Five weeks old Sprague-Dawley rats were anesthetized by isoflurane and perfused with 4% paraformaldehyde. Samples of lumbar segments of the spinal cord and DRG were harvested and quickly frozen. The spinal cord and DRG were cut into 20 and 10 μm sections, respectively. The sections were incubated with polyclonal goat anti-TRPV1 antibody (Calbiochem, PC 420, 1:100) and rabbit anti-TRPV4 antibody (Alomone, Israel, 1:100) for 2 hrs at room temperature, then incubated with Rhodamine Red ™-X donkey anti-goat IgG (Jackson, 711-295-152, 1: 100) and FITC donkey anti-rabbit IgG (Jackson, 715-095-151, 1: 100) for 1 hr at room temperature. Images were taken by a confocal microscope.

DRG, DH and hippocampal neurons were isolated from embryonic day 18 (E18) rat embryos, triturated and cultured in Neurobasal medium (Invitrogen, Carlsbad, CA). The adult DRG neurons were dissociated from 5 weeks old rats. The serum-free medium was supplemented with B-27 and glutamine (Gibco Invitrogen, Grand Island, NY) and the neurons were plated on poly-D-lysine-coated glass coverslips. The neurons were used after 2 weeks growth in culture.

Human embryonic kidney 293T cells were cultured in DMEM with 10% fetal bovine serum and penicillin (50 units/ml)-streptomycin (25 μg/ml) (Gibco Invitrogen, Grand Island, NY). TRPV4 cDNA and GFP cDNA were co-transfected into HEK 293T cells with Lipofectamine 2000 reagent following manufacture's protocol (Invitrogen, Carlsbad, CA). The fluorescent cells were used for recording currents 24 hrs after transfection. The non-fluorescent cells were used as a negative control.

Adult dissociated rat DRG neurons plated on glass coverslips were incubated with 3 μM Fluo-4 AM (Invitrogen, Eugene, OR) for 30 min at 37°C and washed with physiological buffer containing the following (in mM): 140 NaCl, 5 HEPES, 2 CaCl₂, 1 MgCl₂, 2.5 KCl, 2 Lidocaine, pH 7.35. Fluo-4 was excited at 488 nm, and emitted fluorescence was filtered with a 535 ± 25 nm bandpass filter. The ratio of the fluorescence change F/F_o was plotted to represent the change in intracellular Ca²⁺ levels.

DRG neurons grown on poly-D-lysine-coated coverslips were used for recording TRPV1 and TRPV4 currents. For whole-cell patch-clamp recordings, the bath solution contained (in mM): 140 Na gluconate, 5 KCl, 10 HEPES, 1 MgCl₂, 1.5 EGTA, pH adjusted to 7.35 with NaOH and the pipette solution contained (in mM): 140 K gluconate, 5 KCl, 10 HEPES, 2 MgCl₂, 10 EGTA, 2 K₂ATP, 0.5 GTP, pH adjusted to 7.35 with KOH. Currents were recorded using an Axopatch 200B integrating patch-clamp amplifier (Axon Instruments Inc., Union City, CA). Data were digitized (VR-10B; InstruTech, Great Neck, NY) and stored on videotape. For analysis, data were filtered at 2.5 kHz (-3 dB frequency with an eight-pole low-pass Bessel filter; LPF-8; Warner Instruments, Hamden, CT) and digitized at 5 kHz. Current amplitudes were measured using Channel 2 (software kindly provided by Michael Smith, Australian National University, Canberra, Australia).

