From two to three dimensions: The importance of the third dimension for evaluating the limits to neuronal...

Most studies dealing with the limits to miniaturization in insect brains have until now relied on information based on data collected in two dimensions: either histological sections imaged by light microscopy, or electron micrographs of single ultrathin sections imaged by transmission electron microscopy (TEM). To test the validity of transferring information gained from two-dimensional images to the third dimension, we examined a 3D image stack from serial-section TEM (ssTEM) of the optic neuropiles of the miniature parasitic wasp Trichogramma brassicae (Bezdenko, 1968). We reinvestigated the proposed lower limit of 2 µm for the diameters of neuronal somata and found average volumes of 6.5 μm³ for lamina cells and 3.8 μm³ for medulla cells. We likewise found a limiting factor for the volume of nuclei, which averages 41.9% and 49.2% of the cell body volume, respectively, but that in turn the compactness of heterochromatin was not a limiting factor in the minimal volume of the nuclei. Finally, we also found a minimum axon diameter of 98 nm that could nevertheless accommodate axoplasmic mitochondria. Incorporating the third dimension thus proves critically important in avoiding volumetric misinterpretations of these values. We discuss the limitations of analyzing the effects of miniaturization from profile data of neurons and demonstrate that miniaturization within the nervous system can lie beyond previously described limits and in some cases is already present in the optic lobe neurons of T. brassicae.