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Session Schedule & Abstracts

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Saturday 2nd July, 2016

Symposium: Diffusible iodine-based contrast-enhanced computed tomography (diceCT) and related imaging techniques for evolutionary morphology 1

Room: Salon A   9:30 am–11:00 am

Moderator(s): P. M. Gignac, A. N. Herdina, N. J. Kley, A. Morhardt, J. A. Clarke, & M. Colbert
DCT1-1  9:30 am  DiceCTing the future: new horizons for 3-D visualization of vertebrate morphology. Gignac PM*, Oklahoma State University Center for Health Sciences; Herdina AN, Karolinska Institutet; Kley NJ, Stony Brook University; Morhardt AC, Ohio University; Colbert MW, The University of Texas at Austin; Clarke JA, The University of Texas at Austin
Abstract: The ability to rapidly discriminate soft tissues in three dimensions using X-ray computed tomography (CT) has been difficult to realize fully, because of similarities in X-ray attenuation properties among non-mineralized structures. However, recent pioneering work in this area has demonstrated that radiodense contrast agents, such as Lugol's iodine (I2KI), can be highly effective for differentiating many types of soft tissues using CT. Over the last several years, a handful of morphologists the world over have become a driving force in advancing such diffusible iodine-based contrast-enhanced CT (diceCT) imaging and utilizing the remarkable data it generates to reconstruct soft-tissue phenotypes and functional anatomy in three and four dimensions. For the 11th International Congress of Vertebrate Morphology, we have assembled a cadre of well-established researchers and emerging early-career scientists to: (1) highlight recent advances in diceCT imaging, (2) discuss the integration of soft-tissue visualization into existing research toolkits, and (3) lay out the future directions for contrast enhancement in the study of vertebrate soft tissues. In the introduction to our symposium, we provide a critical review of recent contributions to this emerging field and help make sense of its now complex landscape of methodologies. We also report on new initiatives aimed at formally convening the diceCT community for regular exchanges of ideas, methodological advances, and novel research applications. These include published recommendations for best practices, a techniques workshop to help beginners overcome methodological hurdles, and a digital hub to connect researchers ( We aim to grow our community further by spurring the more widespread adoption of these methods and facilitating conversations and collaborations among labs already exploring this powerful new tool with those considering how to apply it to their own research questions for the first time.

DCT1-2  10:00 am  Using the STABILITY protocol prior to IKI staining to provide the first accurate, in situ quantification of mammalian brain proportion scaling using marsupials. Weisbecker V*, School of Biological Sciences, University of Queensland; Carlisle A, School of Biological Sciences, University of Queensland; Hinds L, CSIRO Health and Biosecurity Flagship; Selwood L, University of Melbourne; Whish Sophie, University of Melbourne
Abstract: Iodine-based staining of soft tissue is rapidly gaining popularity in a wide range of applications such as biomedicine, comparative anatomy, and palaeontology. One major downside of this technique is the fact that iodine solution (IKI) stained soft-tissue shrinks substantially and differentially, so that accurate volumetric measurements are not possible. Here we present data on our work on marsupial brain tissue using the STABILITY technique, in which the specimen is hybridized with a hydrogel before IKI staining. The technique worked easily, particularly after we replaced the pre-incubation step of replacing air with nitrogen through a vacuum pump with the simpler protocol of pouring a layer of oil over the hydrogel mixture prior to incubation. Shrinkage in untreated brain tissue stained with 1.75% IKI averaged 35%, whereas in hydrogel-treated specimens shrinkage averaged 11%. Staining took longer in hydrogel-treated specimens, but the contrast was the same. Using STABILITY, we were able to provide the first developmental series of in-situ brain growth in three marsupial species (Macropus eugenii, Trichosurus vulpecula, and Monodelphis domestica), using Mimics as our segmentation software. The volumetric growth of different brain parts (neocortex, hippocampus, cerebellum, and medulla) contradict several hypotheses of brain partition evolution, including that brain partition development in mammals has a uniform slope and intercept, displays the same allometry of adult mammalian brain partition scaling, and contains no phylogenetic signal. We conclude that the STABILITY protocol currently represents a powerful avenue of gaining volumetrically accurate soft-tissue reconstruction, while preserving a near-histological level of resolution on soft-tissue anatomy. This research was supported by Australian Research Council DECRA DE120102034

