Article

Rescuing Perishable Neuroanatomical Information from a Threatened Biodiversity Hotspot: Remote Field Methods for Brain Tissue Preservation Validated by Cytoarchitectonic Analysis, Immunohistochemistry, and X-Ray Microcomputed Tomography

Authors

Daniel Hughes, University of Texas at El Paso
Ellen Walker, University of Texas at El Paso
Paul Gignac, Oklahoma State University Center for Health Sciences
Anais Martinez, University of Texas at El Paso
Kenichiro Negishi, University of Texas at El Paso
Carl Lieb, University of Texas at El Paso
Eli Greenbaum, University of Texas at El Paso
Arshad Khan, University of Texas at El Paso

Publication Date

May 2016

Journal/Book Title/Conference

PLoS ONE

Volume

11

Issue

5

Abstract

Biodiversity hotspots, which harbor more endemic species than elsewhere on Earth, areincreasingly threatened. There is a need to accelerate collection efforts in these regionsbefore threatened or endangered species become extinct. The diverse geographical, ecological,genetic, morphological, and behavioral data generated from the on-site collection ofan individual specimen are useful for many scientific purposes. However, traditional methodsfor specimen preparation in the field do not permit researchers to retrieve neuroanatomicaldata, disregarding potentially useful data for increasing our understanding of braindiversity. These data have helped clarify brain evolution, deciphered relationships betweenstructure and function, and revealed constraints and selective pressures that provide contextabout the evolution of complex behavior. Here, we report our field-testing of two commonlyused laboratory-based techniques for brain preservation while on a collectingexpedition in the Congo Basin and Albertine Rift, two poorly known regions associated withthe Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixationand long-term brain storage, conducted in remote field conditions with no access tocold storage laboratory equipment, had no observable impact on cytoarchitectural featuresof lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixationremained readily compatible with subsequent immunohistochemical detection of neuralantigens, with immunostaining that was comparable to that of laboratory-perfused brain tissue.Third, immersion-fixation of lizard brains, prepared under identical environmental conditions,was readily compatible with subsequent iodine-enhanced X-ray microcomputedtomography, which facilitated the non-destructive imaging of the intact brain within its skull.In summary, we have validated multiple approaches to preserving intact lizard brains inremote field conditions with limited access to supplies and a high degree of environmentalexposure. This protocol should serve as a malleable framework for researchers attemptingto rescue perishable and irreplaceable morphological and molecular data from regions ofdisappearing biodiversity. Our approach can be harnessed to extend the numbers of speciesbeing actively studied by the neuroscience community, by reducing some of the difficultyassociated with acquiring brains of animal species that are not readily available incaptivity.

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