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Comparative Study
. 2007 Sep 19;27(38):10153-64.
doi: 10.1523/JNEUROSCI.1657-07.2007.

Gonadotropin-releasing hormone neurons express K(ATP) channels that are regulated by estrogen and responsive to glucose and metabolic inhibition

Chunguang Zhang  1 , Martha A Bosch, Jon E Levine, Oline K Rønnekleiv, Martin J Kelly
Affiliations
Comparative Study

Gonadotropin-releasing hormone neurons express K(ATP) channels that are regulated by estrogen and responsive to glucose and metabolic inhibition

Chunguang Zhang et al. J Neurosci. .

Abstract

Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner that is dependent on circulating 17beta-estradiol (E2) and glucose concentrations. However, the intrinsic conductances responsible for the episodic firing pattern underlying pulsatile release and the effects of E2 and glucose on these conductances are primarily unknown. Whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons revealed that the K(ATP) channel opener diazoxide induced an outward current that was antagonized by the sulfonylurea receptor 1 (SUR1) channel blocker tolbutamide. Single-cell reverse transcription (RT)-PCR revealed that the majority of GnRH neurons expressed Kir6.2 and SUR1 subunits, which correlated with the diazoxide/tolbutamide sensitivity. Also, a subpopulation of GnRH neurons expressed glucokinase mRNA, a marker for glucose sensitivity. Indeed, GnRH neurons decreased their firing in response to low glucose concentrations and metabolic inhibition. The maximum diazoxide-induced current was approximately twofold greater in E2-treated compared with oil-treated ovariectomized females. In current clamp, estrogen enhanced the diazoxide-induced hyperpolarization to a similar degree. However, based on quantitative RT-PCR, estrogen did not increase the expression of Kir6.2 or SUR1 transcripts in GnRH neurons. In the presence of ionotropic glutamate and GABA(A) receptor antagonists, tolbutamide depolarized and significantly increased the firing rate of GnRH neurons to a greater extent in E2-treated females. Finally, tolbutamide significantly increased GnRH secretion from the preoptic-mediobasal hypothalamus. Therefore, it appears that K(ATP) channels and glucokinase are expressed in GnRH neurons, which renders them directly responsive to glucose. In addition, K(ATP) channels are involved in modulating the excitability of GnRH neurons in an estrogen-sensitive manner that ultimately regulates peptide release.

