Animal treatments were performed according learn more to the guidelines of the International Association for the Study of Pain. Adult C57BL/6J (wild-type) male mice, heterozygous GAD65-EGFP mice, and TRPV1−/− mice of C57BL/6J background were used.

To assess mechanical sensitivities, the withdrawal threshold of the hindpaw was measured using a series of von Frey filaments (Stoelting, Wood Dale, IL). All behavioral testing was performed by an investigator who was blind to the treatment group and genetic background of the mice. Drugs or vehicle (5 μl) was injected at the level of the lumbar enlargement using a 25 μl Hamilton syringe fitted with a 31 gauge needle. Three- to four-week-old mice were intraperitoneally injected with RTX dissolved in a mixture of 10% Tween-80 and 10% ethanol in normal saline or vehicle alone under isoflurane anesthesia as a single bolus

in two injections of 50 μg/kg and 150 μg/kg on days 1 and 2, consecutively. RTX-treated mice were used in experiments at least 7 days after final RTX injection. Real-time PCR was performed using a 7500 Real-Time PCR system (Applied Biosystems). All ΔCt values were normalized to GAPDH. The PCR primer sequences used in this study are listed in Table S1A. Transverse slices (300 μm) were prepared from C57BL/6J or GAD65-EGFP mice (4–6 weeks old). Whole-cell patch clamp recordings of spinal cord SG and STT neurons were performed at room temperature (25°C ± selleckchem 1°C). To prevent spontaneous synaptic activity, a cocktail of neurotransmission inhibitors were added (in μM): 10 CNQX; 50 D-AP5, 10 picrotoxin, 2 strychnine, 0.5 tetrodotoxin. For the composition of all internal and modified aCSF solutions see Supplemental Experimental Procedures. To record EPSCs, SG neurons were held at −70 mV. Electrical stimuli (0.01 ms, 0.066 Hz) were delivered through a bipolar, Teflon-coated tungsten electrode, which was placed in DREZ of spinal cord

and monosynaptic EPSCs were identified on the basis of the absence of conduction failure of evoked EPSCs. To record evoked inhibitory postsynaptic currents (eIPSCs) from STT neurons, the DiI-labeled neurons were held at 0 mV. Under fluorescence microscopy, GAD65-EGFP SG neurons and DiI-labeled STT neurons were verified in spinal cord slices. Identified cells were collected Non-specific serine/threonine protein kinase into a patch pipette with a tip diameter of about 20 μm and gently put into a reaction tube containing reverse transcription reagents. All PCR amplifications were performed with nested primers (Table S1B). Spinal cord slices (700 μm) were incubated in 95% O2/5% CO2 saturated recording aCSF with 5 μM capsaicin for 10 min at 32°C followed by washed out for 30 min. Spinal cord slices were homogenized and centrifuged and protein concentration was determined with BCA assay kit (Pierce). Equal amounts of proteins were separated by SDS-PAGE electrophoresis and transferred onto PVDF membrane.