Conditional expression of TRPV1 by Cre recombinase. (a) Diagram of the conditional stop^flox-TRPV1-IRES-ECFP allele targeted to the ROSA26 locus. SA, splice acceptor; arrowheads, loxP sites. (b–d) TRPV1-IRES-ECFP reporter expression in a brain slice from a ROSA-stop^flox-TRPV1-IRES-ECFP ^+/− mouse after introducing a Cre-expressing plasmid via biolistics, assayed by immunohistochemical detection of the conditionally expressed rat TRPV1 (b) and the CFP reporter (c). Colocalization of TRPV1 and CFP is shown in d. Scale bar, 20 μm. (e–g) Conditional expression of YFP (e) and TRPV1 (f) in cultured neurons from ROSA-stop^flox-TRPV1-IRES-ECFP ^+/− mouse after infection with a lentivirus expressing YFP-IRES-Cre. Colocalization of YFP and TRPV1 is shown in g. Scale bar, 10 μm. (h,i) Capsaicin-evoked currents recorded from a lentivirus-infected (YFP⁺) neuron expressing Cre (h) and a non-infected control cell (YFP⁻; i). Deflections in the top traces indicate capsaicin injections. (j) Currents measured after local application of capsaicin to single neurons expressing TRPV1 (YFP ⁺) or control cells (YFP⁻) not expressing TRPV1. Black bar, mean peak current; error bars, s.e.m. (n = 15,17).

Conditional neuronal expression of TRPV1-IRES-ECFP in vivo by genetic intercross with mice harboring the nestin-Cre driver. (a) A Nissl-stained section of brain. (b–d) Immunohistochemical detection of TRPV1 (b) and CFP (c) and colocalization of the two markers (d) in the neocortex (region boxed in a) of P7 mice harboring both the ROSA-stop^flox-TRPV1-IRES-ECFP (ROSA-TRPV1) and nestin-Cre alleles. DAPI staining indicates nuclei of both neuronal and non-neuronal cells. Insets, immunohistochemical detection of the same markers in ROSA-stop^flox-TRPV1-IRES-ECFP mice not crossed with nestin-Cre. Scale bars, 50 μm (75 μm in inset). (e) Action potentials in TRPV1-expressing cortical neurons in acute brain slices from nestin-Cre∷ROSA-stop^flox-TRPV1-IRES-ECFP mice upon bath application of capsaicin (indicated by the gray bar). (f) Dose-dependent current response to bath applied capsaicin in TRPV1-expressing cells. (g) Bursts of action potentials in TRPV1-expressing cortical neurons in acute brain slices with repeated local injections of capsaicin (deflections in top trace). Inset, magnified view of a capsaicin-evoked burst of action potentials.

In vivo activation of neurons that conditionally express TRPV1. (a) Electrophysiological response from a cortical cell conditionally expressing TRPV1 upon local capsaicin injection (top trace deflection). (b) Peristimulus time histogram of firing rate binned at 1.0-s intervals, assembled from multiple trials of capsaicin-evoked stimulation of neurons conditionally expressing TRPV1. Top, raster sweeps of spikes from individual trials. (c) Capsaicin-induced circling behavior in awake nestin-Cre∷ROSA-stop^flox-TRPV1-IRES-ECFP mice implanted with a ligand delivery cannula unilaterally in the dorsal left striatum. Image of recording arena (left). Tracked locomotor behavior from capsaicin-infused ROSA-stop^flox-TRPV1-IRES-ECFP (control) mice lacking Cre expression (middle), and from capsaicin-infused nestin-Cre∷ROSA-stop^flox-TRPV1-IRES-ECFP conditional expression mice (right). Gray traces, pre-infusion movement; red traces, post-infusion movement; black dotted traces, movement after a 15-min recovery period. Arrowheads indicate the direction of movement (see Supplementary Movies 1 and 2). (d) Number of contralateral circling events made by either TRPV1 conditional or control mice upon infusion with capsaicin. Data represent means ± s.e.m.; n = 6 mice each. **P < 0.001; t-test.