Abstract
High speed imaging of ion concentration changes in neurons is an important and growing tool for neuroscientists. We previously developed a system for simultaneously measuring sodium and calcium changes in small compartments in neurons (Miyazaki and Ross, 2015). We used this technique to analyze the dynamics of these ions in individual pyramidal neuron dendritic spines (Miyazaki and Ross, 2017). This system is based on high speed multiplexing of light emitting diodes (LEDs) and classic organic indicators. To improve this system we made additional changes, primarily incorporating lasers in addition to the LEDs, more sophisticated imaging protocols, and the use of newer sodium and calcium indicators. This new system generates signals with higher signal to noise ratio (S/N), less background fluorescence, and less photodynamic damage. In addition, by using longer wavelength indicators instead of indicators sensitive in the UV range, it allows for the incorporation of focal uncaging along with simultaneous imaging, which should extend the range of experiments.
Highlights
High speed imaging of ion concentration changes has revolutionized our understanding of many aspects of nervous system function
In this article we review the original setup for simultaneous sodium and calcium imaging, pointing out the novel aspects of the system, and we describe the new components, which are responsible for the improvements
Pyramidal neurons were patched on the soma and filled with combinations of sodium and calcium indicators [either SBFI and Oregon Green Bapta-1 (OGB-1; or OGB-5N), or bis-fura-2 and ANG-2]
Summary
High speed imaging of ion concentration changes has revolutionized our understanding of many aspects of nervous system function. Most progress has occurred using calcium indicators because these are easy to design with appropriate sensitivity and because physiological [Ca2+]i changes are large compared to the resting [Ca2+]i in the cell Experiments using this class of indicator have examined the direct physiological role of [Ca2+]i changes in different parts of the cell (e.g., calcium entry in presynaptic terminals, calcium entry through postsynaptic receptors, and calcium release from stores in the soma and dendrites). They used the detected [Ca2+]i changes as an indicator of electrical activity because action potentials almost always cause measurable [Ca2+]i changes in all parts of the cell where they propagate. Some experiments have used voltage sensitive dyes (VSDs) to measure electrical activity directly in small compartments, with some experiments detecting subthreshold potential changes in dendritic spines (Popovic et al, 2015; Acker et al, 2016)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
