Figure 5.

Glutamate does not alter the rate of endocytosis. (A) FM1–43 labels particles endocytosed through a clathrin-dependent pathway. Incubation of cultures with 1 μM FM1–43 causes distinct labeling around the perinuclear region of the cell body (A1). The particles labeled are not synaptic vesicles because they cannot be released on subsequent depolarization with high K⁺ solution (A1, 60 mM K⁺ solution for 1 min, which normally releases all FM1–43 contained in synaptic vesicles, data not shown). Uptake of FM1–43 into the cell body is blocked when cultures were preincubated with hypertonic sucrose solution (A2, 450 mM sucrose solution, total osmolarity was 750 mOsm, treated for 20 min before FM1–43 loading). [Scale bars: 10 μm (A1), 5 μm (A2).] (B) Glutamate does not alter the rate of uptake of FM1–43. The fluorescence intensity of FM1–43 inside the cell body increases roughly linearly with time, and this rate does not differ whether glutamate was present or not. To ensure that the optical section was selected correctly, the fluorescence intensity inside the nucleus was also monitored, which does not change significantly during the same time course (n = 15 cells from 15 experiments). (C) Glutamate does not alter the uptake of Tf. Uptake of Tf was examined with two incubation periods: 5 min or 10 min. In neither case does glutamate alter the uptake significantly. Uptake of Tf depends on a clathrin-mediated pathway because it is severely reduced by prior incubation with hypertonic sucrose solution (n = 26, 24, 29, 29, 14, 16 cells for Tf 5 min, AMPA + Tf 5 min, Tf 10 min, AMPA + Tf 10min, Tf 10 min, and sucrose + Tf 10 min, respectively, from five experiments).