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. 2009 Oct 30;151(1):291–300. doi: 10.1210/en.2009-0692

Kisspeptin Increases γ-Aminobutyric Acidergic and Glutamatergic Transmission Directly to Gonadotropin-Releasing Hormone Neurons in an Estradiol-Dependent Manner

Justyna Pielecka-Fortuna 1, Suzanne M Moenter 1
PMCID: PMC2803153  PMID: 19880809

Abstract

GnRH neurons are the final central pathway controlling fertility. Kisspeptin potently activates GnRH release via G protein-coupled receptor 54 (GPR54). GnRH neurons express GPR54, and kisspeptin can act directly; however, GPR54 is broadly expressed, suggesting indirect actions are possible. Transsynaptic mechanisms are involved in estradiol-induced potentiation of GnRH neuron response to kisspeptin. To investigate these mechanisms, separate whole-cell voltage-clamp recordings were performed of γ-aminobutyric acid (GABA)-ergic and glutamatergic transmission to GnRH neurons in brain slices before and during kisspeptin treatment. To determine whether estradiol alters the effect of kisspeptin on synaptic transmission, mice were ovariectomized and either left with no further treatment (OVX) or treated with estradiol implants (OVX+E). Cells were first studied in the morning when estradiol exerts negative feedback. Kisspeptin increased frequency and amplitude of GABAergic postsynaptic currents (PSCs) in GnRH neurons from OVX+E mice. Blocking action potentials eliminated the effect on frequency, indicating presynaptic actions. Amplitude changes were due to postsynaptic actions. Kisspeptin also increased frequency of glutamatergic excitatory PSCs in cells from OVX+E animals. Kisspeptin did not affect either GABAergic or glutamatergic transmission to GnRH neurons in cells from OVX mice, indicating effects on transmission are estradiol dependent. In contrast to stimulatory effects on GABAergic PSC frequency during negative feedback, kisspeptin had no effect during positive feedback. These data suggest estradiol enables kisspeptin-mediated increases in GABA and glutamate transmission to GnRH neurons. Furthermore, the occlusion of the response during positive feedback implies one consequence of estradiol positive feedback is an increase in transmission to GnRH neurons mediated by endogenous kisspeptin.

Kisspeptin increases fast synaptic transmission directly to GnRH neurons in an estradiol-dependent manner, indicating network as well as direct actions of this neuromodulator.

GnRH neurons are the central gatekeepers of reproduction in all vertebrate species. Over the past few years, much attention has focused on an important upstream neuromodulator of GnRH neurons, kisspeptin. Kisspeptin is a strong stimulator of GnRH release and GnRH neuronal activity (1,2,3,4,5,6,7,8,9). Mutations in the kisspeptin receptor, G protein-coupled receptor 54 (GPR54), are associated with idiopathic hypogonadotropic hypogonadism (10,11). Exogenous GnRH is able to restore normal reproductive hormonal levels in some idiopathic hypogonadotropic hypogonadism patients, suggesting the defect is abnormal GnRH neuron function (12). Consistent with this, mice with knockouts of either GPR54 or Kiss1 are infertile, lack normal pubertal development, and have low levels of gonadotropins and steroids (13).

Kisspeptin and GPR54 are expressed in hypothalamic regions known to regulate GnRH activity (14,15). GnRH neurons express GPR54, and most studies have focused on the mechanisms of direct action of kisspeptin on GnRH neurons (1,2,5). The expression of GPR54 by other brain regions including the hypothalamus (16), however, suggests indirect actions of kisspeptin are also possible. Recent studies showed that estradiol potentiates the GnRH response to kisspeptin and that this potentiation appeared to involve transsynaptic mechanisms (3). Specifically, kisspeptin increased action potential firing activity of non-GnRH neurons in the medial preoptic area of hypothalamus that are potential afferents of GnRH neurons, and blocking ionotropic receptors for γ-aminobutyric acid (GABA) and glutamate eliminated the estradiol-induced potentiation of GnRH neuron response to kisspeptin (3). These studies did not determine whether the estradiol-induced potentiation of GnRH neuron response to kisspeptin involved increased neurotransmission directly to GnRH neurons, an increased response to neurotransmitter receptor activation by GnRH neurons, or other actions within the slice network. They also did not differentiate between the action of GABA and/or glutamate in this potentiation.

