Calcium responses in astrocytes on activation of nAChRs. (A) Fluo-3 fluorescence of a single astrocyte after a 2-s application of 100 μM ACh. Images shown were acquired before (0 s), and 1 s, 3 s, 30 s, and 65s after ACh application. (Bar = 10 μm.) (B) Averaged calcium transients from 31 cells (1 mM ACh) and 42 (100 μM ACh) fluo-3-loaded astrocytes. The averages are not normalized and represent actual increase in fluorescence above baseline. Similar calcium transients were obtained with both agonist concentrations. (C) Calcium response of an astrocyte in the absence (Control) and presence (+MLA) of 10 nM MLA. The third trace (Recovery) shows the ACh response after a 5-min wash to remove the antagonist. MLA reversibly blocked the ACh response. (D) Changes in calcium transients in presence of 100 nM α-Bgt. Cells were preincubated for 30 min with the toxin before application of 100 μM ACh. The lack of response of an astrocyte to ACh application is shown (lower trace). The upper trace (KCl) shows the response of the same cell to a 2-s application of 75 mM KCl, indicating that calcium responses to other stimuli were unperturbed. Cells were loaded with Fluo-3 acetoxymethyl ester for 30 min before being challenged by the agonist. ACh was applied for 2 s as indicated by the arrows, and the cell was imaged at 0.3 Hz. Atropine (500 nM) was present at all times.

αBgt-AChR-mediated calcium response is primarily caused by CICR. (A) Depletion of all intracellular calcium stores by TG abolished response to ACh. Cells were incubated in 1 μM TG for 30 min, washed, and then challenged with 20 mM caffeine (Caffeine) to deplete any residual store calcium. Subsequent application of ACh lead to a small increase in calcium, on average about 2% of control, indicating that most of the rise in cytosolic calcium is due to release from intracellular stores. Data are the mean response from 28 cells. (B) InsP₃ stores contribute to ACh-induced calcium transient. Untreated astrocytes (Control) or astrocytes treated with 20 μM Xe-C for 30 min (Xe-C) were challenged with 100 μM ACh for 2 s. ACh elicited a large calcium transient in Xe-C-treated cells that decayed faster than that obtained from untreated cells. Data are the mean ± SEM from 38 cells (Xe-C) and 42 cells (Control). (C) Caffeine stores are necessary for αBgt-AChR-mediated calcium response. Application of 20 mM caffeine for 2 s depleted calcium stores. Application of ACh 90 s later produced a small response as did a second application of 20 mM caffeine, suggesting that αBgt-AChRs and caffeine share a common pool of intracellular calcium. Data are the mean transient from 38 cells. (D) ACh response can be blocked by ryanodine. Cells were incubated with 100 μM ryanodine for 30 min to block ryanodine receptors. Cells were challenged with 20 mM caffeine for 2 s, followed 5–7 min later by 100 μM ACh for 2 s. Responses to both ACh (lower trace) and caffeine (upper trace) were blocked, suggesting that the rise in cytosolic calcium resulting from activation of αBgt-AChRs is indeed amplified by CICR. Data are the mean response from 40 cells. Basal F/F_Mn levels after incubation with TG and ryanodine were 1.02 ± 0.04 and 1.1 ± 0.05, respectively. All responses were in the presence of 500 nM atropine.

Astrocytes exhibit nicotinic cholinergic current responses. (A) A 300-ms application of 1 mM ACh generated a fast desensitizing response (lower trace). This response is absent when the agonist was applied with 10 nM MLA, an αBgt-AChR-specific antagonist (upper trace). (B) Current responses to 1 mM and 100 μM ACh in the presence of atropine from different cells were normalized (peak current = 1) and averaged. Data from 11 cells (1 mM ACh) and 15 cells (100 μM ACh) are shown. ACh responses decay faster at the higher agonist concentration. (C) Current–voltage relationship from a single cell, obtained in the presence of 100 μM ACh, exhibits an inward rectification. Similar results were obtained from 4 cells. All experiments were done in presence of 100 nM atropine to block muscarinic receptors.

Dependence of the ACh response on extracellular calcium. (A) Response of a single astrocyte to a 2-s application of 100 μM ACh in the presence of normal calcium (2 mM Calcium), where external calcium was substituted with 0.5 mM EGTA; (0 mM Ca) and a second challenge with ACh in 2 mM calcium (Recovery). Removing extracellular calcium abolished the ACh-induced calcium response. Similar results were obtained from 17 cells. (B) Averaged responses to a 2-s application of 100 μM ACh in the presence of 100 μM CdCl₂. Robust ACh responses were observed in the presence of the VGCC blocker (+Cd). These responses were similar to those observed from untreated cells (Control). Data are the mean ± SEM from 40 cells (+Cd) and 42 cells (Control). ACh was applied in the presence of 500 nM atropine.

ACh response in the presence of other divalent cations. (A) Fluorescence response of an astrocyte to a 2-s application of 100 μM ACh in external medium containing no added calcium and 10 mM SrCl₂ (Sr). Response of the same cell to ACh application in the presence of 2 mM calcium (Ca). A robust response to ACh was observed in the presence of strontium comparable to that seen with calcium. (B) No transient was observed when ACh in 10 mM SrCl₂ was applied in the presence of 1 μM TG [compare the control response (Sr) with the response from a treated cell (Sr + TG)]. (C) Rapid imaging in the presence of various divalent cations. Agonist (100 μM ACh) was applied 1 s into the acquisition, for 25 s. Three hundred images were acquired on an average at 17 Hz. At the end of each experiment, a second acquisition was done at the same rate to determine the rate of photobleaching. All data were corrected for photobleaching. Data are the averaged response from 21 cells (Sr), 16 cells (Ca), and 25 cells (Ba). (y-axis bar = 50% ΔF/F0.)