Reassessment of the Effects of Cycloheximide on Mossy Fiber Sprouting and Epileptogenesis in the Pilocarpine Model of Temporal Lobe Epilepsy

Abstract

A feature of animal models of temporal lobe epilepsy and the human disorder is hippocampal sclerosis and Timm stain in the inner molecular layer (IML) of the dentate gyrus, which represents synaptic reorganization and may be important in epileptogenesis. We reassessed the hypothesis that pre-treatment with cycloheximide (CHX) prevents Timm staining in the IML following pilocarpine (PILO)-induced status epilepticus (a multifocal model of temporal lobe epilepsy), but allows epileptogenesis (i.e., chronic spontaneous seizures) after a latent period. Hippocampal slices from PILO-treated rats without Timm stain in the IML after CHX treatment were hypothesized to lack the electrophysiological abnormalities suggestive of recurrent excitation. The primary experimental groups were as follows: 1) CHX (1 mg/kg) 30-45 min prior to administration of PILO (320 mg/kg ip, 2) only PILO, and 3) only saline (0.5 ml, IP). The CHX pre-treatment significantly decreased the number of rats that responded to PILO with status epilepticus compared to rats that received only PILO. Pre-treatment with CHX did not significantly alter the spontaneous motor seizure rate post-treatment compared to treatment with PILO alone in those animals from each group that developed status epilepticus during PILO treatment. Timm stain in the IML was not significantly different between the PILO- and PILO+CHX-treated rats. Using quantitative methods, CHX did not prevent hilar, CA1, or CA3 neuronal loss compared to the PILO-treated rats. Extracellular responses to hilar stimulation in 30 microM bicuculline and 6 mM [K(+)](o) demonstrated all-or-none bursting in both the CHX+PILO- and PILO-treated rats but not in control rats. Whole cell recordings from granule cells, using glutamate flash photolysis to activate other granule cells, showed that both the CHX+PILO- and PILO-treated rats had excitatory synaptic interactions in the granule cell layer, which were not found after saline treatment. Some rats responded to PILO (with or without CHX pre-treatment) with only one or a few seizures at treatment, and some of these animals (n = 4) demonstrated spontaneous motor seizures within 2 mo after treatment. Timm staining and neuron loss in this group were not clearly different from saline-treated rats. These results suggest that in the PILO model, pre-treatment with CHX does not affect mossy fiber sprouting in the IML of epileptic rats and does not prevent the formation of recurrent excitatory circuits. However, the develoment of spontaneous motor seizures, in a small number of rats, could occur without detectable hippocampal neuron loss or mossy fiber sprouting, as assessed by the Timm stain method.

Mossy Fiber Sprouting as a Potential Therapeutic Target for Epilepsy

“…Other groups have challenged these observations. Williams et al (2002) reported that treatment with cycloheximide did not block either hilar cell loss or MF sprouting and that recurrent epileptic seizures can occur even in cycloheximide-treated rats.…”

Modeling epileptogenesis and temporal lobe epilepsy in a non-human primate

“…However, no correlation was found between MFS and seizures frequency and intensity. Whether MFS does or does not seem to be necessary for SRS development is still under debate (Longo and Mello, 1998;Pitkanen et al, 2005;Tauck and Nadler, 1985;Williams et al, 2002).…”

Is Epilepsy a Preventable Disorder? New Evidence from Animal Models

“…However, inhibition of mossy fiber sprouting may not be sufficient for prevention of poststatus epileptogenesis; while administration of the protein synthesis inhibitor cycloheximide blocked mossy fiber sprouting after either pilocarpine or kainite, spontaneous seizures developed with the same frequency (Longo and Mello 1997). While other studies have found cycloheximide to be less efficacious for preventing mossy fiber sprouting, they have also found that poststatus spontaneous seizures could develop without mossy fiber sprouting as detected by Timm staining (Williams and others 2002). …”