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Endocrinology, doi:10.1210/en.2007-1241
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Endocrinology Vol. 149, No. 3 1129-1135
Copyright © 2008 by The Endocrine Society

Norepinephrine Suppresses Gonadotropin-Releasing Hormone Neuron Excitability in the Adult Mouse

Seong-Kyu Han and Allan E. Herbison

Department of Oral Physiology and Institute of Oral Bioscience (S.-K.H.), School of Dentistry, Chonbuk National University, Jeonju 561-756, South Korea; and Centre for Neuroendocrinology and Department of Physiology (S.-K.H., A.E.H.), University of Otago School of Medical Sciences, Dunedin 9054, New Zealand

Address all correspondence and requests for reprints to: Allan E. Herbison, Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin 9054, New Zealand. E-mail: allan.herbison{at}otago.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Norepinephrine (NE) is considered to exert an important modulatory influence upon the activity of GnRH neurons. In the present study, we used a transgenic GnRH-green fluorescent protein mouse model to examine the effects of NE on the electrical excitability of GnRH neurons in male and female mice. Gramicidin-perforated patch recordings demonstrated that NE (10–100 µM) exerted a robust membrane hyperpolarization, with associated suppression of firing, in more than 85% of male prepubertal and adult GnRH neurons (n = 25). The same hyperpolarizing action was observed in female GnRH neurons from diestrous (91%, n = 11), proestrous (50%, n = 14), estrous (77%, n = 13), and ovariectomized (82%, n = 11) mice. A subpopulation (<10%) of silent GnRH neurons in all groups responded to NE with hyperpolarization followed by the initiation of firing upon NE washout. The hyperpolarizing actions of NE were mimicked by {alpha}1-adrenergic (phenylephrine) and β-adrenergic (isoproterenol) receptor agonists, but {alpha}2 receptor activation (guanabenz) had no effect. Approximately 75% of the NE-evoked hyperpolarization was blocked by the {alpha}1 receptor antagonist prazosin, and 75% of GnRH neurons responded to both phenylephrine and isoproterenol. These findings indicate that NE acts through both {alpha}1- and β-adrenergic receptors located on the soma/dendrites of GnRH neurons to directly suppress their excitability throughout the estrous cycle and after ovariectomy. These data force a reanalysis of existing models explaining the effects of NE on gonadotropin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INVESTIGATIONS UNDERTAKEN over many years have implicated norepinephrine (NE) as being one of the key neurotransmitters within the GnRH neuronal network. Pioneering studies by Sawyer and colleagues (1) showed that the administration of adrenergic blockers prevented ovulation in the rabbit in the 1940s, and subsequent investigations have indicated roles for NE in the regulation of LH secretion in multiple species, including primates (2, 3, 4, 5).

It is proposed that NE modulates the activity of GnRH neurons directly to regulate LH release. A solid body of tract-tracing evidence has shown that brainstem NE neurons of the A1, A2, and A6 cell groups provide species-specific inputs to brain regions where GnRH neuron somata are found (6, 7, 8, 9). Early electron microscopic studies identified tritiated NE-containing nerve terminals synapsing on GnRH neurons in the rat (10), although supporting evidence for the direct regulation of GnRH neurons by NE (11) has been slow to emerge in this species. Recent studies in the mouse, however, have shown that that 1) A2 and A6 neurons provide direct inputs to GnRH neurons (12), 2) dopamine-β-hydroxylase-immunoreactive terminals form synapses on GnRH neuron dendrites (13), and 3) adult GnRH neurons express transcripts for {alpha}1-, {alpha}2-, and β1-adrenergic receptors (14). Together, these observations indicate that NE acts directly upon GnRH neurons in the mouse.

