Abstracts

Tuberous Sclerosis complex (TSC) is an autosomal disorder afflicting 1 in 7500 individuals. Nearly 90% of TSC patients experience seizures, and in many cases, seizures are unresponsive to AEDs. Based largely on a limited set of clinical data, cortical tubers are thought to represent epileptogenic foci in some TSC patients. An alternative view suggests that non-tuberous brain regions, characterized by disorganized cortical lamination and dysplastic neurons with aberrant dendritic arbors or axonal projections, play a critical role in generation of abnormal electrical activity. To examine these possibilities, we initiated studies in a conditional knockout mice featuring neuron-selective inactivation of TSC1 i.e., synapsin-TSC1 cKO mice. Mice expressing a floxed TSC1 allele were crossed with genetically modified mice expressing Cre recombinase protein under the control of a neuron-specific promoter (synapsin). Synapsin-TSC1 cKO mice were generated in the laboratory of D. Kwiatkowski (Harvard). Studies combined standard immunohistochemisty, Western blot and acute electrophysiological methods to analyze overall brain structure, c-Fos expression and synaptic excitability in cKO mice (P10-P20). Synapsin-TSC1 cKO mice survive to [sim]6 weeks and frequently, exhibited handling-induced tonic-clonic convulsions, sometimes leading to status epilepticus and death. Light microscopic examination of brains from TSC mutants demonstrated normal cortical and hippocampal architecture; tubers were not observed. Strikingly, SMI311 staining revealed heavily labeled large pyramidal and multipolar neurons in somatosensory cortex. Confocal fluorescence imaging indicated that the majority of SMI311+ cells co-localized with a glutamate transporter (EAAC1) or immediate early gene (c-Fos). SMI311+ cells did not express interneuron-specific markers. vGlut1 and vGlut2 punctate staining was detected only in the proximal dendrites and soma around SMI311+ neurons. Western blot analysis indicated a dramatic increase in cortical expression of c-Fos and c-Jun proteins. Field recording data, obtained from cortical slices of TSC1 mutants bathed in a low concentration of bicuculline (10[micro]m), revealed prolonged field EPSPs, polyspike activity and spontaneous epileptiform discharge. Whole-cell voltage-clamp recording of pyramidal-like neurons in somatosensory cortex revealed long duration epileptiform-like discharges that were blocked by CNQX or TTX. In current-clamp, long bursts in individual cells were accompanied by extracellular field discharge. Neuron-specific deletion of TSC1 in mice, in the absence of tuber formation and architectural disruption, results in activation of SMI311-positive glutamatergic neurons. These neurons are innervated by glutamatergic axon terminals, express IEGs and may form a hyperexcitable cortical network. (Supported by Tuberous Sclerosis Alliance (SCB).)