Development of a contrast‐enhanced micro computed tomography protocol for the oval squid ( Sepioteuthis lessoniana ) brain

Abstract

Coleoid cephalopods (squid, cuttlefish, and octopus) have a well‐developed and complex central nervous system. Its absolute size is the largest among invertebrates, and the brain‐to‐body mass ratio is larger than that of fish and reptiles and equivalent to that of birds and mammals. Although a number of histological studies have been conducted on the brains of cephalopods, most of them used a light microscope or an electron microscope, which show the microstructure of the brain, but often cannot image the whole brain instantaneously. Of late, micro computed tomography (CT) has gained popularity for imaging animal brains because it allows for noninvasive three‐dimensional (3D) reconstruction and preprocessing that are not cumbersome. To perform micro‐CT on cephalopod brains, we first tested conditions suitable for preprocessing, paying special attention to staining conditions that would provide high contrast images. Four agents, iodine in 99.5% ethanol, iodine potassium iodide in water (IKI), phosphotungstic acid in 70% ethanol, and nonionic iodinated contrast agent in water, were tested at various concentrations and durations on brain of juvenile oval squid. To evaluate the quality of staining, we calculated the contrast ratio of the two‐dimensional (2D) images and compared 3D segmentation of the best and worst 2D images. We concluded that 3% IKI staining for 7 days was the best combination to enhance the images contrast of the oval squid brain, in which each brain lobe was clearly detected and 3D segmentation of the whole brain was possible. The wider applicability of this preprocessing method for micro‐CT of the brains of other cephalopods is discussed.

Micro‐CT visualization of the CNS: Performance of different contrast‐enhancing techniques for documenting the spider brain

“…The usefulness of micro-CT for the visualization of internal organs and tissues has been extensively tested and confirmed in arthropods (Lipke et al, 2015;Sombke et al, 2015;Castejón et al, 2018;Dirks-Mulder et al, 2019;Pramanik et al, 2020;Faulwetter et al, 2012;Sakurai & Ikeda 2019;Swart et al, 2016;Lesciotto et al, 2020;Sombke et al, 2019;Dederichs et al, 2019;Steinhoff et al, 2017;Stafstrom et al, 2017;Steinhoff et al, 2018Steinhoff et al, , 2020Alba-Alejandre et al, 2019;Alba-Alejandre et al, 2018). Furthermore, its application for identifying and reconstructing peripheral and central nervous tissue and other systems in spiders has also been explored (Lipke et al, 2015;Dederichs et al, 2019;Steinhoff et al, 2017;Stafstrom et al, 2017;Steinhoff et al, 2018Steinhoff et al, , 2020Lin et al, 2021).…”

Diffusion MRI Connections in the Octopus Brain

“…Following the pioneering work of Young [ 22 ], a large number of anatomical and physiological studies of the octopus and related coleoid cephalopods have been undertaken. In addition to detailed histological studies [ 8 , 12 , 15 , 16 , 23 - 36 ], other efforts have utilized computed tomography [ 37 , 38 ], electron microscopy [ 39 , 40 ], positron emission tomography [ 41 ], fluorescent dye labeling of neuronal connections [ 42 - 44 ] and magnetic resonance imaging [ 45 - 48 ].…”

“…Thus, the octopus is a compelling system for comparison with mammals due to its ancient origin and great anatomical differences combined with fascinating similarities in problem solving skills, learning and memory [11,21] Following the pioneering work of Young [22], a large number of anatomical and physiological studies of the octopus and related coleoid cephalopods have been undertaken. In addition to detailed histological studies [8,12,15,16,[23][24][25][26][27][28][29][30][31][32][33][34][35][36], other efforts have utilized computed tomography [37,38], electron microscopy [39,40], positron emission tomography [41], fluorescent dye labeling of neuronal connections [42][43][44] and magnetic resonance imaging [45][46][47][48].…”

Micro-CT imaging of Thiel-embalmed and iodine-stained human temporal bone for 3D modeling

“…However, most recently, iodine staining was found more efficient compared to PTA for myocardial staining [ 11 ]. Similarly, comparing iodine in ethanol, iodine potassium iodide in water (IKI), PTA in ethanol, and nonionic iodinated contrast agent in water, 3% IKI staining was found best to enhance images contrast for 3D segmentation of oval squid brain [ 12 ]. Iodine has been reported to significantly improve the contrast-to-noise ratio of submillimeter middle ear connective tissue structures [ 6 ].…”