Figure 3: TRPV1-expressing hippocampal neurons are positive for OLM neuron markers and NGF upregulates TRPV1.

(a) Immunostains of hippocampal cultures for TRPV1 and Somatostatin (SOM), reelin, GAD65, VIP or Parvalbumin (PV). (b) Immunostain of hippocampal neuron culture for TRPV1, mGluR7 and MAP2; TRPV1-expressing neurons high mGluR7-positive innervation compared to surrounding neurons. (c) Quantitation of co-localization of immunofluorescence signal of different markers with TRPV1 neurons in hippocampal cultures. VR.5′ sv quantification indicates signal from the C-terminal TRPV1 antibody, compared to the N-terminal TRPV1 antibody (Fig. 1e). Cells/images analysed: TRPV1/VR.5′ sv n=54, TRPV1/mGluR7 n=22, TRPV1/somatostatin n=62, TRPV1/reelin n=52, TRPV1/GAD65 n=60, TRPV1/PV n=26, TRPV1/VIP n=22, from 3 to 6 cultures; scale bar, 20 μm. (d) Immunostain (left) and quantitation (right) of hippocampal cultures showing that NGF is highly expressed in TRPV1-expressing hippocampal neurons, compared to surrounding neurons (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). (e) Images of control hippocampal neuron cultures and cultures treated with 1 ng ml⁻¹ NGF at DIV12–13 for 21–24 h, immunostained for TRPV1 (left). Quantitation of TRPV1 immunofluorescence intensity normalized to control conditions is shown on the right (n=26 (control), and 12 (NGF) from nine cultures; error=s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction, ***P<0.001). (f) Western blot of TRPV1 in three repetitions of treatment of cultured hippocampal neurons with NGF compared to control. The levels of synaptobrevin-2 (used as a negative control) are unchanged. Tubulin serves as a load control. (g) Immunostain of a hippocampal section with TRPV1, vGluT1 and DAPI, showing high vGluT1 innervation of a TRPV1-expressing neuron in the stratum oriens; scale bar=20 μm. (h) Immunostain of a dissociated hippocampal neuron culture with TRPV1, vGluT1 and MAP2 (to mark neuronal cell bodies and processes), showing high vGluT1 innervation of a TRPV1-expressing neuron; scale bar, 20 μm.