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Endocrinology Vol. 145, No. 3 1194-1202
Copyright © 2004 by The Endocrine Society

{gamma}-Aminobutyric Acid Neurons Integrate and Rapidly Transmit Permissive and Inhibitory Metabolic Cues to Gonadotropin-Releasing Hormone Neurons

Shannon D. Sullivan and Suzanne M. Moenter

Department of Internal Medicine and Cell Biology, University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Dr. Shannon M. Moenter, Department of Internal Medicine and Cell Biology, University of Virginia, P.O. Box 800578, Jefferson Park Avenue, Charlottesville, Virginia 22908. E-mail: smm4n{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Negative energy balance inhibits fertility by decreasing GnRH release; however, the mechanisms are not well understood. GnRH neurons can be excited by activation of {gamma}-aminobutyric acid (GABA)A receptors, and GABAergic neurons provide a major synaptic input. We hypothesized that permissive metabolic signals mediated by leptin and inhibitory signals conveyed by neuropeptide Y (NPY) and opiates rapidly alter GABAA receptor-mediated drive to GnRH neurons. In fed and fasted female mice, GABAergic postsynaptic currents (PSCs) were recorded from GnRH neurons before and after in vitro treatment with leptin, NPY, or met-enkephalin. Leptin increased PSC frequency in fed and fasted mice, indicating that it increased presynaptic activity. Leptin also increased PSC size. Inhibiting leptin receptor signaling pathways within GnRH neurons abolished the latter effect, indicating a direct action on these cells. In fed, but not fasted, mice, NPY and met-enkephalin decreased PSC frequency in an antagonist-reversible manner, but did not alter PSC size. NPY-1 receptor antagonists alone increased frequency in fed and fasted mice, as did opiate receptor blockade in fasted animals, suggesting that endogenous NPY and opiates modulate GABAergic drive to GnRH neurons. These data suggest that GABAergic afferents integrate metabolic signals for delivery to GnRH neurons. Decreased sensitivity to NPY and opiates in fasted mice indicate that these peptides send physiologically relevant signals regarding energy balance to GnRH neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH NEURONS form the final central pathway regulating reproduction. Pulsatile release of GnRH stimulates the synthesis and secretion of pituitary gonadotropins and is absolutely required for fertility (1). GnRH pulse patterns are regulated by positive and negative inputs from various sources. Among these, metabolic cues have profound influences on fertility (2, 3, 4). Metabolic signals alter reproduction at least in part by reducing the frequency of pulsatile GnRH secretion (5); however, the precise central mechanisms underlying metabolic regulation of normal fertility as well as forms of hypothalamic infertility in which altered energy balance plays a role (6, 7) are poorly understood.

In this regard, {gamma}-aminobutyric acid (GABA)ergic neurons provide a major input to GnRH neurons (8, 9, 10). A recent report showed that fasting (48 h) decreased the frequency of postsynaptic currents GnRH neurons receive via the GABAA receptor (GABAAR) (11). Unlike most mature neurons, GnRH neurons can be excited by GABAAR activation (12); thus, this fasting-induced decrease in GABAergic drive would decrease the probability for action potential firing and ultimately for GnRH release. Interestingly, in vivo treatment of fasting mice with a leptin regimen known to restore fertility (2, 13) restored GABAergic drive to GnRH neurons to the levels seen in fed animals (11).

These data suggested that modulation of the activity of GABAergic afferents to GnRH neurons may contribute to inhibition of fertility by negative energy balance as well as fertility restoration by permissive leptin signals; however, neither the time course of these effects nor the role of other metabolically important peptides is known. With regard to time course, a short-term fast causes rapid suppression of LH (and presumably GnRH) pulsatility (11), whereas refeeding rapidly reinitiates LH release (14). These observations indicate that metabolic signals act acutely to alter the output of the reproductive axis. The GABAergic system is poised for such rapid communication with GnRH neurons: anatomical and functional evidence suggests an interplay between hypothalamic GABAergic neurons and various metabolic peptides, including leptin (11, 15), which provides a permissive metabolic cue (2), and neuropeptide Y (NPY) (16, 17, 18, 19) and opiate peptides (16, 20), which can act antagonistically to leptin in the regulation of both fertility and food intake (21, 22, 23, 24).

