Figure 7. Axonal CREB is required for CRE-dependent transcription and NGF-mediated DRG survival.

(a) DRG axons in compartmented cultures were transfected with CREB siRNA in the axon compartment only, and CREB levels were detected using a CREB antibody. Axons crossing the compartment divider were retrogradely labelled with WGA-Alexa488. All compartments were maintained in NGF-containing media throughout the experiment. Scale bar, 50 μm. (b) Quantification of data in (a). Immunofluorescence levels in each compartment were normalised to fluorescence signals from cultures treated with non-targeting siRNA. *p<0.0001. Numbers on bars represent n axons per condition. (c) DRGs in compartmented cultures were incubated in NGF-free media, supplemented with BAF to suppress apoptosis. Axon compartments were treated with CREB-specific or non-targeting siRNA. After 48 h, 30 ng/ml NGF was added to the axon compartment for 20 min, after which pCREB levels in nuclei were quantified by immunofluorescence. *p=0.0004. Numbers on bars represent n cells per condition. (d) DRGs in compartmented chambers were infected with adenovirus encoding luciferase under the control of a CRE transcriptional element and treated with NGF as in (c). *p<0.001. Numbers on bars represent n cells per condition. (e) E15 dissociated DRG were cultured in compartmented chambers as in Fig. 1c. NGF-induced neuronal survival at DIV7 was assayed following transfection of control or CREB-specific siRNA into the axon compartment at DIV5. *p<0.001. Numbers on bars represent n cells per condition.