The effects of NE on LH secretion have been assessed by both acute and chronic adrenergic receptor manipulations. In ovariectomized (OVX) rats, the acute infusion of NE (15, 16, 17) or the activation of ascending NE tracts (18) results in the suppression of LH pulse frequency. Interestingly, adrenergic receptor antagonists also suppress pulsatile LH secretion (19), suggesting that a set window of adrenergic receptor activation is essential for pulsatile LH secretion to occur. Importantly, other investigations have shown that pulsatile LH secretion can recover over time after the complete lesioning of NE pathways and inputs (20, 21, 22). Together, these studies suggested that NE exerted a permissive role in the regulation of pulsatile GnRH and LH secretion, whereby a set tone of adrenergic receptor activation is necessary for pulse generation but that this can be replaced under pathological situations (5). A further complexity to the issue of NE actions is that OVX rats treated with estradiol and progesterone (OVX+E+P) respond to NE administration with an increase in LH secretion on the afternoon of the expected LH surge (16, 17). This suggests that gonadal steroids modulate the effects of NE on neural mechanisms regulating gonadotropin secretion.

The recent advent of GnRH transgenic mouse models has enabled the cellular and molecular features of adult GnRH neurons to be examined in situ (23, 24). Whereas the effects of adrenergic receptor manipulations on LH secretion are well characterized, there is presently no information on what actions NE may exert on adult GnRH neurons themselves. In an effort to provide clarity to the precise mechanisms through which NE modulates LH secretion, we have examined here the effects of adrenergic receptor activation on GnRH neuron excitability in male as well as diestrous, proestrous, estrous, and OVX female mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experiments were approved by the University of Otago Animal Welfare and Ethics Committee. Male and female GnRH-green fluorescent protein mice (25) were housed under 12-h light, 12-h dark cycles (lights on at 0700 h) with ad libitum access to food and water. Male (35–100 d) and postpubertal female (>35 d) mice were used for experiments. Vaginal smears were performed to determine the estrous cycle stage for females. One group of adult female mice were OVX under halothane anesthesia and used for experimentation 2 wk later. Animals were killed between 1000 and 1200 h, and recordings were made during the afternoon up to 1900 h.

Brain slice preparation and electrophysiology
Brains were prepared and recordings made as reported previously (25). Brains were rapidly removed and placed in ice-cold bicarbonate-buffered artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 118 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 11 D-glucose, 10 HEPES, 25 NaHCO3 (pH 7.4 when gassed with 95% O2 and 5% CO2). Brains were blocked and glued with cyanoacrylate to the chilled stage of a vibratome (VT1000S; Leica, Nussloch, Germany), and 150- to 200-µm-thick coronal slices containing the rostral preoptic area were cut. The slices were then incubated in oxygenated ACSF at room temperature for at least 1 h before recording.

Slices were transferred to the recording chamber, held submerged, and continuously superfused with ACSF at a rate of 4–5 ml/min. The slices were viewed with an upright microscope (BX51WI; Olympus, Tokyo, Japan) and fluorescent GnRH neurons identified at x10- and x40-objective magnification by brief fluorescence illumination and then viewed and patched under Nomarski differential interference contrast optics. Patch pipettes were pulled from thin-wall borosilicate glass-capillary tubing (PG52151-4; WPI, Sarasota, FL) on a Flaming puller (P-97; Sutter Instruments Co., Novato, CA). The pipette solution was passed through a disposable 0.22-µm filter and contained (in mM) 130 KCl, 5 NaCl, 0.4 CaCl2, 1 MgCl2,10 HEPES, and 1.1 EGTA (pH 7.3 with KOH). Gramicidin (Sigma Chemical Co., St. Louis, MO) was first dissolved in dimethylsulfoxide (Sigma) to a concentration of 2.5–5 mg/ml and then diluted in the pipette solution just before use to a final concentration of 2.5–5 µg/ml and sonicated for 15 min. Before backfilling the electrode with the gramicidin-containing solution, the tip of the electrode was loaded with a small volume of gramicidin-free pipette solution. The tip resistance of the electrodes was 4–7 M{Omega}. In initial experiments, access resistance was monitored and experiments begun when resistance stabilized at 50–90 Mohm. This typically took 15–20 min after gigaseal formation and always corresponded to the resting membrane potential (RMP) of the cell reaching a stable level below –45 mV. In all subsequent cells, experiments were begun when the RMP reached a stable level below –45 mV. Spontaneous rupture of the seal was evident by a sudden overshooting of action potentials above 0 mV. The junction potential between the patch pipettes and bath solution was nulled before gigaseal formation. Spontaneous activities were sampled online using a Digidata 1322A interface (Axon Instruments, Foster City, CA) connected to an IBM personal computer. Signals were filtered (10 kHz, Bessel filter of Multiclamp 700A) before digitizing at a rate of 1 kHz. Acquisition and subsequent analysis of the acquired data were performed using the Clampex9 suite of software (Axon Instruments). Any GnRH neuron that displayed a shift in RMP of more than 2.0 mV was considered to have responded. Traces were plotted using the Origin7 software (MicroCal Software, Northampton, MA). All recordings were made at room temperature.

