July 2, 2012
Glowing Molecules Help Scientists Track Serotonin
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John Neumann for redOrbit.com - Your Universe Online
Researchers have succeeded in tracking a single protein that regulates the neurotransmitter serotonin, making it possible to study the dynamics of the protein which regulates mood, appetite and sleep, at an unprecedented level of detail, reports David Salisbury of Vanderbilt University. Attempts to understand how these transporters work have been limited by the difficulty of studying their dynamic behavior.
The capability, which took nearly a decade to achieve, was reported by an interdisciplinary team of Vanderbilt scientists in the June 27 issue of the Journal of Neuroscience.
The fluorescent tags are nanoscale beads called quantum dots made from a mixture of cadmium and selenium. These beads are only slightly bigger than the proteins they are tagging: stringing 10,000 together would only just span the width of a human hair.
“Now we can follow their motion on the surface of cells in real time and see how their movements relate to serotonin uptake activity,” said chemistry graduate student Jerry Chang, who developed the tagging technique. “In the past, we have been limited to snapshots that show the location of transporter molecules at a specific time.”
When illuminated, quantum dots emit colored light and small changes in their size cause them to glow in different colors. Team member Ian D. Tomlinson, assistant research professor of chemistry, developed a special molecular string that attaches to the quantum dot at one end and, on the other end, attaches to a drug derivative that binds exclusively with the serotonin transporter.
When a mixture that contains these quantum dots is incubated with cultured nerve cells, the drug attaches to the transporter. As the protein moves around, it drags the quantum dot behind it like a child holding a balloon on a string. When the area is illuminated, the quantum dots show up in a microscope as colored points of light.
“Until now, neurobiologists have had to rely on extremely low resolution approaches where it takes the signals coming from thousands to millions of molecules to be detected,” said Randy Blakely, the Allan D. Bass Professor of Pharmacology and Psychiatry. “We really had no idea exactly what we were going to see.”
The researchers examined extensions of the nerve cell that are involved in secreting serotonin on the presumption that transporters would be localized there as well. From previous research, the investigators suspected that the transporters would be concentrated in cholesterol-rich parts of these extensions, termed rafts, although the level of resolution with standard approaches was inadequate to provide any clues as to what they were doing there.
The quantum dot studies demonstrated that there were two distinct populations of transporters in these areas: Those that can travel freely around the membrane and those that act as if they are unable to move. They found that the immobile transporters were located in the rafts. When they stimulated the cell to increase transporter activity, they were surprised at what happened. “We found that the transporters in the rafts began to move much faster whereas the motion of the other population didn´t change at all,” Rosenthal reported.
Since the mobilized transporters do not leave the rafts, they appear to whizz around inside a confined compartment, as if released from chains that normally keep them subdued. These observations suggest it is likely that the two populations are controlled by different regulatory pathways.
“By understanding the basic mechanisms that naturally turn serotonin transporter activity up and down, maybe we can develop medications that produce milder side-effects and have even greater efficacy,” Blakely continued. “Our sights are also focused on transferring what we have learned with normal serotonin transporters to an understanding of the hyperactive transporters we have found in kids with autism.”
Other members of the research team include chemistry graduate student Michael Warnement; Ana Carneiro, assistant professor in pharmacology; graduate student Alessandro Ustione; and David Piston, the Louise B. McGavock Professor from molecular physiology and biophysics.
The research was supported by grants from the National Institutes of Health, the Vanderbilt Institute of Nanoscale Science and Engineering and the NIMH Silvio O. Conte Center for Basic Neuroscience Research. Microscopy support was provided by Vanderbilt´s Cell Imaging Shared Resource.