In vivo patch-clamp analysis of the antinociceptive actions of TRPA1 activation in the spinal dorsal horn
© Yamanaka et al.; licensee BioMed Central. 2015
Received: 27 February 2015
Accepted: 10 April 2015
Published: 21 April 2015
Transient receptor potential (TRP) channels are nonselective cation channels expressed in a variety of sensory structures, and are important molecular mediators of thermal, mechanical, cellular and chemical signals. We investigated the function of one key member of the TRP superfamily, TRPA1, in the spinal dorsal horn using in vivo patch-clamp recordings.
The application of allyl isothiocyanate (AITC), a TRPA1 agonist, significantly increased the frequency and amplitude of inhibitory postsynaptic currents (IPSCs; holding potential (VH) = 0 mV) as well as excitatory postsynaptic currents (EPSCs; VH = −70 mV) in substantia gelatinosa (SG) neurons. The AITC-induced increases in EPSC frequency and amplitude were resistant to the Na+ channel blocker tetrodotoxin (TTX). In the presence of the glutamate receptor antagonists CNQX and AP5, AITC did not generate any synaptic activity. The AITC-induced increases in IPSC frequency and amplitude were abolished by TTX or glutamate receptor antagonists. Moreover, the duration of IPSCs enhanced by TRPA1 activation were significantly longer than those of EPSCs enhanced by activation of this channel in the spinal dorsal horn. AITC induced hyperpolarization of the membrane potential of SG neurons in the spinal cord but depolarized the membrane potential in the presence of TTX. Furthermore, we examined the effects of mechanical stimuli to the skin during TRPA1 activation in the spinal dorsal horn in normal rats in both voltage-clamp and current-clamp modes. In the peripheral tissue stimuli test, AITC significantly suppressed EPSCs evoked by pinch or air puff stimulation of the skin. In current-clamp mode, AITC significantly suppressed excitatory postsynaptic potentials (EPSPs) evoked by pinch stimuli.
TRPA1 appears to be localized not only at presynaptic terminals on SG neurons, enhancing glutamate release, but also in the terminals of primary afferents innervating spinal inhibitory interneurons, which have synaptic interactions with SG neurons. This study offers further insight into the mechanisms underlying the possible antinociceptive actions of TRPA1 activation in the spinal dorsal horn. Our findings suggest that pharmacological activation of spinal TRPA1 channels may have therapeutic potential for the treatment of pain.
KeywordsTRPA1 In vivo patch-clamp Allyl isothiocyanate Antinociceptive action
Transient receptor potential (TRP) channels are tetrameric, nonselective cation channels expressed in a variety of sensory structures. The TRP superfamily can be subdivided into seven families: TRPC, TRPV, TRPM, TRPP, TRPML, TRPA and TRPN [1-3]. Recently, it was shown that TRP channels are expressed at the peripheral terminals of primary afferent fibers. TRPA1, TRPM8 and TRPV1 are well-known molecular transducers of pungent agents, temperature, pain, lipids, acids, shear stress and inflammatory nociceptive signals [4-7]. TRPA1 is activated by noxious cold temperature, reactive oxygen species (ROS) and pungent natural compounds in mustard oil, cinnamon oil, ginger and garlic [8-13]. TRPA1 is found in a subset of primary sensory neurons where it is coexpressed with noxious heat-sensing TRPV1, but not non-noxious cool-sensing TRPM8 [13,14]. It has been found that TRP channels are also localized to the central terminals of primary afferent fibers in the spinal cord; and it is thought that TRP channels in the spinal cord are activated by various endogenous factors. However, the physiological role of spinal TRP channels remains unknown.
Our research group previously examined the function of TRP channels in the dorsal horn of rat spinal cord slices using whole-cell patch-clamp recordings. We found that ROS enhance excitatory synaptic transmission in dorsal horn neurons by activating TRPA1 and TRPV1 channels . We have also reported that the activation of TRPA1 channels facilitates excitatory synaptic transmission in substantia gelatinosa (SG) neurons in the adult rat spinal cord and enhances glutamate release by direct Ca2+ entry through TRPA1 channels in nerve terminals . These findings suggest that TRP channels in the dorsal horn of the spinal cord enhance nociceptive transmission. To date, it has been reported that four TRP channels, TRPA1, TRPM8, TRPV1 and TRPV4 are involved in neuropathic pain [15-18]. However, it was recently reported that intrathecal injections of N-acetyl-P-benzoquinoneimine and P-benzoquinone, which are metabolites of acetaminophen, activate the TRPA1 receptor and have antinociceptive effects . Therefore, TRPA1 may be a promising pharmacological target for the development of new analgesics. In this study, we focus on the TRPA1 channel and investigate its role in pain transmission in the spinal dorsal horn using in vivo patch-clamp recordings.