To determine whether leptin, NPY, and opiates rapidly alter excitatory GABAergic drive to GnRH neurons, we used whole cell electrophysiology to test the response to acute in vitro, rather than in vivo, treatment with each peptide and a respective receptor antagonist when available. During fasting, central NPY (25, 26, 27) and opiatergic (3, 28, 29) tone are increased, whereas leptin signaling is decreased (2); thus, we compared the effects of each peptide in both fed and fasted adult female mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult transgenic female mice in which green fluorescent protein is genetically targeted to GnRH neurons (30) were used. Animals were housed in groups of three to five and were maintained on standard rodent chow (Harlan 7012, Harlan, Bartonsville, IL) and water ad libitum, except for some animals that were fasted for 48 h during diestrus, during which only water ad libitum was given. All animals were held on a 14-h light, 10-h dark light cycle, with lights on at 0500 h eastern standard time. All mice used were between 2 and 4 months of age. Estrous cycle stage was determined by vaginal cytology; all animals were in diestrus on the day of the experiment. When enough serum was available, LH levels were determined on the day of the experiment to verify reproductive inhibition in fasted mice (31). Furthermore, as shown previously (2, 13) fasted mice remained in diestrus at the end of the 48-h fast, e.g. on the day of the experiment, indicating extension of the estrous cycle. Glucose levels were measured on the day of the experiment (One Touch Fast Take glucose monitor, Lifescan, Inc., Milpitas, CA) to verify nutritional state. All procedures were approved by the animal care and use committee of University of Virginia and were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.

Slice preparation and recordings
All reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO). On the day of the experiments, animals were killed by decapitation between 0900–1000 h (all time in this report is given as eastern standard time), and experiments were performed between 1100–1500 h, after slice preparation and equilibration. Coronal sections (200 µm) through the preoptic area and hypothalamus were prepared as previously described (32, 33). Briefly, the brain was rapidly removed and submerged in ice-cold, oxygenated (95% O2-5% CO2) sucrose saline consisting of 250 mM sucrose, 3.5 mM KCl, 26 mM NaHCO3, 10 mM glucose, 1.3 mM Na2HPO4, 1.2 mM MgSO4, and 2.5 mM MgCl2. Coronal slices (200 µm) through the preoptic area and hypothalamus were cut with a Vibratome (Ted Pella, Inc., Redding, CA) and transferred to an oxygenated, 1:1 mixture of sucrose saline and recording solution, which contained 130 mM NaCl, 3.5 mM KCl, 26 mM NaHCO3, 10 mM glucose, 1.3 mM Na2HPO4, 1.2 mM MgSO4, and 2.5 mM CaCl2 for 20 min at room temperature. Slices were then incubated in oxygenated recording solution at 30 C for at least 90 min before recording.

For recording, individual slices were transferred to a recording chamber mounted on the stage of an Olympus BX50WI upright fluorescent microscope (Opelco, Dulles, VA). Slices in the recording chamber were continuously superfused with oxygenated recording saline kept at 30–32 C with an inline heating unit (Warner Instruments, Hamden, CT), with the addition of D(-)2-amino-5-phophonovaleric acid (20 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) to block glutamatergic currents and in some recordings tetrodotoxin (0.5 µM) to block action potentials. In vitro treatments were applied directly to the recording solution and included leptin (50 nM), NPY (100 nM), the NPY1 receptor antagonist BIBP3226 (100 nM), the nonspecific opiate receptor agonist met-enkephalin (1 µM), and the nonspecific opiate receptor antagonist naloxone (10 µM). Doses were chosen to achieve close to maximal responses to in vitro treatment in brain slice preparations, based on either the 50% effective concentration (16, leptin) or on Kd (or Ki) plus previous demonstration of the effectiveness of the dose in similar systems (34, 35, 36, 37). In individual brain slices, the response of only one cell to a single treatment was measured to avoid possible confounds of interaction among in vitro treatments or inadequate washout of treatment effects. From each animal, the response of no more than three (and usually no more than two) cells, each from a different slice, was tested for a particular in vitro treatment. Due to the high degree of variability in baseline GABAergic input among individual cells, the response of each cell compared with its own baseline was used as an individual data point. Experiments were performed using Pulse Control software (Instrutech, Port Washington, NY), and currents were recorded with an EPC-8 amplifier (HEKA, Nova Scotia, Canada), digitized by an ITC-18 acquisition interface (Instrutech), and stored using IGOR PRO software (Wavemetrics, Lake Oswego, OR) on a G4 Macintosh computer (Apple Computer, Cupertino, CA).

