Abstracts

Rationale: Temporal lobe epilepsy is a neurological disorder in which spontaneous seizures are accompanied by neuroanatomical and physiological abnormalities of the hippocampus. Recently, our lab utilized a conditional phosphatase and tensin homolog (PTEN) knockout mouse model to demonstrate that selective deletion of this protein, an inhibitor of mTOR, from 10-25% of granule cells was sufficient to cause epilepsy (Pun et al., Neuron, 2012). Deletion of PTEN from granule cells resulted in somatic hypertrophy, mossy fiber sprouting, hilar basal dendrites and ectopic somata. Here, we extend these studies by examining granule cell mossy fiber axons. Mossy fibers typically collateralize extensively in the hilus, but project only a single branch into the CA3 pyramidal cell layer, where they innervate these neurons via giant mossy fiber boutons. Methods: Gli1-CreER^T2 X PTEN^flox/flox x GFP mice received a subcutaneous injection of 250 mg/kg of tamoxifen at post-natal day 14. Mice were sacrificed for histological studies between the ages of 2.5-7 months. Immunohistochemical techniques were used to determine the percentage of PTEN knockout cells, visualize granule cell axons and reveal giant boutons. Single-cell biocytin fills of granule cells in acute hippocampal slices were also used to reveal axon morphology. Results: Mossy fiber axon diameter was increased in PTEN knockout mice compared to controls. Stratum lucidum was also thicker in the knockout mice, with axons sprouting into stratum oriens in some animals. Interestingly, the number of giant mossy fiber boutons per length of axon was reduced in the knockout mice compared to controls. Importantly, however, reconstruction of biocytin-filled PTEN KO cells occasionally revealed multiple axon collaterals projecting into CA3 (2 of 6 neurons), while all control cells examined had only a single collateral in this region (n=5). Mossy fiber sprouting, in which granule cell axons project back into the molecular layer, was also observed. Conclusions: Mossy fiber axons from PTEN knockout cells grow abnormally, with thicker axons and decreased bouton density. The increased axonal thickness and sprouting could support enhanced communication efficiency between dentate granule cells and CA3 pyramidal cells, while the decrease in bouton density could reflect a compensatory mechanism to suppress enhanced excitatory output. Additional axon collaterals in CA3, however, might offset the reduction in bouton density, preserving or even increasing CA3 innervation ratios. These findings suggest that PTEN knockout cells may influence a larger network of cells in CA3 with greater efficiency, consistent with a pro-excitatory effect of PTEN deletion.