NE, phenylephrine (PE), guanabenz, DL-2-amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and isoproterenol were obtained from Sigma and dissolved directly in the ACSF. Prazosin and picrotoxin were obtained from Sigma and were first dissolved in dimethylsulfoxide and then diluted by 103 in ACSF before use. Tetrodotoxin (TTX) was obtained from Tocris (Ellisville, MO) and dissolved directly in the ACSF. Drugs were tested on cells held at their RMP by applying to the bathing solution for 1 or 2 min.

Statistical analysis
Experimental data were expressed as mean ± SEM. Because more than one acceptable recording was only occasionally recorded from the same mouse brain, the number (n) for GnRH neurons is equivalent to the animal number (n). Mann-Whitney U test was used to examine differences between two experimental groups. One-way ANOVA was performed to test changes in membrane potential and RMP differences between more than two experimental groups. Changes in membrane potential were calculated by comparing pretest levels (30 sec before drug) with the 30-sec period at the time of maximal hyperpolarization. The {chi}2 test was used to examine differences in the percentage of responding cells from different groups. A level of P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In total, perforated-patch recordings were made from 88 GnRH neurons of which 80 were located in the rostral preoptic area and eight in the medial septum (MS). Under perforated-patch, RMPs (in mV) were –59.4 ± 1.0 (males, n = 39), –55.5 ± 1.5 (diestrous females, n = 11), –59.3 ± 1.2 (proestrous females, n = 14), –55.6 ± 2.5 (estrous females, n = 13), and –58.8 ± 1.1 (OVX females, n = 11).

NE hyperpolarizes GnRH neurons in male mice
In the first series of experiments, the effects of 1, 10, 30, and 100 µM NE on GnRH neuron excitability were examined in male mice. At 1 µM, NE had no effect on five of six cells (RMP, –56.8 ± 1.4 mV) with the remaining cell responding with a 3-mV hyperpolarization. At 10 µM, NE generated a robust 6.7 ± 1.0-mV hyperpolarization in nine of 10 (90%; RMP, –58.0 ± 1.1 mV) GnRH neurons (Fig. 1Go, A and B). The remaining cell did not respond. The 30-µM dose of NE was examined on two cells and both showed a 6- to 8-mV hyperpolarization (Fig. 1AGo). At 100 µM, NE generated a similar 6.6 ± 0.6-mV hyperpolarization in 17 of 20 GnRH neurons (85%; RMP, –60.2 ± 1.3 mV) (Fig. 1Go, A and B), with one neuron exhibiting a depolarizing response and the other two not responding. After rupture of the membrane, indicated by the sudden overshoot of the action potential, NE failed to evoke a response in adult male GnRH neurons. No differences in RMP existed between the GnRH neurons tested with the different concentrations of NE (ANOVA), and no relationship was found between the RMP of a cell and the degree of hyperpolarization evoked by NE (r = –0.297; P = 0.13; linear regression analysis; n = 27). In GnRH neurons exhibiting spontaneous firing, the NE hyperpolarization was associated with a cessation or marked reduction in firing rate with a return to the pretest pattern of firing upon washout (Fig. 1AGo). Three medial septum GnRH neurons were recorded, and two of these were hyperpolarized by NE.