Recording postsynaptic currents (PSCs)
Electrodes (2–4 M{Omega}) were filled with the high chloride pipette solution with the addition of 4 mM MgATP and 0.4 mM NaGTP before adjusting to pH 7.2 with NaOH. In a subset of recordings, the Janus kinase-2/3 inhibitor AG490 (0.2 µM) or dimethylsulfoxide (DMSO) vehicle (0.02%) was added to the internal solution. Green fluorescent protein-GnRH neurons were identified, and the whole cell recording configuration was achieved. Membrane potential was clamped at -60 mV, and signals were filtered at 5–7 kHz with gain set at 10 mV/pA for 120-sec recording periods. The liquid junction potential of 3 mV (38) was not corrected. PSCs were stored as Event Tracker files using Pulse Control and IGOR PRO software. Input resistance (Rin), series resistance (Rs), and membrane capacitance (Cm) were continually monitored as previously described (39). Only recordings with Rin greater than 500 M{Omega} and Rs less than 20 M{Omega} were included for analysis. Mean Rin, Rs, and Cm were not different (P > 0.05) among or within cells where comparisons were made. In a subset of recordings in each treatment group, e.g. in the presence of in vitro leptin, NPY, or met-enkephalin, elimination of all PSC activity after bath application of the GABAAR antagonist bicuculline (20 µM) confirmed that PSC events were GABAAR mediated. Individual cells were further examined to determine that changes in the PSC properties examined were not due to alterations in passive properties or Rs within the acceptable ranges defined above.

PSC analysis
Stored 120-sec traces of current activity were analyzed off-line using custom event detection software to identify PSCs (events). The threshold for event detection was set manually for each 120-sec record and was typically 5 pA or greater. Events were confirmed by eye, and detection errors were corrected manually. The mean event frequency from two to four 120-sec records was calculated for each cell during baseline and in vitro treatment periods to obtain the mean PSC frequency before and after treatment. Within each in vitro treatment group, the mean PSC frequencies from individual cells before and during treatments were compared by paired Student-Newman-Keuls test when appropriate. The rate of rise (a measure of receptor on-rate), peak amplitude (a measure of conductance), and 10–90% decay time (a measure of off-rate/affinity) for every event detected were calculated by the program and exported for further analysis in a spreadsheet (Microsoft Excel, Microsoft, Redmond, WA). For each cell, probability distributions for each parameter were generated during baseline and treatment periods using all events recorded during the respective periods, as previously described (11, 40, 41). Probability distributions among within-cell treatments were compared with the Komolgorov-Smirov Goodness of Fit (KS) test (SPLUS Professional 2 data analysis software, MathSoft, Inc., Cambridge, MA). Within individual cells, the mean percent change from baseline after in vitro treatment was then calculated for each parameter. Group means for each miniature PSC (mPSC) parameter were compared among treatment groups using one-way ANOVA, followed by post hoc analysis with Fisher’s protected least significant difference test and Student-Newman-Keuls test for pairwise comparisons when appropriate. For each 120-sec recording period, an average mPSC waveform was generated from all mPSC events detected. For each cell, averaged mPSC waveforms were aligned on the rising phase for comparison between baseline and treatment conditions. These averaged mPSCs were used to illustrate changes in mPSC amplitude before and after treatment(s). The averaged mPSC waveforms were then normalized by amplitude to illustrate differences in mPSC decay time between treatments. All values are reported as the mean ± SEM, and significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overall decrease in GABAergic PSC frequency in fasted compared with fed females
Because levels of and sensitivity to metabolic peptides are altered during fasting, we compared responses of GnRH neurons to in vitro leptin, NPY, and opiate treatments between fed and fasted mice. To confirm that a 48-h fast altered the reproductive and nutritional status of mice, we assessed estrous cyclicity and measured LH (when enough serum was available) and glucose levels before each experiment. As shown previously (2, 13), 48 h of fasting beginning during diestrus extended reproductive cyclicity such that all mice remained in diestrus at the end of the fast (the day of the experiment). LH levels were decreased in fasted compared with fed females (fed, 0.23 ± 0.09 ng/ml; n = 13); in all but one fasted female (LH, 0.102 ng/ml) LH levels were below the detection sensitivity of the assay (<0.04 ng/ml; n = 7). Interestingly, this animal had the highest level of GABAergic PSC frequency among fasted animals (1.23 Hz). Blood glucose levels in fed animals (7.2 ± 0.6 mmol/liter; n = 23) were significantly greater than those in fasted animals (3.8 ± 0.1 mmol/liter; n = 17; P < 0.001).

