1. Computational and Systems Biology
  2. Neuroscience

Estradiol elicits distinct firing patterns in arcuate nucleus kisspeptin neurons of females through altering ion channel conductances

  1. Jian Qiu  Is a corresponding author
  2. Margaritis Voliotis
  3. Martha A Bosch
  4. Xiao Feng Li
  5. Larry S Zweifel
  6. Krasimira Tsaneva-Atanasova
  7. Kevin T O'Byrne
  8. Oline K Rønnekleiv
  9. Martin J Kelly  Is a corresponding author
  1. Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, United States
  2. Department of Mathematics and Statistics, University of Exeter, United Kingdom
  3. Living Systems Institute, University of Exeter, United Kingdom
  4. Department of Women and Children’s Health, School of Life Course and Population Sciences, King’s College London, United Kingdom
  5. Department of Psychiatry and Behavioral Sciences, University of Washington, United States
  6. Depatment of Pharmacology, University of Washington, United States
  7. Division of Neuroscience, Oregon National Primate Research Center, United States
https://doi.org/10.7554/eLife.96691.4
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Cite this article
  1. Jian Qiu
  2. Margaritis Voliotis
  3. Martha A Bosch
  4. Xiao Feng Li
  5. Larry S Zweifel
  6. Krasimira Tsaneva-Atanasova
  7. Kevin T O'Byrne
  8. Oline K Rønnekleiv
  9. Martin J Kelly
(2024)
Estradiol elicits distinct firing patterns in arcuate nucleus kisspeptin neurons of females through altering ion channel conductances
eLife 13:RP96691.
https://doi.org/10.7554/eLife.96691.4
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15 figures, 3 tables and 1 additional file

Figures

Figure 1
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Properties of the firing of Kiss1ARH neurons from ovariectomized (OVX) and E2-treated OVX mice.

(A–E) Representative whole-cell, current-clamp recordings of spontaneous phasic burst firing (A, which is only seen in E2-treated mice with clear up and down states), irregular burst firing (B), tonic firing (C), irregular firing (D), and silent (E) in Kiss1ARH neurons from E2-treated OVX mice. (F, G) Summary pie chart illustrating the distribution of firing patterns in Kiss1ARH neurons from OVX + E2 (F, from six mice) or OVX (G, from eight mice) mice (OVX versus OVX + E2, X2(3) = 6.05, p=0.0011). (H) Current-clamp recording in a Kiss1ARH neuron from an OVX + E2 female demonstrating the response to glutamate (100 μM). RMP = –70 mV. Similar responses were observed in all recorded female Kiss1ARH neurons (n = 7 from four mice). (I) The spiking activity above the bar in (H) was expanded to highlight the pronounced effects of glutamate on burst firing activity of Kiss1ARH neurons, characterized by an ensemble of spikes riding on top of low-threshold spikes (arrows). Drugs were rapidly perfused into the bath as a 4 μl bolus. (J) Spontaneous AP frequency of Kiss1ARH neurons before, during, and after glutamate application (n = 7 cells). Data are presented as mean ± SEM. Statistical comparisons between different treatments were performed using one-way ANOVA (F(2, 18) = 14.60, p=0.0002) followed by Bonferroni’s post hoc test. ***p<0.005. (K, L) Representative traces showing the amplitude of slow excitatory postsynaptic potential (EPSP) induced by high-frequency photostimulation in Kiss1ARH neurons in the presence of ionotropic glutamate receptor antagonists CNQX and AP5 from OVX (K) and OVX + E2 (L) females. The arrows indicate the measurements of slow EPSP amplitude after low-pass filtering (shown in K). (M) Bar graphs summarizing the slow EPSPs in the Kiss1ARH neurons from OVX and E2-treated OVX mice in the presence of CNQX and AP5. Statistical comparisons between the two groups were performed using an unpaired t-test (t(12) = 5.181, p=0.0002). Data are expressed as mean ± SEM, with data points representing individual cells.

Figure 1—source data 1

Data presented in Figure 1.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig1-data1-v1.xlsx
Figure 2
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Relative contribution of voltage-gated calcium currents in Kiss1ARH neurons from OVX mice.