Many studies indicate estradiol feedback regulation of GnRH neurons is mediated by afferent inputs to GnRH neurons that express ERα (14,17,18,19,20). ERα is expressed by neurons that synthesize kisspeptin (14), and estradiol regulates kisspeptin1 gene expression (14,15). Here, we tested the hypothesis that estradiol enables kisspeptin to alter transsynaptic regulation of GnRH neurons by GABA and glutamate. We examined the effects of kisspeptin on GABAergic and glutamatergic transmission to GnRH neurons, the response of GnRH neurons to these transmitters, and whether or not these actions are estradiol regulated.

Materials and Methods

Animals

Adult transgenic female mice in which green fluorescent protein (GFP) is genetically targeted to GnRH neurons (GnRH-GFP mice) were used in all experiments (21). Mice were housed on a 14-h light, 10-h dark cycle, with lights off at 1630 h, and were maintained on Harlan 2916 rodent chow (Harlan, Bartonsville, IL) and water ad libitum. The Animal Care and Use Committee of the University of Virginia approved all procedures, which were conducted within the guidelines of the National Research Council’s Guide for the Care and Use of Laboratory Animals. Female GnRH-GFP mice were ovariectomized (OVX) under isoflurane (Abbott Laboratories, North Chicago, IL) anesthesia and either received sc SILASTIC brand (Dow Corning, Midland, MI) capsules containing 0.625 μg estradiol in sesame oil (OVX+E) or were left with no further treatment. These implants produce a constant physiological level of estradiol in the circulation that induces diurnal shifts between negative feedback in the morning and positive feedback in the night (22). Postoperative analgesia was provided by a long-acting local anesthetic (0.25% bupivacaine; 7.5 μl/site; Abbott Laboratories). All experiments were performed 2–4 d after surgery. Most recordings were done during negative feedback; a subset was conducted during positive feedback as specified below and in Results.

Brain slice preparation

All reagents were from Sigma Chemical Co. (St. Louis, MO) unless noted. Brain slices were prepared using modifications (23) of a previously described method (24). Briefly, all solutions were bubbled with a 95% O2/5% CO2 mixture throughout the experiments and for at least 15 min before exposure to the tissue. The brain was rapidly removed and placed in ice-cold, high-sucrose saline solution containing 250 mm sucrose, 3.5 mm KCl, 26 mm NaHCO3, 10 mm glucose, 1.25 mm Na2HPO4, 1.2 mm MgSO4, and 2.5 mm MgCl2. Sagittal 300-μm brain slices were cut with a Vibratome 3000 (Technical Products, International, Inc., St. Louis, MO). Slices were incubated for 30 min at 30–32 C in a solution of 50% high-sucrose saline and 50% artificial cerebrospinal fluid (ACSF) containing (in mm) 135 NaCl, 26 NaHCO3, 3.5 KCl, 10 glucose, 1.3 Na2HPO4, 1.2 MgSO4, and 2.5 CaCl2 (pH 7.4) and were then transferred to a solution of 100% ACSF at room temperature and kept at least 30 min and no more than 6 h before recording.

Electrophysiological recordings

Brain slices were transferred individually and placed in a recording chamber superfused continuously at 5–6 ml/min with oxygenated ACSF solution and kept at 29–31 C with an inline heating unit (Warner Instruments, Hamden, CT). Cells were visualized with an Olympus BX50WI upright fluorescent microscope with infrared differential interference contrast (Olympus, Central Valley, PA). GnRH-GFP neurons were identified by brief illumination at 470 nm to visualize the GFP signal. Recording pipettes were pulled from borosilicate glass capillaries (1.65 mm outer diameter; 1.12 mm inndr diameter; World Precision Instruments, Inc., Sarasota, FL) using a Flaming/Brown P-97 (Sutter Instrument, Novato, CA) and were 2–4 MΩ. Pipettes were placed in contact with a GnRH neuron using an MP-285 or MP-225 micromanipulator (Sutter Instruments), a gigaohm seal was formed between the pipette and the cell membrane, and the whole-cell configuration was obtained. During all recordings, input resistance (Rin), series resistance (Rs), and membrane capacitance (Cm) were continually measured. Only recordings with Rin higher than 500 MΩ, Rs lower than 20 MΩ, and stable Cm were used for analysis. Currents were obtained using an EPC-8 amplifier (HEKA, Mahone Bay, Nova Scotia, Canada) with the PulseControl XOP (Instrutech, Port Washington, NY) running in Igor Pro (Wavemetrics, Lake Oswego, OR) or with PatchMaster (HEKA).