DCT1-3  10:15 am  Mind the gap: ontogenetic shape differences between brains and endocasts in archosaurs. Watanabe A*, American Museum of Natural History; Gignac PM, Oklahoma State University Center for Health Sciences; Norell MA, American Museum of Natural History
Abstract: The braincase is a crucial osteological correlate for neuroanatomy and an indispensable resource for inferring the brain morphology of extinct vertebrates. As an internal mold of this space, endocasts provide size and shape approximations, allowing the exploration of neurological structures, capacities, and evolution. Nevertheless, the validity of such investigations pivots on the accuracy of these estimations. In mammals and birds, volumetric studies have shown that endocasts closely reflect the size of actual brains, which occupy nearly the entire braincase. Although size is an important metric, volumetric measurements are limited in their characterization of morphology and may obscure localized morphological biases. Here we test shape differences between endocasts and brains reconstructed from computed-tomography imaging of model archosaurs—the American alligator and the chicken. Using a dense ontogenetic sampling of each taxon, we evaluated whether endocast-brain shape discrepancies (1) exist, (2) change through ontogeny, and (3) are greater than intra- and interspecific variation in brain shape. Alarmingly, the results show that endocasts are significantly distinct from brains in shape due to lack of furrows, relative mediolateral reduction in the cerebrum, and less dorsoventral flexion in both the cerebellum and medulla. In both taxa, these discrepancies generally decrease through ontogeny. However, the endocast-brain difference is still greater than shape differences charted across all of ontogeny. Moreover, even with a broad taxonomic sampling, we find that endocranial shapes are collectively shifted to a more "juvenilized" morphology relative to brain shape. Researchers should be aware, therefore, that endocasts contain critical systematic biases in their characterization of brain morphology and that these artifacts may substantially impact the results of comparative neuroanatomical study.

DCT1-4  10:30 am  Incorporating diceCT into multi-scale structural studies of the brain for highly divergent lineages of acrodont lizards: validation of preservation methods conducted in the field. Hughes DF*, University of Texas at El Paso; Walker EM, University of Texas at El Paso; Gignac PM, Oklahoma State University Center for Health Sciences; Khan AM, University of Texas at El Paso
Abstract: Biodiversity hotspots, which harbor more endemic species than elsewhere on Earth, are increasingly threatened. There is a need to accelerate collection efforts in these regions before poorly understood or undocumented species become extinct. However, traditional specimen preparations do not permit researchers to retrieve neuroanatomical data at high resolution. We field-tested two traditional laboratory-based techniques for brain preservation (transcardial perfusion and immersion fixation) while collecting specimens of Agamidae and Chamaeleonidae in the Eastern Afromontane biodiversity hotspot of Central Africa. Field- and laboratory-perfused brain samples were compared for tissue cytoarchitecture and chemoarchitecture using Nissl-based staining and fluorescence immunocytochemistry, respectively. We found that transcardial perfusion fixation and long-term storage, conducted under remote field conditions without access to cold storage, had no observable impact on cytoarchitectural integrity or stain evenness. Further, immunostaining for small neurotransmitter and neuropeptide biomarkers was similar between our comparisons. With respect to immersion-fixation methods, field-preserved chameleon brains were readily compatible with subsequent diffusible iodine-based contrast-enhanced computed tomography (diceCT) imaging, which facilitated the non-destructive imaging of the intact brain within the skull. In particular, diceCT images revealed excellent contrast of brain tissue structures, including myelinated and unmyelinated portions of the brain. When paired with cytoarchitectural and immunocytochemical techniques, the use of diceCT allows for the neuroanatomical study of poorly known and often inaccessible species across micro- to macroscopic scales of analysis. Our protocol serves as a malleable framework intended for future researchers attempting to rescue irreplaceable neuroanatomical information from disappearing regions.

DCT1-5  10:45 am  Applying diceCT to PET: new tools for correlating morphology to function in living animals. Gold MEL*, Stony Brook University; Schulz D, Yeditepe University; Budassi M, Stony Brook University; Gignac PM, Oklahoma State University Center for Health Sciences; Vaska P, Stony Brook University; Norell MA, American Museum of Natural History
Abstract: The evolution of flight-related features in theropod dinosaurs is iconic and well documented in the fossil record. Although much of the morphological adaptations for flight precedes crown-group birds in non-avian dinosaurs, the precise origin of powered flight has eluded paleontologists because of the difficulty in directly linking morphology to flight capacity. Recently, endocranial data have demonstrated that a highly encephalized brain evolved in non-avian theropods, but whether these neuroanatomical changes reflect behavioral transformations is untested. To explore brain function during locomotion we used positron emission tomography (PET) scanning to record brain activity in flying starlings. Diffusible iodine-based contrast-enhanced computed tomography (diceCT) scans of an intact starling head provided the pivotal neuroanatomical detail used to identify which nuclei of the brain were activated during flight. By overlaying PET data onto diceCT scans, we identified the active brain regions as the entopallium and anterior Wulst, which are involved in visual processing and somatosensory integration. These results imply that the visual and somatosensory processing systems mainly used for flight are completely separate from the optic tectum—a structure thought to be in control of all visual processing in avians. The entopallia and anterior Wulst may work together to create a short-term conflict avoidance system to bridge fast approaching visual input to locomotor controls, allowing for rapid interpretation of the flightscape. Expansion of the Wulst through increased use during volancy may have driven the dorsal expansion of this structure along theropod evolution. Combined with fossil data, these findings point towards the evolution of volant behaviors at Avialae, with the appearance of the Wulst. Brain activation maps in starlings represent a first step in a new aspect of paleontology, bridging the gap between fossil morphology and living behaviors.

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