What you are looking at above is something scientists couldn’t even dream of imaging less than a decade ago: bundles of neurons in the brainstem of an adult mouse. These bundles are randomly labeled with various colors that enable researchers to trace the course of each as it projects from the brainstem areas to other parts of the brain. Until recently, such a view would have been impossible because, like other organs, the brain is opaque and had to be sliced into thin, transparent sections of tissue to be examined under a light microscope. These sections forced a complex 3D structure to be visualized in 2D, losing critical detail about the connections.
But now, researchers have developed innovative approaches to make organs and other large volumes of tissue transparent when viewed with standard light microscopy . This particular image was made using the Passive CLARITY Technique, or PACT, developed by the NIH-supported lab of Viviana Gradinaru at the California Institute of Technology (Caltech), Pasadena. Gradinaru has been working on turning tissues transparent since 2010, starting as a graduate student in the lab of CLARITY developer and bioengineering pioneer Karl Deisseroth at Stanford University. PACT is her latest refinement of the concept.
So, exactly how does PACT make a brain transparent? It removes the lipids, the light-scattering molecules that shield and structurally support our cells. The researchers do so by using lipid-dissolving detergents and hydrogel, a water-based polymer gel. The detergent “passively” dissolves lipids throughout the tissue, while the hydrogel fills in for these molecules to provide structural support and leave a cell’s 3D structure intact. The technique can turn whole organs—and even entire organisms—transparent when perfused through the vasculature. That technique, invented by the Gradinaru group, is termed Perfusion-assisted Agent Release in Situ (PARS).
For the labeling of individual neurons throughout the adult brain, Gradinaru and her team administer into the circulatory system a specially engineered virus that bears a cassette of fluorescence genes . The systemically delivered viruses randomly infect neurons or other cells and uniquely color-code them by expression of varying levels of the red, green, and blue fluorescent proteins. The different ratios of these colorful proteins mix to give each cell a distinctive hue when imaged under a microscope.
In recent work, Gradinaru used PACT in rats to map a set of neurons involved in gait and other motor skills that are often impaired in people with Parkinson’s disease. Specifically, they were able to track the arm-like extensions, or axons, of such neurons as they extend from the brainstem to the midbrain. Employing optogenetics, which uses light to control the activity of brain cells, they showed that those axons connected to the lateral and medial parts of the ventral midbrain played a key role in regulating motor function. In the quest to use deep-brain stimulation to help people with Parkinson’s disease maintain their ability to walk, this is the precise type of information needed to stimulate the right parts of the brain and thereby improve treatment .
 Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors. Treweek JB, Gradinaru V. Curr Opin Biotechnol. 2016 Jul 6;40:193-207.
 Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Deverman BE, Pravdo PL, Simpson BP, Kumar SR, Chan KY, Banerjee A, Wu WL, Yang B, Huber N, Pasca SP, Gradinaru V. Nat Biotechnol. 2016 Feb;34(2):204-209
 Cholinergic Mesopontine Signals Govern Locomotion and Reward through Dissociable Midbrain Pathways. Xiao C, Cho JR, Zhou C, Treweek JB, Chan K, McKinney SL, Yang B, Gradinaru V. Neuron. 2016 Apr 20;90(2):333-347.
Parkinson’s Disease Information Page (National Institute of Neurological Disorders and Stroke/NIH)
Gradinaru Lab (Caltech, Pasadena)
Beckman Institute Resource Centers (Caltech)
NIH Support: Common Fund; National Institute on Aging; National Institute of Neurological Disorders and Stroke; National Institute of Mental Health; National Institute of General Medical Sciences.