Abstract
Three-dimensional (3-D) mapping of brain circuits controlling retrocerebral complex of the cockroach, Diploptera punctata, was presented. Using enzyme-aided penetration and microwave-aided fixation, an intact adult brain of more than 500 mm in thickness was stained with NBD C6-ceramide, cleared by Confocal Clear and reconstructed from the stack of confocal images. Mushroom bodies (MB) and central body (CB) were demarcated and digitally dissected as spatial landmarks. Additionally, developmental changes of allatostatinergic (ASTergic) and leucokininergic (LKergic) neurons in the brain were examined from embryos to adults. ASTergic neurons first appeared at pars intercerebralis (PI) on day-20 old embryo (E20, 26% of embryonic development completed) and first occurred at pars lateralis (PL) with axons terminated at corpus allatum (CA) on E25 (32%). Whereas, LKergic neurons first emerged at PL on E35 (45%) and first presented at PI with axon terminated at the corpus cardiacum (CC) on E55 (71%). During postembryonic development, the number and spatial distribution of ASTergic and LKergic neurons showed less change. Since the entire brain was reconstructed, NBD C6-ceramide-stained spatial landmarks, ASTergic and LKergic neurons and retrocerebral complex-innervated neurons were all performed as modules. As a result, a 3-D virtual brain containing landmarks and three modules was built up to demonstrate correlations among neuronal circuits.