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Departments of Internal Medicine (C.X., X.-Z.X., C.S.N., S.M.M.) and Cell Biology (S.M.M.), University of Virginia, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Suzanne M. Moenter, Department of Internal Medicine, P.O. Box 800578, University of Virginia, Charlottesville, Virginia 22908. E-mail: smm4n{at}virginia.edu.
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Abstract |
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Introduction |
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One possible mechanism for synchronizing GnRH neurons is through synaptic interactions among these cells (8, 9). GnRH itself is a candidate for mediating such communication. The type I GnRH receptor (GnRHR-1) binds mammalian GnRH with high affinity (10) and is expressed within the hypothalamus (11, 12, 13), although the phenotype(s) of GnRHR-1-expressing cells in this brain region has not been determined. Intraventricular treatment with low doses of GnRH inhibits LH release (14, 15). Furthermore, GnRH agonists reduce GnRH release both in vivo (16) and in vitro (17). Whether these effects occur directly on GnRH neurons or are mediated by other cell types could not be determined from those studies. In this regard, an immortalized GnRH cell line (GT1–7) (18) has been shown to express GnRHR-1 under some conditions (19), as have cultured embryonic GnRH neurons (20), suggesting direct action is possible. In the former, GnRH agonists alter GnRH release and intracellular calcium levels (19, 21, 22). The direct effects of GnRH on GnRH neurons from reproductively mature animals, however, have yet to be investigated. To address this, we examined GnRH and non-GnRH (control) neurons for both GnRHR-1 expression and electrophysiological responses to GnRH using acutely prepared brain slices from castrate adult male mice.
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Materials and Methods |
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Preparation of brain slices
All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted. Brain slices were prepared as previously described (24, 25). Briefly, the brain was rapidly removed and placed in oxygenated (95% O2-5% CO2), ice-cold, high-sucrose saline containing 250 mM sucrose, 3.5 mM KCl, 26 mM NaHCO3, 10 mM glucose, 1.3 mM NaHPO4, 1.2 mM MgSO4, and 2.5 mM MgCl2. Coronal 200-µm slices through the preoptic area and anterior hypothalamus were cut with a vibratome (Ted Pella, Inc., Redding, CA) in the same solution, then slices were transferred to and incubated for 15 min at room temperature in an oxygenated solution of 50% high-sucrose saline and 50% normal saline (NS) solution, which contained 130 mM NaCl, 3.5 mM KCl, 26 mM NaHCO3, 10 mM glucose, 1.3 mM NaHPO4, 1.2 mM MgSO4, and 2.5 mM CaCl2. Slices were then transferred to 100% NS at 30–32 C for at least 90 min before recording. During experiments, brain slices were transferred to a recording chamber and perfused continuously with oxygenated NS at 5–6 ml/min. The temperature of the perfusion solution was maintained at 30–32 C with an inline-heating unit (Warner Instruments Corporation, Hamden, CT). GFP-expressing neurons were identified by brief illumination at 470 nm using an upright fluorescent microscope (Olympus BX51WI; Opelco, Dulles, VA) equipped with infrared differential interference contrast and a video monitoring system to aid visualization of neurons; non-GFP-positive neurons were found using infrared differential interference contrast alone. GFP-positive neurons with bipolar processes were preferentially targeted to increase the probability of interactions with other cells.
Cell harvesting and single-cell RT-PCR
Single-cell RT-PCR was conducted in the following three reaction steps: cDNA-synthesis-primer annealing, RT, and PCR. Pipettes were made from capillary glass (Type 7052, OD/ID 1.65/1.1 mm; World Precision Instrument, Sarasota, FL) with a two-stage pipette puller (PP-830; Narishige, Tokyo, Japan). Pipettes were targeted to neurons with an MP-285 micromanipulator (Sutter Instrument Company, Novato, CA). Slight positive pressure was applied before the patch pipette was lowered into the NS solution in the recording chamber. When the target cell was reached, positive pressure was released and a small amount of negative pressure was applied to aspirate the target cell into the pipette. The pipette tip was broken into a sterilized, UV light-treated microcentrifuge tube, and the contents were expelled. This tube contained 14 µl cDNA-synthesis primer annealing mixture composed of 0.5 µl dithiothreitol (100 mM), 1 µl deoxynucleotide triphosphates (10 mM each; Invitrogen, Carlsbad, CA), 1 µl random hexamer primer (50 µM; Roche, Indianapolis, IN), 1 µl oligo deoxythymidine primer (500 µg/ml; Roche), 0.5 µl RNase inhibitor (20 U/ml; Roche), 0.5 µl tRNA (10 µg/µl; Roche), 0.5 µl BSA (2 mg/ml; Sigma), and 9 µl RNase-free water. This mixture was heated for 10 min at 70 C and then placed on ice, and 4.5 µl RT reaction mixture [containing 1.5 µl dithiothreitol (100 mM), 2 µl 10 x PCR buffer without Mg2+ (Invitrogen), and 1 µl MgCl2 (50 mM; Invitrogen)] plus 0.5 µl RNase inhibitor and 1 µl Superscript II RT (200 U/µl; Invitrogen) were added. The entire mixture was then incubated for 90 min at 45 C to synthesize first-strand cDNA. The mixture was then heated at 70 C for 10 min to inactivate reverse transcriptase and placed on ice.
