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. 2008 Aug 21;150(1):333–340. doi: 10.1210/en.2008-0962

Suppression of Basal Spontaneous Gonadotropin-Releasing Hormone Neuronal Activity during Lactation: Role of Inhibitory Effects of Neuropeptide Y

Jing Xu 1, Melissa A Kirigiti 1, Michael A Cowley 1, Kevin L Grove 1, M Susan Smith 1
PMCID: PMC2630892  PMID: 18719019

Abstract

Increased neuropeptide Y (NPY) activity drives the chronic hyperphagia of lactation and may contribute to the suppression of GnRH activity. The majority of GnRH neurons are contacted by NPY fibers, and GnRH cells express NPY Y5 receptor (Y5R). Therefore, NPY provides a neurocircuitry for information about food intake/energy balance to be directly transmitted to GnRH neurons. To investigate the effects of lactation on GnRH neuronal activity, hypothalamic slices were prepared from green fluorescent protein-GnRH transgenic rats. Extracellular loose-patch recordings determined basal GnRH neuronal activity from slices of ovariectomized control and lactating rats. Compared with controls, hypothalamic slices from lactating rats had double the number of quiescent GnRH neurons (14.51 ± 2.86 vs. 7.04 ± 2.84%) and significantly lower firing rates of active GnRH neurons (0.25 ± 0.02 vs. 0.37 ± 0.03 Hz). To study the NPY-postsynaptic Y5R system, whole-cell current-clamp recordings were performed in hypothalamic slices from control rats to examine NPY/Y5R antagonist effects on GnRH neuronal resting membrane potential. Under tetrodotoxin treatment, NPY hyperpolarized GnRH neurons from −56.7 ± 1.94 to −62.1 ± 1.83 mV; NPY’s effects were blocked by Y5R antagonist. To determine whether increased endogenous NPY tone contributes to GnRH neuronal suppression during lactation, hypothalamic slices were treated with Y5R antagonist. A significantly greater percentage of GnRH cells were activated in slices from lactating rats (52%) compared with controls (28%). These results suggest that: 1) basal GnRH neuronal activity is suppressed during lactation; 2) NPY can hyperpolarize GnRH neurons via postsynaptic Y5R; and 3) increased inhibitory NPY tone during lactation is a component of the mechanisms responsible for suppression of GnRH neuronal activity.

Neuropeptide Y (NPY) directly hyperpolarizes GnRH neurons via postsynaptic NPY Y5 receptor. Increased inhibitory NPY tone during lactation is an important component of the suppression of GnRH neuronal activity.

Lactation is a critical physiological state in which various adaptations occur, including an increase in food/water intake (1,2), energy sparing in response to negative energy balance (3,4), and cessation of reproductive cyclicity (1,5). Although it is well established that negative energy balance is associated with suppression of reproductive function and ovarian cyclicity (6,7), the signals that convey information about energy balance to hypothalamic GnRH neurons to control LH secretion remain elusive.

Many orexigenic neuropeptide systems, whose activities increase during negative energy balance, also participate in the regulation of reproductive function. These include neuropeptide Y (NPY) (8), agouti-related protein (AGRP) (9), melanin concentrating hormone (10), and orexin (11). These systems make direct connections with GnRH neurons, and respective receptors are expressed in GnRH cell bodies (9,12,13,14). Therefore, they provide a neuroanatomical framework by which information about food intake/energy balance can be directly transmitted to GnRH neurons.

