Adult male Sprague–Dawley rats (Harlan Sprague Dawley, Indianapolis IN) were used for all experiments. Rats were housed in an AAALAC approved animal facility with a 12:12 light/dark cycle (lights off at 7 PM) with food and water available ad libitum. All procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in according with the recommendations of the National Institutes of Health and the Committee for Research and Ethical Issues of the International Association for the Study of Pain. All efforts were employed to minimize the number of animals used in this study.
Retrograde labeling and inflammation
Cutaneous afferents were retrogradely labeled with DiI as previously described [5, 8]. Briefly, rats were anesthetized with isofluorane and 10 μl of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI, Invitrogen, Carlsbad CA, 17 mg/ml in saline diluted from a stock of 170 mg/ml in DMSO) was injected at 3–5 sites (with 1.5 - 2 μl per site) with a 30 g needle directed into the epidermis. Rats were studied 14 to 17 days post DiI injection. Inflammation was induced in a subgroup of rats with a 100 μl subcutaneous injection of complete Freund’s adjuvant (CFA, mixed 1:1 in saline) into the same site previously labeled with DiI. This injection was also made under isofluorane-induced anesthesia. This group of rats was studied 3 days after CFA injection.
Preparation of isolated cutaneous neurons
Acutely dissociated cutaneous neurons were obtained as previously described [5, 8]. Briefly, rats were deeply anesthetized with 1 ml/kg rat cocktail (55 mg/ml ketamine, 5.5 mg/ml xylazine and 1.1 mg/ml acepromazine) and L4 and L5 dorsal root ganglion (DRG) were harvested, enzymatically treated, mechanically dissociated and plated onto laminin-ornithine coated cover slips. After 2 hrs of incubation at 37°C/3% CO2, cover-slips were flooded with HEPES buffered L-15 media and stored at room temperature during the period of recording (< 8 hrs after removal from the animal).
Patch clamp recording
Isolated cutaneous neurons were studied with conventional whole-cell and perforated patch configurations with an Axopatch 200B (Medical Devices Sunnyvale CA) controlled with pClamp (v 8.2, Molecular Devices) or a HEKA EPC9 amplifier (HEKA Electronik, Lambrecht/Pfalz Germany) controlled with Pulse software (V8.8, HEKA). The conventional whole cell configuration was used for current clamp recording and for voltage-clamp analysis of Kv currents while the perforated patch configuration was used for the analysis of BKCa currents. For current clamp recording, the bath solution contained (in mM): NaCl, 130; KCl, 5; CaCl2, 2.5; MgCl2, 0.6; HEPES, 5; and glucose, 10; pH adjusted to 7.4 with Tris-Base, and osmolality adjusted to 320 mOsm with sucrose. For voltage-clamp recording of Ca2+ dependent K+ currents, NaCl was replaced with choline-Cl to eliminate voltage-gated Na+ currents. This same solution was used for voltage clamp recording of voltage-gated K+ currents, except that CaCl2 was replaced with CoCl2 to eliminate voltage-gated Ca2+ currents. The electrode solution used for current clamp and to record voltage-gated K+ currents contained (in mM): KCl, 30; K-methanesulfonate (MES), 110; MgCl2,1; CaCl2, 0.1; EGTA, 1; HEPES, 10; ATP-Mg, 2; GTP, 1; pH adjusted to 7.2 with Tris-Base, and osmolality adjusted to 310 mOsm with sucrose. The electrode solution used for perforated patch clamp recordings contained (in mM): KCl, 30; K-MES, 110; MgCl2, 1; HEPES, 10; EGTA, 0.1; pH adjusted to 7.2 with Trisbase, and osmolality adjusted to 310 mOsm with sucrose. Amphotericin B, used to obtain whole cell access for perforated patch recording, stock solution was prepared in DMSO (90 mg/ml) then diluted to a final concentration (600 μg/ml) in electrode solution immediately prior to use. All salts used for electrophysiology were obtained from Sigma-Aldrich (St Louis MO).
