All animals were housed in the centralized animal facilities as assigned by the University of California Berkeley and were provided food and water ad libitum. Animal care and experimental protocols were approved by the University of California Berkeley Animal Care and Use Committee. All chemicals were purchased from Sigma-Aldrich, except BCTC ((N-(4-Tertiarybutylphenyl)-4(3-cholorphyridin-2-yl)-tetrahydro-pyrazine1(2H)-carboxamide)) and AITC (Allyl isothiocyanate) that was purchased from Tocris.
QAQ synthesis and spectroscopic characterization
The synthesis of QAQ was performed as previously described5
. UV-Vis spectra of QAQ were measured using a smartSpec Plus spectrophotometer (Bio-Rad) in combination with illumination using the Polychrome V (Till Photonics), through an optic fiber positioned perpendicular to the detection beam of the spectrophotometer.
HEK-293 cells were cultured under standard conditions (DMEM containing 10% FBS). We grew GH3 cells in F-12K medium containing 15% horse serum and 2.5% FBS. NG108-15 cell medium contained 95 % DMEM mixed with HAT (0.1 mM Hypoxanthine, 400 nM Aminopterin, 0.016 mM Thymidine) and 5% FBS. Cells were plated on poly-L-lysine (0.1 mg/ml) treated coverlips in a density of 12,000 cells per cm2
for electrophysiological measurements. Dissociated hippocampal neuronal preparations were performed from neonatal Sprague Dawley rats according to standard procedures1
. Hippocampi were dissected, dissociated and cells were plated on poly(L-lysine)-coated coverslips at a density of 100,000/cm2
. We grew hippocampal neurons in minimum essential medium containing 5% FBS, 20 mM glucose, B27 (Invitrogen), glutamine and Mito+ Serum Extender (BD Biosciences). TG neurons from neonatal rats were prepared as previously described30
. TG were dissected, dissociated (with collagenase and trypsin) and plated on poly(L-lysine)-coated coverslips. We grew TG neurons in minimum essential medium containing 5% horse serum, MEM vitamins (Invitrogen), glutamine and Penn/Strep. HEK-293 cells were transfected using calcium phosphate precipitation and measured after 24-48 h4
. GH3 and NG108-15 cells were recorded 24 h after plating. Hippocampal neurons were transfected at d7 and measured at d10-14. TG cells were measured 12-48 h after plating.
Dorsal root ganglia (DRG) preparation
Mice, C57/BL6 WT or TRPV1−/− aged 1-6 months of either sex, were deeply anaesthetized with isoflurane and killed by cervical dislocation. The spinal column and surrounding muscle, from the sacral to cervical regions, was removed from the mouse and dissected in cold ACSF (in mM: NaCl 124, KCl 4, MgCl2 2, CaCl2 2, NaHCO3 26, glucose 20, sodium pyruvate 2, ascorbic acid 0.4,pH 7.3) equilibrated with 95% O2-5% CO2. A laminectomy was performed from the thorax to the sacrum, and the spinal cord was gently removed, exposing the DRG. The DRG and attached nerves (10-20 mm in length) were removed from the lumbar region, and incubated for at least 30 minutes at room temperature in an oxygenation chamber on a nitrocellulose membrane (Sartorius Stedim Biotech) moistened with ACSF.
Spinal-cord slices preparation
C57/BL6 mice were deeply anesthetized with isoflurane and quickly beheaded. The spinal column and surrounding muscles were removed and dissected in ice-cold oxygenated low calcium/low magnesium ACSF (in mM: NaCl 101; KCl 3.8; MgCl2 18.7, MgSO4 1.3; KH2PO4 1.2; HEPES 10; CaCl2 1; Glucose 1). After laminectomy, the spinal roots were cut, the spinal cord was gently removed and its lumbar part was placed into a small agarose block. 300 μm thick slices were prepared using a Leica VTS 1000 vibratome. The slices were then transferred in warm (31°C) ASCF equilibrated with 95% O2 - 5% CO2 for at least one hour before starting patch-clamp recordings.