One orexigenic neuropeptide that likely plays a role in integrating energy balance and reproduction is NPY. NPY neurons in the arcuate nucleus (ARH) and dorsomedial hypothalamic nucleus (DMH) are activated and provide the drive for sustained hyperphagia during lactation (15). Besides its role in regulation of food intake, NPY also modulates reproductive function (16,17). Although both stimulatory (17,18) and inhibitory (19,20) effects of NPY on GnRH/LH have been reported, it is likely that inhibitory effects predominate during lactation and other states of negative energy balance because of the chronic elevation in NPY and low levels of estradiol (2,8). The majority of GnRH neurons are directly contacted by NPY fibers, including those originating from ARH NPY/AGRP neurons (13,21). In addition, the Y1 receptor subtype (Y1R) has been shown to be expressed on presynaptic terminals making contact with GnRH neurons (13), whereas the NPY Y5 receptor subtype (Y5R) is expressed directly on GnRH cell bodies (21). Therefore, the NPY system is a likely candidate for integrating the regulation of food intake/energy balance and GnRH neuronal activity (5).

Previous studies have provided evidence for an inhibitory role of NPY on GnRH neurons during states of negative energy balance such as fasting; the inhibitory effects of NPY were mediated by presynaptic Y1R. Based on electrophysiological recordings of green fluorescent protein (GFP)-GnRH neurons from hypothalamic slices obtained from control and fasted mice, the group of Moenter and colleagues (22,23) suggested that increased NPY during fasting may contribute to a decreased excitatory secretion of γ-aminobutyric acid (GABA) drive onto GnRH neurons.

The present study focuses on the Y5R subtype because it has been shown to be involved in mediating NPY’s stimulatory effects on food intake and inhibitory effects on GnRH/LH (24,25). Furthermore, the expression of Y5R in GnRH cell bodies provides a mechanism by which NPY could have direct postsynaptic effects to alter GnRH neuronal activity (21). Postsynaptic NPY receptor subtypes, through activation of G protein-coupled inwardly rectifying K+ channels (GIRK) and voltage-dependent inhibition of Ca2+ channels (26), would be predicted to have direct inhibitory effects.

Electrophysiology recordings were performed on hypothalamic slices obtained from GnRH-GFP transgenic rats to examine the role of NPY on GnRH neuronal activity, especially during lactation. The hypotheses were: 1) basal GnRH activity is suppressed during lactation, 2) NPY has direct inhibitory effects on GnRH neurons through postsynaptic Y5R, and 3) there is increased inhibitory NPY tone during lactation.

Materials and Methods

Experimental animals

GnRH neurons were recorded from transgenic female Wistar rats (kindly provided by Dr. M. Kato, Nippon Medical School, Tokyo, Japan) (27,28), in which GFP is genetically targeted to GnRH neurons. All animals were maintained under a 12-h light, 12-h dark (lights on at 0700 h) cycle and constant temperature (23 ± 2 C). Rodent chow and water were provided ad libitum. Pregnant rats were housed individually and checked for birth of pups every morning. The day of birth was described as d 0. The lactating rats were ovariectomized and litters were adjusted to eight pups on d 2 postpartum. Control rats were virgin and ovariectomized during random days of the estrous cycle and used 7–10 d later. Ovariectomized animals were used in these studies to eliminate any possible effects of varying levels of estradiol on responses to NPY (17,18). All protocols were approved by the Oregon National Primate Research Center Animal Care and Use Committee and conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

GnRH-GFP transgene expression in rats

Double-label immunofluorescence coupled with confocal microscopic analysis was performed to optimize detection of GFP expression in rats to confirm that the GFP transgene expression was confined to a high percentage of GnRH cells. Control GnRH-GFP heterozygous rats under pentobarbital anesthesia were cardiac perfused with 4% paraformaldehyde/PBS buffer [0.1 m (pH 7.4)]. The brain was removed, cryoprotected in 25% sucrose/PBS at 4 C overnight, and stored at −80 C until being sectioned coronally on a microtome (25 μm). Free-floating sections were incubated with a blocking solution containing 2% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and 0.4% Triton X-100/PBS for 30 min at room temperature. Sections were then incubated with anti-GnRH monoclonal antibody (Hu4H, a kind gift from Dr. Henryk Urbanski, Oregon National Primate Research Center) at a concentration of 1:5,000, and rabbit-antihuman recombinant GFP antibody (Stratagene, La Jolla, CA) at a concentration of 1:20,000 in the blocking solution for 48 h at 4 C. After washes in PBS, sections were incubated for 1 h at room temperature with secondary antibodies: 1:200 donkey-antimouse Alexa 568 (Invitrogen Corp., Carlsbad, CA) and donkey-antirabbit fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories) in the blocking buffer. Sections were then washed, mounted onto gelatin-coated slides, and coverslipped with buffered glycerol mountant. Confocal microscopic analysis was performed as previously described (21,29).

