Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats: Visualization after retrograde transport of biocytin

Abstract

In complex partial epilepsy and in animal models of epilepsy, hippocampal mossy fibers appear to develop recurrent collaterals that invade the dentate molecular layer. Mossy fiber collaterals have been proposed to subserve recurrent excitation by forming granule cell-granule cell synapses. This hypothesis was tested by visualizing dentate granule cells and their mossy fibers after terminal uptake and retrograde transport of biocytin. Labeling studies were performed with transverse slices of the caudal rat hippocampal formation prepared 2.6-70.0 weeks after pilocarpine-induced or kainic acid-induced status epilepticus. Light microscopy demonstrated the progressive growth of recurrent mossy fibers into the molecular layer; the densest innervation was observed in slices from pilocarpine-treated rats that had survived 10 weeks or longer after status epilepticus. Thin mossy fiber collaterals originated predominantly from deep within the hilar region, crossed the granule cell body layer, and formed an axonal plexus oriented parallel to the cell body layer within the inner one-third of the molecular layer. When sprouting was most robust, some recurrent mossy fibers at the apex of the dentate gyrus reached the outer two-thirds of the molecular layer. The distribution and density of mossy fiber-like Timm staining correlated with the biocytin labeling. When viewed with the electron microscope, the inner one-third of the dentate molecular layer contained numerous mossy fiber boutons. In some instances, biocytin-labeled mossy fiber boutons were engaged in synaptic contact with biocytin-labeled granule cell dendrites. Granule cell dendrites did not develop large complex spines ("thorny excrescences") at the site of synapse formation, and they did not appear to have been permanently damaged by seizure activity. These results establish the validity of Timm staining as a marker for mossy fiber sprouting and support the view that status epilepticus provokes the formation of a novel recurrent excitatory circuit in the dentate gyrus. Retrograde labeling with biocytin showed that the recurrent mossy fiber projection often occupies a considerably greater fraction of the dendritic region than previous studies had suggested.

The pilocarpine model of epilepsy: what have we learned?

“…The effect of synaptic sprouting in the hippocampal function in the epilepsy depends, in part, of the balance between the new innervations of granule cells and inhibitory interneurones (Okasaki et al 1995). However, there are controversies regarding the hippocampal damage, which concerns if it is a cause or consequence of seizures.…”

Changes in Intracellular Chloride after Oxygen–Glucose Deprivation of the Adult Hippocampal Slice: Effect of Diazepam

“…In parallel to the imaging and the electrophysiology experiments, slices were collected 2 hr after reoxygenation-glucose and fixed in phosphate-buffered 4% paraformaldehyde, pH 7.4, overnight. The fixed slices were embedded in gelatin/albumin as described by Okazaki et al (1995). Immediately after embedding, the slices were cut into 30 m sections with a vibrotome and either stained with cresyl violet for histological observation or subjected to cytochrome c immunohistochemistry.…”

Actions of Brain-Derived Neurotrophic Factor in Slices from Rats with Spontaneous Seizures and Mossy Fiber Sprouting in the Dentate Gyrus

“…This is followed after ϳ1-2 weeks by reorganization of the mossy fibers (Mello et al, 1993). The reorganized mossy fibers innervate novel target neurons, including the granule cells (Okazaki et al, 1995;Kotti et al, 1997). We hypothesized that if BDNF's effects were specific for mossy fibers, then responses of granule cells to their new mossy fiber input would be enhanced by BDNF.…”