Simultaneous Sodium and Calcium Imaging from Dendrites and Axons

Dynamic calcium imaging is a major technique of neuroscientists. It can reveal information about the location of various calcium channels and calcium permeable receptors, the time course, magnitude, and location of intracellular calcium concentration ([Ca(2+)](i)) changes, and indirectly, the occurrence of action potentials. Dynamic sodium imaging, a less exploited technique, can reveal analogous information related to sodium signaling. In some cases, like the examination of AMPA and NMDA receptor signaling, measurements of both [Ca(2+)](i) and [Na(+)](i) changes in the same preparation may provide more information than separate measurements. To this end, we developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiologic events. This approach has advantages over sequential imaging because the preparation may not respond identically in different trials. We designed custom dichroic and emission filters to allow the separate detection of the fluorescence of sodium and calcium indicators loaded together into a single neuron in a brain slice from the hippocampus of Sprague-Dawley rats. We then used high-intensity light emitting diodes (LEDs) to alternately excite the two indicators at the appropriate wavelengths. These pulses were synchronized with the frames of a CCD camera running at 500 Hz. Software then separated the data streams to provide independent sodium and calcium signals. With this system we could detect [Ca(2+)](i) and [Na(+)](i) changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal-to-noise ratio (S/N).