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A model fish

More practical and less expensive than rodents, the zebrafish is beginning to be used in neuroscience research and drug studies in Brazil

Filling a need: the zebrafish, a native of Asia, is an intermediate animal model between fruit flies and rodents

NATALIA ELTZ SILVAFilling a need: the zebrafish, a native of Asia, is an intermediate animal model between fruit flies and rodentsNATALIA ELTZ SILVA

There is a strange matchmaking agency in the basement of the Science and Technology Museum at the Rio Grande do Sul Pontifical Catholic University (PUC-RS), in Porto Alegre. Every afternoon some couples—trios, actually—are conducted into a quiet room where, in the dark, they spend some hours getting to know each other at a certain distance. In the morning, when the lights are turned on and the participants are free to interact, they begin a relationship of just 15 minutes that usually results in many descendants. A notice posted outside the door keeps the curious at bay: “Do not enter: coupling in process.” In this PUC-RS lab, biologist Monica Ryff Moreira Vianna, following strategies she optimized, controls the reproduction of a small silver and black striped fish known as the zebrafish, increasingly used in neuroscience research around the world and, now, in Brazil.

“In some tests, the zebrafish can serve as an alternative to rodents; in others, it can provide complementary information,” says biologist Denis Rosemberg, who recently participated in the construction of a zebrafish vivarium at the Chapecó Region Community University (Unochapecó), in the state of Santa Catarina. He began working with the fish in the Neurochemistry and Psychopharmacology Laboratory of pharmacologist Carla Bonan, during his undergraduate studies at PUC-RS. He investigated the harmful effects of alcohol on the brain and demonstrated the neuroprotective action of taurine, which is naturally produced by the body and found in energy drinks, when he moved to the Federal University of Rio Grande do Sul (UFRGS).

In Santa Catarina, Rosemberg and pharmacologist Angelo Piato are now beginning to use the fish to investigate the effects of stress on the central nervous system and on behavior. This, incidentally, is one of the situations in which the zebrafish (Danio rerio) may offer advantages over rodents. This is because cortisol is the hormone that controls stress in the fish, the same that is released by glands located on the kidneys in humans when faced with a real or imagined threat to a person’s life. In rodents, the hormone corticosterone—which occurs in very low concentrations in the human body—is produced in similar situations.

At UFRGS, biologist Diogo Losch de Oliveira’s group managed to go one step further. In an article published earlier this year in the journal PLoS One, he and his Master’s student Ben Hur Mussulini described in detail the behavioral changes that characterize the epileptic stages of adult zebrafish. “The scientific literature had only detailed descriptions for the model in larvae, which have a more restricted behavioral repertoire,” says Oliveira.

Almost transparent: zebrafish embryos 24 hours after fertilization

LAURA ROESLER NERY Almost transparent: zebrafish embryos 24 hours after fertilizationLAURA ROESLER NERY

More recently, his group began testing the first of a group of 30 compounds developed in partnership with Grace Grosmann, of the UFRGS School of Pharmacy. These compounds attempt to explore a biochemical pathway different from those targeted by currently available drugs, which are unable to control epilepsy in 30% of cases. Of the three compounds tested, only one has been shown to reduce the intensity of crises and is expected to undergo further evaluation.

Note that international studies consider the zebrafish a promising tool for analysis and selection of candidate drug compounds. This fish is expected to accelerate the process and decrease costs. One of the advantages is that its life cycle is quick—several of its organs are formed within four days—and its larvae, which are born by the hundreds after each mating, are only a few millimeters in length and can be placed in test tanks with very low doses of chemical compounds. With this selection, it is thought to be possible to reduce the number of molecules that continue on to the next phase of experiments with rodents. “With the zebrafish, we can run a test in a few months for a few thousand dollars, while the same test would take years with rodents and cost millions,” says biochemist Diogo Onofre Souza, coordinator of the National Institute of Science, Technology and Innovation in Excitotoxicity and Neuroprotection, which is performing research with zebrafish at UFRGS. Overseas, some drug companies have begun to use them for their tests.

Pioneers
Native to Southwest Asia, where it is found in calm, shallow rivers and flooded rice and jute plantations, this fish was introduced to research laboratories in the late 1960s by the American biologist George Streisinger, of the University of Oregon. He worked alone for a decade to select lines that allow us to understand how defects in different genes affect development. His efforts only managed to reduce the skepticism of colleagues in 1981, when he published an article in the journal Nature presenting a consolidated model. In the following years, the number of scientific papers that used the fish as a biological model grew rapidly, especially in genetic and development studies, but the zebrafish has begun to be used in neuroscience only in the last decade.

