Na gtp

AlexaFluor-594 hydrazide, AlexaFluor-594 dextran (10,000 MW), Fluo-4 and Fluo-4FF pentapotassium salts were purchased from Invitrogen. CsCl and NaHCO₃ were obtained from ACP Chemicals, tetrodotoxin (TTX) from Alomone Labs, N-ethyllidocaine bromide (QX-314, Br-) and bicuculline from Tocris Bioscience, SP20 antibody from Santa Cruz Biotechnology (Cat# sc-65512, Lot# K1407, RRID:AB_1129364), paraformaldehyde from Electron Microscopy Sciences. We ordered NaCl, KCl, glucose, NaH₂PO₄, Na-pyruvate, myo-inositol, l-ascorbic acid, MgCl₂, CaCl₂, K-gluconate, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), HEPES, phosphocreatine, Na-ATP, Na-GTP, phosphocreatine di(tris) salt, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and Strychnine from Sigma-Aldrich.

Polymerization of tubulin was performed as previously described (Chae et al., 2009 (link); Wilson et al., 2012a (link)) with modifications. Polymerization was performed in 0.1 M G-PEM buffer (1 mM GTP, 80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, pH 7.0), 1 mM Na-GTP (Sigma), and 2 mg/ml tubulin (Cytoskeleton, Inc). CRMP2 proteins (0.2 μM) as well as 3 μM of (R)-LCM, (S)-LCM or 0.01% DMSO were added to the samples and pipetted onto a 96-well plate at 4°C. Following a 30 min incubation on ice, turbidity changes were assessed at 412 nm using a Synergy™ 2 Multi-Detection Microplate Reader (BioTek Instruments, Inc., San Diego, CA) which had previously been pre-warmed to 37°C. Absorbances were measured over time and compared to background samples which contained only buffer + GTP.

Using the whole-cell voltage-clamp configuration of the patch-clamp technique, ionic currents were recorded from isolated neurons at -80 mV holding potential after 18–32 hours as previously published
[15 (link), 58 (link)]. The external solution (ECS) contained (in mM): 145 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂ (all Sigma), 10 glucose and 10 HEPES (Merck, Darmstadt, Germany), at pH 7.3 adjusted with NaOH (Merck). Borosilicate glass micropipettes (Science Products, Hofheim, Germany) pulled with a horizontal puller (Sutter Instruments Company, Novato, CA, USA) were filled with internal solution (ICS, in mM): 148 KCl, 2 MgCl₂, 2 Na-ATP, 0.2 Na-GTP, 0.1 CaCl₂, 1 EGTA (all Sigma) and 10 HEPES (Merck), at pH 7.3 adjusted with KOH (Merck). After filling, electrode resistance was 4–6 MΩ. Currents were filtered at 2.9 kHz, sampled at 3 kHz and recorded using an EPC-9 (HEKA, Germany) and the Pulse v8.74 software (HEKA) without Rs compensation. Experiments were performed at room temperature and only one neuron was tested per Petri dish. An automated seven-barrel system with common outlet positioned at 100 μm distance from the recorded cell was used for fast drug administration
[15 (link)]. S1P (1.0 μM) was used as intermittent conditioning stimuli (60s). S1P, capsaicin, PTX and GDPβS were purchased from Sigma Aldrich. All other chemicals were purchased from Merck-Calbiochem.

D2 receptor-mediated outward currents were recorded in the presence of the following receptor antagonists (all from Sigma-Aldrich): MK-801 (30 µM added to the recovery bath, NMDA), picrotoxin (100 μM, GABA_A), CGP 56999a (100 nM, GABA_B), DNQX (6,7-dinitroquinoxaline-2,3(1H,4H)-dione) (10 μM, AMPA), and hexamethonium (100 μM, nicotinic acetylcholine). For bath application experiments, drug concentrations were as follows: dopamine hydrochloride (Sigma-Aldrich, 100 μM), CRF (Tocris, 100 nM), and baclofen (GABA_B agonist, Sigma-Aldrich, 3–30 μM). GABA, Mg ATP, Na GTP, Na HEPES, K HEPES, and BAPTA were obtained from Sigma-Aldrich. K-methyl sulfate was from MP Biomedicals, LLC.

The chemicals used to make the ACSF were purchased from Panreac or Merck. Mg‐ATP, Na‐GTP, and phosphocreatine disodium salt hydrate for the internal solution were obtained from Sigma‐Aldrich. Stock solutions of 5‐CT (5‐carboxamidotryptamine maleate salt; Sigma‐Aldrich, Madrid, Spain), baclofen [(R)‐4‐Amino‐3‐(4‐chlorophenyl)butanoic acid; Abcam, Cambridge, UK], CGP55845 (CGP55845 hydrochloride; Abcam, Cambridge, UK), GABA (4‐aminobutanoic acid; Abcam), tertiapin‐Q (Tocris, Bristol, UK), and WAY100635 (N‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐2‐pyridinylcyclohexanecarboxamide maleate salt; Sigma‐Aldrich) were prepared in distilled water. DNQX [6,7‐dinitroquinoxaline‐2,3‐dione; Abcam] and MK‐801 [(+)‐MK‐801 maleate; Abcam] were prepared in DMSO (dimethyl sulfoxide). Drug stocks were diluted in artificial cerebrospinal fluid (ACSF) immediately before application. The highest experimental concentration of DMSO was 0.01%. Isoflurane was purchased from Abbott.