Abstracts

Following status epilepticus, there is a large proliferation of dentate granule cells, many of which develop ectopically in the hilus. Despite evidence that these EGCs contribute to increased excitability after status, it is still unclear whether this population is substantial enough to affect hippocampal network physiology.
To quantify this population and its persistence, the total number of EGCs was estimated, using unbiased stereology, at different times after PILO. Adult male Sprague Dawley rats (200 [plusmn] 20g; n=5) received 1 mg/kg atropine methylbromide, s.c., followed by 380mg/kg pilocarpine HCl i.p. (or 1 ml/kg saline in controls; n=4). Diazepam (10 mg/kg, i.p.) was administered after 1 hr to attenuate status, and [sim]5 hrs later animals received 1 ml castor oil (orally), and 2 ml dextrose-lactate Ringer[apos]s Solution (s.c.). Diet was supplemented with apples for [sim]1 wk.
Rats were perfused with PBS, 2-18 mon after PILO. One hippocampus was embedded in agarose, post-fixed (4% PFA), and chopped longitudinally (McIlwain) into 6 equal-length slabs. A randomly chosen slab was Vibratome resectioned (75 [mu]m) in its entirety. Free-floating immunocytochemistry used Prox-1 for EGC identification (1:60,000; Covance).
The number of Prox-1 immunopositive neurons in the hilus was estimated for each animal using the optical fractionator method. Samples were taken at systematic points along a randomly placed grid using software (StereoInvestigator). The hilus was defined as the region between the apex of the granule cell layer and the tip of the individual blades, with a border 100[mu]m away from the granule cell layer along each blade. When every section was counted, the total number of counted cells was multiplied by the reciprocal value of the sampling probabilities. After PILO, mean EGC number per hippocampus was 17770 [plusmn] 5023 (sem), which was significantly higher than controls (1613 [plusmn] 248; [underline]t[/underline](4)=3.2, [underline]p[/underline] [lt] 0.05). The total number of EGCs after PILO ranged from 6,300 to 32,250. The survival time did not appear to correlate with EGC number, but animals with few behavioral spontaneous seizures appeared to have fewer EGCs. These data are the first quantify hilar EGCs after PILO. The use of a granule cell specific antibody and unbiased stereology ensures maximal accuracy. EGCs were found to be at least 3 times higher in pilocarpine-treated rats than control rats at all time points tested (2, 14, and 18 months), showing that EGC formation represents a long-lasting change in the [quot]epileptic[quot] rat hippocampus. Together with their physiological abnormalities, the high number of EGCs suggests that they may contribute to hippocampal hyperexcitability and epileptogenesis in the PILO model. (Supported by NIH NS 41490, Epilepsy Foundation, and Helen Hayes Hospital Foundation.)