Almost transparent: zebrafish larva 5 days old

LÉO RAMOS Almost transparent: zebrafish larva 5 days oldLÉO RAMOS

Complementary models
“The zebrafish is beginning to fill the gap that existed between different animal models for studying human diseases,” says neurophysiologist Luiz Eugenio Mello, of the Federal University of São Paulo (Unifesp), who studies changes in the central nervous system, including those caused by epilepsy, using rats as an animal model. Mello reminds us of an axiom of science that says that the best model to investigate a thing is the thing itself. According to this reasoning, the ideal for studying human diseases would be humans themselves. But this is rarely possible. “Most of the time there are ethical constraints and limitations of time, space and cost to carry out research,” he says. “This is why we need several experimental models, from the simplest to the most complex, to understand the source of some problems.”

When you cannot investigate a problem in human beings, medicine and biology adopt a kind of preferential hierarchy of models, which take into account factors such as evolutionary, anatomical, physiological and genetic similarity. Based on this system, the animals that would allow us to extrapolate the results more confidently to human beings would be the other primates, such as chimpanzees, whose use in research is banned in Brazil and is being banned in the United States, and other monkeys. “Only researchers with plenty of money and space work with primates,” says molecular biologist João Bosco Pesquero, also at Unifesp, and creator of one of the first Brazilian transgenic mice lines. “For this reason, many people choose rodents, which are mammals like humans,” he says.

When technical difficulties prevent working with rodents, which sometimes occurs in genetics—for example, transgenic mice have been produced only very recently—the solution is to work with models that are more evolutionarily distant from humans, but easier to manipulate, such as fruit flies. And more recently the zebrafish.

Most important, however, is that from the evolutionary point of view the zebrafish is closer to humans than fruit flies, which have been used as a model organism in genetics for almost a century. The zebrafish genome, completed earlier this year, indicates that 70% of its 26,000 genes are similar to human genes. This is greater than the similarity to fruit flies, and less than the similarity to mice and rats, which served as the basis for much of what is known of human physiology.

“Historically, neuroscience research has used rodents as the animal model, but this scenario is beginning to change,” says Vianna, who is also a member of the board of the Latin American Zebrafish Network (Lazen). This consortium brings together researchers from seven countries that use the fish in their studies and provides training for those interested in adopting the zebrafish as an experimental model—generally early in their careers. Of the 39 groups that make up the network, 11 are Brazilian and almost half of these are in Rio Grande do Sul.

Brazilian scientific research using the zebrafish, which did not exist just over a decade ago, has been growing quickly in recent years, at a rate higher than in the rest of the world. The biologist Luciana Calabró, a specialist in scientometrics and a member of one of the groups that perform studies with zebrafish at UFRGS, reached this conclusion in a recent search of one of the principal international databases of scientific articles, Scopus. “The number of articles published by Brazilian authors grew from 2 articles per year in 1999 to 36 in 2012, and represented about 2% of articles published worldwide using the zebrafish,” she says.

The number of national articles using this fish is still modest in an international context, since nearly 2,000 articles are being published yearly in recent years. But Brazil has been making a name for itself in the neurosciences. “The zebrafish is a new model in this area and the community working with it is still small,” says Vianna.

In Brazil
The first Brazilian studies carried out using this fish were in the laboratory of researcher Rosana Mattioli, of the Federal University of São Carlos (UFSCar), in the state of São Paulo. At that time, the zebrafish was just beginning to be used in research in neuroscience, but the normal behavior of the species was still little known. Mattioli then conducted a series of simple experiments that helped identify the fish’s preference for living in dark environments. She placed the zebrafish in a tank painted with two colors—half black and half white—and measured the average time they spent in each half. Thus, she observed that they spent most of the time (about 80%) in the dark side. She also saw that, when placed in the light side, they quickly swam to the dark region. This article, published in 1999 in the Brazilian Journal of Medical and Biological Research, began to lay the foundation for an important anxiety test, which she and other researchers then enhanced, and is now used to assess the effect of compounds that fight depression and anxiety.

Anxiety
When the psychologist Amauri Gouveia Junior, then at the Universidade Estadual Paulista (UNESP) in Bauru, saw this article, he noticed a great similarity between the light/dark test for zebrafish and an experiment that evaluates rodent anxiety levels. In the latter, the rodent is placed on an X-shaped platform about 60 cm above the ground. In two of the arms, the walking space is protected by walls, while the other two are open. Once in the maze, the curious rats tend to go exploring. However, they avoid the open part. Anxiety results from a conflict between curiosity and fear. “The time that the fish spent in the dark side was very similar to that which the rodents spent in the protected part of the maze,” says Gouveia. “So, I thought that the two tests might both measure anxiety, in different animals.” Since then, he has applied the light/dark test to 12 species of fish, including zebrafish, to assess anxiety in fish. “It is one of the most widely adopted tests today in laboratories studying fish all over the world,” says Gouveia, now a researcher at the Federal University of Pará.

