Long-term time-lapse imaging of developing hippocampal neurons in culture

Dissociated zell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Studying developmental processes that occur over many hours or days-for example, dendritic branching-involves capturing images of a cell at regular intervals without compromising cell survival. This approach is also useful for studying events of short duration that occur asynchronously across the cell population. For such studies, it is highly desirable to use a computer-controlled microscope with an automated stage, to follow multiple cells at different locations in the culture, moving sequentially from one to the next and capturing an image at each location. A method to correct for focal drift is also required. For long-term imaging, we culture neurons in a medium without phenol red, which is thought to give rise to toxic substances following exposure to light. To label cells with GFP-tagged proteins for long-term imaging, we usually use nucleofection (rather than lipid-mediated transfection); this gives a high transfection efficiency, which makes it easier to find the right cell for imaging. Our protocol for long-term imaging is given here, along with appropriate methods to express GFP-tagged proteins. Examples illustrate how the protocol can be used to image cytoskeletal dynamics during axon specification and to study kinesin motor dynamics in stage 2 neurons (when minor neurites extend).