Abstracts

Rationale: Epileptogenesis describes the complex, plastic changes that alter a normal brain into a hyperexcitable network debilitated by recurrent seizures. The dentate gyrus (DG) and granule cell neurons control excitatory inputs into the hippocampus. Voltage-gated KCNQ2/3 K+ channel (M-channel) current maintains homeostatic control over neuronal resting membrane potential and firing frequency, acting as a "brake" on excitability. M-current is sensitive to suppression by muscarinic acetylcholine receptor activation. Cholinergic input to M1 muscarinic receptors and the dominant function of M-current in neuronal excitability suggest involvement in epileptogenesis pathways. We hypothesized that in DG granule cells, muscarinic receptor activity induces maladaptive changes in which M-current is more susceptible to muscarinic depression, conferring hyperexcitability and recurrent seizures. Methods: We induced epileptogenesis by transgenic expression of muscarinic "Designer Receptors Exclusively Activated By Designer Drug" (DREADD) exclusive to the DG-CA3 circuit using Cre-loxP mice. Mice were administered the synthetic designer drug clozapine-N-oxide (CNO) to elicit remote activation of the muscarinic DREADDs in DG. EEG electrodes were implanted in mice to record electrographic seizure activity. Stimulation of the muscarinic DREADD by CNO delivery was repeated once daily, and seizure behavior was scored. After one week of stimulation, active and passive discharge properties of DG granule cells were characterized with patch-clamp electrophysiology in the brain slice. M-channel currents were also quantified. Results: Selective muscarinic stimulation originating from the DG circuit was sufficient to induce epileptogenic activity in vivo. DREADD-expressing mice displayed focal seizure activity after systemic administration of CNO. CNO potency was decreased in Cre-POMC mice compared with Cre-CamKII expressing mice, and Cre-POMC mice had delayed progression of seizure onset. Repeated kindling of seizures resulted in generalized clonic seizures that persisted for 30-60 seconds. DG granule cells exhibited robust M-current suppression and greater hyperexcitability upon DREADD receptor stimulation. Conclusions: Muscarinic acetylcholine receptor stimulation in the DG-CA3 circuit is sufficient to induce epileptiform excitation that later progressed into network seizures. Chemogenetic spatial and temporal control represents a promising advancement in understanding epileptogenesis over traditional chemoconvulsant models. The interaction of muscarinic receptors and M-channels may strongly contribute to maladaptive, epileptogenic changes in the DG to promote seizure. Funding: Work supported by UTHSCSA School of Medicine Pilot Grant (to MSS) and NIH institutional training grant T32-HL007446 (to CMC).