(A–E) Representative current–voltage relationships showing that Cd2+ (non-selective blocker of calcium channels)-sensitive peak currents were inhibited by different calcium channel blockers: (A) nifedipine; (B) ω-conotoxin GIVA; (C) ω-agatoxin IVA; (D) SNX-482; (E) TTA-P2. (F) The maximum peak currents were measured at –10 mV. The proportions of Ca2+ currents inhibited by nifedipine (L type), ω-conotoxin GVIA (N type), ω-agatoxin IVA (P/Q), SNX-482 (R type), and TTA-P2 (T type). Data are expressed as mean ± SEM, with data points representing individual cells.

Figure 2—source data 1

Data presented in Figure 2.

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Figure 3
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Blockade of voltage-activated Ca2+ channels decreases the slow excitatory postsynaptic potential (EPSP) in Kiss1ARH neurons.

(A–D) Representative traces showing that the slow EPSPs induced by high-frequency photostimulation were abolished by perfusing the blocker of the L-type calcium channel, nifedipine (A) or N- and P/Q-type calcium channels, ω-conotoxin MVIIC (B), or the R-type calcium channel, SNX 482 (C), or the T-type calcium channel, TTA-P2 (D), respectively. The arrows indicate the measurements of slow EPSP amplitude, denoted as R1 and R2, after low-pass filtering (shown in C). (E) Bar graphs summarizing the effects of drugs on the R2/R1 ratios. The slow EPSP was generated in OVX Kiss1-Cre::Ai32 mice. Comparisons between different treatments were performed using a one-way ANOVA (F (3, 51) = 14.36, p<0.0001) with the Bonferroni’s post hoc test. *, **, **** indicate p<0.05, 0.01, 0.001, respectively versus control. (F, G) Representative traces show that in voltage clamp senktide induced a significant inward current in Kiss1ARH neurons (F) in the presence of TTX. This current was inhibited by the calcium channel blocker CdCl2 (200 μM) in another cell (G). (H) Bar graphs summarize the effect of the calcium channel blocker CdCl2 on the senktide-induced inward currents. Unpaired t-test for (F) versus (G): t(20) = 2.575, p=0.0181; *p<0.05. Data are expressed as mean ± SEM, with data points representing individual cells.

Figure 3—source data 1

Data presented in Figure 3.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig3-data1-v1.xlsx
Figure 4
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E2 increases the mRNA expression of volatge-activated Ca2+ channels and Hcn1 channels in Kiss1ARH neurons.

(A) E2 increases the expression of low and high voltage-activated calcium channels in Kiss1ARH neurons. Kiss1ARH neurons (three 10-cell pools) were harvested from each of five vehicle- and five E2-treated, OVX females to quantify ion channel mRNA expression of low and high voltage-activated calcium channels as described in the ‘Materials and methods’. The analysis included T-type (Cacna1g) low voltage-activated, as well as the following high voltage-activated channels: R-type (Cacna1e), L-type (Cacna1c), N-type (Cacna1b), and P/Q-type (Cacna1a) calcium channels. Interestingly, all of these channels were upregulated with E2 treatment, which significantly increased the whole-cell calcium current (see Figure 5). Bar graphs represent the mean ± SEM, with data points representing individual animals (oil versus E2, unpaired t-test for Cacna1g, t(8) = 7.105, ***p=0.0001; for Cacna1e, t(8) = 3.007, *p=0.0169; for Cacna1c, t(8) = 2.721, *p=0.0262; for Cacna1b, t(8) = 4.001, **p=0.0039; for Cacna1a, t(8) = 4.225, **p=0.0028). (B) The same Kiss1ARH neuronal pools were also analyzed for mRNA expression of Hcn1 ion channels, and E2 also increased the expression of hyperpolarization-activated, cyclic-nucleotide gated Hcn1 channels in Kiss1ARH neurons. Hcn1 channel mRNA expression was the most highly upregulated by E2 treatment in Kiss1ARH neurons. The expression values were calculated via the ΔΔCT method, normalized to Gapdh and relative to the oil control values (oil versus E2, unpaired t-test, t(8) = 11.450, ****p<0.0001).

