Odors activate dual pathways, a TRPC2 and a AA-dependent pathway, in mouse vomeronasal neurons

Abstract

Located at the anterior portion of the nose, the paired vomeronasal organs (VNO) detect odors and pheromones. In vomeronasal sensory neurons (VSNs) odor responses are mainly mediated by phospholipase C (PLC), stimulation of which elevates diacylglycerol (DAG). DAG activates a transient receptor potential channel (TRPC2) leading to cell depolarization. In this study, we used a natural stimulus, urine, to elicit odor responses in VSNs and found urine responses persisted in TRPC2(-/-) mice, suggesting the existence of a TRPC2-independent signal transduction pathway. Using perforated patch-clamp recordings on isolated VSNs from wild-type (WT) and TRPC2(-/-) mice, we found a PLC inhibitor blocked urine responses from all VSNs. Furthermore, urine responses were reduced by blocking DAG lipase, an enzyme that produces arachidonic acid (AA), in WT mice and abolished in TRPC2(-/-) mice. Consistently, direct stimulation with AA activated an inward current that was independent of TRPC2 channels but required bath Ca(2+) and was blocked by Cd(2+). With the use of inside-out patches from TRPC2(-/-) VSNs, we show that AA activated a channel that also required Ca(2+). Together, these data from WT and TRPC2(-/-) mice suggest that both DAG and its metabolite, AA, mediate excitatory odor responses in VSNs, by activating two types of channels, a TRPC2 and a separate Ca(2+)-permeable channel.

Fig. 1.

Diluted urine elicited odor responses in wild-type (WT, +/+) vomeronasal sensory organs (VSNs). A: in current-clamp mode, a 2-s stimulation of the 1:500-diluted urine depolarized the membrane potential and elicited repetitive APs in a WT neuron (resting membrane potential = −53 mV, n = 9). B: in comparison, in voltage-clamp mode, a 0.5-s application of the 1:500-diluted urine induced an inward current in a cell held at −80 mV (n = 85/145), whereas the application of Ringer (control) had no effect on the same cell. C: magnitude of urine-induced inward currents from the 85 urine-responsive cells. D: phospholipase C (PLC) inhibitor (10 μM U73122) abolished the urine response in a cell. E: U73122 significantly reduced the urine-induced currents (n = 6, P < 0.001, paired Student's t-test). F: U73343 (10 μM, the inactive analog of U73122) failed to abolish the urine response in a cell. G: U73343 did not affect the urine-induced currents (n = 8, P = 0.83, paired Student's t-test). For each cell, the currents were normalized to the peak responses in Ringer. Data are expressed as means ± SE. *P < 0.05.

Fig. 2.

Urine activated a transient receptor potential channel (TRPC2)-independent pathway in WT and TRPC2^−/− VSNs. A and B: putative TRPC2 channel blocker (100 μM SKF-96365) significantly decreased but did not block the urine (1:500) responses in WT neurons (n = 8, P < 0.001, paired Student's t-test). C and D: second putative TRPC2 channel blocker (50 μM 2-APB) inhibited the urine responses (1:500) in WT neurons (n = 5, P < 0.001, paired Student's t-test). However, 2-APB failed to completely block the odor responses to a more concentrated urine solution (1:100). E: in voltage-clamp mode, a 0.5-s stimulation of urine (1:500) induced an inward current at −80 mV in a TRPC2^−/− VSN (−/−, n = 6). F: in current-clamp, a 2-s stimulation of urine (1:500) depolarized the membrane potential and elicited repetitive action potentials in a TRPC2^−/− VSN (resting membrane potential = −55 mV, n = 12). *P < 0.05.

Fig. 3.

Diacylglycerol (DAG) lipase inhibitor decreased urine responses in WT and TRPC2^−/− VSNs, supporting a role for arachidonic acid (AA) in the odor responses. A and B: DAG lipase inhibitor (50 μM RHC80267) significantly decreased odor responses in WT VSNs by ∼60% (n = 7, P < 0.001), suggesting that AA mediates at least part of urine responses in WT VSNs. C and D: with DAG lipase inhibited with RHC80267 more than 90% of urine responses in TRPC2^−/− neurons were blocked (n = 6, P < 0.001, paired Student's t-test), suggesting that AA mediates most if not all urine responses in TRPC2^−/− VSNs. *P < 0.05.

Fig. 4.

