Abstracts

Rationale: Chloride ions play an important role in the inhibitory function through chloride-permeable GABA receptor channels. In epilepsy, the brain network becomes hyper-excitable, partly due to the loss of proper inhibitory function. Dysregulation of chloride ions and the resulting elevated intracellular chloride levels in principal neurons could be a cause of this loss. Specifically, dentate granule neurons in the hippocampus have been the focus of epilepsy research, as the dentate gyrus acts as a gatekeeper for incoming excitatory input to the hippocampus.



Methods: To this end, we evaluated the intracellular chloride levels in dentate granule neurons, as well as in pyramidal neurons in the CA1 and subiculum, using two-photon excitation fluorescence lifetime imaging microscopy (2P-FLIM) with the chloride-sensitive probe Clomeleon in the intrahippocampal kainate (IHK) model of epilepsy.


Results: In areas ipsilateral to the kainate injection site, significant portions of the CA1 pyramidal cell layer and subicular structure were lost, while contralateral structures were preserved. The dentate gyrus in the ipsilateral hippocampus was present but exhibited a significantly thickened granule cell layer. Cellular resolution images showed that the dentate granule cells were swollen and disorganized. Contrary to expectations of elevated intracellular chloride levels in the affected area, the CFP fluorescence lifetime of Clomeleon in dentate granule cells in the ipsilateral hippocampus was equal to or even shorter than those in the contralateral hippocampus, indicating that chloride levels in the ipsilateral dentate granule cells were equal to or lower than those on the contralateral side (N=110 ipsilateral granule cells vs. 188 contralateral granule cells; mean 1.70 ± 2.8 ns vs. 1.74 ± 1.7 ns, p=0.14). We observed longer CFP lifetimes, or higher chloride levels, in subicular neurons compared to contralateral dentate granule cells (91 subicular neurons, mean 1.94 ns vs. 188 contralateral granule cells, mean 1.74 ± 1.7 ns, p< 0.0001).


Conclusions: This suggests that subicular neurons may play a role in the hyperexcitable hippocampal network in epileptic brains. We discuss the possibilities and limitations of using the Clomeleon-2PFLIM approach and extend our method to include FLIM with pH-insensitive dyes like MQAE. We also explore a transgenic approach to label specific neuronal subpopulations with SuperClomeleon probes using Cre-expressing transgenic lines.

Funding: R01NS082046-10A1
1P50HD105354-03