PMC

Quantitative analysis of dendritic BC1 delivery in hippocampal neurons developing in culture. The number of cells analyzed for each time point was as follows: 2 DIV, 214; 4 DIV, 187; 7 DIV, 171; 9 DIV, 200; 11 DIV, 298; 14 DIV, 219; 21 DIV, 238.

Development of somatodendritic BC1 expression in hippocampal neurons in culture. Cells were grown at medium density (6,000 cells per cm²) for 2 d in vitro (DIV; A and B); 4 DIV (C and D); 7 DIV (E and F); 9 DIV (G and H); and 14 DIV (I and J). (Left) Dark-field photomicrographs; (right) DIC photomicrographs. Arrows indicate axonal (B) or dendritic (D) growth cones. Bar, 50 μm.

Localization of BC1 RNA to dendrites of mature hippocampal neurons in culture. Cells were probed for BC1 RNA by in situ hybridization; labeling signal is indicated by autoradiographic silver grains (white in dark-field photomicrographs A, C, and D). (A and B) BC1 labeling signal is observed over somata and dendrites, but not over axons (black arrows in DIC photomicrograph B) of hippocampal neurons in culture. Labeling was discontinuous over some dendrites: gaps are indicated by white arrows in A. A higher power photomicrograph (C) reveals clustering (white arrowheads) of the BC1 labeling signal over dendrites. (D) Sense strand control experiments did not produce significant labeling over either cell bodies (white arrowheads) or neurites. (A, B, and D) Bar, 50 μm; (C) bar, 25 μm.

Regulation of BC1 expression by neuronal activity. Hippocampal neurons were grown in culture for 14 d in the absence of TTX (A–C), in the presence of 1 μM TTX (D–F), or in the presence of 1 μM TTX for the first 9 d, and in the absence of TTX for the following 5 d (G–I). Left (A–G): BC1 RNA, dark-field photomicrographs; middle (B–H), BC1 RNA, phase contrast photomicrographs, corresponding to dark-field photomicrographs in left column; right (C–I): 7SL RNA, dark-field photomicrographs. Dark field photomicrograph D (BC1 RNA in the presence of TTX) was overexposed to reveal absence of any significant labeling over cells or neurites. All cultures were grown at medium density. Bar, 50 μm. (J) Cultured hippocampal neurons on coverslips were exposed to autoradiographic film. Autoradiographs show BC1- and 7SL-labeling signals for cells that were grown for 14 d in the absence of TTX (1), in the presence of 1 μM TTX (2), or in the presence of 1 μM TTX for the first 9 d and in the absence of TTX for the following 5 d (3). (4) Sense strand control.

Expression of BC1 RNA in cultured hippocampal neurons developing at different cell densities. Hippocampal neurons were grown for 7 d in culture and were double-labeled for BC1 RNA (in situ hybridization; green) and synaptophysin (immunocytochemistry; red). A green filter was used for visualizing autoradiographic silver grains in double-exposure photomicrographs in order to differentiate them from the red immunofluorescence signal. (A and B) 1,000 cells per cm²; (C) 4,000 cells per cm²; (D) 16,000 cells per cm². At low density, beginning BC1 expression can be observed in a few cells (B) while the majority shows no labeling (A). At medium density (C), BC1 labeling was often observed over proximal segments of dendrites that were lined with synaptophysin puncta. At high density (D), BC1 labeling was frequently observed over dendrites at considerable distances from somata. Bar, 25 μm.