Slimy Fish and the Origins of Brain Development

Lamprey—slimy, eel-like parasitic fish with tooth-riddled, jawless sucking mouths—are rather disgusting to look at, but thanks to their important position on the vertebrate family tree, they can offer important insights about the evolutionary history of our own brain development, a recent study suggests.

The work appears in a paper in the September 14 advance online issue of the journal Nature.

"Lamprey are one of the most primitive vertebrates alive on Earth today, and by closely studying their genes and developmental characteristics, researchers can learn more about the evolutionary origins of modern vertebrates—like jawed fishes, frogs, and even humans," says paper coauthor Marianne Bronner, the Albert Billings Ruddock Professor of Biology and director of Caltech's unique Zebrafish/Xenopus/Lamprey facility, where the study was done.

The facility is one of the few places in the world where lampreys can be studied in captivity. Although the parasitic lamprey are an invasive pest in the Great Lakes, they are difficult to study under controlled conditions; their lifecycle takes up to 10 years and they only spawn for a few short weeks in the summer before they die.

Each summer, Bronner and her colleagues receive shipments of wild lamprey from Michigan just before the prime of breeding season. When the lamprey arrive, they are placed in tanks where the temperature of the water is adjusted to extend the breeding season from around three weeks to up to two months. In those extra weeks, the lamprey produce tens of thousands of additional eggs and sperm, which, via in vitro fertilization, generate tens of thousands of additional embryos for study. During this time, scientists from all over the world come to Caltech to perform experiments with the developing lamprey embryos.

In the current study, Bronner and her collaborators—who traveled to Caltech from Stower's Institute for Medical Research in Kansas City, Missouri—studied the origins of the vertebrate hindbrain.

The hindbrain is a part of the central nervous system common to chordates—or organisms that have a nerve cord like our spinal cord. During the development of vertebrates—a subtype of chordates that have backbones—the hindbrain is compartmentalized into eight segments, each of which becomes uniquely patterned to establish networks of neuronal circuits. These segments eventually give rise to adult brain regions like the cerebellum, which is important for motor control, and the medulla oblongata, which is necessary for breathing and other involuntary functions.

However, this segmentation is not present in so-called "invertebrate chordates"—a grouping of chordates that lack a backbone, such as sea squirts and lancelets.

"The interesting thing about lampreys is that they occupy an intermediate evolutionary position between the invertebrate chordates and the jawed vertebrates," says Hugo Parker, a postdoc at Stower's Institute and first author on the study. "By investigating aspects of lamprey embryology, we can get a picture of how vertebrate traits might have evolved."

In the vertebrates, segmental patterning genes called Hox genes help to determine the animal's head-to-tail body plan—and those same Hox genes also control the segmentation of the hindbrain. Although invertebrate chordates also have Hox genes, these animals don't have segmented hindbrains. Because lampreys are centered between these two types of organisms on the evolutionary tree, the researchers wanted to know whether or not Hox genes are involved in patterning of the lamprey hindbrain.

To their surprise, the researchers discovered that the lamprey hindbrain was not only segmented during development but the process also involved Hox genes—just like in its jawed vertebrate cousins.

"When we started, we thought that the situation was different, and the Hox genes were not really integrated into the process of segmentation as they are in jawed vertebrates," Parker says. "But in actually doing this project, we discovered the way that lamprey Hox genes are expressed and regulated is very similar to what we see in jawed vertebrates." This means that hindbrain segmentation—and the role of Hox genes in this segmentation—happened earlier on in evolution than was once thought, he says.

Parker, who has been spending his summers at Caltech studying lampreys since 2008, is next hoping to pinpoint other aspects of the lamprey hindbrain that may be conserved in modern vertebrates—information that will help contribute to a fundamental understanding of vertebrate development. And although those investigations will probably mean following the lamprey for a few more summers at Caltech, Parker says his time in the lamprey facility continually offers a one-of-a-kind experience.

"The lamprey system here is unique in the world—and it's not just the water tanks and how we've learned to maintain the animals. It's the small nucleus of people who have particular skills, people who come in from all over the world to work together, share protocols, and develop the field together," he says. "That's one of the things I've liked ever since I first came here. I really felt like I was a part of something very special.

These results were published in a paper titled "A Hox regulatory network of hindbrain segmentation is conserved to the base of vertebrates." Robb Krumlauf, a scientific director at the Stower's Institute and professor at the Kansas University Medical Center, was also a coauthor on the study. The Zebrafish/Xenopus/Lamprey facility at Caltech is a Beckman Institute facility.

