FIGURE 11.

Blockade of KCa3.1 attenuated Aβ-induced, indirect, astrocyte-mediated damage to dendrites and synapses. Levels of dendritic and synaptic markers were compared between hippocampal neurons treated with solvent only (control treatment), 5 μM Aβ, Aβ-CM, or CM from astrocyte cultures in which Aβ-induced activation was inhibited for 24 h by TRAM-34 (TRAM-34 + Aβ-CM). A Cellomics KineticScan HCS Reader was used to image the primary cerebral neurons. (A) For better visualization of individual dendrites, dendrites of the sparsely plated neurons were immunostained with MAP2 (red), and nuclei were stained with DAPI (blue). Extended Neurite Outgrowth bioapplication software was used to analyze (B) neurite length and (C) branch point counts. Data represent mean ± SEM (n = 3). ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared with Aβ-CM. ^##p < 0.01 compared with control. Aβ-CM treatment significantly reduced neurite length and branch point counts. This reduction was prevented by inhibition of astrocyte activation by TRAM-34 (1 and 10 μM; n = 3). (D) Western blot analysis with the following primary antibodies: neuronal marker NeuN, dendritic marker MAP2, post-synaptic proteins PSD95, and pre-synaptic protein synaptophysin. (E) Results are presented as mean ± SEM (n = 3). ^#p < 0.05 compared with con-CM. ^∗p < 0.05 compared with Aβ-CM. CM, conditioned medium.