Three-dimensional mapping of brain neuropils in the cockroach, Diploptera punctata

Herein, we present a complete three-dimensional (3D) map of major neuropil structures in the central brain of the cockroach Diploptera punctata. The p ositions of the structures have been ascertained by confocal microscopy, wh ich, until now-for reasons of tissue opacity and nonhomogeneity-has been th ought impractical in imaging fluorescently labeled structures thicker than 150 mum. In this report, however, we have used digestive enzymes and microw aveaided fixation to stain, clear, and optically section, in its entirety, an intact central brain more than 500 mum thick. The central brain from an adult female cockroach was stained thoroughly with the membrane probe NBD-c eramide and the DNA probe propidium iodide. The central brain as well as su ch neuropil regions as mushroom bodies, central complex, antennal glomeruli , and lobus glomerulati were individually outlined, segmented, and reconstr ucted in three dimensions to a spatial resolution of approximately 1 mum in the X-Y plane and 3 mum in the Z plane. The volume and surface area of eac h neuropil compartment were determined, and Kenyon cells of the mushroom bo dies were counted. We determined that each brain hemisphere contains about 230,000 Kenyon cells, 99 antennal lobe glomeruli, and 40 lobus glomerulatus glomeruli. Segmented compartments were assigned as separate channels and m erged into a single data base to reconstruct a 3D central brain containing eight different channels. This is the first 3D map at submicron resolution of an entire animal's brain that measures more than 500 mum in thickness. ( C) 2001 Wiley-Liss, Inc.