evidence for this hypothesis and show that stimulation of bARs reduces CDI in TC neurons via a protein complex including AKAP and PKA. Materials and Methods Ethics Statement All experiments were carried out in accordance with the European Committees Council Directive (86/609/EEC) and approved by the local animal care committee (Landesverwaltung- samt Sachen-Anhalt, AZ: 42502/5.19 UNI MD). Reverse transcription-polymerase chain reaction (RT-PCR) assays Poly A+ mRNA was prepared from freshly dissected thalamic tissue (the ventrobasal thalamic complex (VB) and dLGN were visually identified) or whole brain by extraction with Trizol reagent according to the manufacturer’s instruction (Oligotex Direct mRNA, Qiagen, Hilden, Germany). First-strand cDNA synthesis was primed with oligo (dT) from 0.5–1 mg of mRNA, using the SuperScript II enzyme (Invitrogen Life Technologies). Specific primers for calcium channels a1C-a1E, G-proteins, adenylatecyclase, and b-receptors were used according to manufacturer description (Figure 1A–1F). Accession numbers for used primers were: Alpha1S (nucleotides 445–828) No. L04684; Alpha1C (nucleotides 2624–3033) No. NM012517; Alpha1D (nucleotides 3691–4200) No. NM017298; Alpha1F (nucle- otides 3196–3608) No. NM0053701; Alpha1A (nucleotides 5115– 5387) No. NM012918; Alpha1B (nucleotides 1742–2051) No. NM147141; Alpha1E (nucleotides 5831–6377) No. NM019294; Galpha q (nucleotides 188–560) No. AF234260; Galpha11 (nucleo- tides 139–554) No. AF239674; Galpha i–1 (nucleotides 669–1198) No. NM013145; Galpha i-2 (nucleotides 1125–1520) No. 031035; Galpha i-3 (nucleotides 1993–2367) No. NM013106; b1–AR (nucleotides 742–1104) No. NM012701; b2-AR (nucleotides 676– 964) No. NM012492; b3-AR (nucleotides 398–797) No. NM013108; AC1 (nucleotides 1361–1863) No. XM223616; AC6 (nucleotides 1581–1940) No. NM012812; AC8 (nucleotides 3001–3480) No. NM017142; AKAP5 (nucleotides 122–589 No. NM 133515; AKAP7 (nucleotides 494–1059) No. NM 001001801; Gs (nucleotides 332–756) No. NM 019132. Cell type-specific RT - PCR Brain tissue from P14–P24 Long Evans rats consisting of dLGN was sliced using a vibratome to 500 mM. After try- psinization of tissue blocks cells were visually identified under the microscope. Complete cells were sucked into the pipette and transferred into 3 ml carrier RNA buffer (RNeasy Micro Kit, QIAGEN) by breaking the tip of the pipette and expelling 3 ml of solution with positive pressure. The pipette solution (6 ml) was supplemented with a recombinant ribonuclease inhibitor (0.24 U/ml; RNasin; Promega, Madison, WI, USA). Cytoplasm from single, identified cells were pooled (interneurons and TC neurons, respectively), RNA was isolated without DNase treat- ment using an RNA isolation kit (RNeasy Micro Kit, QIAGEN), and reverse transcription (RT) protocol was used for cDNA according to manufactures protocol. Integrity of all obtained cDNAs was confirmed by using primers for the housekeeping gene GAPDH. After confirmation of integrity, cDNAs were used for standard RT-PCRs with specific primers for each gene of interest (AKAP 5; AKAP 7; AC stimulatory G-protein, Gs; Figure 2A). PCR products were separated by size on 1% agarose gels and visualised using an Eagle eye system after ethidium bromide staining. Modulation of Calcium-Dependent Inactivation Quantitative Real – time PCR Real-time PCR was performed using the Real PCR Master Mix 2.5X (Eppendorf) and the ABI Prism 7000 Sequence Detection System (Applied Biosystems); PCR program