Evaluation of standard imaging techniques and volumetric preservation of nervous tissue in genetically identical offspring of the crayfishProcambarus fallaxcf.virginalis(Marmorkrebs)

Abstract

In the field of comparative neuroanatomy, a meaningful interspecific comparison demands quantitative data referring to method-specific artifacts. For evaluating the potential of state-of-the-art imaging techniques in arthropod neuroanatomy, micro-computed X-ray microscopy (μCT) and two different approaches using confocal laser-scanning microscopy (cLSM) were applied to obtain volumetric data of the brain and selected neuropils inProcambarus fallaxformavirginalis(Crustacea, Malacostraca, Decapoda). The marbled crayfishP. fallaxcf.virginalisfeatures a parthogenetic reproduction generating genetically identical offspring from unfertilized eggs. Therefore, the studied organism provides ideal conditions for the comparative analysis of neuroanatomical imaging techniques and the effect of preceding sample preparations of nervous tissue. We found that wet scanning of whole animals conducted with μCT turned out to be the least disruptive method. However, in an additional experiment it was discovered that fixation in Bouin’s solution, required for μCT scans, resulted in an average tissue shrinkage of 24% compared to freshly dissected and unfixed brains. The complete sample preparation using fixation in half-strength Karnovsky’s solution of dissected brains led to an additional volume decrease of 12.5%, whereas the preparation using zinc-formaldehyde as fixative resulted in a shrinkage of 5% in comparison to the volumes obtained by μCT. By minimizing individual variability, at least for aquatic arthropods, this pioneer study aims for the inference of method-based conversion factors in the future, providing a valuable tool for reducing quantitative neuroanatomical data already published to a common denominator. However, volumetric deviations could be shown for all experimental protocols due to methodological noise and/or phenotypic plasticity among genetically identical individuals. MicroCT using undried tissue is an appropriate non-disruptive technique for allometry of arthropod brains since spatial organ relationships are conserved and tissue shrinkage is minimized. Collecting tissue-based shrinkage factors according to specific sample preparations might allow a better comparability of volumetric data from the literature, even if another technique was applied.