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Endocrinology, doi:10.1210/en.2007-1260
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Endocrinology Vol. 149, No. 2 587-596
Copyright © 2008 by The Endocrine Society

Activation of Neuronal Nitric Oxide Release Inhibits Spontaneous Firing in Adult Gonadotropin-Releasing Hormone Neurons: A Possible Local Synchronizing Signal

Jérôme Clasadonte, Pierre Poulain, Jean-Claude Beauvillain and Vincent Prevot

Inserm, Jean-Pierre Aubert Research Center, Unité 837, Development and plasticity of the postnatal brain, Place de Verdun, 59045 Lille cedex, France; and University of Lille 2, School of Medicine, Institut de médecine prédictive et recherche thérapeutique, 59046 Lille, France

Address all correspondence and requests for reprints to: Vincent Prevot, Ph.D., Institut National de la Santé et de la Recherche Médicale Unité 837, Place de Verdun, 59045 Lille Cedex, France. E-mail: prevot{at}lille.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activation of nitric oxide (NO) signaling pathways in hypothalamic neurons plays a key role in the control of GnRH secretion that is central to reproductive function. It is unknown whether NO directly modulates the firing behavior of GnRH neurons in the preoptic region of the mature brain. Using patch-clamp recordings from GnRH neurons expressing green fluorescent protein in adult mice brain slices, we demonstrate that the NO precursor, L-arginine (Arg), or the NO donor, diethylamine/NO, induced a robust and reversible reduction in the spontaneous firing activity of GnRH neurons, including bursting activity. The effects of L-Arg were prevented by the NO synthase inhibitor N{omega}-nitro-L-Arg methyl ester hydrochloride. Histochemical studies revealing a close anatomical relationship between neurons producing NO and GnRH perikarya, together with the loss of the L-Arg-mediated inhibition of GnRH neuronal activity via the selective blockade of neuronal NO synthase, suggested that the primary source of local NO production in the mouse preoptic region was neuronal. Synaptic transmission uncoupling did not alter the effect of NO, suggesting that NO affects the firing pattern of GnRH neurons by acting at a postsynaptic site. We also show that the NO-mediated changes in membrane properties in the GnRH neurons require soluble guanylyl cyclase activity and may involve potassium conductance. By revealing that NO is a direct modulator of GnRH neuronal activity, our results introduce the intriguing possibility that this gaseous neurotransmitter may be used by the sexual brain to modulate burst firing patterns. It may set into phase the bursting activity of GnRH neurons at key stages of reproductive physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE COORDINATED and timely activation of GnRH neurons is essential for adult reproductive function. It represents the final common pathway for the neural control of LH secretion. In rodents, the localization of GnRH neuronal cell bodies is scattered throughout the preoptic region. Despite considerable research, the intimate mechanism by which the neuroendocrine brain synchronizes the activity of these neurons, to provide physiologically relevant episodes of GnRH secretion, remains unsolved. However, substantial evidence indicates that the increase in GnRH release, signaling the onset of the preovulatory GnRH surge, requires changes in transsynaptic communication within the neuronal networks associated with the GnRH neurons in the preoptic region (1, 2, 3). These changes involve, at least in part, an increase in N-methyl-D-aspartate acid (NMDA) receptor-dependent glutamatergic neurotransmission (4, 5), and require the production of nitric oxide (NO) (6, 7). NO is a gaseous neurotransmitter generated by nitric oxide synthase (NOS) via the de-amination of L-arginine (Arg) (8).

NO travels readily across cellular membranes, and enters both presynaptic and postsynaptic sites. This atypical transmitter with a range of action delimited by its half-life and diffusion constant, is thus poised to play a major role in coordinating neuronal inputs in a restricted brain volume (9). In the rat, GnRH neuronal cell bodies are surrounded by neurons that express neuronal NOS (nNOS) but do not express nNOS themselves (10). NO-producing neurons relay both stimulatory and inhibitory influences to GnRH neurons (for review, see Refs. 11 and 12). In vitro studies using mouse GnRH-secreting GT1 cell lines suggest that NO acts directly on immortalized GnRH neurons to either inhibit their secretory activity (13), or stimulate the secretion of GnRH and synchronize its pulsatile release (14, 15, 16, 17). We recently showed that the precise regulation of nNOS activity in the vicinity of GnRH perikarya, during fluctuating physiological conditions in vivo, plays a critical role in the central control of adult female reproduction (7). Therefore, the NO produced locally within the preoptic region may be a synchronizing agent, coordinating a functionally meaningful pattern of GnRH activity. However, whether NO has neurophysiological effects on native hypothalamic GnRH neurons is unknown. Accordingly, we sought to determine whether exogenous and endogenous sources of NO modulate the electrical activity of GnRH neurons in brain slices from transgenic mice expressing the green fluorescent protein (GFP) under the control of the GnRH promoter, using patch-clamp recordings. In addition, we examined the relative distribution of GnRH-GFP neurons and NOS-synthesizing cells in the mouse forebrain. Our results indicate that NO acutely inhibits the discharge of GnRH-GFP neurons via a direct postsynaptic action. This raises the intriguing possibility that pinpoint regulation of nNOS activity may be a key mechanism used by the neuroendocrine brain to both modulate bursting firing patterns, and set into phase the bursting activity of GnRH neurons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic mice
The generation of animals expressing GFP in GnRH neurons has been previously reported (18). Homozygous GnRH-GFP C57Bl6 mice were bred and housed in a room with controlled photoperiods (14-h light and 10-h darkness) and temperature (21–23 C), with food and water ad libitum. Both males and females were used for the experiments. Vaginal smears were examined at the time of death. Smears were transferred onto microscopic slides and dried. They were fixed in absolute ethanol for 30 sec, and stained with hematoxylin-eosin and a Papanicolaou method (Merck, Darmstadt, Germany) to identify the cycle phases (19). All experiments were performed in accordance with the European Communities Council Directive of November 24th, 1986 (86/609/EEC) regarding mammalian research, and the experimental protocols were approved by the local institutional research animal committee.