Brain slice preparation for electrophysiology recording

Brain slices for electrophysiological recording were prepared as previously described with a slight modification (30). All solutions were continuously saturated and bubbled with a 95% O2-5% CO2 mixture throughout the experiments and at least 15 min before exposure to the tissue. Rats were anesthetized with 5% ketamine/5% xylazine/5% acepromazine/saline solution and decapitated. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid [aCSF (pH 7.2–7.4); osmolarity 295–305 osmol/liter] containing (in millimoles) 126 NaCl, 2.5 KCl, 1.2 Na2HPO4, 24 NaHCO3, 2.4 CaCl2, 1.2 MgSO4 · 7H2O, and 11.1 glucose. Coronal hypothalamic slices were cut at 200 μm with a vibratome (Leica VT1000 S; Leica Microsystems Inc., Bannockburn, IL) in ice-cold aCSF. Slices were incubated in the aCSF at room temperature for 1 h and then transferred to a submerged chamber for recording. The slices were bathed in a continuous flow of oxygenated normal aCSF at 1–1.5 ml/min during recording. All recordings were made from hypothalamic GnRH neurons identified by bright green fluorescence, as described by others (22,23,31).

Basal spontaneous GnRH neuronal activity during lactation in rats

Extracellular loose-patch recording was used to determine the basal spontaneous GnRH neuronal activity in hypothalamic slices from ovariectomized control (n = 70 cells from seven rats) and lactating (n = 70 cells from seven rats) rats. The lactating rats were suckling eight-pup litters and were studied on d 9–11 postpartum. The recordings were performed under a Axioskop 2 FS plus (Carl Zeiss MicroImaging, Inc., Thornwood, NY) outfitted with epifluorescence (fluorescein isothiocyanate filter set) and infrared-differential interference contrast video microscopy. The area containing GnRH neurons was initially identified under a ×5 objective with UV illumination. Then the GFP-tagged GnRH neurons were visualized through a ×40 water immersion objective. The GFP-GnRH neuron position was marked on a monitor and compared carefully with its position under infrared-differential interference contrast imaging. Patch borosilicate pipettes (World Precision Instruments, Inc., Los Angeles, CA) ranging from 3 to 5 mΩ were filled with solution containing (in millimoles): 130 K gluconate, 5 EGTA, 10 HEPES, 3 KCl, 2 MgCl2, 4 Na2 ATP, 5 Na2 phosphocreatine, and 0.4 Na2 GTP [(pH 7.4) osmolarity, 280 ± 10 osmol/liter]. Pipettes were targeted to GnRH neurons using an MP-285 micromanipulator (Sutter Instruments, Novato, CA). Seal resistances ranged from 5 to 19 mΩ and either remained stable or increased during recording up to as high as 36 mΩ. The standard whole-cell patch recording procedures were performed as described preciously (32,33) using an Axopatch 700B amplifier (Axon Instruments Inc., Union City, CA). Minimal amounts of pipette drift were manually compensated to maintain contact with the cell. The recordings of membrane potential were done in I = 0 mode, filtering at 10 kHz. The recording was continued for up to 10 min and pClamp 9 software (Molecular Devices, Union City, CA) was used to detect the cell membrane currents associated with the action potential firing. In general, a similar number of cells were recorded from each rat for a total of 70 cells/group.

