Implementation of pigment-dispersing factor-immunoreactive neurons in a standardized atlas of the brain of the cockroach Leucophaea maderae.

The cockroach Leucophaea maderae is an established model in circadian rhythm research. Its circadian clock is located in the accessory medulla of the brain. Pigment-dispersing factor-immunoreactive (PDF-ir) neurons of the accessory medulla act as circadian pacemakers controlling locomotor activity rhythms. To characterize the neuronal network of the circadian system in L. maderae, the PDF-ir neurons were implemented into a standardized three-dimensional atlas of the cockroach brain. Serial confocal images from 20 wholemount brains were used for the construction of the atlas comprising 21 neuropils. Two different standardization protocols were employed: the iterative shape averaging (ISA) procedure using an affine transformation followed by iterative non-rigid registrations, and the virtual insect brain (VIB) protocol employing local non-rigid transformations after global and local rigid transformations. Quantitative analysis of the 20 brains revealed that volumes of the accessory medulla are directly correlated with the volumes of the medulla, the protocerebral bridge, and the upper division of the central body, suggesting functional connections among these neuropils. For a standardized reconstruction of the circadian pacemaker network, the ISA protocol was used to register PDF-ir neurons in the standard cockroach brain. The registration revealed that two PDF-ir arborization areas in the brain are highly interconnected with other PDF-ir projection sites and appear to be contacted both by fibers in the posterior and the anterior optic commissures. The distances between PDF-ir branching areas show specific numerical relationships that might be physiologically relevant for temporal encoding.