Scientists researching the many mechanisms behind learning, memory and other activities of the brain have a new tool that promises to enable tracking of some of the most basic processes. It eventually could help unravel some of the mysteries of thought.A group at the University of California, San Diego, in La Jolla, developed a new genetically expressed glutamate-sensitive fluorescent reporter. They dubbed it SuperGluSnFR because it’s an optimized version of the group’s original GluSnFR glutamate-sensing fluorescent reporter. The new version exhibits a 6.2-fold increase in response magnitude over its predecessor. It could help researchers make direct, quantitative, spatially and temporallyresolved measurements of glutamate release, which should help settle the debate on how synaptic connections pass information on to their neighbors, said team member Andrew Hires.Researchers performed a linker truncation screen when improving their glutamate-sensitive fluorescent reporter. Each axis is the number of residues removed from termini of glutamate binding domain. Color indicates the percentage of maximal ratio change relative to SuperGluSnFR. Black indicates improper construct folding. AAs = amino acid. Images reprinted with permission from PNAS.Hires was a neuroscience graduate student working with Roger Y. Tsien at the time the reporter was developed. He is now doing postdoctoral work at the Ashburn, Va.-based Janelia Farm Research Campus of Howard Hughes Medical Institute.Glutamate is the primary neurotransmitter excreted by neurons as they communicate. Consequently, tracking the substance’s concentration as it varies in space and time could provide important clues about the working of neurons. The problem has been that available methods and techniques provide some information but lack the necessary spatial and temporal resolution.In response to that, the team developed genetically encoded sensors of glutamate concentration based on Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins that bracket a bacterial glutamate-binding protein. The GluSnFR sensor it developed, with an enhanced cyan fluorescent protein and a Citrine yellow fluorescent protein, had a weak response and, thus, a poor signal-to-noise ratio. Additionally, its glutamate affinity had to be improved, which the group set out to do.According to Hires, the process took several years, with two distinct phases. First the researchers performed a few changes using a piecemeal optimization process that involved tinkering with the cyan and yellow fluorescent proteins, with the binding pocket that attaches to the glutamate or with the linkers that tie the proteins to the rest of the molecule.This exploration continued in a methodical way until the researchers understood the impact of each change on the reporter’s response to glutamate. They then performed a comprehensive linker screen to increase the dynamic range of the reporter, a process that took a couple of months. The result of the second phase was SuperGluSnFR, a much more responsive fluorescent reporter that had a stronger affinity for the target, with a 44 percent change in emission ratio upon glutamate binding when expressed on the extracellular surface of neurons.Further optimizations are possible and might even be necessary — particularly with regard to response — for in vivo imaging use. On the other hand, Hires noted, “Perfect is the enemy of good enough. SuperGluSnFR’s signal-to-noise ratio was good enough to make clean, quantitative measurements of synaptic glutamate release in neuronal culture.”Spatially resolved glutamate-mediated FRET changes on the surface of a dendrite are shown. Color indicates percent change in FRET ratio before (left), during (middle) and 1 s after (right) a 10-action potential, 30-Hz field stimulus in Ringer’s solution (upper) and with 100 μM of the glutamate transporter blocker TBOA (lower).The researchers used the new reporter to detect glutamate transmission on the surface of cultured dissociated hippocampal neurons. They used a mercury arc lamp with appropriate filters for an excitation source, carefully limiting the light to minimize exposure and bleaching during the experiment. To capture the data and characterize the fluorescent reporter, they employed a Hamamatsu CCD camera and a Roper high-speed camera, along with imaging optics and the right filters. Because the reporter is a ratiometric indicator, they had to average the cyan and the yellow fluorescent proteins’ emission intensities, subtract the background and ratio the emissions pixel by pixel in regions of interest when examining the neuronal response.As detailed in the March 18 issue of PNAS, they followed the time course of synaptic glutamate release, spillover and reuptake with a temporal resolution in the milliseconds. That response time was fast enough to follow the waveform of the spillover glutamate decay. They found that, during burst firing, functionally significant spillover persisted for hundreds of milliseconds.The fluorescent response to neural stimulation was expected, given the tuning of the affinity of the sensor to glutamate. However, the researchers were surprised by how long the glutamate, once freed, remained so. It was thought that diffusion or uptake by astrocytes, star-shaped subtypes of glial cells in the brain, would have done a better job absorbing the spilled glutamate, Hires said.Because SuperGluSnFR is genetically expressed, it allows cell-specific or even subcellular targeting. A single fluorescent protein version of the reporter also could be developed for two-photon imaging or for simultaneous use of multiple optical sensors.Hires said that research into applying the reporter will develop in different directions, driven by the interests of the investigators. “Potential applications range from the study of biophysical properties of synaptic transmission, mapping the functional connectivity of brain circuits, how activity impacts development of sensory organs like the retina, and the central role that glutamate plays in neuronal injury during a stroke.”