Description DTI allows visualization of the actual paths of connectivity taken by white matter tracts (Fig. 9.2). It takes advantage of the fact that within a white matter tract, the diffusion of water is largely constrained along the direction of the axons. This means that diffusion within an axon tract is anisotropic, or more probable along a given direction. Fractional anisotropy is thought to be related to the degree of axon myelination, the diameter of axons, and/or the density of a fiber tract. Comparative DTI/immunohistochemistry and dissection studies have confirmed that DTI can accurately retrace the actual routes taken by white matter tracts (Schmahmann et al. 2007).

Strengths Non-invasive in living humans and great apes. It can be performed at high (~100–300 micron) resolution in fixed post mortem brains, which can be acquired after natural death from humans or great apes. Connectivity throughout the entire brain can be assessed from a single scan. When performed in fixed, post-mortem brains, scanning does not alter the tissue so it is still available for other forms of analysis (e.g., histology).

Weaknesses: DTI has relatively lower resolution than injection tract-tracing. False positives and false negatives are likely without proper controls and analysis techniques. Tracking white matter connections through areas of crossing fiber tracts and into gray matter is problematic, but continuing increases in scanner technology and analysis algorithms are addressing this obstacle.

Relevant uses for neuroarchaeology DTI is invaluable for studying brain anatomy in species in which the gold-standard technique, injection of axonal tracers followed by post-mortem histological examination of connectivity, is not possible (this technique is invasive and terminal). Because neuroarchaeology is concerned with the evolution of the human brain, DTI is an important technique for this field.

Description Brain tissue is exposed to an agent that binds to or is taken up by a particular structure of interest (e.g., a particular neurotransmitter receptor type, such as 5HT1A serotonin receptors; cell type, such as interneurons or astrocytes; or cell compartment, such as axons or dendrites) (Fig. 9.3). This exposure can occur in fixed, post mortem brain tissue, or in vivo, as in the case of injection tract tracing, where a tracing agent is injected into an area of the brain, allowed to be transported along axons for a period of time. The animal is then sacrificed to obtain the brain, which now contains tracer along the axon pathways connected to the injection site. The brain then undergoes a series of processing steps to allow the location of the agent to be visualized in slices of tissue. Tissue is placed on slides and can be examined with either a light microscope or an electron microscope.

Strengths Histological analysis is generally considered to be a “gold standard” which is more accurate and reliable than neuroimaging. It has very high spatial resolution. Light microscopy allows visualization at the level of cells and large cell compartments, such as dendritic arbors; electron microscopy allows visualization of even smaller features, such as synapses and synaptic vesicles. Many different reagents have been developed allowing high specificity for visualization of particular receptor classes, cell types, cell compartments, etc. These reagents can even be used in tandem on the same tissue, allowing for visualization of co-localization of particular features, e.g., a particular neurotransmitter receptor on a particular type of cell. Different reactions can also be carried out on alternating slices of tissue, allowing for multiple experiments to be carried out in the same brain.

Weaknesses Histological analysis requires brain tissue to be extracted and preserved immediately after death, which can be difficult to accomplish in great apes and humans, and histological experiments on connectivity involve in vivo injection of tracers, which is not ethically possible in great apes and humans. Well-preserved, high-quality human and great ape brain tissue is a rare and valuable resource, and histology studies in these species are relatively rare. Therefore, structural MRI and DTI provide important sources of information about brain anatomy in these species. Additionally, brain tissue shrinks after fixation, so volumetric measurements in preserved post mortem tissue may not accurately reflect the in vivo condition. Once tissue is processed, it is largely unavailable for other kinds of techniques. Histological processing is extremely time-intensive, so most experiments focus on only a small portion of a few subjects’ brains.

Relevant uses for neuroarchaeology This family of techniques is typically used in rodents and macaque monkeys. Injection tract tracing is not used in humans or other great apes, since it is invasive and requires the non-natural death of the subject. However, post mortem histological analysis has been used in brains from humans and other great apes that can be removed and preserved immediately after death. This type of research has identified uniquely human features in the cellular organization of several brain regions (e.g. Barger et al. 2012; Bryant et al. 2012).