Abstracts

Rationale: A common animal model of temporal lobe epilepsy employs the pro-convulsant muscarinic acetylcholine receptor (mAChR) agonist pilocarpine, yet the mechanisms of pilocarpine-induced epileptogenesis are poorly understood. Knockout mice that lack M1 mAChRs are resistant to pilocarpine-induced seizures (Hamilton et al. 1997). M1 mAChRs can activate both glutamatergic and GABAergic microcircuits, yet it is not clear how pilocarpine activation of M1 mAChRs disrupts excitation/inhibition balance. Gamma oscillations, generated by fast-spiking parvalbumin-positive (PV) inhibitory interneurons, are associated with seizure onset and are susceptible to depolarization block during epileptiform discharges in vitro (Cammarota et al. 2013). Here, we test the hypothesis that M1 mAChR activation of PV interneurons contributes to pilocarpine-induced seizures.Methods: For electrophysiological studies, PV cells were visualized in acute mouse hippocampal slices by stereotaxic injection of a floxed YFP adeno-associated virus (AAV) or by crossing PV-CRE mice with the RosaYFP reporter line (PV-Rosa mice). PV-M1KO mice were generated by crossing PV-CRE and floxed M1 mice. Action potential (AP) frequency was monitored in loose patch mode before and during bath application of mAChR agonists (200 M pilocarpine or 10 M muscarine). In some experiments synaptic blockers (DNQX, APV, and gabazine) were used. For behavioral experiments, mice were injected with atropine (1 mg/kg i.p.) 30 minutes prior to pilocarpine injection (155 mg/kg i.p.). Pilocarpine-induced seizures were recorded for 45 minutes with position tracking software and video cameras. Seizure severity was scored blind in 5 minute intervals on a modified Racine scale (0-6).Results: In PV-Rosa mice, AP frequency increased in CA1 PV cells after 3 min. application of muscarine (from 4.2 2.3 Hz to 16.6 4.7 Hz, p=0.016, n=7) or pilocarpine (from 0.9 0.4 Hz to 15.1 5.0 Hz; p=0.035, n=5). In the presence of synaptic blockers, pilocarpine increased AP frequency within 90 sec (from 3.3 2.8 Hz to 14.2 5.4 Hz, p=0.002, n=9), consistent with a direct postsynaptic effect on M1 mAChRs. However, in the continued presence of pilocarpine, AP firing was not sustained in a subset of PV+ cells (n=6). The mAChR-induced increases in AP frequency were prevented by pretreatment with atropine (n=9) or in PV-M1KO mice (n=7, p>0.05). In behavioral experiments, seizures were less severe in PV-M1KO (3.9 0.5, n=10) than WT (5.4 0.3, n=10, p=0.027) mice at 30 min., which remained significant at 45 min. (p=0.004). Conclusions: Pilocarpine directly excites PV+ cells via M1 mAChR activation but a subset of these cells progress to depolarization block. Consistent with this mechanism, the elimination of M1 mAChRs from PV cells reduces seizure severity. Therefore, we propose that pilocarpine activation of M1 mAChRs on PV cells induces the collapse of PV-mediated GABAergic inhibition, leading to disruption of excitation/inhibition balance and the promotion of epileptogenesis.