The cortex of the human brain holds more than 100 trillion neural connections, or synapses, packed into a layer of tissue just 2 to 4 millimeters thick. Visualizing these densely packed units individually has proved extremely challenging. Synapses in the brain are crowded in so close together that they cannot be reliably resolved by even the best of traditional light microscopes, explains Stanford neuroscientist Stephen Smith in a press release from the university.

Smith and collaborators have developed a new technology that highlights all the synapses in the mouse cortex. “Now we can actually count them and, in the bargain, catalog each of them according to its type,” he says.

The ability to study synapses en masse could help scientists understand Alzheimer’s disease, autism and other neurological and developmental disorders. Many of these have been shown to be linked to dysfunction or degeneration of the synapses.

Smith and collaborators first sliced thin pieces of brain tissue and then stained the slices with groups of three different antibodies. Each antibody was designed to bind to a specific type of protein found in different types of synapses and to glow a certain color, with 17 different antibodies in all.

Sections of neurons are shown in green. The protruding bulbs are the parts of the neuron that receives a connection from another cell, highlighted in blue, yellow and pink.

After each application huge numbers of extremely high-resolution photographs were automatically generated to record the locations of different fluorescing colors associated with antibodies to different synaptic proteins. The antibodies were then chemically rinsed away and the procedure was repeated with the next set of three antibodies, and so forth. Each individual synapse thus acquired its own protein-composition “signature,” enabling the compilation of a very fine-grained catalog of the brain’s varied synaptic types.

These individual pictures were then stitched together using specialized software also designed in Smith’s lab, creating a three-dimensional image of the section of brain tissue. The research was published this week in the journal Neuron.

The researchers were able to “travel” through the resulting 3-D mosaic and observe different colors corresponding to different synaptic types. Observed in this manner, the brain’s overall complexity is almost beyond belief, said Smith. “One synapse, by itself, is more like a microprocessor —with both memory-storage and information-processing elements — than a mere on/off switch. In fact, one synapse may contain on the order of 1,000 molecular-scale switches. A single human brain has more switches than all the computers and routers and Internet connections on Earth,” he said.

His group is now focused on using array tomography to tease out more such distinctions, which should accelerate neuroscientists’ progress in, for example, identifying how many of which subtypes are gained or lost during the learning process, after an experience such as traumatic pain, or in neurodegenerative disorders such as Alzheimer’s. With support from the National Institutes of Health, Smith’s lab is using array tomography to examine tissue samples from Alzheimer’s brains obtained from Stanford and the University of Pennsylvania.

“I anticipate that within a few years, array tomography will have become an important mainline clinical pathology technique, and a drug-research tool,” Smith said. He and Micheva are founding a company that is now gathering investor funding for further work along these lines.