However an alternative pos sibility is that p75ntr is

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reflects the lack of trophic factors. However, an alternative pos- sibility is that p75NTR is aggregated and consequently activated by the antibody used in the panning procedure, thereby resulting in the slow cell death seen in culture. We consider this possibility highly unlikely because the monoclonal antibody used in panning [mAb 192 (Chandler et al., 1984)] does not affect survival of subplate neurons when either added to the culture medium or used as a substrate for cell attachment (data not shown). Addition of exogenous BDNF increases survival approximately twofold over control cultures (Fig. 3 a , b ). Survival was dose dependent and required a concentration of 0.3 ng/ml for maximal response (Fig. 3 b ). To test directly the role of p75NTR in BDNF-mediated survival, FAb fragments of a polyclonal antibody to the extracel- lular domain of p75NTR were used to block BDNF binding (Weskamp and Reichardt, 1991). Anti-p75NTR FAb fragments completely inhibit BDNF-mediated survival but have no effect on the basal survival of subplate neurons (Fig. 3 a ). Normal rabbit FAbs are without effect. Thus, BDNF binding to p75NTR is necessary for BDNF-mediated survival. Because p75NTR is known to bind all neurotrophins with similar affinity (Chao et al., 1998), the effects of NGF and NT3 on subplate neuron survival were also examined. NT3, like BDNF, increases subplate neuron survival approximately twofold (Fig. 3 c ). NGF, on the other hand, has no significant effect on subplate neuron survival. However, when used in excess, NGF blocks BDNF-dependent survival (Fig. 3 d ). This observation suggests that NGF competes with BDNF for binding to p75NTR and is consistent with the model that BDNF binding to p75NTR is required for BDNF-dependent survival. Importantly, NGF does not cause significant cell death, despite the fact that subplate neurons do not express the Trk receptor for NGF, TrkA. These observations differ significantly from results in sympathetic neu- rons and oligodendrocytes, in which p75NTR signaling promotes cell death in the absence of activated TrkA receptors (Bamji et al., 1998; Yoon et al., 1998). Coaddition of saturating amounts of both BDNF and NT3 does not increase subplate neuron survival above that with either factor alone (Fig. 3 c ). Therefore, BDNF and NT3 must act on the same population of neurons, possibly via similar mechanisms. Figure 1. Subplate neurons, but not neurons of the cortical plate, express p75NTR. Localization of mRNA for p75NTR, TrkA, TrkB, and TrkC on parasagittal sections of E17 rat brain. For localization of the subplate neurons, comparable sections were stained with cresyl violet or with an antibody to BrdU. BrdU was given at E12 to label earliest-generated subplate and mar- ginal zone neurons. Heaviest BrdU- labeled cells are restricted to marginal zone and the subplate. Note that sub- plate neurons are the only p75NTR- expressing neurons at this age. m , Me- ninges; MZ , marginal zone; CP , cortical plate; SP , subplate. Scale bar, 100 m m.
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