Electrophysiological recordings
Mice were used for experimentation between 64 and108 d of age (14 males and 46 females: 21 in diestrus, 21 in proestrus, and four in estrus). The animals were deeply anesthetized with a mixture of ketamine hydrochloride and xylazine hydrochloride. They were perfused via the left ventricle of the heart with 15 ml of a chilled artificial cerebrospinal fluid (ACSF) solution [117 mM NaCl, 4.7 mM KCl, 1.2 mM NaH2PO4, 23 mM NaHCO3, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM glucose, bubbled with 95% O2-5% CO2 (pH 7.4), and osmolarity 304 mOsm]. The brains were harvested, and 150-µm frontal brain slices were cut with a Vibratome (Leica, Wetzlar, Germany) throughout the preoptic region. The slices were stored for 35 min at 32 C in the ACSF. For recording, slices were transferred onto the stage of an upright fluorescent Leica DL MFSA microscope (Wetzlar, Germany) in a recording chamber that was continuously perfused with 2 ml/min ACSF, at 25 C. GnRH-GFP neurons were visualized by green fluorescence under a GFP filter (Leica), and the chosen cell body was observed under infrared differential interference contrast. Patch pipettes were produced from borosilicate capillaries and filled with an intracellular solution containing 135 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 2 mM ATP-Mg, 1 mM EGTA, and with a pH of 7.3 and an osmolarity of 274 mOsm. Whole-cell and loose patch-clamp recordings were performed with an Axoclamp 2A amplifier (Axon Instruments, Sunnyvale, CA) in the bridge mode for current-clamp recording, and displayed on a pen recorder (Gould Windograph, Royston, UK). Acquisition and analysis of potentials were performed with pClamp8 software (Axon Instruments). During whole-cell recordings, the liquid junction potential of –12 mV was corrected. A low-Ca2+/high-Mg2+ solution [117 mM NaCl, 4.7 mM KCl, 1.2 mM NaH2PO4, 23 mM NaHCO3, 0.05 mM CaCl2, 14 mM MgSO4, 25 mM glucose, bubbled with 95% O2-5% CO2 (pH 7.4), osmolarity 300 mOsm] was used to eliminate synaptic transmission. Pharmacological agents were added to the perfusion system at final concentrations. L-Arg hydrochloride, N{omega}-nitro-L-Arg methyl ester hydrochloride (L-NAME), 8-(4-chlorophenylthio)guanosine 3' 5'-cyclic monophosphate sodium (8-pCPT-cGMP), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and (4S)-N-(4-amino-5-[aminoethyl] aminopentyl)-N'-nitroguanidine (AAANG) were obtained from Sigma-Aldrich (St Louis, MO). Tetrodotoxin citrate (TTX) was obtained from Tocris (Ellisville, MO), and 2-(N, N-diethylamino)-diazenolate-2-oxide.diethyl-ammonium salt [diethylamine (DEA)/NO: half-life of 16 min in our experimental conditions] was from Alexis (San Diego, CA).

Mean firing rate values were obtained before, during, and after L-Arg and DEA/NO treatments. Neurons were considered responsive when a change of more than 20% in firing rate was observed. The peak response was determined, and the number of spikes was counted 2 min before and after the peak effect. Basal and recovery firing rate values were obtained by counting the number of spikes during a 4-min period before and after treatment, respectively.

Statistical analysis was performed using Sigma-Stat software (Jandel, San Raphael, CA). The differences between several groups were analyzed by one-way ANOVA, followed by a Student-Newman-Keuls multiple comparison test. The Student’s t test was used to compare two groups. Before statistical analysis, percentages were subjected to arc-sine transformation to convert them from a binomial to a normal distribution. The level of significance was set at P < 0.05.

