Abstracts

Rationale: Our understanding of neuronal network malfunction and its relation to seizure evolution and progression in intact cortical circuitry is currently limited. A new cellular imaging tool enables the first interrogation of dynamic signaling patterns in cortical neurons during generalized spike-wave seizures in vivo. We examined the stargazer mouse, where a genetic mutation in Cacng2 leads to dendritic AMPA receptor trafficking defects and rhythmic 6-7/sec cortical spike-wave discharges. Methods: Using in vivo two-photon confocal microscopy and simultaneous intracranial EEG in awake mice, we are able to identify calcium activity in individual neurons in layer 2/3 of somatosensory and visual cortex during spontaneous spike-wave seizures. Calcium activity in the visualized population of neurons is averaged over 150-300 seizures per field of view and imaging session after temporal alignment of the seizures at their onset. Either Oregon-green BAPTA-1 or Gcamp6 are used for calcium imaging. Use of the latter allows us to follow the activity of the same population of cells over months, enabling the evolution of seizure participation of cortical networks to be mapped over time. Cell activity is also monitored before and after i.p. administration of 1 mg/kg MK-801, an NMDA receptor antagonist which paradoxically induces spike-wave status epilepticus in stargazer mice. Results: Preliminary data (n=3 animals) show that <20% of the total layer 2/3 neurons display significant changes in calcium activity during the seizures. Among those neurons, three types of temporally progressing activity changes appear to stand out: (1) several seconds before seizure onset, (2) at seizure onset, and (3) several seconds following seizure onset. These classes are observed using both the short-lasting calcium indicator Oregon-green BAPTA-1 and the chronic indicator Gcamp6. Within 30 minutes of MK-801 administration, there is a pronounced shift of calcium activity patterns, both in neurons and surrounding neuropil from largely uncorrelated, sparse activity to highly synchronous firing. One possible explanation for this paradoxical synchronization by an NMDA receptor antagonist might be that the excitation of AMPA receptor-deficient stargazer PV+ interneurons is critically dependent on NMDA-receptors, such that their inhibitory impact on the network is greatly reduced with MK-801. Conclusions: We show for the first time that small subgroups of superficial layer cortical neurons display significant activity changes before, during or immediately after seizures in stargazer mice. Identification of the specific cell types using CLARITY will facilitate the evaluation of cell-specific network mechanisms in these mice with in vivo two-photon microscopy. Combining these techniques thus offers a unique opportunity to bridge the gap between cellular and network behavior in this genetic epilepsy model.