Teaching an old dog new tricks: Neuroscience research at IU combines centuries-old methods with modern technology

This post is the second installment in a two part series. Check out last week’s post here.

Thanks to modern technology, the field of cellular neuroscience has become illuminated with brightly colored images – tissue samples, cells, and individual molecules have been stained, photographed, colorized, and even reconstructed in three dimensions. A Google Image search quickly provides thousands of examples, and a walk through the research wing in IU’s Multidisciplinary Sciences Building II is no different. The images that IU neuroscientists have collected are proudly displayed on posters and signs that line the hallways.

However, the field of neuroscience hasn’t always been so bright. Before the late 1800s, scientists could look at samples of brain tissue through the lens of a microscope, but there was no way to pick out individual brain cells, or neurons, from the background–the detailed structure of individual neurons was essentially invisible. That all changed when Camillo Golgi, an Italian biologist, developed a technique that he called “the black reaction.” He found a way to stain entire neurons black against a brown background. For the first time, neuroscientists were able to see individual neurons. His technique, now called Golgi histology, was quickly adopted by Spanish scientist Santiago Ramon y Cajal, who created the first maps of how neurons are organized in the brain.

Left panel: Two black and white portraits, one of Camillo Golgi and another of Santiago Ramon y Cajal. Each individual’s signature is shown below his portrait. Right panel: An illustration of four different neuron types. A few neurons of each type are drawn in black on brown paper. All cell bodies are depicted as black ovular shapes, but vary in size, depending on the type of neuron. Dendritic branches, depicted as sinuous lines extending from each cell body vary in length and thickness, and some neurons have more densely packed dendrites than others. Axons, shown as long, thin lines with only a few branches, extend from neurons in the top left and bottom middle areas of the page to form a loose, net-like pattern. Descriptions of neuron types: Top row, left: Two large neurons, each with only one dendritic “tree” (one dendritic “trunk” extends from the cell body, then branches many times). Branches are long and densely packed, and cross over one another to form a net-like pattern. Top row, right: Nine neurons, each with approximately six dendritic trees with “trunks” that extend from all sides of the cell body. Dendrites are long, thin, and sparsely distributed throughout the area surrounding the cell body. Bottom row, middle: A neuron with seven dendritic trees extending from all sides of the cell body. Dendrites are thicker and shorter than those previously described. Bottom row, right: Twenty small neurons, each with only three short, thick dendrites projecting from each side of the cell body.
Left: Camillo Golgi and Santiago Ramon y Cajal. Right: Original drawing by Cajal, depicting various types of neurons and their organization in the chick brain. From: “Estructura de los centros nerviosos de las aves”, Madrid, 1905.

You might be surprised to learn that Golgi histology is still in use today. By leafing through virtually any neuroscience textbook, you’ll find images of Golgi-stained neurons and, many times, copies of Cajal’s original illustrations. Microscope slides containing samples of Golgi-stained brain tissue can be found in research labs across the globe and, as you might have guessed, here at IU as well.

Myself and other members of Dr. Cara Wellman’s Neurobiology of Stress Lab use Golgi histology to study the rat brain. Using a process derived from Golgi’s original methods, we soak whole brains in solutions that contain microscopic metallic crystals. After soaking for a few weeks in solution, neurons become filled with metal. At this point, we cut the brains into thin sections and place the sections onto microscope slides. We run the slides through a series of solutions that are similar to those used to develop black and white photographs: they cause the metallic crystals inside neurons to turn black. Any metal that is not contained within neurons is washed away, leaving clear images of entire neurons. Under a microscope, it is easy to see large neuronal cell bodies, long branch-like projections which extend from each cell body, called dendrites, and microscopic protrusions on the surface of each dendrite, called dendritic spines.

Three photographs of neurons, each image at a higher magnification than the previous. The image background is brown. Cell bodies are black and ovular. Dendritic branches look like sinuous lines extending from each cell body. Left panel: Lowest magnification. Approximately 200 neurons are shown. Neurons in the lower portion of the image are densely packed. A long dendrite extends from the top of each neuron, in parallel with those from neighboring neurons. Dendrites at the base of each neuron extend in all directions, overlapping with those from neighboring neurons. Neurons in the upper portion are less densely packed, and have fewer dendritic branches that extend from all sides of the cell body. Middle panel: Higher magnification. A single neuron is shown. The neuron looks similar to those in the lower portion of the left panel. Right panel: Highest magnification. A segment of a single dendrite is shown. Small bumps and short mushroom-shaped projections (dendritic spines) cover the surface of the dendrite.
Left: Pyramidal neurons within the medial prefrontal cortex. Middle: A single pyramidal neuron. Right: Dendritic spines. Image credit: Rachel Skipper, Dr. Cara Wellman, and Katie Morales, Neurobiology of Stress Lab, Indiana University.

While these images look similar to what Golgi and Cajal saw over 200 years ago, today’s neuroscientists study neurons a bit differently. Thanks to modern microscopy tools, I can photograph neurons that I find in my tissue samples rather than draw them by hand (and, yes, I proudly display them in the halls of MSBII). I also use advanced methods to measure and analyze the shape and size of Golgi-stained neurons. Using neuron tracing software, I can superimpose a computer screen onto the image I see under the microscope. I then select neurons that are completely stained and click along their dendrites to reconstruct them in three dimensions. I save these reconstructions as digital files, and can automatically measure the length of a neuron’s dendrites, the number of dendritic spines on each dendrite, and many other features of the neuron’s overall structure.

A photograph of a neuron (left panel) is shown next to an illustration of the same neuron (right panel). Left panel: The image background is brown. A single neuron is shown. The cell body is black and ovular, and dendritic branches look like sinuous lines extending from the cell body. A single dendrite extends from the top of the cell body, and multiple dendrites extend from the base. Right panel: A multicolor illustration of the neuron pictured in the left panel. The image background is black. The cell body and each dendritic tree is shown in a different color (dark blue, light blue, pink, green, and yellow). Parts of the dendritic tree that were out of focus in the left panel are clearly reconstructed in the right panel; the size and shape of each dendrite is otherwise identical between the two panels.
Left: A neuron stained using Golgi histology. Right: The same neuron, reconstructed using neuron tracing software. Image credit: Rachel Skipper, Neurobiology of Stress Lab, Indiana University.

These methods are helpful for studying how dendrites change over time or after a given experience. For example, my colleagues in the Wellman lab study how stressful experience affects dendritic structure. They have found that neurons in the medial prefrontal cortex, a brain region that controls emotional reactions, shrink in response to stress. I have found that neurons in the amygdala, another brain region that is important for emotional behavior, are affected by early life stress. Meanwhile, others in the lab are studying how neurons recover following stressful experiences. Better understanding the effects of stress on neurons could be an important key to understanding how stress-induced psychological disorders arise and, importantly, how they may be treated. As we gain insights into the effects of stress on neurons, we will have Camillo Golgi and Santiago Ramon y Cajal to thank.

Want to read more about how this technology is used in neuroscience research? Check out my previous post!

Edited by Karna Desai and Anna Jessee

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