Histochemistry
NOS-synthesizing cells were labeled using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry. This was performed on free floating sections from five GnRH-GFP mice to combine the mapping of NADPHd-positive neurons and GnRH-GFP fluorescent neurons. After deep anesthesia, the mice were perfused sequentially via the left ventricle with 20 ml chilled ACSF and 20 ml cold 4% paraformaldehyde in a 0.1 M sodium phosphate buffer (PB) (pH 7.4). The brains were removed and postfixed in the same fixative for 24 h at 4 C. After fixation, they were soaked overnight in 20% sucrose in PB at 4 C. The brains were cut on a freezing microtome into frontal sections at a thickness of 60 µm. Sections were washed three times for 10 min in 0.1 M PB and incubated in 0.1 M PB containing 1 mM β-NADPH (Roche Molecular Biochemicals, Meylan, France), 0.1 M nitroblue tetrazolium (Sigma-Aldrich), and 0.3% Triton X-100, at 37 C for 1 h. After incubation, the sections were rinsed three times in 0.01 M PB containing 0.9% NaCl, put on gelatin-coated slides, air-dried overnight, and coverslipped. NADPHd-positive neurons were initially viewed under bright-field microscopy, whereupon the GnRH-GFP neurons were observed under fluorescence. Images were acquired with Leica FW4000 software, and composites were realized to allow superposition of the NADPHd-positive neurons and the GnRH-GFP fluorescent neurons. Nomenclature of the structures was taken from Paxinos and Franklin (20). All compounds were purchased from Sigma-Aldrich.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH and nNOS neurons intermingling in the mouse forebrain
To examine the anatomical relationship of NO-synthesizing neurons with GnRH neurons in the mouse brain, a histochemical method (using β-NADPH as a substrate) was used to detect NADPHd activity. The distribution of NADPHd strictly correlates with the localization of nNOS (21). With reference to the areas of the brain where GnRH-GFP neurons are found, NADPHd histochemistry labeled neuronal cell bodies and fibers in the diagonal bands of Broca (DBs), the medial septum, and the preoptic region. In these areas, NADPHd staining was confined to the cytoplasm, except around the anterior commissure, where some cells displayed labeled dendritic trees (data not shown). No GnRH neuron exhibited NADPHd staining. In the DBs (Fig. 1AGo), NADPHd-labeled cell bodies were packed in narrow clusters, running parallel to the external edges of the vertical limb. At this level, the GnRH cell bodies were arranged in parallel but laid more medially in the vertical limb. Thus, the two populations of neurons appeared rather separated, although sporadic, close contacts were observed (Fig. 1BGo). In the medial septal nucleus (Fig. 1AGo), a similar arrangement was observed, with NADPHd cell bodies located at the periphery of the nucleus, and GnRH cell bodies dispersed more centrally inside the nucleus. The anteroventral periventricular nucleus (AVPe) contained a conspicuous population of densely packed NADPHd cell bodies (Fig. 1AGo). They formed an inverted V that covered the vascular organum of the lamina terminalis, and extended dorsally in the median preoptic nucleus (MnPO). At this anatomical level, GnRH cell bodies observed laterally or dorsally to the AVPe in the medial preoptic area (MPA) and the MnPO were near, but not completely surrounded by, NADPHd-labeled cells (Fig. 1CGo). More caudally, at the level where the anterior commissure merges, the population of GnRH cell bodies was sparse and scattered throughout the medial preoptic nucleus and MPA. NADPHd cell bodies were extremely abundant along the third ventricle. They formed a narrow cluster of positive cells laid in a periventricular position in the AVPe, periventricular hypothalamic nucleus (Pe), and MnPO (cut into two parts by the anterior commissure at this level) (Fig. 1CGo). Some close contacts between NADPHd-stained cells and GnRH cells were observed at these caudal levels (data not shown). As a rule, direct anatomical contacts between the two types of cells were not abundant, but GnRH neurons were rarely separated from NADPHd-positive cells by more than 200 µm. This distance corresponds to the physiological sphere of influence of a single point source of NO that emits for 1–10 sec (22).


Figure 1
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  		FIG. 1. The relationship of NADPHd staining to GnRH neurons in GnRH-GFP transgenic mice. A, Schematic brain maps demonstrating GnRH-GFP neurons (green circles) and NADPHd-stained cells (black circles) detected in individual 60-µm thick coronal brain sections, at three levels in the forebrain. B and C, GFP-fluorescence (green) and dark-field histochemistry for NADPHd (dark precipitate) in the caudal DB (B) and the MnPO (C). Arrows show close relationships between NADPHd-containing neurons and GnRH-GFP neurons. Scale bar in B represents 40 µm, whereas that in C is 80 µm. ac, Anterior commissure; cc, corpus callosum; HDB, nucleus of the horizontal limb of the DB; LS, lateral septal nucleus; LV, lateral ventricle; MPMO, medial preoptic nucleus, medial part; MS, medial septal nucleus; 3V, third ventricle; VDB, nucleus of the vertical limb of the DB; VOLT, vascular organum of the lamina terminalis.