Effects of NPY through postsynaptic Y5R on GnRH neurons

Whole-cell recordings in current-clamp mode were performed on hypothalamic slices from control ovariectomized rats (n = 7 cells from three rats) to determine whether NPY can act through postsynaptic Y5R in GnRH neurons to inhibit neuronal activity. In general, a similar number of cells were recorded from each rat. The electrodes had resistances of 3–5 mΩ after being filled with the same internal solution as in the previous experiment. The patch pipettes were advanced with a MP-285 micromanipulator. The change in resting membrane potential (millivolts) for GnRH neurons was measured using a Axopatch 700B amplifier and digitized with an ITC-18 computer interface (InstruTEC Corp., Port Washington, NY). The GFP-GnRH neurons were approached with slight positive pressure and offset potentials were corrected. After forming a high-resistance seal (>10 GΩ) by applying negative pressure, a second pulse of negative pressure was used to rupture the membrane. Data were collected if series resistance was less than 35 mΩ. Bath application of 1 μm tetrodotoxin (TTX) was used to block voltage-gated sodium channels. After recording of basal membrane potential for 10 min, 300 nm NPY (Bachem Bioscience, Inc., King of Prussia, PA) was perfused into the bath solution. After 5 min recording, the slice was washed for 10 min. NPY or NPY/Y5R antagonist (300 nm, L-152,804; Tocris Bioscience, Ellisville, MO) (34) was added for 5 min of additional recording. Then data were recorded for another 10 min after the drug was discontinued. The pClamp 9 software was used for data acquisition and analysis.

Effects of endogenous inhibitory NPY tone on GnRH neuronal activity during lactation in rats

To determine whether a change of inhibitory NPY tone contributes to the suppression of GnRH neuronal activity during lactation, recordings from hypothalamic slices were made using the same whole-cell patch method, as described above, for control (n = 14 cells from four rats) and lactating rats (n = 23 cells from six rats). In general, a similar number of cells were recorded from each rat.

After recording of basal cell activity for 10 min, 300 nm Y5R antagonist was applied for 5 min recording. Then data were recorded for another 10 min after the drug was discontinued. Any cell that displayed a change in resting membrane potential of greater than 2.5 mV or a change in firing rate greater than 25% was considered to have responded (35,36).

Statistical analysis

Statistical evaluation of mean differences in membrane potential after NPY/Y5R treatment was performed by one-way ANOVA, with a significance level set at P = 0.05. To identify significant differences between groups, the Student-Newman-Keuls post hoc test was used for pairwise multiple comparisons. Data are presented as mean ± sem. A Student’s t test was used to compare firing rate and quiescent cell percentage of GnRH neurons between control and lactating animals.

Results

Specific GFP expression in GnRH neurons of transgenic rats

Double-label immunofluorescence in fixed brain sections from transgenic rats revealed a normal distribution of GnRH-containing cell bodies and fibers in the preoptic area of the hypothalamus (Fig. 1A). Single-labeled GnRH-ir neurons, shown as red, were visualized using confocal microscopy. With GFP coexpression, GnRH cells were double labeled with red and green, shown as yellow (Fig. 1, A and B). Of the several hundred GnRH neurons examined, greater than 80% could be identified by their expression of the GFP transgene, in agreement with a previous report (27). There were no GFP+/GnRH− neurons observed.

Figure 1.

Figure 1

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Double-label immunofluorescence of the GnRH-GFP transgenic rat hypothalamus. GnRH-immunoreactive staining is shown as red and GFP-immunoreactive staining is shown as green. GFP-GnRH double labeling is shown as yellow. Bar, 50 μm (A); 10 μm (B).