Previous work in our lab indicates that when ionotropic receptors for GABA and glutamate are blocked, no postsynaptic currents are detectable at the cell body (23,25). This indicates GABA and glutamate provide the vast majority of fast synaptic transmission to these cells, and we limited our studies to these two transmitters as a result. To record GABA postsynaptic currents (PSCs) ionotropic glutamatergic currents were blocked by inclusion of 20 μm APV [d(−)-2-amino-5-phosphonovaleric acid] and 20 μm CNQX (6-cyano-7-nitroquinoxaline) in the ACSF recording solution. Recording pipettes were filled with isotonic chloride pipette solution (140 mm KCl, 10 mm HEPES, 5 mm EGTA, and 0.1 mm CaCl2) with the addition of 4 mm MgATP and 0.4 mm NaATP before adjusting to pH 7.2 with NaOH. Membrane potential of GnRH neurons was held at −60 mV. Under these experimental conditions, either 20 μm bicuculline (GABAA receptor blockers) eliminated all PSCs, implicating they are mediated through the activation of GABAA receptors on GnRH neurons (23,25). Miniature PSCs (mPSCs) were recorded as above with the addition of 0.5 μm tetrodotoxin (TTX) to the ACSF.

To record glutamatergic excitatory PSCs (EPSCs), GABAA receptors were blocked with 100 μm picrotoxin. Recording pipettes were filled with internal solution containing 125 mm d-gluconic acid, 125 mm CsOH, 25 mm CsCl, 10 mm HEPES, 1 mm EGTA, 4 mm Mg-ATP, 0.4 mm Na-GTP, and 0.1 mm CaCl2, with pH adjusted to 7.4 by CsOH. Membrane potential of GnRH neurons was held at −70 mV. Under these experimental conditions, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor-mediated currents are recorded, because N-methyl-d-aspartic acid receptors are blocked by Mg2+ and inclusion of the AMPA/kainate receptor blocker CNQX (20 μm) blocks all PSCs (26).

Kisspeptin treatment

After a 5-min stabilization period, GABA PSCs or glutamate EPSCs were recorded for a 5-min control period, and then kisspeptin [KiSS-1 (112-121)-amide/kisspeptin-10/metastin (45-54)-NH2; Phoenix Pharmaceuticals, Burlingame, CA] was bath-applied to a final concentration of 10 or 100 nm for 5 min and washed out for an additional 10 min with continued recording.

Local application of GABA

To determine whether the effects of kisspeptin on GABAergic PSC amplitude are in part mediated by postsynaptic mechanisms, we recorded currents generated by local GABA application. To isolate currents mediated through GABAA receptor, ionotropic glutamatergic receptors, GABAB receptors (10 μm SCH50911), and sodium-dependent action potential generation (0.5 μm TTX) were blocked. GABA (0.1 mm) was applied to GnRH neurons by a pressure pipette located near the recorded cell. Six applications were made at 30-sec intervals during the control period, and this was repeated after washing 10 nm kisspeptin into the bath. The advantage of this approach is that the GABA signal from the pipette, although high and unlikely physiological is constant and is not altered by kisspeptin treatment, is thus experimentally useful in isolating postsynaptic effects on amplitude.

Analyses

Sixty-second traces of spontaneous GABAergic PSCs/glutamate EPSCs were analyzed for control and kisspeptin treatment period using custom event detection software in Igor Pro (25). All events were confirmed by eye, and all errors were corrected manually. For each cell, averaged GABA PSC/glutamate EPSC frequency (Hz) was calculated before kisspeptin was added (control) and during kisspeptin treatment. Criteria to define cells as responding to treatment were a 30% change in frequency and a 20% change in amplitude of PSCs. This information was used to calculate a response rate (i.e. percent of cells responding), but data from all cells, both responders and nonresponders, were included in the analysis. Amplitude of GABA-induced current events was measured from the recording baseline just before the GABA puff to the peak current. For each cell, the amplitude of six GABA-induced currents was averaged for the control period before kisspeptin treatment and six events during kisspeptin treatment. All data were transferred to Excel (Microsoft, Redmond, WA), InStat, or Prism (GraphPad Software, San Diego, CA) for statistical analysis. Group means for control and kisspeptin treatment were compared within OVX and OVX+E treatments using two-way mixed-model (repeated-measures pairing before and during kisspeptin treatment in the same cell) ANOVA followed by Bonferroni post hoc test. Amplitude, decay time, and interevent interval probability distributions were compared using two-sample Kolmogorov-Smirnov goodness-of-fit tests. Significance was set at P < 0.05, and all data are reported as mean ± sem.