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Figures

Figure 1.
Figure 1.
Characteristics of mouse GnRH neurons in hypothalamic slices. A, Identification of EGFP-labeled GnRH neurons by immunocytochemical staining for GnRH peptide. Aa, EGFP-GnRH neurons in a thin section (15 μm) of a coronal preoptic slice from a female mouse. Ab, The immunocytochemical staining of same section. Ac, Overlay of Aa and Ab. Scale bar, 25 μm. B, B1, Representative whole-cell voltage-clamp recordings showing that female mouse EGFP-GnRH neurons express transient outward potassium current (IA). Whole-cell currents were elicited by holding the cell at −60 mV and giving a series of 1 s prepulses ranging from −50 to −120 mV (in 5 mV increments) and then stepping back to −50 mV for 0.5 s to activate IA. B2, Expanded time scale of A-current activated at −50 mV after leak subtraction (see Materials and Methods). C, C1, A representative whole-cell voltage-clamp recording showing that female mouse EGFP-GnRH neurons express a hyperpolarization-activated cation current (Ih). Whole-cell currents were elicited by holding the cell at −60 mV and giving a series of 1 s prepulses ranging from −50 to −120 mV (in 5 mV increments) and measuring the slow activating steady-state current at the last 100 ms of the 1 s pulse. C2, ZD 7288 (50 μm)-sensitive component of the whole-cell currents shown in C1.
Figure 2.
Figure 2.
RT-PCR identification of KATP channel subunits and GK transcripts in GnRH neurons. A, A representative gel illustrating that harvested EGFP-GnRH neurons express mRNA for Kir6.2, SUR1, and GK (a). However, nonfluorescence (non-FL) cells (n = 12) expressed Kir6.2 (50%) and SUR1 (25%), but not GnRH or GK transcripts (b). In addition, the following controls were included: a tissue control and a cell without MuLV RT, aCSF from the dispersed cellular milieu, and water blank, all of which were negative after RT-PCR (data not shown). A base pair ladder (L) is given for determining the relative size of the transcripts. B, Quantitative analysis of Kir6.2, SUR1, and GK mRNA expression in individual GnRH neurons from eight animals, 8–14 cells per animal. Percentage expression was determined for each animal, and the mean ± SEM was calculated (n = 8). C, Quantitative analysis of Kir6.2, SUR1, and GK mRNA expression in GnRH neuronal pools from eight animals, two to three pools per animal. Percentage expression was determined for each animal, and the mean ± SEM was calculated (n = 8).
Figure 3.
Figure 3.
qPCR analysis of Kir6.2 and SUR1 mRNA expression in GnRH neuronal cell pools. A–E, Kir6.2 expression was determined using the SYBR green method (A–C), and SUR1 expression was determined using the Taqman gene expression method (D, E). A, D, For both, cycle number is plotted against the normalized fluorescence intensity (Delta Rn) to visualize the PCR amplification. The cycle threshold (Ct) value (line with arrows) is the point in the amplification at which the sample values were calculated. A, D, POA cDNA serial dilutions and one representative GnRH neuronal pool (■-■) as well as the corresponding cycle number when the fluorescent signal was detected. B, The superimposed melting curves for Kir6.2 depict a single product. A, D, The standard curve regression line (inset) produced a slope of −3.3 for Kir6.2 and −3.2 for SUR1, which translates into similar efficiencies of 100%. C, E, Quantitative analysis of Kir6.2 and SUR1 mRNA expression in GnRH neuronal cell pools from oil- and E2-treated animals (mean ± SEM; n = 4 for each group).
Figure 4.
Figure 4.
Pharmacological identification showed that female mice GnRH neurons mainly express SUR1-containing KATP channels. A, A representative recording showing that GnRH neurons express diazoxide- and tolbutamide-sensitive KATP channels. Diazoxide induced an outward current (76 pA) in a GnRH neuron, which was reversed by tolbutamide. Vhold = −60 mV. B, A representative recording showing that MCC-134 (300 μm), a selective SUR2-containing KATP channel opener, did not induce an outward current in a GnRH neuron, but diazoxide (300 μm) induced an outward current of 20 pA that was antagonized by tolbutamide (200 μm). Vhold = −60mV. C, Current–voltage plot taken from another cell at the time immediately before (control) and after application of diazoxide and tolbutamide. The diazoxide-induced outward current had a reversal potential of −80 mV, which is close to the predicted EK+. The voltage protocol consisted of 1 s steps every 5 mV from −50 to −120 mV. Vhold = −60 mV.
Figure 5.
Figure 5.