To determine whether GnRHR-1 is expressed by GnRH neurons and other neurons in the preoptic area and anterior hypothalamus, three types of samples were tested: GFP-expressing neurons (n = 42), non-GFP-expressing preoptic and anterior hypothalamic neurons (n = 22), and false harvests (n = 13). For false harvests, pipettes were lowered into the slice, but positive pressure was maintained, and no cell was harvested. Identity of GFP-expressing neurons was confirmed by using 8 µl of the RT product from each cell for PCR amplification with primers specific to the coding sequence for the entire GnRH peptide and GnRH-associated peptide (GnRH-GAP; see Table 1
for primer sequence, product size, and conditions). For determination of GnRHR-1 expression, a primary amplification was performed on all samples with 9 µl of the RT product using GnRHR primers (Table 1). Primers were directed toward the 3' end of the cDNA and designed to yield short products to increase the success of single-cell amplification. For primary amplification of GnRH-GAP or GnRHR, the final composition of the PCR was cDNA product (volume specified above), 2.5 µl 10 x PCR buffer without Mg2+, 0.75 µl MgCl2 (50 mM), 0.5 µl Platinum Tag DNA polymerase (5 U/µl; Invitrogen), 0.25 µl primer mix (20 µM each primer; Integrated DNA Technologies Inc., Coralville, IA), 0.5 µl deoxynucleotide triphosphate (10 mM), and water to a total volume of 25 µl. A subset of samples (detailed in Results) was reamplified using a nested primer pair (GnRHR nest, Table 1) to increase sensitivity. For this reamplification, 5 µl of primary GnRHR PCR product plus 4 µl of water were substituted for cDNA product. The positive control for RT-PCR was RNA prepared from GT1–1 cells and hypothalamus for GnRH and from pituitary for GnRHRs; negative PCR control substituted water for sample. PCR (42 cycles for primary amplification and 35 cycles for reamplification) was performed with a licensed Perkin-Elmer 9600 PCR machine (Perkin-Elmer Corporation, Norwalk, CT). PCR products were resolved on 2% agarose gels (Life Technologies, Inc., Rockville, MD) containing 1.27 µM ethidium bromide (Sigma) for visualization and photographed using a Foto/phoresis I camera (Fotodyne Inc., Hartland, WI). PCR product identity was confirmed by sequencing.
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) were filled with normal HEPES-buffered solution (pH 7.4) containing 150 mM NaCl, 10 mM HEPES, 10 mM glucose, 2.5 mM CaCl2, 1.3 mM MgCl2, and 3.5 mM KCl. When the target cell was reached, positive pressure was released, and a low resistance seal (8–18 M initial) was formed between the recording pipette and the cell. Seal resistance either remained stable or increased slowly over time to as high as 30 M. Effects of both native mammalian GnRH (2–2000 nM) and a GnRH agonist (100 nM, [D-Ala6, Des-Gly10]LHRH ethylamide; both from Peninsula Laboratories, Belmont, CA) on firing rate were examined. Test substances were both applied by addition to the NS recording solution. Solutions containing GnRH agonist were recirculated because this substance is metabolically stable; solutions containing GnRH peptide were not recirculated due to the short half-life of GnRH in tissue (26, 27). Once the recording configuration was established, spontaneous firing was recorded for 5 min to ensure recording stability. Data collection was then initiated and consisted of a 5-min control period, a 5-min treatment period to mimic a pulse of GnRH release, and a 5-min wash-out period. The following treatments were evaluated: GnRH neurons treated with GnRH agonist (n = 8), GnRH neurons treated with native GnRH (total, n = 55; 2 nM, n = 10; 20 nM, n = 22; 200 nM, n = 10; or 2000 nM, n = 13), untreated GnRH neurons (n = 13), non-GnRH neurons treated with 20 nM GnRH (n = 12), and GnRH neurons pretreated with GnRH antagonist ([D-pGlu1, D-Phe2, D-Trp3,6]LHRH; 10 nM) then treated with either 20 or 2000 nM GnRH (n = 8 and n = 10, respectively). No more than three cells per treatment per animal were included for analyses.