Figure 1
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  		FIG. 1. NE suppresses the excitability of GnRH neurons. A, Perforated-patch recording from a GnRH neuron of a 35-d-old male mouse in which three concentrations of NE were applied. B, Perforated-patch recording from a GnRH neuron of a 35-d-old male mouse in which 0.5 µM TTX was applied before NE. C, Perforated-patch recording from a GnRH neuron of a 45-d-old male mouse in which 0.5 µM TTX and the amino acid receptor antagonists (AAA) CNQX, APV, and picrotoxin were applied before NE. D, Dose-response relationship showing the effect of increasing NE concentrations upon evoked membrane hyperpolarization in male mice (1 µM, only one of six cells responded; 10 µM, n = 9; 100 µM, n = 16). E, Bar graphs show the mean + SEM membrane hyperpolarization evoked by 100 µM NE in GnRH neurons of peripubertal (35–45 d old, n = 5) and adult (>60 d old, n = 12) male mice.

 
Responses to NE were short-lived, repeatable (Fig. 1AGo), and dose dependent. A dose-response relationship existed in terms of the percentage of GnRH neurons that responded to NE (1 µM, 17%; 10 µM, 90%; 100 µM, 85%; P < 0.01; {chi}2 test). Because only one cell responded to NE at 1 µM (3-mV hyperpolarization), it was not possible to test for significant differences across the NE concentrations (Fig. 1DGo). A dose-response relationship also existed in terms of the duration of the hyperpolarization evoked by NE (Fig. 1AGo). The duration of hyperpolarization in response to 10 µM NE was 2.8 ± 0.3 min (n = 9) and 9.2 ± 0.9 min after 100 µM NE (n = 11; P < 0.001, Mann-Whitney U test). In all subsequent experiments, 100 µM NE was used.

The possibility that NE may exert direct actions on GnRH neurons was assessed by undertaking recordings in the presence of TTX, a sodium channel antagonist that blocks action potential-dependent transmission. TTX quickly blocked the occurrence of action potentials but did not inhibit the hyperpolarizing action of NE on GnRH neurons (Fig. 1BGo; n = 3). Because GnRH neurons are subjected to action potential-independent GABA (and possibly glutamate) release in the brain slice preparation (26, 27), experiments were also undertaken in the presence of TTX plus the amino acid receptor antagonists CNQX, APV, and picrotoxin. NE continued to exert hyperpolarizing actions upon GnRH neurons under these conditions (Fig. 1CGo; n = 3).

Because mice from peripubertal (postnatal d 35–45) to adult (>60 d) ages were investigated, the effects of NE on GnRH neurons from these two age groups was compared. The responses were the same in both age groups with five of six (83%; RMP, –59 ± 1.2 mV) peripubertal GnRH neurons responding to 100 µM NE with a 6.3 ± 1.4-mV hyperpolarization compared with 12 of 14 (86%; RMP, –60.8 ± 1.9 mV) adult GnRH neurons that displayed a 6.7 ± 0.6-mV hyperpolarization (Fig. 1EGo).

NE hyperpolarizes GnRH neurons in OVX and cycling female mice
The effects of NE in the female were examined by making recordings from GnRH neurons obtained from diestrous, proestrous, estrous, and OVX mice. NE was found to exert hyperpolarizing effects similar to those recorded from male mice in each of the female groups (Figs. 2Go and 3Go). Ten of 11 (91%; RMP, –56.3 ± 1.7 mV) diestrous GnRH neurons responded to 100 µM NE with a mean membrane hyperpolarization of 6.8 ± 0.5 mV (Fig. 3Go) with the remaining cell not responding. Seven of 14 (50%; RMP, –61.4 ± 1.1 mV) proestrous GnRH neurons responded to 100 µM NE with a mean membrane hyperpolarization of 7.6 ± 1.2 mV (Fig. 3Go) with the remaining cells (RMP, –59.9 ± 2.3 mV) not responding. Ten of 13 (77%; RMP, –56.0 ± 2.1 mV) estrous GnRH neurons responded to 100 µM NE with a mean membrane hyperpolarization of 6.9 ± 0.9 mV (Fig. 3Go) with the remaining cells not responding. Nine of 11 (82%; RMP, –58.8 ± 1.1 mV) GnRH neurons in OVX mice responded to 100 µM NE with a mean membrane hyperpolarization of 7.0 ± 1.1 mV (Fig. 3Go) with the remaining cells not responding. Spontaneously active neurons stopped firing during the NE-induced hyperpolarization and then continued their pattern of firing upon washout (Fig. 2Go, A and C).


