Home Abstract Introduction
Construction of glutamate sensors Initial testing of sensors in vitro Optimization of sensor characteristics Expression of sensor in living cells Glutamate measurement in dendrites Glutamate measurement in response to frequency of action potentials
Glutamate function Sensor design
Protein purification through His tags Overexpression of protein using pRSETB Directing proteins to a membrane by signal peptides Transmembrane regions of proteins Expression of proteins on the surface of mammalian cells through pDISPLAY

Journal of Comprehensible Results

Hires SA, Zhu Y, Tsien R (2008)
Optical measurement of synaptic glutamate spillover and reuptake
by linker optimized glutamate-sensitive fluorescent reporters
Proc Natl Acad Sci USA 105:4411-4416

(Translated by Jeff Elhai)

Experiment: Initial testing of sensors in vitro

Purified soluble glutamate sensor was tested for its ability to detect glutamate by irradiating it with violet light to excite ECFPenhanced cyan fluorescent protein and measuring fluoresence emission (Fig. 5). In the presence of glutamate, the emission of blue-green light (476 nm) was increased and the emission of yellow-green light (526 nm) was decreased. This was because less blue-green light was captured by Citrinea type of yellow fluorescent protein and so less yellow-green light was emitted by that protein.

The difference can be expressed as a ratio (called CFP/YFP) of the fluorescence at 476 nm (the maximum emission of ECFPenhanced cyan fluorescent protein) to the fluorescence at 526 nm (the maximum emission of Citrinea type of yellow fluorescent protein). When glutamate is absent, this value is:

CFP/YFP [- glutamate] = 1.08/1.38 = 0.78
CFP/YFP [+ glutamate] = 1.12/1.31 = 0.85
The addition of glutamate therefore increases the CFP/YFP ratio by 0.07/0.78 = 9%. This isn't that much, but it's a start. The authors sought to increase the sensitivity of the sensor.
Fig. 5: Glutamate-dependent fluorescence from soluble glutamate sensor. Fluorescence emitted from the soluble glutamate sensor in vitro in response to violet light (excitation at 420 nm).
 
In order to determine the level of glutamate to which the sensor responds, the protein was tested at a wide range of glutamate concentrations (Fig. 6). There is no change in fluorescence, measured as the CFP/YFP ratio (described above), when the concentration of glutamate is below 10 nM, nor is there a change when the concentration exceeds 1 uM. The concentration of greatest sensitivity (steepest change in response) is about 150 nM.

Unfortunately, this concentration is likely below what is physiologically relevant in brain. The authors sought to increase the concentration of glutamate at which the sensor was maximally responsive.

Fig. 6: Response to different levels of glutamate by soluble sensor. Ratio of fluorescent emission at 476 nm (blue-green light) to emission at 526 nm (yellow-green light).