Figure 3. Memory tests and immunoblotting analysis: postsynaptic protein levels were decreased in hippocampus of polypharmacy fed mice. (A) Y maze test: graphs indicate the number of entries and percentage of spontaneous alternations performed by control and polypharmacy mice. Note that while 90% of control animals alternated above the 50% chance level, only 30% of polypharmacy mice did so, thus indicating a possible impairment in spatial working memory. *p<0.05, t-Student test. (B) NOR test (day 3, test day): histograms on the left show average of exploration time spent on the two objects, for control and polypharmacy group, *p<0.05; the statistical comparison between time spent on novel object vs familiar in control or polypharmacy group was analyzed with paired t-Student test. The respective heatmaps at the bottom visually represent the area explored around the objects, highlighting a clear preference for the novel object in both the groups (in red color the most visited zones). Dot graph shows the calculated discrimination index, where a score above 0 indicates that animals spent more time exploring the novel object than the familiar one. (C) Contextual FC test: percentage of freezing time measured on day 1 (habituation phase) and day 2 (context testing). ***p<0.001, two-way ANOVA repeated measurements test. (D–F) Hippocampal tissue samples from control and polypharmacy mice were analyzed by western blotting experiments. Dot plots show quantification of NMDAR1, phospho-NMDAR2A and PSD95 protein levels, which were significantly decreased in polypharmacy mice compared to control animals; *p<0.05, **p<0.01; t-Student test. Total protein levels were normalized with respect to α tubulin. On the right, representative examples of immunoblots for NMDAR1, phospho-NMDAR2A and PSD95 proteins. All data are presented as mean ± SEM. Animals per group: n= 9 control, 10 polypharmacy. Ctrl= control, Poly= polypharmacy.