Fig 1: EPA impaired behaviors and synaptic plasticity via the 5-HT6R on GABAergic interneurons.a Representative fluorescence images showing most of the cells infected with pAAV-CAG-DIO-5-HT6R-EGFP (shRNA) vectors are GABAergic neurons in the CA1 region of GAD-Cre mice. Scale bar: 100 µm. b Representative fluorescence images showing the knockdown of 5-HT6R in CA1 GABAergic neurons infected with pAAV-CAG-DIO-5-HT6R-EGFP (shRNA) vectors in GAD-Cre mice. Scale bar: 50 µm. c Histogram showing average fluorescence intensity (red) in the CA1 neurons from GAD-Cre mice that were injected with pAAV-CAG-DIO-EGFP (control) and shRNA virus (Ctrl group: n = 20 cells from four mice; shRNA group: n = 32 cells from five mice; two-tailed Student’s t-test, P = 0.002). The fluorescence intensity of the 5-HT6R-positive neurons (red) merged with GFP (green) was plotted using the same imaging conditions for every slice. d, e Western blots showing 5-HT6R reduction after shRNA virus injection (n = 4 experiments/group; two-tailed Student’s t-test, P = 0.032). f–i Knocking down 5-HT6R in the GABAergic interneurons prevented EPA-induced enhancement of sIPSC frequency (n = 8 cells/group; two-tailed Student’s t-test; f: Ctrl, P = 0.021 and shRNA, P = 0.505, g: Ctrl, P = 0.789 and shRNA, P = 0.917) and EPA-impaired LTP (h, i: n = 7–8 slices/group; two-tailed Student’s t-test, P = 0.001). Scale bars: 0.5 mV, 5 ms. (j-p) The i.g. administration of EPA did not impair LTP (j, k: n = 7 slices/group; two-tailed Student’s t-test; P < 0.0001) or learning and memory (MWM test: n = 9–10 mice/group; l: repeated measures two-way ANOVA, F(1, 68) = 43.369, P = 0.003; m: two-tailed Student’s t-test; P = 0.029; n: two-tailed Student’s t-test; P = 0.045; o: two-tailed Student’s t-test; P = 0.002. fear conditioning test in p: n = 10–11 mice/group; two-tailed Student’s t-test; P = 0.038) after knockdown of 5-HT6R in the CA1 GABAergic interneurons. Data show mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: EPA modulated GABAergic transmission via the 5-HT6R.a Coronal sections of the hippocampal CA1 of GIN-GFP mice were stained with anti-5-HT6R antibody. The arrows indicate neurons double positive for both 5-HT6R and GAD67. Scale bar: 100 µm. b–d Effects of EPA on sIPSCs frequency and amplitude (n = 9 cells from five mice; two-tailed Student’s t-test, c, P = 0.013; d, P = 0.85). Scale bars: 20 pA, 2 s. e, f HFS-evoked IPSCs in hippocampal CA1 neurons with or without EPA treatment (n = 9–11 cells from four mice; two-tailed Student’s t-test, P = 0.016). g–i EPA was unable to enhance sIPSCs in the presence of the 5-HT6R antagonist (n = 10 cells; two-tailed Student’s t-test; f, P = 0.868; g P = 0.798).Scale bars: 20 pA, 2 s. j, k Increased frequency of sIPSCs induced by EPA disappeared in 5-HT6R KO mice. The average sIPSC frequency is shown in h (two-tailed Student’s t-test; WT, P = 0.043 and KO, P = 0.604), and the amplitude is shown in i (two-tailed Student’s t-test; WT, P = 0.546 and KO, P = 0.907) (n = 9 cells from four mice/group). l, m The 5-HT6R agonist EMD and the GABAA receptor agonist diazepam mimicked the impairing effect of EPA on LTP, but the GABAA receptor antagonist BMI at 10 µM can block the EPA effect on LTP (n = 5–11 slices/group; one-way ANOVA, F(8, 47) = 40.19, P = 0.021). Data show mean ± s.e.m. *P < 0.05, ***P < 0.001.
Fig 3: The expression of 5-HT6 receptors protein level was specifically decreased in the contralateral VLO after SNI. (A) Representative Western blotting and (B) summary scatter plots showing the 5-HT6 receptors of the VLO in control (sham) and SNI rats. **p < 0.01 and ##p < 0.01 vs corresponding control and contralateral group, respectively. n = 4 rats/group. 5-HT6R, 5-HT6 receptors.
Fig 4: 5-HT6R controls PN migration through an agonist-independent mechanism. (A) In utero electroporation of (h)5-HT6R-Gs dead or (h)5-HT6R-D106A significantly rescues 5-HT6R-shRNA1-induced mispositioning of PNs compared with scram-shRNA (***P<0.001, **P<0.01, one-way ANOVA, Tukey's post hoc test). Data are mean±s.e.m. Scale bars: 100 µm in A. (B) 5-HT6R signalling pathways and their role in migration of PNs. Cdk5/p35 overexpression, 5-HT6R plasmids abolishing serotonin-induced cAMP signalling [(h)5-HT6R-D106A] and Gs-dependent cAMP signalling [(h)5-HT6R-Gs-dead] rescue the 5-HT6R-shRNA1-induced mispositioning phenotype. PNs, pyramidal neurons; IZ, intermediate zone; CP, cortical plate; AC, adenylyl cyclase.
Fig 5: The impairing effect of EPA disappeared in 5-HT6R KO mice.a, b Western blots of 5-HT6R in the hippocampus from 5-HT6R KO (5-HT6R−/−) mice and their WT (5-HT6R+/+) littermates (n = 3–4 experiments/group; two-tailed Student’s t-test; P < 0.0001). Full-length blots are presented in Supplementary Fig. 9. c Specificity characterization of the anti-5-HT6R antibodies. Scale bars: 100 μm. d–k The impairing effect of EPA on learning and memory in the MWM (d–g: n = 10–12 mice/group; d: repeated measures two-way ANOVA, F(3, 200) = 41.279, P = 0.053; e: one-way ANOVA, F(3, 39) = 43.379, P = 0.028; f: one-way ANOVA, F(3, 39) = 41.662, P = 0.02; g: one-way ANOVA, F(3, 39) = 42.726, P = 0.057), contextual fear conditioning (h: n = 9–12 mice/group; one-way ANOVA, F(3, 38) = 46.588, P = 0.001), NOR (i: n = 9–11 mice/group; one-way ANOVA, F(3, 35) = 34.161, P = 0.005), and LTP (j, k, n = 5–8 slices/group; one-way ANOVA, F(3, 24) = 38.908, P < 0.0001) disappeared in 5-HT6R KO mice. Data show mean ± s.e.m. Scale bars: 0.5 mV, 5 ms. *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-5HT6 Receptor antibody