Real-time Imaging of the Glutamine-Glutamate Shuttle in Epilepsy

Richard J. Reimer, M.D.

Stanford University School of Medicine

Funded in December, 2006: $100000 for 1 years
LAY SUMMARY . HYPOTHESIS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Testing the Ability of FRET Imaging to Identify Neurotransmission Imbalance in Epilepsy

Researchers will test if their newly developed molecular imaging biosensor, used in combination with electrical measurements, can identify whether excess amounts of the excitatory neurotransmitter glutamate build up in brain tissue in an animal model of epilepsy.  

Epileptic seizures involve the excessive excitability of neurons, and while the excitatory neurotransmitter glutamate is probably involved, scientists do not know how glutamate triggers seizures. A better understanding of this could help the 600,000 people, out of three million in this country, whose epilepsy does not respond to current treatments, and the large number of treated patients who suffer major side-effects. The researchers hypothesize that excess levels of glutamate are produced by a dysfunctional “glutamate-glutamine shuttle.”  Ordinarily, via this shuttle, glutamate is released by neurons to be recycled by glia (supportive tissue that nourishes neurons). The glia break down glutamate into glutamine and release it to neurons, which then convert it back to glutamate. The researchers hypothesize that this process is altered in epilepsy, where excessive glutamate builds up and triggers seizures.

They are developing biological sensors that can be used with the molecular imaging technique FRET (fluorescence resonance energy transfer) to detect levels of glutamate, and particularly levels derived from the shuttle process.  They plan to further develop the FRET biosensors.  Then they will combine the FRET biosensor imaging with measures of electrical signals from glutamate-using neurons in tissue of animals with epilepsy, and determine whether they can obtain direct evidence of alterations in the glutamate-glutamine shuttle.

Significance:  If this technology is shown to be feasible, and eventually demonstrates that epileptic seizures occur from an imbalance in the glutamate-glutamine shuttle, the research will have identified a potential new therapeutic target for controlling epileptic seizures.

HYPOTHESIS

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Hypothesis: 
A primary mechanism underlying the development of epilepsy is increased availability and release of the excitatory neurotransmitter glutamate

Goals:
1. to use recently developed fluorescence-based biosensors to determine if the development of epilepsy is associated with increased glutamate release.
2. to develop and validate a glutamine biosensor to use in parallel with the glutamate biosensor and electrophysiology to determine if availability of glutamine regulates glutamate release in normal and epileptic brain.

Methods:
Incubating brain slices in purified bacterially-produced glutamate biosensor allows the protein to permeate the tissue and leads to a stable fluorescent signal with retention of a glutamate sensitive FRET response. Since the biosensor remains in the extracellular space and retains its sensitivity, we can detect submicromolar concentrations of glutamate released from cells and sample a two-dimensional region of interest in the slice at ~50 Hz, giving resolution of glutamate dynamics in the tens of milliseconds range. We are now combining imaging with electrophysiology and pharmacology in a slice model of epilepsy to determine how glutamate release is altered in this setting.

SELECTED PUBLICATIONS

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Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc Natl Acad Sci. 2005 Jun 14;102(24):8740-5.

Tani H, Bandrowski AE, Parada I, Wynn M, Huguenard JR, Prince DA, Reimer RJ. Modulation of epileptiform activity by glutamine and system A transport in a model of post-traumatic epilepsy. Neurobiol Dis. 2007 Feb;25(2):230-8.