 
Electrophysiological properties of GnRH-GFP neurons
Patch-clamp recordings were obtained in the whole-cell configuration from 75 GnRH-GFP neurons in 69 brain slices. Recorded neurons displayed a resting membrane potential of –64.39 ± 0.33 mV (n = 74) and a membrane resistance of 1729.11 ± 49.07 M{Omega} (n = 71). In response to hyperpolarizing current pulses, the majority of neurons (67 of 71) exhibited an instantaneous inward rectification, indicating the activation of an IKIR -type conductance, whereas time-dependant inward rectification was never recorded. A prominent transient outward rectification, indicating the activation of an IA-type conductance, was also observed in 35 of 71 neurons (not illustrated). These properties are in accordance with observations made in other models of GnRH-GFP neurons (23, 24). Three main types of spontaneous discharges were observed at the resting potential. The majority of neurons (51 of 75) displayed an irregular discharge pattern with sporadic bursts of several action potentials. The remaining neurons discharged in an irregular manner without bursts (10 of 75 neurons), or discharged in repetitive bursts of action potentials (13 of 75 neurons), and one neuron was silent. Bursts were of highly variable durations (from 0.4–15 sec) and contained a variable number of action potentials. They were superimposed on long duration, depolarized plateau potentials with amplitude of 3–12 mV that sustained discharge throughout the burst and were still apparent at the end of the discharge (Fig. 2AGo). At resting membrane potential, 20-msec current pulses of +200 pA triggered bursts similar to spontaneous bursts, whereas they evoked single-action potentials when the membrane was hyperpolarized (Fig 2AGo). Noticeably, 300-msec current pulses triggered depolarized plateau potentials, outlasting the repetitive discharge of action potentials evoked by these pulses in 37 of 63 GnRH neurons (Fig 2Go, B and C).


Figure 2
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  		FIG. 2. L-Arg alters bursting discharge patterns in GnRH neurons. A, At the resting potential, 20-msec (ms) current pulses (+200 pA) (asterisk) elicited bursts similar to those recorded during spontaneous bursting activity in GnRH neurons (left panel). Bursts were sustained by plateau potentials (arrows). When the membrane was hyperpolarized (right panel), a single-action potential was evoked, followed by a depolarizing after potential (arrowhead). B and C, L-Arg reversibly reduced the amplitude (B) and duration (C) of the plateau potentials evoked by 300-msec current pulses, and reduced the associated discharge of action potentials. Whole-cell patch-clamp recordings were used in all the experiments. s, Seconds.

 
Activation of endogenous NO secretion inhibits spontaneous firing in GnRH neurons via a postsynaptic effect
To determine whether NO elicits changes in GnRH neuron activity, we studied the effect of endogenous NO production on their firing rate. L-Arg, a natural substrate for NOS-mediated NO production, was perfused, at 10 and 20 mM, to 24 neurons. Markedly, L-Arg treatment resulted in the inhibition of spontaneous discharge in 22 GnRH neurons (Fig. 3Go), whereas two neurons were unresponsive. The L-Arg inhibitory effects were fully reversible (Fig. 3BGo) and were not monitored at lower doses. These effects were unlikely to be due to putative dilution of the intracellular compartment by the medium contained in the patch electrode because the L-Arg-promoted inhibition of firing in the GnRH neurons was also monitored using loose patch-clamp recording (Fig. 3DGo). No difference in L-Arg effects was monitored between genders or between stages of the estrous cycle (Table 1Go). The L-Arg-mediated inhibition was monitored in neurons displaying irregular firing (three of three neurons, Fig. 3DGo), irregular firing and bursts (12 of 14 neurons, Figs. 3Go, C, and 4Go, A and B), or bursts (seven of seven neurons, Fig. 3AGo). Interestingly, L-Arg reduced the amplitude (Fig. 2BGo) and duration (Fig. 2CGo) of the plateau potentials induced by 300-msec current pulses, and dramatically reduced the number of associated spikes. L-Arg inhibitory effects were accompanied by a mean membrane depolarization of 4.17 ± 0.42 mV (n = 15) (Figs. 3Go, A, and 4Go, A and B), as previously reported by others in a different model system (25), and/or a 52.72 ± 3.93% increase in membrane resistance (n = 5) (data not shown). To determine whether NO synthesis was required by L-Arg to promote discharge inhibition in GnRH neurons, brain slices were incubated with L-NAME (1 mM), a broad spectrum NOS inhibitor, for 15 min before exposing them to L-Arg. As shown in Fig. 4AGo, inhibition of NOS activity obliterated the ability of L-Arg to decrease the GnRH neuron firing rate (n = 4), whereas L-NAME alone had no effect on GnRH neuron bursting activity (data not shown). In addition, slice-perfusion (15–75 min) with a medium containing low-Ca2+/high-Mg2+, to uncouple synaptic transmission, failed to modify the L-Arg-mediated inhibitory effects (firing rate of GnRH neurons in low-Ca2+/high-Mg2+ medium, 0.71 ± 0.08 Hz before treatment vs. 0.05 ± 0.02 Hz with L-Arg, paired t test; P < 0.01; n = 4; Fig. 3CGo). Thus, the inhibitory action of NO on GnRH neuron discharge may be postsynaptic in nature.


