While figuring these numbers out is a prodigious feat no matter how you cut it, determining glial cell counts has been particularly challenging due to the small size of glia and the difficulty in telling them apart from other small cells. Despite technical limitations like poor microscope resolution and undeveloped approaches to staining cells, early neuroscientists sometimes still managed to arrive at credible counts of neurons in the brain.
Helen Bradford Thompson, for example, published an estimate in of the number of neurons in the cerebral cortex about 9 billion that matches up well with current estimates of about billion. Early neuroscientists like Helen Bradford Thompson arrived at neuronal numbers by actually counting neurons. In fact, this approach is still used today, just in a more refined manner. But the overall idea is the same: count the number of cells in various samples of brain tissue and extrapolate the numbers obtained to a larger brain region, or the whole brain.
A more recently developed method of cell counting uses some additional steps to make the process a bit easier and more precise. It involves taking a sample of brain tissue and homogenizing it—destroying the cell membranes, leaving the nuclei intact, and creating a soup-like mixture of liquefied brain. The nuclei can be stained with a fluorescent dye, antibodies can be used to differentiate between neural and non-neuronal cells, and then the nuclei can be counted.
This process is called isotropic fractionation. Isotropy is uniformity, and refers to the mixture formed after homogenization of the brain tissue.
And fractionation indicates that cells are counted in a fraction of the whole tissue, and then the results are used to infer numbers in the rest of the brain region. Isotropic fractionation is a relatively new method.
Before it was developed, finding accurate cell numbers in the brain was more painstaking and susceptible to errors. And, as mentioned above, glial cells were especially problematic. This difficulty in counting glia was represented in some of the uncertainty researchers expressed about the number of glial cells in the brain before the s. Although it was widely believed that the tiny glia outnumbered neurons, there was not a lot of hard evidence to prove this was the case.
A common estimate at this time was that there were "perhaps" ten times as many glial cells as neurons. Gray matter is largely made up of the unmyelinated parts of neurons—neurons that are not sheathed by glial cells—whereas white matter is comprised of axons wrapped in insulating oligodendrocytes. These results might explain why so many early counting studies that only sampled cortical gray matter found a roughly or slightly higher glia to neuron ratio. Overall the cerebral cortex—including both gray and white matter—contains far more glia than neurons, but its outermost gray layer is more balanced.
And the cerebellum's incredible density of neurons balances out the glia to neuron ration throughout the whole brain. When Herculano-Houzel first published her innovative technique in , the main objection was that she had not directly compared it to more typical stereological methods, in which cells are counted in slices of brain tissue.
When her results with whole brains matched counts from different brain regions in previous stereological studies, however, Herculano-Houzel says most critics backed off.
Some researchers remain concerned that grinding and dissolving the brain destroys a significant number of nuclei. Herculano explains, however, that the saline detergent she uses Triton X destroys fatty tissues, like cell membranes, but preserves the protein-rich nuclear membrane. Furthermore, she says, fixing brain tissue in formaldehyde before grinding strengthens the bonds between proteins, making them especially difficult to break.
Other researchers say they are hesitant to trust a method that has not been widely used outside of a single research group. So far, however, at least seven different research teams in the U. Neurobiologist Ben Barres of Stanford University says he never believed the widely parroted glia to neuron ratio—until he looked into the matter himself.
Now, he is certain that glia make up at least 80 percent of cells in the human brain. Here is his main reasoning. The human brain contains a finite number of cells, each of which holds the same amount of DNA about 6. The developing human brain produces most of its neurons within the first trimester of pregnancy, but glia do not finish growing in number until a few years after birth. By comparing the total amount of DNA in a week-old human brain to the total amount of DNA in an infant's brain, Barres reasoned, one could figure out the glia to neuron ratio.
Barres found a study published in that analyzed DNA levels in human brains ranging in age from 10 weeks to seven years. The forebrains which do not include the cerebellum contained about 0. Based on these numbers—and accounting for DNA from blood vessel cells—Barres concludes that growing numbers of glia explain the increase in total forebrain DNA and that glia therefore make up at least 80 percent of cells in the human brain. Even though Barres is confident in his own unpublished calculations—and intends to write that glia far outnumber neurons in the newest edition of the Principles of Neural Science —he argues that no one has conducted the kind of rigorous study that would definitively answer the question of the glia to neuron ratio once and for all.
Barres envisions a study in which researchers stain whole human brains with just about every known marker for both neurons and glia—making sure to capture as many of the different cell types as possible—before slicing up the brains and meticulously counting the cells in each section.
He says all the necessary tools are available. It is only a matter of funding the project and finding the time for all that counting. Let's say scientists figure out exactly how many glia and neurons the brain contains and everyone agrees on the numbers—what will that accomplish?
Why does it matter? Some scientists think that the glia to neuron ratio is just about one of the least important questions you can ask about the brain. Instead, they argue, scientists should focus on how brain cells behave. Other scientists point out that aging, as well as many neurological diseases, involve the loss of brain cells.
Understanding exactly which brain cells die and which survive could spur the development of new treatments. Some biologists and neuroscientists are also very interested in whether the glia to neuron ratio has changed over the course of evolution and whether, for example, animals with large brains—or brains that are large for their body size—have unusually high or low numbers of glia.
In a study , scientists sliced up five minke whale brains, counted the cells with the help of computers and found However, the study did not include the cerebellum, which contains most of the mammalian brain's neurons according to Herculano-Houzel's work. Many researchers have argued that glia deserve more attention in part because they are so numerous. But prevalence is not equivalent to significance. Scientists no longer need to depend on the alleged ratio to justify glia research.
Glial cells are fascinating and important because of their structural diversity, functional versatility and the fact that they can change the behavior of firing neurons even though they cannot discharge electrical impulses of their own. They guide early brain development and keep their fellow brain cells healthy throughout life.
Glia are not mere structural filler, but—as the origin of their name implies Greek for glue —they help keep things together. Regardless of the true glia to neuron ratio, scientists have already shown that glia are, functionally, the brain's other half.
Azevedo, Frederico A. Carvalho, Lea T. The placement and relative strengths of the trillion-plus synapses in our brain are intimately associated with what defines us—learning, thinking, feeling, remembering and forgetting. And, it turns out, our very synaptic architecture is profoundly influenced by the glial cells that envelop, ensheathe and communicate with neurons everywhere one looks in the brain. Besides astrocytes, two other glial-cell types have important, if not exotic, functions in the brain.
Astrocytes are so named because their many long, thin projections cause them to appear star-shaped under a microscope. And to do that, they first had to be able to tease them apart.
Accomplishing that task was no mean feat. But Barres was persistent. Since his graduate school days in neurobiology, Barres now aided by numerous postdocs and grad students has figured out how to separate the brain into its cellular building blocks. That purification advance allows for gene profiling. This has allowed the Barres group to harvest very specific markers for all those cell types. One astrocyte can envelop thousands of them, even tens of thousands, at a time. In a study published in , Barres and his co-investigators showed astrocytes are necessary for synapse generation.
To show this, they cultured highly purified mouse neurons in the total absence of glial cells. Everything looked good. Except for one thing—they were hardly making any synapses. They produced what at least looked like synapses, but only at one-tenth the number normally seen. He found that astrocytes produce it only during brain development, just at a time and a place synapses are sprouting up all over.
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