Common inducers of autophagy in non-neuronal cells fail to stimulate autophagy in primary neurons. Relative intensities of LC3-I and LC3-II bands reflect levels of autophagy. (A) Bafilomycin A₁ (bafA; 4 h, 1 nM) induced LC3-II accumulation in striatal and cortical neurons and in HeLa cells. (cont), control untreated cells. (B) Striatal and cortical neurons were incubated in Hanks' solution. Starvation (2 h in Hanks' solution) induced autophagy in HeLa cells but not neurons. Longer incubations gave similar results. (C) Pretreatment with rapamycin (2 μM) blocked BDNF-induced phosphorylation of p70S6K in striatal neurons. (D) Striatal and cortical neurons incubated in medium with the mTOR inhibitors 2 μM rapamycin (rap) or everolimus (everol) for 48 h. Shorter or longer incubations and higher and lower concentrations (2 nM to 20 μM) gave similar results (not shown). Rapamycin (2 μM, 24 h) induced autophagy in HeLa cells. (E) Lithium chloride (LiCl, 10 mM) induced autophagy in COS-7 cells but not neurons. (F) Autophagy inducers did not increase the flux through the autophagic pathway. In striatal neurons, inhibition of lysosomal degradation with bafilomycin A₁ (overnight) led to LC3-II accumulation (compare lanes 1 and 2). Starvation of these treated cells did not increase LC3-II levels (compare lanes 2, 3, and 4). Incubation of these treated cells with 2 μM rapamycin (compare 2, 5, 6), 2 μM everolimus (compare 2, 7, 8), or 10 mM LiCl (compare 2, 9, 10) did not increase LC3-II levels. (G) Striatal and cortical neurons were incubated in medium with niguldipine (nig, 4 μM), trifluoperazine (3F, 8 μM), or loperamide (lop, 5 μM) (overnight). Note LC3-II accumulation. (H) Starvation (starv, Hanks' solution, 8 h), rapamycin (rap, 2 μM, 24 h), and lithium chloride (LiCl, 10 mM) induced autophagy in astrocytes.