Figure 2
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  		FIG. 2. NE suppresses the excitability of GnRH neurons throughout the cycle and after OVX in female mice. Representative perforated-patch recordings from GnRH neurons of diestrous (A), proestrous (B), and OVX (C) female mice show the hyperpolarizing action of 100 µM NE.

 

Figure 3
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  		FIG. 3. NE exerts a similar effect on GnRH neurons across the estrous cycle. A, Bar graphs showing the mean + SEM hyperpolarization evoked by 100 µM NE from GnRH neurons in male and female diestrous (D), proestrous (Pro), estrous (E), and OVX mice; B, bar graphs showing the percentage of GnRH neurons responding to NE in male and female diestrous (D), proestrous (Pro), estrous (E), and OVX mice. The total number of GnRH neurons tested is given in brackets for each group. No significant differences were detected.

 
Across the four female groups, no significant differences were detected in the RMP (P > 0.05, ANOVA), percentage of GnRH neurons responding to NE (P = 0.10, {chi}2), degree of membrane hyperpolarization evoked by NE (P > 0.05, ANOVA; Fig. 3AGo), or the duration of the NE-induced hyperpolarization (P > 0.05, ANOVA; diestrus 11.0 ± 1.7 min; proestrus 10.1 ± 0.4 min; estrus 10.6 ± 1.4 min; OVX 9.6 ± 1.4 min; n = 11–14 per group).

In both males and females, a small subpopulation of silent GnRH neurons was found that responded to NE with hyperpolarization followed by phasic or continuous firing after NE washout (Fig. 2BGo). In total, nine of 88 (10%) GnRH neurons responded in this way and were evenly distributed among the experimental groups (percent silent cells responding to NE in this way was 25% males, 22% diestrus, 20% proestrus, and 28% estrus). The RMP of silent GnRH neurons that were activated after NE hyperpolarization (–60.6 ± 1.2 mV; n = 9) was not different to the RMP of silent GnRH neurons that remained silent after NE hyperpolarization (–57.6 ± 1.2 mV; n = 20; P > 0.05; Mann-Whitney U test). However, silent GnRH neurons with rebound activation exhibited a significantly greater NE-evoked hyperpolarization (8.2 ± 0.5 mV; n = 9) compared with silent GnRH neurons that remained silent after NE washout (6.0 ± 0.4 mV; n = 20; P < 0.01, Mann-Whitney U test).

The hyperpolarizing actions of NE are mediated by {alpha}1- and β-adrenergic receptors
The adrenergic receptor types underlying the effects of NE were examined using a range of agonists and antagonists. In each case, a GnRH neuron was tested with NE (100 µM) followed by the {alpha}1-adrenergic receptor agonist PE (30–100 µM), {alpha}2-adrenergic receptor agonist guanabenz (10 or 20 µM), or β-adrenergic receptor agonist isoproterenol (100 µM). If a good quality recording was still possible after this, tests were made examining the presence of multiple adrenergic receptors and/or the effects of prazosin, the {alpha}1-aderenergic receptor antagonist (10 µM). The concentrations of agonists and antagonists used in this study have been proven to be effective in previous electrophysiological experiments (28, 29).

