Whole-cell patch clamp experiments

The cells were voltage clamped at -70-mV holding potential. Pipette offset potential, series resistance (R_s) and capacitance were compensated before recording. Only cells with low holding current (<≈10 pA) and stable baseline were used. Input resistance (R_in), R_s, and membrane capacitance (C_m) were also measured before each recording by using 5-mV hyperpolarizing pulses. To ensure consistent recording qualities, only cells with R_s < 20 MΩ, R_in > 500 MΩ, and C_m > 10 pF were accepted.

There is a widely accepted consensus that GABA is excitatory via GABA_A-R in GnRH neurons due to the high intracellular chloride concentration in these cells (DeFazio et al., 2002; Moenter and DeFazio, 2005; Herbison and Moenter, 2011). Our main goal was to mimic the physiologic conditions of GnRH neurons as much as possible during the entire experiment, therefore, high concentration of chloride in the intracellular solution was indispensable. According to this requirement, the intracellular pipette solution contained high amount of chloride: 10 mM HEPES, 140 mM KCl, 5 mM EGTA, 0.1 mM CaCl₂, 4 mM Mg-ATP, and 0.4 mM Na-GTP (pH 7.3). The resistance of the patch electrodes was 2–3 MΩ. For the miniature PSC (mPSC) measurements, 10 min before the start of recording, the spike-mediated transmitter release was blocked by adding the voltage sensitive Na-channel inhibitor tetrodotoxin (TTX; 646 nM; Tocris) to the aCSF. In the experiments, where the involved neurotransmitter receptors were examined, the GABA_A-Rs were blocked by picrotoxin (100 μM), the ionotropic glutamate-receptors (Glu-Rs) were inhibited by the Glu-R inhibitor kynurenic acid (2 mM), and the AMPA/kainate-Rs were antagonised by the AMPA/kainate-R antagonist NBQX (10 μM), added to the aCSF during the respective recordings. To demonstrate the effect of E2, the recordings were conducted with an initial control recording (5 min), then high physiologic dose of E2 (200 pM, Sigma), characteristic for the proestrous phase of the estrous cycle in rodents (Nelson et al., 1992; Christian et al., 2005; Freeman, 2006; Chu et al., 2009), was added to the aCSF in the recording chamber and the recording continued for a subsequent 10 min. Stock of E2 (10 mM) was prepared in DMSO, which was diluted in aCSF to reach the final working concentration (200 pM) pipetted onto the slice in a single bolus. Instantaneous volume of the aCSF in the chamber was 1.5 ml. A single bolus of 3 µl of 100 nM E2 (diluted in aCSF) was pipetted to the inlet of the chamber. Using these volume and concentration data, 3 µl of 100 nM E2 in 1.5 ml aCSF gives the actual dose of E2 (200 pM) in the chamber. Flow rate of perfusion was 3 ml/min. When the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; 1 μM) was used extracellularly, it was added to the aCSF 10 min before starting the recording, whereas in case of its intracellular application (intraPHTPP) the drug was added to the intracellular solution in the pipette. The membrane-impermeable NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO; 1 mM), the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine,4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-]pyrimidine (PP2; 10 µM) or the phosphatidylinositol 3 kinase (PI3K) blocker 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002; 50 µM) were added to the intracellular solution in the pipette to block NO production and release in the measured GnRH neuron. The pipette solution containing intracellularly applied drug was allowed to equilibrate with the intracellular milieu of the cell for 15 min before starting recording.

Time distribution graphs of frequencies were generated by using 1-min time bins to show time courses of effect of E2.

To show action of E2 onto the firing, resting potential (V_rest), R_in, and C_m in GnRH neurons of proestrous mice, current clamp measurements were conducted. Three 900-ms-long current steps were applied (-25, 0, and +25 pA). V_rest was evaluated from the 0-pA step, firing was analyzed during the depolarizing step. The R_in was determined from the voltage response to the application of hyperpolarizing current. The time constant was the time required to reach 63% of the maximum voltage response to hyperpolarizing current (Spergel et al., 1999). The C_m was then calculated by dividing the time constant by the R_in. After control recording, E2 was pipetted into the measurement chamber and 1, 3, 5, and 10 min later, the three current steps were repeated.