Figure 8: TRPV1-induced excitatory synaptogenesis requires calcium influx, NGF and BDNF.

(a) Immunostain of rat hippocampal cultures with TRPV1 and BDNF. (b) Quantitation of BDNF signal in somas of TRPV1-expressing neurons normalized to surrounding non-TRPV1-expressing cells (n=15 images from three cultures; error=s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction, **P<0.01). (c) Immunostain of WT and TRPV1 knockout mouse cultures with the C-terminal TRPV1 antibody (which detects a remainins splice isoform in the TRPV1 knockouts and marks TRPV1-expressing cells—Fig. 1e,f), BDNF, and DAPI. (d) Quantitation of BDNF puncta/μm (left panel) and BDNF puncta intensity (right panel) on OLM neurons marked by the C-terminal TRPV1 antibody, in WT and TRPV1 knockout cultures, normalized to WT. (e) Immunostains of TRPV1, vGluT and MAP2 in cultures in control conditions, and in cultures treated with capsaicin in the presence of absence of 2 mM EGTA to block calcium influx, 1 μg ml⁻¹ TrkA-Fc to scavenge NGF, or 0.4 μg ml⁻¹ TrkB-Fc to scavenge BDNF. (f) Quantitation of excitatory synapse number (vGluT puncta number) on TRPV1-expressing hippocampal neurons in the indicated conditions. Images used for quantitation were: control n=28, 1 μM cap. n=14, 2 mM EGTA n=21, 2 mM EGTA + 1 μM cap. n=21, 1 μg ml⁻¹ TrkA-Fc n=21, 1 μg ml⁻¹ TrkA-Fc + 1 μM cap. n=21, 0.4 μg ml⁻¹ TrkB-Fc n=19, 0.4 μg ml⁻¹ TrkB-Fc + 1 μM cap. n=20; from four cultures. Error=s.e.m., significance determined by one way ANOVA with Tukey's post hoc test for multiple comparisons, ***P<0.001. (g) Immunostains of EGFP and TRPV1-IRES-GFP transfected hippocampal neurons with NGF. Quantitation of NGF intensity in somas of transfected neurons is indicated on the right (n=12 images from three cultures; error=s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction). (h) Immunostains of EGFP and TRPV1-IRES-GFP transfected hippocampal neurons with BDNF. Quantification of BDNF intensity in somas of EGFP and TRPV1-IRES-GFP transfected neurons with and without addition of 1 μM SB-366791 2 days after transfection, is indicated on the right (n=13 images from three cultures; error=s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction, ***P<0.001). Scale bars, 20 μm in all panels.