Figure 3
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  		FIG. 3. L-Arg alters spontaneous firing in GnRH neurons. Whole-cell patch-clamp (A and C) and loose patch-clamp (D) recordings from representative GnRH neurons exhibiting either spontaneous bursts (A and C) or irregular firing (D) are used for illustration. A, L-Arg provoked a slowing down of spontaneous burst firing in GnRH neurons. This effect was reversible and repeatable. Note that the L-Arg-mediated inhibitory effects were accompanied by very slight membrane depolarization. B, A bar graph illustrating the firing rate of GnRH neurons before (control), during (L-Arg 10 mM), and after (Wash) L-Arg application (*, P < 0.05 compared with all other groups, one-way repeated measures ANOVA; n = 7 neurons). Error bars indicate SEM. C, Suppression of synaptic transmission using a low-Ca2+/high-Mg2+ solution did not alter the L-Arg inhibitory effect on GnRH neuron spontaneous firing. D, A representative loose patch-clamp recording illustrating the L-Arg inhibitory effect on GnRH neuron firing discharge.

 

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  		TABLE 1. Percent changes in the firing rate of GnRH neurons under L-Arg and DEA/NO treatments in male and female mice and in mice at different stages of the estrous cycle

 

Figure 4
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  		FIG. 4. L-Arg requires nNOS activity to inhibit spontaneous firing in GnRH neurons. A, Application of L-NAME, a NOS inhibitor, prevented L-Arg-mediated inhibition of spontaneous discharges in L-Arg-responsive GnRH neurons. B, Application of AAANG, a selective nNOS inhibitor, abrogated L-Arg inhibition of firing in L-Arg-responsive GnRH neurons. Note that neither of the NOS inhibitors prevented the membrane depolarization observed with L-Arg perfusion (A and B). C, A bar graph illustrating changes in the firing rate of GnRH neurons under L-Arg and L-Arg plus AAANG treatments (**, P < 0.01 between the two groups). Error bars indicate SEM. Whole-cell patch-clamp recordings were used in all the experiments.

 
L-Arg-mediated inhibition of GnRH neuron firing requires nNOS activity
Having identified a close anatomical relationship between nNOS neurons and GnRH-GFP neuronal cell bodies (Fig. 1Go), we investigated whether activation of nNOS is required for the L-Arg-mediated inhibition of GnRH neuron firing activity. We used AAANG, a selective inhibitor of nNOS (26). A 20- to 50-min bath application of 10 µM AAANG resulted in a loss of L-Arg-mediated inhibition of action potential discharge in GnRH neurons (Fig. 4Go, B and C). Interestingly, AAANG did not prevent the membrane depolarization that accompanies L-Arg-mediated activity (Fig. 4BGo), suggesting that this effect is not a consequence of NO production but is mediated by L-Arg itself. This depolarization resulted in an increased firing rate of the neurons being treated with AAANG (Fig. 4Go, B and C).

NO inhibition of GnRH neuron firing requires guanylyl cyclase activation
Cyclic GMP production, resulting from the activation of soluble guanylyl cyclase (sGC), is the classical signal transduction mechanism mediating NO activity within the central nervous system (27). To determine whether the inhibition of GnRH neurons by NO requires cGMP-dependent signaling pathways, the effect of L-Arg was tested in the presence of the sGC specific inhibitor, ODQ (Fig. 5Go, A and B). The reduced firing observed with L-Arg was abolished by ODQ, when applied for 25 min to 1 h at 30 or 40 µM (three of five neurons). ODQ alone had no effect on GnRH neuron firing (data not shown). As observed with AAANG, in the presence of ODQ, the membrane depolarization evoked by L-Arg also resulted in an increased firing rate in the GnRH neurons (Fig. 5Go, A and B). To determine whether cGMP is able to inhibit the activity of GnRH neurons on its own, brain slices were treated with a membrane permeable cGMP analog. Application of 8-pCPT-cGMP, at 200 µM, resulted in a significant reduction in the firing rates of GnRH neurons (0.82 ± 0.25 Hz before treatment vs. 0.13 ± 0.04 Hz with 8-pCPT-cGMP, paired t test; P < 0.05; n = 6; Fig. 5CGo), thus mimicking L-Arg inhibitory effects (Fig. 3Go). These effects were fully reversible in half of the neurons that were studied.


Figure 5
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  		FIG. 5. L-Arg requires guanylyl cyclase (sGC) activity to inhibit spontaneous firing in GnRH neurons. A, The loss of sGC activity with the sGC specific inhibitor, ODQ, eliminated the L-Arg-mediated inhibitory effects on firing. Downward deflections correspond to voltage responses to 300-msec hyperpolarizing pulses used to test membrane resistance. Note that the membrane depolarization elicited by L-Arg subsisted under ODQ treatment and accelerated discharge of action potentials. B, A bar graph illustrating changes in the firing rate of GnRH neurons under L-Arg and L-Arg plus ODQ treatments (*, P < 0.05 between the two groups). Error bars indicate SEM. C, 8-pCPT-cGMP, a permeable analog of the by-product of sGC, mimicked the L-Arg-mediated inhibitory effects on GnRH neuron firing. Whole-cell patch-clamp recordings were used in all the experiments.