Results

Kisspeptin increases GABAergic PSC frequency and amplitude in GnRH neurons in an estradiol-dependent manner at the time of estradiol negative feedback

To test the hypothesis that kisspeptin alters GABAergic transmission directly to GnRH neurons, we performed whole-cell voltage-clamp recordings of spontaneous GABAA receptor-mediated PSCs in GnRH neurons from OVX and OVX+E mice. Representative traces are shown in Fig. 1, A and B. In cells from OVX+E mice, kisspeptin increased frequency of GABAergic transmission to a majority of GnRH neurons tested (71%, 15 of 21 cells responding, all cells included in statistical analyses; 10 nm kisspeptin, n = 13 cells from eight mice, P < 0.03; 100 nm kisspeptin, n = 8 cells from five mice, P < 0.02). Response rates were 69 and 75% for 10 and 100 nm kisspeptin treatments, respectively. Among those cells meeting the criteria to be considered as responding cells, 10 nm kisspeptin induced a 90 ± 16% increase in frequency, and 100 nm kisspeptin induced a 90 ± 25% increase (not shown). Consistent with these observations, kisspeptin decreased interevent interval (10 nm P < 0.03, 100 nm P < 0.001; Fig. 1E).

Figure 1.

Figure 1

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Kisspeptin increases GABAergic transmission to GnRH neurons in an estradiol-dependent manner. A and B, Representative recordings of GABAergic PSCs in GnRH neurons from OVX+E (A) and OVX (B) mice; C and D, mean ± sem PSC frequency in cells from OVX+E (C) and OVX (D) mice, with white bars showing control period and black bars kisspeptin treatment; E and F, cumulative probability distribution plots of all events for interevent interval; kisspeptin (100 nm in this example) shortens interevent interval in GnRH neurons from OVX+E (E) mice, but there is no change in cells from OVX mice (F). *, P < 0.05.

Kisspeptin also increased GABAergic PSC amplitude in most GnRH neurons tested from OVX+E mice (62%, 13 of 21 cells responding, all cells included in statistical analyses; 10 nm kisspeptin, P < 0.02, n = 13; 100 nm kisspeptin, P < 0.002, n = 8). There was no effect of kisspeptin on decay time of GABAergic PSCs (control 23.9 ± 2.1 msec, 10 nm kisspeptin 24.8 ± 1.6 msec, P > 0.6; control 24.3 ± 1.9 msec, 100 nm kisspeptin 24.2 ± 2 msec, P > 0.9). Representative averaged PSC traces are shown in Fig. 2A, overall group means and event distribution in Fig. 2, C and E. Response rates for changes in amplitude were 46 and 88% for 10 and 100 nm kisspeptin treatment, respectively. Among those cells meeting the criteria to be considered responding cells, 10 nm kisspeptin induced a 39 ± 6% increase in amplitude, and 100 nm kisspeptin induced a 35 ± 10% increase.

Figure 2.

Figure 2

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Kisspeptin (kiss) increases the amplitude of spontaneous GABAergic PSCs in GnRH neurons from OVX+E mice. A and B, Representative averaged PSC traces from cells from OVX+E (A) and OVX mice (B); C and D, mean ± sem PSC amplitude in cells from OVX+E (C) and OVX (D) mice, with white bars showing control (con) period and black bars kisspeptin treatment; E and F, cumulative probability distribution plots of all events comparing control period and kisspeptin treatment in cells from OVX+E (E) and OVX (F). *, P < 0.05.

In contrast to the effects of kisspeptin on GABAergic transmission to GnRH neurons from OVX+E mice, in GnRH neurons from OVX mice, there was no effect of kisspeptin on PSC frequency (Fig. 1, B and D; 10 nm kisspeptin, n = 7 cells from four mice, P > 0.2; 100 nm kisspeptin, n = 8 cells from five mice, P > 0.2), interevent interval (Fig. 1F, P > 0.3), amplitude (Fig. 2, B, D, and F; 10 nm kisspeptin, P > 0.4 control; 100 nm kisspeptin, P > 0.9) or decay time (control 24 ± 2.6 msec, 10 nm kisspeptin 24.4 msec, P > 0.7; control 24.4 ± 1.9 msec, 100 nm kisspeptin 23.1 ± 1.5 msec, P > 0.4). Of the 15 cells examined from OVX mice, only one cell exhibited a change in frequency (∼40% increase) of GABAergic transmission that met the criterion to be classified as responding; there were no cells that met the criterion for changes in amplitude. As previously reported, frequency of GABAergic transmission to GnRH neurons at the time these recordings were made (in the morning) is greater in cells from OVX mice than in cells from OVX+E mice (P < 0.05), an effect attributed to negative feedback action of estradiol (27). There was no difference (P > 0.5, both doses) in the PSC frequency during kisspeptin treatment between cells from OVX and OVX+E mice. Together these results suggest exogenous kisspeptin increases GABAergic transmission to GnRH neurons in an estradiol-dependent manner at the time of negative feedback.

