Article Figures & Data
Figures
- Figure 1.
Total voltage-dependent K+ current in AVPV kisspeptin neurons has three distinct components. A, Representative total K+ current in response to the voltage-clamp protocol shown. B, Representative recording of total K+ current from a different cell in response to the voltage protocol shown; both a slowly-inactivating and residual (sustained) component are evident during the extended prepulse. C, Expansions of the areas within the dashed boxes in B, showing the change in inactivation rates as more depolarizing prepulses are applied (left), and how this affects both activation and inactivation during the test pulse (right). D, Mean ± SEM peak current density in cells from mice in diestrus (black symbols) and proestrus (magenta symbols). Error bars are smaller than symbols for some values.
- Figure 2.
Potassium current recorded from three different cells before and during application of potassium channel blockers. A, 20 mm TEA. B, 5 mm 4-AP. C, Time control.
- Figure 3.
4-AP-resistant slow-transient voltage-dependent K+ current is larger on diestrus. A, Representative traces (top) in response to the activation voltage-clamp protocol (bottom) in the presence of 5 mm 4-AP. B, Representative traces (top) in response to the inactivation voltage-clamp protocol (bottom). Offline leak subtraction was applied to test pulses only, hence the Cm transient is visible at the start of the recording. C, Mean ± SEM peak current density from cells in mice in diestrus (black symbols) and proestrus (magenta symbols). D, Mean ± SEM normalized conductance. Solid lines are Boltzmann fits to the mean data. E, Individual values and mean ± SEM parameters obtained from Boltzmann fits to normalized conductance curves for each cell. F, G, Representative traces (top) in response to the voltage-clamp protocol (bottom) used to measure the time dependence of inactivation (F) and recovery (G). Arrow denotes peak of fast transient current. H, Mean ± SEM normalized peak current versus prepulse duration for time dependence of inactivation (inact) and recovery from inactivation. Error bars are smaller than symbols for some values; *p < 0.05.
- Figure 4.
TEA-resistant fast-transient voltage-dependent K+ current has a depolarized inactivation curve during proestrus. A, left, Representative unsubtracted K+ current (top) in response to the voltage-clamp protocol (bottom) in presence of 20 mm TEA. Middle, Residual current in the same cell with a −30-mV prepulse. Right, Fast transient current yielded by subtracting residual current from raw current. B, Mean ± SEM peak current density in cells from mice in diestrus (black symbols) and proestrus (magenta symbols). C, Mean ± SEM normalized conductance. Solid lines represent Boltzmann sigmoidal fits to the mean data for the fast transient, dashed lines are the fits for the residual current. D, Individual values and mean ± SEM parameters obtained from Boltzmann fits to normalized conductance curves for each cell. E, F, Representative traces (top) in response to the voltage-clamp protocol (bottom) used to measure the time dependence of inactivation (E) and recovery (F). G, Mean ± SEM normalized peak current versus prepulse duration for time dependence of inactivation (inact) and recovery from inactivation. Error bars are smaller than symbols for some values; *p < 0.05.
- Figure 5.
Potassium conductance model output versus experimental data for voltage steps. Rainbow colors indicate mean ± SEM current traces recorded at different test potentials; black lines show model simulations. In each panel, diestrus is on the left and proestrus on the right. A, Slow (voltage protocol as in Fig. 3A). B, Fast (voltage protocol as in Fig. 4A). C, Residual (voltage protocol as in Fig. 4A).
- Figure 6.
Conductance models versus data for steady state activation/inactivation and current density experimental data. A–C, Steady state activation/inactivation curves calculated from voltage-clamp simulations (dashed lines) compared with corresponding mean ± SEM experimental data (symbols) from diestrous (black) and proestrous (magenta) groups. D–F, Peak current density for various conductances. Experimental data for K+ current subcomponents are the same as in Figures 3, 4 and replotted here for ease of comparison. Data points for NaP (E) and CaT (C, F) are adapted from Wang et al. (2016).
- Figure 7.
Reconstruction of the total K+ current from the sum of the three subcomponents. A, Peak current density when Ifast, Islow, and Iresid are simulated together in the same model (dashed lines) after
- Figure 8.
Simulations of firing from a baseline of −70 mV. A, Performance of diestrous (black) and proestrous (magenta) models in response to −30 pA (top), +6 pA (middle), and +28 pA (bottom) applied current. Boxed regions are shown with expanded axes in the indicated subfigure. B, Posthyperpolarization rebound of diestrous and proestrous models from panel A. C, Rheobase action potentials for both models (dashed lines) from A (middle) and experimental counterparts (solid lines, mean ± SEM). D, F-I curves for diestrous and proestrous models (lines) and experimental counterparts (circles are means, shading is SEM). E, F-I performance of hybrid models in which one or multiple K+ conductances in the diestrous model was substituted for a proestrous counterpart. Non-hybrid models from part D (di full and pro full) are reproduced here to facilitate comparison in this panel as well as F, G. F, F-I performance of hybrid models in which one or multiple subthreshold depolarizing currents in the diestrous model were replaced with proestrous counterparts. G, Rebound bursting performance of hybrid models in response to −30-pA current injection.
