Figure 1. OPHN1 is Rapidly Synthesized in CA1 Dendrites in Response to Group I mGluR Stimulation.

(A₁) DHPG treatment (100 µM) of acute hippocampal slices increases OPHN1 immunofluorescence (red) in dendrites of CA1 neurons 10 min after DHPG onset. Pretreatment with anisomycin (20 µM, 30 min) blocks DHPG-induced increases of OPHN1. βIII-tubulin immunoreactivity (green) indicates the presence of dendrites. Scale bar, 50 µm. s.p. stratum pyramidale; s.r. stratum radiatum. (A₂) Quantification of OPHN1 immunofluorescence in dendrites. Mean OPHN1 fluorescence intensity was expressed as percentage of control. n = 27–36 slices (40 µm) from 4–5 animals per condition, *p < 0.01 (unpaired t-test), as compared with control. Error bars represent SEM in all panels.

(B₁) Western blot of OPHN1 in acute hippocampal slices (CA1 regions) from rats pretreated with anisomycin (20 µM) or control vehicle for 30 min prior to DHPG (100 µM, 10 min) or ACSF treatment. γ-tubulin was used as loading control. (B₂) Mean OPHN1 levels in drug treated slices expressed as percentage of control treated slices. OPHN1 levels were normalized to γ-tubulin levels in the same sample. n = 3–6 slices (400 µm) from 3–6 animals per condition. *p < 0.01 (unpaired t-test), as compared with control.

(C) Western blots of OPHN1 in acute hippocampal slices (CA1 regions) from rats treated with DHPG (100 µM, 10 min), NMDA (20 µM, 3 min), or ACSF. ERK2 was used as loading control. NMDA treatment does not increase OPHN1 levels; n = 3 slices (400 µm) from 3 animals; p > 0.05.

(D₁) Representative differential interference contrast (DIC) (left) and OPHN1 immunofluorescence (middle) images from CA1 regions where a cut severed the dendrites from the pyramidal cell layer. βIII-tubulin immunoreactivity (right) indicates the presence of dendrites. Top: vehicle treated; bottom: DHPG treated (100 µM, 10 min). Scale bar, 20 µm. (D₂) Quantification of OPHN1 immunofluorescence in dendrites that were severed (cut) from the soma and in neighboring uncut dendrites, akin as in A₂. n = 5 slices (40 µm) from 5 animals per condition. *p < 0.01 (unpaired t-test), as compared with control. Quantification of βIII-tubulin immunofluorescence reveals no difference in βIII-tubulin levels in cut and uncut dendrites treated with DHPG or control vehicle; p > 0.05.

(E₁) Western blots of synaptoneurosome preparation (SN) reveals enrichment of PSD-95 and Synaptophysin (Syn) and a reduction in βIII-tubulin and Histone H3 (H3) in comparison to whole homogenate (Input) or supernatant (Sup). (E₂) Western blot of synaptoneurosome preparation pretreated with anisomycin (20 µM) or control vehicle 30 min prior to DHPG treatment (100 µM, 15 min). (E₃) Mean OPHN1 levels in drug treated synaptoneurosomes expressed as percentage of control vehicle treated synaptoneurosomes. OPHN1 levels were normalized to ERK2 levels in the same sample. n = 3 independent preparations from 3 animals. *p < 0.01 (unpaired t-test), as compared with control.

(F₁) Western blot of OPHN1 in acute hippocampal slices (CA1 regions) from rats pretreated with LY367385 (100 µM), MPEP (10 µM), or control vehicle 30 min before DHPG treatment (100 µM, 10 min). ERK2 was used as loading control. (F₂) Mean OPHN1 levels are presented as in E1 above. n = 3–6 slices (400 µm) from 3–6 animals per condition. *p < 0.01 (unpaired t-test), as compared with control.

(G₁) Western blot of OPHN1 in acute hippocampal slices (CA1 regions) from 4 to 6 weeks old Fmr1 KO mice and corresponding WT mice treated with DHPG (100 µM, 10 min) or ACSF. βIII-tubulin was used as loading control. (G₂) Mean OPHN1 levels are presented as above. n = 4 slices (400 µm) from 4 animals per condition. *p < 0.01 (unpaired t-test), as compared with control treated WT slices.