We recently reported that this fasting regimen caused a significant decrease in GABAergic PSC frequency in GnRH neurons compared with the frequency in fed mice (11). Here we confirm these data, showing that PSC frequency is decreased in fasted compared with fed diestrous females [fed, 0.91 ± 0.12 Hz (n = 44 cells); fasted, 0.38 ± 0.06 (n = 28 cells); P < 0.001], supporting our previous finding that fasting-induced inhibition of fertility may be communicated to GnRH neurons at least in part via decreased excitatory GABAergic drive.

Experiment 1: acute effects of leptin on GABAergic input to GnRH neurons
To determine whether leptin can rapidly alter GABAergic drive to GnRH neurons, we recorded PSCs from these cells in brain slices before and after in vitro treatment with leptin. In both fed and fasted animals, bath application of leptin (50 nM) to the slice rapidly (2–5 min) increased PSC frequency (n = 5 cells for fed; n = 7 cells for fasted; P < 0.01 compared with pretreatment frequency; Fig. 1Go). PSC frequency was restored to pretreatment levels by washout (Fig. 1AGo; n = 4; P > 0.6 vs. control frequency) or by subsequent treatment with tetrodotoxin (TTX; 0.5 µM), which minimizes action potential-dependent presynaptic activity (Fig. 1BGo; n = 6; P > 0.7 vs. control frequency). Reversal by TTX suggests that leptin rapidly increased the activity of GABAergic neurons that are presynaptic to the GnRH neuron. Figure 2CGo illustrates PSC frequency from individual GnRH neurons from fed and fasted mice before and after in vitro leptin treatment to show variation in baseline frequency among cells as well as individual responses to leptin. The mean percent change from baseline PSC frequency (Fig. 1DGo) illustrates that the sensitivity to bath-applied leptin was similar in fed and fasted mice. In both fed and fasted mice and in the presence of in vitro leptin, all PSC activity was eliminated by bicuculline (20 µM), confirming that events were GABAAR mediated (not shown).



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  		FIG. 1. Leptin acutely increases GABAergic PSC frequency in GnRH neurons. A, Recordings of GABAergic PSCs from a representative GnRH neuron from a fasted female under control conditions (top), during leptin treatment (50 nM; middle), and after washing out leptin (bottom). B, Recordings of PSCs from a representative GnRH neuron from a fasted female under control conditions (top), during leptin treatment (center), and during subsequent treatment with leptin and TTX (0.5 µM; bottom). C, PSC frequency in individual GnRH neurons before (control) and after (leptin) in vitro leptin treatment, illustrating cell to cell variation in initial frequency and direction of response. Values from fed animals are on the left ({bullet}), and values from fasted animals are on the right ({circ}). D, Percent change in PSC frequency from baseline after in vitro leptin treatment in GnRH neurons from fed (left, {blacksquare}) and fasted (right, {square}) mice. *, P < 0.05 vs. control in respective metabolic state.

 


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  		FIG. 2. Evidence for direct leptin action on GnRH neurons to modify the postsynaptic response to activation of GABAARs. A, Average mPSC traces recorded in the presence of TTX from a representative GnRH neuron show mPSC amplitude under control conditions (con) and during in vitro leptin treatment (leptin) when DMSO vehicle (vehicle) is present in the pipette solution or when the Janus-2/3 inhibitor AG490 is present in the pipette (AG490). B, Normalized average mPSC traces from a representative GnRH neurons show mPSC decay time; see A for details. C, Percent change from baseline in mPSC rate of rise, amplitude, and decay time after in vitro treatment with leptin with vehicle or AG490 inside the pipette. *, P < 0.05 vs. vehicle.

 
Experiment 2: postsynaptic effects of leptin treatment
We have previously shown that in the presence of TTX to minimize activity-dependent effects, in vitro leptin treatment also increased the magnitude of the postsynaptic response (i.e. rate of rise, amplitude, and duration) of GABAergic mPSCs by GnRH neurons (11). In that study, bath application of AG490, an inhibitor of Janus kinase-2/3, the kinase through which the leptin receptor signals (42, 43), reversed these leptin effects. These results suggested that leptin may also act directly on the postsynaptic GnRH neuron to increase the response of the cell to GABAAR activation. Although leptin receptors have not been detected in GnRH neurons using molecular approaches (44, 45), these results provided the first functional assay demonstrating the possibility of their existence in these cells. In those experiments, however, both leptin and AG490 were applied directly to the entire slice via the recording solution; thus, activity-independent presynaptic actions, such as changes in size or spontaneous release of GABA-containing vesicles, could not be discounted.