Figure 4—source data 1

Data presented in Figure 4.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig4-data1-v1.xlsx
Figure 5
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E2 treatment (positive feedback regimen) increases the Ca2+ currents in Kiss1ARH neurons.

(A, B) Ca2+ currents in Kiss1ARH neurons with the same membrane capacitance from oil-treated (A) or E2-treated (B) animals. (C) The maximum peak currents were measured at –10 mV. The current amplitudes were normalized to the cell capacitance in all cases to calculate current density. The bar graphs summarize the density of Ca2+ current in Kiss1ARH neurons from oil-treated and E2-treated animals. The mean density was significantly greater in E2-treated (13.4 ± 0.9 pA/pF, n = 11) than in oil-treated OVX females (7.2 ± 0.5 pA/pF, n = 40) (unpaired t-test, t(49) = 5.75, ****p<0.0001). (D) Relative contribution of voltage-gated calcium currents in Kiss1ARH neurons from OVX, E2-treated mice. The maximum peak currents were measured at –10 mV. The proportions of Ca2+ currents inhibited by nifedipine (L type), ω-conotoxin GVIA (N type), ω-agatoxin IVA (P/Q), SNX-482 (R type), and TTA-P2 (T type). Data are expressed as mean ± SEM, with data points representing individual cells. (E) The modeling predicts that E2-treated, OVX females exhibit a significantly greater inward Ca2+ current (red trace) than the vehicle-treated females (black trace). The conductance of the modeled voltage-gated calcium current (L-, N-, P/Q-, and R-type) is set to 2.1 nS in the OVX state and 2.8 nS in OVX + E2 state, while for the T-type is set to 0.66 nS in the OVX state and 5 nS in OVX + E2 state.

Figure 5—source data 1

Data presented in Figure 5.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig5-data1-v1.xlsx
Figure 6
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Voltage dependence of ICa in Kiss1ARH neurons from OVX and OVX + E2 mice.

(A, B) Top panels: activation and inactivation protocol. Bottom: representative traces. (C, D) The mean V1/2 values for calcium channel activation were not significantly different for cells from controls versus cells from E2-treated females. Similarly, the V1/2 values for channel steady-state inactivation were similar for both groups. Data are expressed as mean ± SEM.

Figure 6—source data 1

Data presented in Figure 6.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig6-data1-v1.xlsx
Figure 7
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Contribution of small conductance, calcium-activated K+ (SK) channel to Kiss1ARH neuronal excitability.

(A) Representative traces of the inhibition of outward currents before (left, control) and after the specific SK blocker apamin (500 nM, middle). Apamin-sensitive currents were calculated from the subtraction of control and apamin at depolarized potentials (right). Cells were clamped at –70 mV and given 500 ms voltage pulses from –60 mV to +40 mV in 10 mV steps at 0.2 Hz, as shown in (A) at the bottom. (B) Mean current density–voltage relationships measured at the end of the 500 ms voltage step ranging from –60 mV to +40 mV were obtained in the absence and presence of apamin (two-way ANOVA: main effect of treatment [F(1, 8) = 22.69, p=0.0014], main effect of voltage [F(10, 80) = 306.0, p<0.0001] and interaction [F(10, 80) = 24.76, p<0.0001]; mean ± SEM; n = 5; post hoc Bonferroni test, ***p<0.005, ****p<0.001). (C) Apamin-sensitive current densities were obtained from (B) (mean ± SEM, n = 5). (D) Representative traces of the inhibition of outward currents before (left, control) and after the specific SK blocker apamin (500 nM, middle). Apamin-sensitive currents resulted from the subtraction of control and apamin at depolarized potentials (right). (E) Mean current density–voltage relationships measured at the end of the 500 ms voltage step ranging from –60 mV to +40 mV were obtained in the absence and presence of apamin (two-way ANOVA: main effect of treatment [F(1, 10) = 12.85, p=0.0050], main effect of voltage [F(10,100) = 264.1, p<0.0001] and interaction [F(10, 100)=11.93, p<0.0001]; mean ± SEM, n = 6; post hoc Bonferroni test, **p<0.01, ****p<0.001). (F) Apamin-sensitive current densities were obtained from (C) and (E) (ns; two-way ANOVA followed by Bonferroni post hoc test; mean ± SEM; OVX, n = 5; OVX + E2, n = 6). (G) Kiss1ARH neurons (three 10-cell pools) were harvested from each of five vehicle- and five E2-treated, OVX females to quantify the mRNA expression of Kcnn3 ion channel. E2 did not increase the mRNA expression of small conductance calcium-activated K+ (SK3) channels in Kiss1ARH. Bar graphs represent the mean ± SEM, with data points representing individual animals, oil versus E2, Unpaired t-test, t(8) = 0.551, p=0.5967. The expression values were calculated via the ΔΔCT method, normalized to Gapdh and relative to the oil control values. (H) The mathematical model was calibrated on the electrophysiology data from Kiss1ARH neurons before and after treatment with the specific SK blocker apamin, left panel versus middle panel, respectively. For the calibration, it was assumed that the applied concentration of apamin (500 nM) completely blocked the SK current. The modeled apamin-sensitive current with gSK = 28.1 nS (right panel) matches the electrophysiological data from OVX animals.

