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Electrodes spy serotonin in vivo The mood-regulating neurotransmitter 5-hydroxytryptamine (5-HT), otherwise known as serotonin, has achieved notoriety in recent years as antidepressants that target its level in the brain have become pervasive. Yet, measuring the neurotransmitter in vivo has remained an elusive research goal, until now. In a paper in AC (2009, DOI 10.1021/ac9018846), Mark Wightman and colleagues from the University of North Carolina Chapel Hill used cyclic voltammetry (CV) to make the first measurements of serotonin release in live animals, paving the way for future studies on how the neurotransmitter bridges the gap between environmental stimuli and behavior. “Nobody has ever had an opportunity to look at serotonin on a rapid time scale,” says Wightman. Luckily, “serotonin is easily oxidized, which means you can use electrochemistry to study it.” CV is typically used to detect analytes in solution by measuring current as a function of increasing and then decreasing voltage. This cycle is repeated as the analyte of interest comes and goes, giving a characteristic trace or signature that depends on the analyte’s oxidative tipping point. The neurotransmitter dopamine has been studied extensively in vivo with CV, but serotonin has remained a challenge because of so-called microelectrode fouling. This defilement occurs if too many molecules in the vicinity of the electrode get stuck to its surface, gumming up the works. “It’s been one of those very tricky problems for a number of years. There are a lot of people who want to measure serotonin in the brain the way they measure dopamine,” says Paul Garris of Illinois State University. “The one thing that impressed me about this paper was that I always assumed it was serotonin doing the fouling.” But, as it turns out, serotonin was a red herring. The real culprit was a metabolite of serotonin, 5-hydroxyindole acetic acid (5-HIAA). 9534
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This discovery required a bit of detective work. According to Margaret Rice of the New York University School of Medicine, serotonin had for some time been readily measurable in brain slices, but somehow switching to a live animal always led to fouling. “There has been quite a bit of work in
(Top) A color plot of all the cyclic voltammograms recorded in vivo. The electrical stimulation was delivered at the time indicated by the bar at the top. The white dashed line is the time section shown in the bottom panel. (Bottom) The cyclic voltammogram at the end of the stimulation (solid) is shown superimposed with a 5-HT measurement recorded in vitro (dashed).
vitro in brain slices, but the novelty here is being able to monitor 5-HT in vivo,” says Rice. A major difference between brain slices and live brains is the concentration of extracellular metabolites, such as 5-HIAA. During slice preparation, 5-HIAA is washed away, whereas in vivo this metabolite is abundant; its concentration can be 200⫺1000⫻ that of serotonin. Wightman and colleagues hypothesized that, given its abundance and colocalization with serotonin, 5-HIAA must be the compound causing the fouling. So they turned to the popular surface modifier Nafion.
DECEMBER 1, 2009
“Nafion is chock-full of negative charges and was widely used for measuring dopamine several years ago,” says Garris. “Now, they’ve dusted it off and used it for serotonin. It puts up a fence of negative charges and so repels 5-HIAA, which is also negatively charged. Serotonin is positively charged and so is drawn in.” Using a method described by Rice and colleagues in 1989, the researchers deposited a thin, uniform layer of Nafion onto a microelectrode and verified its placement by scanning electron microscopy. Voltammograms that had been ugly were suddenly transformed and displayed the chemical signature of serotonin. “This Nafion coating allows reproducibly beautiful voltammograms,” says Rice. To verify that the signal they were measuring was really coming from serotonin and not some other metabolite, the researchers made measurements in the presence of a dopamine reuptake inhibitorOleading to no change in signal and ruling out dopamineOand then a serotonin reuptake inhibitorOcausing a greatly increased signal. “That’s pretty convincing,” says Rice. One potential drawback to using the coating is that it slows the response time of the electrodes. And serotonin signaling is rapid. “I’d be very curious to see if this approach has the temporal response to catch these very fast signals,” says Garris. After further characterizing the stimulated release of serotonin and fully understanding what happens to it once released from a neuron, Wightman wants to move on to bigger questions. “The long-term goals are to study a behavior that you would expect serotonin to be involved with.” —Erika Gebel
10.1021/AC9024297 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 11/09/2009