Decrease in basal spontaneous GnRH neuronal activity during lactation

To determine whether basal spontaneous GnRH neuronal activity was suppressed during lactation, we used the cell-attached patch configuration. We observed spontaneously active (cells with spontaneous action potentials) and quiescent GnRH neurons in slices obtained from lactating and control rats. In this configuration, the unclamped action potential arising from intact cells was recorded without alteration of intracellular constituents. Figure 2 shows examples of recordings from spontaneously active GnRH neurons in slices from control (Fig. 2Aa) and lactating rats (Fig. 2Ab). Analysis of the frequency distribution histogram, represented in Fig. 2, Ac and Ad, by a slice from a control and a lactating rat, respectively, indicated that the firing rate (Hertz) of active GnRH neurons was significantly lower (P < 0.05) in hypothalamic slices of lactating rats (0.25 ± 0.02 Hz) compared with controls (0.37 ± 0.03 Hz) (Fig. 2B). Furthermore, there were twice as many quiescent GnRH neurons (no firing) in slices from lactating animals (14.51 ± 2.89%) compared with controls (7.04 ± 2.8%) (Fig. 2C).

Figure 2.

Figure 2

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Basal spontaneous GnRH neuronal activity is reduced during lactation. A, Representative examples of action potentials observed during loose-patch extracellular recordings of GnRH-GFP neurons in hypothalamic slices obtained from control (a) and lactating (b) rats with respective frequency distribution histograms shown below (c and d). B, Average firing rate of spontaneously active GnRH neurons in hypothalamic slices obtained from control (n = 65 cells) and lactating rats (n = 60 cells); the firing rate was significantly decreased in slices from lactating rats compared with controls. C, Percent of GnRH neurons that are quiescent in hypothalamic slices from control and lactating rats (n = 70 cells for each group); there was a doubling of the number of quiescent GnRH neurons in slices from lactating animals compared with controls. *, Significant difference (P < 0.05).

Inhibitory Y5R postsynaptic effects of NPY on GnRH neuronal activity

We performed whole-cell, current clamp recordings to examine the postsynaptic effects of NPY on GnRH neurons and its actions through Y5R. Addition of TTX to the bath resulted in a complete block of spike activity, demonstrating the inhibition of most potential-dependent stimulatory or inhibitory presynaptic input. In the presence of TTX, addition of NPY to the bathing medium induced a significant hyperpolarization of the GnRH neuron membrane (Fig. 3A). It should be noted that in the presence of TTX, no stimulatory effects of NPY, only inhibitory effects, were observed. NPY’s effects could be washed out completely and the membrane potential returned back to baseline. Applying NPY again yielded the same effect (Fig. 3A). After a washout of NPY, simultaneous perfusion of Y5R antagonist with the NPY blocked the inhibitory effects of NPY and maintained the membrane potential at baseline levels (Fig. 3B). Summary data in Fig. 3C show that NPY treatment significantly decreased (P < 0.05) the membrane potential of GnRH neurons from −56.7 ± 1.94 to −62.1 ± 1.83 mV, and the hyperpolarizing effects of NPY were completely blocked by Y5R antagonist, as the membrane potential returned to −57.5 ± 1.82 mV.

Figure 3.

Figure 3

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NPY induces hyperpolarization of the GnRH neuron through actions on the postsynaptic Y5R. Whole-cell patch recordings of GnRH-GFP neurons were made in the presence of 1 μm TTX. A, Representative recording of a GnRH-GFP neuron in response to NPY treatment. Basal membrane potential (−55 mV) was stable until NPY treatment induced a potent hyperpolarization of the GnRH neuronal membrane. After a washout, the membrane potential returned back to baseline, and reapplication of NPY yielded the same hyperpolarization. B, Representative recording of a GnRH-GFP neuron in response to NPY followed by a washout of NPY and then the simultaneous addition of a Y5R antagonist with the NPY. The presence of the Y5R antagonist blocked the hyperpolarizing effects of NPY and maintained the membrane potential at baseline level (−55 mV). C, Average membrane potential changes under NPY and NPY/Y5R antagonist treatment. NPY treatment significantly decreased the membrane potential of GnRH neurons. The inhibitory effect of NPY was blocked by addition of the Y5R antagonist to the bath. *, Significant difference (P < 0.05).

