Abstracts

Rationale: Granule cell axon (mossy fiber) sprouting creates an aberrant positive-feedback circuit that might be epileptogenic. Presumably, injury-related changes in molecular signals initiate mossy fiber sprouting, but it is unclear whether they are expressed transiently or persistently. If transient, there might be a critical period during which time short preventative treatments could permanently block mossy fiber sprouting. Alternatively, if signals persist, continuous treatment would be necessary. We tested whether systemic treatment with rapamycin for 2 months persistently reduced mossy fiber sprouting after pilocarpine-induced status epilepticus.Methods: Male and female mice (FVB strain) were treated systemically (ip) with 5 mg/kg atropine methyl bromide followed by 300 mg/kg pilocarpine to induce status epilepticus when they were ~45 d old. Diazepam (10 mg/kg) was administered 2 h after the onset of convulsions and repeated as necessary. Systemic 1.5 mg/kg rapamycin or vehicle began 24 h after status epilepticus and continued daily for 2 months. One set of mice was evaluated at the end of the 2 month treatment period ('0 delay'). Another set was evaluated 6 months later ('6 mo delay'). At both time-points mice were perfused for Timm staining. Hippocampi were isolated, gently straightened, and sectioned transversely from the septal to the temporal pole. 1-in-12 series of sections were developed for Timm staining. Images of each section were evaluated with ImageJ to measure the total area of the granule cell layer plus molecular layer (gcl + ml) and the subarea that was stained black by the Timm stain. For each hippocampus the percentage of the total volume of the gcl + ml that was Timm-positive was calculated. An adjacent series of Nissl stained sections was evaluated with the optical fractionator method to estimate the total number of hilar neurons/hippocampus. Results: Naive control mice (n=6) had 11,500 200 hilar neurons/hippocampus. All groups that experienced status epilepticus lost an average of ~50% of their hilar neurons, with no significant differences between them. Naive control mice had only 2.5 0.3% of the gcl + ml that was Timm-positive, which was significantly less than all other groups. Vehicle-treated mice had high levels of mossy fiber sprouting at 0 delay (20.4 1.7%, n=10) and at 6 mo delay (18.6 1.4%, n=18, no significant difference). Rapamycin-treated mice had significantly less Timm staining at 0 delay (11.5 0.7%, n=17). However, by 6 mo delay (n=21), rapamycin-treated mice had 21.7 1.3% Timm staining, which was significantly more than at 0 delay and similar to that of vehicle-treated mice.Conclusions: Timm stain results suggest mossy fiber sprouting was suppressed by systemic treatment with rapamycin but resumed after treatment ceased. Neuron counts suggest differences in Timm staining among mice that experienced status epilepticus were not attributable to differences in hilar neuron loss. Together, these findings suggest signals that trigger mossy fiber sprouting persist longer than 2 months. Supported by NIH/NCRR&NINDS.