Abstracts

Rationale: Altered inhibition is postulated to contribute to mechanisms underlying epilepsy. This study focused on changes in inhibitory innervation of CA1 pyramidal cell bodies in a mouse model that exhibits spontaneous seizures. Basket cells, which strongly inhibit pyramidal cell bodies, can be delineated into functionally distinct subtypes by their immunoreactivity (ir) for either parvalbumin (PV) or cholecystokinin (CCK). In order to uncover changes that may be overlooked by considering GABAergic innervation as a whole, protein expression profiles of discrete subpopulations of interneurons were investigated. Methods: Young adult C57BL/6 male mice were injected with pilocarpine, a cholinergic agonist (320 mg/kg), to induce status epilepticus (SE). Behavioral seizures were aborted with diazepam after 2 hours. The mice developed spontaneous seizures within 3 weeks. For light microscopy, mice were studied at time points from 9 hours to 2 months after SE for immunohistochemical localization of glutamate decarboxylase (GAD), PV, CCK and cannabinoid receptor type 1 (CB1). For electron microscopy, mice were studied at time points from 9 hours to 7 days after SE; sections were prepared for analysis of degenerating terminals and for peroxidase or gold-enhanced nanogold immunohistochemistry for CCK. Results: Boutons expressing either GAD or PV were not strikingly altered in the CA1 pyramidal cell layer of pilocarpine-treated mice. However, CCK-expressing boutons were dramatically reduced in this region, as were boutons expressing CB1 receptors, which are specific to CCK-ir neurons. Electron microscopy of the CA1 pyramidal cell layer at short intervals following SE revealed dark terminals consistent with electron dense degeneration. Furthermore, pre-embedding immunogold assay established the presence of CCK in some degenerating terminals. In CA1 strata oriens and radiatum, light microscopy demonstrated reduced punctate labeling of CB1 receptors and GAD after SE (using CB1 antibodies both selective and nonselective for inhibitory boutons), but diffuse expression of CCK was increased. Conclusions: The contrasting effects of SE on CCK- versus PV-expressing boutons in the CA1 pyramidal cell layer suggest that subsets of basket cells are differentially affected in mice with spontaneous seizures. The parallel decrease of both CCK and CB1 receptors, along with electron microscopic evidence for degeneration of CCK-ir terminals following SE, favors loss of terminals rather than loss of expression. A potential decrease of inhibition of CA1 pyramidal cell bodies by CCK-ir basket cells could be compounded by reduced CCK-mediated enhancement of GABAergic release from PV-ir basket cells. The reduced terminal-like labeling of CB1 receptors and GAD in strata radiatum and oriens suggests a further loss of inhibitory innervation to dendrites. In the broader context of changes in GABAergic innervation, these data support a selective loss of inhibitory innervation to CA1 pyramidal cells by specific cell types in mice that experience spontaneous seizures.