Abstracts

Rationale: Hippocampal mossy fibers exhibit complex structural rearrangements and abnormal synaptic function in mesial temporal lobe epilepsy (MTLE). During epileptogenesis, axons of dentate gyrus granule cells sprout and establish new excitatory synapses onto abnormal targets, including granule cells, interneurons, and basal dendrites of CA3 pyramidal neurons Such synaptic reorganization and abnormal sprouting are thought to play a critical role in the development of recurrent excitatory circuits, hyperexcitability and ultimately seizures in MTLE. While many studies have shown alterations in postsynaptic function of both excitatory and inhibitory circuits in MTLE, far less attention has been paid to the role of presynaptic dysfunction in the pathogenesis of epilepsy. Methods: By using two-photon laser scanning confocal microscopic imaging of presynaptic vesicular release in mice expressing a fusion protein of green fluorescent protein and the SNARE protein synaptobrevin (SynaptopHluorin, SpH), we assessed whether the organization of functional synaptic vesicle pools at dentate granule cell mossy fiber bouton (MFB) synapses contacting spines in the CA3 region is altered in chronic pilocarpine-induced epilepsy. Results: A 20Hz/600 mossy fiber stimulus train evoked peak SpH fluorescence in MFB s from epileptic slices significantly higher than control MFB (epileptic 2.02 0.15, n=8; control 1.47 0.03, n=10, P<0.05). Control MFB peak fluorescence at the end of the stimulus train showed a normal distribution (mean normalized fluorescence = 1.41 0.025), while MFB in slices from epileptic mice showed a bimodal ( 1 = 1.76 0.04 and 2 = 3.89 0.10; P<0.05) suggesting two different functional vesicle pools. A single exponential decay fit to SpH fluorescence decay after cessation of stimulation showed epileptic MFB had significantly faster rates of endocytosis (tau = 7.2 0.32s) compared to control MFB (tau = 10.4 0.76s, P<0.05). Neurotransmitter release from the MFB readily-releasable vesicle pool (RRP) was estimated by destaining kinetics of the styryl dye FM1-43 selectively loaded into MFB RRP by hypertonic shock. FM1-43 destaining from the RRP was significantly faster from MFB of 1-2 month chronic epileptic animals compared to controls (P<0.05). In contrast, FM1-43 destaining was significantly slower in MFB of >11 chronic epileptic rat slices compared to age matched controls. These findings suggest seizure-related plasticity of presynaptic elements in mossy fiber-CA3 pyramidal cell synapses during the course of epileptogenesis leads to enhanced neurotransmitter release and vesicle recycling in an early phase of chronic epilepsy, but later exhibits reduced rates of release, perhaps due to either compensatory changes or widespread presynaptic damage. Conclusions: Our findings indicate that persistent alterations in presynaptic vesicular exocytosis and endocytosis correlate with structural plasticity of MFB terminals at mossy fiber-CA3 synapses in chronic epilepsy, suggesting an under appreciated role in development of MTLE.