Gabazine

All drugs were prepared as stock solutions, aliquoted and frozen until use. The following pharmacological agents from Abcam (Cambridge, United Kingdom) were used: gabazine (GABA_A antagonist; 1 μM), a group I/II metabotropic glutamate receptor (mGluR) antagonist MCPG (methylene cyclopropyl glycine; 200 μM); a group I mGluR agonist DHPG (3,5-dihydroxy- phenylglycine; 5 μM); a group II mGluR agonist LY354740 [(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; 0.5 μM] and a group III mGluR agonist L-AP4 (l-2-amino-4-phosphonobutylate; 1 μM). The concentration of the different mGluR agonists used in the present study was determined by performing dose-response curves to reach similar inhibitory effects on the glutamatergic synaptic transmission (data not shown, see section “RESULTS”). The glutamate transporter inhibitor TBOA (DL-threo-benzyloxyaspartic acid; 5 μM) was purchased from Tocris (Bristol, United Kingdom), and the glycine receptor antagonist strychnine (1 μM) from Sigma-Aldrich (St. Louis, MO, United States).

The standard ACSF for acute brain slices contained (in mM): NaCl 125, KCl 2.5, CaCl₂ 2, MgCl₂ 1, D-glucose 25, NaHCO₃ 26, NaHPO₄ 1.25, gassed during the entire experiment with carbogen to adjust the pH to 7.4. Phosphate buffered solution (PBS) contained (in mM): NaCl 130, Na₂HPO₄ 7, NaH₂PO₄ 3. Norepinephrine and 2-APB (2-aminoethyl diphenylborinate) were obtained from Sigma-Aldrich (Darmstadt, Germany). ICI 118,551 hydrochloride (ICI), MPEP, CGP55845, MRS 2179, and ZM241385 were obtained from Tocris (Bristol, UK). BTP2 (YM-58483), D-APV, NBQX, gabazine, TTX (tetrodotoxin), CPA (cyclopiazonic acid), prazosin, and rauwolscine were obtained from Abcam (Cambridge, United Kingdom). All substances were stored as stock solutions according to the manufacturers' description.

Solutions were applied by gravity from a glass capillary (1.2 mm outer diameter) placed 1–2 mm from the neuron under study. Solutions were switched manually using a low dead volume manifold upstream of the glass capillary. CNQX and Gabazine were supplied by Abcam. Creatine Phosphate was supplied by Santa Cruz Biotech. Cinacalcet was supplied by Toronto Research Chemicals. All other reagents were supplied by Sigma-Aldrich.

Membrane potential fluctuations were recorded for a 3 s period at a holding potential of −80 mV. Voltage fluctuations consisted of a mixture of excitatory and inhibitory post-synaptic potentials observed as outward deflections. Voltage fluctuations were blocked by 1 mM kynurenic acid (Sigma-Aldrich), 10 μM NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione, Ascent Scientific), 10 μM gabazine (Abcam) and 10 μM strychnine (Abcam) (n = 4, data not shown). Membrane potential fluctuations were quantified by obtaining the baseline to peak amplitude for each time point during a 3 s recording. The baseline was taken as the steady state voltage (absence of voltage fluctuations) at −80 mV and was visually determined. Cumulative distribution plots of the voltage amplitudes were then constructed and differences were assessed using Kolmogorov-Smirnov tests.

Unless otherwise indicated, all reagents were acquired from Sigma (St. Louis, MO, United States).
Adenosine-5′-triphosphate was from Invitrogen (Waltham, MA, United States). α-Cyclopiazonic Acid (CPA) a reticulum Ca²⁺ ATPases inhibitor was from Tocris (Avonmouth, United Kingdom). Dimethylsulfoxide (DMSO), muscimol (a GABA_AR agonist), Nocodazole (which interferes with microtubules polymerization) and strychnine (a GlyR antagonist) were from Sigma. Gabazine (GABA_AR antagonist) and glycine (GlyR agonist) were purchased from Abcam (Cambridge, MA, United States). N,N′-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] (BAPTA-AM) was obtained from Molecular Probes (Eugene, OR, United States). Fura-2 acetoxymethyl ester (Fura-2AM) was from Calbiochem (Darmstadt, Germany).
Nocodazole stock solution (10 mM) was prepared in 1% of DMSO. All aliquots were stored at -20°C until further use. Working aqueous dilution was prepared at the day of the experiment. This drug was used in calcium imaging experiments. The maximum amount of DMSO present in the bath (0.0001% v/v) was devoid of influence in astrocytic calcium transients, when compared with controls (p > 0.05).