Abstracts

RATIONALE: In many animal models of epilepsy, the axons of dentate gyrus granule cells (mossy fibers) develop collaterals that innervate an abnormal lamina, the inner molecular layer. Whether sprouting leads to excitatory monosynaptic connections among granule cells has been a subject of great interest. This study was undertaken to examine the question with direct methods, using simultaneous recordings of granule cells in a slice preparation with mossy fiber sprouting. At the end of this activity the participants should be able to discuss new evidence for excitatory monosynaptic connections among granule cells in rats with mossy fiber sprouting.
METHODS: The pilocarpine model was used to examine mossy fiber sprouting in adult male Sprague-Dawley rats. Pilocarpine was administered (380mg/kg i.p.) 30 min after atropine (1mg/kg). After 1 hr of status epilepticus, diazepam was injected (5mg/kg i.p.). All animals had spontaneous behavioral seizures. After 2-8 months, slices from the ventral hippocampus of one hemisphere were prepared with conventional methods. Simultaneous recordings of granule cells were made in a semi-submerged chamber using sharp microelectrodes (60-80 megohms) filled with 4% Neurobiotin in 1M Kacetate. The opposite hemisphere was immersed in 4% paraformaldehyde. It was subsequently sectioned and stained with antisera to neuropeptide Y to confirm that sprouting occurred.
RESULTS: Of 873 pairs of granule cells, 6 pairs demonstrated monosynaptic connectivity. Thus, an action potential evoked in one cell by current injection through the recording electrode depolarized (up to 3.7 mV) the second cell. The depolarization began during the repolarization of the action potential of the first cell, a latency that is consistent with a monosynaptic event. These putative monosynaptic EPSPs appeared to be excitatory because they failed to invert at membrane potentials depolarized to the reversal potential for chloride, and in some cases could trigger action potentials at depolarized potentials. Interestingly, the failure rate was extremely high (45-63%) when compared to other monosynaptic pathways examined with the same methods (0-40%). Pairs of presynaptic action potentials with short interspike intervals led to paired-pulse depression. When the recorded cells were filled with Neurobiotin, their morphology confirmed that they were granule cells. Several of the filled neurons had axon collaterals in the inner molecular layer with many varicosities.
CONCLUSIONS: The results suggest that there are functional excitatory monosynaptic connections among granule cells in epileptic rats with mossy fiber sprouting. They also suggest that these synapses may be functionally weak because of their relatively high failure rate and frequency depression. These data support the hypothesis that new recurrent excitatory circuits develop in rats with mossy fiber sprouting, and may contribute to the hyperexcitability that often occurs when sprouting is present. The results are also consistent with studies that have demonstrated strong inhibition in the dentate gyrus despite the presence of mossy fiber sprouting.
[Supported by: NINDS 38285 to H.E.S.]; (Disclosure: Grant - Contract with Neuropace Inc.)