Rats used in this study remained in a stable condition for over 10 h, comparable to previous experiments using an artificial ventilator. Whole-cell patch-clamp recordings were made from 182 SG neurons. All neurons studied had membrane potentials more negative than −50 mV. All SG neurons tested exhibited excitatory postsynaptic currents (EPSCs) at a VH of −70 mV, and no inhibitory postsynaptic currents (IPSCs) were observed because the reversal potential for IPSCs was near −70 mV [20,21]. Furthermore, SG neurons exhibited IPSCs at a VH of 0 mV, and no EPSCs were observed because the reversal potential for EPSCs was near 0 mV .
The effect of TRPA1 activation on excitatory synaptic transmission
The effect of TRPA1 activation on inhibitory synaptic transmission
AITC (300 μM) robustly increased IPSC frequency and amplitude in 7 of the 9 neurons recorded (77.7%) (Figure 2A). When measured for 30 s in the presence of AITC, the average increase in IPSC frequency and amplitude, including both responsive and non-responsive neurons, were 151.5 ± 23.3% and 129.6 ± 8.6% (n = 9), respectively (Figure 2C). Figure 2B demonstrates the effects of AITC (300 μM) on the cumulative distribution of the interval and amplitude of IPSCs. AITC increased the proportion of IPSCs having a shorter inter-event interval (p < 0.05) and a larger amplitude (p < 0.05) when compared with control (same neuron as in Figure 2A). Figure 2D demonstrates the frequency of IPSCs following application of AITC plotted against time (same neuron as in Figure 2A). Interestingly, IPSCs induced by AITC had a significantly longer duration (average; 377.1 ± 87.8 s) compared with EPSCs (average; 274.2 ± 20.8 s) induced by AITC (Figure 2E).
Characterization of EPSCs and IPSCs induced by AITC
The effect of TRPA1 activation on membrane potential
Effect of AITC on responses to noxious and innocuous stimuli
The central terminals derived from primary afferent fibers make their first sensory synapses with spinal dorsal horn neurons. At these synapses, glutamate is used as a fast excitatory neurotransmitter to convey sensory signals from the periphery . The superficial laminae of the spinal dorsal horn, particularly the SG, receive nociceptive information from the viscera, skin and other organs through primary afferent fibers . The pain-sensing channels, including TRP channels, are not only expressed at peripheral nerve endings, but also in central terminals of nociceptive primary afferent fibers innervating SG neurons . TRPA1 is present mainly in small cells in sensory ganglia [13,29,30] and on peptidergic TRPV1-expressing primary sensory neurons [13,31]. It is thought that TRPA1 is upregulated in primary sensory neurons after nerve injury and inflammation [15,32]. In one study, TRPA1 antisense oligodeoxynucleotides decreased the induction of TRPA1 and suppressed inflammation and nerve injury-induced cold hyperalgesia . However, it is unknown how TRPA1 activation of SG neurons influences responses to the noxious stimuli.
In this study, we examined the actions of AITC, an agonist of the TRPA1 channel, on synaptic transmission in SG neurons in the spinal cord using in vivo patch-clamp analysis. AITC-induced an increase in EPSC frequency and amplitude that was resistant to TTX. The EPSCs were also suppressed by the addition of CNQX and AP5. This indicates that TRPA1 appears to be localized at presynaptic terminals innervating SG neurons in the spinal cord, and that AITC enhances glutamate release, and the frequency and amplitude of EPSCs in SG neurons, similar to the findings of previous reports [14,22,33]. However, the present study also demonstrated that AITC remarkably increased GABAergic and glycinergic IPSCs in SG neurons. The AITC-induced increase in IPSC frequency and amplitude was abolished in the presence of TTX, as well as with a mixture of CNQX and AP5. Therefore, TRPA1 is likely to be localized not only at presynaptic terminals innervating SG neurons, but also in primary afferent fibers innervating spinal inhibitory interneurons that synapse with SG neurons. These findings are similar to our previous in vitro study . However, the TRPV1 agonist capsaicin does not affect inhibitory synaptic transmission in SG neurons . The reason for this is thought to be that the expression of TRPA1 does not appear to fully overlap with that of TRPV1 at the central terminals of primary afferent fibers. Our results could indicate that the activation of TRPA1 has the possibility of antinociceptive actions in the dorsal horn of the spinal cord. A recent study reported that metabolites of acetaminophen activate TRPA1 and have an antinociceptive effect . However, it remains unclear whether the excitatory or inhibitory effect was more robust because TRPA1 activation enhanced both EPSCs and IPSCs in SG neurons. Initially, we analyzed the duration of EPSCs and IPSCs induced by TRPA1 activation to determine this. The duration of enhanced IPSC frequency by TRPA1 activation was significantly longer than that of enhanced EPSC frequency by TRPA1 in SG neurons in this study. Each showed periods of enhanced glutamate release and GABA/glycine release. We interpret the longer duration of the IPSCs to mean that inhibitory interneurons react to TRPA1 fibers activated by AITC more than excitatory neurons. This suggests that the AITC-sensitive pathways may recruit polysynaptic inhibitory inputs onto SG neurons, and that nociceptive transmission may be suppressed by TRPA1 activation on presynaptic terminals of primary afferent fibers in the spinal cord. We also investigated changes in membrane potential, and showed that hyperpolarization occurred in the presence of AITC. However, in presence of TTX, AITC induced depolarization of the membrane potential. These results indicate that the cellular mechanism of AITC-induced hyperpolarization is the summation of enhanced IPSCs, more than EPSCs, by AITC. Taken together, our results suggest that under normal conditions the activation of TRPA1 channels facilitates inhibitory neurotransmission more than excitatory neurotransmission in the dorsal horn, and that the overall effects of TRPA1 activation are antinociceptive.
