Confocal microscopy and three-dimensional reconstruction of electrophysiologically identified neurons in thick brain slices.

Three-dimensional morphology and electrophysiology were correlated from individual neurons in a thick brain slice preparation. The hippocampal formation from immature and adult rats was cut transverse to the longitudinal axis into 500 microns-thick slices which were maintained under physiologic conditions. Individual neurons were impaled and physiologically characterized using microelectrodes. Recordings were made from the soma and in some cases from a dendrite. The impaled neurons were filled through the microelectrode with the fluorescent dye lucifer yellow and imaged by confocal scanning laser microscopy using an analog preprocessor. As many as 180 optical sections were recorded as a function of depth through the slices. Images are presented as a series of optical sections, stereo pairs, or three-dimensional reconstructions. Both stereo contouring and volume rendering methods were employed, and the reconstructions were viewed from any arbitrary perspective. Dendritic and axonal fields were separated from each other and displayed separately or as different pseudocolors. The three-dimensional reconstructions provided perspectives that were difficult or impossible to appreciate by viewing the optical sections or conventionally formed stereo pairs.