Tables
By cycle stage Diestrus Proestrus Mann–Whitney U Diff p Rs (MΩ) 15.8 ± 0.3 15.5 ± 0.4 2601 −0.1774 0.57 Rinput (MΩ) 1045 ± 46.3 917.1 ± 59.4 2265 −86.68 0.07 Cm (pF) 17.3 ± 0.5 18.1 ± 0.7 2525 0.6704 0.4 Ihold (pA) −21.6 ± 2 −19.7 ± 2.9 2601 0.0917 0.57 By drug None (total) 4-AP (slow) TEA (fast) Kruskal–Wallis Diff p 16.58; Rs (MΩ) 15.7 ± 0.5 16.4 ± 0.4 15 ± 0.5 4.87 −6.137 0.09 10.41 Rinput (MΩ) 816.8 ± 50.0 1158 ± 86.4 1179 ± 84.4 16.31 −22.91 <0.001 −25.98 −3.074; Cm (pF) 16.6 ± 0.7 18.2 ± 0.9 17.4 ± 0.8 1.53 −8.139 0.47 −6.148 1.991; Ihold (pA) −18.7 ± 4.1 −26.6 ± 2.2 −18.9 ± 3.3 3.154 −0.7543 0.21 −12.02 −11.27 n cells,
n animalsEstrous stage Estrous stage × Vm or
duration interactionTotal di pro Density 10, 4 17,5 Diff, −15.42 [CI −40.49, 9.660] F(1,25) = 1.603; p = 0.217 F(11,275) = 0.6496 p = 0.785 Slow Density 12, 5 13, 5 Diff, 24.07 [CI −5.523, 53.66] F(1,23) = 6.503; p = 0.106 F(8,184) = 10.03 p < 0.001 Activation 12, 5 13, 5 Diff, −0.01901 [CI −0.06921, 0.03120] F(1,25) = 5.283; p = 0.442 F(11,275) = 3.336 p = 0.169 Inactivation 9, 4 12, 5 Diff, −0.0009547 [CI −0.06269, 0.06078] F(1,20) = 0.1782; p = 0.975 F(12,240) = 0.1988 p > 0.999 Rate of inactivation 10, 7 9, 4 Diff, −0.03472 [CI −0.08026, 0.01082] F(1,17) = 2.587; p = 0.126 F(14,238) = 1.336 p = 0.187 Rate of recovery 9, 5 13, 5 Diff, −0.05901 [CI −0.09461, −0.02342] F(1,20) = 10.52; p = 0.002 F(14,280) = 4.006 p < 0.001 Fast Density 11, 4 13, 4 Diff, −6.607 [CI −22.53, 9.317] F(1,22) = 0.7404; p = 0.399 F(11,242) = 0.9366 p = 0.506 Activation 11, 4 13, 4 Diff, 0.01876 [CI −0.03188, 0.06940] F(1,23) = 0.6133; p = 0.450 F(8,184) = 1.475 p = 0.981 Inactivation 11, 4 12, 4 Diff, −0.05682 [CI −0.1119, −0.001691] F(1,21) = 4.594; p = 0.044 F(9,189) = 4.041 p < 0.001 Rate of inactivation 10, 4 12, 4 Diff, −0.0085 [CI −0.03704, 0.02004] F(1,20) = 0.3859; p = 0.541 F(11,220) = 0.4253 p = 0.944 Rate of recovery 10, 4 12, 4 Diff, −0.01081 [CI −0.03695, 0.01532] F(1,20) = 0.7448; p = 0.398 F(11,220) = 0.3670 p = 0.967 Residual Density 11, 4 13, 4 Diff, 0.07334 [CI −3.263, 3.409] F(1,22) = 0.002079; p = 0.964 F(11,242) = 0.05676 p > 0.999 Activation 11, 4 13, 4 Diff, 0.01068 [CI −0.03932, 0.06068] F(1,22) = 0.1962; p = 0.662 F(11,242) = 0.2095 p = 0.997 Bold font indicates p < 0.05.
