Abstracts

Rationale: Interictal spikes are a diagnostic feature of EEG recordings from epileptic patients, but their role in ictogenesis and epileptogenesis remains elusive. Understanding the basic circuit and molecular mechanisms underlying interictal spikes will offer new insight into how the epileptic brain can transition to pathological activity but isolating key elements responsible for the generation of network rhythms is technically challenging. In vivo and in vitro recordings suggest that interictal spikes may be generated primarily by synchronous interneuron activity(Michelson and Wong, 1991; Lillis et al., 2012; Muldoon et al., 2015; Bohannon and Hablitz, 2018), while recent modeling studies suggest that this activity may be generated by more widespread network activity(Jacob et al., 2019). Methods: In this work, we imaged the earliest emergence of interictal spikes in an in vitro model of post-traumatic epileptogenesis: the organotypic hippocampal slice culture. Slices were prepared from P7 DLX-cre mice that were transduced intracerebroventricularly on P0 with two AAV vectors: FLEX-tdTomato and syn-GCaMP7f, producing pan-neuronal expression of a green calcium sensor and interneuron-specific expression of a red fluorescent protein. We then used a novel imaging system constructed inside of a tissue culture incubator, to image slices continuously beginning shortly after the injury and continuing through the onset of spontaneous recurrent seizures (after ~7 days in vitro). Every 4 hours during this latent period, a movie of calcium dynamics with cellular resolution and a field of view spanning the entire epileptic network was acquired. For each detected interictal spike, all imaged neurons (typically n~=1000) were quantified by onset time and amplitude of calcium transient (ΔF/F).Next, we sought to test the hypothesis that synchronous interneuron activation is sufficient to trigger interictal spikes. We co-transfected DLX-cre animals with FLEX-CoChR and syn-jRCaMP1a, producing pan-neuronal expression of a red calcium indicator and interneuron-specific expression of a high-current channelrhodopsin variant. We then used a digital micromirror device (DMD) to pattern light to selectively stimulate selected groups of interneurons. Results: Preliminary data suggest that there is a bimodal distribution of onset times and peak ΔF/F amplitude, with the earliest firing and highest amplitude activity occurring in interneurons. Furthermore, preliminary data using selective stimulation with a DMD indicate that the number of interneurons stimulated is directly proportional to the amplitude of the network-wide calcium response. Conclusions: Together, these results suggest that the earliest synchronous activity in post-traumatic epileptogenesis is interneuron-driven interictal spiking. Uncovering the mechanisms of this synchronization and characterizing the emergence of pathological activity from physiological surges of interneuron activity will provide fundamental insight into epileptogenesis and potentially stimulus paradigms capable of disrupting it. Funding: NIH 5R01NS034700, 5R37NS077908, 5R01NS040109, CURE W81XWH-15-2-0069