Abstracts

Rationale: Neurovascular coupling based technologies (e.g., intrinsic optical imaging, fMRI and SPECT) are often used to estimate the spatial distribution of epileptic activity in clinical practice. However, the mechanisms of neurovascular coupling during pathological circumstances such epilepsy are largely unknown. Previous data were mainly obtained on anesthetized animals little data exist in awake models. We investigated the spatiotemporal dynamics of epileptic neurovascular coupling using calcium imaging to generate spatial maps of neuronal activity and intrinsic optical spectroscopy (IOS) to measure oxy-hemoglobin (Hbo), deoxy-hemoglobin (Hbr) and total hemoglobin (Hbt), in vivo during interictal spikes (IIS) in awake mouse neocortex to examine their spatiotemporal correlations. Methods: Seven transgenic mice expressing GCaMP6f in subset of excitatory neurons (Jackson Lab, #024276) were employed in the study. A 5x7mm craniotomy window was created over both hemisphere and the window was sealed with a clear silicone-based polydimethylsiloxane (PDMS) film for long term imaging. Three weeks after the surgery, the mice will be fixed on a clamp for imaging. 5mM bicuculline (0.5ul) was injected in the neocortex to induce interictal spikes. We used simultaneous calcium (illumination at 470 nm) and intrinsic signal at 530 nm and 610 nm imaging to record the neuronal and hemodynamic changes.  Results: The IIS showed a monophasic increase in calcium concentration peaking at 98+-24 ms with an amplitude of 159+-60% dF/F. Hbr and Hbt also showed monophasic waveform but profound delay, with the peak times of 1076+-156 ms (Hbt) and 1364+-169 ms (Hbo) and amplitudes of 1.24+-0.62 µM (Hbt) and 1.53+-0.82 µM (Hbo). The Hbr, on the contrary, showed a bi-phasic waveform, with an early increase (initial dip) peaking at 414+-114 ms, with an amplitude of -0.77+-0.47 µM, and a late decrease (overshoot) peaking at 1963+-154 ms, with an amplitude of 0.65+-0.24 µM. Interestingly, we found that the amplitude of Hbr and Hbt during individual IIS was linearly correlated with the interval before IIS (F test, p<0.01). The maximal calcium area detected with Chen-Bee method (20% of maximal amplitude) was 6.10+-2.25 mm2. The maximal hemodynamic signals showed bi-lateral distribution with the area of 11.92+-3.71 mm2, 14.95+-5.74 mm2and 22.60+-6.56 mm2 for Hbt, Hbo and Hbr dip, respectively. Although hemodynamic signals overestimated the neuronal activity, a high correlation could be found at specific time points in the evolution and dissolution of the hemodynamic signals.  Conclusions: Our data suggest that the maximal hemodynamic changes overestimate the neuronal activity during IIS in awake mice. However, hemodynamic signals can better define the spatial extent of excitatory IIS activity at specific time points in their evolution.  Funding: No funding