Figure 7—source data 1

Data presented in Figure 7.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig7-data1-v1.xlsx
Figure 8
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E2 upregulates Kcnma1 mRNA and BK current in Kiss1ARH neurons.

(A) Representative traces of the inhibition of outward currents before (left, control) and after the specific BK blocker iberiotoxin (IbTx; 200 nM, middle). IbTx-sensitive currents were calculated from the subtraction of control and IbTx at depolarized potentials (right). Cells were clamped at –70 mV and given 500 ms voltage pulses from –60 mV to +40 mV in 10 mV steps at 0.2 Hz, as shown in (A) at the bottom. (B) Mean current density–voltage relationships measured at the end of the 500 ms voltage step ranging from –60 mV to +40 mV were obtained in the absence and presence of IbTx (two-way ANOVA: main effect of treatment [F(1, 8) = 0.8841, p=0.3746], main effect of voltage [F(10, 80) = 71.56, p<0.0001] and interaction [F(10, 80) = 1.127, p=0.3528]; mean ± SEM, n = 5; post hoc Bonferroni test, p>0.05). (C) IbTX-sensitive current densities were obtained from (B) (mean ± SEM, n = 5). (D) Representative traces of the inhibition of outward currents before (left, control) and after the specific BK blocker iberiotoxin (IbTx; 200 nM, middle). IbTx-sensitive currents resulted from the subtraction of control and IbTx at depolarized potentials (right). (E) Mean current density–voltage relationships measured at the end of the 500 ms voltage step ranging from –60 mV to +40 mV were obtained in the absence and presence of IbTX (two-way ANOVA: main effect of treatment [F(1, 10) = 4.660, p=0.0562], main effect of voltage [F(10, 100) = 63.98, p<0.0001] and interaction [F(10, 100) = 4.907, p<0.0001]; mean ± SEM, n = 6; post hoc Bonferroni test, *p<0.05, **p<0.01, ****p<0.001). (F) IbTx-sensitive current densities were obtained from (C) and (E) (two-way ANOVA: main effect of treatment [F(1, 9) = 22.04, p=0.0011], main effect of voltage [F(10, 90) = 78.26, p<0.0001] and interaction [F(10, 90) = 17.84, p<0.0001]; mean ± SEM, OVX, n = 5; OVX + E2, n = 6; Bonferroni post hoc test, *p<0.05, ***p<0.005, ****p<0.001). (G) Kiss1ARH neurons (three 10-cell pools) were harvested from each of five vehicle- and five E2-treated, OVX females to quantify the mRNA expression of Kcnma1 (BKα1) channel. E2-treatment increased the mRNA expression of Kcnma1. The expression values were calculated via the ΔΔCT method, normalized to Gapdh and relative to the oil control values. Bar graphs represent the mean ± SEM, with data points representing individual animals (unpaired two-tailed t-test for BK, t(8) = 3.479, **p=0.0083). (H) The mathematical model was calibrated to reproduce the current–voltage relationship observed in Kiss1ARH neurons before and after treatment with IbTx. For the calibration, it was assumed that the applied concentration of IbTx (200 nM) completely blocked the BK current. The modeled IbTx -sensitive current with gBK = 20.0 nS (right panel) matches the electrophysiological data from OVX + E2 animals.

