Abstracts

Rationale: Currently available anti-seizure therapies broadly affect activity in the brain by altering ion channels/neurotransmission (drugs), metabolic processes (diet), or regional activity (electrical or regional optogenetic stimulation). These treatments often produce incomplete seizure control and, due to their lack of specificity, may result in undesirable neurological side effects. In this work we are determining the minimum intervention that is sufficient to prevent ictogenesis, using targeted optogenetic activation of individual neurons. Methods: Organotypic hippocampal slice cultures, which generate spontaneous periodic seizures after approximately one week in culture, were prepared from animals transduced with AAV vectors coding for the light-sensitive, high-current channelrhodopsin CoChR and the red-shifted calcium indicator jRCaMP1a. When imaging relatively thin samples with sparse expression, it is possible to resolve activity in individual neurons using standard single photon widefield fluorescence microscopy with a low-magnification objective lens, whose field-of-view spans the entire slice culture. Furthermore, we incorporated a digital micromirror device (DMD) in the conjugate image plane of the blue excitation light pathway, which enabled projection of high resolution patterns onto the slice. By co-registering the camera and DMD, we could target any subset of individually imaged neurons for optical stimulation. Results: While optically monitoring seizure activity using the jRCaMP1a signal, we disrupted activity by periodically stimulating neurons in one of three hippocampal subregions: s. radiatum, s. pyramidale, or s. oriens. For each region 360 selected cells were divided into 6 groups of 60 cells. The 60-cell patterns were cycled through at 6 Hz, resulting in 1Hz stimulation per targeted cell. Preliminary data show that stimulation of cells in s.p. reliably prevents seizures for > 30 minutes in slices that were undergoing 60 second seizures every 3 minutes at baseline. Immediately after turning off the stimulation light, slices returned to their baseline seizure pattern. Stimulation of s.o. and s.r. were less effective, preventing seizures for a maximum of 15 minutes before seizures emerged despite ongoing optical stimulation.  Conclusions: In these experiments, chronic patterned stimulation, predominantly of pyramidal neurons, prevents ictogenesis. We hypothesize that depletion of glutamate from stimulated neurons decreases the likelihood that the network will enter a state of seizure. In ongoing experiments, we are characterizing the impact of different stimulation strategies on spontaneous network activity, and exploring the vast stimulation parameter space to identify the smallest population of neurons whose manipulation prevents seizures, with minimal disruption of baseline activity.  Funding: NIH R01 NS034700