Patch clamp recordings of mammalian cells were performed at room temperature. Bath solution for K+
current contained in mM: NaCl 138, KCl 1.5, MgCl2
2.5, tetrodotoxin 0.001 (for hippocampal neurons only), HEPES 5 and glucose 10. Bath solution for Na+
current contained in mM: NaCl 145, CdCl2
2, HEPES 5 and glucose 5. Bath solution for Ca2+
current contained in mM: NaCl 138, KCl 5.4, MgCl2
20, tetrodotoxin 0.001 (for GH3 cells only), HEPES 10 and glucose 5. Bath solution for current clamp experiments contained in mM: NaCl 138, KCl 1.5, MgCl2
2.5, HEPES 5 and glucose 10. Pipette solution for K+
current contained in mM: NaCl 10 mM, K+
gluconate 135 mM, HEPES 10, MgCl2
2, MgATP 2, EGTA 1. Pipette solution for Na+
current contained in mM: NaCl 30, CsCl 100, HEPES 10, MgCl2
1, MgATP 2, NaGTP 0.05, EGTA 10, glucose 5. Pipette solution for Ca2+
current contained in mM: CsCl 120, HEPES 20, CaCl2
1, MgATP 2, EGTA 11, glucose 5. Pipette solution for current clamp experiments contained in mM: NaCl 38, Kgluconate 97, HEPES 20, MgATP 4, NaGTP 0.35, EGTA 0.35. All solutions were adjusted to pH 7.4. Electrophysiological measurements were performed with an Axopatch 200A (Molecular Devices) or a Patch-Clamp PC505B (Warner) amplifier. Patch pipettes resistances varied from 2-4 MΩ. Sodium channel currents in NG108-15 cells and calcium channel currents in GH3 cells were corrected by P/N leak subtraction. pClampex 8.2 software (Molecular Devices) in combination with a Digidata 1200 interface (Molecular Devices) were used to create and apply pulse protocols. Voltage clamp recordings were low-pass filtered at 2 kHz while current clamp measurements were low-pass filtered at 5 kHz. Illumination of cells was based on a xenon lamp either in combination with narrow band-pass filters or with a monochromator Polychrome V (Till Photonics), as described5
. For direct internal application through the patch pipette, QAQ was dissolved to a final concentration of 100 μM. Measurements were started after 5-10 min of equilibration time for HEK-293, NG108-15, GH3 cells and TG neurons, and after 15-20 min for hippocampal neurons. For bath incubation, cells were incubated with QAQ (classically 1 mM) in the presence or absence of agonist (ATP 1-2.5 mM or capsaicin 1 μM) at 37°C in the dark. Loading solution is similar to K+
current recording solution but with no calcium. Pretreated coverslips were rinsed with regular calcium-containing recording solution before measurement.
For spinal slice electrophysiology, slices were placed in a recoding chamber bathed with warmed (31°C) ACSF (in mM: NaCl 130.5; KCl 2.4;CaCl2 2.4; NaHCO3 19.5; MgSO4 1.3; KH2PO4 1.2; HEPES 1.25; glucose 10; pH 7.4) equilibrated with 95% O2 - 5% CO2.
Electrophysiological measurements were performed under the control of an Olympus BX51 microscope using an Axoclamp 2B (Molecular devices). Patch pipettes (7-11 MΩ) were filled with appropriate pipette solution (in mM: KGluconate 120; KCl 20; CaCl2 0.1; MgCl2 1.3; EGTA 1; HEPES 10; GTP 0.1; cAMP 0.2; Leupeptin 0.1; Na2ATP 3; D-Manitol 77; pH 7.3). Illumination of preparations was performed using two different wavelength diodes (380 and 500nm) controlled by Transistor-Transistor Logic (TTL) pulses. A glass suction electrode connected to Master 8 (A.M.P.Instrument Ltd) stimulator was used to stimulate dorsal roots. Non-nociceptive primary afferent fibers were specifically recruited using low-threshold stimulations (50μs, less than 100μA) whereas nociceptive fibers were recruited using high-intensity stimulations (500μs, more than 250μA).