Kisspeptin does not alter frequency of miniature GABAergic PSCs

To determine whether the exogenous kisspeptin-induced increase in frequency of GABAergic transmission to GnRH neurons from OVX+E mice was due to increased activity of presynaptic GABAergic neurons, mPSCs were recorded before and during kisspeptin treatment (10 nm, Fig. 3, A and B). Kisspeptin had no effect on the frequency of GABAergic mPSCs (one of seven cells from five mice responding, P > 0.8; Fig. 3B) or interevent interval (P > 0.5; Fig. 3C). Of interest, kisspeptin still slightly but significantly increased the amplitude of mPSCs in 57% of cells (four of seven; P < 0.05 for n = 7 cells). Kisspeptin increased decay time of GABAergic mPSCs (control 21 ± 1.6 msec, 10 nm kisspeptin 25 ± 2.3 msec); although this was statistically significant (P < 0.05), it was due to alterations in only two of seven cells studied and thus may be of limited biological impact. Moreover, no change in decay time after kisspeptin treatment was observed in the recordings of spontaneous PSCs. This suggests the effects of kisspeptin on frequency of transmission are action potential dependent and presynaptic, whereas alterations in amplitude may be attributed at least in part to postsynaptic action.

Figure 3.

Figure 3

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Kisspeptin (kiss) does not alter frequency of mPSCs but does increase amplitude in cells from OVX+E mice. A, Representative traces during the control (con; left) and kisspeptin (right) treatment in the presence of TTX; B, mean ± sem mPSC frequency showing lack of effect of kisspeptin when action potential generation is blocked; C, cumulative probability distribution of mPSC interevent intervals of all events during control and kisspeptin treatments; D, averaged mPSC during control (black) and kisspeptin (gray) treatment; E, probability distribution of all events of mPSC amplitude. *, P < 0.05.

Kisspeptin acts postsynaptically to increase GABA PSC amplitude, and this effect is estradiol-dependent

To test further whether kisspeptin acts directly on GnRH neurons to increase response to GABAA receptor activation, we performed experiments in which GABA was locally applied from a pipette onto GnRH neurons in slices from OVX+E and OVX mice before and during treatment with 10 nm kisspeptin (Fig. 4). Kisspeptin increased GABA-induced current amplitude in GnRH neurons from OVX+E mice (n = 7 cells from five mice, P < 0.006) but had no effect in cells from OVX mice (n = 6 cells from five mice, P > 0.5). These data suggest that kisspeptin acts at least in part directly on GnRH neurons to increase response to GABAA receptor activation and thereby GABAergic PSC amplitude.

Figure 4.

Figure 4

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Kisspeptin (kiss) increases response of GnRH neurons to GABAA receptor activation. A and B, Representative current induced by a brief, local puff of GABA (0.1 mm) during control (con) or kisspeptin treatment of cells from OVX+E (A) and OVX (B) mice; C, mean ± sem GABA-induced currents during control (white bars) and kisspeptin (black bars) treatment periods from cells from OVX+E (left) and OVX (right) mice. *, P < 0.05.

Kisspeptin increases glutamatergic EPSC frequency in GnRH neurons in an estradiol-dependent manner

To test whether kisspeptin affects glutamatergic transmission to GnRH neurons, the membrane potential of GnRH neurons was clamped at −70 mV, and spontaneous AMPA/kainate receptor-mediated EPSCs were recorded in cells from OVX and OVX+E mice (Fig. 5). In previous work, the negative feedback effects of estradiol suppressed glutamatergic transmission to GnRH neurons (26); in the present experiment, the data showed the same trend but did not quite achieve statistical significance (P = 0.056, control in Fig. 5, A vs. B). In cells from OVX+E mice, kisspeptin (10 nm) treatment significantly increased the frequency of AMPA/kainate receptor-mediated EPSCs in 88% of GnRH neurons (seven of eight) examined (n = 8 cells from six mice, P < 0.002; Fig. 5, A and C). Kisspeptin also decreased interevent interval (P < 0.05) in cells from OVX+E mice (Fig. 5E). As with GABAergic transmission, kisspeptin treatment did not alter glutamatergic transmission to GnRH neurons in slices from OVX mice (n = 9 cells from seven mice, P > 0.2; Figure 5, B, D, and E). There was no change in the EPSC amplitude either in OVX+E or OVX cells after kisspeptin treatment (OVX+E: control 26 ± 2 pA, 10 nm kisspeptin 25 ± 1.7 pA, P > 0.1; OVX: control 29 ± 2.9 pA, 10 nm kisspeptin 29 ± 4.1 pA, P > 0.9), implicating that the observed effect is presynaptic to GnRH neurons.

