FIGURE 3.

NE-mediated hyperpolarization in layer III pyramidal neurons of the EC requires the functions of inhibitory G-proteins (Gα_i), AC, and PKA. A, intracellular application of GDP-β-S (4 mm) via the recording pipettes blocked NE-induced increases in outward holding currents (n = 5). NE was applied after the formation of whole cell configuration for >20 min to allow the diffusion of GDP-β-S into cells. B, application of NE (100 μm) failed to induce an outward holding current in slices pretreated with pertussis toxin for ∼8 h (n = 5). C, intracellular dialysis of MDL-12,330A (2 mm) via the recording pipettes generated an outward holding current per se, and subsequent application of NE (100 μm) in the presence of MDL-12,330A did not significantly alter the holding currents (n = 8). Holding currents were recorded immediately after the formation of whole cell configuration and zeroed by subtracting the holding current at 1 min prior to the application of NE. D, intracellular dialysis of (R_p)-cAMPS (1 mm) via the recording pipettes generated an outward holding current per se and blocked NE-induced increases in outward holding currents (n = 6). Holding currents were recorded immediately after the formation of whole cell configuration and zeroed by subtracting the holding current at 1 min prior to the application of NE. E, bath application of forskolin (20 μm) and 3-isobutyl-1-methylxanthine (IBMX, 1 mm) to increase intracellular cAMP concentration induced an inward holding current, and subsequent application of NE (100 μm) in the presence of forskolin and 3-isobutyl-1-methylxanthine induced a smaller outward holding current (n = 5). Holding currents were zeroed by subtracting the current recorded at 1 min before application of NE. F, bath application of 8-CPT-cAMP (500 μm), a membrane-permeable cAMP analog, induced an inward holding current and reduced NE-induced increases in outward holding current (n = 6).