Fluorescent sensors are important tools that can allow real-time, live, single molecule imaging of microscopic millisecond scale events. It is even better if these sensors are genetically encoded sensors (i.e. fluorescent proteins). We have already encountered the pH sensors pHluorin and pHTomato and the Ca2+ sensor GCaMP. There have been a few others, such as HyPer that detects H2O2 or ArcLight and ElectrikPk which are voltage sensors.
Now, the group of Loren Looger from HHMI Janelia developed a sensor for a very important molecule: L-glutamate. L-glutamate is an essential amino acid, but it is also a very important signaling molecule in most organisms. It is of particular interest due to its pivotal role in neuronal communication. For instance, glutamate released from pre-synaptic vesicles activates post-synaptic glutamate gated ion channels, which causes Ca2+ increase, and action potential propagation. Therefore, detecting local and timely glutamate increase by live imaging in neurons can advance our understanding of neurobiology.
The authors fused a bacterial periplasmic component of a glutamate transporter to a circularly permutated (cp) GFP. In cpGFP, the N & C termini are fused by a flexible linker and new N & C termini are formed in the “middle” of GFP. The sensor peptide (in this case the GltI protein from E. coli) is fused to these new termini. In the absence of glutamate, a domain near the chromophore is presumably distorted, leading to dim fluorescence of the cpGFP. In the presence of bound glutamate the correct structure of cpGFP is restored and brightness increases. The resulting sensor was named iGluSnFr.
The purified protein had no affinity to a panel of other amino acids (except aspartate) or other neurotransmitters, which is very good. They also show it to be insensitive to glutamate receptor agonists and antagonists or to glutamate transporter inhibitor.
I am actually not sure if this is good or not, since this sensor will detect glutamate even at (pharmacological) conditions when the endogenous glutamate receptors will not. I guess it depends on whether we want to measure the amount of glutamate released (which will always be detected by iGluSnFr) or the amount of glutamate that is activating the cell (i.e. glutamate that activates receptors & ion channels).
Another problem with their system is that their sensor is of bacterial origin and not based on endogenous neuronal glutamate receptor. Therefore, the sensor must first be expressed in neurons and targeted to the neuronal membrane. They do that by fusion a neuron specific promoter (synapsin) or glia specific promoter (GFAP) and the protein is fused to a platelet-derived growth factor receptor transmembrane helix – hardly specific to certain membranal regions (e.g. synapse). This protein will actually be all over the cell membrane.
A second problem that I can see is that it may compete with other glutamate receptors/channels for glutamate, particularly when local concentration may be low and limiting. Thus, just having this sensor may affect the behavior of the neuron, or the entire animal. Just recently we learnt that long term expression of bacterial channelrhodopsin 2 (chR2), a very common optogenetic tool, causes neuronal abnormalities. How would iGluSnFr affect the neurons? The authors do not mention any abnormalities, but this does not mean these are absent. Just that the authors didn’t look for them, or didn’t notice them, or didn’t think of mentioning them.
[Note 22.4.2014: see discussion at the article’s page at PubPeer concerning the above issue.]
Having said all that, I think they did a phenomenal work to show that this sensor works in a wide range of animals (worms, fish, mammals), tissues (cell culture, hippocampal slices, retina and in vivo imaging of awake mouse brain) and imaging methods and scenarios (one or two photon, uncaging, two-color with RCaMP, in vivo imaging), and that it shows what is expected – i.e. glutamate peaks just prior to Ca2+ peaks and action potential. Moreover, this sensor is definitely superior to previously developed glutamate sensors which they mention in the text (such as microdialysis, enzymes or dyes).
Their in vivo experiment is particularly cool and you should check it out.
This could be a good tool, at least until a better sensor, based on neuronal glutamate receptors, will be developed.
Marvin JS, Borghuis BG, Tian L, Cichon J, Harnett MT, Akerboom J, Gordus A, Renninger SL, Chen TW, Bargmann CI, Orger MB, Schreiter ER, Demb JB, Gan WB, Hires SA, & Looger LL (2013). An optimized fluorescent probe for visualizing glutamate neurotransmission. Nature methods, 10 (2), 162-70 PMID: 23314171