Abstracts

This abstract has been invited to present during the Basic Science Poster Highlights poster session

Rationale: A significant challenge to studying chronic seizures is due to their inherent nature of occurring spontaneously. This necessitates a way to identify cells specifically active during seizures, which typically occur within a relatively short temporal window, often on a time-scale of seconds to minutes. Previous methods for identifying active cells relied on either light-based assays that used application of damaging UV light to trigger labeling or the expression of immediate early genes and other activity-dependent regulators within a time window of several hours. Here, we have utilized a newly developed light and calcium-gated transcription factor, which operates on the scale of a few minutes, in conjunction with a closed-loop system for seizure detection and light delivery in order to identify and manipulate seizure active neurons.

Methods: Adult male C57BL/6 mice received unilateral intrahippocampal kainic acid (IHKA) injection to induce epilepsy. At least 3 weeks later, hippocampal injection of AAV vectors encoding the light and calcium gated transcription factor (TF) and a TF-gated reporter of either mCherry, halorhodopsin (eNpHR3.0), or bReaChes was performed. Mice were then implanted with optical fibers and EEG electrodes and monitored for the occurrence of chronic spontaneous seizures. One week after viral injection, a single bout of pulsed (2s on, 4s off) blue light was delivered in response to seizure detection to induce TF release. For intervention experiments, red light was delivered in response to subsequent seizure detection.

Results: We were able to label seizure active neurons with just a single 10-minute pulsed delivery of blue light in response to a detected seizure. We found that select hippocampal neuronal ensembles were reliably labeled, including CA2 associated neurons. Using our gated TF to drive expression of the inhibitory opsin eNpHR3.0, we found that inhibiting these seizure active neuronal ensembles was able to shorten the duration of subsequent detected seizures. We further developed our gated TF to have Cre-dependent expression. Utilizing this tool in transgenic mice allowed us access to a genetically restricted subset of these neuronal ensembles.

Conclusions: Our data show that our newly developed light and calcium gated transcription factor can be used to allow targeted control of functionally relevant seizure active neurons. The development of a Cre-dependent version of our transcription factor has allowed us unprecedented access to functional subsets of genetically defined seizure active neurons. We continue to explore the mechanism of how these ensembles drive seizure development and how altering their activity can pave the way for more targeted seizure therapy.

Funding: NIH F32NS106764, K99NS121399 (Q.A.N.), R01MH119353 (A.Y.T), R01NS094668 (I.S.); EMBO long-term postdoctoral fellowship (M.I.S); Chan Zuckerberg Biohub (A.Y.T); Beckman Technology Development Seed Grant (A.Y.T).