Multi-electrode array (MEA) Recordings
A DRG was placed onto a 3D multi-electrode array chip (MEA60 200 3D GND, Ayanda Biosystems) and secured in place with a “harp” made from dialysis membrane stretched over thick platinum wire and bonded with superglue; the wire was U-shaped to allow the nerve to exit without being crushed. The MEA chip was mounted on an MEA1060-Up amplifier (Multi Channel Systems), and placed on the stage of an IX71 inverted microscope (Olympus). The nerve was led into a manipulator-mounted glass suction electrode of appropriate size driven by a DS2 stimulus isolator (Digitimer Ltd) triggered by pClamp v10.0 software through a Digidata 1440A data acquisition system (Molecular Devices). Except during drug incubations, the MEA chamber was continuously perfused with oxygenated ACSF at ~2 ml/minute. Recordings were performed at 30°C.
Before the drug incubation, the DRG was checked for response to stimulation at 1 Hz. If the signal was acceptable, the MEA chamber solution was replaced with oxygenated ACSF containing QAQ with or without other drugs, and incubated for 5 minutes. When using blockers, the DRG was pre-incubated with the blocker for 5 minutes before the application of QAQ with the blocker. The DRG was then washed with ACSF for 10 minutes before performing the experiment.
Recordings were done at a stimulation rate of 10 Hz while illuminating the DRG with 380 nm or 500 nm light. Each experiment consisted of 5 cycles of: 30 seconds under 380 nm light followed by 30 seconds under 500 nm light. The DRG was stimulated with 1 ms pulses at 10 Hz for the last 5 seconds under each wavelength of light, allowing 25 seconds to recover from adaptation in between stimulation episodes. Illumination was provided by a U-LH100HGAPO mercury lamp (Olympus) through a 4x objective, resulting in intensities of 17-28 mW/mm2. Filters for 380 nm and 500 nm were switched by a Lambda 10-3 system (Sutter Instrument Company) under the control of Metamorph v22.214.171.124 software (Molecular Devices). Evoked responses were recorded at 20 KHz with MC_Rack v4.0 software (Multi Channel Systems). Pictures were taken using Metamorph with a CoolSNAP HQ2 camera (Photometric) connected to the microscope.
MEA Data Analysis
Data was recorded in 40 ms-long sweeps synced to stimulation pulses, so that the stimulation produced an artifact at the beginning of the sweeps. Evoked spikes were detected by a negative threshold manually set beyond the noise level. For each detected spike, the first millisecond before the peak and the two milliseconds after were extracted into text files by MC_DataTool software (Multi Channel Systems), for processing with a custom Matlab program.
Our custom Matlab program calculated the area under each spike to the threshold level. A “region of interest” (ROI) was also set manually for each recording to exclude the stimulus artifact. The total integrated area of all spikes was calculated for each sweep, and averaged over the 5 cycles in each wavelength (). This averaged area per sweep was summated over the 5 seconds of stimulation to quantify the total evoked response in each wavelength of light. For each active channel, the normalized photosensitization was calculated as: (area380nm − area500nm) / (area380nm + area500nm). Channels with excessively small and/or irregular signals were conservatively culled. The per-channel photosensitization values, generated from at least three separate DRGs per drug condition, were pooled by condition and compared for significance using a Mann-Whitney U test (5% significance level).
Cornea-evoked reflex blinks
Sprague-Dawley rats (3-6 weeks old of either sex) were sedated using intraperitoneal injection of xylazine (9 mg/kg) and ketamine (60 mg/kg). Animals were then placed on a warming pad and behavioral testing was initiated when spontaneous movements ceased, while pinching the paw with a pair of forceps elicited a brisk withdrawal reflex. We used a series of von Frey hairs, nylon fibers of increasing diameter, which are pressed against the cornea to impart increasing force of high accuracy. We held von Frey hairs perpendicular to the cornea for approximately 2 s, or until a blink is initiated, using progressive increase in force from 8 mg to a maximal value of 1 g. Stimuli were presented three times for each stiffness, at intervals of several seconds. Both eyes were tested, and a positive response was noted if the animal blinks two or three times for a given force. Capsaicin (10 μM) was then topically applied on one cornea using a pipette (10 μl volume), while the contralateral cornea was treated with a mixture of capsaicin (10 μM) and QAQ (20 mM). Von Frey testing was done again 10-15 minutes after drug application. Immediately after von Frey testing, light was applied using an LED (Prizmatix, λmax = 385 nm, 30 mW/cm2) for one minute and von Frey hair were tested again.
Unless otherwise noted, all data are presented as ± s.e.m and statistics were analyzed with a Student t-test.