Increased inhibitory NPY tone acting through Y5R on GnRH neurons during lactation

The increase in NPY expression in neurons that project to GnRH neurons (13,21) suggests that there might be increased inhibitory tone on GnRH neurons during lactation. The results from the experiments shown in Fig. 3 suggest that the increased inhibitory tone could result from NPY acting through the postsynaptic Y5R. To test this hypothesis, whole-cell current-clamp recordings of GFP-identified GnRH neurons were obtained before and after addition of Y5R antagonist to the bathing medium. In hypothalamic slices from control and lactating rats, recordings from GnRH neurons showed quiescent and spontaneously active firing patterns, similar to the data reported in Fig. 2. Responses to the administration of the Y5R were varied. Approximately 50% of GnRH cells in slices from either control (n = 14 cells) or lactating (n = 23 cells) rats had no response to application of the Y5R antagonist (Δ −1 to +1 mV in membrane potential or no change in firing rate). Modest responses to the Y5R antagonist (Δ +1 to +2.5 mV in membrane potential or 0–25% increase in firing rate) were observed in about 15 and 5% of cells from control and lactating animals, respectively. Figure 4 summarizes the significant activational responses of GnRH neurons to application of the Y5R antagonist (>+2.5 mV in membrane potential and >25% increase in firing rate). Figure 4, Aa and Ab, show recordings from inactive GnRH neurons in which application of the Y5R antagonist alone caused a depolarization of the GnRH neuron membrane (Fig. 4Aa) or a depolarization followed by spontaneous high frequency bursts of action potentials (Fig. 4Ab). Figure 4Ac shows a spontaneously active GnRH neuron with higher frequency of action potentials after the Y5R antagonist administration. The data in Fig. 4B summarizes the significant activational responses to application of the Y5R antagonist. Whereas 28% of GnRH neurons were activated in slices from control animals, 52% of GnRH neurons were activated in slices from lactating animals.

Figure 4.

Figure 4

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Increased endogenous inhibitory NPY tone on GnRH neurons during lactation. A, Representative recordings of GnRH-GFP neurons in response to Y5R antagonist treatment of hypothalamic slices from lactating rats (resting membrane potential = −55 mV); the Y5R antagonist caused a depolarization (>2.5 mV) of the cell membrane in a quiescent GnRH neuron (a), activation of a quiescent GnRH neuron with spontaneous high-frequency bursts of action potentials (b), or increase in frequency (>25%) of action potentials in an active GnRH neuron (c). B, Percent of GnRH neurons that are activated by Y5R antagonist in hypothalamic slices from control and lactating rats. Application of the Y5R antagonist caused a greater activation of GnRH neurons in slices from lactating animals (52%) compared with slices from control animals (28%).

Discussion

These studies provide the first direct measurements of decreased basal spontaneous GnRH neuronal activity during lactation (Fig. 2). Previous studies have used indirect measures to show that pulsatile LH secretion, the most accepted indirect measure of GnRH secretion, is greatly suppressed during lactation in both ovarian intact and ovariectomized lactating rats (37,38,39,40). The deficits in pituitary gonadotrope function during lactation, including the absence of postcastration-induced increases in LH and GnRH receptors (37), also indirectly reflect an inhibition of GnRH. Studies in rhesus monkeys showed that multiunit activity in the hypothalamus, an indirect measure of GnRH pulse generator activity, is remarkably suppressed during lactation (41). Thus, our direct measurements of suppressed GnRH neuronal activity, based on electrophysiological recordings in hypothalamic slices from lactating rats, add validity to the results from earlier studies using indirect measures. Our use of the GnRH-GFP-expressing rat makes these studies particularly relevant because much of our physiological knowledge of reproductive function, particularly during lactation, has been obtained from the rat.

