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. 2003 Jun 15;23(12):4967–4974. doi: 10.1523/JNEUROSCI.23-12-04967.2003

Figure 5.

Figure 5.

A, Oligodendrocyte-conditioned medium contains GDNF. Immunoblots of nonconditioned medium (N) are shown; astrocyte-conditioned medium (A), OCM1(1), and OCM4 (4) are blotted for antibodies against GDNF (representative blots of three independent experiments).B, GDNF production by astrocytes is not influenced by the maturation state of astrocytes. Immunoblots of medium conditioned by highly purified astrocytes were cultured for 1 DIV (A1) and 4 DIV (A4), blotted for antibodies against GDNF and showing comparable levels of GDNF are shown (equal amounts of protein loaded per lane; see Materials and Methods; representative blots of three independent experiments). C, GDNF staining within a differentiated oligodendrocyte. Immunocytochemical staining for GDNF within OPCs (left) and a differentiated oligodendrocyte (right) are shown. Phase contrast picture is shown with superimposed rhodamine-labeled GDNF staining of the same field, showing that differentiated oligodendrocytes contain GDNF. D–F, Neutralizing antibodies to GDNF reduce SMI312-positive process length per cell in neurons exposed to OCM4. D, Graph showing effect of OCM4 and OCM4 plus neutralizing antibodies to GDNF (20 μg/ml;4+aG) on length of SMI312-stained processes per live neuron in aged neurons (cultured for 120 hr and exposed to test conditions for 72 hr; expressed as a percentage of values obtained culturing neurons in B27 throughout) (n = 6; **p < 0.01). E, F, Representative immunomicrograph showing the effect of OCM4 (E) and OCM4 plus neutralizing antibodies (F) to GDNF (20 μg/ml) on SMI312 (phosphorylated neurofilament; green) staining in aged neuronal cultures. Nuclei stained with Hoechst are shown (blue). G, GDNF increases MAPK/Erk1/2 phosphorylation in aged neurons. Immunoblot showing MAPK/Erk1/2 phosphorylation in aged neurons (cultured for 120 hr) and exposed to DMEM/10% fetal calf serum (F), nonconditioned media (D), and nonconditioned media plus recombinant GDNF (10 ng/ml; G) for 30 min. Bottom panel, Phospho-Erk1/2 immunoblot. Top panel, Total-Erk1/2 immunoblot of the same membrane (representative immunoblot of four independent experiments).