How does the human brain develop? The study of neurodevelopment is critical to our understanding of how the brain is structured, how it works, and how it may be affected by disease. But it’s a tricky subject for study: there’s only so far sophisticated scanning and imaging of live brains can take us. Developmental neuroscientists also need to make detailed observations of sections of the brain halted at multiple, precise stages of development – and for obvious reasons, human samples are not easy to come by. A human analogue
So, scientists often turn to the next best thing: the brains of some of our closest evolutionary and anatomical relatives, the non-human primates (apes, monkeys, lemurs and lorises). In the 1960s, Dr Pasko Rakic, then working at Harvard University, initiated a programme of developmental neuroscience research focused on a captive colony of almost 200 rhesus macaques1. These monkeys are perhaps the most widely used primates in research, due to being relatively easy to keep (compared to other primates) and anatomically and physiologically similar to humans.
Over the next fifty years – and joined in 1979 by his wife, Dr Patricia Goldman-Rakic – Dr Pasko Rakic established at the Yale School of Medicine one of the few non-human primate breeding colonies in the world, and the only one with the capacity for timed breeding, still operational today. Timed breeding is essential for studies of pre- and postnatal brain development where the age of each monkey embryo needs to be known precisely to the day.
Establishment of such a breeding colony is nowadays almost unthinkable. As Drs Duque and Selemon explain, “Non-human primate research has become exponentially more difficult,” due to many factors, including the purchase cost of animals, housing, veterinary care, complex regulations, and growing public concern over animal research. Maintaining a breeding colony is particularly challenging, as specialised facilities are needed to care for pregnant, infant, and juvenile members of the colony. Preserving the past
As primate research is in decline worldwide, Drs Duque and Selemon are determined that material still available from the half-century of work carried out by the Rakic/Goldman-Rakic labs to elucidate primate brain architecture and development shall not go to waste. Thankfully, slides and sections from hundreds of monkey brains have been carefully preserved over the years, forming an immense archive of material for future research. Dr Duque and Dr Selemon’s mission – inspired by their concern over escalating costs, logistical obstacles, and ethical objections to animal experimentation – is to make this archive available to researchers from all over the world, both in person and remotely, via the world-wide-web. As well as providing scientists with a unique resource for neurological research, this will greatly reduce the need to sacrifice any more live macaques in the name of much-needed scientific study. As Duque puts it, “The goal is to facilitate non-human brain research while decreasing animal use and increasing cost efficiency and data sharing.”
Dr Duque and Dr Selemon, funded by the US National Institute of Mental Health, have created the Macaque Brain Resource (‘MacBrainResource’ for short), available online at https://medicine.yale.edu/neuroscience/macbrain/ or macbrainresource.org. Thanks to the generous donation by Dr Rakic of his private collections, the MacBrainResource aims to provide access to five distinct sets of material derived from the Rakic/Goldman-Rakic labs’ wide-ranging neuroscience research on their macaque colony.
Under the microscope
Four of the five collections comprise microscope slides – that can be digitised and placed online as high-resolution images – pertinent to a wide range of neurological studies. These include slides generated using Dr Rakic’s once-pioneering technique of treating pregnant macaques with a radioactive molecule, tritiated thymidine, to mark newly-generated nerve cells, hence facilitating the study of ‘neurogenesis’ (where and when new nerve cells are created). The embryos were then sacrificed at times ranging from one hour to several years after treatment, giving insights into the staggered, temporal development of different brain regions.
A second collection of slides, emanating from the neuroanatomical work of the late Dr Goldman-Rakic, is comprised of brains in which radioactive labelling was injected into specific cortical areas or subcortical nuclei in order to reveal the complex connections between them. Fortunately, in almost every case, the entire brain was preserved so that even those connections not initially targeted for study have been preserved for analysis. Both Dr Rakic and Dr Goldman-Rakic were skilled brain surgeons and carried out intricate procedures involving the removal and replacement of a macaque foetus from the womb in order to surgically resect precise regions of the brain during specific developmental stages. Postnatal sacrifice of these animals enabled assessment of morphologic changes in other brain regions consequent to loss of the resected area. These prenatally lesioned brains, and in addition brains that were lesioned at various postnatal stages of development including full maturation at adulthood, form the basis of the third collection of slides.