Electrophysiological recordings from hypothalamic slices have been used in several studies to show changes in the regulation of GnRH neurons resulting from in vivo manipulation of the animal, such as fasting or ovarian steroid treatment (22,23,42). Our studies add to these data and demonstrate that changes in the regulation of GnRH neurons during different physiological states, such as lactation, can be assessed under the ex vivo conditions of the hypothalamic slice. Ultimately, measurements of the pattern of GnRH secretion in hypophysial portal blood are needed to fully describe the suppression of GnRH neuronal activity during lactation.

Based on the electrophysiological recordings made in the present studies, NPY can have direct inhibitory effects on GnRH neurons through hyperpolarization of the membrane potential. These inhibitory effects of NPY were demonstrated in the presence of TTX, which blocks many presynaptic actions, suggesting the inhibitory effects of NPY were postsynaptic on GnRH cell bodies. The ability of the Y5R antagonist to block the effects of NPY strongly suggests that the inhibitory effects of NPY are acting through the postsynaptic Y5R (Fig. 3). These results are the first demonstration of direct postsynaptic effects of NPY on GnRH neurons and of inhibitory effects through the postsynaptic Y5R. These results are in agreement with earlier pharmacological studies showing that the inhibitory effect of NPY on LH secretion is mediated by the Y5R (25). Although these studies did not examine the mechanism by which activation of the Y5R might lead to hyperpolerization of the GnRH neuronal membrane, work of others provides a likely explanation (26,43). There is general agreement that activation of multiple postsynaptic NPY receptor subtypes activates GIRK and voltage-dependent inhibition of Ca2+ channels (26). In other hypothalamic neuropeptide systems, such as orexin neurons in the lateral hypothalamic area or GABA neurons in the arcuate nucleus, NPY’s actions through postsynaptic NPY receptor subtypes result in activation of GIRK channels and attenuation of Ca2+ channels (44,45). Our data showing the postsynaptic hyperpolarizing effects of NPY (Fig. 3) and the depolarizing effects of the Y5R antagonist (Fig. 4) are consistent with these findings.

These electrophysiology data confirm our previous neuroanatomical characterization of NPY fibers making close contacts with GnRH neurons (13) and the Y5R being present on approximately 50% of GnRH neurons (21). The presence of the Y5R on only a subset of GnRH neurons most likely accounts for our observation that only about 50% of the GnRH cells had any response to the application of the Y5R antagonist. In those cells that did respond, application of the Y5R antagonist alone increased GnRH neuronal activity in both control and lactating animals, suggesting that an endogenous inhibitory NPY tone is a component of the regulation of GnRH neurons under stimulatory or inhibitory conditions (Fig. 4). The higher percentage of activated GnRH cells in slices from lactating rats in response to the Y5R antagonist supports the notion of an increase in inhibitory NPY tone during lactation (Fig. 4). It should be noted that Y5R effects could have involved other neuronal systems in addition to direct effects on GnRH cells, because presynaptic inputs were not blocked for these studies, so as to be able to observe action potentials in response to application of the Y5R antagonist (Fig. 4).

Our data extend those of Sullivan and colleagues (22,23), who showed the involvement of the Y1R in mediating NPY’s inhibitory effects during states of negative energy balance, resulting in a reduction in excitatory GABA input to GnRH neurons. Earlier studies from our group showed that Y1R was expressed on presynaptic inputs making contact with GnRH neurons (13). Sullivan et al. (23) also reported data suggesting the presence of an endogenous inhibitory NPY tone acting on GnRH neurons, in that administration of a Y1R antagonist alone increased postsynaptic current frequency in GnRH neurons from fed and fasted mice. Thus, under conditions of negative energy balance, NPY may have inhibitory effects on GnRH through presynaptic and postsynaptic sites of action. In the present study, we tested only the direct postsynaptic effects via the Y5R.