The cell-attached patch-clamp technique was used to record single-channel currents. The bath solution contained (mM): K gluconate 140, KCl 2.5, MgCl₂ 1, HEPES 5, EGTA 1.5, pH adjusted with KOH to 7.3. The patch pipettes were made from glass capillaries (Drummond, Microcaps), coated with Sylgard (Dow Corning, Midland, MI, USA). The patch pipettes were filled with a solution that contained (mM): Na gluconate 140, MgCl₂ 2, HEPES 10, EGTA 1, pH adjusted with NaOH to 7.35. Currents were recorded using an Axopatch 200B integrating patch-clamp amplifier (Axon Instruments Inc., Union City, CA). Data were digitized (VR-10B; InstruTech, Great Neck, NY) and stored on videotape. For analysis, data were filtered at 10 kHz (-3 dB frequency with an eight-pole low-pass Bessel filter; LPF-8; Warner Instruments, Hamden, CT) and digitized at 50 kHz. The data were analyzed using Channel 2 and QUB (State University of New York at Buffalo, NY)

DRG, DH and hippocampal pyramidal neurons were identified by their morphology. The extracellular bath solution contained (in mM): 130 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, 5 D-glucose, pH 7.35 and the pipette solution contained (in mM): 140 K gluconate, 2 MgCl₂, 1 EGTA, 10 HEPES, 1 ATP, pH adjusted to 7.35 with KOH. In order to record fast excitatory postsynaptic currents, neurons were voltage-clamped (Axopatch 200B, Axon Instruments Inc., Union City, CA) at -60 mV (close to E_Cl). While recording mEPSCs the bath solution contained lidocaine (10 mM). The data were filtered at 2.5 kHz and digitized at 5 kHz. Data were analyzed using Mini Analysis Program (Synaptosoft, Decatur, GA). The amplitude and frequency of the events were determined from 30 s data segments.

B27, glutamine, fetal bovine serum and penicillin-streptomycin were obtained from Gibco Invitrogen, Grand Island, NY. Fluo-4 AM was obtained from Invitrogen, Eugene, OR. Lipofectamine 2000 reagent was obtained from Invitrogen, Carlsbad, CA. All other chemicals used in this study were obtained from Sigma (St. Louis, MO).

Data are represented as mean ± S. E. M. (Standard Error of the Mean) Kolmogorov-Smirnov (KS) test was used to compare the cumulative probability for inter-events and amplitude between different groups. Student's t-test and one way analysis of variance (ANOVA) were used for statistical comparisons and the significance is considered at P < 0.05.

Using immunohistochemical, patch-clamp and Ca²⁺ fluorescence imaging techniques, we studied the expression and co-expression of TRPV4. Double immunostaining of sections from whole DRG revealed that 33.7 ± 2.4% of neurons expressed TRPV1, 88.5 ± 4.8% of the neurons expressed TRPV4, and 27.9 ± 2.8% of the neurons expressed both TPRV4 and TRPV1 (n = 4 rats, at least 3 slices from each animal) (Fig. 1A, B, C, D, E). The specificity of antibodies has been confirmed by a single band in Western blots from samples of HEK 293T cells heterologously expressing TRPV1 or TRPV4 (data not shown). Similarly, in Ca²⁺ fluorescence imaging from adult dissociated neurons, 37.6 ± 3.9% of neurons responded to capsaicin, 64.2 ± 5.4% of neurons responded to 4α-PDD and 29.5 ± 3.2% of neurons responded to both, which is consistent with the immunostaining data (Fig. 1F). Mostly, the co-expression of TRPV1 and TRPV4 was observed in small and medium diameter neurons.

Then, we used whole-cell current recordings and determined whether TRPV1- and TRPV4-mediated currents can be elicited in the same neuron. In embryonic rat DRG neuronal cultures, 51/73 neurons responded to both capsaicin (100 nM) and 4α-PDD (10 μM) (Fig. 1G). These results indicate that TRPV1 and TRPV4 are co-expressed in DRG neurons. A higher number of neurons co-expressing TRPV1 and TRPV4 in whole-cell current recording as compared to immunostaining or Ca²⁺ fluorescence imaging techniques may be due to the bias of selecting small diameter neurons or differential expression of these two channels between embryonic and adult DRG neurons.