Semi-quantitative RT-PCR (sqRT-PCR)
Dorsal root ganglia (DRG) from anesthetized male rats were harvested in a manner identical to that used for neuron isolation and plating. mRNA was extracted and cDNA synthesis performed as previously described , except that that random hexomers were used to prime the reverse transcription reaction. SYBR Green was used to monitor amplification of template with primers on a real-time thermal cycler (Life Technologies, Grand Island NY) controlled by a PC running Prism 7000 SDS software. A melting curve was generated at the end of each experiment to assess for the presence of contamination. Amplification efficiency was determined for each target gene. The ΔΔCT method was used to assess differences in relative expression levels. Primers for amplification of the core BKCa α subunit were: F - TGTCATGATGACGTCACAGATCC, R -TTTTTTTGGTGACAGTGTTGGC; those for amplification of the BKCa α subunit with the STREX insert were: F - AGCCGAGCATGTTGTTTTGAT, R- ACGCACACGGCCTGACA; while those for GAPDH were: F - GGCCTACATGGCCTCCAA, R -TGGAATTGTGAGGGAGATGCT. Commercially available primer sets were used for the amplification of β2, 3 and 4 (Qiagen).
Single cell PCR
Single cell PCR was performed as previously described . Because perforated patch recording is relatively slow and many neurons are needed from a single animal to obtain a reasonable estimate of the proportion of neurons from a single animal, a different set of neurons was used for single cell PCR analysis. Following identification of cutaneous neurons under epifluorescence illuminations, neurons were collected with large bore (~30 μm) glass pipettes and expelled into microcentrifuge tubes containing reverse transcriptase (RT) mix. RT-PCR was performed as described previously  utilizing an anchored primer (5’-ttttttttttttttttttvn-3’; v = a, c, or g; n = a, c, g, or t, from Life Technologies) for the RT reaction and a nested PCR amplification strategy for the PCR reaction. rslo primer sequences were identical to those described previously . For each cell preparation, at least two tubes were run in which no cell was collected (although the electrode was manipulated in a manner identical to that used for cell collection) and at least two additional tubes were run in which no reverse transcriptase was added to the RT mix prior to the RT reaction. Cyclophillin (0.5 μl of cDNA) was used to monitor the success of the cell collection/RT reaction: only neurons in which cyclophillin was detected were used for further analysis. 5 μl of PCR products were loaded onto 2% agorase/TAE gel.
DRG (L4/L5) were rapidly removed from deeply anesthetized rats and homogenized in solubilization buffer (50 mM Tris.HCl, pH8.0; 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 1 U/ml aprotinin, 20 μg/ml leupetin, 20 μg/ml pepstatin A). The homogenate was centrifuged at 20,000 X g for 10 min at 4°C. The supernatant was removed. Protein (50–120 μg) was separated on a 7.5-10% SDS-PAGE gel and blotted to nitrocellulose membrane (Amersham) with a Trans-Blot Transfer Cell system (Bio-Rad). Blots were blocked with 5% milk in TBS buffer (20 mM Tris, 150 mM NaCl pH 7.4) at room temp for 1 hour. After decanting the blocking buffer, the blots were incubated with primary antibodies. These included: the α subunit of the BKCa channel (AKA slo1, NeuroMab clone L6/60, NeuroMab, Davis CA: 1:200), BKCa β2 (NeuroMab clone N53/32: 1:200), BKCa β3 (NeuroMab clone N40B/18: 1:200), BKCa β4 (NeuroMab clone L18A/3: 1:200), and GAPDH (sc-25778, Santa Cruz Biotechnology: 1:1000). The specificity of all BKCa subunit antibodies has been confirmed in heterologous expression systems where there was no evidence of cross reactivity with other BKCa subunits or KV2.1. Membranes were incubated with BKCa subunit antibodies overnight at 4°C, and GAPDH for 1 hour at room temperature. Membranes were washed with TBS buffer and incubated for 1 hour with anti-goat IgG horseradish peroxidase (1:3000, Santa Cruz) in 5% milk/TBS. Membranes were then washed with TBS buffer. The immunoreactivity was detected using Enhanced Chemiluminescence (ECL, Amersham). Chemiluminescence was captured with a CCD camera (Las-3000, Fujifilm) and analyzed with Fuji software Multi Gauge. The relative protein levels were obtained by comparing target protein to loading control (GAPDH) in the same membrane.
Data are expressed as mean ± S.E.M unless otherwise stated. Student’s t test, one- and two-way ANOVA with the Holm-Sidak post hoc test were used for comparisons of parametric data between groups. For single cell PCR analysis, between 30 and 40 cutaneous neurons were collected from each animal, although only 29 neurons were collected for one of the 4 naïve animals used to assess changes in α-subunit splice variants. The proportion of the total number of neurons from each animal in which a splice variant or β-subunit was detected was used as the “statistic” for that animal, where the mean proportion of expression in naïve animals was compared to that in inflamed animals. Statistical significance was assessed at p < 0.05.