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Numbers under review

Recounting neurons puts ideas about neuroscience in check

Leo RamosOn the afternoon of Wednesday January 11, researchers Frederico Casarsa de Azevedo and Carlos Humberto Moraes carried out an unusual task for neuroscientists. They covered a masonry bookcase with white cardboard to hide the window at the back, cleaned a granite table and removed glass recipients, pipettes and reagents to a bench alongside that already held more glassware, pipettes and reagents. They were preparing a laboratory, which is headed up by a physician, Roberto Lent, from the Federal University of Rio de Janeiro (UFRJ) for a photo and filming session. They wanted to record in detail the functioning of a machine that they started creating seven years ago and that is now ready: the automatic cell fractionator, on which they intend taking out a patent – and the scenery could not interfere.

The equipment with its complicated name and almost one meter tall is a type of family-sized waste disposal unit. It has electric motors that make six plastic pistons, fixed to a mobile base, rotate at 400 rpm. Each piston works immersed in a glass recipient containing samples of brain tissue bathed in a solution with detergent. Once the fractionator has been switched on, its pistons stir the colorless liquid, creating vortexes that break up the samples. Two pieces of brain tissue are dissolved in a milky-colored mixture. This is what the researchers affectionately called brain juice.

The machine undergoing tests in UFRJ’s Neuroplasticity Laboratory at the Institute of Biomedical Sciences (ICB) is a turbo-charged version of a very much simpler fractionator, a tube and a piston, both made of glass and manually activated, which Lent and neuroscientist Suzana Herculano-Houzel have used since 2004 for breaking up pieces of brain and counting the cells. The technique they have created has allowed them to get to know more precisely something that was already known, supposedly: how many neurons there are in the brain and in other encephalon organs that are housed in the skull.

Today it is known, in part thanks to the work of the group from Rio, that there are 86 billion neurons in the human brain and not the 100 billion that used to be talked about years ago. It can also be stated with more assurance that these neurons are accompanied by 85 billion glial cells, the other type of cell that comprises the brain, a number far lower than the previously touted one trillion.

These are not just details. Checking more accurately how many brain cells there are and where they are is important to understand how the brain functions and to try and get to know the strategies adopted by nature for constructing an organ that is so complex that in the human case it has allowed for the self-conscious mind to appear. It can also help identify characteristics that distinguish a normal brain from one that is sick.

However, looking just at the number of cells is not enough to unveil one of the most intriguing and fascinating body organs. Today, neuroscience considers the brain to be much more than just a collection of neurons, cells that communicate by means of electricity. Just as important, or even more, than the total number of neurons, are the effective connections they establish amongst themselves, creating networks that process the information in a broader way, according to Italian neuro-anatomist Alessandro Vercelli, from the University of Turin. “The number, pattern and quality of these connections vary in space and time,” says Martín Cammarota, a neuroscientist from the Pontifical Catholic University of Rio Grande do Sul, who studies the formation and recall of memories. “Having more or fewer neurons does not necessarily make an individual or a species more or less intelligent than another,” he says.

018-023_CAPA_DogmasNeuro3_192-2Despite these considerations, the results that Suzana and Lent have been collecting since 2005 led them to question some ideas that were held as absolute truths about the composition and structure of the brain. Last year, Lent considered that the data generated by his group and that of Suzana were already sufficiently consistent to be consolidated into a more direct critique. With three researchers from his laboratory, he wrote the review published in January in the European Journal of Neuroscience, stating that at least four basic concepts of neuroscience need to be rethought.

The first dogma discussed in the article is that the human brain and the rest of the encephalon together have 100 billion neurons. Known even by those who are not specialists, this number has been circulating in scientific articles and textbooks for almost 30 years. Lent himself has a book published in 2001 and used in graduate courses, entitled ‘Cem bilhões de neurônios’ [One hundred billion neurons].

The origin
This book, by the way, is to a certain extent at the origin of the doubts that drove the researchers from UFRJ to investigate how many cells there are in the brain. A little before its launch, Suzana had started a study to evaluate knowledge about neuroscience among high school and university students. One of the 95 statements they had to say was right or wrong was: we only use 10% of our brain.

Almost 60% of the 2,200 people interviewed replied that yes it was correct. This statement, which is wrong because we use all of the brain all the time, results from another statement, made in 1979 by the Canadian neurobiologist David Hubel, who received the Nobel Prize in Medicine or Physiology in 1981. He stated that there were 100 billion neurons in the brain and 1 trillion glial cells. Repeated in other publications, the information spread. As the neurons are units that process information and represent only one tenth of the brain cells, it was concluded that the other 90% of the brain was not used when walking, planning a trip or sleeping.