To more rigorously test the hypothesis that leptin acts directly on GnRH neurons via leptin receptors expressed in these cells, we examined the response to in vitro leptin when AG490 (0.2 µM in 0.02% DMSO vehicle) was included in the internal recording solution, so that its effects would be confined to the postsynaptic GnRH neuron. As in the previous study (11), these experiments used fed females and were performed entirely in the presence of TTX to minimize presynaptic effects. In contrast to the increase in mPSC size in response to in vitro leptin under normal recording conditions (11) or when DMSO (vehicle) alone was included in the pipette solution (n = 3; P < 0.05 for all mPSC properties, leptin vs. control), leptin had no effect on mPSC rate of rise, amplitude, or decay time when AG490 was present in the internal recording solution (n = 6; P > 0.2 for all mPSC properties, leptin vs. control; Fig. 2Go). This lack of response when the effects of AG490 were essentially limited to the postsynaptic GnRH neuron provide stronger evidence that leptin acted directly on GnRH neurons to alter their response to GABAAR activation.

Experiment 3: acute effects of NPY on GABAergic input to GnRH neurons
We next asked whether NPY, which has been demonstrated to act centrally in an antagonist manner to leptin (34, 46, 47), also rapidly alters presynaptic GABAergic drive to GnRH neurons and/or the size of spontaneous postsynaptic GABAAR-mediated events. Because NPY is an inhibitory metabolic signal to the reproductive axis in some animals models (21, 22, 23), we hypothesized that NPY would decrease excitatory GABAergic input to GnRH neurons. We recorded GABAergic PSCs from GnRH neurons before and after in vitro treatment with NPY (100 nM). In GnRH neurons from fed females, NPY rapidly decreased PSC frequency (n = 6; P < 0.01; Fig. 3Go, A–C). Frequency was restored to control levels by subsequent treatment with the NPY-1 receptor antagonist BIBP3226 (100 nM; n = 3; P > 0.1 vs. control; Fig. 3AGo). Furthermore, in the presence of TTX to minimize activity-dependent presynaptic input, NPY had no effect on frequency (TTX control, 0.38 ± 0.18 Hz; TTX plus NPY, 0.41 ± 0.24 Hz; P = 0.36; n = 4; not shown), indicating the presynaptic action of this peptide. TTX eliminates action potential-dependent transmitter release; thus, in its presence only spontaneous release of vesicular GABA occurs (mPSCs). The lack of action potential-dependent release probably explains the reduced baseline PSC frequency seen in experiments in which effects were tested in the presence of TTX.



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  		FIG. 3. NPY acutely reduces PSC frequency only in GnRH neurons from fed animals. A, Recordings of PSCs from a representative GnRH neuron from a fed female (top) and a fasted female (bottom) under control conditions (left), during NPY treatment (middle), and after washout (fasted) or subsequent treatment with the NPY-1 receptor antagonist BIBP3226 (fed; right). B, PSC frequency in individual GnRH neurons before (control) and after (NPY) in vitro NPY treatment, illustrating cell to cell variation in initial frequency and direction of response. Values from fed animals are on the left ({bullet}), and values from fasted animals are on the right ({circ}). C, Percent change in PSC frequency from baseline after in vitro NPY treatment in GnRH neurons from fed (left, {blacksquare}) and fasted (right, {square}) mice. D, Percent change in PSC frequency from baseline after in vitro treatment with the NPY-1 receptor antagonist BIBP3226 alone in GnRH neurons from fed (left, {blacksquare}) and fasted (right, {square}) mice. E, Percent change from baseline in mPSC rate of rise, amplitude, and decay time after in vitro NPY treatment of GnRH neurons from fed mice. Experiments shown in E were performed entirely in the presence of TTX to minimize presynaptic input and thus isolate activity-independent postsynaptic effects. *, P < 0.05 vs. control in respective metabolic state.

 
Interestingly, NPY had no effect on PSC frequency in GnRH neurons from fasted mice (Fig. 3Go, B and C; n = 6; P > 0.3), suggesting that maximal reduction of GABAergic drive was achieved by the increased endogenous NPY tone during fasting or that changes in other fasting-induced neural pathways diminished responsiveness to NPY. In GnRH neurons from both fed and fasted mice, the NPY-1 receptor antagonist BIBP3226 alone caused a rapid increase in PSC frequency (Fig. 3DGo; n = 7 for fed; n = 5, for fasted; P < 0.05), suggesting that NPY may exert a tonic inhibitory influence, at least in part via the NPY-1 receptor subtype, on presynaptic GABAergic drive to GnRH neurons during both fed and fasted states.