The activation of TRP channels enhances glutamate release from presynaptic terminals on SG neurons [14,34,35], and TRPA1 and/or TRPV1 activation completely blocks monosynaptic C-fiber-evoked EPSCs in vitro [36,37]. Voltage-gated Na+ and voltage-gated Ca2+ channels are inactivated by the depolarization of C-fiber terminals and axons, and the activation of Ca2+-permeable TRP channels causes a robust elevation of Ca2+ concentration in C-fiber terminals and enhances EPSCs in SG neurons . These events are thought to block the conduction of action potentials transmitted from the periphery . Similarly, our in vivo results suggest that spinal TRPA1 activation decreases the transmission of excitatory input through primary afferent fibers to SG neurons in the spinal cord. It is thought that TRPA1 channels play the role of a low-pass filter and convert excessive high-frequency noxious input through primary afferent fibers into low-frequency activity. In addition, we showed responses to pinch stimuli decrease in the presence of AITC in current-clamp mode. This result indicates that TRPA1 channels play not only the role of a low-pass filter, but also play GABAergic and glycinergic inhibitory roles in the spinal cord, thereby inhibiting excessive pain transmission to the central nervous system.
In the present study, we demonstrate for the first time that both EPSCs and IPSCs are enhanced by TRPA1 activation in the spinal dorsal horn using in vivo patch-clamp methods. We also found that the duration of IPSCs induced by TRPA1 activation are significantly longer than that of EPSCs induced by activation of these channels, and that TRPA1 activation hyperpolarized the membrane potential. Furthermore, we show for the first time that the nociceptive transmission of peripheral tissue stimuli is attenuated by TRPA1 activation under normal conditions. Collectively, our findings suggest that spinal TRPA1 activation may have an antinociceptive effect under normal conditions.
All experimental procedures involving the use of animals were approved by the Ethics Committee on Animal Experiments, Wakayama Medical University, and were in accordance with the UK Animals (Scientific Procedures) Act, 1986, and associated guidelines.
The methods used for in vivo patch-clamp recordings were similar to those described previously [22-24,39]. Male Sprague–Dawley rats (5 weeks of age, 150–180 g) were anesthetized with urethane (1.2–1.5 g/kg, intraperitoneally). Artificial ventilation of the pneumothorax was not performed as the rats could be maintained in good condition by supplying oxygen through a nose cone . If a withdrawal reflex occurred, a supplemental dose of urethane was given during surgery and the data collection period. A heating pad was placed beneath the rat to maintain body temperature. The lumbar spinal cord was exposed from L3 to L5 following thoraco-lumbar laminectomy from Th12 to L2, and the rat was placed in a stereotaxic apparatus (Model STS-B & SR-5R-HT, Narishige, Tokyo, Japan). Under a binocular microscope with × 8–40 magnification, the dura was cut and removed. The dorsal root that enters the spinal cord above the level of the recording site was gently moved bilaterally using a small glass retractor to expose Lissauer’s tract so that a recording electrode could be advanced into the SG from the surface of the spinal cord. The pia-arachnoid membrane was removed using microforceps to make a window large enough to allow the patch electrode to enter the spinal cord. The surface of the spinal cord was irrigated with 95% O2/5% CO2-equilibrated Krebs solution (10–15 mL/min; mM: NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, NaH2PO4 1.2, glucose 11 and NaHCO3 25) through a glass pipette at 36.5 ± 0.5°C. At the end of the experiment, the rats were given an overdose of urethane and then sacrificed by exsanguination.
