Consistent with the action of Homer1a to compete with Homer1c, Homer1a expression reduced the coimmunoprecipitation (co-IP) of mGluR5 with Homer1c compared to GFP-expressing neurons (Figure 4F). We monitored Homer1a mRNA and protein in DIV 14 cortical neurons treated with TTX or bicuculline for 3 hr, 6 hr, 12 hr, 24 hr, and 48 hr (Figure 5A). Galunisertib price Bicuculline produced a time-dependent increase that was maximum at 6hrs for mRNA and 12 hr for protein (each ∼8-fold), and returned to basal levels at 48 hr. By

contrast, TTX treatment reduced Homer1a mRNA ∼5-fold by 24 hr and protein by ∼4-fold at 48 hr. To assess how Homer1a KO affects homeostatic scaling, we examined the surface levels of AMPARs after chronic TTX or bicuculline treatment. Biotinylation and IHC assays revealed an absence of homeostatic adaptations of GluA2/3 in Homer1a KO neurons (Figures 5B–5E). Homeostatic adaptations of GluA1 were significantly reduced in Homer 1a KO neurons, but not as strikingly disrupted as GluA2. Homeostatic adaptations of mGluR5 were not significantly different in Homer1a KO neurons. Disruption

of homeostatic scaling in Homer1a KO neurons was also evident in mEPSCs recordings (Figures 5F and 5G). In contrast to WT neurons where TTX resulted in an increase of mEPSC (WT-control Epacadostat mouse 20.9 ± 1.1 pA; n = 24 cells; TTX-treated 30.1 ± 2.2 pA; n = 15 cells, ∗∗∗p < 0.001), mEPSC amplitudes of TTX-treated Homer1a KO neurons

(31.4 ± 2.6 pA; n = 20 cells) were not significantly greater than untreated Homer1a KO neurons (28.9 ± 1.3 pA; n = 33 cells) (Figure 5G). Similarly, chronic bicuculline treatment reduced mEPSC amplitudes in WT neurons (14.1 ± 0.2 pA; n = 28 cells; ∗∗∗p < 0.001), but did not produce a significant decrease in mEPSC amplitudes in Homer1a KO neurons (27.2 ± 1.9 pA; n = 35 cells) compared to untreated Homer1a KO neurons. Comparison of Homer1a KO neurons treated with bicuculline versus TTX suggested a small difference but was not statistically significant below (27.2 ± 1.9 pA compared to 31.4 ± 2.6 pA; p = 0.19, not significant); this is dramatically different than WT neurons (14.1 ± 0.2 pA compared to 30.1 ± 2.2 pA). There was no difference in the frequency of mEPSCs between TTX-treated WT neurons (24.4 ± 2.6 Hz; n = 24 cells), bicuculline-treated WT neurons (22.2 ± 1.7 Hz; n = 28 cells), untreated WT neurons (23.4 ± 2.6 Hz; n = 24 cells), or similarly treated Homer1a KO neurons (TTX-treated, 24.9 ± 2.6 Hz; n = 20 cells; bicuculline-treated 27.6 ± 2.8 Hz; n = 35 cells; untreated 25.3 ± 2.9 Hz; n = 33 cells) (Figure 5G). These observations confirm that homeostatic changes of synaptic strength are markedly disrupted in Homer1a KO neurons.