Optical imaging of neuronal activity in tissue labeled by retrograde transport of Calcium Green Dextran.

In many neurophysiological studies it is desirable to simultaneously record the activity of a large number of neurons. This is particularly true in the study of vertebrate motor systems that generate rhythmic behaviors, such as the pattern generator for locomotion in vertebrate spinal cord. Optical imaging of neurons labeled with appropriate fluorescent dyes, in which fluorescence is activity-dependent, provides a means to record the activity of many neurons at the same time, while also providing fine spatial resolution of the position and morphology of active neurons. Voltage-sensitive dyes have been explored for this purpose and have the advantage of rapid response to transmembrane voltage changes. However, voltage-sensitive dyes bleach readily, which results in phototoxic damage and limits the time that labeled neurons can be imaged. In addition, the signal-to-noise ratio is typically small, so that averaging of responses is usually required. As an alternative to voltage-sensitive dyes, calcium-sensitive dyes can exhibit large changes in fluorescence. Most neurons contain voltage-sensitive Ca2+ channels, and numerous reports indicate that neuronal activity is accompanied by increased intracellular Ca2+ concentration. In this protocol we describe a method to use retrograde transport of the dextran conjugate of a calcium-sensitive dye (Calcium Green Dextran) to label selectively populations of brain and spinal interneurons in a primitive vertebrate (lamprey), for subsequent video-rate imaging of changes in intracellular fluorescence during neuronal activity. Although described with specific reference to lampreys, the technique has also been applied to embryonic chick spinal cord and larval zebrafish preparations and should be easily adaptable to other systems. The most significant novel feature of the protocol is the use of retrograde axonal transport to selectively fill neurons that have known axonal trajectories. Using lampreys, we have obtained activity-sensitive labeling across longer distances and over a longer transport time (up to 14 mm and 4 days) than has been reported in other species. In addition, retrograde transport allows filling of neurons more deeply within tissue than would be possible with bath application of calcium-sensitive dyes. Furthermore, the dyes are readily taken up by adult tissues, while bath application is usually limited to embryonic and neonatal vertebrate nervous tissues (although the reasons for this limitation are not clear). Attempts to load the AM (acetomethoxy) esters of calcium-sensitive dyes into lamprey spinal cord neurons by bath application have been unsuccessful (McPherson, unpublished observations, and).