was: 2 min at 50uC, 10 min at 95uC, 50 cycles: 15 s at 95uC and 1 min at 60uC. Analysed probes were AKAP 5, AKAP 7, and b-2 microglobulin as the internal reference gene used for normalisation (Figure 2B). All primers were purchased from Applied Biosystems. Results were analysed with the ABI Prism 7000 SDS software. The efficiency of real-time primer/probes was nearly identical. Quantification was done using the comparative Ct method as described earlier [21]. In a standard PCR GAPDH (nucleotides 789–1028, accession No. NM017008) expression was checked as a positive control to confirm the integrity of transcribed cDNA. The PCR protocol for GAPDH amplification was: 3 min 94uC; 50 cycles (30 s 94uC, 1 min 61uC, 1 min 72uC); 7 min 72uC. Tissue Preparation Thalamic slices were prepared from juvenile postnatal day (P) 12–P24 Long–Evans rats. After anaesthesia with isoflurane, animals were decapitated and a block of tissue containing the dLGN was rapidly removed and placed in chilled (2–4uC), oxygenated slicing solution (pH 7.35, with NaOH) containing the following (in mM): sucrose, 195; glucose, 11; Pipes, 20; KCl, 2.5; MgSO4, 10; and CaCl2, 0.5. Coronal slices of the thalamus were cut at 300 mm on a vibratome and kept submerged in artificial cerebrospinal fluid (ACSF; pH 7.35, with 95% O2-5% CO2) containing the following (in mM): NaCl, 125; KCl, 2.5; NaH2PO4, 1.25; NaHCO3, 22–26; MgSO4, 2; CaCl2, 2; and glucose, 10. Slices were kept for 20 min at 34uC before being cooled to room temperature, and allowed to rest for 60 to 90 min. Patch clamp recordings Whole-cell recordings under voltage clamp condition were performed on visually identified TC neurons of the dLGN at room temperature (21–23uC) using borosilicate glass pipettes (GC150TF-10, Clark Electromedical Instruments, Pangbourne, UK) connected to an EPC-9/2 amplifier (HEKA Electronics, Lamprecht, Germany). Typical electrode resistances amounted to 2–4 MV, while access resistance was 5–15 MV. Series resistance compensation was routinely used (30%). Voltage-clamp experiments were governed by Pulse software (HEKA, Germany). For standard recordings the following solutions were used: (i) extracellular solution (in mM): NaCl, 125; KCl, 2.5; NaH2PO4, 1.25; NaHCO3, 22–26; MgSO4, 2; CaCl2, 2; and glucose, 10; TTX, 0.001; 4-AP, 4; pH 7.35 with NaOH. (ii) Intracellular solution: Cs-gluconate, 85; Cs3-citrate, 10; NaCl, 10; KCl, 1; EGTA. 1.1; CaCl2, 0.1; MgCl2, 0.25; HEPES, 10; TEA-Cl, 15; Mg-ATP, 3; Na2-GTP, 0.5; pH 7.25 with CsOH. In some experiments equimolar concentrations of ascorbic acid were added to the bath solution in order to prevent oxidation of the drugs. HVA Ca2+ currents were evoked from a holding potential of 240 mV by a double-pulse protocol in which conditioning pulses to varying potentials (240 to +60 mV, 200 ms duration) were followed by a brief gap (240 mV, 50 ms) and a subsequent analyzing test pulse to a fixed potential of +10 mV (200 ms) (Figure 3A). For standard recordings Ca2+ was used as a charge carrier and 1.1 mM EGTA was included to the intracellular solution. The degree of inactivation (Dinact) was determined by dividing the current amplitudes elicited by a test pulse to +10 mV with maximal and minimal preceding conditioning current amplitude (Figure 3B–3C, Dinact = [12(A2/A1)]6100(%)). A2 (Imin, Figure 3C) represents the minimal current amplitude after a preceding conditioning pulse, as measured at the beginning of PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e27474