Figure 8—source data 1

Data presented in Figure 8.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig8-data1-v1.xlsx
Figure 9
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E2 upregulates Kcnq2 channels and M current in Kiss1ARH neurons.

(A, B) Representative current traces of the M current inhibition caused by 40 µM XE-991 perfused for 10 min in (A) OVX oil and (B) OVX + E2-treated female mice. Inset: M current deactivation protocol. (C, D) Current density–voltage plots from –75 to –30 mV of vehicle and XE-991 perfusion in (C) OVX oil and (D) OVX + E2-treated mice. Two-way ANOVA for (C): main effect of treatment (F(1, 17) = 1.908, p=0.1851), main effect of voltage (F(9, 153) = 187.1, p<0.0001) and interaction (F(9, 153) = 3.901, p=0.0002); Veh, n = 11; XE-991, n = 8; Bonferroni post hoc test, p>0.05. For (D): main effect of Veh and XE-991 (F(1, 24) = 24.92, p<0.0001), main effect of voltage (F(9, 216) = 174.5, p<0.0001) and interaction (F(9, 216) = 52.75, p<0.0001); Veh, n = 13; XE-991, n = 13; Bonferroni post hoc test, a = p<0.05, b = p<0.001. (E) Treatment with E2 elevated, while XE-991 diminished the maximum peak current density elicited by a –30 mV step in OVX- and OVX + E2-treated mice. Two-way ANOVA: main effect of Veh and XE-991 (F(1, 41) = 47.59, p<0.0001), main effect of OVX and OVX + E2 (F(1, 41) = 15.76, p=0.0003), and interaction F(1, 41) = 18.2, p=0.0001; Bonferroni post hoc test, Veh: OVX vs. OVX + E2, a = p<0.001. XE-991: OVX vs. OVX + E2, p>0.05. Data are expressed as mean ± SEM, with data points representing individual cells. (F) Kiss1ARH neurons (three 10-cell pools) were harvested from each of five vehicle- and five E2-treated, OVX females to quantify the mRNA expression of Kcnq2. E2 treatment increased the mRNA expression of Kcnq2. Unpaired t-test, t(8) = 4.850, **p=0.0013. Data are expressed as mean ± SEM, with data points representing individual animals. (G) Percent contribution of the different K+ currents to the repolarization current during burst-type firing activity in the OVX + E2 state. At each time point, the length of each color bar denotes the percent contribution of the corresponding current to the total outward current.

Figure 9—source data 1

Data presented in Figure 9.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig9-data1-v1.xlsx
Figure 10
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Estradiol decreases Tac 2, Trpc5, and Kcnj6 but increases Slc17a6 mRNA expression in Kiss1ARH neurons.

(A) qPCR amplification curves illustrating the cycle threshold (CT) for Tac2, Gapdh, Kiss1, Trpc5, Slc17a6 (Vglut2), and Kcnj6 (Girk2) in Kiss1ARH ten cell neuronal pools (3–6 pools from each animal) in OVX Oil-treated and (B) OVX E2-treated females. (C) Quantitative real-time PCR analysis of Tac2 mRNA, (D) Slc17a6, (E) Trpc5, and (F) Kcnj6. Comparisons were made between oil-treated and E2-treated, OVX females using the comparative 2-ΔΔCT method. Bar graphs represent the mean ± SEM, with data points representing individual animals (unpaired t-test for Tac2, t(6) = 10.670, ***p<0.0001; for Slc17a6, t(7) = 9.678, ***p<0.0001; for Trpc5, t(6) = 5.774, **p=0.0012; unpaired t-test for Kcnj6, t(8) = 3.457, **p=0.0086).

Figure 10—source data 1

Data presented in Figure 10.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig10-data1-v1.xlsx
Figure 11
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CRISPR mutagenesis of Trpc5 channels in Kiss1ARH neurons.