Analysis
Cell location was mapped to an atlas of the mouse brain (28) to determine whether there was a correlation between anatomical region and expression of GnRH receptor. Action currents were detected using Pulse Control Event Tracker software (Instrutech), with the threshold set between -50 and -150 pA depending on noise levels and initial action current amplitude. For each event, the time of the event and 10 msec centered on the event were digitized and stored to a data file. Artifacts were eliminated using a program designed to detect spurious events using a minimum event width of 0.05 msec at half-peak amplitude. Action currents were counted and binned at 1-min intervals to identify changes in firing rate using custom programs written for Igor Pro, and binned data were evaluated for mean firing frequency using Microsoft Excel (Microsoft, Redmond, WA). To allow time for solution change and drug effects, only the last 3 min of each 5-min recording period were evaluated. The frequency of action currents before, during, and after treatment was calculated, and raw data were compared by ANOVA for repeated measures followed by post hoc analysis with Fisher’s protected least significant difference test. All values in graphs are normalized means ± SEM, and the significance was set at P < 0.05.
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Results |
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, top, illustrates representative examples). Previous work with dual-label immunocytochemistry indicates that GFP identifies GnRH neurons with high fidelity (>99%) (23), suggesting the lack of GnRH message in the two GFP-positive neurons was due to mRNA degradation rather than misidentification; these two neurons were excluded from further analysis. Among the 42 GnRH neurons, 16 (38%) expressed GnRHR-1 when PCR was performed with the GnRHR primer pair (Fig. 1A, bottom). Of the 26 GnRH neurons that were negative for GnRHR-1, the PCR product from the GnRHR primer pair of 16 of these cells was reamplified using the GnRHR nest primer pair. Of these 16, six were positive for GnRH receptor upon reamplification with the nested primer pair (product from the other 10 cells was used to optimize reamplification conditions; Fig. 1B). Thus, evidence for GnRHR-1 was found by single-cell RT-PCR in 22 of 42 GnRH neurons (52%; if one assumes the same rate of reamplification in the 10 samples used for optimization, the frequency would be 61%). Mapping of GnRHR-1-positive vs. GnRHR-1-negative GnRH neurons revealed receptor expression was not localized to cells in a particular region of the preoptic area and anterior hypothalamus (data not shown).
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). The 20 negative PCR products were reamplified with the nested primer pair, but no additional GnRHR-1-positive cells were identified (Fig. 1B).
Because the pipette used to harvest the cell passes through several cell layers, damaging cells en route, it is possible that mRNA from cells along the path could adhere to the pipette and be transferred to the sample tube. Contamination of samples with extraneous mRNA is likely to be a random occurrence. Therefore, it is important to perform a number of false harvests, in which the pipette is lowered into the slice but no cytoplasm is intentionally removed, so that a frequency of contamination can be determined and compared with the frequency of true harvests that are positive. When there is a clear increase in percentage of cells of interest that are positive for a message relative to the false harvest percentage, one can conclude with confidence that the message of interest is expressed in the harvested cell. In the present study, none of these false harvests (n = 13) were positive for GnRHR-1 after single amplification (Fig. 1A) or after reamplification with the nested primer pair (Fig. 1B), indicating the validity of the observation that roughly half of adult GnRH neurons express GnRHR-1.
Effects of GnRH agonist on GnRH neuron firing rate
Targeted single-unit extracellular recordings were used to examine the effects of activation of GnRHR-1 on the firing pattern of neurons. This method was chosen because it does not disrupt the intracellular milieu and allows the integrated response of the cell to treatment to be monitored (25). Initially, the effect of a GnRH agonist (100 nM) on GFP-expressing neurons was tested. Firing rate was markedly suppressed during treatment (Fig. 2) in six of eight neurons tested (by 61 ± 12%, P < 0.05; response defined as a change in firing rate of > 25%). This corresponds roughly with the percentage of GnRH neurons that expressed GnRHR-1. The effect of GnRH agonist was reversible in three of the six cells that responded. We hypothesized that the lack of reversibility in some cells was due to the long-acting nature of this drug.
