Preservation of α5 GABAARs in CA1 pyramidal cells and 3 sub-types of interneurons in the AD model
Using immunofluorescence and confocal microscopy analysis in the CA1 region of the hippocampus, we investigated α5 subunit-containing GABAAR expression in three sub-types of modulatory inhibitory interneurons, CR-, SST- and CCK-expressing interneurons, as well as in pyramidal cells (stained for CaMKII-α) in theAPPNL-F/NL-F mouse model and wild-type animals (Figure 3(A-D)). The imaged area in each case is shown in Figure 3(E).
This was measures in three different ways, quantification of the total intensity of α5 signal in CA1 measured from confocal Z-stacks, followed by the quantification of α5 expression from individual cell populations measured from their somata and dendrites, using Pearson’s correlation coefficient R with Fisher’s transformation.
We also quantified the total intensity of α5 signal in CA1 confocal Z-stacks and observed no differences in the AD model compared to wild-type (P > 0.05, n =5 wild-type animals and 6 APPNL-F/NL-F animals), suggesting a preservation of α5 expression in theAPPNL-F/NL-F animals.
The α5 subunits expressed on all three interneuron subtypes were analysed further from somata of the different cell types (Figure 3 (F)). There was no significant change in α5 expression on CR cells inAPPNL-F/NL-F animals compared to wild-type (only a slight increase of 11.86 ± 3.14 %, P > 0.05, n =6 wild-type animals and 7APPNL-F/NL-F animals). Similarly, there was no change in the expression of α5 expression in SST or CCK interneurons between wild-type and APPNL-F/NL-F mice (only insignificant changes of; 27.35 ± 12.61 % and 36.09 ± 12.45 % observed in SST and CCK cells, respectively, inAPPNL-F/NL-F animals compared to wild-type animals, P > 0.05, n =6). Thus, the three interneuron subtypes studies showed no significant differences in α5 subunit expression between wild-type animals andAPPNL-F/NL-F animals, highlighting a preservation of the α5 subunit in AD.
Analysis of CaMKII-α and α5 co-staining (Figure 3 (F)) showed no significant differences in the expression of α5 expression on the pyramidal cells in APPNL-F/NL-F animals compared to wild-type (P > 0.05, n =5). This observation is consistent with previous studies, which reported α5 expression on pyramidal cells (Brünig et al, 2002).
Next, we investigated the expression of the α5 subunit on CR, SST, and pyramidal cell dendrites (Figure 3 (G)), as the subunit has been reported to be located postsynaptically at dendritic sites where presynaptic CR cells target SST interneurons (Magnin et al., 2019) and on postsynaptic dendrites of pyramidal cells (Ali and Thomson, 2008). CCK cells also receive input-from dendrite-targeting interneurons (Ali, 2007), but their dendrites could not be investigated in detail here, due to the unavailability of specific anti-CCK anitbody that shows good expression of CCK in dendrites in mouse tissue. We investigated up to 5 cells in each animal, and observed no significant difference in the α5 expression between the genotypes or neuron subtypes in their dendrites (P > 0.05, one-way ANOVA with post-hoc Tukey’s test for multiple comparisons).