(A) Structure of AAV1-FLEX-SaCas9-U6sgTrpc5-exon2. Exon 2 of Trpc5 is denoted with guide sequence highlighted in red, the PAM is underlined. (B) Structure of AAV1-FLEX-SaCas9-U6sgTrpc5-exon7. Exon 7 of Trpc5 is denoted with guide sequence highlighted in red, the PAM is underlined. (C1) Image of coronal section through the ARH from Kiss1-Cre::Ai32 mouse with dual co-injections of AAV-DIO-mCherry and AAV1-FLEX-SaCas9-U6-sgTrpc5. Scale = 200 µm. (C2, C3) Higher-power overlays of epifluorescence (EYFP and mCherry) images with recording pipette patched onto a Kiss1ARH-Cre:mCherry cell (C3). Scale = 40 µm. (D) Quantitative PCR measurements of Trpc5 transcripts from 10-cell neuronal pools (three pools from each animal) in double sgRNA mutagenesis of Trpc5 (second sgRNA against pore-forming region) in Kiss1ARH neurons. Primers were targeted to first (left panel) or second (right panel) guide, respectively (unpaired t-test for first, t(10) = 10.67, ****p<0.0001; for second, t(10) = 10.79, ****p<0.0001). Data are expressed as mean ± SEM, with data points representing individual animals.

Figure 11—source data 1

Data presented in Figure 11.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig11-data1-v1.xlsx
Figure 12
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Double CRISPR mutagenesis of Trpc5 attenuates slow excitatory postsynaptic potential (EPSP), increases rheobase, and shifts the F-I curve.

(A) High-frequency photostimulation (20 Hz) generated slow EPSP in Kiss1ARH neuron from ovariectomized, control mouse. Red trace is slow EPSP after low-pass filtering. (B) Slow EPSP in Kiss1ARH neuron from OVX, double sgTrpc5 -targeted (Trpc5 mut) mouse. (C) Summary of the effects of Trpc5 mutagenesis on slow EPSP amplitude in female mice. Unpaired t-test, t(27) = 5.916, ****p<0.0001. Data are expressed as mean ± SEM, with data points representing individual cells. (D) Double sgRNA mutagenesis of Trpc5 channels in Kiss1ARH neurons significantly increased the RMP (control: –64.7 ± 1.4 mV versus Trpc5 mut: –71.1 ± 1.2 mV, unpaired t-test, t(73) = 3.524, ***p=0.0007). (E) Current ramp showing the increased rheobase in a Kiss1ARH neuron from Trpc5 mut mice (control: 31.1 ± 1.2 pA, n = 31, versus Trpc5 mut, 35.3 ± 1.0 pA, n = 33, unpaired t-test, t(62) = 2.777, **p=0.0073). (F) Firing frequency vs. current (F-I) curves for control versus Trpc5 mut (two-way ANOVA: main effect of treatment [F(1,52) = 13.04, p=0.0007], main effect of injected current [F(8,416) = 291.3, p<0.0001] and interaction [F(8,416) = 6.254, p<0.0001]; control, n = 26, Trpc5 mut, n = 28; post hoc Bonferroni test, *p<0.05; **p<0.01, and ***p<0.005, respectively). (G) Model simulations of the effects of a current ramp (50 pA/s) for OVX (left panel) and OVX female with reduced (muted) TRPC5 conductance (right panel), and (H) the associated firing frequency versus current curves. In the latter case, the TRCP5 conductance was halved, which is a conservative estimation of the CRISPR state in which the Trpc5 is much more mutated in Kiss1ARH neurons (Figure 11D).

Figure 12—source data 1

Data presented in Figure 12.

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Figure 13
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Computational modeling of a Kiss1ARH neuron in the OVX and OVX + E2 state demonstrates its distinct dynamic responses.

A model of the Kiss1ARH neuron was developed and calibrated using molecular data and electrophysiological recordings of Kiss1ARH neurons from OVX and OVX + E2 mice. (A) Simulations of the OVX-parameterized model demonstrating high-frequency activity in response to saturating levels of NKB stimulation. The balance between GIRK and TRCP5 conductance controls the response of the neuron to NKB stimulation, with neuronal response eliminated when TRPC5 conductance is low (red triangle) relative to the GIRK conductance. (B) The OVX + E2 parameterized models demonstrate sustained burst firing activity. The bursting activity that is supported by elevated h- and Ca2+ currents (red square) as observed in OVX + E2 mice. (C) In the OVX + E2 state, burst firing activity is also supported by high conductance of HVA Ca2+ channels relative to the conductance of TRPC5 channels. Representative points in the parameter space giving rise to burst firing activity are marked with red squares, whereas red triangles are used for points resulting in regular spiking. The black line separates these two regions of activity.