Several TRP channels have been shown to be modulated by PKC, which contributes to inflammatory pain 31. There are suggestions that TRPV4 may be modulated/activated by PKC 3233. In order to determine the modulation of 4α-PDD-induced TRPV4 currents by PKC, we used patch-clamp technique and recorded whole-cell and single-channel currents.

In whole-cell current recordings, 4α-PDD (10 μM)-induced current was robustly potentiated following incubation with PDBu (1 μM, for 2 min), a PKC activator (4.00 ± 0.65-fold, n = 11, P < 0.05, Fig. 2A, B). As a positive control, we determined the potentiation of TRPV1 currents by PKC. As expected, capsaicin-evoked TRPV1 currents were significantly potentiated by PDBu (2.37 ± 0.45-fold, n = 11, P < 0.05, Fig. 2A, C).

Then, we recorded single-channel currents in cell-attached patches in DRG neurons and TRPV4 heterologously expressed in HEK 293T cells. Single-channel currents were recorded at positive and negative potentials. In DRG neurons at a holding potential of +60 mV, single-channel current had an amplitude of 4.40 ± 0.10 pA corresponding to a chord conductance of 73.3 ± 1.6 pS and at -60 mV single-channel current had an amplitude of 1.76 ± 0.15 pA corresponding to a chord conductance of 29.3 ± 2.5 pS (n = 3, Fig. 3). In similar experimental conditions, single-channel currents recorded at +60 mV from HEK cells had an amplitude of 5.70 ± 0.04 pA corresponding to a chord conductance of 95.0 ± 7.1 pS and at -60 mV had an amplitude of 3.81 ± 0.04 pA corresponding to a chord conductance of 63.5 ± 6.3 pS (n = 5, Fig. 3). It is clear from these experiments that TRPV4-mediated currents exhibited strong outward rectification. The outward rectification observed is similar to that observed with TRPV1 currents and we have described that outward rectification is due to both lower single-channel conductance and decreased open probability (P_o) at negative membrane potentials 34. In order to determine the mechanism of rectification, we have constructed current-voltage relationship by recording currents at different voltages form DRG neurons and HEK cells expressing TRPV4. The data points of outward and inward current limbs were fitted with linear functions to determine the slope conductance. The currents recorded from DRG neurons exhibited slope conductances of 135 ± 32 pS at positive potentials and 22 ± 4 pS at negative potentials (Fig. 3D). In similar experimental conditions, currents recorded from HEK cells expressing TRPV4 exhibited slope conductances of 110 ± 5 pS at positive potentials and 76 ± 6 pS at negative potentials (Fig. 3B). These results show that TRPV4 channels exhibit a strong outward rectification. The outward rectification exhibited by TRPV4 channels recorded from DRG neurons is more pronounced. The reason for this difference is not known, but it is possible that associated proteins in native TRPV4 channels 35 as reported in native TRPV1 channels 36 may contribute to rectification pattern.

In order to determine voltage dependency of the channel activity, we analyzed single-channel open probability at different membrane potentials from apparent single-channel patches. Single-channel patches were confirmed in patches that exhibited high P_o by using the criteria of non-overlapping events when the P_o was > 0.7. The P_o at +60 mV and -60 mV is 0.84 ± 0.11 and 0.57 ± 0.11 (n = 3) in DRG neurons and 0.14 ± 0.04 and 0.07 ± 0.03 (n = 4) in HEK cells expressing TRPV4, respectively. These studies suggest that TRPV4 exhibits a milder voltage dependency in P_o as compared to TRPV1.

