Journal: Biomedicines

Article Title: Human iPSC Modeling of Genetic Febrile Seizure Reveals Aberrant Molecular and Physiological Features Underlying an Impaired Neuronal Activity

doi: 10.3390/biomedicines10051075

Figure Lengend Snippet: Xenopus oocytes injected with membranes from idNs incorporated functional neurotransmitter receptors. ( A ) Sample currents evoked by 500 μM GABA or ( B ) 20 μM AMPA on oocytes microinjected with membranes extracted from cultured idNs obtained from a patient carrying the M145T mutation of the SCN1A gene. ( A ) GABA currents were completely inhibited by a brief pre-incubation (30 s) with bicuculline (100 μM) and subsequently recovered following the washout of the inhibitor. ( B ) AMPA currents were completely inhibited by co-administration of NBQX (50 μM), and they recovered to the original amplitude once NBQX administration was interrupted. AMPA currents were recorded in presence of CTZ (20 μM). Black bars = GABA; gray bars = AMPA; white bars in ( A ) bicuculline; in ( B ) NBQX. ( C ) Time course of the GABA current rundown evoked by six consecutive GABA applications (500 μM, 10 s) interspaced by a 40 s washout, in oocytes injected with membranes from control (black dots; ●) and M145T idNs (magenta; ● ; p > 0.05). The dots represent GABA currents expressed as a percentage of the first evoked response (● = 16.6 ± 1.0 nA, n = 8; ● = 23.7 ± 1.1 nA, n = 10).

Article Snippet: GABA, AMPA, CTZ, Bicuculline methochloride and NBQX were purchased from Tocris Bioscience (Bristol, UK) and dissolved in sterile water (GABA, AMPA and Bicuculline methochloride) or DMSO (CTZ, NBQX) before final dilution to the desired concentration in OR.

Techniques: Injection, Functional Assay, Cell Culture, Mutagenesis, Incubation, Control

Journal: Diabetes

Article Title: γ-Aminobutyric Acid (GABA) Is an Autocrine Excitatory Transmitter in Human Pancreatic β-Cells

doi: 10.2337/db09-0797

Figure Lengend Snippet: Quantal release of GABA from human β-cells. A : A β-cell overexpressing α 1 /β 1 GABA A R was held at −70 mV and infused with intracellular solution containing 2 μmol/l free Ca 2+ (at 5 mmol/l extracellular glucose). SR-95531 (10 μmol/l) was applied as indicated by the bar. The inset shows a part of the trace (indicated by *) on an expanded time scale. B: A train of 10 500-ms voltage-clamp depolarizations from −70 to 0 mV was applied to a cell overexpressing α 1 /β 1 GABA A R (with intracellular solution containing 50 μmol/l EGTA). GABA-induced transient currents are indicated by arrows. The inset shows a part of the trace (marked by the dotted rectangle) on an expanded time base. Note that the direction of transient currents is outward at 0 mV. The initial downward component represents the opening of the voltage-gated Na + and Ca 2+ currents triggered by the depolarization. The activation of the GABA A R accounts for the outward current. The recording shown is representative of seven experiments.

Article Snippet: Sections were then incubated with antibodies directed against GABA A R subunits α 1–6 (1:50, H-300; Santa Cruz Biotechnology, Santa Cruz, CA), β 2/3 (1:100, MAB341, Millipore, Hampshire, U.K.) or γ 2 (1:100, LS-C14, Life span, Seattle, WA) dissolved in REAL antibody diluent (Dako, Cambridge, U.K.) at 4°C overnight.

Techniques: Activation Assay

Journal: Diabetes

Article Title: γ-Aminobutyric Acid (GABA) Is an Autocrine Excitatory Transmitter in Human Pancreatic β-Cells

doi: 10.2337/db09-0797

Figure Lengend Snippet: Storage and secretion of GABA by insulin-containing LDCVs in human β-cells. A : GABA release was detected by patch-clamping in an identified β-cell overexpressing α 1 /β 1 GABA A R ( upper trace ). The cell was held at −70 mV and infused with intracellular solution containing 2 μmol/l free Ca 2+ . Serotonin release was measured simultaneously in the same cell by carbon fiber amperometry ( lower trace ). The dashed lines indicate simultaneous occurrence of GABA-induced TICs and amperometric currents. B : Immunogold labeling of GABA in a human β-cell. Scale bar: 250 nm.

