Molecular mechanisms that drive estradiol-dependent burst firing of Kiss1 neurons in the rostral periventricular preoptic area - PubMed (original) (raw)

Molecular mechanisms that drive estradiol-dependent burst firing of Kiss1 neurons in the rostral periventricular preoptic area

Chunguang Zhang et al. Am J Physiol Endocrinol Metab. 2013.

Abstract

Kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3V) provide excitatory drive to gonadotropin-releasing hormone (GnRH) neurons to control fertility. Using whole cell patch clamp recording and single-cell (sc)RT-PCR techniques targeting Kiss1-CreGFP or tyrosine hydroxylase (TH)-EGFP neurons, we characterized the biophysical properties of these neurons and identified the critical intrinsic properties required for burst firing in 17β-estradiol (E2)-treated, ovariectomized female mice. One-fourth of the RP3V Kiss1 neurons exhibited spontaneous burst firing. RP3V Kiss1 neurons expressed a hyperpolarization-activated h-current (Ih) and a T-type calcium current (IT), which supported hyperpolarization-induced rebound burst firing. Under voltage clamp conditions, all Kiss1 neurons expressed a kinetically fast Ih that was augmented 3.4-fold by high (LH surge-producing)-E2 treatment. scPCR analysis of Kiss1 neurons revealed abundant expression of the HCN1 channel transcripts. Kiss1 neurons also expressed a Ni(2+)- and TTA-P2-sensitive IT that was augmented sixfold with high-E2 treatment. CaV3.1 mRNA was also highly expressed in these cells. Current clamp analysis revealed that rebound burst firing was induced in RP3V Kiss1 neurons in high-E2-treated animals, and the majority of Kiss1 neurons had a hyperpolarization threshold of -84.7 mV, which corresponded to the V½ for IT de-inactivation. Finally, Kiss1 neurons in the RP3V were hyperpolarized by μ- and κ-opioid and GABAB receptor agonists, suggesting that these pathways also contribute to rebound burst firing. Therefore, Kiss1 neurons in the RP3V express the critical channels and receptors that permit E2-dependent rebound burst firing and provide the biophysical substrate that drives the preovulatory surge of GnRH.

Keywords: RP3V; T-type calcium channel; burst firing; kisspeptin; pacemaker current.

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Figures

Fig. 1.

Fig. 1.

LH levels in two-dose 17β-estradiol (E2) treatment regimen of ovariectomized (OVX) Kiss1-CreGFP mice. LH (ng/ml) levels from serum of Kiss1-CreGFP mice at ZT4 (E2, n = 8) and ZT12 (n = 8). Lights went off at ZT12. LH levels of E2-treated animals at ZT12 (4.69 ± 1.5 ng/ml) were significantly higher than LH levels in E2-treated animals at ZT4 (0.35 ± 0.10 ng/ml); *P < 0.05 (Student's _t_-test).

Fig. 2.

Fig. 2.

Single-cell RT-PCR identification of Kiss1 mRNA and Th mRNA. A: expression of kisspeptin (Kiss1) and tyrosine hydroxylase (Th) mRNA in Kiss1-CreGFP neurons in the periventricular POA. Representative gels illustrating coexpression of Kiss1 mRNA and Th mRNA in dispersed and harvested Kiss1-GFP neurons from high-E2 (surge-level)-treated animals. Expected sizes for the PCR products are 120 bp for Kiss1 and 131 bp for Th. As a negative control, cells reacted without reverse transcriptase (−RT) did not express any of the transcripts (a total of 6 cells were reacted −RT). POA tissue RNA was also included as a positive control (+RT) and negative control (−RT). MM, molecular markers. Kiss1 mRNA was expressed in 94% of Kiss1-GFP harvested neurons (n = 139); 88% of _Kiss1-_positive cells coexpressed Th mRNA. B: expression of Kiss1 and Th mRNA in Kiss1-CreGFP neurons harvested in the POA slice. Representative gels illustrating coexpression of Kiss1 and Th mRNA in Kiss1-GFP neurons harvested in the slice following whole cell recording for 30–40 min. Expected sizes for PCR products are 120 bp for Kiss1 and 131 bp for Th. POA tissue RNA was included as a positive control (+RT) and negative control (−RT). Kiss1 mRNA was expressed in 100% of Kiss1-GFP neurons (n = 27) that were located in the periventricular POA of high-E2-treated mice. Th mRNA was expressed in 75% of these neurons.

Fig. 3.

Fig. 3.