Figure 13—source data 1

Data presented in Figure 13.

https://cdn.elifesciences.org/articles/96691/elife-96691-fig13-data1-v1.xlsx
Appendix 1—figure 1
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Schematic diagram of the conductance-based mathematical model of arcuate nucleus Kiss1 neurons.
Author response image 1
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Tables

Table 1
Primer table.
Gene name (encodes for)Accession
number
Primer
location (bp)
Product
length (bp)
Annealing
temperature (°C)
Efficiency
slope
r2%
Cacna1c
(Cav 1.2,L-type)
NM_0097811331–1348
1390–1407
7760–3.4780.98994
Cacna1a
(Cav 2.1,P/Q-type)
NM_0075786035–6054
6090–6109
7560–3.4980.98093
Cacna1b
(Cav 2.2,N-type)
NM_0010425285405–5426
5467–5488
8460–3.4830.99294
Cacna1e
(Cav 2.3,R-type)		NM_009782530–549
617–636
10760–3.4410.98395
Cacna1g
(Cav 3.1,T-type)		NM_0097835004–5025
5060–5083
8060–3.3720.96898
Hcn1 (HCN1)NM_0104081527–1546
1641–1662
13660–3.2530.958100
Hcn2 (HCN2)NM_0082261122–1143
1199–1218
9760–3.2790.969100
Kcnd2 (KCND2)NM_0196972135–2156
2217–2238
10460–3.3120.972100
Kcnn3 (SK3)NM_0804661259–1277
1352–1370
11260–3.3690.95298
Kcnma1 (BKα1)NM_0012533582745–2762
2833–2852
10860–3.3380.97199
Kcnb1 (KCNB1)NM_008420691–710
759–780
11960–3.3750.98398
Kcnq2 (KCNQ2)NM_0106111079–1098
1151–1170
9260–3.3670.97898
Tac2 (NKB)NM_009312368–389
490–511
14460–3.4400.98995
Slc17a6 (VGLUT2)NM_0808531275–1296
1371–1390
11660–3.3740.99798
Trpc5 (TRPC5)*NM_009428734–753
832–851
11860–3.1610.952100
Trpc5 (TRPC5)†NM_009428616–637
772–792
17760–3.4070.98797
Trpc5 (TRPC5)†NM_0094282083–2100
2162–2179
9760–3.2550.963100
Kcnj6 (GIRK2)NM_001025584488–507
592–609
12260–3.3680.91998
Gapdh (GAPDH)NM_008084689–706
764–781
9360–3.3520.99899
  1. *

    Trpc5 primers (Figure 10E).

  2. †

    Trpc5 primers flanking sgRNA and PAM sites in mutagenesis (Figure 11D).