Thirteen of 21 (62%) GnRH neurons responding to NE also responded to PE with membrane hyperpolarization (5.5 ± 0.6 mV) (Fig. 4AGo and Table 1Go). The eight other cells did not respond. It was possible to test three NE- and PE-sensitive GnRH neurons further with prazosin. Prazosin alone was found to increase cell excitability in two of the three cells and significantly attenuate the NE-induced hyperpolarization by 75% (before prazosin, 8.6 ± 0.5 mV; during prazosin, 2.0 ± 0.3 mV; P < 0.01, paired t test) in all three GnRH neurons (Fig. 4AGo).


Figure 4
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  		FIG. 4. NE acts through {alpha}1- and β-adrenergic receptors to suppress GnRH neuron excitability. A, Perforated-patch recording from a GnRH neuron of a 33-d-old male mouse showing hyperpolarizing responses to NE (100 µM) and PE (30 µM) followed by a partial suppression of the NE hyperpolarization by prazosin (Praz; 10 µM) and then recovery of the NE action. B, Perforated-patch recording from a GnRH neuron of a 60-d-old male mouse showing lack of effect of guanabenz (GBZ; 10 µM). C, Perforated-patch recording from a GnRH neuron of a 42-d-old female mouse showing hyperpolarizing responses to isoproterenol (Iso; 100 µM), PE (30 µM), and NE (100 µM).

 

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  		TABLE 1. Summary showing numbers of GnRH neurons hyperpolarized by {alpha}1 (PE), {alpha}2 (guanabenz), and β (isoproterenol) adrenergic receptor agonists

 
None of seven NE-sensitive GnRH neurons responded to the selective {alpha}2-adrenergic receptor agonist guanabenz (Fig. 4BGo and Table 1Go).

Seven of 12 (58%) NE-sensitive GnRH neurons also responded to isoproterenol with a membrane hyperpolarization (6.1 ± 0.7 mV) (Fig. 4CGo and Table 1Go). Eight GnRH neurons were tested with NE, PE, and isoproterenol, and six were found to respond to all three agonists (Fig. 4CGo), and the remaining two cells responded to NE and PE but not isoproterenol, or NE and isoproterenol but not PE.

The hyperpolarizing responses of male and female GnRH neurons to agonists, either alone or after NE treatment, were similar (Table 1Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show here that GnRH neurons respond directly to NE. These effects are mediated by both {alpha}1- and β-adrenergic receptors on the soma and/or dendrites and generate a robust suppression of excitability and firing in the great majority of GnRH neurons. The effects of NE persist in the presence of TTX and amino acid receptor antagonists, indicating that NE acts directly upon GnRH neurons. This corroborates tract-tracing (12), immunocytochemical (13, 30), and receptor-profiling (14) studies that have all suggested a direct NE input to GnRH neurons in the mouse. We found no differences in the effects of NE on GnRH neurons recorded from males or females across the cycle or after ovariectomy. Together, these results demonstrate that direct adrenergic receptor activation exerts a potent, gonadal steroid-independent, suppressive influence upon GnRH neurons.

Eighty to 90% of GnRH neurons were found to respond to NE, and the majority (75%) tested with PE and isoproterenol responded to both agonists, suggesting the coexistence of {alpha}1- and β-adrenergic receptors. Furthermore, the hyperpolarizing action of NE was significantly reduced, but not completely abolished, by the {alpha}1 receptor antagonist prazosin, suggesting cooperation between the two receptors in hyperpolarizing GnRH neurons. This indicates a unique scenario in which either or both {alpha}1- or β-adrenergic receptors function to hyperpolarize GnRH neurons. Whereas β-adrenergic receptors are known to mediate the suppression of neuronal excitability, for example in hypothalamic paraventricular neurons (31), to our knowledge, this is the first time an {alpha}1-receptor-mediated hyperpolarizing response has been recorded from central neurons. To date, we have been unable to establish the signaling pathway underlying this unique response.

