Tadpoles don’t behave, but zebra fish do, and that’s why newly promoted Associate Professor Florian Engert is switching the focus of his neuroscience experiments to fish. In the “wild,” the small, silvery zebra fish avoid large, dark looming objects that might be predators and they hunt tiny moving objects that might be food. They can also learn to avoid completely neutral visual cues, such as a geometrical pattern on the bottom of their tank, should Engert surprise them with a mild electric shock when they swim over it.
The point of this fish obedience school is to link behavior to learning, and learning to synaptic plasticity, which refers to the changes in the structural connections among neurons. It’s generally believed that strengthening those connections is the underlying basis of learning and memory, and that a “lesson”—whether associating a shock with the color red or learning the ABCs—causes the neurons to fire in such a way that strengthens synapses.
Researchers study the synaptic strength by measuring the electrical impulses that one neuron receives when another one is stimulated. Typically, they use microelectrodes inserted directly into neurons to record those impulses. But inserting the electrodes inside the neuron is technically difficult in intact and awake animals. And stimuli delivered through these electrodes do not necessarily simulate the physiologically relevant neuronal activity.
Just as the big push in neuroscience is to study the human brain noninvasively with fMRI brain scans, Engert also wants to spare his fish the invasive electrode in the brain. To that end, he has developed light-based technologies that he credits to his early background in physics as an undergraduate at the Ludwig-Maximilians University of Munich, Germany.
“The big questions in modern biology can often be narrowed down to the underlying principles of physics,” Engert explains. “In many cases, we’re no longer going out into the field to observe organisms. We’re creating concise models of the systems we want to understand, and we probe these models with specifically designed experiments. That requires the quantitative and modeling skills of physics.”
Physics Shines the Light on Neurons
Engert began applying the principles and skill of physics while investigating the role of synaptic plasticity in memory during his PhD studies with Tobias Bonhoeffer at the Max Plank Institute in Munich. Using 1/2-millimeter-thick slices of the rat’s hippocampus, the brain region where new memories form, he used electrodes to measure the connections between two individual neurons. “We could record from one and stimulate another, and then do experiments to study the changes in their synaptic strength,” he recalls.
Engert’s early claim to fame was to supplement electrodes with a light-based method for recording neurons, two-photon laser scanning microscopy (TPLSM), which he developed during his postdoctoral studies with Bonhoeffer.
“We could look at many intact cells at the same time, deep in the tissue, and see what’s changing over time, like in a movie,” Engert explains. “We could also see morphological changes, such as how, after stimulation, a new spine would grow on a neuron’s dendrite. It was the birth of a new synapse, most likely.”
The next challenge was to apply this optophysiological technique to a living organism, which Engert undertook during a second postdoctoral project with Mu-Ming Poo, then at the University of California–San Diego and soon at UC–Berkeley. Poo had pioneered the study of synaptic plasticity in the visual system of living tadpoles—using electrodes. The visual system has long been a beloved model for studying synaptic development in other organisms, including mammals, since vision is such an important means for learning about the world in many animals and because visual signals are easy to control. In the tadpole, the retinal ganglion cells are just one synapse away from the optic tectum, which is one of the major visual processing regions in this animal.
For Engert, tadpoles were a great system because their basic neuronal setup is very similar to mammalian in vivo systems, but they are as easy to handle for recording and stimulation as the rat brain slices. “The big advantage is that you can study the intact neural network and you can use physiological stimulation—light—instead of electrodes to stimulate your network,” Engert says. After two and half years, he successfully refined the laser scanning microscopy technique for studying synaptic plasticity in the tadpole’s visual system.
That accomplished, Engert went looking for another challenge—and he does love a challenge. When he skis, he prefers to hike up to untracked peaks and only takes lifts when necessary. Engert took up surfing in San Diego—with the difficult short board. And he has climbed the 7,300-meter (23,950-foot!) Mt. Barun Tse in the Himalayas.
His next career Himalaya? “I wanted to move from looking at synaptic plasticity to the visual process itself: how visual information is processed and how we store visual images in the brain,” Engert says.
Training Zebra Fish
To make that leap, Engert needed to make the move from tadpoles to an animal that could form visual memories—zebra fish. When the Department of Molecular and Cellular Biology “made me an offer I couldn’t refuse,” as Engert jokes, he brought his surfboard, tennis and squash rackets, and backcountry skis to Harvard Square in early 2002 to begin the winter semester. He flew back to California in the spring so he could ride his Honda Shadow motorcycle from Berkeley to Cambridge.
Already, Engert has accomplished his first aim at MCB, establishing in vivo electrophysiology and two-photon microscopy in the zebra fish’s central nervous system. He has also established the zebra fish obedience school. One of his pupils swims in a small, glass-bottom tank placed on top of an upturned computer screen, which displays squares of red and white. An infrared camera constantly monitors the fish’s position. When it swims over a red square, a mild electric shock pulses on. A quick study in aversive training, the fish learns within a minute or two to avoid the red and seek the safety of white.
“It’s harder to do positive reinforcement,” Engert explains. “What do they really like? We haven’t figured that out.” But he envisions short-circuiting that problem—again using a physicist’s skill with light, along with a little molecular biology. He is working with Professor Alexander Schier, MCB’s resident expert in zebra fish genetics, to develop transgenic fish that will express channelrhodopsin-2, a light-sensitive algae protein, in just the neurons that produce dopamine. Dopamine is the “reward” neurotransmitter, the one that gets released from neurons when we experience something pleasant, be it chocolate ice cream, sex, or cocaine.
When they succeed in creating fish with light-activated dopamine neurons, Engert can watch them swimming around and flash light when the fish see a visual cue he wants to positively reinforce. In that way, he could study reward learning—another type of learning most likely analogous to the way we form the bulk of our memories.
“My long term goal is to use light instead of electrodes to stimulate the neurons, and to use laser microscopy to record from them,” Engert says. “Then we can use this optophysiology to do functional imaging and behavioral assays. We can ask: What is the underlying neuronal activity that induces fish to respond to certain visual stimuli?” He continues: “I hope to find out how visual information is processed in the brain. What changes in the brain when animals learn something and how is behavior different?” Eventually, Engert will study “learning disabilities” in zebra fish, screening for mutants with visually impaired behavior—or developing them through genetic manipulation.
Since his arrival at Harvard, Engert has traded his Honda motorcycle for a BMW 1200 CL motorcycle. “I only get around by motorcycle or rollerblading,” he says—except when he rents a car to take the three postdocs, six graduate students in his lab, and an assortment of MCB neuroscientists up to Sunday River, Maine, for the lab’s winter retreat or to Sutton Island in Acadia National Park for their summer one.
In his four and a half years at MCB, Engert garners the highest rating in the Cue Guide’s student evaluations of faculty. He has found MCB to be “wonderful, supportive, and inspiring.” His colleagues have become good friends, and he praises the high-quality students and postdocs. The admiration is mutual. “Florian is a gregarious and creative colleague who has the courage to develop a new system to address fundamental questions in neurobiology,” says Alexander Schier, one of his many friends and collaborators here.