The final set of slides uses an alternative to surgery, x-rays, to reduce the number of neurons generated in targeted brain regions at pre-defined stages of gestation. Some of these macaques were allowed to mature to full adulthood before being sacrificed, with the aim of investigating potential neurological causes for disorders such as schizophrenia. The slides in this collection of irradiated and control macaques are particularly useful because they were processed to allow stereological cell counting, a method for unbiased assessment of the number of neurons in a brain structure. In addition, some brains in this collection are accompanied by matching magnetic resonance imaging (MRI) scans, allowing researchers today to capitalise both on the undistorted morphology provided by
The fifth collection in the MacBrainResource is made up of macaque brain samples embedded in small plastic blocks, suitable for high-powered, high-resolution electron microscopy. The samples cover many different parts of the brain and were taken from monkeys ranging from embryos to mature twenty-year-olds (rhesus macaques typically live to about twenty-five years of age). High-resolution electron microscopy allows, for example, the visualisation of cellular organelles and synaptic membrane specialisations, subcellular morphology that permitted analysis of synaptic generation and pruning in the cerebral cortex by Dr Rakic and his associates. However, many other brain regions, not included in these studies, are available.
Taken together, the collections form a remarkable archive which would be impossible to duplicate today. But they are not just of historical interest: The collections housed in MacBrainResource are already forming the basis of cutting-edge neuroscience research.
Contemporary uses of archived brains
The web-based part of the MacBrainResource project is in the relatively early stages, with data from over 100 brains catalogued and uploaded so far. The goal is to provide images of slides in a high-resolution format that enables researchers to zoom right in on individual cells of interest. While only a few slides have been digitised to date, additional digitised images are available to researchers around the world on request. Furthermore, researchers from Illinois, California, and as far away as Europe and Australia, have visited Yale in person to initiate studies on parts of the brain, such as the amygdala and the retina, brain areas that were preserved in the collections but not previously investigated.
Dr Duque and colleagues have already published a paper3 in the prestigious journal Proceedings of the National Academy of Sciences of the United States of America, using more than thirty specimens and hundreds of slides from the Macaque Brain Resource. The work uses slides from the brains of tritiated thymidine-labelled embryos to explore the development of the ‘subplate zone,’ a crucial but transient part of the primate (and human) brain. Their findings not only expand on the current understanding of the development of this structure, but may help explain the differences underlying certain developmental brain disorders in humans. Duque is keen to emphasise how significant it is to be able to do new and noteworthy research on macaque brains without subjecting any further animals to experimentation or death. Crucially, comparisons have shown that conclusions drawn from the archive material are corroborated by modern imaging studies, suggesting that any research drawn from the MacBrainResource will be as reliable as that based on new specimens.
Most users of the resource to date have focused on Dr Rakic’s tritiated thymidine collection. However, Drs Duque and Selemon believe that the other four collections may be equally useful and that their potential has not yet been fully appreciated. According to Duque, “These collections hold a wealth of data that has yet to be tapped.” His hope is that, through the MacBrainResource, they will be used for new neuroscience projects around the world.
- Dove, A. 2005. Profile: Pasko Rakic. Nature Medicine 11(4): 362. http://dx.doi.org/10.1038/nm0405-362.
- Selemon , L.D., Ceritoglu, C., Ratnanather, J.T., Wang, L., Harms, M.P., Aldridge, K., Begovic’, A., Csernansky, J.G., Miller, M.I., Rakic, P. 2013. Distinct abnormalities of the primate prefrontal cortex caused by ionizing radiation in early or midgestation. Journal of Comparative Neurology 52 (5):1040-1053.
- Duque, A., Krsnik, Z., Kostović, I. & Rakic, P. 2016. Secondary expansion of the transient subplate zone in the developing cerebrum of human and nonhuman primates. Proceedings of the National Academy of Sciences of the United States of America 113(35): 9892 –9897.
Macaque Brain Resource at Yale (MacBrainResource) has been created to make five distinct collections of macaque brain tissue available to investigators worldwide and thereby provide a cost-effective means for researchers to conduct de novo studies on the non-human primate brain without exorbitant costs and without having to sacrifice additional animals. Research on the non-human primate brain is critical to understanding human brain development, architecture, connectivity, and disease.
- Aviva Rabin-Court: Undergraduate student, Yale University
- Philip M. Barello: Computer Systems Manager, Yale University School of Medicine – Neuroscience
- Jose Andrade: Sr. Solution Architect, Yale University ITS
- Dr Pasko Rakic, Dorys McConnell Duberg Professor of Neuroscience and Professor of Neurology
Dr Duque and Dr Selemon, neuroanatomists trained at Rutgers University and the University of Rochester, respectively, held postdoctoral fellowships in the laboratory of the late Dr Patricia Goldman-Rakic and have worked with Dr Pasko Rakic on primate neurodevelopmental studies. Currently both hold Research Scientist appointments in the Department of Neuroscience.
Dr Alvaro Duque and Dr Lynn Selemon
Yale School of Medicine
Department of Neuroscience
PO Box 208001
333 Cedar Street
New Haven, CT 06520-8001