Blockade of excitatory synaptogenesis with proximal dendrites of dentate granule cells following rapamycin treatment in a mouse model of temporal lobe epilepsy

Abstract

Inhibiting the mammalian target of rapamycin (mTOR) signaling pathway with rapamycin blocks granule cell axon (mossy fiber) sprouting after epileptogenic injuries, including pilocarpine-induced status epilepticus. However, it remains unclear whether axons from other types of neurons sprout into the inner molecular layer and synapse with granule cell dendrites despite rapamycin treatment. If so, other aberrant positive-feedback networks might develop. To test this possibility stereological electron microscopy was used to estimate the numbers of excitatory synapses in the inner molecular layer per hippocampus in pilocarpine-treated control mice, in mice 5 days after pilocarpine-induced status epilepticus, and after status epilepticus and daily treatment beginning 24 hours later with rapamycin or vehicle for 2 months. The optical fractionator method was used to estimate numbers of granule cells in Nissl-stained sections so that numbers of excitatory synapses in the inner molecular layer per granule cell could be calculated. Control mice had an average of 2,280 asymmetric synapses in the inner molecular layer per granule cell, which was reduced to 63% of controls 5 days after status epilepticus, recovered to 93% of controls in vehicle-treated mice 2 months after status epilepticus, but remained at only 63% of controls in rapamycin-treated mice. These findings reveal that rapamycin prevented excitatory axons from synapsing with proximal dendrites of granule cells and raise questions about the recurrent excitation hypothesis of temporal lobe epilepsy.

Keywords: electron microscopy; hippocampus; mossy cell; mossy fiber sprouting; pilocarpine; stereology.

Figure 1

Nissl stained dentate gyrus of a pilocarpine-treated control mouse (A), a mouse 5 d after status epilepticus (B), a mouse that was treated with vehicle for 2 months after status epilepticus (C), and a mouse that was treated with rapamycin for 2 months after status epilepticus (D). m=molecular layer, g=granule cell layer, h=hilus, CA3=CA3 pyramidal cell layer. Scale bar (in D)=250 µm.

Figure 2

Group averages and septotemporal distributions of synapses, inner molecular layer volume and area, number of granule cells, and number of hilar neurons in pilocarpine-treated control mice, mice 5 d after status epilepticus, and mice 2 months after status epilepticus and daily treatment with vehicle or rapamycin. A1 Number of asymmetric synapses counted per sample site. Values represent mean ± s.e.m. Average number of synapses was larger in control mice compared to mice 5 d after status epilepticus (p=0.033, one way ANOVA with Holm-Sidak method). A2 Septotemporal distribution. B1 Volume of the inner molecular per hippocampus. Average volume of the inner molecular layer was larger in vehicle-treated mice compared to controls (p=0.031). B2 Inner molecular layer area per section. C1 Number of Nissl-stained granule cells per hippocampus. No significant differences between groups. C2 Number of granule cells per section. D1 Number of Nissl-stained large (>10 µm soma diameter) hilar neurons per hippocampus. Control mice had more hilar neurons than all other groups (p<0.003). D2 Large hilar neurons per section.

Figure 3

An asymmetric (arrow) and symmetric synapse (arrowhead) with a dendritic shaft in the inner molecular layer of the dentate gyrus in a mouse 5 d after status epilepticus. Scale bar=0.25 µm.

Figure 4

Asymmetric synapses in serial electron micrographs of the inner third of the dentate gyrus molecular layer in a pilocarpine-treated control mouse. An axospinous synapse (arrow) with a spine neck connected to a dendritic shaft in the plane of section. Other axospinous synapses are indicated by arrowheads. Scale bar (in E)=0.5 µm.

Figure 5

Asymmetric synapses in serial electron micrographs of the inner third of the dentate gyrus molecular layer in a mouse that experienced pilocarpine-induced status epilepticus and then was treated for 2 months with 3 mg/kg/d rapamycin. An axospinous synapse (arrow) with a spine neck connected to a dendritic shaft in the plane of section. Other axospinous synapses are indicated by arrowheads. Scale bar (in E)=0.5 µm.

Figure 6

Number of asymmetric synapses in the inner molecular layer of pilocarpine-treated control mice, mice 5 d after pilocarpine-induced status epilepticus, and mice 2 months after status epilepticus and daily treatment with vehicle or rapamycin. Values represent mean ± s.e.m. A Average numbers of asymmetric synapses in the inner molecular per mouse of control group and vehicle-treated mice 2 months after status epilepticus were larger than those of 5 d and rapamycin-treated groups (p<0.008, one way ANOVA with Holm-Sidak method). B The average number of asymmetric synapses per granule cell was larger in the control group than in the 5 d and rapamycin-treated group (p<0.05).

Figure 7

Ultrastructural characteristics of asymmetric synapses in the inner molecular layer in pilocarpine-treated control mice, mice 5 d after status epilepticus, and mice that experienced stats epilepticus and then were treated with vehicle or rapamycin for 2 months. A Number of shaft synapses per granule cell. Values represent mean ± s.e.m. The average value for mice 5 d after status epilepticus is larger than that of controls (p=0.039, ANOVA with Holm-Sidak method). B Number of perforated synapses per granule cell. No significant differences. C Synapse size. No significant differences.