Making Zebrafish Move Like Icelandic Horses to Explore Motor Control

The transparent larval zebrafish (Danio rerio) is only 4mm long, but for NJIT biologist Kristen Severi, the vertebrate offers an expansive window for exploration into how the brain and nervous system controls behavior.

"Like putting a big puzzle together, we're trying to understand all the circuits in the brain that control locomotion," said Severi. "If the spinal cord is damaged, it can result in paralysis, but we still don't understand all the pieces we'd need to restore full function.

"The same types of neurons that form the circuits for locomotion in humans can be found in zebrafish, and because they only have about 80,000 neurons at the larval stage, it's a much smaller puzzle to study," Severi added. "Zebrafish are see-through, so we can image their brains fully under a microscope to see the neuronal activity involved in controlling the animal's movement while they perform behaviors in real time."

Sharing more 70% of genetic material with humans, zebrafish have been a well-studied model organism since the 1960s. However, Severi's team has put an unusual twist on the fish in her lab and has adopted a novel combination of techniques to study them.

Her lab is using a genetically encoded Botulinum toxin, a form of Botox, to inhibit the synaptic output of certain neuronsin zebrafish. Severi says the non-destructive, gene-silencing technique is being used to create a line of transgenic zebrafish that possess a gait-altering genetic mutation found in Icelandic horses.

"We're basically making Icelandic horses out of zebrafish," said Severi. "We want to learn more about the function of neurons comprising circuits for gait and speed control, and this gene was implicated in exactly that."

Unlike most horses with four gait patterns (walk, trot, canter and gallop), Icelandic horses have two others ("tölt" and "flying pace") due to a mutation in a gene, called DMRT3. The mutation enables lateral gaits, ambling and pace, but inhibits the ability to transition from trotting to galloping.

"Typically, mouse models are used to study how tetrapods transition from walking to running, but fish also use limb-like pectoral fins to change speeds," explained Severi. "They have equivalents to our deltoid and adductors, and those muscles have motor neurons that sit in the spinal cord. The timing of the activation of those motor neurons are controlled by interneurons, like DMRT3, making it a great candidate for study."

The lab is using high-speed cameras capable of capturing up to 1,000 frames per second, as we as an artificial neural network - called DeepLabCut - to capture micro-movements of the animal's tail and fins, and study behavioral changes that occur in the experimental group.

"We can analyze the difference between fish that are 'control' group and fish that have Botox in this group of neurons in the spinal cord to better understand the mechanisms under which this circuit is behaving inappropriately," said Severi. "We are trying to approach one cell type at a time with the zebrafish, where we have amazing genetic and optical tools to dissect these different neuronal classes that humans share.

"Ultimately, we hope our studies will help us understand the role of the different players in the spinal cord that control locomotion."