Figure 2

Effects of NH₄Cl on intracellular calcium ([Ca²⁺]_i). (A) Sample images on neurons preloaded with Fluo-4-AM and sequentially exposed to different concentrations of NH₄Cl. (B) Fluo-4 fluorescence change (normalized to the pretreatment baseline) in response to different concentrations of NH₄Cl as indicated (n = 300 randomly selected synaptic boutons from 6 independent trials). (B1) In somatodendritic regions 5 mM NH₄Cl caused strong (~34.2%) but transient increase of Fluo-4 fluorescence. The response to subsequent 10 mM NH₄Cl application was smaller, whereas 50 mM NH₄Cl lead to a [Ca²⁺]_i increase comparable to that of 5 mM NH₄Cl but lasted even after the 5-min wash-off. (B2) [Ca²⁺]_i change in SypHTm-defined synaptic areas resembled that of somatodendritic areas. In both cases, a gradual decrease of Fluo-4 fluorescence occurred likely due to photobleaching and dye loss. (C) NH₄Cl-induced [Ca²⁺]_i changes during 0 Ca²⁺ or after thapsigargin pretreatment. (C1) The prolonged [Ca²⁺]_i increase (white circles) caused by 50 mM NH₄Cl was diminished with 0 Ca²⁺ (gray squares), and the resupply of 2 mM extracellular Ca²⁺ led to a large [Ca²⁺]_i increase with or without thapsigargin pretreatment (black triangles). For each group, 10 ROIs/FOV were randomly selected and 4 FOVs were analyzed. (C2) The transient [Ca²⁺]_i increase caused by 5 mM NH₄Cl was significantly reduced with 0 Ca²⁺ (gray squares), and the resupply of 2 mM extracellular Ca²⁺ led to additional [Ca²⁺]_i increase. Thapsigargin pretreatment eliminated those two [Ca²⁺]_i increases (black circles). For each group, 10 ROIs/FOV were randomly selected and 4 FOVs were analyzed. (D) Cytosolic pH changes upon various concentrations of NH4Cl were measured by ratiometric imaging of BCECF fluorescence (R = F₄₈₀/F₄₄₀, i.e. excited at 440 and 480 nm, and fluorescence imaged with 510 ± 10 nm band-pass emission filter), which was further normalized to the initial R₀ before any treatments. The cytosolic pH changes are very different from [Ca²⁺]_i change reported by Fluo-4 (B). (E) Spectrofluorometry scan of F_{Fluo -4} emission with 480 nm excitation in the presence of different concentrations of NH₄Cl as indicated. Four repeats for every concentration.