A single cell death is disruptive to spontaneous Ca2+ activity in astrocytes

Abstract

Astrocytes in the brain are rapidly recruited to sites of injury where they phagocytose damaged material and take up neurotransmitters and ions to avoid the spreading of damaging molecules. In this study we investigate the calcium (Ca²⁺) response in astrocytes to nearby cell death. To induce cell death in a nearby cell we utilized a laser nanosurgery system to photolyze a selected cell from an established astrocyte cell line (Ast1). Our results show that the lysis of a nearby cell is disruptive to surrounding cells' Ca²⁺ activity. Additionally, astrocytes exhibit a Ca²⁺ transient in response to cell death which differs from the spontaneous oscillations occurring in astrocytes prior to cell lysis. We show that the primary source of the Ca²⁺ transient is the endoplasmic reticulum.

Keywords: Ca2+; astrocyte; calcium; cell death; laser; photolysis; spontaneous activity.

Figure 1

Spontaneous activity is disrupted in response to a single cell death. (A) Spontaneous vs. photolysis induced transients. The frequency (Hz) of spontaneous oscillations decreases following a photolysis from 0.033 + 0.02 to 0.020 + 0.001. N = 40 for Spontaneous and N = 54 for post lysis spontaneous. The amplitude and area of spontaneous oscillations also decreases after a cell death. The duration (s) increases. ****p ≤ 0.0001, ***p < 0.001, **p < 0.01. (B) Spontaneous oscillations vs. photolysis transients. Some cells did not show spontaneous activity before a photolysis transient and were separated and labeled as “not active.” In the graph, spontaneous refers to the Ca²⁺ characteristics of the spontaneous oscillations. First photolysis active refers to the Ca²⁺ transient in cells with previous spontaneous activity. First photolysis not active is for cells that had no previous spontaneous activity. Spontaneous post photolysis are spontaneous Ca²⁺ oscillations which occurred after a photolysis. ****p ≤ 0.0001, ***p < 0.001, **p < 0.01. Spontaneous oscillations: N = 81, 1st photolysis active: N = 33, 1st photolysis not active: N = 12, Spontaneous post-photolysis: N = 72.

Figure 2

Extracellular Ca²⁺ does not contribute to the transient observed after a photolysis. (A) Cells were treated with the NMDAR inhibitor MK801 (10 μm). No statistical differences were found for amplitude, duration or area under the curve. Control: N = 23, MK801: N = 45. (B) Cells in medium without Ca²⁺ in 1M EGTA did not show any statistical differences in amplitude, duration (FWHM) or area. Control: N = 24, EGTA: N = 36. (C) Ca²⁺ traces of cells treated with BAPTA AM did not show a photolysis transient. Vertical lines depict the time at which a photolysis occurred. Control: N = 68, BAPTA AM: N = 23. (D) Treatment with Thapsigargin, 2-APB or Apyrase affected the ability of the cells to have a cytosolic Ca²⁺ transient. ****p ≤ 0.0001, *p < 0.05 Thapsigargin: Control N = 46, Drug N = 19. 2-APB: Control N = 49, Drug N = 20. Apyrase: Control N = 33, Drug, N = 56.

Figure 3

Ryanodine receptors contribute to the ER release of Ca²⁺following a photolysis. (A) Amplitude, duration (FWHM) and area of the transient for cells treated with Ryanodine. ****p ≤ 0.0001, ***p < 0.001 Control: N = 78, Ryanodine: N = 87. (B) Amplitude and time to peak plotted by distance to lysed cell. (C) FWHM of transient plotted by distance to lysed cell.