Because GnRH neurons receive innervation from NPY-containing afferents (48), we also looked for evidence suggesting that direct effects of NPY on the postsynaptic GnRH neuron alter the response to GABA. In the presence of TTX to minimize presynaptic effects, there was no effect of NPY on GABAergic mPSC properties, i.e. rate of rise, amplitude, or duration (Fig 3EGo). This indicates that NPY does not alter the response of the postsynaptic GnRH neuron to GABAA receptor activation and/or presynaptic vesicular release of GABA (Fig. 3EGo; n = 4 all from fed mice; P > 0.3 for all properties, control vs. NPY) under the conditions examined.

Experiment 4: acute effects of opiates on GABAergic input to GnRH neurons
Similarly to NPY, endogenous opiates are potent inhibitors of fertility (49, 50, 51, 52, 53); thus, we next hypothesized that in vitro opiate treatment would rapidly decrease GABAergic drive to GnRH neurons and, further, that sensitivity to opiate treatment would be decreased during fasting, when endogenous opiatergic tone is elevated (29, 54).

We recorded GABAergic PSCs from GnRH neurons before and after in vitro treatment with the nonspecific opiate receptor agonist met-enkephalin (2 µM). In GnRH neurons from fed females, met-enkephalin rapidly decreased PSC frequency (Fig. 4Go, A–C; n = 6; P < 0.02). Frequency was restored toward pretreatment levels after subsequent treatment with the nonspecific opiate receptor antagonist naloxone (10 µM; n = 5; P < 0.05 vs. met-enkephalin; Fig. 4AGo). Met-enkephalin did not alter PSC frequency in the presence of TTX in fed mice (TTX control, 0.53 ± 0.26 Hz; TTX plus met-enkephalin, 0.50 ± 0.25 Hz; P = 0.2; n = 5), indicating that this peptide acted presynaptically to reduce the activity of GABAergic neurons afferent to GnRH neurons. Interestingly, in GnRH neurons from fasted females, met-enkephalin had no effect on PSC frequency (Fig. 4Go, B and C; n = 4; P > 0.3), again suggesting that maximal reduction of GABAergic drive was achieved by the increased endogenous opiate tone during fasting (29, 54). Consistent with this observation, naloxone alone induced a rapid increase in PSC frequency in GnRH neurons from fasted animals (n = 8; P < 0.01), but had no effect in GnRH neurons from fed mice (n = 6; P > 0.1; Fig. 4DGo), providing further evidence for tonic inhibitory opiatergic tone on afferent GABAergic input to GnRH neurons during fasting.



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  		FIG. 4. Met-enkephalin acutely reduces PSC frequency only in GnRH neurons from fed animals. A, Recordings of PSCs from a representative GnRH neuron from a fed female (top) and a fasted female (bottom) under control conditions (left), during met-enkephalin treatment (middle), and after washout (fasted) or subsequent treatment with the opiate receptor antagonist naloxone (fed; right). B, PSC frequency in individual GnRH neurons before (control) and after (met-enk) in vitro met-enkephalin treatment, illustrating cell to cell variation in initial frequency and direction of response. Values from fed animals are on the left ({bullet}), and values from fasted animals are on the right ({circ}). C, Percent change in PSC frequency from baseline after in vitro met-enkephalin treatment in GnRH neurons from fed (left, {blacksquare}) and fasted (right, {square}) mice. D, Percent change in PSC frequency from baseline after in vitro treatment with the opiate receptor antagonist naloxone alone in GnRH neurons from fed (left, {blacksquare}) and fasted (right, {square}) mice. E, Percent change from baseline in mPSC rate of rise, amplitude, and decay time after in vitro met-enkephalin treatment to GnRH neurons from fed mice. Experiments shown in E were performed entirely in the presence of TTX to minimize presynaptic input and thus isolate activity-independent postsynaptic effects. *, P < 0.05 vs. control in the respective metabolic state.

