3.7.1 Electrophysiological recordings analysis
Analysis of electrophysiological recordings was performed using Clampfit 10.6 (Molecular Devices).
3.7.2 Steroid effect assessment
The degree of steroid modulation (𝐸; either potentiation or inhibition) was calculated using the following equation:
𝐸 = (𝐼𝑆− 𝐼𝐴)/𝐼𝐴× 100, Equation 1
where 𝐼𝐴 is the current amplitude of the response to glutamate application and 𝐼𝑆 is the current amplitude of the response to the co-application of glutamate and the steroid (Figure 3.1A-B).
Glutamate concentration was equal to 1 μM in the experiments performed at GluN2B- containing recombinant NMDARs or 3 μM in the experiments performed at GluN2A- containing recombinant NMDARs.
The relative degree of potentiation (𝐸) induced by different steroid doses in individual HEK293T cells was fit to the following equation:
𝐸 = 𝐸𝑚𝑎𝑥/(1 + ( 𝐸𝐶50/[𝑠𝑡𝑒𝑟𝑜𝑖𝑑])ℎ Equation 2 where 𝐸𝑚𝑎𝑥 is the maximal degree of steroid-induced potentiation, 𝐸𝐶50 is the steroid concentration that produces half-maximal modulation, [𝑠𝑡𝑒𝑟𝑜𝑖𝑑] is the concentration of the steroid, and ℎ is the Hill coefficient.
3.7.3 Agonist dose-response analysis
To fit the normalized currents (I) measured in individual HEK293T cells, the following logistic equation was used:
𝐼 = 1 (1 + (𝐸𝐶⁄ 50⁄[𝑎𝑔𝑜𝑛𝑖𝑠𝑡])ℎ), Equation 3 were 𝐸𝐶50 is the agonist concentration that produces a half-maximal NMDAR response, [𝑎𝑔𝑜𝑛𝑖𝑠𝑡] is the concentration of agonist (glycine or glutamate), and ℎ is the Hill coefficient.
43 Glutamate dose-response assessment was made in the presence of 30 μM glycine and glycine dose-response assessment was made in the presence of 1 mM glutamate.
Figure 3.1. (A-B) Representative recordings demonstrate the effect of potentiating (A) and inhibitory (B) steroids on GluN1/GluN2B receptor responses to 1 μM glutamate in the presence of 30 μM glycine. 𝐼𝐴 indicates the amplitude of the current response to glutamate alone and 𝐼𝑆 is the current amplitude of response to the co-application of glutamate and steroid. (C) Representative recording demonstrates the response of GluN1/GluN2B receptors induced by 1 mM glutamate in the presence of 30 μM glycine. 𝐼𝑃 indicates the peak response and 𝐼𝑆𝑆 indicates the steady-state response.
3.7.4 Current density analysis
The current density (𝑗) was determined using the following formula:
𝑗 = 𝐼𝑚𝑎𝑥 / 𝐶𝑚, Equation 4 where 𝐼𝑚𝑎𝑥 is the maximal peak amplitude of NMDAR response to 1 mM glutamate and 30 μM glycine and Cm is the membrane input capacitance.
3.7.5 Open probability analysis
The NMDAR open probability (Po) was estimated from the kinetics of MK-801 block onset. 1 μM MK-801 was used to inhibit the NMDAR responses to 1 mM glutamate and 30 μM glycine. Gepasi 3.21 (Mendes, 1993; Mendes, 1997; Mendes & Kell, 1998) was used to fit the data to the following kinetic model:
,
44 where D, R, O, and B indicate desensitized, closed, open, and MK-801 blocked state of the receptor, respectively. Calculations based on the rate constants determined earlier (Cais et al., 2008) predict a high probability of the existence of the NMDAR in the doubly liganded state in the presence of 1 mM glutamate; therefore, glutamate binding steps are neglected and not considered in the scheme. The fitting was implemented by a two-step procedure (Turecek et al., 2004). In the first step, the NMDAR desensitization and desensitization kinetics were characterized. Desensitization (D) was calculated as:
𝐷 = 1 − (𝐼𝑆𝑆/ 𝐼𝑃), Equation 5
where 𝐼𝑆𝑆 is the steady-state response and 𝐼𝑃 is the peak response to 1 mM glutamate (Figure 3C). The kinetic constants characterizing the onset of desensitization (𝑘𝑑) and resensitization (𝑘𝑟) were calculated by the following equations:
𝑘𝑑 = D/ 𝜏𝑑 , Equation 6
𝑘𝑟 = (1 − D)/ 𝜏𝑑 . Equation 7
The rate of desensitization onset (𝜏𝑑) was calculated in Clampfit 10.6 by a single exponential function using the Levenberg–Marquardt algorithm.
In the second step, the kinetic constants 𝑘𝑟 and 𝑘𝑑 were fixed at values determined in the first step. The close rate constant (𝑘𝑐) was set at an arbitrary value of 200 s-1. The opening rate constant (𝑘𝑜) was set as a free parameter. The time course of MK-801 inhibition was fitted to the kinetic model mentioned above. The MK-801 blocking rate constant (𝑘𝑏) was set at the value of 25 μM-1 s-1 (Huettner & Bean, 1988; Jahr, 1992; Rosenmund et al., 1995).
3.7.6 Comparative analysis of MK-801 blocking rate
The NMDAR responses to 1 mM glutamate were inhibited by the application of MK- 801 (1 μM) in the continuous presence of glutamate. The time course of the MK-801 blockade was fitted to a double-exponential function to determine time constants, 𝜏𝑠𝑙𝑜𝑤 and 𝜏𝑓𝑎𝑠𝑡. The weighted time constant 𝜏𝑤 was calculated using the following equation:
𝜏𝑤 = (𝜏𝑓𝑎𝑠𝑡 × 𝐼𝑓𝑎𝑠𝑡 + 𝜏𝑠𝑙𝑜𝑤× 𝐼𝑠𝑙𝑜𝑤)/ (𝐼𝑓𝑎𝑠𝑡 + 𝐼𝑠𝑙𝑜𝑤) , Equation 8
45 where 𝐼𝑓𝑎𝑠𝑡 is the current amplitude of the fast component and 𝐼𝑠𝑙𝑜𝑤 is the current amplitude of the slow component.
3.7.7 Statistical analysis
Data was compared as an effect in control conditions versus an effect in experimental conditions. Statistical analysis was performed with Sigma Plot 14.0 software package (Systat Software Inc., Palo Alto, USA). All data are presented as the mean ± standard error of the mean (SEM), n indicates the number of cells that have been tested. Data distribution was evaluated by the Shapiro-Wilk normality test and the equality of group variances was assessed by the Brown–Forsythe test. Parametric statistics (Student's t-test, paired t-test, or ANOVA) was used for data sets with normal distribution whereas nonparametric statistics (Wilcoxon-signed rank test, Mann–Whitney rank-sum test, or Kruskal–Wallis one-way ANOVA on ranks) was used for data sets without normal distribution. At multiple group comparisons, post hoc tests were performed when a significant difference had been found.
Correlation between data sets was assessed with the Pearson product-moment correlation coefficient. The p-value ≤ 0.05 was considered significant in the study.
46 4 Results
4.1 Functional and pharmacological properties of disease-associated de novo