Mossy fibre sprouting (MFS) is characterised by an abnormal projection of granule cell axon collaterals into the molecular layer of the dentate gyrus, where they form a recurrent excitatory network capable of promoting seizure-like activity.

This study aims to analyse the changes in granule cell excitability that occur in MFS and to investigate the involvement of presynaptic receptors at the sprouted synapses. We used organotypic slice cultures (obtained from 7-day-old rats killed according to Home Office regulations) since these cultures can display MFS (Zimmer & Gèhwiler, 1984). First, we analysed the extent of MFS in cultures incubated for 7, 14 and 21 days in vitro (DIV), and then in cultures treated with the neurotoxic convulsant pilocarpine (0.5 mM) for 48 h at 5 DIV and tested at 21 DIV.

Timm staining revealed the presence of mossy terminals in the hilus and in a compact band along the stratum lucidum (n = 45). The density of staining in the hilus and molecular layer increased as a function of DIV. Pilocarpine treatment greatly enhanced the Timm stain in the molecular layer.

Whole-cell voltage-clamp recording of granule cells showed mEPSCs in the presence of 0.5 µM TTX and 30 µM bicuculline. The frequency of mEPSCs increased with DIV and was highest in pilocarpine-treated cultures (n = 29) (7 DIV, 0.28 ± 0.04; 14 DIV, 0.72 ± 0.08; 21 DIV*, 0.73 ± 0.05, pilocarpine treated*, 1.09 ± 0.03 (data shown as mean (Hz) ± S.E.M., * denotes significance with respect to 7 DIV, considered significant if P < 0.05, Student’s t test)). Recorded granule cells were visualised by biocytin and a morphometric analysis revealed that the number of axon collaterals in the hilus was positively correlated with DIV, with the largest number in pilocarpine-treated cultures. 21 DIV control and treated cultures contained collaterals that extended into the granule and molecular layers.

Paired recordings of granule cells showed that pilocarpine-treated cultures had a larger proportion of monosynaptic and polysynaptic connections (n = 59). Monosynaptic unitary EPSCs had short latencies with a small jitter, were reliable and displayed paired-pulse depression. Application of the group II metabotropic glutamate receptor (mGluR) agonist LY354740 (0.5 µM) decreased the amplitude of EPSCs, elicited paired-pulse facilitation and increased the number of failures.

The excitability of granule cells in slice cultures is positively correlated with DIV, and pilocarpine treatment further enhances MFS and excitability. This study provides direct evidence for active connections between granule cells and demonstrates that presynaptic group II mGluRs are functional in the sprouted synapses.