Abstracts

Rationale: The mechanism of how seizures impair consciousness has yet to be elucidated. Recent evidence suggests that ictal suppression of subcortical cholinergic arousal circuits, such as the nucleus basalis (NB) and pedunculopontine tegmental nucleus (PPTg), may play a role. Functional imaging and extracellular recordings show suppression of neuronal in the NB and PPTg during seizures, but the synaptic mechanisms of this depressed firing rate are not known. This investigation aims to determine whether reduced neuronal firing in the subcortical cholinergic arousal system during seizures is associated with reduced excitatory input or increased inhibitory input to cholinergic neurons. Whole cell recordings are commonly used to access cortical neurons in vivo, but have not been previously reported from the PPTg. Methods: In this study, whole-cell recordings were obtained in neurons of the PPTg, accessed 5.4 – 6.2 mm deep from the cortical surface, in head-fixed, anesthetized rats during focal limbic seizures. Micropipettes were fabricated with a ~10-mm taper and a resistance of 4-6 MΩ. To allow the brain to form a narrow, low-resistance canal through which subsequent pipettes may pass, the first pipette in each recording session was lowered to the target region and left in place for 30-60 minutes. Positive pressure was maintained at 500 mbar in the pipette throughout descent, and dropped to < 30 mbar at the target region. All recordings used a K-gluconate-based internal solution. Seizures were triggered with a 2 second, 60Hz stimulation of the hippocampus through a twisted bipolar electrode, with current titrated to seizure threshold. Continuous recordings were made using Spike2 and digitized using a Micro1401 (CED). Whole cell recordings in current clamp mode were obtained during seizures. Results: A subset of neurons (n=5), histologically identified as cholinergic, showed hyperpolarization of membrane potential associated with decreased action potential firing. In addition, moment-to-moment input-resistance at baseline and during seizures was measured using 5-10Hz, low amplitude hyperpolarizing square current steps. Input resistance showed an increase during seizures concomitant with hyperpolarization (n=3). Non-cholinergic neurons (n=11) in the same region did not show these consistent changes during seizures. Conclusions: These results are consistent with a mechanism of decreased firing through reduced excitatory input. The identification of the synaptic mechanisms of depressed subcortical arousal during seizures may lead to new treatments targeting these changes to improve ictal and postictal cognition. Funding: Howard Hughes Medical InstituteCitizens United for Research in Epilepsy