Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • Initially we carried out gap FRAP

    2022-08-03

    Initially, we carried out gap-FRAP experiments using the two-photon configuration of the confocal microscope. A SR101-positive astrocyte was targeted and a 5 × 5 μm square area was exposed to a laser illumination (5 flashes of 5 s each during 25 s). This procedure resulted in a bleaching of this astrocyte as indicated by a reduction in the recorded fluorescence level. With time, a recovery of fluorescence was observed and after 8 min it stabilized to a fluorescent level corresponding to 74 ± 4% (n = 6) of the basal fluorescence level recorded before bleaching (with a percentage of recovery 64 ± 3% obtained by subtracting the background after bleaching) (Fig. 3A1 and A2). When the gap junction channel inhibitor carbenoxolone (CBX, 200 μM) was applied 15 min before, during and after the laser pulses, the level of fluorescence recovery was reduced by 24 ± 3%, n = 4 (39 ± 3% of the recovery level compared to control condition) (Fig. 3A2). These results indicated the occurrence of a reduction in GJC between the bleached astrocyte and its neighbors. However, as CBX used at this concentration is well known to totally abolish GJC in hippocampal astrocytes studied in acute slices (see for instance Wallraff et al., 2004; Rouach et al., 2008) the sensitivity of the two-photon configuration was not considered as satisfactory. In fact, in this configuration, the thickness of the laser illumination was very limited in the Z axis (Fig. 2A1 and B1), hence the poor resolution of the GJC component in the recovery phase was interpreted as the consequence of a low proportion of SR101 bleached Isochlorogenic acid C in the targeted astrocyte compared to the total amount of SR101 molecules contained in its cytoplasm, including the soma and the primary processes (Fig. 2A1 and B1). In order to increase the proportion of SR101 molecules available for photolysis, we decided to use the same confocal microscope but in a one-photon configuration which results in a much larger thickness of the laser stimulation in the Z axis (Fig. 2A2 and B2) and consequently in a higher number of SR101 bleached molecules, in particular in the soma. Moreover, a larger square area with the size 15 × 15 μm targeting a SR101-positive astrocyte was exposed to a laser illumination (15 flashes of 0.684 s each during 10.26 s). By using the one-photon configuration, the recovery level in control condition is 65 ± 3% (n = 5), while negative feedback is only 23 ± 4% (n = 4) in presence of CBX, so the CBX-sensitive component is 42± 3% (n = 4) (Fig. 3B1 and B2). Fig. 4 illustrates examples of gap-FRAP experiments in SR101 loaded populations of hippocampal astrocytes using the one-photon configuration of the microscope. In control condition, after selection of a targeted SR101-positive astrocyte (Fig. 4A, left image) in the CA1 region, fluorescence disappeared after the laser bleaching in the soma (Fig. 4A, middle image) and reappeared progressively with time after laser illumination (20 min, Fig. 4A, right image). In contrast, when the slice was pre-incubated with CBX (200 μM) the recovery of fluorescence monitored after bleaching was much weaker (sequential images in Fig. 4B taken at similar time than in Fig. 4A). Quantification of these two conditions is illustrated in Fig. 4C. We also noted that fluorescence recovery appeared faster with CBX than in control condition. Then we took advantage of knock out mice for the two major Cxs expressed in astrocytes, i.e. KO Cx43 and KO Cx30 (Ransom and Giaume, 2013), as well as the Double KO mice (KO for both Cxs, see Methods) to obtain conditions in which GJC is partly or totally abolished. Using these animals we observed that the recovery after photo-bleaching was reduced by 21 ± 3% (n = 12) for the KO Cx43, 15 ± 3% (n = 10) for the KO Cx30 and 38 ± 3% (n = 9) for the Double KO compared to the recovery value in control condition, i.e. wild type. These values were determined by subtracting the plateau value measured at t = 20 min after bleaching to the value measured immediately after bleaching. In this set of experiments the recovery for hippocampal slices from wild type mice was 65 ± 3% (n = 5) (Fig. 4D). It is noteworthy that the inhibition of fluorescence recovery was the same for CBX-treated wild type mice and the Double KO mice (Fig. 4C and D). Altogether, these results indicate that gap-FRAP applied in the one-photon configuration allows assessing the level of GJC between astrocytes in various situations.