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Tipping the Balance of Behavior

Humans with autism often show a reduced frequency of social interactions and an increased tendency to engage in repetitive solitary behaviors. Autism has also been linked to dysfunction of the amygdala, a brain structure involved in processing emotions. Now Caltech researchers have discovered antagonistic neuron populations in the mouse amygdala that control whether the animal engages in social behaviors or asocial repetitive self-grooming. This discovery may have implications for understanding neural circuit dysfunctions that underlie autism in humans.

This discovery, which is like a "seesaw circuit," was led by postdoctoral scholar Weizhe Hong in the laboratory of David J. Anderson, the Seymour Benzer Professor of Biology at Caltech and an investigator with the Howard Hughes Medical Institute. The work was published online on September 11 in the journal Cell

"We know that there is some hierarchy of behaviors, and they interact with each other because the animal can't exhibit both social and asocial behaviors at the same time. In this study, we wanted to figure out how the brain does that," Anderson says.

Anderson and his colleagues discovered two intermingled but distinct populations of neurons in the amygdala, a part of the brain that is involved in innate social behaviors. One population promotes social behaviors, such as mating, fighting, or social grooming, while the other population controls repetitive self-grooming—an asocial behavior.

Interestingly, these two populations are distinguished according to the most fundamental subdivision of neuron subtypes in the brain: the "social neurons" are inhibitory neurons (which release the neurotransmitter GABA, or gamma-aminobutyric acid), while the "self-grooming neurons" are excitatory neurons (which release the neurotransmitter glutamate, an amino acid).

To study the relationship between these two cell types and their associated behaviors, the researchers used a technique called optogenetics. In optogenetics, neurons are genetically altered so that they express light-sensitive proteins from microbial organisms. Then, by shining a light on these modified neurons via a tiny fiber optic cable inserted into the brain, researchers can control the activity of the cells as well as their associated behaviors.

Using this optogenetic approach, Anderson's team was able to selectively switch on the neurons associated with social behaviors and those linked with asocial behaviors.

With the social neurons, the behavior that was elicited depended upon the intensity of the light signal. That is, when high-intensity light was used, the mice became aggressive in the presence of an intruder mouse. When lower-intensity light was used, the mice no longer attacked, although they were still socially engaged with the intruder—either initiating mating behavior or attempting to engage in social grooming.

When the neurons associated with asocial behavior were turned on, the mouse began self-grooming behaviors such as paw licking and face grooming while completely ignoring all intruders. The self-grooming behavior was repetitive and lasted for minutes even after the light was turned off.

The researchers could also use the light-activated neurons to stop the mice from engaging in particular behaviors. For example, if a lone mouse began spontaneously self-grooming, the researchers could halt this behavior through the optogenetic activation of the social neurons. Once the light was turned off and the activation stopped, the mouse would return to its self-grooming behavior.

Surprisingly, these two groups of neurons appear to interfere with each other's function: the activation of social neurons inhibits self-grooming behavior, while the activation of self-grooming neurons inhibits social behavior. Thus these two groups of neurons seem to function like a seesaw, one that controls whether mice interact with others or instead focus on themselves. It was completely unexpected that the two groups of neurons could be distinguished by whether they were excitatory or inhibitory. "If there was ever an experiment that 'carves nature at its joints,'" says Anderson, "this is it."

This seesaw circuit, Anderson and his colleagues say, may have some relevance to human behavioral disorders such as autism.

"In autism," Anderson says, "there is a decrease in social interactions, and there is often an increase in repetitive, sometimes asocial or self-oriented, behaviors"—a phenomenon known as perseveration. "Here, by stimulating a particular set of neurons, we are both inhibiting social interactions and promoting these perseverative, persistent behaviors."

Studies from other laboratories have shown that disruptions in genes implicated in autism show a similar decrease in social interaction and increase in repetitive self-grooming behavior in mice, Anderson says. However, the current study helps to provide a needed link between gene activity, brain activity, and social behaviors, "and if you don't understand the circuitry, you are never going to understand how the gene mutation affects the behavior." Going forward, he says, such a complete understanding will be necessary for the development of future therapies.

But could this concept ever actually be used to modify a human behavior?

"All of this is very far away, but if you found the right population of neurons, it might be possible to override the genetic component of a behavioral disorder like autism, by just changing the activity of the circuits—tipping the balance of the see-saw in the other direction," he says.

The work was funded by the Simons Foundation, the National Institutes of Health and the Howard Hughes Medical Institute. Caltech coauthors on the paper include Hong, who was the lead author, and graduate student Dong-Wook Kim.