Properties of spontaneous firing of Kiss1 neurons in the rostral periventricular area of the 3rd ventricle (RP3V) from E2-treated OVX mice. A–C: representative whole cell, current clamp recordings of spontaneous burst firing (A), irregular firing (B), and tonic firing (C) in RP3V Kiss1 neurons. D: summary of percentage of cells showing different firing pattern in whole cell and loose patch recordings. E and F: representative recordings showing glutamate- or NMDA-induced firing in Kiss1 neurons. G: hyperpolarization-induced spontaneous rebound burst firing recorded from a RP3V Kiss1 neuron. The cell was hyperpolarized by constant current injection of −15 pA.

Fig. 4.

Fig. 4.

Kiss1 neurons in the RP3V expressed intrinsic conductances for postinhibitory rebound burst firing. A: representative recording showing a series of hyperpolarizing/depolarizing pulses induced current (inset: voltage protocol: holding potential = −60 mV; steps from −45 to −120 mV, duration 500 ms). Single-head arrows indicate where instantaneous and steady-state hyperpolarization-activated h current (_I_h) were measured. Steady-state current (_I_steady state) was taken as an average from the arrow to the end of the pulse. Double-head arrow indicates peak T-type calcium current (_I_T), which was activated when the voltage was stepped back to −60 mV. B: representative recording showing persistent sodium current (_I_NaP) in RP3V Kiss1 neurons. _I_NaP was activated by a slow ramp of voltage (20 mV/s) from −80 to −20 mV (see inset protocol). Measurement of _I_NaP was indicated as the difference between the inward peak at −50 mV and the extrapolated leak current from −80 mV (dashed line). C: scatter plot of amplitude distribution of the _I_T, _I_h, and _I_NaP that were larger than 5 pA in RP3V Kiss1 neurons. (_I_T: 42.9 ± 4.6 pA, n = 30; _I_h: 39.4 ± 4.9 pA, n = 36; _I_NaP: 35.3 ± 5.2 pA, n = 13, respectively). _I_h and _I_NaP were expressed in 100% of Kiss1 neurons; _I_T was expressed in 83% of cells.

Fig. 5.

Fig. 5.

RP3V Kiss1 neurons express ZD-7288-sensitive _I_h and mRNA for HCN channels. A: representative voltage-clamp recording of _I_h with a hyperpolarizing voltage step protocol. Top: characteristic _I_h are visible as slowly activated inward currents at hyperpolarized membrane potentials. Inset: voltage clamp protocol: Vhold = −60 mV; range from −40 to −120 mV, step size 5 mV, step duration 1 s. Middle: _I_h is blocked by 50 μM ZD-7288, evidenced by lack of slowly developed inward current. Bottom: ZD-7288-sensitive current, which represents the isolated _I_h. B: film image of representative Kiss1 neurons that expressed HCN1 and Cav3.1 transcripts following whole cell recording for 30–40 min. C: I/V relationship of I_h from A, showing voltage-dependent activation and inward rectification at increasingly hyperpolarized voltages. D: mean I/V relationship of 16 Kiss1 neurons showing instantaneous (filled circles) and steady-state (open circles) whole cell currents. Vhold = −60 mV. Protocol is indicated in Fig. 4_A. The difference between instantaneous and steady-state I/V was due to activation of _I_h. E: relative levels of mRNA for HCN1, -2, and -3 in pools of Kiss1 neurons within the RP3V (n = 4–5 animals, 3 pools/animal). Levels of HCN2 and HCN3 mRNA were significantly lower than HCN1 mRNA. Relative expression was calculated by ΔΔCT method and normalized to the mean ΔCT of HCN1. ***P < 0.001 for HCN2 vs. HCN1 and HCN3 vs. HCN1 (ANOVA).

Fig. 6.

Fig. 6.