Turbo-charged fractionator: it transforms brain pieces into a soup of neuron nuclei

Leo RamosTurbo-charged fractionator: it transforms brain pieces into a soup of neuron nucleiLeo Ramos

The result bothered Suzana, who looked for the original source of these figures in scientific literature and did not find it. She had collaborated with Lent on his book and raised her doubt with him: “How do you know that there are 100 billion neurons?” Lent replied: “Look, everybody knows, every book says so.” Many articles and books carried the information, but did not state its source. “They were apparently intuitive data that had become consolidated and people cited them without thinking,” comments Lent.

One of the reasons why these numbers are not easily found is that counting brain cells is no easy matter. In addition to being a large organ (the human brain weighs around 1,200 grams and the encephalon, 1,500), its architecture is complex. Different areas have varied cell concentrations and the technique available for counting them previously, stereology, only works well for small regions with a homogenous cell distribution. Its application in counting brain cells generated unreliable estimates, which varied by as much as 10 times for some regions and left the human brain with something between 75 billion and 125 billion neurons.

Having been recently hired by UFRJ, Suzana told Lent that she had a “bold and half crazy” idea of how to count neurons, but she had no laboratory. He invited her to work with him. Suzana’s proposal was simple: make the brain regions homogenous before counting their cells. How? By breaking up the cells.

The main reason for the heterogeneity of the encephalon is that the cells and the space between them vary in size. By dissolving the cells, the question would be resolved, provided it was possible to preserve their nuclei, the most central part that houses the DNA. As each brain cell has just one nucleus, the count is simple. The sum of the nuclei would give the total number of cells. Dyes marking the neurons only then allowed them to be distinguished from other brain cells.

Using chemical compounds that preserve cell structures, Suzana managed to destroy just the external membrane without damaging the nucleus, and with Lent, she described the technique in 2005 in the Journal of Neuroscience. “It’s an intelligent, simple and easy method to use and replicate,” comments Vercelli. “I wonder why no one ever thought of this before.” In the opinion of Zoltan Molnar, a neuroscientist from the University of Oxford, in England, it was an important advance. “Genomics, trasnscriptomics and proteomics are quantitative and accurate areas that have made a lot of progress, while we anatomists have remained in the dark ages. We haven’t developed methods that can measure the number, density and variations in cell architecture,” he says.

The first test was on the brains of rats. The total number of cells in the encephalon was 300 million, of which 200 million were neurons.  Unexpectedly, only 15% of them were in the brain, the most voluminous part. Most (70%) were found in a smaller organ in the back of the skull, the cerebellum.

This was how it was in rats. But what about other species? Suzana then analyzed the brain of another five rodents (mice, hamsters, guinea pigs, pacas and capybaras). As was already known, the bigger the animal the bigger the brain and the number of neurons. The mouse, weighing just 40 grams, is the smallest and has 71 million neurons, stored in a brain weighing 0.4 grams. Almost 1,200 times heavier, the capybara has an encephalon 183 times bigger (76 grams), but only 22 times more neurons (1.6 million).

The human brain
Supervised by Suzana and Lent, biologist Frederico Azevedo counted the cells in human brains. First, however, he had to adapt the technique. “What functioned with rodents didn’t work for humans,” he says. It took months before he discovered that the problem lay in the way the tissue was fixed before fractioning it. When the brain was immersed too long in compounds that avoid its deterioration the researcher was unable to color the neurons in order to be able to count them by microscope. Frederico fractioned by hand the brain samples of four people (ranging in age from 50 to 71), which were supplied by the brain bank of the University of São Paulo (USP). “This was when I began thinking of a way to make this work automatic,” says the biologist, who is doing a PhD at the Max Planck Institute in Germany.

The cell count revealed that the human brain has on average 86 billion neurons, or 14% less than the previous estimate and close to the number suggested in 1988 by Karl Herrup, from Rutgers University. “There are those who say that the difference is small, but I disagree,” says Suzana. “It corresponds to the brain in a baboon or half the brain of a gorilla, one of the primates that in evolution terms is closest to human beings,” explains the neuroscientist, who is head of the Compared Neuroanatomy Laboratory of ICB-UFRJ.

Lent comments cautiously: “We can’t state that these numbers are representative of the human species. It’s probable that they’re representative of mature adults.” Or not even this, since only four brains were analyzed. In younger people, it might also be different. “Who knows whether individuals in the 20-year old age group might not have 100 billion neurons that they lose over time?” asks the researcher. His group is now studying the brain of younger people and comparing brains of men and women. Until he answers this question, Lent has altered the title of the second edition of his book, published in 2010, to Cem bilhões de neurônios? [One hundred billion neurons?], with a question mark at the end.

