TY - JOUR
T1 - Rescuing perishable neuroanatomical information from a threatened biodiversity hotspot
T2 - Remote field methods for brain tissue preservation validated by cytoarchitectonic analysis, immunohistochemistry, and x-ray microcomputed tomography
AU - Hughes, Daniel F.
AU - Walker, Ellen M.
AU - Gignac, Paul M.
AU - Martinez, Anais
AU - Negishi, Kenichiro
AU - Lieb, Carl S.
AU - Greenbaum, Eli
AU - Khan, Arshad M.
N1 - Funding Information:
For their companionship and assistance in the field, we are grateful to: Dr. Mathias Behangana, Lukwago Wilber, Dr. Chifundera Kusamba, Mwenebatu M. Aristote, Wandege M. Muninga, and Jean-Pierre Mokanse Watse. Dr. Baluku Bajope of the Centre de Recherche en Sciences Naturelles (CRSN) provided project support and permits, and the Institut Congolais pour la Conservation de la Nature (ICCN) kindly granted permits to work in protected areas in DRC. We thank the Uganda Wildlife Authority (UWA) of Kampala for necessary permits to work in Uganda, and Mr. James Lutalo, Commissioner of Wildlife Conservation and CITES Authority for Uganda. We are also grateful to Dr. Xiaogang Su, Director of the BBRC Statistical Consulting Laboratory (SCL), for assistance with the statistical analyses used in this study. The SCL is supported by grant 5G12MD007592 from the National Institute on Minority Health and Health Disparities (NIMHD), a component of the National Institutes of Health (NIH). For support throughout this project, we owe special thanks to all members of the UTEP Systems Neuroscience Laboratory, especially Berenise De Haro. We also thank Morgan Hill and Henry Towbin of the American Museum of Natural History’s Microscopy and Imaging Facility for assistance. EG and DFH are supported by grant DEB-1145459 from the National Science Foundation (NSF) of the United States. Work in the UTEP Systems Neuroscience Laboratory is supported by NIH grant GM109817 awarded to AMK and a Grand Challenges Grant awarded to AMK by the UTEP Office of Research and Sponsored Projects. Additionally, research work by AMK and EG is supported by a Howard Hughes Medical Institute STEM Education Grant (UTEP PERSIST; awarded to AMK and EG as co-PIs; PI = Dr. Stephen Aley). DFH and EMW are supported by Keelung Hong Graduate Fellowships and AM is supported by an NSF STEM Fellows in K-12 Education (GK-12) Fellowship. PMG is supported by grants DEB-1457180 and EAGER 1450850 from NSF and the Oklahoma State University Center for Health Sciences Department of Anatomy and Cell Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2016 Hughes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2016/5
Y1 - 2016/5
N2 - 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 threatened or endangered species become extinct. The diverse geographical, ecological, genetic, morphological, and behavioral data generated from the on-site collection of an individual specimen are useful for many scientific purposes. However, traditional methods for specimen preparation in the field do not permit researchers to retrieve neuroanatomical data, disregarding potentially useful data for increasing our understanding of brain diversity. These data have helped clarify brain evolution, deciphered relationships between structure and function, and revealed constraints and selective pressures that provide context about the evolution of complex behavior. Here, we report our field-testing of two commonly used laboratory-based techniques for brain preservation while on a collecting expedition in the Congo Basin and Albertine Rift, two poorly known regions associated with the Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixation and long-term brain storage, conducted in remote field conditions with no access to cold storage laboratory equipment, had no observable impact on cytoarchitectural features of lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixation remained readily compatible with subsequent immunohistochemical detection of neural antigens, 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 microcomputed tomography, 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 in remote field conditions with limited access to supplies and a high degree of environmental exposure. This protocol should serve as a malleable framework for researchers attempting to rescue perishable and irreplaceable morphological and molecular data from regions of disappearing biodiversity. Our approach can be harnessed to extend the numbers of species being actively studied by the neuroscience community, by reducing some of the difficulty associated with acquiring brains of animal species that are not readily available in captivity.
AB - 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 threatened or endangered species become extinct. The diverse geographical, ecological, genetic, morphological, and behavioral data generated from the on-site collection of an individual specimen are useful for many scientific purposes. However, traditional methods for specimen preparation in the field do not permit researchers to retrieve neuroanatomical data, disregarding potentially useful data for increasing our understanding of brain diversity. These data have helped clarify brain evolution, deciphered relationships between structure and function, and revealed constraints and selective pressures that provide context about the evolution of complex behavior. Here, we report our field-testing of two commonly used laboratory-based techniques for brain preservation while on a collecting expedition in the Congo Basin and Albertine Rift, two poorly known regions associated with the Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixation and long-term brain storage, conducted in remote field conditions with no access to cold storage laboratory equipment, had no observable impact on cytoarchitectural features of lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixation remained readily compatible with subsequent immunohistochemical detection of neural antigens, 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 microcomputed tomography, 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 in remote field conditions with limited access to supplies and a high degree of environmental exposure. This protocol should serve as a malleable framework for researchers attempting to rescue perishable and irreplaceable morphological and molecular data from regions of disappearing biodiversity. Our approach can be harnessed to extend the numbers of species being actively studied by the neuroscience community, by reducing some of the difficulty associated with acquiring brains of animal species that are not readily available in captivity.
UR - http://www.scopus.com/inward/record.url?scp=84971232293&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0155824
DO - 10.1371/journal.pone.0155824
M3 - Article
C2 - 27196138
AN - SCOPUS:84971232293
SN - 1932-6203
VL - 11
JO - PLoS ONE
JF - PLoS ONE
IS - 5
M1 - e0155824
ER -