Figure 5.

Figure 5

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Kisspeptin (kiss) increases AMPA/kainate receptor-mediated glutamatergic transmission to GnRH neurons in estradiol-dependent manner. A and B, Representative recordings of AMPA/kainate receptor-mediated EPSCs in GnRH neurons from OVX+E (A) and OVX (B) mice; C and D, mean ± sem EPSC frequency with kisspeptin (kiss) treatment; E and F, cumulative probability distributions plots of all events for interevent interval; kisspeptin decreases interevent interval in GnRH neurons from OVX+E mice (E) but not OVX mice (F). *, P < 0.05. con, Control.

Kisspeptin does not alter frequency or amplitude of GABAergic PSCs at the time of estradiol positive feedback

GABAergic transmission to GnRH neurons is increased during estradiol positive feedback (27). This observation, along with recent work suggesting a role for kisspeptin in LH surge generation (28,29), raised the question of a possible role for kisspeptin in increasing GABAergic transmission during positive feedback. If this were one mechanism of kisspeptin action, we hypothesized that the response to exogenous kisspeptin would be blunted at this time. We tested this by examining GABAergic transmission in OVX+E animals during positive feedback when the frequency of GABAergic transmission is higher and more dramatically altered by estradiol than glutamatergic transmission. Supporting our hypothesis, during positive feedback, kisspeptin (10 nm) had no effect on frequency of GABAergic transmission (n = 9, P > 0.3), interevent interval (P > 0.05), amplitude (P > 0.8), or decay time (P > 0.7) (Fig. 6). The characteristics of GABAergic PSCs during the control period of the positive feedback recordings were similar to the effects achieved with kisspeptin during negative feedback, suggesting a possible role for endogenous kisspeptin in increasing GABAergic transmission during positive feedback actions of estradiol.

Figure 6.

Figure 6

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Kisspeptin (kiss) has no effect on GABA transmission in GnRH neurons from OVX+E mice during estradiol positive feedback. A, Representative traces during the control (con; left) and kisspeptin (right) treatment; B, mean ± sem PSC frequency showing lack of effect of kisspeptin; C, cumulative probability distribution of PSC interevent intervals of all events during control and kisspeptin treatments; D, averaged PSC during control (black) and kisspeptin (gray) treatment; E, probability distribution of all events of PSC amplitude.

Discussion

The discovery of dysfunctional mutations in the kisspeptin receptor GPR54 in patients with hypothalamic infertility stimulated considerable research on the role of this neuropeptide and its receptor in reproduction. Although the intrinsic mechanisms of kisspeptin action directly on GnRH neurons have been probed (1,2,30), recent work indicates estradiol potentiates the effect of kisspeptin on LH release in vivo (31) and on GnRH neuron activity in vitro, possibly via changes in GABAergic/glutamatergic transmission (3). Here we extend our previous findings of the effects of interactions between kisspeptin and estradiol milieu on GnRH neuron activity (3). The present data provide evidence for network as well as direct regulation of GnRH neurons by kisspeptin in the presence of estradiol. Specifically, estradiol not only enables exogenous kisspeptin to increase GABAergic and glutamatergic transmission to GnRH neurons and potentiates the postsynaptic response to GABA but also may play an inhibitory/excitatory role in the release of endogenous kisspeptin at the time of estradiol negative/positive feedback, respectively, and thereby alter synaptic transmission to GnRH neurons.

Both GABAergic and glutamatergic transmission play important roles in the control of GnRH neuronal activity and in steroid feedback regulation of GnRH/LH secretion (26,27,32,33,34,35). GABAA receptor activation can be excitatory to GnRH neurons (36,37,38), and GABAergic afferents are known to be among the major synaptic inputs to GnRH neurons (33,34,39). GABAergic and glutamatergic transmission to GnRH neurons are regulated by steroids and metabolic cues that affect fertility (25,26,40,41,42). As demonstrated here, exogenous kisspeptin increases both GABAergic and glutamatergic transmission directly to GnRH neurons at the time of estradiol negative feedback. Changes in the PSC frequency detected at the postsynaptic cell typically reflect changes in release from the presynaptic cell; therefore, the ability of kisspeptin to increase GABAergic and glutamatergic transmission is most likely attributed to kisspeptin action on neurons afferent to GnRH neurons. In this regard, blockade of action-potential-dependent transmitter release also eliminated the change in the frequency of transmission induced by kisspeptin, suggesting kisspeptin increases the activity of presynaptic neurons (3). Consistent with this, kisspeptin increased firing activity of non-GnRH neurons in the medial preoptic area, and blocking GABAergic and glutamatergic transmission reduced GnRH neuron response to kisspeptin (3).

