Abstracts

Rationale: Many patients with temporal lobe epilepsy display neuron loss in the dentate gyrus. One possible epileptogenic mechanism is the loss of GABAergic interneurons, which could reduce inhibition of dentate granule cells. Consistent with this hypothesis, frequencies of mIPSCs in granule cells are reduced shortly after epileptogenic injuries and remain low in epileptic rats. These findings suggest that loss of inhibitory interneurons permanently reduces numbers of GABAergic synapses with granule cells, which reduces mIPSC frequency. However, despite interneuron loss and reduced mIPSC frequency, it remains unclear whether numbers of GABAergic synapses with granule cells are reduced in temporal lobe epilepsy. Some measures of granule cell inhibition in epileptic dentate gyrus appear normal or enhanced. Surviving interneurons might sprout axon collaterals and develop new GABAergic synapses with granule cells. Methods: Stereological techniques were used to estimate numbers of gephyrin-positive punctae (postsynaptic markers of GABAergic synapses) in the dentate gyrus of a rat model of temporal lobe epilepsy. A confocal laser-scanning microscope equipped with a 100X objective was used to collect images from which 58,140 gephyrin-positive punctae were counted at 1469 sample sites in 162 sections from 18 rats. To evaluate GABAergic synapses and their targets more specifically, stereological techniques were used to estimate numbers of synapses in electron micrographs of serial sections processed for post-embedding GABA-immunoreactivity. A total of 16,335 GABA-positive synapses were counted at 252 sample points consisting of over 29,000 electron micrographs from 14 rats. Adjacent sections were used to estimate numbers of granule cells and glutamic acid decarboxylase-positive neurons per dentate gyrus. Results: Gephyrin-positive punctae were significantly reduced to 70% of control levels short-term (5 d after pilocarpine-induced status epilepticus) but later rebounded significantly (to 126% of controls) in epileptic rats. In another set of rats evaluated for cell counts and EM, GABAergic neurons were reduced to 70% of control levels short-term, where they remained in epileptics. Integrating synapse and cell counts revealed that control granule cells receive an average of 2982 GABAergic synapses: 12 axo-axonic, 190 axo-somatic, 1490 axo-shaft, and 1290 axo-spinous. Average numbers of GABAergic synapses per granule cell decreased short-term (to 71% of controls), and rebounded in epileptics (to 139% of controls). Numbers of axo-shaft and axo-spinous GABAergic synapse in the outer molecular layer changed most. Conclusions: These findings suggest interneuron loss initially reduces numbers of GABAergic synapses with granule cells, but later, synaptogenesis by surviving interneurons overshoots control levels. Thus, reports of reduced functional inhibitory synaptic input to granule cells in epileptic rats might be attributable not to fewer but instead to abundant but dysfunctional GABAergic synapses.