 
Exogenous NO evokes membrane hyperpolarization by activating potassium conductance and inhibits spontaneous firing in GnRH neurons
To investigate further the effects of NO on GnRH neuron firing, brain slices were perfused with the NO donor DEA/NO. One hundred micromoles of DEA/NO reduced significantly the spontaneous discharge of 21 (of 23) GnRH neurons (Fig. 6Go). The remaining neurons were unresponsive. The effects were completely reversible (Fig. 6Go). No difference in DEA/NO effects on GnRH neuron firing was monitored between genders or between stages of the estrous cycle (Table 1Go). Lower concentrations of DEA/NO (30–50 µM) had no significant effect on neuronal activity (not illustrated). At 100 µM DEA/NO, the reduction in the firing rate was accompanied by a membrane hyperpolarization ranging from 2.5–10 mV. This was observed in neurons displaying irregular firing (n = 1, not illustrated), irregular firing plus bursts (n = 18, Fig. 6AGo), or bursts (n = 2, Fig. 6BGo). Interestingly, in the bursting neurons, DEA/NO decreased both the burst frequency and the number of action potentials per burst (Fig. 6BGo). This strengthens the idea that NO acts on depolarizing plateau potentials to reduce spontaneous firing in GnRH neurons. Because the amount of NO released from DEA/NO depends on the time after dissolution (28), we tested sequential applications of 100 µM DEA/NO at different times after dissolution (<10, 10–20, and 45 min). Reductions in the firing rate, accompanied by light membrane hyperpolarization (4.53 ± 0.28 mV, n = 16), were observed with the shorter times after dissolution (Fig. 6Go, B and C). Full activity, with total suppression of the discharge and strong membrane hyperpolarization (7.92 ± 1.10 mV, n = 3), was observed between 10 and 20 min after dissolution of the DEA/NO. No significant effect was obtained with DEA/NO at the 45-min time point after dissolution (Fig. 6Go, B and C). Because the effect of DEA/NO reflects the known time course of NO release from this compound (28), our data indicate that the inhibition of firing promoted by DEA/NO in GnRH neurons is a true response to NO, rather than a nonspecific effect of the NO donor.


Figure 6
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  		FIG. 6. The effect of NO donors on bursting discharges in GnRH neurons. A, Application of DEA/NO, a NO donor, caused membrane hyperpolarization and concomitant discharge suppression in GnRH neurons, as shown in this trace. B, A representative recording illustrating the effect of the sequential application of DEA/NO at different times after its dissolution (3, 12, and 45 min) on the firing pattern of a GnRH neuron. At dissolution times less than 10 min, DEA/NO application caused an increase in the delay between individual bursts and a decrease in the number of action potentials per burst. Between 10 and 20 min after dissolution, DEA/NO application caused membranes to hyperpolarize, and the firing rate decreased markedly. DEA/NO became inefficient at longer times after dissolution (45 min). C, Quantitative analysis of these changes is reported in the bar graph (*, P < 0.05 when compared with the control, one-way ANOVA; n = 3–24 neurons). Error bars indicate SEM. Whole-cell patch-clamp recordings were used in all the experiments.

 
DEA/NO-induced membrane hyperpolarization persisted under bath application of TTX (0.5 µM) (n = 3, Fig. 7AGo). These results suggest that, as in the case of NOS-originating NO (Fig. 3CGo), exogenous NO inhibits spontaneous firing in GnRH neurons via postsynaptic effects. With TTX, 100 µM DEA/NO induced a mean membrane hyperpolarization of 11.67 ± 4.64 mV (n = 3) with a 55.50 ± 10.39% decrease in the membrane resistance (n = 3) (Fig. 7AGo). Recording the voltage responses in two neurons, before and during application of DEA/NO, revealed a linearization in the current-voltage relationship curve. The two neurons demonstrated a reversal potential of –84.65 and –88.95 mV, respectively, both near the equilibrium potential for potassium ions, which is –88.63 mV (as calculated in our experimental conditions) (Fig. 7BGo). Previous studies have shown that high doses of NO may promote the release of adenosine (29, 30), which is known to depress neuronal activity (31, 32, 33, 34). Thus, we investigated the putative effects of adenosine on GnRH neurons. Bath applications of 100 µM to 2 mM adenosine, with TTX, neither affected membrane potential nor membrane resistance in the GnRH neurons (n = 6, Fig. 7CGo).