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shows representative examples; Fig. 4A illustrates group mean of responding cells only). The remaining 10 GnRH neurons were unaffected (<25% change, Fig. 3B). Responsiveness did not appear to depend on firing rate during the control period because an equivalent number of responding GnRH neurons had control firing rates above and below the median. Despite the short half-life of GnRH, the inhibitory action was reversible in just 50% of responding cells (Fig. 3A, far right, illustrates cell in which effect did not reverse). Observation of some neurons for a longer recovery failed to reveal a consistent washout response after treatment with 20 nM GnRH. It is important to bear in mind that the endogenous activity pattern of these neurons make longer duration applications and washouts difficult to interpret because it becomes difficult to parse out treatment effects from the underlying rhythm. One possibility for the lack of reversibility within the time examined in some cells may be continued signaling through downstream effectors induced by receptor occupancy, which has been shown to persist for up to 15 min in the pituitary (29).
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and 4B). The firing rate remained unchanged in most (11 of 13) untreated GnRH neurons; the firing rate spontaneously increased in one untreated GnRH neuron and decreased in another. The lack of change in firing rate in the vast majority of GnRH neurons over the short time period used in these experiments indicates that the suppression of firing rate observed in the presence of GnRH was due to GnRH action rather than a spontaneous shift in firing rate.
Dose response to GnRH
It is not known what levels of GnRH are achieved in the vicinity of receptors. Furthermore, GnRH release changes markedly during the female reproductive cycle, reaching substantially higher levels for much longer periods during the GnRH surge. Therefore, we tested additional doses, including 2, 200, and 2000 nM (Fig. 5). There was no response to the 2-nM treatment (n = 10, raw data not shown), likely due to being below the sensitivity of the receptor in a brain slice preparation with active proteases that would degrade GnRH (26, 27). Interestingly, the percentage of cells showing the dominant response to a dose from 20–2000 nM was similar (Fig. 6A). In marked contrast, however, to the inhibition seen with 20 nM GnRH, doses of 200 and 2000 nM stimulated firing activity of GnRH neurons in a majority of the GnRH neurons tested (n = 10 and n = 13, respectively, Fig. 6).
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). Of those in which a change in firing rate was observed, the direction of change was not consistent (increased firing, n = 4; decreased firing, n = 3). Subsequent addition of either 20 or 2000 nM GnRH did not further change the firing rate in any GnRH neuron tested. This suggests that both the inhibition observed with low doses and the stimulation observed with high doses are specific to activation of GnRHR-1.
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Discussion |
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The concept that GnRH may serve in an autocrine manner to control its own release was first advanced when administration of low levels of GnRH into the third ventricle was shown to suppress LH secretion (14, 15). That this effect was centrally mediated was confirmed by the inhibition of GnRH release both in vivo (16) and in vitro (17) by GnRH agonists. The previous approaches importantly revealed a biological effect, but the site of GnRH action within the brain could only be narrowed down to the regions near the ventricular system. Later, it was shown that both cultured embryonic GnRH neurons and immortalized GnRH cell lines expressed GnRHR-1 (19, 20), suggesting a direct effect was possible. The detection of GnRHR-1 message in the cytoplasm from individual cells harvested from acutely prepared brain slices in the present study supports this hypothesis and extends it to reproductively mature animals. Furthermore, with the present approach, the possibility that GnRHR-1 expression is due to adaptation to culture is largely eliminated. Taken together, the present work in adults and previous work in immature GnRH neurons may suggest that the expression of GnRHR-1 by these cells is not developmentally regulated to a great extent.
Firing rate is positively correlated with peptide release in neuroendocrine systems (30, 31); therefore, the changes in firing rate observed in the present study likely reflect downstream changes in GnRH secretion. The dose dependence of the firing response of GnRH neurons to GnRH is particularly intriguing given the variations in the release of this hormone over the female reproductive cycle. The reduction in firing rate induced by a low-dose GnRH signal designed to mimic a pulse supports the hypothesis that GnRH exerts an ultrashort negative feedback loop upon its own release. This notion is consistent with the findings in whole-animal and tissue perfusion studies in which LH or GnRH secretion was suppressed (14, 15, 16). Our data extend these findings by showing that this inhibition in response to low doses of GnRH likely occurs through direct action on GnRH neurons and specifically through activation of GnRHR-1.
In contrast to the inhibition produced by low doses of GnRH, high doses increased firing rate. This suggests that autoregulation of GnRH neurons in vivo would vary depending on the level of GnRH near the receptor. In females, the amount of GnRH present will be much greater during the preovulatory surge. In this case, such a switch from autoinhibition to autoexcitation would be physiologically advantageous for propagation of the surge. That said, it is important to point out that the present study was performed in males that were castrated as adults; such animals cannot mount a surge response. This fact, by itself, does not invalidate the above hypothesis and, indeed, suggests an additional one. Specifically, it suggests that induction of the GnRH surge may involve both transmitted signals that are sexually differentiated and changes intrinsic to the GnRH neuron that may not be sex specific.
