Surprisingly, however, they were also eliminated between P15 and P25 (Figure 3G). The rate of inactive DG axon elimination was not affected by the percentage of Epacadostat ic50 mature axons that were inactivated (Figure 3H). This elimination was not just a developmental retraction but was induced by synaptic inactivity, because in DG-A::tau-lacZ mice (no TeTxLC), DG axons were maintained at P25 (Figure 3I; quantification at P15 and P23 is shown in Figure 3J). To further characterize the inactive axon elimination in DG-A::TeTxLC-tau-lacZ mice, we visualized

TeTxLC/tau-lacZ-expressing axons by staining with the antibody to β-gal. Confocal microscopy revealed that at P15, TeTxLC-expressing axons were intact at both the proximal and distal regions (Figure 4A). At P20, the proximal region of TeTxLC-expressing axons appeared still intact (Figure 4B, proximal). However, OTX015 order at the distal region of TeTxLC-expressing axons, there were clear signs of axon retraction: swollen axonal tips (retraction bulbs) and remnants of axons (axosomes) (Figure 4B, distal). These features resemble axon retraction observed during synapse elimination at the neuromuscular junction (Bishop et al., 2004). Without TeTxLC (DG-A::tau-lacZ), the distal region of DG axons remained intact at P23 (Figure 4C). We further examined the morphology of active and inactive DG axons in

DG-A::TeTxLC-tau-lacZ mice by sparsely labeling DG axons with a lipophilic tracer, DiI. Inactive (TeTxLC/tau-lacZ-expressing) axons were identified by staining for β-gal. At P18, active (β-gal−/DiI+) DG axons formed many large mossy fiber boutons with long extensions in CA3 (Figure 4D) (Amaral and Dent, 1981). In

contrast, inactive (β-gal+/DiI+) DG axons did not have any large mossy fiber boutons (Figure 4D). These results indicate that synaptic transmission plays an important role in the refinement of mossy fiber synapses and that inactive axons are retracted during development. Is the elimination of inactive DG axons initiated by the death of DG neurons? To address this question, we injected 5-bromo-2-deoxyuridine (BrdU) into DG-A::TeTxLC-tau-lacZ from mice at P7–8 to label newborn DGCs (Kee et al., 2002 and Kokoeva et al., 2005) and examined the number of surviving BrdU-positive cells at P15, P20, and P25 (Figure 4E). All of the BrdU-labeled neurons became NeuN positive mature neurons by P15 (Figure 4F), consistent with earlier reports showing that DG neurons differentiate very quickly during development (Amaral and Dent, 1981 and Hastings and Gould, 1999; also see Figure 7). The total number of BrdU-positive cells in DG-A::TeTxLC-tau-lacZ mice was similar to that in wild-type mice at P15 and P20 (Figure 4E). In addition, at P20 there was no apparent increase in the number of DG cells that are positive for activated caspase 3, a marker for apoptosis (Figure S2).