Project #1: Elimination of mossy cell NMDA receptors results in impaired emotional reactivity. Previously, we subjected mossy cell NR1-KO mice and their control littermates to a battery of behavioral tasks including fear conditioning, a task that involves associating a particular context with an aversive stimulus. We found that the mutants show reduced freezing compared to their control littermates in response to a single foot shock. Further, they are unable to discriminate training and novel contexts 3 hours after fear conditioning. Interestingly, when the foot shock was intensified, these phenotypes disappeared, suggesting that impaired context discrimination is due to a deficit in immediate post-shock freezing. Collectively, these results indicate that the behavioral deficit observed in the mutants is perhaps due to an impaired sensorimotor integration of weak stimuli. This year, we explored the mechanisms underlying these impaired behaviors, and found a profound impairment in dentate neurogenesis. Interestingly, a decrease in the expression of neurogenesis markers was detected after postnatal 15 weeks, 5-6 weeks after completion of NR1 knockout in the mossy cells, but temporally correlated with the emergence of the behavioral deficits. Currently, we are trying to elucidate the underlying mechanisms that lead to impaired neurogenesis in the mossy cell-NR1 knockout. Project #2: Elimination of mossy cells impairs contextual pattern separation but does not induce epileptogenesis. Earlier, we found that intraperitoneal injection of diphtheria toxin (DT) to mossy cell-DTR mice induces significant mossy cell death within a week. Subsequently, approximately 90% of mossy cells were selectively eliminated by 4 weeks after the treatment. We further found no obvious or sustained epilepsy-like discharges in the hippocampus as measured by in vivo local field potential recordings. Interestingly, no mossy fiber sprouting was detected by Timm staining. These results suggested that, in contrast to previous reports showing that lesions of the entire hilar region induce massive mossy fiber sprouting and epilepsy, selective in vivo elimination of mossy cells does not trigger behavioral epilepsy or mossy fiber sprouting. This year, we found that GAD67-positive neurons slowly innervate the vacated area after the mossy cell loss, i.e., inner molecular layer of dentate gyrus. We explored the underlying mechanisms of this slow inhibitory refinement by slice physiology, and found that dentate granule cells are hyperexcitable immediately after the mossy cell ablation, which disappeared along with the absence of GABAergic sprouting. In summary, we concluded that mossy cell loss in vivo renders the granule cells hyperexcitable, which is insufficient to trigger the mossy fiber sprouting and epileptic discharges. Perhaps, in addition to the loss of mossy cells, neurodegeneration of other limbic areas, such as entorhinal cortex, is necessary to induce medial temporal lobe epilepsy. These findings provide new insights into the mechanisms of epileptogenesis in the limbic cortex.