The ultimate goal of this project is to understand the role of axon sprouting, in particular recurrent mossy fiber sprouting, in the propagation and pathophysiology of hippocampal seizures. Recurrent mossy fiber sprouting occurs in the hippocampus of persons with complex partial epilepsy and in several animal models of epilepsy. At least one effect of this seizure-induced growth is to create a recurrent excitatory circuit in the dentate gyrus that is either not present normally or is normally very sparse. It has been proposed that such a circuit would contribute to epileptogenesis by diminishing the resistance of the dentate gyrus toward seizure propagation. In contrast, some evidence suggests that recurrent mossy fibers also project to inhibitory neurons and serve to restore synaptic inhibition that was lost as a result of seizure-induced damage. There is also disagreement about the stimulus that triggers mossy fiber growth. Some studies indicate that the seizure-induced degeneration of hilar neurons, especially of mossy cells, opens up synaptic territory that is then occupied by mossy fibers. Other studies, however, suggest that recurrent mossy fiber sprouting might be triggered by seizures per se. These issues will be investigate with use of two animal models. The pilocarpine-treated rat is the only available model that is characterized by both consistently robust recurrent mossy fiber sprouting and spontaneous seizures. Mice genetically engineered to carry a null mutation for the alpha subunit of CaM kinase II also develop spontaneous seizures, accompanied by a lesser number of recurrent mossy fibers that develop in the rat pilocarpine model. Recurrent excitation, polysynaptic inhibition and monosynaptic inhibition will be tested in dentate granule cells by simultaneous recording of field potentials and whole cell currents. Recordings will be made at different times after pilocarpine-induced status epilepticus, after different durations of status epilepticus and at different times in the seizure history of the mutant mice. The hypothesis underlying this work predicts that the magnitude of excitatory and inhibitory synaptic currents, as well as the granule cell firing pattern, will correlate with the extent of recurrent mossy fiber sprouting. The recurrent mossy fiber EPSC will be characterized unambiguously by activating single granule cells with glutamate uncaged by highly focused laser UV light. An in vitro model of """"""""maximal dentate activation"""""""" will be used to determine the extent to which recurrent mossy fiber sprouting accounts for enhanced propagation of seizure discharge through the dentate gyrus. Excitatory hilar neurons will be labeled by their immunoreactivity for glutamate and their abundance will be compared tot he extent of recurrent mossy fiber sprouting. This study will determine whether or not the occurrence of sprouting depends on loss of these neurons. Finally, we will investigate the possibility that axons of CA3 hippocampal pyramidal cells also sprout in these epilepsy models and form a second type of recurrent excitatory circuit in the dentate gyrus.
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