Most importantly, our observations demonstrate that there is a higher level of endogenous inhibitory NPY tone during lactation, based on the following findings: 1) fewer GnRH neurons were spontaneously active in hypothalamic slices from lactating rats compared with controls; 2) in those GnRH neurons that were spontaneously active, there was a lower firing rate in GnRH neurons from slices of lactating rats; and 3) in response to the Y5R antagonist treatment alone, a significantly greater percentage of GnRH neurons were activated in slices from lactating rats compared with controls. Taken together, these data provide convincing evidence that the higher level of endogenous inhibitory NPY tone is one neuronal pathway that contributes to the suppression of GnRH neuronal activity during lactation. It is likely that the inhibitory effects of NPY on GnRH neuronal activity are not sufficient to account for the suppression of GnRH during lactation. Another factor may be a decrease in excitatory input to GnRH neurons during lactation. Kisspeptin has been reported to be greatly suppressed during lactation (46). Thus, the combination of increased inhibitory NPY tone plus a decrease in excitatory kisspeptin tone could account for the suppression of GnRH activity during lactation.

In Fig. 5, we present a hypothesized model of inhibitory effects of NPY on GnRH neurons during lactation or other states of negative energy balance. The model is based on several years of data published by our laboratory and others on the role of Y5R and Y1R. GnRH neurons are innervated by NPY fibers, although the proportion of NPY fibers coming from all of the various sources has not been determined (Fig. 5). NPY neurons from the ARH make direct connections with about 50% of GnRH neurons in the preoptic area (13,47). Others have shown that DMH neurons send projections to the preoptic area (48). There is also NPY produced by brain stem catecholaminergic neurons that send projections to the preoptic area and contact GnRH neurons (48,49,50). NPY neurons in the ARH and DMH are activated during lactation (8,9) and provide chronically elevated basal NPY tone (our unpublished observations suggest that brain stem NPY expression does not change during lactation). During states of negative energy balance, such as during lactation or fasting, the presynaptic and postsynaptic inhibitory effects of NPY, acting through Y1R and Y5R, respectively, would be greatly enhanced on GnRH neurons (Fig. 5) (13,21). NPY may also alter GnRH release through actions on Y1R expressed on GnRH terminals and fibers in the median eminence (13).

Figure 5.

Figure 5

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Proposed model of NPY regulation of GnRH neuronal activity during lactation or other states of negative energy balance through actions via Y5R and Y1R. The increase in NPY in response to peripheral metabolic signals denoting negative energy balance or the neural suckling stimulus would result in an increase in NPY release in the area of GnRH cell bodies. NPY actions through presynaptic Y1R and postsynaptic Y5R would result in increased inhibition of the GnRH neuron.

In summary, this study strongly supports the conclusion that NPY is a significant contributor to the suppression in GnRH neuronal activity during lactation. NPY has direct inhibitory effects by acting through the postsynaptic Y5R in GnRH cell bodies. Thus, the increased NPY activity during lactation appears to have a duality of function: the support of hyperphagia and the suppression of GnRH secretion. It will be important to perform similar electrophysiological studies to examine the potential redundancy of Y5R and Y1R inhibitory effects as well as the effects of other orexigenic neuropeptides, such as AGRP, melanin concentrating hormone, and orexin, on GnRH neuronal activity.

Acknowledgments

We are grateful to members of the Division of Animal Resources and the Electrophysiology Core at the Oregon National Primate Research Center for their technical assistance. We also thank Dr. Martin J. Kelly (Oregon Health and Sciences University) for his helpful suggestions during the preparation of this manuscript.

Footnotes

This work was supported by National Institutes of Health Grants HD14643, DK60685, and DK62202, through a cooperative agreement (U54 HD18185) as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research and the Oregon National Primate Research Center base Grant RR00163.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 21, 2008

Abbreviations: aCSF, Artificial cerebrospinal fluid; AGRP, agouti-related protein; ARH, arcuate nucleus hypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus; GABA, γ-aminobutyric acid; GFP, green fluorescent protein; GIRK, G protein-coupled inwardly rectifying K+ channels; NPY, neuropeptide Y; TTX, tetrodotoxin; Y1R, Y1 receptor subtype; Y5R, Y5 receptor subtype.

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