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(Mus musculus)		C57BL/6JThe Jackson LaboratoryRRID:IMSR_JAX:000664
Genetic reagent
(M. musculus)		Kiss1Cre:GFP version 2 (V2)Dr. Richard D. Palmiter; University of Washington;
PMID:29336844		RRID:IMSR_JAX:033169
Genetic reagent
(M. musculus)		Ai32The Jackson LaboratoryRRID:IMSR_JAX:024109
Genetic reagent (Adeno-associated virus)AAV1-FLEX-SaCas9-U6-sgTrpc5-exon2Dr. Larry S. Zweifel; University of Washington
Genetic reagent (Adeno-associated virus)AAV1-FLEX-SaCas9-U6-sgTrpc5-exon7Dr. Larry S. Zweifel; University of Washington
Genetic reagent (Adeno-associated virus)AAV1-FLEX-SaCas9-U6-sgRosa26Dr. Larry S. Zweifel; University of Washington
Genetic reagent (Adeno-associated virus)AAV1-Ef1α-DIO-ChR2:YFPDr. Stephanie L. Padilla; University of Washington; PMID:25429312
Genetic reagent (Adeno-associated virus)AAV1-Ef1α-DIO-ChR2:mCherryDr. Stephanie L. Padilla; University of Washington; PMID:25429312
Appendix 1—table 1
Table of model parameters.
Model parameters
Cm23.13 nF (19.5 in OVX + E2)
Current parameters*Reference
INaTτh,NaT4.5 msVh,NaT–43.2 mVFry et al., 2007
Vm,NaT–25 mVkm,NaT3 mV
kh,NaT–8.2 mVgNaT90 nS
ENa66.1 mV
INaPτh,NaP250 msVh,NaP–47.4 mVZhang et al., 2015; Moran et al., 2016
Vm,NaP–41.5 mVkm,NaP3 mV
kh,NaP–8.5 mVgNaP3.37 nS
ENa66.1 mV
IAτh,A10 msVh,A–55.1 mVMendonça et al., 2016; Mendonça et al., 2018
τm,A20 msVm,A–30 mV
kh,A–11.4 mVkm,A10 mV
gA60 nS in OVX
35 in OVX + E2		EK–81 mV
IBKkBK10 mVkshift18 mVTsaneva-Atanasova et al., 2007
VBK,0–22.52 mVkc,BK1.5 μM
gBK13.50 nS in OVX;
20 nS in OVX + E2		EK–81 mV
ISKKSK0.45 μMn4Bond et al., 1999
gSK28.13 nS in OVX;
26.05 nS in OVX + E2		EK–81 mV
IMτm,M10 msVm,M–50 mVNowacki et al., 2011; Conde and Roepke, 2020
km,M20 mVgM0.23 nS in OVX
1.23 ns in OVX + E2
EK–81 mV
Ihτm,h,180 msVm,h,1–102 mVGottsch et al., 2011; Booth et al., 2016; Qiu et al., 2018
τm,h,1310 msVm,h,2–102 mV
ph0.85km,h,1–10 mV
km,h,2–10 mVgh0.56 nS (11.23 nS in OVX + E2)
Eh–27.8 mV
ITτh,T,11.94 msVh,T,1–69.1 mVZhang et al., 2015; Wang et al., 2016
τh,T,286.3 msVh,T,2–69.1 mV
ph0.5Vm,T–54 mV
kh,T,1–5.3 mVkh,T,2–5.3 mV
km,T3.3 mVgT0.66 nS (5 nS in OVX + E2)
ICaτh,Ca10 msVh,Ca–48.9fitted
τm,Ca300 msVm,Ca–27.3 mV
kh,Ca–18.2km,Ca4.62 mV
gCa2.1 nS (2.8 nS in OVX + E2)ECa121.6 mV
ITRPC5cTRPC50.6 μMkTRPC50.33 μMQiu et al., 2021
kRTRPC50.006 ms–1k-RTRPC50.002 ms–1
kRTRPC5,00.002 ms–1gTRPC58.4 nS (1.68 nS in OVX + E2)
KNKB32 μMn22
RTRPC5,T1 μMETRPC5–15 mV
IGIRKVGIRK–70 mVkGIRK,120 mVTian et al., 2022
α0.8ατ0.0061
kGIRK,2100 mVβτ0.0818
gGIRK0.4 nSEK–81 mV
kRGIRK,00 ms–1kRGIRK10–3 ms–1
RGIRK,T1 μMKS1 μM
k-RGIRK3.3⋅10–6 ms–1n32
Ileakgleak,13.5 nS in OVX
3.65 nS in OVX + E2		gleak,24 nSFitted
Intracellular calcium
γ1.96 ⋅10–5μM nA–1 ms–1dca0.008 ms–1Tsaneva-Atanasova et al., 2007
r0.9
  1. *

    Parameters g∙ denote the maximum conductance associated with each current.

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  1. Jian Qiu
  2. Margaritis Voliotis
  3. Martha A Bosch
  4. Xiao Feng Li
  5. Larry S Zweifel
  6. Krasimira Tsaneva-Atanasova
  7. Kevin T O'Byrne
  8. Oline K Rønnekleiv
  9. Martin J Kelly
(2024)
Estradiol elicits distinct firing patterns in arcuate nucleus kisspeptin neurons of females through altering ion channel conductances
eLife 13:RP96691.
https://doi.org/10.7554/eLife.96691.4