Abstracts

Rationale: Mechanisms of temporal lobe epilepsy remain unclear. Involvement of the hippocampus has long been suspected because it displays severe neuropathology in patients. Dramatic changes in the cellular composition of the hippocampal dentate gyrus include loss of hilar neurons, especially mossy cells, generation of ectopic granule cells, and loss of inhibitory interneurons. Each of these cellular changes has been proposed to be epileptogenic. To help prioritize potential mechanisms for further experimental manipulation and testing we asked which cellular abnormalities correlate best with the frequency of spontaneous seizures in an animal model of temporal lobe epilepsy.Methods: Mice (FVB, both sexes) were treated systemically with pilocarpine to induce at least 2 h of status epilepticus when they were 58 ± 2 d old (mean ± sem). One month later they began to be video-monitored for spontaneous convulsive seizures that involved forelimb clonus, at least. Monitoring was conducted 9 h/d every day for a month. Then, mice were perfused with fixative, and the left hippocampus was sectioned transversely from the septal pole to the temporal pole. Mossy cells, ectopic granule cells, and interneurons were visualized with GluR2-immunolabeling, Prox1-immunolabeling, and in situ hybridization for glutamic acid decarboxylase 65, respectively. The optical fractionator method was used to estimate numbers of mossy cells, ectopic hilar granule cells, and GABAergic interneurons per dentate gyrus in seizure-monitored epileptic mice (n=128) and in controls (n=10).Results: Mice were observed to have an average of one seizure every 7 h (0.14 ± 0.02 seizures/h, range 0.01-0.47). There was no significant difference in seizure frequency of male (n=45) and female mice (n=83, p=0.68, t test). Control mice had 3300 ± 250 mossy cells, 600 ± 30 ectopic granule cells, and 7780 ± 310 interneurons per dentate gyrus. Average values from epileptic mice revealed loss of mossy cells (1270 ± 60, 38% of controls), generation of ectopic granule cells (2180 ± 80, 360% of controls), and loss of interneurons (5830 ± 80, 75% of controls), all significant (p<0.0001). Scatter plots of cell number versus seizure frequency were evaluated by linear regression. Generation of ectopic granule cells was not significantly correlated with seizure frequency (p=0.30, R=0.09). Mossy cell loss correlated with seizure frequency (p=0.04, R=0.18), but interneuron loss correlated even more (p=0.01, R=0.23). To begin identifying which subpopulations might be most important, interneurons were divided according to cell body location. Loss of interneurons in the hilus and molecular layer did not correlate with seizure frequency (p=0.90 and 0.13, R=0.01 and 0.14, respectively). Loss of interneurons in or adjacent to the granule cell layer correlated with seizure frequency more than any other parameter evaluated (p=0.005, R=0.25).Conclusions: These findings prioritize loss of granule cell layer interneurons for further testing as a potential mechanism of temporal lobe epileptogenesis.