendency of synthetic dyes might fail when only fluorescence emission data are considered. We propose to refer instead to the energetic difference of the 0-0 transition in absorption together with the solvatochromism of the acidic form in aprotic solvents of similar polarity.
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