 
In the presence of TTX, there was no effect of met-enkephalin on postsynaptic mPSC properties, i.e. rate of rise, amplitude, or duration, indicating that this opiate did not alter the response of the postsynaptic GnRH neuron to GABAAR activation and/or presynaptic vesicular release of GABA (Fig. 4EGo; n = 5 for fed; P > 0.6 for all properties, baseline vs. met-enkephalin).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fertility is clearly linked to energy intake and utilization, as evidenced by numerous animal models and clinical syndromes in which energy imbalance is associated with infertility (6, 7). Important permissive and inhibitory metabolic signals, including leptin, NPY, and endogenous opiates, help mediate this link. The present data demonstrate that inputs from these cues are communicated to GnRH neurons at least in part via the GABAergic system. As the majority of both anatomical and functional synaptic inputs to GnRH neurons demonstrated to date are GABAergic (8, 10), this system may serve as a primary integrating center for many different inputs to GnRH neurons. In that regard, signals from steroid hormones (55) and fasting (11) have been shown to alter afferent GABAergic drive to GnRH neurons, which in the whole animal may contribute to changes in GnRH neuron firing activity and hormone output. The goal of the present study was to determine whether specific metabolic signals important in fertility regulation rapidly signal via the GABAergic system to GnRH neurons.

Leptin, a permissive signal to fertility, rapidly increased PSC frequency, indicating that this hormone increased the activity of GABAergic neurons presynaptic to GnRH neurons. Leptin also increased the postsynaptic GnRH neuron response, as measured by the size of mPSCs recorded under minimal influence from presynaptic inputs (e.g. in the presence of TTX). Leptin receptors have been identified on hypothalamic GABAergic neurons (15), providing a mechanism for presynaptic leptin actions; however, molecular approaches have not yet identified these receptors on GnRH neurons (44, 45). The possibility that leptin can act directly on GnRH neurons to increase the postsynaptic response to GABA was suggested by a previous study (11). In that study, however, presynaptic effects could not be entirely ruled out. In the present study, signaling through the leptin receptor was blocked intracellularly to isolate any effects of leptin on the postsynaptic GnRH neuron. Under these conditions, leptin had no effect on mPSC size, providing strong evidence that leptin acted directly at the postsynaptic GnRH neuron to increase responsiveness to GABA. Together, these data show leptin acts acutely via both pre- and postsynaptic mechanisms to enhance afferent GABAergic drive to GnRH neurons and the response of these cells to GABAAR activation, respectively.

For the GnRH neuron, these pre- and postsynaptic leptin effects should increase the probability that GABAAR activation will drive the membrane potential to the threshold for action potential firing, possibly leading to increased GnRH neuron firing activity. In that regard, leptin treatment in vivo has been shown to increase GABAergic drive to GnRH neurons from fasted animals (11) as well as to stimulate GnRH secretion (56, 57, 58, 59). These observations are in accordance with the present findings, in which in vitro leptin acutely increased excitatory GABAergic input to GnRH neurons, an effect that should result in increased GnRH output in the whole animal. These results together with the previous work (11, 56, 57, 58, 59) suggest that permissive actions of leptin on fertility are mediated at least in part via the GABAergic system. Furthermore, these data suggest that in fed animals, central leptin tone is submaximal, such that exogenous application of leptin is able to enhance PSC frequency above normal fed levels.

NPY, which can act in an antagonist manner to leptin in the regulation of both fertility and satiety (21, 22, 23, 60), had opposite effects to leptin on GABAergic drive to GnRH neurons. In GnRH neurons from fed animals, in vitro NPY induced a rapid decrease in GABAergic PSC frequency. NPY-induced changes in PSC frequency were reversed by subsequent treatment with the NPY-1 receptor antagonist BIBP3226, indicating that this NPY effect was mediated at least in part via NPY-1 receptors. In that regard, the NPY-1 subtype has been specifically implicated in the metabolic regulation of fertility (61); thus, these results suggest that the GABAergic system is a possible mechanism for this.

Interestingly, NPY had no effect on PSC frequency in GnRH neurons from fasted females. During fasting, endogenous NPY tone is increased to stimulate feeding and suppress fertility (25, 26, 27); thus, it is possible that in fasted mice, endogenous NPY exerts maximal effects on the GABAergic system, such that we did not see an increased response to exogenously applied peptide. Another possibility is that fasting-induced increases in glucocorticoids, which may also contribute to fasting-induced reproductive inhibition (62), caused the decreased response to NPY in fasted mice. In accordance with the former hypothesis, the NPY-1 receptor antagonist BIBP3226 alone caused a rapid increase in PSC frequency in GnRH neurons from fasted mice, indicating that increased NPY tone during fasting (25, 26, 27) may contribute to fasting-induced infertility by decreasing excitatory GABAergic drive onto GnRH neurons. BIBP3226 also increased PSC frequency in GnRH neurons from fed animals, suggesting that even in a fed state, the output of these cells may be kept in check by inhibitory NPY signals. NPY had no effect on mPSC size in fed or fasted animals, indicating no postsynaptic effects with regard to GABAAR activation.