Approximately 20% of silent GnRH neurons responding to NE were noted to fire in a rebound manner after the NE-induced hyperpolarization. GnRH neurons showing rebound firing were significantly more hyperpolarized by NE than silent cells that remained silent. This suggests the involvement of an intrinsic hyperpolarization-activated conductance. Previous studies have indicated that a subpopulation of GnRH neurons exhibit the hyperpolarization-activated cation current (32) that is known mediate rebound activation (33). However, it is important to note that the number of GnRH neurons showing this response was small, representing less than 10% of all recorded GnRH neurons, and that they had a similar incidence in males and females throughout the cycle.

To examine the possibility that gonadal steroids modulated the effects of NE on GnRH neurons, we evaluated GnRH neurons taken from diestrous, proestrous, estrous, and OVX mice. Surprisingly, considering the clear steroid dependency of NE actions on LH secretion (16, 17), we found that NE exerted a consistent suppressive effect on GnRH neuron excitability throughout the cycle and in OVX mice. Whereas the robust suppression of LH release by NE activation in OVX rats (16, 17) is compatible with the present electrophysiological data, the stimulatory effects of NE on LH are not explained by the direct effects NE on GnRH neurons. There are several explanations for this apparent incongruence. It is possible that the NE component of the GnRH neuronal network operates differently in the mouse compared with the rat, where the LH secretion data have been obtained. Another explanation is that the reported effects NE on LH secretion result from widespread activation of adrenergic receptors throughout the GnRH neuronal network rather than reflecting actions solely at the GnRH neuron somata/dendrites. Also, it is important to recognize that this switch from an inhibitory LH response in OVX animals to a stimulatory LH response in OVX+E+P rats has been observed with a wide range of neurotransmitters (see Ref. 34). Thus, it is very likely that the switch to a stimulatory action reflects more the state of the GnRH neurons in OVX+E+P rats rather than any special feature of the individual neurotransmitter or receptor (34).

Brought together, we propose that under physiological situations, direct inputs from brainstem NE neurons to GnRH neuron somata/dendrites suppress firing throughout the estrous cycle in females and in males. We find no evidence that gonadal steroid status influences the functioning of adrenergic receptors expressed by GnRH neurons. However, other actions of NE within the GnRH neuronal network may modulate, and even override, this direct suppressive influence. As such, we speculate that that gonadal steroid influences occur either indirectly through the regulation of adrenergic receptor activity on afferent neurons within the network or through the modulation of NE release (Fig. 5Go).


Figure 5
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  		FIG. 5. Schematic diagram showing proposed NE connections within the GnRH neuronal network of the mouse. Direct NE inputs from brainstem A2 and A6 cell groups suppress GnRH neuronal firing through {alpha}1- and β-adrenergic receptors. Indirect modulation through the regulation of afferent inputs (light gray) is also likely. Open arrows indicate possible sites through which gonadal steroids may act to modulate NE influences within the network.

 
In summary, we provide here evidence that NE evokes a direct and robust suppressive influence on GnRH neuron firing in male as well as female mice throughout the estrous cycle. These actions are mediated through both {alpha}1- and β-adrenergic receptors on the GnRH neuron soma and/or dendrite. The suppressive influence of {alpha}1-adrenergic receptor activation on GnRH neurons is unique within the brain. Coming many years after the description of the effects of NE on LH, the identification here that NE potently inhibits GnRH neuron firing calls for a reassessment of current models explaining NE’s actions within the brain to regulate LH secretion.


   Acknowledgments
 
We thank Drs. Grattan, Jasoni, Lee, and Liu of the Centre for Neuroendocrinology for helpful comments on the draft of the manuscript.


   Footnotes
 
First Published Online December 13, 2007

Abbreviations: ACSF, Artificial cerebrospinal fluid; APV, DL-2-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; NE, norepinephrine; OVX, ovariectomized; OVX+E+P, OVX rats treated with estradiol and progesterone; PE, phenylephrine; RMP, resting membrane potential; TTX, tetrodotoxin.

This work was supported by The Wellcome Trust (UK) and the Health Research Council of New Zealand.

Disclosure Statement: The authors have nothing to disclose.

Received September 7, 2007.

Accepted for publication December 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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