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  • Thyroid Hormone Receptor Antagonist (1-850) By using pharmac

    2021-10-21

    By using pharmacological treatment with CBX or transgenic animals lacking the two major astroglial Cxs, the Double KO mouse, we demonstrated that the gap-FRAP technique was able to detect the total inhibition of GJC with a reduction of about 39% and 38% compared to control, respectively. The residual resting fluorescence was interpreted as the return of non-bleached molecules of SR101 from distant processes of the targeted astrocyte located outside the bleached zone. Based on this statement, it is expected that, using CDCF for cell loading (see Cotrina et al., 2001), this component that will also include neuronal processes and endothelial Thyroid Hormone Receptor Antagonist (1-850) present in the bleached zone, will be largely increased and thus will reduce the signal-to-noise ratio. Using single astroglial Cx knock out, Cx43KO and Cx30KO, we demonstrated that the gap-FRAP was enough sensitive to detect changes in the level of GJC. The lack of Cx43 was found to reduce by 21% fluorescence recovery compared to control condition, while the absence of Cx30 was associated to a 15% reduction. These data indicate that Cx43 contributed to 55% of GJC and Cx30 to 40%. Interestingly, these changes in GJC associated to each Cx are very close to those reported previously using patch-clamp recording and dye coupling experiments in 3 week-old hippocampal slices which established that Cx43 and CX30 contribute to 50% and 35% of GJC, respectively, with the same single Cx knockout mice (Rouach et al., 2008). In addition, in another study (Wallraff et al., 2006) in the Cx43KO it was reported that Cx30 accounts for 50% of coupling. Finally, we demonstrated that while fluorescence recovered in the bleached astrocyte, those located close to this target cell loosed fluorescence while those located far did not show fluorescence loss. These features are expected when considering the processes of gap junction-mediated diffusion involved using the gap-FRAP technic (see Delèze et al., 2001). A mathematical model based on experimental data obtained along our study was also carried out to validate the gap-FRAP technique. To reach this aim, we considered a two-dimension model that takes into account two compartments in the targeted cell, the central part that will be bleached and the distal processes that are out of this zone. Our model-based estimation of the apparent intracellular diffusion coefficient of SR101 in astrocytes is 3.0 μm2/s in control conditions and 1.3 in CBX-treated cells. Albeit the diffusion coefficient of SR101 has thoroughly been measured in water (in the [200,400] μm2/s range, Gendron et al., 2008), quantitative studies of its value in living cells are very rare. In rod photoreceptors, SR101 diffusion coefficient was estimated to around 6 μm2/s (Chen et al., 2002). Given that astrocyte processes are expected to be highly tortuous (Rusakov, 2015), our estimations fall within the expected range of values. Inspecting the fitting of experimental recovery curves with those given by the model, we were able to distinguish two components in the recovery, one rapid and one slower. This was the case when using data from control (wild type), KO Cx43 and KO Cx30. In these cases, the rapid component was associated to the recovery due to SR101 molecules coming from the non-bleached distal processes of the targeted cells while the second component was associated to SR101 molecules contributed by adjacent cells through gap junction channels. The difference between KO and control mice was that in KO astrocytes, the slow intercellular component brought less SR101 in the bleached zone, thus confirming a weaker coupling. With the Double KO, half of the cells exhibited exponential fit results identical to those obtained with CBX, as intuitively expected. For the other half however, the fit results were close but not identical to single KOs. We cannot exclude that such discrepancy was due to alterations of morphology and physiology of Double KO astrocytes, reported as hypertrophic and reactive compared to wild type (Pannasch et al., 2011), a situation that was not taken into account in our model. However, it should be kept in mind that in the Double KO the lack of coupling is permanent during all animal life, while CBX-induced uncoupling only results from short time exposure and in this case morphological changes have not been studied so far. Finally, the analysis of recovery and our mathematical model indicated that the apparent diffusion coefficient of SR101 within the distal processes of the photo-bleached cell is strongly reduced in the presence of CBX. Our interpretation is that this reduction of apparent diffusion is caused by a global increase of the intracellular tortuosity of the astrocytes with respect to SR101. We can however only speculate on the cause of such a CBX-induced increase of intracellular tortuosity. For instance, this could be due to CBX-induced changes in the cell or process morphology. Alternatively, it could be due to the inhibition by CBX of reflexive gap junctions, i.e. the gap junctions between processes belonging to the same astrocyte. Indeed, the occurrence of gap junctions between astroglial processes of the same cell has been evidenced by electron microscopy data (Wolff et al., 1998; Genoud et al., 2015)