Figure 7
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  		FIG. 7. DEA/NO, a NO donor, hyperpolarizes GnRH neurons via a postsynaptic action. A, Application of DEA/NO with TTX resulted in a reversible membrane hyperpolarization. Constant current injection (asterisk), to bring the membrane back to its resting potential, showed that DEA/NO application decreases membrane resistance (downward deflections). The arrowheads mark the voltage response to the current injection and illustrate the current-voltage relationship. B, An illustration of representative voltage responses to current steps (10-pA increments) obtained before (control) and during DEA/NO application. Note that GnRH neurons exhibit instantaneous membrane inward rectification in control conditions. The current-voltage relationship indicates reversal potential of the hyperpolarizing response, i.e. –84.65 mV, which is close to the equilibrium potential for potassium ions. C, Application of adenosine did not mimic the DEA/NO effects on GnRH, DEA/NO-responsive neurons (the trace used for illustration is from the same cell as used in A). Whole-cell patch-clamp recordings were used in all the experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Data accumulated over the last 15 yr have clearly established that NO contributes to the control of GnRH neurosecretion (6, 35, 36, 37). We recently demonstrated that timely activation of NO production plays a key role in the neuroendocrine control of female reproductive function during adulthood. This was the case both in the preoptic region (where GnRH neuron perikarya reside) (7) and in the median eminence of the hypothalamus (the projection field of GnRH neurons) (38, 39). We now show that an intimate anatomical relationship exists between GnRH neurons and NADPHd/nNOS-expressing neurons in the mouse brain. We also provide the first electrophysiological evidence that local NO release in the preoptic region functions as an acute modulator of GnRH neuron excitability. Importantly, both endogenous and exogenous sources of NO restrict the electrical activity of GFP-identified GnRH neurons, via postsynaptic mechanisms involving activation of cytosolic enzymes. By demonstrating the ability of NO to directly modulate GnRH neuronal activity, our results suggest that this short-acting, rapidly diffusible mediator of volume transmission (40) may be capable of coordinating the activity of GnRH neuronal perikarya in the restricted volume of the preoptic region. Thus, NO may define functional microdomains in the control of reproduction in the neuroendocrine brain.

Previous studies performed in rats have described the relationship that exists between GnRH neurons and NO-producing neurons in the preoptic region (10, 41, 42, 43). The present study confirms that the general distribution pattern of these two neuronal populations in the mouse forebrain is similar to that of the rat. Our results provide direct evidence that mouse GnRH neurons do not express NADPHd activity, which strictly correlates with the localization of NOS (21), although a close anatomical relationship between NADPHd-stained neurons and GnRH cell bodies has been identified. Because NO may be effective in influencing neural elements as far away as 200 µm from its point source (22), neurons capable of releasing NO are well placed to influence GnRH neuronal activity. Our studies demonstrating that the perfusion of brain slices with L-Arg results in a NOS-dependent reduction of firing in most adult male and female GnRH neurons provide direct evidence for an involvement of endogenous NO in modulating GnRH electrical activity. Because the selective inhibition of nNOS with AAANG (26) prevented the L-Arg-mediated inhibitory effect, local NO production is likely to occur from neurons expressing NADPHd-dependent NOS activity in the neighborhood of GnRH cell bodies. This idea is consistent with our histochemical analysis that did not detect any extraneuronal expression of NADPHd activity in the surroundings of the GnRH-GFP neurons. However, because brain astrocytes and endothelial cells are able to synthesize NO, both in vitro (38, 44) and in vivo (45), the possible participation of nonneuronal sources of NO in the electrophysiological changes we observed during L-Arg application cannot be excluded.

Importantly, when the NO donor, DEA/NO, was applied to brain slices, it mimicked L-Arg-mediated effect by inducing a NO-dependent reduction of firing in GnRH neurons. NO physiological concentrations within the brain are expected to range from nanomolar (46) to micromolar (47). In this study the most consistent results where obtained when DEA/NO was used at 100 µM, a concentration shown by Bon and Garthwaite (29) to deliver 100 nM NO within the superfused tissue. These electrophysiological data are likely to be of physiological significance since we recently showed that the magnitude of NO release across the reproductive cycle varies between 20 and 70 nM in the preoptic region (7).

The finding that NO’s ability to promote the inhibition of GnRH neurons is retained during synaptic uncoupling with a medium containing low-Ca2+ and high-Mg2+, or TTX, indicates that this gaseous transmitter acts directly at a postsynaptic site to change membrane properties in GnRH neurons. Previous studies also show that the reversible alteration of cell metabolism with high doses of NO may cause an extracellular accumulation of adenosine (29), shown to hyperpolarize neurons by activating K+ currents (32, 34). Although our results suggest that the profound membrane hyperpolarization evoked by DEA/NO involves K+ conductance, adenosine is unlikely to mediate this effect because DEA/NO-sensitive GnRH neurons appeared unresponsive to adenosine.

Our results show that bath application of sGC antagonist blocks the inhibition of firing promoted by NO in GnRH neurons, and the exogenous cGMP analog mimics the action of NO. Thus, NO appears to require the sGC-cGMP signaling cascade to modulate neuronal excitability in GnRH neurons. However, recent findings that NO can modify proteins, including ion channels, via direct chemical reactions such as S-nitrosylation (48), suggest that direct posttranslational modification may provide an alternative route by which NO may regulate electrical activity in GnRH neurons. The NO-cGMP signaling cascade and S-nitrosylation are two effector pathways shown to work independently, or in concert, to modulate the firing behavior of neurons. Nonetheless, additional studies are required to characterize the downstream effectors activated by cGMP and the precise nature of the NO-modulated ion conductances responsible for the inhibitory effects on the firing discharge in GnRH neurons.

