Neurons perform their assigned duties by firing or turning silent in response to chemical neurotransmitters, depending on what types of ion channels they contain. Neuroscientists can study how isolated neurons work in a lab dish. However, to connect neural circuitry to complex behaviors, Sternson wanted to probe the neuronal wiring in living mice. To do that, he needed a way to “re-ticket” individual ion channels within a neuron or small group of neurons by forcing them to respond to a unique, synthetic neurotransmitter—one not normally seen in nature.

Sternson began by collaborating with Looger, a protein chemist, to exploit the modular structure of ligand-gated ion channels. In these channels, the ion pore domain (IPD) is tethered to an independently functioning ligand-binding domain (LBD). Scientists had previously engineered “chimeric” ion channels by genetically splicing the LBD from one type of channel to the IPD from another. Such hybrid channels transport ions specified by the IPD but in response to the neurotransmitter recognized by the grafted LBD.

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To create an ion channel that could respond to a novel neurotransmitter, Sternson and Looger needed to design a new LBD and synthesize a neurotransmitter that could bind and activate it—two tall orders, Sternson says. “The thinking was that it would be very difficult. It was an uncertain challenge for us whether or not we could even modify these complex ligand-binding domains.” Nonetheless, they persevered.

Starting with the LBD that recognizes acetylcholine, the team applied a combination of protein structural chemistry and molecular modeling to predict which parts of the LBD were most likely to contact this natural neurotransmitter. They then created dozens of channels, each containing a mutation at one or more of the 19 positions on the LBD predicted to be important for acetylcholine binding.

Next, they designed a compound that would unlock their newfangled ion channels. Starting with a chemical analog of acetylcholine, the researchers synthesized a collection of 71 slightly modified versions of the compound. When they tested each one for its ability to selectively activate or silence each of the hybrid ion channels, they found many combinations that worked.

The ultimate test of the artificial ion channel system, the researchers knew, would be trying it out in a living organism. So Sternson inserted a neuron-silencing version of one of his designer ion channels into specific hypothalamus neurons, called AGRP neurons, in mice. His team had previously tailored these neurons to stimulate voracious appetites in the mice. When Sternson injected the animals with the appropriate synthetic neurotransmitter, their overindulging habits subsided dramatically.

The effects of AGRP neuron stimulation on the appetite of a mouse. Video is at 5x speed.

With their toolbox of new ion channels and neurotransmitters in hand, the Janelia Farm researchers intend to examine the role of specific neurons in other complex behaviors. “To me, that’s a real frontier for neuroscience and will ultimately allow us to demystify some of the processes that underlie why we do what we do,” Sternson says.

Video: Yeka Aponte

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