In order to determine the role of PKC, cell-attached patches were formed with the pipette containing 4α-PDD (1 μM) and the cells were exposed to a PKC activator, PDBu (1 μM). In cell-attached patches from TRPV4 expressing HEK 293T cells, single-channel activity induced by 4α-PDD (1 μM) was significantly enhanced by the incubation of the cells with PDBu (1 μM). The open probability was significantly increased as compared to patches, in which the cells were not incubated with PDBu (control, 0.096 ± 0.023; PDBu, 0.507 ± 0.086; n = 7, P < 0.05, Fig. 4). In order to confirm the specificity of PKC action, the cells were incubated with a selective PKC inhibitor, BIM (500 nM). BIM significantly reduced the increase in the P_o induced by PDBu (PDBu, 0.476 ± 0.113; BIM, 0.074 ± 0.035, n = 5, P < 0.05). These results indicate that TRPV4 can be sensitized by activation of PKC.

Immunohistochemical data showed that TRPV1 and TRPV4 are expressed not only in the DRG neuronal cell bodies, but also expressed in central sensory nerve terminals at laminae I and II of the spinal dorsal horn (Fig. 5). In order to determine the role of TRPV4 expressed in the spinal cord, we studied the modulation of synaptic transmission by TRPV4 activation in DRG-DH neurons grown in culture. In DRG-DH co-cultures, synaptic connections are formed between DRG and DH neurons and synaptic currents can be recorded by voltage clamping the DH neurons. In a previous study from our lab has shown that TRPV1 activation increases mEPSC frequency without affecting the amplitude at the first sensory synapse 25. We have also reported that only DRG-DH co-cultures responded to capsaicin application by increasing the frequency of mEPSCs. In similar experimental conditions, application of capsaicin to DH-only cultures did not induce a response suggesting the expression of TRPV1 only at the sensory nerve terminals. Application of 4α-PDD (40 μM) increased the frequency of mEPSCs significantly (2.33 ± 0.39-fold, n = 5, P < 0.05, Fig. 6), while there was no change in the amplitude of mEPSCs, suggesting that TRPV4 activation causes enhanced glutamate release from the presynaptic terminals. To determine whether TRPV4-mediated increase in mEPSCs is modulated by PKC, the neurons were incubated with PDBu (1 μM). A confounding issue is that PDBu by itself has been shown to increase the mEPSC frequency by binding to a site, independent of phosphorylation 2537383940; therefore, first we quantitated PDBu-mediated increase in the frequency of mEPSCs. PDBu alone significantly increased the frequency (2.58 ± 0.25-fold, n = 3, P < 0.05, Fig. 6). However, application of 4α-PDD + PDBu synergistically increased the frequency of TRPV4-induced mEPSCs (7.51 ± 0.99-fold, n = 3, P < 0.05, Fig. 6). These results suggest that TRPV4 activation modulates synaptic transmission, which is further enhanced by activation of PKC.

TRP channels are also expressed in certain regions of the CNS. TRPV1 and TRPV4 expression has been shown in the hippocampus 29414243. Rat embryonic cultured hippocampal neurons were used to study the modulation of synaptic transmission by activation of TRPV4. We have selectively voltage-clamped hippocampal pyramidal neurons identified by their morphology. Using whole-cell recordings, application of 4α-PDD (10 μM) induced an increase in the frequency of mEPSCs, which was not accompanied by a change in the amplitude. The frequency of mEPSCs was increased 2.40 ± 0.26-fold (n = 7, P < 0.05) following application of 4α-PDD (Fig. 7). As observed in DRG-DH cultures, application of 4α-PDD + PDBu synergistically potentiated TRPV4-mediated response (10.06 ± 1.91-fold, n = 7, P < 0.05, Fig. 7). These experiments suggest that TRPV4 activation exerts an effect presynaptically and modulates synaptic transmission. Intriguingly, application of capsaicin (1 μM) in voltage-clamped cultured hippocampal pyramidal neurons neither changed the frequency (1.11 ± 0.10-fold, n = 6) nor the amplitude of mEPSCs. Although TRPV1 expression has been shown in the hippocampus 4142, the function has not been studied extensively. Recently, a form of long term depression (LTD) has been attributed to activation of TRPV1 in the hippocampus 29.