Article Snippet: Sections were then incubated with antibodies directed against GABA A R subunits α 1–6 (1:50, H-300; Santa Cruz Biotechnology, Santa Cruz, CA), β 2/3 (1:100, MAB341, Millipore, Hampshire, U.K.) or γ 2 (1:100, LS-C14, Life span, Seattle, WA) dissolved in REAL antibody diluent (Dako, Cambridge, U.K.) at 4°C overnight.

Techniques: Labeling

Journal: Diabetes

Article Title: γ-Aminobutyric Acid (GABA) Is an Autocrine Excitatory Transmitter in Human Pancreatic β-Cells

doi: 10.2337/db09-0797

Figure Lengend Snippet: Functional detection of endogenous GABA A R Cl − channels in human islet cells. A : Patch-clamp recording of currents evoked by puffer application of GABA (1 mmol/l, as indicated by the bars) to an identified β-cell in the absence ( black trace ) and presence ( gray trace ) of 50 μmol/l SR-95531 in the same cell. The cell was held at −70 mV throughout the experiment. B : As in A , showing a δ-cell (note the difference in scale bars). C : As in A , showing an α-cell. The cell had been incubated in the presence of 0.5 μmol/l insulin for 1 h before the experiment.

Article Snippet: Sections were then incubated with antibodies directed against GABA A R subunits α 1–6 (1:50, H-300; Santa Cruz Biotechnology, Santa Cruz, CA), β 2/3 (1:100, MAB341, Millipore, Hampshire, U.K.) or γ 2 (1:100, LS-C14, Life span, Seattle, WA) dissolved in REAL antibody diluent (Dako, Cambridge, U.K.) at 4°C overnight.

Techniques: Functional Assay, Patch Clamp, Incubation

Journal: Diabetes

Article Title: γ-Aminobutyric Acid (GABA) Is an Autocrine Excitatory Transmitter in Human Pancreatic β-Cells

doi: 10.2337/db09-0797

Figure Lengend Snippet: Glucose- and tolbutamide-induced GABA release from human β-cells. Experiments were performed in small clusters of islet cells overexpressing α 1 /β 1 GABA A R. The patch-clamped cell was held at −70 mV and infused with pipette solution containing 10 mmol/l EGTA. A : The glucose concentration in the bath was increased from 1 to 6 mmol/l as indicated. B ( upper ): The extracellular glucose concentration was increased from 1 to 20 mmol/l. SR-95531 (10 μmol/l) was included as indicated. B ( lower ): Sections of top trace (as indicated by letters i and ii ) shown on an expanded time base. C : Summary of observed frequencies of GABA release (TICs/min) at the indicated glucose concentrations (* P < 0.05). The measurements were made at steady state (1–5 min after addition of glucose). D : Tolbutamide (100 μmol/l) was applied as indicated (extracellular glucose concentration 4 mmol/l). E : SR-95531 (10 μmol/l) was applied as indicated at 1 mmol/l extracellular glucose.

Article Snippet: Sections were then incubated with antibodies directed against GABA A R subunits α 1–6 (1:50, H-300; Santa Cruz Biotechnology, Santa Cruz, CA), β 2/3 (1:100, MAB341, Millipore, Hampshire, U.K.) or γ 2 (1:100, LS-C14, Life span, Seattle, WA) dissolved in REAL antibody diluent (Dako, Cambridge, U.K.) at 4°C overnight.

Techniques: Transferring, Concentration Assay

Journal: Diabetes

Article Title: γ-Aminobutyric Acid (GABA) Is an Autocrine Excitatory Transmitter in Human Pancreatic β-Cells

doi: 10.2337/db09-0797

Figure Lengend Snippet: Expression of GABA A R subunits in human islets. A : Expression profiling of GABA A R subunits in islets by quantitative RT-PCR ( n = 3 preparations from three donors). B–D : Co-labeling of pancreatic tissue sections with anti-insulin and anti-GABA A R β 2/3 ( B ), anti-GABA A R α 1–6 ( C ), or anti-GABA A R γ 2 ( D ). Insulin is shown in red and GABA A R subunits in green; co-localization results in yellow labeling (scale bars = 10 μm). a.u., arbitrary units.

Article Snippet: Sections were then incubated with antibodies directed against GABA A R subunits α 1–6 (1:50, H-300; Santa Cruz Biotechnology, Santa Cruz, CA), β 2/3 (1:100, MAB341, Millipore, Hampshire, U.K.) or γ 2 (1:100, LS-C14, Life span, Seattle, WA) dissolved in REAL antibody diluent (Dako, Cambridge, U.K.) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Labeling