The electrophysiological properties of neuroendocrine cells that generate bursts of action potentials are essential for neuropeptide secretion (49). Although the precise pattern of neuronal firing resulting in release of the GnRH peptide is unknown, trains of action potentials in mouse GnRH neurons likely contribute to the episodic release of GnRH into the portal blood vessels of the median eminence (50). In this study most of the monitored GnRH neurons exhibited spontaneous burst firing. These rhythms were sustained by long-duration depolarizing plateau potentials, reminiscent of the depolarizing after potentials, or the slow after-depolarization potentials described by others in GnRH-GFP neurons (24, 51). The currents underlying plateau potentials in GnRH neurons are not fully understood. They appear to involve, at least in part, TTX-sensitive sodium influxes (51), which is a persistent conductance modulated by NO/cGMP signaling (48). Our findings that NO both increases intervals between spontaneous bursts and reduces the number of action potentials per bursts on depolarizing plateau potentials (by reducing their duration and/or their amplitude) show that this gaseous neurotransmitter acutely and dramatically modulates the pattern of bursting activity in GnRH neurons. This would suggest that NO impacts on GnRH secretion in vivo. The effects of NO in GnRH neurons are markedly different from those in the magnocellular neurons in the supraoptic nucleus, in which NO-induced cGMP increased burst duration (52). Strikingly, our results show that NO-promoted reduction of firing in GnRH neurons is very robust because it was monitored consistently in both genders and at all stages of the estrous cycle. At first glance our results showing that NO decreases, rather than increases, the excitability of GnRH neurons may appear intriguing. They are in apparent contradiction to the accumulating evidence showing that nNOS-derived NO plays a critical role in the onset of the preovulatory GnRH surge (6, 7, 35, 53). However, equally intriguing are the results of others showing that estradiol treatment reduces the activity of GnRH neurons just before the surge release of GnRH/LH in ovariectomized mice (54). This reduction of activity may involve a lengthening of the interval between bursts (55), which may play a critical role in the shift of fertility during the reproductive cycle. Interestingly, these estrogen effects on GnRH neuron rhythms were mediated, at least in part, through the activation of NMDA receptors (56). We recently showed that estrogens promote NMDA receptor-nNOS complex assembly in neurons (7), thus potentiating NO secretion by coupling nNOS to its main stimulatory calcium influx pathway (8, 57). These findings, together with our present data, raise the intriguing, although speculative, possibility that estrogens promote changes in the long-term firing patterns of GnRH neurons through the stimulation of NO release from neighboring neurons. This may constitute one of the key mechanisms by which estrogens mediate their positive feedback on the function of GnRH neurons.

Our histochemical analysis showed that a significant subset of the mouse hypothalamic NADPHd/nNOS neurons is contained in the periventricular zone of the preoptic region within the AVPe, Pe, and MnPO. A recent study elegantly demonstrated that estrogen receptor {alpha}-expressing neurons of the AVPe, Pe, and MnPO are critical for estrogen-positive feedback to the GnRH neurons in mice (58). Because up to 90% of nNOS neurons of the preoptic region express estrogen receptor {alpha} (59, 60), these studies, combined with ours, raise the exciting possibility that nNOS neurons of the preoptic region may be directly targeted by estrogens during its positive feedback on the sexual brain. The resulting production of NO would then act on GnRH neurons to synchronize their activity and adjust their firing behavior to enable peak release of GnRH.


   Acknowledgments
 
We thank D. J. Spergel (University of Chicago, Chicago, IL) for his generous gift of the GnRH-green fluorescent protein mice.


   Footnotes
 
This research was supported by Inserm Grants U816 and U837, the University of Lille 2, the Institut Fédératif de Recherche (IFR114), the Agence Nationale pour la Recherche (ANR, France), and the Fondation pour la Recherche Médicale (Equipe FRM, France). J.C. was a Ph.D. student supported by a fellowship from Institut National de la Santé et de la Recherche Médicale and the Région Nord Pas de Calais.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 15, 2007

Abbreviations: AAANG, (4S)-N-(4-amino-5-[aminoethyl] aminopentyl)-N'-nitroguanidine; ACSF, artificial cerebrospinal fluid; Arg, arginine; AVPe, anteroventral periventricular nucleus; DB, diagonal band of Broca; DEA, diethylamine; GFP, green fluorescent protein; L-NAME, N{omega}-nitro-L-arginine methyl ester hydrochloride; MnPO, median preoptic nucleus; MPA, medial preoptic area; NADPHd, nicotinamide adenine dinucleotide phosphate-diaphorase; NMDA, N-methyl-D-aspartate acid; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PB, phosphate buffer; 8-pCPT-cGMP, 8-(4-chlorophenylthio)guanosine 3' 5'-cyclic monophosphate sodium; Pe, periventricular hypothalamic nucleus; sGC, soluble guanylyl cyclase; TTX, tetrodotoxin citrate.

Received September 12, 2007.

Accepted for publication November 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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