Abstracts

Rationale: Seizure perpetuation in epilepsy might originate from long term neuroinflammatory responses associated with lesions. It has been demonstrated that astrocytes are among the critical cell types mediating the pathway between microglial immunological responses and gene transcription in neurons. Astrocytic calcium activity is believed to be the regulatory mechanism underlying such multicellular interactions. Optogenetics, a modern technique in neuroscience, has recently been expanded from the conventional method of controlling excitable cells with light to control electrically non-excitable cells, e.g. astrocytes. However, the mechanisms by which light-activated channelrhodopsins affect the behavior of such non-excitable cells have not been clearly identified or quantified. Also, a methodology to control calcium signaling in astrocytes and their implications in the neuroinflammatory environment is required. The goal of this study is to develop a technique to quantify and validate calcium signaling in astrocytes using light, to elucidate their role in the case of epilepsy. We hypothesize that reducing abnormal calcium signaling in astrocytes reacting to the lesion may help delay the non-desirable transcription of genes in neurons. This neuronal environment might be prompted to hyperexcitability, otherwise. Methods: We have established a protocol to stimulate astrocytes with light in vitro, to find the ideal light stimulation parameters to achieve calcium signals in transfected astrocytes. A biophysical model (Stefanescu et al., 2012) was employed to quantify the spontaneous calcium oscillations in astrocytes. To validate the expression of the optogenetic virus which confers light sensitivity to astrocytes, in vivo, a serotype evaluation of the construct was performed.The viral construct of interest, to target astrocytes in our experiments is AAV-GFAP-hChR2 (H134R)-mCherry. The plausible serotypes for the study were narrowed down to serotypes 1, 5 and 8, based on previous studies in the spinal cord and the rat brain targeting neurons. The validation of viral expression has been performed by post mortem histological analysis. Results: We were able to evoke and quantify Ca2+ signaling in cultured astrocytes and found that Ca2+ signaling is highly dependent on light stimulation parameters. As predicted by our biophysical model, low percentage of sustained light was not enough to reach the calcium basal level needed for firing Ca2+ spikes, whereas high percentage brought Ca2+ baseline to a level that blocked Ca2+ activity.From our preliminary data in vivo, serotype 8 of the virus shows promising transduction patterns in astrocytes in the cerebral cortex, in terms of the highest transverse and radial spread. Conclusions: We concluded that in order to induce sustained excitability in astrocytes, the overall proportion of sustained light (d/D %) should be kept within the range of 5-25%. These light stimulation parameters are crucial, as it can adversely affect cellular activity by different mechanisms (e.g. phototoxicity, IP3R saturation, ER Ca2+ depletion), if subjected to high frequencies/ time periods of stimulation. The in vivo evaluation of the serotypes revealed that serotype 8 shows better transduction patterns in astrocytes in the rat brain cortex.This evaluation would help us understand the expression of the gene conferring light sensitivity to the astrocytes, and thereby allowing us to control them using light. The optogenetic methodology will be employed to delve into the response of astrocytes to light stimulation allowing the study of neuroinflammatory and functional changes associated with glioreactivity. Funding: N/A