Abstracts

Rationale: Epilepsy is a neurological disorder characterized by aberrant neuronal excitability. Glutamate is the major excitatory neurotransmitter in the brain and plays an important role in the initiation and spread of epileptic activity. However, little data exists on the utility of in vivo real-time glutamate neurotransmission mapping of epileptic activity and the impact of anesthesia on glutamate concentration and release. Furthermore, glutamate has been hypothesized to play a role in neurovascular coupling.  Methods: We performed in vivo real time imaging of interictal spikes using local field potential, glutamate, blood flow and oxygenation imaging in unanaesthetimzed and anesthetized transgenic mice.  We generated Emx-CaMKII-iGluSnFR transgenic mice to produce expression of iGluSnFR within all excitatory neurons across all layers of the cortex, but not in GABAergic neurons. For the anesthetized experiments, 1.5% isoflurane was used. We induced acute focal interictal discharges in the neocortex by injection of bicuculline methiodide (BMI, 5mM, 0.5μl). Glutamate fluorescence imaging and intrinsic optical imaging including 530nm and 617nm wavelengths were used to measure glutamate transmission and hemodynamic signals. The local field potential was recorded to identify the epileptic discharges.  Results: Single interictal spike induced a strong change in iGlu fluorescence in the area of the interictal spike focus. The maximum amplitudes of fluorescence changes in anesthetized and awake mice were 17.1±5.7% (n=178 spikes, n=3 animals) and 42.7±9.9% (n=284 spikes, n=3 animals; p=0.044) at 0.033±0.010s and 0.027±0.006s (p=0.322), respectively. The intrinsic optical imaging showed that Hbt concentration increase peaks at 0.57±0.13 µm and 1.56±0.45μm (p=0.049) with latencies of 1.95±0.32 s and 1.21±0.02s (p=0.042) in anesthetized and awake mice, respectively, while Hbr concentration decreases by 0.37±0.15 µm and 0.51±0.19 µm (p=0.304) and at 0.37±0.31 s and 1.07±0.63 s (p=0.148).  Conclusions: Our data suggests that genetically encoded glutamate sensor imaging might provide a powerful tool to map neuronal activity in neocortical epilepsy. The glutamate neurotransmission and hemodynamic responses measured in awake mice are substantially different from anesthetized animals. These results may have important implications for the neuroimaging research in awake epileptic humans and animals.  Funding: The Cornell University Ithaca-WCMC seed grant and the Daedalus Fund for Innovation