RP3V Kiss1 neurons express nickel- and TTA-P2-sensitive _I_T and mRNA for CaV3 channels. A: representative traces showing I-V relationship of _I_T before (left, control) and after exposure to 50 μM Ni2+ for 10 min (middle, Ni2+), and the subtracted Ni2+-sensitive _I_T (right) recorded from a RP3V Kiss1 neuron. Traces were truncated to highlight inward _I_T. The insert panel shows the voltage-clamp protocol used to measure _I_T. The pulse protocol consisted of 5-mV steps, from −40 to −120 mV, before returning to −60 mV. Pulses were of 1-s duration to ensure complete de-inactivation of the T-channel. B: representative traces showing I-V relationship of _I_T before (left, control) and after exposure to 5 μM TTA-P2 for 10 min (middle) and the subtracted, TTA-P2-sensitive _I_T (right) recorded from a RP3V Kiss1 neuron. Traces were truncated to highlight the inward _I_T following de-inactivation steps. Bottom: voltage-clamp protocol used to measure _I_T in Kiss1 neurons. C: analysis of time course of TTA-P2 blockade of _I_T in a Kiss1 neuron. D: summary of effects of TTA-P2 on maximum peak amplitude of _I_T at −50 mV. **P < 0.01 vs. control, with Student's unpaired _t_-test; n = 5 cells. Mean inhibition of _I_T by 5 μM TTA-P2 was 90.9 ± 5.3%. E: Boltzmann equation fit of the voltage-dependent de-inactivation of the T-channel. Half-maximum de-inactivation voltage is indicated: V1/2 = −86.4 ± 0.2 mV (n = 5, r_2 = 0.999). F: levels of mRNA for Ca v 3.1, −3.2, and −_3.3 derived from qPCR (n = 3 animals; 3 pools/animal). Relative expression was calculated by ΔΔCT method and normalized to mean ΔCT of Ca V 3.1. *P < 0.05 for Ca V 3.3 vs. Ca V 3.1; **P < 0.01, Ca V 3.2 vs. Ca V 3.1 (ANOVA).

Fig. 7.

Fig. 7.

Hyperpolarization-induced rebound burst firing in RP3V Kiss1 neurons. A: hyperpolarization-induced rebound burst firing from a Kiss1+/TH+ cell that expressed _I_T of 52 pA (activated at −60 mV) and had input resistances of 0.53 and 2.0 GΩ between −80 and −60 mV and between −70 and −50 mV, respectively. B: analysis of the effect of hyperpolarizing steps on maximal burst frequency in 11 cells. Dashed line, mean hyperpolarization threshold for burst firing of 82% of neurons. C: example recording showing that T-channel blocker TTA-P2 (5 μM) blocked the rebound burst firing. This cell had _I_T of 67 pA and _I_h of 46 pA. Current injections of −30 and −90 pA yielded hyperpolarizations of −89 and −109 mV, respectively. D: summary of effects of TTA-P2 on spike number of rebound burst firing. **P < 0.01, paired Student's _t_-test. E: example recording showing that h-channel blocker ZD-7288 (50 μM) did not block the rebound burst firing but increased the delay to the first spike. This cell had _I_T of 30 pA and _I_h of 38 pA. Current injection of −60 pA hyperpolarized the cell membrane to −98 mV.

Fig. 8.

Fig. 8.

Expression of _I_T and _I_h is estrogen state dependent in RP3V Kiss1 neurons. A and B: 2 representative recordings showing that _I_T and _I_h greatly increased in high-E2-treated vs. low-E2-treated mice. C: summary of current density of _I_T and _I_h in RP3V Kiss1 neurons from low-E2 and high-E2 animals. All cells were included for statistical analysis (n = 10 for low-E2 group, 5 animals; n = 36 for high-E2 group, 16 animals). **P < 0.01, unpaired Student's _t_-test.

Fig. 9.

Fig. 9.

Effects of μ- and κ-opioid receptor and GABAB receptor agonists on Kiss1 neurons in the RP3V. A: in voltage-clamp and in the presence of TTX (0.5 μM), the selective μ-opioid receptor agonist DAMGO (1 μM) induced an outward current. Vhold = −60 mV. B and C: selective κ-opioid receptor agonist U-69593 (1 μM) induced an outward current (B), which reversed near −90 mV (C), indicating activation of a K+ current. Effects of U-69593 were antagonized by naloxone Vhold = −60mV. D: summary of outward K+ current amplitude induced by DAMGO and U-69593 at −60 mV (n = 9, *P < 0.05). E: relative expressions of μ-, κ-, and δ-opioid receptor mRNAs based on qPCR (n = 4–5 animals, 3 pools/animal). Relative expression was calculated by ΔΔCT method and normalized to mean ΔCT of μ-opioid receptors; a-a, b-b, P < 0.05; c-c, P < 0.001 (one-way ANOVA). F and G: selective κ-opioid receptor agonist U-69593 (1 μM)-induced and GABAB receptor agonist baclofen (20 μM)-induced hyperpolarization of RP3V Kiss1 neurons. H: summary results of U-69593- and baclofen-induced hyperpolarization.

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