The next phase is to test compounds that interfere with this behavior to try to figure out how they alter the fish. Using some of these standardized tests, Brazilian researchers have already identified chemical and cellular changes in the brain, caused either by epileptic seizures or by compounds that control depression and anxiety. At the State University of Campinas (Unicamp), which set up a zebrafish laboratory two years ago, the geneticist Cláudia Maurer-Morelli and her Master’s degree student Patrícia Barbalho saw that the levels of an inflammatory molecule, interleukin-1 beta, increased after an induced epileptic seizure. Seizures also increase the production and activity of brain-derived neurotrophic factor (BDNF), a protein that is altered in epilepsy in humans, as shown by the results published by Fernanda Reis-Pinto in 2012 in the Journal of Epilepsy and Clinical Neurophysiology. In a line of research in its initial phase, Maurer-Morelli plans to produce fish with the genetic alterations found in people with epilepsy in order to investigate the role of these mutations in the disease. The work is being done at the Brazilian Institute for Neuroscience and Neurotechnology, coordinated by Fernando Cendes, which is one of the Research, Innovation and Dissemination Centers funded by FAPESP.

Although the first studies with zebrafish were carried out in São Paulo, about half of Brazilian articles in recent years are the product of teams in Rio Grande do Sul, most of them in neuroscience. According to Monica Vianna, there is a historical reason why most Brazilian studies involving zebrafish are in neuroscience. Both she and Carla Bonan, at PUC-RS, one of the first to create a zebrafish laboratory in Brazil, had trained in Iván Izquierdo’s group at UFRGS. He is one of the leading memory researchers in the world. After working with rodents in their Master’s and PhD programs, Bonan and Vianna decided to invest in zebrafish. In recent years, Bonan has shown that the levels of some molecules that are involved in communication between brain cells—adenosine triphosphate and one of its components, adenosine—play a protective role against epilepsy, stress and neurotoxicity induced by metals in these fish.

Reproduction
At PUC-RS, Vianna and her team worked for months to determine the most efficient strategy to promote fish coupling in the Laboratory of Biology and Development of the Nervous System. She only managed to increase the reproductive rate when participants were placed in groups of three (one female and two males) and kept separated by a transparent divider—males on one side and female on the other—for a whole night before they could finally make contact. “If you do not separate them, each female produces less than a dozen eggs,” says the biologist. But with isolation and 12 hours at a distance to create interest, this number can increase to about 200. About ten matings occur each day and, on average, 2,000 fish are born per month. In an exceptionally productive morning in May of this year, Vianna and her team spent hours collecting the approximately 1,800 embryos that resulted from a single mating of a few dozen trios of zebrafish, one by one, with a pipette, and she is using them to investigate the biochemistry of memory and neurodegenerative diseases such as Alzheimer’s.

Expecting that the demand for the fish might grow in the coming years, Vianna, Bonan and their colleagues, who share the vivarium at PUC-RS, are working to expand it. The 5,000 fish kept there today are only enough for their studies and those conducted by some of their team members. The goal is to make these laboratories a leading supplier of zebrafish for research in Brazil, together with the National Laboratory for Biosciences (LNBio), in Campinas, where staff biologist José Xavier Neto built a vivarium last year to produce zebrafish with genetic alterations in order to study vertebrate development. Comparing the embryonic development of fish, chickens and mice, Xavier’s team has recently begun to clarify the role of some factors involved in the differentiation of the vertebrate heart and the development of sensory neurons.

One reason to increase fish production is that there is a potential market. The Arouca Law, which regulates the use of animals in research, states that beginning in 2014 only animals with certified origin, quality and uniformity may be used. “In principle,” says Vianna, “you can no longer do research with fish bought from pet stores.”

Scientific articles
MUSSULINI, B.H. et al. Seizures Induced by Pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization. PLos One. v. 8. Jan. 2013.
REIS-PINTO, F. C. et al. Análise temporal dos transcritos dos genes bdnf e ntrk2 em cérebro de zebrafish induzido à crise epiléptica por pentilenotetrazol. Journal of Epilepsy and Clinical Neurophysiology. v. 18, n. 14, p. 107-13. 2012.
CASTILLO, H. A. et al. Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer. Development. v. 137, p. 507-18. 2010.

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