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. 2017 May 30;8:15592. doi: 10.1038/ncomms15592

Figure 5. Pyk2 modulates glutamate-induced PSD-95 accumulation in dendritic spines.

Figure 5

(a) Hippocampal neurons were cultured for 3 weeks and treated for 15 min with vehicle or glutamate (Glu, 40 μM) without or with MK801 (MK, 10 μM), added 30 min before. PhosphoTyr402-Pyk2 (pY402-Pyk2), Pyk2 and α-tubulin as a loading control were analysed by immunoblotting. Molecular weight markers position is indicated in kDa. (b) Densitometric quantification of results as in a. One-way ANOVA (F(2,13)=8.02, P=0.005, n=4–7 per group) and post hoc Holm-Sidak's test for multiple comparisons. (c) Cultured hippocampal neurons were treated with vehicle or glutamate (40 μM) without or with MK801 (10 μM) for 3 h, fixed and labelled for PSD-95 immunoreactivity and rhodamine–phalloidin (an F-actin marker) to identify PSD-95-positive puncta localized in dendritic spines (arrows). (d) The size of these PSD-95-positive puncta was measured and analysed with one-way ANOVA (F(2,30)=15.37, P<0.0001, n=10–12 per group) and Holm-Sidak's test. (e) Hippocampal neurons from wild-type (WT) or Pyk2 KO mice were treated for 3 h with vehicle (Veh) or glutamate (40 μM) and immunostained for PSD-95. (f) The size of spine-associated PSD-95-positive puncta was measured in Pyk2+/+ and Pyk2−/− hippocampal cultures treated as in e and quantified (n=18–27 per group). Statistical analysis with two-way ANOVA (interaction F(1,89)=12.42, P=0.0007, glutamate effect, F(1,89)=1.84, P=0.18, genotype effect, F(1,89)=35.29, P<10−4) and post hoc multiple comparisons Holm-Sidak's test. In d,f, one–two dendrites per neuron from two to three independent experiments were measured. In b,d,f, data are means+s.e.m., *P<0.05, **P<0.01, ***P<0.001, as compared to vehicle-treated Pyk2+/+ cultures; °P<0.05, °°°P<0.001 and °°°°P<10−4, as compared to glutamate-treated Pyk2+/+ cultures. Scale bars, 5 μm (c and e). Uncropped blots for a are shown in Supplementary Fig. 8.