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Wednesday, September 24, 2014

A chance to meet Pasadena Unified School District Leadership

Wednesday, September 10, 2014
Avery Dining Hall – Avery House

RESCHEDULED to Sept 24th: A chance to meet Pasadena Unified School District Leadership

Biology Made Simpler With "Clear" Tissues

In general, our knowledge of biology—and much of science in general—is limited by our ability to actually see things. Researchers who study developmental problems and disease, in particular, are often limited by their inability to look inside an organism to figure out exactly what went wrong and when.

Now, thanks to techniques developed at Caltech, scientists can see through tissues, organs, and even an entire body. The techniques offer new insight into the cell-by-cell makeup of organisms—and the promise of novel diagnostic medical applications.

"Large volumes of tissue are not optically transparent—you can't see through them," says Viviana Gradinaru (BS '05), an assistant professor of biology at Caltech and the principal investigator whose team has developed the new techniques, which are explained in a paper appearing in the journal Cell. Lipids throughout cells provide structural support, but they also prevent light from passing through the cells. "So, if we need to see individual cells within a large volume of tissue"—within a mouse kidney, for example, or a human tumor biopsy—"we have to slice the tissue very thin, separately image each slice with a microscope, and put all of the images back together with a computer. It's a very time-consuming process and it is error prone, especially if you look to map long axons or sparse cell populations such as stem cells or tumor cells," she says.

The researchers came up with a way to circumvent this long process by making an organism's entire body clear, so that it can be peered through—in 3-D—using standard optical methods such as confocal microscopy.

The new approach builds off a technique known as CLARITY that was previously developed by Gradinaru and her collaborators to create a transparent whole-brain specimen. With the CLARITY method, a rodent brain is infused with a solution of lipid-dissolving detergents and hydrogel—a water-based polymer gel that provides structural support—thus "clearing" the tissue but leaving its three-dimensional architecture intact for study.

The refined technique optimizes the CLARITY concept so that it can be used to clear other organs besides the brain, and even whole organisms. By making clever use of an organism's own network of blood vessels, Gradinaru and her colleagues—including scientific researcher Bin Yang and postdoctoral scholar Jennifer Treweek, coauthors on the paper—can quickly deliver the lipid-dissolving hydrogel and chemical solution throughout the body.

Gradinaru and her colleagues have dubbed this new technique PARS, or perfusion-assisted agent release in situ.

Once an organ or whole body has been made transparent, standard microscopy techniques can be used to easily look through a thick mass of tissue to view single cells that are genetically marked with fluorescent proteins. Even without such genetically introduced fluorescent proteins, however, the PARS technique can be used to deliver stains and dyes to individual cell types of interest. When whole-body clearing is not necessary the method works just as well on individual organs by using a technique called PACT, short for passive clarity technique.

To find out if stripping the lipids from cells also removes other potential molecules of interest—such as proteins, DNA, and RNA—Gradinaru and her team collaborated with Long Cai, an assistant professor of chemistry at Caltech, and his lab. The two groups found that strands of RNA are indeed still present and can be detected with single-molecule resolution in the cells of the transparent organisms.

The Cell paper focuses on the use of PACT and PARS as research tools for studying disease and development in research organisms. However, Gradinaru and her UCLA collaborator Rajan Kulkarni, have already found a diagnostic medical application for the methods. Using the techniques on a biopsy from a human skin tumor, the researchers were able to view the distribution of individual tumor cells within a tissue mass. In the future, Gradinaru says, the methods could be used in the clinic for the rapid detection of cancer cells in biopsy samples.

The ability to make an entire organism transparent while retaining its structural and genetic integrity has broad-ranging applications, Gradinaru says. For example, the neurons of the peripheral nervous system could be mapped throughout a whole body, as could the distribution of viruses, such as HIV, in an animal model.

Gradinaru also leads Caltech's Beckman Institute BIONIC center for optogenetics and tissue clearing and plans to offer training sessions to researchers interested in learning how to use PACT and PARS in their own labs.

"I think these new techniques are very practical for many fields in biology," she says. "When you can just look through an organism for the exact cells or fine axons you want to see—without slicing and realigning individual sections—it frees up the time of the researcher. That means there is more time to the answer big questions, rather than spending time on menial jobs."

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Friday, October 10, 2014
Center for Student Services 360 (Workshop Space) – Center for Student Services

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Wednesday, January 7, 2015
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Head TA Network

Thursday, September 25, 2014

Head TA Network

Wednesday, November 5, 2014
Center for Student Services 360 (Workshop Space) – Center for Student Services

HALF TIME: A Mid-Quarter Meetup for TAs

Thursday, April 9, 2015
Center for Student Services 360 (Workshop Space) – Center for Student Services

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