Fig. 6. Caspases are not required for the death of GDNF-deprived enteric neurons. (A) Enteric neurons from distinct gut regions respond differentially to GDNF deprivation. Enteric neurons isolated from mouse E15.5 colon and small intestine were cultured for 2 days and then switched to GDNF-deprived conditions. The ratio of surviving neuron numbers (48 hours after GDNF deprivation) to initially plated cell numbers is shown. (B) Timecourse analysis of colonic neuron survival. TuJ1-positive neurons were counted at 0, 12, 24 and 48 hours after GDNF deprivation (-) or in the presence of GDNF (+). (C) Nuclear staining by Hoechst 33342 (upper panels) and TUNEL (lower panels) of enteric neurons 12 hours after GDNF deprivation (left panels) or of SCG neurons 24 hours after NGF deprivation (right panels). Condensed chromatin and nuclear fragmentation associated with strong TUNEL-reactivity, salient features in dying SCG neurons (arrowheads in right panels), were never observed in GDNF-deprived enteric neurons (arrowheads in left panels). (D) Treatment with the broad-range caspase inhibitor zVAD-fmk (100 µM) rescues NGF-deprived SCG neurons (right) but not GDNF-deprived enteric neurons (left). Survival of enteric neurons and sympathetic neurons was examined 2 days after GDNF deprivation or 5 days after NGF deprivation, respectively. (E) Responses of Bax-deficient enteric neurons to GDNF withdrawal. No significant differences were observed in the survival of colonic neurons between Bax-deficient and wild-type embryos (E15.5). Values represent the percentages of living cells normalized to the GDNF-maintained neurons. (F) Bcl-X_L overexpression rescues the survival of GDNF-deprived enteric neurons. Enteric neurons were infected with lentivirus expressing Bcl-X_L or vector alone several hours after plating. Surviving neurons (TuJ1⁺) were counted 48 hours after GDNF deprivation. Scale bar: 10 µm in C. Co, colon; SI, small intestine; WT, wild type.