Figure 9.

Integrity of F-actin is critical for SP-mediated caffeine-induced increase in GluR1 in spine. A, Latrunculin causes a large reduction in rhodamine-phalloidin staining of F-actin. Top, Control neuron transfected with GFP for imaging morphology, and stained with phalloidin, which labels primarily dendritic spines. Bottom, 2 h of exposure to latrunculin produced a highly significant reduction in phalloidin staining (bar graph below, averages of 50 spines sampled from five cells each). Scale bar, 5 μm. B, In the same dendritic segment, transfected with GFP-SP and DsRed for morphology, imaged before (top) and after (bottom) 2.5 h of exposure to latrunculin A (20 μm). Green arrowheads point to persistent spines; white arrowheads point to modified (removed, elongated) spines. C, Summary diagram of the proportion of persistence among SP(+) and SP(−) spines, indicating a clear correlation between the persistence of spines and the presence of SP puncta. D, E, Latrunculin does not affect the ability of caffeine to induce a rise of [Ca²⁺]_i in dendrites and spines. D, Consecutive responses of the same dendritic segment to a pulse application of caffeine applied before, 1.5 h, and 2 h after exposure to latrunculin. E, Averaged responses of five fields in two cells to caffeine, before and 2.5 h after the onset of exposure to latrunculin. F–H, Exposure of control (F) and latrunculin-treated (H) cultures to caffeine causes a change in GluR1 puncta, such that in the control there is a rise in spine GluR1 fluorescence (green arrowheads), while in the toxin-exposed culture the density of GluR1 was reduced significantly. In both F and H cultures, the three images were taken before (left), after 1.5 h of caffeine (middle), and in retrospective immunocytochemistry (immuno) for SP (blue arrowheads) (right). G, The difference between control and latrunculin-treated cultures was highly significant.