Yesterday I went to a UPENN neuroscience retreat at The College of Physicians of Philadelphia, which happens to be the same building that houses the Mütter Museum. The highlight was an introductory lecture by Dr. Roger Tsien, inventor of calcium-sensitive dyes and Nobel favorite.
Recently his lab developed two novel techniques for imaging brain activity. First, Tsien discussed the Glutamate Sensitive Fluorescent Reporter (GluSnFR aka “Glue Sniffer”). The probe works by Fluroresence Resonance Energy Transfer (FRET). This technique involves two fluoresecent proteins that are matched so that the emission wavelength of the first is the excitation wavelength of the second. If the molecules are close together and oriented properly, then exciting the first protein (say, one that normally fluoresces blue) will in turn excite the second protein (say, one that normally fluoresces yellow). When the fluorescent proteins are separated, only blue light is emitted because FRET cannot take place. When they are oriented in the right way, only yellow light is emitted because all the energy is transferred via FRET.
GluSnFR works by linking blue and yellow fluorescent proteins with a third protein that changes conformation when it binds glutamate. Normally the two fluorescent proteins are close enough to engage in FRET, so excitation results in only yellow light. But glutamate released from neurons can bind the linker domain in GluSnFR and disrupt FRET, causing only blue light to be emitted. The ratio of blue to yellow light emission can be measured with high spatial resolution, facilitating time-lapse movies of glutamate spillover from synapses. This is a cool new tool for imaging neuronal activity. The graduate student who performed the work has a better explanation than I can provide. Paper here.
Tsien began the second half of his lecture by discussing competing theories for memory storage in the brain. The dominant theory has been that learning involves modulation of synaptic strength. However, Tsien stressed new evidence showing that learning depends on the formation of new synapses. TimeSTAMP is a new way of monitoring synapse formation.
Tsien’s lab engineered animals to express modified versions of the proteins normally expressed at the synapse, such as PSD-95. This version of the protein is linked to a hemagglutinin (HA) tag via another protein. The linker protein is actually a cis-acting protease, meaning that it spontaneously cleaves itself. Normally, the linker cleaves immediately after PSD-95 is translated, thus separating it from the HA tag. But when a protease inhibitor is added, all the newly translated PSD-95 will have the HA tag. After the experiment is over, the brain can be stained with anti-HA antibodies to see only the synapses that formed after the addition of the protease inhibitor. The ultimate goal is to administer the inhibitor before a learning experience and then observe the when and where of synapse formation compared to an animal that did not have that learning experience.
TimeSTAMP isn’t quite as cool as GluSnFR becasue the results are obtained retroactively with immunohistology instead of real time optical imaging. However, this approach is advantageous because it allows you to view the whole brain instead of just the superficial areas that light can penetrate. Another problem is that there may be high turnover of proteins like PSD-95 even at old synapses. The perfect marker will use a synaptic protein that is only translated during synapse formation, although it is unclear whether such a master molecule exists.
Needless to say, Tsien’s research is pretty awesome.