RT Journal Article SR Electronic T1 diceCT: a valuable technique to study the nervous system of fishes JF eneuro JO eNeuro FD Society for Neuroscience SP ENEURO.0076-20.2020 DO 10.1523/ENEURO.0076-20.2020 A1 Victoria Camilieri-Asch A1 Jeremy A. Shaw A1 Andrew Mehnert A1 Kara E. Yopak A1 Julian C. Partridge A1 Shaun P. Collin YR 2020 UL http://www.eneuro.org/content/early/2020/05/29/ENEURO.0076-20.2020.abstract AB Contrast-enhanced X-ray imaging provides a non-destructive and flexible approach to optimising contrast in soft tissues, especially when incorporated with Lugol’s solution (1% I2, 2% KI), a technique currently referred to as diffusible-iodine contrast-enhanced computed tomography (diceCT). This stain exhibits high rates of penetration and results in excellent contrast between and within soft tissues, including the central nervous system. Here, we present a staining method for optimising contrast in the brain of a cartilaginous fish, the brownbanded bamboo shark, Chiloscyllium punctatum, and a bony fish, the common goldfish, Carassius auratus, using diceCT. The aim of this optimisation procedure is to provide suitable contrast between neural tissue and background tissue(s) of the head, thereby facilitating digital segmentation and volumetric analysis of the central nervous system. Both species were scanned prior to staining and were rescanned at time (T) intervals, either every 48h (C. punctatum) or every 24h (C. auratus), to assess stain penetration and contrast enhancement. To compare stain intensities, raw X-ray computed tomography (CT) data were reconstructed using air and water calibration phantoms that were scanned under identical conditions to the samples. Optimal contrast across the brain was achieved at T = 240h for C. punctatum and T = 96h for C. auratus. Higher resolution scans of the whole brain were obtained at the two optimised staining times for all the corresponding specimens. The use of diceCT provides a new and valuable tool for visualising differences in the anatomical organisation of both the central and peripheral nervous systems of fishes.SIGNIFICANCE STATEMENT This study uses an emerging bioimaging technique based on X-ray CT, to advance our visualisation, and thus understanding, of brain organisation and structure in a cartilaginous and bony fish model species. diceCT is a relatively new approach to improving contrast between/within soft tissues, and is becoming increasingly popular across many fields of research, including evolutionary biology, biomechanics, palaeontology and comparative anatomy. Few studies have undertaken a rigorous assessment of stain penetration using Lugol’s iodine, especially in the brain. Here, we present the first staining optimisation using diceCT in any species of fish. There is therefore a vital need to standardise the methodology used, to collect comparable, replicable data, and thus further develop its usage and value across disciplines and taxa.