Pyridostatin So far several methods have been used
So far, several methods have been used to monitor the level of GJC and its regulation in astrocytes studied in acute Pyridostatin slices (see Giaume et al., 2012). This mainly includes approaches based either on electrophysiological properties of gap junction channels or on “dye coupling” experiments that consist in injecting into one astrocyte a membrane non-permeable molecule with a low molecular weight (<700–800 Da such as Lucifer yellow, sulforhodamine B, biocytin, …) that diffuses with time to neighboring coupled cells. In astrocytes, the double patch-clamp technique allows recording single-channel junctional currents in cultured cells (Giaume et al., 1991; Dermietzel et al., 1991) as well as monitoring macroscopic junctional currents in acute slices (Meme et al., 2009; Zhong et al., 2016), while dye injections visualize astroglial and panglial networking (Blomstrand et al., 2004; Houades et al., 2006, Houades et al., 2008; Roux et al., 2011; Griemsmann et al., 2015). Alternatively, the intercellular diffusion of dyes can also by studied by adding Lucifer yellow in the external solution during the slicing procedure (Menezes et al., 2000) or by applying sulforhodamine 101 (SR101) either in vivo on the pial surface (Nimmerjahn et al., 2004) or by intravenous injection (Appaix et al., 2012). However, these methods suffer from some limitations. Although dual patch clamp recording and dye injections can be routinely performed in slices from juvenile animals, they become difficult in the adult. Alternatively, approaches based on dye loading, mainly SR101, after deposition on the pia or passage through the BBB can be used in vivo and in adults, are difficult to control from one experiment to the other. This strongly jeopardizes the comparison of GJC levels in different conditions. Interestingly, non-invasive techniques based on the gap-Fluorescent Recovery After Photobleaching (gap-FRAP) (Delèze et al., 2001) and its derivative the Local Activation of Molecular fluorescent probes (LAMP) (Yang and Li, 2009) have been developed in cultured cells. They consist in loading a population of coupled cells with a cell-permeable and photoactivatable fluorophore, then photolyzing locally the dye in one cell and recording fluorescence recovery. We have adapted the gap-FRAP technique to study astroglial GJC in acute hippocampal slices from old mice. Indeed experiments were performed at 9 months, an age at which very few analysis of GJC has been reported in the literature due to the difficulty to apply classical double patch-clamp recording or dye coupling experiments in acute brain slices (see Discussion). In order to load solely astrocytes, we took advantage of sulforhodamine 101 (SR101), a fluorescent low molecular weight (607 Da) dye that is selectively taken up by astrocytes and passes through gap junction channels (Nimmerjahn et al., 2004; Schnell et al., 2015).
Results A gene profile study from CNS cells has identified Aldh1L1 as an astroglial specific marker in the CNS (Cahoy et al., 2008). In the present study, adult Aldh1L1-eGFP transgenic mice were used to detect astrocytes in the CA1 area of the hippocampus (Fig. 1A1, B1 and C1). Using 9 month-old mice, we found that eGFP-expressing cells in Aldh1L1-eGFP mice were always characterized by a soma size <10 μm and a typical rich arborization (Fig. 1C1) defining the so-called astroglial domain as previously reported (Yang et al., 2011; Zhong et al., 2016). In addition, no cell with a neuronal morphology was identified based on the presence of eGFP fluorescent signal. Similar observations were made using the fluorescent dye SR101 (Fig. 1A2, B2 and C2) for astrocyte loading (Nimmerjahn et al., 2004). Consequently, as illustrated in Fig. 1, we examined the co-localization of eGFP with SR101 staining. Quantification of dual staining for eGFP and SR101 (Fig. 1A3, B3 and C3) in the adult mouse hippocampus indicated that 93 ± 1% (n = 809 cells from 4 mice) of SR101-positive cells were eGFP-positive and 99 ± 1% (n = 754 cells from 4 mice) of eGFP-positive cells were also SR101-positive. These numbers are in agreement with previous studies using SR101 staining in the hippocampus of young mice expressing eGFP driven either by hGFAP (Schnell et al., 2012) or Aldh1L1 (Zhong et al., 2016) (see also Nimmerjahn et al., 2004). Consequently, we concluded that SR101 is a reliable and specific marker for the identification and loading of adult astrocytes in the CA1 region, and that this marker can be used for gap-FRAP functional study of GJC in hippocampal astrocyte networks.