In addition to changing the frequency of transmission from presynaptic cells, kisspeptin increased the amplitude of GABAergic postsynaptic currents in GnRH neurons. It is important to point out that changes in amplitude of a postsynaptic response measured with the whole-cell approach must be interpreted with caution. Amplitude can be altered by presynaptic actions (altered vesicular GABA concentration or vesicle release) and/or postsynaptic actions (number and composition of channels and channel open time). The ability of kisspeptin to increase both the amplitude of GABA mPSCs and the response of GnRH neurons to a fixed GABA signal indicates the observed effect on GABA PSC amplitude is most likely through a postsynaptic action on GnRH neurons. A recent study demonstrated opposing actions of kisspeptin and GABAB receptor activation in GnRH neurons; simultaneous activation of GABAB receptors thus may blunt the effect of kisspeptin on PSC amplitude (43). Mechanistically, activation of kinases downstream of GPR54 in GnRH neurons could alter phosphorylation of GABAA receptors and/or alter GABAA receptor trafficking, insertion into the cell membrane, and/or removal from the membrane (44). GnRH neurons express estrogen receptor-β (45,46,47); thus, the kisspeptin-estradiol interaction that facilitates amplitude of GABAergic PSCs could occur within the GnRH neuron itself. In agreement with this, recent studies have shown that rat GnRH neurons coexpress GABAA receptor and Gec1, an estrogen-regulated protein that is involved in GABAA receptor trafficking from intracellular stores to the membrane (48). In contrast to data from hippocampal granule cells (49), no effect of kisspeptin on amplitude of AMPA receptor-mediated EPSCs was detected in GnRH neurons.

A role for kisspeptin in estradiol feedback regulation of GnRH neuronal activity is suggested by several studies. First, kisspeptin neurons express estrogen receptors α and β (50). Second, estradiol differentially regulates Kiss-1 mRNA expression in specific brain nuclei involved in feedback regulation of GnRH/LH secretion (14,15). Specifically, in the anteroventral periventricular nucleus (AVPV) Kiss-1 mRNA expression decreased after ovariectomy and increased after estradiol replacement, whereas the opposite effect was observed in the arcuate nucleus. Third, in female mice during induced LH surges, there is increased coexpression of kisspeptin and the transcription factor Fos, a marker of neuronal activity, that is correlated with increased Fos expression in GnRH neurons (29). Fourth, recent studies have investigated the role of GPR54 in estradiol-dependent GnRH/LH surge generation making use of GPR54 knockout mice (28,29). Dungan et al. (28) showed GPR54 knockout mice still exhibit LH surges; however, surge amplitude was decreased compared with control mice. These findings suggest GPR54 is important for surge generation and point out the importance of other mechanisms that may also be involved. In contrast, Clarkson et al. (29) showed that GPR54 knockout mice are not able to generate an LH surge. The differences between these studies might be attributed to methods used in the generation of the knockout mice and/or the steroid treatment paradigm used to generate the surge. Despite these differences, these findings indicate the likely importance of kisspeptin and GPR54 signaling in GnRH/LH surge generation.

Likewise, recent studies indicate diurnal and estradiol-dependent changes in GABA and glutamate transmission to GnRH neurons involved in surge generation (27,32). GABAergic and glutamatergic transmission to GnRH neurons are reduced compared with that in OVX mice during estradiol negative feedback (27,32). The present data, in agreement with these studies, showed that GABAergic and glutamatergic transmission are lower in GnRH neurons from OVX+E than in cells from OVX mice during estradiol negative feedback. During estradiol positive feedback, GABA transmission is increased to a subpopulation of GnRH neurons (27), and blocking the receptors for GABA and glutamate at this time decreases GnRH firing activity and presumably GnRH release (32). Although glutamatergic transmission to GnRH neurons from estradiol-treated mice is not increased relative to that in cells from OVX mice during estradiol positive feedback (26), it is increased compared with that observed in estradiol-treated mice at the time of negative feedback. Together these findings show the important correlations between GnRH activity and GABA/glutamate transmission during the time of estradiol negative and positive feedback.