Property n cells, n animals Difference (Diff), CI, t or U, df, p Notes di pro Slow V50 activation 12, 5 13, 5 Diff, −1.888 [CI −6.587, 2.811] t = 0.8428, df = 18.42, p = 0.410 Welch’s correction Slow activation slope 12, 5 13, 5 Diff, −0.2234 [CI −0.6005, 0.1537] t = 1.225, df = 23, p = 0.233 Slow V0 inactivation 9, 4 12, 5 Diff, 1.42 [CI −7.327, 10.17] t = 0.3386, df = 20, p = 0.738 Slow inactivation slope 9, 4 12, 5 Diff, 0.7169 [CI −0.8095, 2.243] t = 1.038, df = 10.67, p = 0.322 Welch’s correction Fast V50 11, 4 13, 4 Diff, 2.201 [CI −4.110, 8.513] t = 0.7233, df = 22, p = 0.477 Fast activation slope 11, 4 13, 4 Diff, 0.451 [CI −0.5518, 1.454] t = 0.9327, df = 22, p = 0.361 Fast V50 inactivation 11, 4 12, 4 Diff, 6.23 [CI 0.09700, 12.36] t = 2.180, df = 13.92, p = 0.047 Welch’s correction Fast inactivation slope 11, 4 12, 4 Diff, −0.1266 [CI −0.9059, 0.6526] t = 0.3379, df = 21, p = 0.739 Residual V50 activation 11, 4 13, 4 Diff, 1.329 [CI −4.612, 7.269] t = 0.4638, df = 22, p = 0.647 Residual activation slope 11, 4 13, 4 Diff, 0.1930 U = 61; p = 0.569 Mann–Whitney Bold font indicates p < 0.05. Differences shown for means for normally-distributed data and medians for non-normally-distributed data.
Slow Fast Residual NaP CaT h Leak E (mV) −92.00 −92.00 −92.00 50.00 155.00 −19.90 −70.00 37.4 36.9 29.7 0.14 1.60 0.11 1.06 m h m h m h m h m h m V50 −1.70 −43.84 −19.22 −57.95 −5.54 −39.16 50.45 31.84 −55.61 −76.02 −97.55 K −7.73 8.41 −7.84 6.33 −8.85 11.29 −3.72 3.20 −5.45 10.90 4.19 h1 = 14.55
h2 = 113.55tau Eq. 8 Eq. 8 Eq. 9 Eq. 8 Eq. 8 0.40 Eq. 10 Eq. 9 Eq. 9 Eq. 9 va 1.40 64.50 0.53 0.93 64.50 67.30 1.85 15.58 201.00 b 1.54 1401.00 0.00 16.39 1401.65 −27.50 1.65 70.15 0.00 c 15.81 59.70 −8.68 44.02 59.77 67.30 −60.60 −57.66 −2.20 d −0.27 −6.42 7.88 −0.33 −6.42 27.50 5.00 3.61 −5.95 e 8.67 1.34 0.13 1.34 4650.15 f 2.89 −8.26 −6.77 −8.26 62.48 g 8.50 1.82 7.76 1.82 r 1 2 1 n/a Slow Fast Residual NaP CaT h Leak E (mV) −92.00 −92.00 −92.00 50.00 155.00 −19.90 −70.00 42.0 45.7 32.9 0.20 2.0 0.64 0.88 m h m h m h m h m h m V50 −3.71 −40.51 −19.22 −57.13 −5.54 −39.16 50.45 31.84 −54.87 −74.00 −97.55 K −9.01 8.41 −7.84 6.33 −8.85 11.29 −3.72 3.20 −5.45 10.90 4.19 h1 = 14.55
h2 = 113.55tau Eq. 8 Eq. 8 Eq. 9 Eq. 8 Eq. 8 0.40 Eq. 10 Eq. 9 Eq. 9 Eq. 9 a 1.40 64.50 0.47 0.93 64.50 67.30 1.85 15.58 201.00 b 0.66 873.09 0.00 16.39 1401.65 −27.50 1.65 70.15 0.00 c 20.02 46.89 −8.68 44.02 59.77 67.30 −60.60 −57.50 −2.20 d −0.20 −6.42 7.88 −0.33 −6.42 27.50 5.00 3.61 −5.95 e 8.67 1.34 0.13 1.34 3980.90 f −8.10 −8.26 −6.77 −8.26 62.48 g 10.50 1.82 7.76 1.82 r 1 2 1 Di/Pro NaT E (mV) 50.00 68.12 α(V) β(V) r1(V) r3(V) s 65.24 48.59 9.52 12.68 k −6.05 5.09 −4.66 3.08 r 38.40 391.84 1.36 0.014
Extended Data 1
CaTcustom: hpp file containing code for T type calcium current; HCurrentcustom: hpp file containing code for hyperpolarization-activated current; markovNA: hpp file containing code for transient Na current; NaP_V2: hpp file containing code for persistent Na current; optifastK_GHK: hpp file containing code for fast K current; optiSlowKV5: hpp file containing code for slow K current; testcond: hpp file containing code for residual K current. These conductance files are written in C++ and can be added to xolotl neuron objects. Parameter values used for models are listed in Tables 4-Tables 6. Download Extended Data 1, ZIP file.
In this issue