The patch-electrodes were pulled from thin-walled borosilicate glass capillaries (OD: 1.5 mm) using a P-97 puller (Sutter Instrument, Novato, CA, USA). For whole-cell recordings of EPSCs, electrodes were filled with a patch-pipette solution composed of the following (mM): potassium gluconate 135, KCl 5, CaCl2 0.5, MgCl2 2, EGTA 5, ATP-Mg 5 and HEPES-KOH 5; pH 7.2 (305 mOsm). Recording of IPSCs was performed using an electrode solution composed of the following (mM): Cs2SO4 110, tetraethylammonium 5, CaCl2 0.5, MgCl2 2, EGTA 5, ATP-Mg 5 and HEPES-KOH 5; pH 7.2 (305 mOsm). The electrode, with a resistance of 8–12 MΩ, was advanced at an angle of 30–45° into the SG through a window in the pia-arachnoid membrane using a micromanipulator (Model MWS-32S, Narishige). A tight seal (resistance of at least 10 GΩ) was then formed with neurons at a depth of 30–150 μm. Membrane potentials were held at −70 mV in voltage-clamp mode. After forming the seal, the membrane patch was ruptured by a brief period of more negative pressure, thus resulting in a whole cell configuration. Signals were collected using an Axopatch 200B amplifier in conjunction with a Digidata 1440A A/D converter (Molecular Devices, Sunnyvale, CA, USA) and stored on a personal computer using the pCLAMP 10 data acquisition program (Molecular Devices). Recordings were analyzed using Mini Analysis 6.0 software (Synaptosoft, Fort Lee, NJ, USA) and pCLAMP 10. Membrane potential recordings were made in current-clamp mode.
The methods used for peripheral tissue stimulation were similar to those described previously . Noxious and innocuous mechanical stimuli were applied to the receptive field of the ipsilateral hindlimb using toothed forceps or air puffs (Pressure system IIe, Toohey Company, Fairfield, NJ, USA), respectively. To maintain fixed-strength noxious stimulation, the toothed forceps were clamped during skin pinching. Synaptic responses in SG neurons from animals stimulated with air puffs did not differ between those given a preceding pinch stimulus and those not given a preceding pinch stimulus.
The drugs used in this study were AITC, CNQX, AP5, bicuculline and strychnine. AITC, CNQX, bicuculline and strychnine were first dissolved in dimethyl sulfoxide at 1,000 × the concentration to be used, while AP5 and TTX were dissolved in distilled water at 1,000 × final concentration. All drugs were then diluted to final concentrations in Krebs solution immediately before use and applied by perfusion via a three-way stopcock without any change in perfusion rate or temperature. The time necessary for the solution to flow from the stopcock to the surface of the spinal cord was approximately 40 s.
Rats, from which recordings had been made, were perfused through the ascending aorta with saline followed by 4% paraformaldehyde with 1.5% picric acid in 0.16 M phosphate buffered saline (PBS), pH7.2–7.4 (4°C). After fixation, spinal cords were removed and the L4-L5 spinal cord segments were dissected. Spinal cord slices were stained with neurobiotin to label neurons in the SG. Neurobiotin was detected using the avidin-biotin complex (ABC) system (Elite ABC kit; Vector Laboratories, Burlingame, CA, USA). The sections were rinsed in PBS with 0.3% Triton X-100 and 1% normal bovine serum, then incubated with ABC solution diluted with PBS for 2 hours. After three 20-minute rinses in PBS, the sections were reacted with 0.05% diaminobenzidine (DAB) and 0.003% H2O2 in PBS to visualize injected neurons. After washing, staining was observed by microscopy (Olympus, Tokyo, Japan).
All numerical data were expressed as the mean ± S.E.M. For electrophysiological data, n refers to the number of neurons studied. For analysis of the change in frequency and amplitude of postsynaptic currents following the application of AITC, the time course of postsynaptic current frequency before and after AITC application was first constructed with a time bin of 30 s using the Mini Analysis Program 6.0 (Synaptosoft, Decatur, GA). The average response in 30 s of the peak was then used to calculate the percentage change from control. P < 0.05, evaluated using Student’s t-test or Student’s paired t-test, was considered to indicate statistical significance. Similar to previous studies [36,40], cells were deemed to be responsive to the testing compounds when there was a > 20% decrease or increase in the frequency of EPSCs or IPSCs. The Kolmogorov-Smirnov test was used to compare the cumulative distributions of the postsynaptic current parameters in the absence and presence of the test drugs. The membrane potentials were not corrected for the liquid junction potential between the Krebs and patch-pipette solutions.
We are grateful to S. Ito and T. Kagiya for histological support. This work was supported in part by MEXT KAKENHI 30597084 to M.Y., MEXT KAKENHI 24791558 to W.T., MEXT KAKENHI 25460730 to T.N., MEXT KAKENHI 23592173 to M.Y. and Grant of Japan Orthopaedics and Traumatology Foundation, Inc.No.274. to W.T.. There are no conflicts of interest regarding this study for any of the authors.
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