The mechanisms by which estradiol drives these changes in fast synaptic transmission between negative and positive feedback states is unknown. Estradiol may act directly on GABAergic and/or glutamatergic cells (51). Alternatively, estradiol may engage other neurotransmitters or neuromodulators that subsequently affect the GABAergic and glutamatergic afferents of GnRH neurons. The present data indicate that kisspeptin is able to increase GABA and glutamate transmission to GnRH neurons from estradiol-treated but not OVX mice at the time of estradiol negative feedback. This suggests a role for estradiol in altering synaptic connectivity between kisspeptin-sensitive afferents and GnRH neurons. In this regard, estradiol increases hippocampal synapse density and regulates synaptic plasticity (52,53). In female rats, estradiol induced an increase in GABAergic axosomatic synapses onto neurons within the AVPV, with an opposite effect observed within the arcuate nucleus, and modulates the number of dual GABA-glutamate terminals on subpopulations of GnRH neurons (54,55,56). Furthermore, there is a trend for higher frequency of mPSCs in GnRH neurons from estradiol-treated mice than from OVX mice during the time of positive feedback, indicative of activity-independent modulation of GABAergic transmission to these neurons by estradiol (27). Finally, the ability of kisspeptin to increase activity of nonidentified neurons in the medial preoptic area does not appear to be dependent upon estradiol (3). The present observations may thus be explained by an estradiol-mediated increase in synaptic connectivity to GnRH neurons, so that kisspeptin activation of afferent GABA and glutamate neurons results in increased synaptic transmission to GnRH neurons when this steroid is present. Neurons in the AVPV project to GnRH neurons and use both GABA and glutamate as neurotransmitters, and their connectivity with GnRH neurons is altered during the estradiol-induced surge (17,56). Together with findings that GPR54 is expressed in the AVPV (16) and the present data, this suggests the AVPV as one possible location for cells that respond to exogenous, and perhaps endogenous, kisspeptin by altering fast synaptic transmission to GnRH neurons.

The present data also provide indirect support for alterations in endogenous kisspeptin release being a component of the mechanism by which estradiol exerts feedback upon GnRH neuron activity. Specifically, during negative feedback, estradiol may reduce endogenous kisspeptin release, enabling us to observe an increase in synaptic transmission in response to exogenous kisspeptin at that time. Reduced endogenous kisspeptin release would affect both direct and transsynaptic activation of GnRH neurons by kisspeptin. Although estradiol up-regulates kisspeptin expression in the AVPV, nothing is known about its effects on release of this neuropeptide. Fos is expressed in kisspeptin neurons during positive feedback, suggesting increased activity, but there is no fos expression during negative feedback, suggesting little neuronal activity (50). In this case, exogenous kisspeptin would be expected to exert a greater effect in a preparation in which there was less endogenous release. Of interest in this regard, kisspeptin treatment increased the frequency of GABAergic synaptic transmission only during estradiol negative feedback and only to the level observed in OVX mice at that time. With regard to positive feedback, kisspeptin had no effect on GABAergic transmission. This suggests that an endogenous increase in kisspeptin may have already signaled to these cells, precluding a further increase. Of note, the control values of GABAergic transmission in positive feedback were similar to the post-kisspeptin values during negative feedback.

In summary, our data extend previous findings in which estradiol was shown to potentiate GnRH neuron response to kisspeptin, and this potentiation appeared to involve changes in GABA and glutamate transmission. Here we showed that kisspeptin increases GABAergic and glutamatergic transmission directly to GnRH neurons in an estradiol-dependent manner and can increase GnRH neuron response to activation of GABAA receptors. Moreover, our data implicate that the release of endogenous kisspeptin may be inhibited by estradiol during negative feedback and increased during positive feedback, which in turn may alter synaptic transmission to GnRH neurons. All together, the present data indicate that kisspeptin can act via both direct and transsynaptic mechanisms to modulate GnRH neuronal activity.

Acknowledgments

We thank Debra Fisher for excellent technical assistance and Kimberly Cox, Catherine Christian, Alison Roland, Jianli Sun, and Pei-San Tsai for useful editorial comments.

Footnotes

This work was supported by National Institutes of Health Grant HD41469.

Portions of this work were presented in abstract form at the 2008 Endocrine Society and 2008 Society for Neuroscience Meetings.

Disclosure Summary: J.P.-F. and S.M.M. have nothing to disclose.

First Published Online October 30, 2009

Abbreviations: ACSF, Artificial cerebrospinal fluid; AVPV, anteroventral periventricular nucleus; EPSC, excitatory PSC; GABA, γ-aminobutyric acid; GFP, green fluorescent protein; GPR54, G protein-coupled receptor 54; mPSC, miniature PSC; OVX, ovariectomized; OVX+E, OVX plus treated with estradiol implants; PSC, postsynaptic current; TTX, tetrodotoxin.

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