Dpni caged gaba

Anti-vGAT-Oyster 550 antibody was purchased from Synaptic System (Goettingen, Germany). Anti-HA antibody was from Roche (Milan, Italy). BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), L-NAME (L-NG-Nitroarginine methyl ester), Nifedipine (1,4-Dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethylester), and Bicuculline were purchased from Sigma (Milan, Italy). KN-93 and KN-92 were acquired from Millipore Merck (Darmstadt, Germany). APV (D-(−)-2-Amino-5-phosphonopentanoic acid), CNQX (6-Cyano-7-nitroquinoxaline-2,3-dione), ω-conotoxin MVIIC, ω-conotoxin GVIA, DPNI-caged-GABA (1-(4-Aminobutanoyl)-4-[1,3-bis(dihydroxyphosphoryloxy)propan-2-yloxy]-7-nitroindoline) and MNI-caged-L-glutamate ((S)-α-amino-2,3-dihydro-4-methoxy-7-nitro-δ-oxo-1H-indole-1-pentanoic acid) were purchased from Tocris (Bristol, UK). Rhod-2 tripotassium salt was purchased from AAT Bioquest (Sunnyvale, CA, USA).

Acute 400-μm coronal neocortical slices were used as before 5 (link). To measure KCC2-mediated Cl⁻ extrusion, we used our standard assay where a somatic Cl⁻ load (19 mM) is imposed on the neuron via a whole-cell patch pipette. The ensuing bumetanide-insensitive somatodendritic E_GABA gradient, ΔE_GABA, determined as the difference between the E_GABA at the soma and at 50 μm away along the apical dendrite, provides an accurate estimate of the net Cl⁻ extrusion 13 (link). Whole-cell patch clamp recordings were performed as before 13 (link) in an experiment-blind manner from EGFP-positive layer 2/3 pyramidal neurons from slices of P6-7 mice co-electroporated in utero with plasmids bearing constructs encoding for EGFP and one of the following: KCC2-WT, KCC2-R952H or rKCC2-ΔNTD. Some animals were electroporated with the EGFP plasmid alone. DPNI-caged GABA (Tocris) was used to elicit GABA_AR-mediated currents 31 (link). All recordings were performed in the presence of 10 μM bumetanide (Tocris), 0.5 μM TTX (Abcam), 10 μM CNQX (Abcam) and 1 μM CGP 55845 (Abcam) in the standard extracellular solution 13 (link).