Figure 7: TRPV1 overexpression in neurons increases excitatory innervation.
From: TRPV1 regulates excitatory innervation of OLM neurons in the hippocampus
(a) Immunostains of TRPV1-IRES-GFP and EGFP transfected neurons in the indicated conditions with vGluT1 and lumenal syt1 antibody internalized by recycling synaptic vesicles during depolarization; scale bars, 10 μm. (b) Quantitation of excitatory synapse number (number of vGluT1 puncta) on TRPV1-expressing cells with and without treatment with 1 μM SB-366791 to block TRPV1 channels, compared to EGFP controls. (c) Quantitation of vGluT1 signal intensity (corresponding to number of vGluT1 vesicles per terminal) in the indicated conditions. (d) Quantitation of lumenal syt1 antibody uptake in depolarizing conditions (corresponding to the total number of recycling synaptic vesicles in excitatory terminals) in vGluT1-positive terminals (neurons/images quantified were: EGFP n=60, TRPV1-IRES-GFP n=30, TRPV1-IRES-GFP + 1 μM SB-366791 DIV4 n=15; from 5 to 20 different neuronal cultures). (e) TRPV1-IRES-GFP-expressing neurons have increased mEPSC frequency compared to control EGFP-expressing neurons, and unchanged amplitude, consistent with an increase in synapse number in TRPV1-over-expressing neurons (cells used for analysis: EGFP n=29, TRPV1-IRES-GFP n=27). (f) Immunostain of TRPV1-IRES-GFP or EGFP transfected neurons in the indicated conditions with vGAT and syt1 internalized by recycling synaptic vesicles during depolarization; scale bars, 10 μm. (g) Quantitation of inhibitory synapse number (number of vGAT puncta) on TRPV1-expressing cells with and without treatment with 1 μM SB-366791 to block TRPV1 channels, compared to EGFP. (h) Quantitation of vGAT signal intensity (corresponding to number of vGAT vesicles per terminal). (i) Quantitation of lumenal syt1 antibody uptake in depolarizing conditions (corresponding to the total number of recycling synaptic vesicles in inhibitory terminals) in vGAT-positive terminals (neurons/images quantified were: EGFP n=60, TRPV1-IRES-GFP n=27, TRPV1-IRES-GFP + 1 μM SB-366791 DIV4 n=15; from five cultures). (j) TRPV1-IRES-GFP-expressing neurons have no change in mIPSC frequency or amplitude, compared to control EGFP-expressing neurons (cells used for analysis: EGFP n=27, TRPV1-IRES-GFP n=24). Error=s.e.m.; significance determined by unpaired Student’s t-test *P<0.05, **P<0.01, ***P<0.001.
