Rapamycin suppresses axon sprouting by somatostatin interneurons in a mouse model of temporal lobe epilepsy

Abstract

Purpose: In temporal lobe epilepsy many somatostatin interneurons in the dentate gyrus die. However, some survive and sprout axon collaterals that form new synapses with granule cells. The functional consequences of γ-aminobutyric acid (GABA)ergic synaptic reorganization are unclear. Development of new methods to suppress epilepsy-related interneuron axon sprouting might be useful experimentally.

Methods: Status epilepticus was induced by systemic pilocarpine treatment in green fluorescent protein (GFP)-expressing inhibitory nerurons (GIN) mice in which a subset of somatostatin interneurons expresses GFP. Beginning 24 h later, mice were treated with vehicle or rapamycin (3 mg/kg intraperitoneally) every day for 2 months. Stereologic methods were then used to estimate numbers of GFP-positive hilar neurons per dentate gyrus and total length of GFP-positive axon in the molecular layer plus granule cell layer. GFP-positive axon density was calculated. The number of GFP-positive axon crossings of the granule cell layer was measured. Regression analyses were performed to test for correlations between GFP-positive axon length versus number of granule cells and dentate gyrus volume.

Key findings: After pilocarpine-induced status epilepticus, rapamycin- and vehicle-treated mice had approximately half as many GFP-positive hilar neurons as did control animals. Despite neuron loss, vehicle-treated mice had over twice the GFP-positive axon length per dentate gyrus as controls, consistent with GABAergic axon sprouting. In contrast, total GFP-positive axon length was similar in rapamycin-treated mice and controls. GFP-positive axon length correlated most closely with dentate gyrus volume.

Significance: These findings suggest that rapamycin suppressed axon sprouting by surviving somatostatin/GFP-positive interneurons after pilocarpine-induced status epilepticus in GIN mice. It is unclear whether the effect of rapamycin on axon length was on interneurons directly or secondary, for example, by suppressing growth of granule cell dendrites, which are synaptic targets of interneuron axons. The mammalian target of rapamycin (mTOR) signaling pathway might be a useful drug target for influencing GABAergic synaptic reorganization after epileptogenic treatments, but additional side effects of rapamycin treatment must be considered carefully.

Figure 1

GFP-immunostaining of the dentate gyrus in a control mouse (A) and in mice that had experienced pilocarpine-induced status epilepticus and then were treated daily for two months with vehicle (B) or 3 mg/kg rapamycin (C). Asterisks in left panels indicate areas shown at higher magnification in right panels. m = molecular layer, g = granule cell layer, h = hilus, CA3 = CA3 pyramidal cell layer.

Figure 2

GFP-positive axons could be distinguished from a dendrite (arrows) in the dentate gyrus molecular layer in a vehicle-treated mouse that experienced status epilepticus.

Figure 3

Number of GFP-positive hilar neurons, length of GFP-positive axon in the dentate gyrus, and related parameters of control mice and mice that experienced pilocarpine-induced status epilepticus and then were treated daily for two months with vehicle or 3 mg/kg rapamycin. A Vehicle- and rapamycin-treated mice had fewer GFP-positive hilar neurons compared to controls (*p < 0.001, ANOVA with Student-Newman-Keuls Method). There was no significant difference between vehicle- and rapamycin-treated mice. Error bars indicate s.e.m. Each group consisted of 6 mice. B Vehicle-treated mice had more GFP-positive axon per dentate gyrus compared to control and rapamycin-treated mice (*p < 0.001). There was no significant difference between control and rapamycin-treated mice. C Average GFP-positive axon density was slightly elevated in vehicle-treated mice but not significantly different among experimental groups (p = 0.068). D Average number of GFP-positive axon crossings of the granule cell layer was similar among experimental groups. E GFP-positive axon length correlated with number of granule cells per dentate gyrus (p = 0.006, R² = 0.385). F GFP-positive axon length correlated with volume of the dentate gyrus (p < 0.001, R² = 0.871).