Abstract
Temporal Lobe Epilepsy (TLE) represents the most frequent form of epilepsy in adults and is characterized by hippocampal sclerosis and glial reactivity. The latter triggers an inflammatory response contributing to hippocampal remodeling that leads to spontaneous recurrent seizures. ATP and purinergic signaling are key components of glial reactivity and we already demonstrated that an enhanced purinergic signaling in astrocytes, driven by the release of TNFa, is responsible for the increased excitatory hippocampal synaptic transmission during epileptogenesis (1). The lack of appropriate tools to study ATP has so far limited the understanding of its origin, release mechanisms and spatiotemporal dynamics. To overcome these limitations and to clarify the role of ATP during epilepsy progression we took advantage of a fluorescent ATP biosensor based on the engineering of the P2Y1 receptor (2). Mice were injected with Adeno-Associated-Virus (AAV) to drive the expression of the ATP-P2Y1 biosensor in hippocampal astrocytes. The specific expression of the biosensor in astrocytes was first confirmed by immunohistochemistry. We then tested the proper functioning of the biosensor by combining local field potential and optical recordings on acute hippocampal slices. We observed that the induction of epileptiform activity by bath application of 4-Aminopyridin was accompanied by phasic fluorescent signals in astrocytes that were time locked with neuronal population bursts. These fluorescent signals were specifically related to the biosensor activation since they were drastically reduced by applying the P2Y1 receptor inhibitor MRS 2179 (10 µM). Moreover, blocking neuronal activity by means of TTX application also blocked the fluorescence oscillations in astrocytes. These data indicate that the enhanced epileptiform neuronal activity triggered a concomitant release of ATP nearby the astrocytes. To check for ATP biosensor activity in vivo, mice injected with the same AAV in the hippocampus were implanted with a grin lens placed 150 µm above the AAV injection site and equipped with a miniscope to perform high resolution imaging of fluorescent signal dynamics in freely behaving animals. Three weeks after surgery, we recorded fluorescent basal activity of the biosensor in the infected astrocytes for 1h. Mice were then intraperitoneally injected with Kainate to induce status epilepticus. During epileptic seizures the fluorescent signals in the infected astrocytes were clearly increased, demonstrating also in vivo that neuronal activity was able to trigger a concomitant release of ATP near astrocytes. These results confirm the validity of the biosensor to better investigate extracellular purine dynamics and mechanisms of release in mouse models of TLE.References1. Nikolic L, Shen W, Nobili P, Virenque A, Ulmann L, Audinat E. Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis. Glia. 2018 Dec;66(12):2673-2683. doi: 10.1002/glia.23519. Epub 2018 Nov 5. PMID: 30394583.2. Wu Z, He K, Chen Y, Li H, Pan S, Li B, Liu T, Xi F, Deng F, Wang H, Du J, Jing M, Li Y. A sensitive GRAB sensor for detecting extracellular ATP in vitro and in vivo. Neuron. 2022 Mar 2;110(5):770-782.e5. doi: 10.1016/j.neuron.2021.11.027. Epub 2021 Dec 22. PMID: 34942116.