Together these data suggest that one mechanism by which NPY may inhibit fertility is by decreasing the activity of GABAergic neurons afferent to GnRH neurons, an effect probably mediated via the NPY-1 receptor subtype. This NPY effect together with our demonstration that leptin rapidly increased GABAergic input to these cells suggest a neural mechanism by which these metabolic signals may act antagonistically with one another in the regulation of fertility and possibly satiety. In some animal models, NPY has a stimulatory effect on the reproductive axis (63, 64); thus, the precise role of this peptide in fertility regulation in the whole animal remains unclear and appears to be state dependent.

In addition to leptin and NPY, opiatergic peptides are important signals in the metabolic regulation of fertility (49, 50, 53). Here we demonstrate that the nonselective opiate met-enkephalin rapidly decreased PSC frequency in GnRH neurons from fed, but not fasted, mice. This opiate effect was reversed by washout or subsequent treatment with the nonselective opiate receptor antagonist naloxone, confirming the specificity of the response to the opiatergic system. The lack of effect of met-enkephalin on PSC frequency in fasted mice suggests that, similarly to NPY, endogenous opiatergic tone is increased during fasting, such that there is no further response to exogenous treatment. It is also possible that stress-induced increases in glucocorticoid signaling during fasting (62) acted independently to decrease GnRH neuron responsiveness to opiates. Despite this possibility, the present results together with whole animal studies (3, 54) suggest that opiates at least in part mediate reproductive suppression during fasting. Furthermore, these data suggest a possible mechanism (decreased excitatory GABAergic drive onto GnRH neurons) by which opiates may contribute to fasting-induced infertility. In accordance with this hypothesis, the opiate receptor antagonist naloxone increased PSC frequency in fasted, but not fed, animals, providing further functional evidence that increased endogenous opiatergic tone contributes to reproductive inhibition induced by negative energy balance. Similarly to NPY, met-enkephalin had no effect on mPSC size in fed or fasted animals.

Although both met-enkephalin and naloxone bind opiate receptors nonselectively at the doses used in this study, they exhibit highest affinity for the µ-opiate receptor subtype (35). The µ-opiate receptor has been implicated in the regulation of both fertility (65) and food intake (24); thus, it is possible that the inhibitory effects of met-enkephalin on GABAergic drive to GnRH neurons in fed mice were mediated primarily via the µ receptor subtype. Studies using specific agonists and antagonists are needed to determine the precise receptor mechanism(s) for this communication.

Together these data indicate that metabolic cues from leptin, NPY, and endogenous opiates, potent regulators of both food intake and fertility, are communicated to GnRH neurons via GABAergic afferents. A majority of the functional synaptic input demonstrated to GnRH neurons to date is GABAergic (8, 10); thus, these signals are poised to exert significant effects on GnRH neuron output and its regulation by energy availability. In this regard, it would be predicted that when energy requirements are satisfied, a balance of inhibitory (e.g. NPY and opiatergic) and permissive (e.g. leptin) metabolic signals maintains normal GnRH neuron pulsatility, and that when energy availability is either in excess or insufficient, an acute imbalance of metabolic signals would perturb GnRH neuron output until normal energy homeostasis was restored. Furthermore, it is interesting that metabolic signals with both inhibitory and stimulatory effects on fertility are communicated to GnRH neurons via a common, afferent neurotransmitter system that elicits an excitatory response at the postsynaptic cell. The lack of direct inhibitory action at the GnRH neuron under the conditions tested by opiates or NPY in conjunction with the ability of these peptides to rapidly decrease presynaptic drive via GABAARs suggest that metabolic inhibition of GnRH neuron output is primarily mediated transsynaptically via reductions in excitatory drive.


   Acknowledgments
 
We thank Dr. Xu-Zhi Xu for expert technical assistance.


   Footnotes
 
This work was supported by the NICHHD, NIH, through Cooperative Agreement U54-HD-28934, the Ligand Assay and Analysis Core, and Individual Predoctoral National Research Service Award NS-43871 (to S.D.S.).

Abbreviations: Cm, Membrane capacitance; DMSO, dimethylsulfoxide; GABA, {gamma}-aminobutyric acid; GABAAR, {gamma}-aminobutyric acidA receptor; mPSC, miniature PSC; NPY, neuropeptide Y; PSC, postsynaptic current; Rin, input resistance; Rs, series resistance; TTX, tetrodotoxin.

Received October 14, 2003.

Accepted for publication November 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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