Male GnRH neurons express diazoxide/tolbutamide-sensitive KATP current, and tolbutamide stimulates GnRH release from superfused POA-MBH tissues of adult male rats. A, Representative recording of diazoxide-induced outward current (30 pA) in a GnRH neuron that was reversed by tolbutamide. Vhold = −60 mV. B, Current–voltage plot taken from the recording shown in A at the time immediately before (Control) and after application of diazoxide. C, GnRH release rates (mean ± SEM) in successive 10 min superfusate collections, in which tissues were exposed to blank medium only, or diazoxide-containing medium at the times indicated. All tissues were exposed to isotonic medium containing 60 mm K+ after the drug challenges to confirm the viability of the preparations. D, GnRH release from tissues that were superfused with tolbutamide-containing medium, or tolbutamide and diazoxide-containing medium during the times indicated (* p < 0.05).
Figure 6.
Figure 6.
GnRH neurons are responsive to changes in glucose concentrations. A, Top, A representative loose cell-attached record showing the time course of the effect of low glucose (0.1 mm) on high K+ (15 mm) induced firing in a GnRH neuron. High Mg2+/low Ca2+ aCSF containing blockers for GABAA (100 μm picrotoxin) and ionic glutamate receptors (20 μm CNQX plus 20 μm d-APV) were used to isolate the cell from presynaptic input. Bottom, Firing frequency-time curve corresponding to the top record. B, Expanded recording traces indicated in the top panel of A. C, Summary of the effects of low glucose (0.1 mm) on the firing rate of GnRH neurons (n = 5 per group; **p < 0.01, *** p < 0.001).
Figure 7.
Figure 7.
GnRH neurons are sensitive to metabolic inhibition. A–C, Loose cell-attached patch recordings in the presence of 5 nm Kisspeptin-10, which was used to stimulate the firing activity. A, A representative recording showing that sodium azide (NaN3) strongly inhibited the kisspeptin induced firing of a GnRH neuron (top trace), and this inhibition was reversed after washout of sodium azide (bottom trace). B, The NaN3 inhibition was reversed by tolbutamide. C, Summary of the effects of sodium azide on the firing rate of GnRH neurons (n = 4; *p < 0.05, **p < 0.01). D, Whole-cell, voltage-clamp recording with 3 mm ATP in pipette showing that NaN3 strongly hyperpolarized a GnRH neuron by 7 mV, which reversed when returned to control conditions (washout). Vhold = −60 mV.
Figure 8.
Figure 8.
Estrogen enhanced the diazoxide-induced KATP channel currents in female GnRH neurons. Female mice were ovariectomized and implanted with oil or estrogen capsule for 4–7 d (see Materials and Methods). A, Whole-cell voltage-clamp recording in a GnRH neuron from an oil-treated female. Diazoxide (300 μm) induced an outward current of 16 pA that was antagonized by tolbutamide (200 μm). Vhold = −60 mV. B, Whole-cell voltage-clamp recording from a GnRH neuron from an estrogen-treated female. Diazoxide induced an outward current of 38 pA that was antagonized by tolbutamide. Vhold = −60 mV. C, Summary of the differences in the diazoxide-induced current in GnRH neurons between oil- and estrogen-treated females (**p < 0.01).
Figure 9.
Figure 9.
Tolbutamide differentially depolarizes and diazoxide hyperpolarizes GnRH neurons from oil- and estrogen-treated, ovariectomized female mice. A, Representative recordings from estrogen-treated female showing that tolbutamide induced a depolarization of 6 mV in one cell and diazoxide a hyperpolarization of 12 mV in another cell. B, Summary of the effects of tolbutamide and diazoxide on membrane potentials in GnRH neurons from oil- and estrogen-treated females (n = 5; **p < 0.01).
Figure 10.
Figure 10.
Tolbutamide differentially increased the firing rate of GnRH neurons from estrogen- and oil-treated, ovariectomized females. A, A representative recording from E2-treated female mouse showing spontaneous burst firing. Tolbutamide increased the firing rate and caused continuous firing. Resting membrane potential (RMP), −65 mV. B, Another representative recording from an oil-treated female showing a higher baseline firing rate. Tolbutamide also caused continuous firing. RMP, −63 mV. C, Summary of the effects of tolbutamide on the firing rate of GnRH neurons from oil- and E2-treated females. GnRH neurons from E2-treated females had significantly lower basal firing rates than GnRH neurons from oil-treated females (++p < 0.01; n = 5). Tolbutamide significantly increased the firing rate in both groups (oil, **p < 0.01, n = 5; E2, ***p < 0.001, n = 5). D, Tolbutamide increased the firing rate of GnRH neurons more in E2-treated versus oil-treated females (*p < 0.05).
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