Figure 1.
Effects of 2-BFI and NMDA on rat cortical neuronal excitability.
(A) Representative current traces induced by 3–1000 µM 2-BFI and 30 µM NMDA plus 1 µM glycine at −50 mV are shown. (B) The current amplitude is normalized to the current activated by NMDA and the summary data is shown. (C) Representative membrane potential (MP) change induced by 3–1000 µM 2-BFI and 30 µM NMDA plus 1 µM glycine is shown. (D) The membrane potential change (ΔVm) is normalized to that of NMDA and the summary data is shown. Data in B and D represents the mean ± SEM.
Figure 2.
Inhibition of NMDA-activated current by 2-BFI in rat cortical neurons.
(A) Representative currents activated by 30 µM NMDA plus 1 µM glycine and their inhibition by 10–2000 µM 2-BFI. 2-BFI was first applied for 30 s, and then simultaneously applied with NMDA. Membrane potential was clamped at −70 mV. (B) 2-BFI concentration-response relationship for inhibition of NMDA currents activated by 30 µM NMDA plus 1 µM glycine. Concentration-response curve was fitted by the logistic equation, with the IC50 of 124.33±13.11 µM and a slope factor of 1.2±0.1. Data presented in B represents the average of 5–6 cells ± SEM.
Figure 3.
The binding kinetics of 2-BFI on NMDA receptors.
(A) Representative current traces showing the kinetic protocol, where a single concentration of 2-BFI was applied in the constant presence of 30 µM NMDA plus 1 µM glycine. (B) The onset and offset rates of 2-BFI were measured from the recordings by the kinetic protocol. Tauon and Tauoff were obtained by a single exponential function fit. Mean 1/tau values were plotted against the corresponding 2-BFI concentration to determine Kon and Koff. The fitted kinetic values were Kon = 2.19±0.33×10−9 M−1sec−1, Koff = 0.67±0.02 sec−1, Kd = Koff/Kon = 305.94±0.3 µM.
Figure 4.
Effects of membrane potential on inhibition of NMDA-activated current by 2-BFI in rat cortical neurons.
(A) Current-voltage relationship for currents activated by 30 µM NMDA and 1 µM glycine in the absence or the presence of 200 µM 2-BFI at membrane potentials between −60 mV and +60 mV. Current amplitude is normalized to the current activated by 30 µM NMDA and 1 µM glycine in the absence of 2-BFI at −60 mV. (B) The average reversal potential of current activated by 30 µM NMDA and 1 µM glycine was 4.2±3.2 mV in the absence and 2.8±3.2 mV in the presence of 200 µM 2-BFI (paired t-test with P>0.05, n = 6). (C) Summary data showing inhibition of NMDA-activated current by 200 µM 2-BFI at membrane potentials from −60 mV to +60 mV. The percentage inhibition of NMDA-activated current by 2-BFI was not significantly different as determined using one way ANOVA with post hoc Tukey’s analysis (P>0.05, n = 6).
Figure 5.
Effect of NMDA concentration on 2-BFI inhibition of NMDA-activated current in rat cortical neurons.
(A) Representative currents activated by 1000 µM NMDA and 30 µM NMDA in the presence of 1 µM glycine before and after application of 200 µM 2-BFI in a single cell. 2-BFI was first applied for 30 s, and then simultaneously applied with NMDA. (B) Concentration-response relationship for NMDA in the absence (○) and presence (•) of 200 µM 2-BFI. Current amplitude is normalized to the current activated by 1000 µM NMDA and 1 µM glycine. Concentration-response curves were fitted by logistic equation, with the EC50 of 33.67±4.3 µM and the slope factor of 1.25±0.1 in the absence of 2-BFI and EC50 of 29.44±10.5 µM and the slope factor of 1.25±0.6 in the presence of 2-BFI (n = 6–8).
Figure 6.
Effect of glycine concentration on 2-BFI inhibition of NMDA-activated current in rat cortical neurons.
(A) Representative currents activated by 100 µM glycine and 1 µM glycine in the presence of 30 µM NMDA before and after the application of 200 µM 2-BFI in a single cell. 2-BFI was first pre-applied for 30 s, and then simultaneously applied with glycine. (B) Concentration-response relationship for glycine in the absence (○) and presence (•) of 200 µM 2-BFI. Current amplitude is normalized to the current activated by 100 µM glycine and 30 µM NMDA. Concentration-response curves were fitted by the logistic equation, with the EC50 of 1.52±1.6 µM and the slope factor of 0.39±0.2 in the absence of 2-BFI and EC50 of 1.68±1.2 µM and the slope factor of 0.58±0.6 in the presence of 2-BFI (n = 6∼8).
Figure 7.
Effect of 2-BFI on AMPA-activated current in rat cortical neurons.
(A) Representative currents activated by 100 µM AMPA and 30 µM NMDA plus 1 µM glycine and their inhibition by 200 µM 2-BFI. (B) Summary data for inhibition of AMPA-activated current and NMDA-activated current by 2-BFI. 2-BFI at 200 µM significantly reduced NMDA-activated current, but had no effect on AMPA-activated current.
Figure 8.
Inhibition of NMDA-evoked [Ca2+]i in cortical neurons.
(A) Relative [Ca2+]i level in the presence of various doses of 2-BFI and 100 µM NMDA was normalized to NMDA evoked [Ca2+]i. Data represents the average of measurements of at least 20 cells. (B) Representative traces of [Ca2+]i in cortical neurons treated with MK801 (10 µM), 2-BFI (200 µM) or memantine (10 µM) in the presence of NMDA (100 µM). A brief wash with PSS removed the inhibition of NMDA-induced [Ca2+]i in 2-BFI and memantine treated cells, but not in MK801 treated cells. (C) Adding 2-BFI and MK801 together completely inhibited NMDA induced [Ca2+]i and such an inhibition can not be removed by washing. Adding KCl depolarized the membrane and showed a non-regulated calcium influx.
Figure 9.
Neuroprotection by 2-BFI against NMDA toxicity.
(A–E) Photomicrographs of nuclei of cortical neurons treated with or without NMDA and other indicated inhibitiors. Arrows show condensed nuclei, indicating dead cells. Scale bar = 100 µm. (F) Treated cortical neurons were subjected to Western blotting to show inhibition of the production of spectrin breakdown product (SBP) induced by NMDA excitotoxicity. GAPDH was used as an internal protein loading control. (G) Dose-dependent neuroprotection against NMDA by 2-BFI was measured using an Alamar blue assay the mean ± SEM was plotted (n = 3). (H) Time-dependent protection by 2-BFI against NMDA toxicity to cortical neurons was measured using an Alamar blue assay. The data represents mean ± SEM of at least three repeats. ** indicates statistical significant using paired t-test (P<0.01).