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

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 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.

The Utility of DiceCT Imaging for High-Throughput Comparative Neuroanatomical Studies

“…; all other specimens were received as previously preserved.) Although numerous studies demonstrate research-quality visualizations using specimens not initially acquired or prepared specifically for diceCT imaging (i.e., vouchered museum specimens) [Herdina et al, 2010;Cox and Jeffery, 2011;Jeffery et al, 2011;Cox and Faulkes, 2014;Herdina et al, 2015a, b], the best contrasted -and, thus, easiest to model digitally -neural structures are those associated with specimens fixed immediately upon death [Hughes et al, 2016b; see also Fig. 1-4] (see online suppl.…”

“…Hughes et al [2016b] recently developed field fixation techniques that are effective for preserving neural tissues in remote conditions without laboratory facilities or cold storage. This approach uses both liquid (37% volume/volume [v/v] formaldehyde) and solid (100% formaldehyde powder) stocks to mix buffered formalin solutions in the field.…”

“…This approach uses both liquid (37% volume/volume [v/v] formaldehyde) and solid (100% formaldehyde powder) stocks to mix buffered formalin solutions in the field. The taxa collected by Hughes et al [2016b] were chameleons from Uganda and the Democratic Republic of the Congo. Whole-body fixation was achieved via transcardial perfusion.…”

“…Whole-body fixation was achieved via transcardial perfusion. Specimens were stored in a 10% buffered formalin solution containing 12% sucrose (weight by volume [w/v]) for up to 54 days in the field before reaching laboratory facilities [Hughes et al, 2016b]. (Note: Sucrose was added as a cryoprotectant in anticipation of future freezing upon return to the laboratory.)…”

Longitudinal study of rat volar fat pad fixation and ethanol storage: implications for the use of fluid‐preserved specimens in morphological studies

“…; Hughes et al. ). This combination of chemical fixation and subsequent transfer to an antimicrobial media has proven highly effective at halting metabolic and autolytic processes and providing a sterile environment for long‐term storage (Hopwood, ; Fox et al.…”