AA activated a TRPC2-independent current in both WT and TRPC2^−/− VSNs. A: 50 μM AA elicited an inward current in about half of the WT VSNs tested (33/68, −80 mV), whereas the application of Ringer had no effect. B: AA-induced currents from the 33 responsive WT VSNs. C and D: AA-induced inward current in WT VSNs was not blocked by the putative TRPC2 channel blocker 50 μM 2-APB (n = 5, P = 0.47, paired Student's t-test). E: AA activated a similar inward current in TRPC2^−/− VSNs (n = 5), suggesting that AA activated a TRPC2-independent channel.

Fig. 5.

AA-induced inward current was a Ca²⁺ or Ca²⁺-dependent current. A and B: AA-induced current was abolished in a Ca²⁺-free Ringer (n = 5, P < 0.001, paired Student's t-test). C and D: responses to AA were blocked by the addition of 1 mM Cd²⁺ to bath (n = 9, P < 0.001, paired Student's t-test). E and F: Ca²⁺ imaging, with fura-2 as the Ca²⁺ indicator, was performed to measure the intracellular Ca²⁺ of the vomeronasal and olfactory neurons. In a WT VSN (5E), the intracellular Ca²⁺ ([Ca²⁺]_i) was increased by depolarization (with a high K⁺ Ringer for 3 s), 20 μM 1-oleoyl-2-acetyl-sn-glycerol (OAG, 30 s, n = 34/60), or 50 μM AA (30 s, n = 6/15). In contrast, in an OSN (5F), application of cAMP pathway activators (I/F: 100 μM IBMX and 30 μM Forskolin, 8 s), but not AA or OAG (30 s) induced the increases of [Ca²⁺]_i. *P < 0.05.

Fig. 6.

AA activated a Ca²⁺-dependent channel in WT VSNs. A: in an excised inside-out patch from a WT VSN, no spontaneous channel activity was observed with <0.01 μM [Ca²⁺]_i (holding potential = +60 mV). The increase of [Ca²⁺]_i to 0.15 or 50 μM increased the channel activities without the presence of AA. The same patch was then exposed to 50 μM AA (intracellular side) for 1 min, which greatly potentiated the channel activities (n = 20/36). The dashed line indicates the close state (c). B: left, panels 1–4, expanded areas of single-channel recordings on excised patch in underlined region of A; right, all-point amplitude histograms are constructed from the corresponding traces in the left and show the regulatory effect of both Ca²⁺ and AA on the channel opening. C: close state; O: the opening state. C: excised patches were exposed to AA for 1 min and mean currents at +60 mV were calculated in 10-s windows. The mean currents vs. time plot (n = 4) shows a relative rapid stimulatory effect of AA on the channel opening, whereas the stimulatory effect remained after AA was washed off. D: AA-activated channels had a single channel conductance of 23.1 ± 1.6 pS (means ± SE of 4 patches).

Fig. 7.

AA activated a Ca²⁺-dependent channel in TRPC2^−/− VSNs. A: in a representative inside-out patch obtained from a TRPC2^−/− VSN, increases of bath (intracellular) Ca²⁺ concentration from <0.01 to 50 μM slightly increased the single-channel activity of a channel, whereas the exposure to 50 μM AA for ∼1 min further potentiated the opening of the channel (+60 mV, n = 17). B: left, panels 1–3, the expanded tracings from underlined regions of A show the stimulatory effect of both Ca²⁺ and AA on channel activities. (right, panels 1–3), the corresponding all-point amplitude histograms show the effect of Ca²⁺ and AA on single channel currents. C: close state; O₁: the opening state of one channel; O₂: the opening state of two channels. C: averaged opening probabilities (NP_o) under different concentration of Ca²⁺ and AA, show that both Ca²⁺ and AA stimulated the activity of the channel. Data are averaged from 11 (−AA, <0.01 μM Ca²⁺), 6 (−AA, <0.15 μM Ca²⁺), 11 (−AA, 50 μM Ca²⁺), 6 (+AA, 0.15 μM Ca²⁺) and 13 (+AA, 50 μM Ca²⁺) instances from 13 inside-out patches, respectively. Data are expressed as means ± SE. *P < 0.05, **P < 0.01 with Student's t-test. D: averaged current voltage (I-V) relationship of the single channel currents of the AA-activated channels, indicates a single-channel conductance of 27.1 ± 3.8 pS (means ± SE, n = 9).

Fig. 8.

Proposed signal transduction of odor responses in VSNs. Odor (urine) stimulation activates the PLC pathway and elevates DAG, which is converted to AA by a DAG lipase. DAG activates TRPC2, whereas AA activates a Ca²⁺-dependent channels. Both pathways play the excitatory role in the odor responses.