Figure 6. Hyperpolarization voltage protocol impacts the relationship between input current and threshold voltage. A, Threshold voltage plotted as a function of input current for APD, APRE (double square pulse), and absolute current values for APR show different resolution, based on the slopes from linear fits: black (m = 0.595, R2 = 0.74, n = 90), gray (m = 0.1198, R2 = 0.795, n = 38), and dashed (m = 0.0222, R2 = 0.599, n = 77) lines, respectively. B, Threshold sweeps from a single neuron when holding potential is hyperpolarized from −60 to −80 mV in 5 mV increments. Inset, dV/dt (mV/ms) plotted as a function of voltage for each high-resolution action potential stimulated with 24 ms square pulses. C1-C3, Overlapping sweeps from low, middle, and high threshold neurons, respectively. Bottom right, Calibration bars apply to all sweeps. Symbols represent the time where measurements were made. D, Voltage to current relationships at −60 mV holding potential for the three recordings shown in C1-C3. Fits were made to the linear regions (thick lines) of the function that extended from just below threshold, through holding potential to ∼−70 mV. The resulting conductance values were 57.7, 72.7, and 95.7 mΩ for the high, middle, and low threshold neurons, respectively. E, Measurements from the three example neurons of APD at −60 to −80 mV holding potentials (colored diamond symbols) are superimposed on average linear fits of APD, APR, and APRE population data replotted from A (gray lines). Colored lines fitted to a larger dataset ranging from 7 to 9 measurements from 9 different neurons had relatively stable slopes (red: −60 mV, m = 0.051, R2 = 0.77, n = 9; amber: −65 mV, m = 0.058, R2 = 0.77, n = 9; light green: −70 mV, m = 0.062, R2 = 0.77, n = 9; light blue: −75 mV, m = 0.062, R2 = 0.78, n = 8; purple: −80 mV, m = 0.061, R2 = 0.82, n = 7). To show the consistency in measurements for each category of response, APRE (gray triangles) and APR (gray squares) measurements shown for the three example neurons aligned closely with the population data replotted from A (top and bottom light gray lines, respectively). APD at −60 mV holding potential for the three neurons (red diamonds), similarly aligned with the population data replotted from A (middle light gray line).