The cerebellum
Just as in rodents, most of these neurons are not in the brain but in the cerebellum. The brain (more specifically the cerebral cortex, until quite recently considered primarily responsible for cognitive functions such as attention, memory and language) is the part of the encephalon that has grown largest throughout evolution. In the human case, it weighs 1,200 grams and occupies more than half the skull, but houses just 16 billion neurons. The cerebellum, on the other hand, weighing 150 grams, has 69 billion cells.

How can we explain the different sizes of these organs? The reply is manifold. First, the brain has fewer neurons than the cerebellum, but almost four times more other cell types, such as the glial cells. These cells, previously seen merely as the physical support for neurons, perform other essential functions: they help in the transmission of impulses, feed the neurons and defend the central nervous system from invading microorganisms; and, of course, they occupy space. Second, the brain and the cerebellum are formed from different types of neurons, which are connected in a different way.

In doing this work, the group from Rio also found that evolution did not favor just the development of the brain. Among mammals, the class of animals to which human beings belong, the brain and the cerebellum gained neurons at the same pace. This result, according to Vercelli, corroborates that of research, indicating that the role of the cerebellum does not merely control movement. It is fundamental for learning, memory, the acquisition of language and control of behavior and emotions. “It’s being increasingly shown that the cerebellum participates in processes that we previously only associated with the cerebral cortex,” comments Herrup from Rutgers.

The strategies
Since developing the technique, Suzana has already applied it to the study of the encephalon of 38 species of mammals and found that over the last 90 million years nature has adopted at least two strategies to build brains; one for rodents and another for primates.

In rodents, the increase in the number of neurons in the encephalon occurs logarithmically. Generally speaking, as the size of the species increases, the encephalon becomes bigger and the absolute number of neurons does as well. However, the bigger the rodent, it gains proportionally fewer neurons. On the other hand, among primates, which include monkeys and human beings, the increase is linear: the number of neurons increases in proportion to cerebral volume. “There was an abrupt transition between lower mammals, like rodents, and the higher mammals, like primates,” comments Vercelli. According to Lent, this change allowed the brain of primates to group more neurons in a smaller volume and to accumulate more cells than rodent brains.

This pattern of encephalic development in primates led Suzana and Lent to question another idea in force for nearly 40 years: that the human brain is exceptionally large. In 1973, American paleoneurologist, Harry Jerison, said that our brain was seven times bigger than what one might expect of a 70-kilogram mammal. Neuroscientist Lori Marino later said that it was large even for a primate. At almost 1,500 grams, the human encephalon is the biggest among all the primates – the gorilla, the biggest primate, weighs 200 kilos and has a 500-gram encephalon. However, the starting point of this notion is the principle that body size is a good indicator of brain dimensions. It now seems that it is not.

Setting aside body mass and analyzing the number of cells, it is seen that the human brain does not differ from the pattern for primates. “Our brain has the number of cells to be expected for a primate of this size,” says Suzana.

018-023_CAPA_DogmasNeuro3_192-3Based on this rule and the volume of the skull, in 2011 Suzana and neuroscientist Jon Kaas, from Vanderbilt University in the United States, published in Brain, Behavior and Evolution an estimate of the number of brain cells of another nine hominids. As was to be expected, the species that is closest to humans (Homo sapiens) in terms of neuron numbers is Neanderthal man (Homo neanderthalensis), who lived between 30,000 and 300,000 years ago in the region where Europe is today. They had 85 billion neurons, according to Suzana and Kaas’ estimate. With the help of bioanthropologist Walter Neves, from USP, Lent expanded the projection to include other species of primates that belong to the super-family of hominids and calculated that the Neanderthals may have had 100 billion neurons.

Another dogma being questioned is that the total number of glial cells exceeds the number of neurons ten-fold. This is the source of the idea that only 10% of the brain is used. “This high rate of glial cells was taught in text-books, although experiments were already indicating that the proportion was 1 to 1,” says Helen Barbas, from the University of Boston.

Rather than the number of glial cells (there are 85 billion in human beings, and they are concentrated more in the brain than in the cerebellum) what most surprised Suzana is that they underwent practically no morphological changes during evolution. Their size is almost constant among the primates, while the size of neurons has varied by a factor of 250. “The functioning of glial cells should be adjusted in such a fundamental way that nature would eliminate any change arising,” she comments.

It is expected that more exciting results will appear as the Brazilian technique spreads. “If it’s widely used it can simplify the tedious process of counting brain cells,” says Herrup. Perhaps more hours can be saved if the turbo-charged version of the fractionator is as efficient as expected.

Scientific articles
LENT, R. et al. How many neurons do you have? Some dogmas of quantitative neuroscience under revision. European Journal of Neuroscience. v 35 (1). Jan. 2012.
HERCULANO-HOUZEL, S.; LENT, R. Isotropic fractionator: a simple, rapid method for the quantification of total cell and neurons in the brain. Journal of Neuroscience. v. 25(10), p. 2.518-21. 9 Mar. 2005.