Abstracts

Rationale: Synaptic pruning is a developmental CNS regulatory process by which excessive synapses are eliminated to establish mature patterns of neuronal connectivity. A complement cascade protein, C1q, has been shown to be necessary for synaptic elimination during development. Mice deficient in this protein demonstrate failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons (Stevens et al., 2007). We hypothesized that C1q knockout (KO) mice would also exhibit defects in neocortical synapse elimination, which would result in enhanced excitatory synaptic connectivity and epileptiform activity. Methods: Using standard techniques, we recorded spontaneous and evoked field potential activity in neocortical slices and obtained video-EEG recordings from unanesthetized implanted C1q KO and wild type (WT) mice. We also used laser scanning photostimulation of caged glutamate to map excitatory and inhibitory synaptic connectivity. Glutamate uncaging was accomplished using 300-600 μs UV laser flashes. Cortical layers II ~ VI were stimulated in grids of 600~650 x 1000~1200 µm with 50 µm spacing. Evoked EPSCs and IPSCs were recorded from layer V pyramidal neurons that were voltage clamped at -70 mV and +20 mV respectively. Results: Spontaneous and evoked interictal epileptiform field potentials occurred at multiple sites in C1q KO but not in WT neocortical slices. In laser mapping experiments, most EPSCs in pyramidal neurons were evoked from layer V, within a radius of 150 μm of somata in WT mice. In C1q-KO slices, significant increases in composite EPSC amplitudes were observed in an area 200~300 µm superficial to layer V, corresponding to layer IV. The fraction of “hotspots” from which EPSCs could be evoked also increased significantly in layer IV. There were no significant differences in maps of evoked IPSCs in C1q vs WT mice. Implanted KO mice had frequent behavioral seizures consisting of freezing associated with bihemispheric spikes and slow wave activity lasting from 5-30 sec. Conclusions: Results indicate that C1q KO mice represent a model of epileptogenesis due to a genetically-determined failure to prune excessive excitatory synaptic connectivity during development. Failure to prune may be an epileptogenic mechanism in other types of neocortical disorders. Supported by NIH grants 5K99NS57940-2, NS12151 and DA15043.