The current observations of dose dependence are of interest with regard to findings in the immortalized GnRH neural GT1 cell line and in primary culture. On one hand, treatment of these cells with GnRH agonists alters the pattern of secretion, increasing amplitude and decreasing frequency, with a net effect of increasing the amount of GnRH release (19, 20, 32), which is consistent with the high-dose excitation observed in the present study. On the other hand, GnRH antagonists eliminate pulsatile release and also cause an increase in basal release. This latter result appears more consistent with the suppression of GnRH neuron activity observed in response to low doses in the present study. Despite differences in approach and duration of treatment, there is an underlying consistency between the present data and previous studies; specifically, in all cases, the pattern of either secretion or firing activity was altered. This strongly suggests an important autocrine role for GnRH.
The postreceptor mechanisms in adult GnRH neurons that are initiated by GnRH action remain to be determined. In GT1 cells, GnRH has been shown to cause a transient increase in intracellular calcium levels that is accompanied by hyperpolarization of the membrane potential and a reduction in firing rate secondary to opening of small conductance calcium-activated potassium channels. Within a short time (
1 min), intracellular calcium levels attained a plateau slightly elevated over baseline; this was associated with an increase in firing rate compared with the pretreatment period (21, 22). This increase in firing rate occurred despite the continued presence of GnRH and did not appear to be due to changes in either voltage-dependent sodium or calcium channels. A recent study of GnRH action in GT1 cells is of particular interest with regard to the dose response in the present study. GnRH action was shown to be mediated by different G protein subunits depending on time and dose of exposure (32). The doses examined in that study (1 nM vs. 1 µM) are similar to the doses producing the most consistent inhibition (20 nM) and stimulation (2000 nM) in the present study. This finding suggests a plasticity in downstream signaling that will likely be important in understanding the role of autocrine GnRH action, particularly with regard to the switch from an episodic to a surge mode of release. Of note, if different downstream signaling pathways are engaged, this may contribute to the difference in reversibility between the stimulatory and inhibitory doses.
Activation of GnRHR-1 led to a change in the GnRH neuron firing rate in approximately one half to three quarters of the cells tested. Although consistent with the percentage that expresses GnRHR-1, this raises the question of why only a portion of GnRH neurons were found to use this feedback mechanism. Several explanations might account for this. First, subpopulations of GnRH neurons with different functions have been hypothesized (33, 34, 35, 36). Cells that respond may belong to one or another functional subpopulation. Although we observed no anatomical localization of receptor-expressing or responding GnRH neurons, functional subpopulations may not be anatomically distinct with regard to the location of the cell body. Second, GnRH neurons may use other transmitters contained within small clear vesicles (37) to communicate with one another, such as 
-aminobutyric acid (38, 39, 40, 41, 42), galanin (43, 44), or glutamate (39, 45), so that the GnRHR-1 is not required by all GnRH neurons to coordinate activity. Third, the phase of the pulse cycle when the treatment is applied could alter the cell’s ability to respond. The present study was not designed to examine long-term firing patterns, so the stage of the pulse cycle was unknown. If a GnRH neuron had recently been exposed to an endogenous GnRH signal, this could have set a series of events in motion and the cell might be refractory to a further stimulus. Fourth, the localization of the receptor within the cell would impact on our ability to monitor a response. For example, if a proportion of GnRH neurons targeted receptor to terminals rather than the cell body, recording from the cell body several millimeters distant and likely even in a separate slice would preclude observation of a functional response in those cells. Finally, although GnRHR-1 expression and function were found in a similar percentage of GnRH neurons, both could be underestimates.
Within the preoptic area and anterior hypothalamus, only a low percentage of non-GnRH neurons expressed GnRHR-1, and those neurons tested did not alter firing rate in response to GnRH treatment. Consistent with this, in autoradiographic studies of GnRHR-1 within the brain, little detectable signal was observed in these areas (11). Audioradiographic signal and GnRHR-1 mRNA were evident in other brain regions, including the arcuate region of the hypothalamus (11, 13). The phenotype of these latter cells remains unknown. One possibility is that the binding in this region is due to interaction of the probe with GnRHR-1 on the axons of GnRH neurons that pass through this region en route to the median eminence. The paucity of GnRHR-1, particularly within the regions containing the majority of GnRH cell bodies (1, 2), indicates that the expression of this receptor may be somewhat confined to GnRH neurons. This suggests that GnRH could serve as a relatively selective intercellular messenger among the neurons that produce it. Such a characteristic would be ideal for regulation of the GnRH neuronal network without affecting the activity of other cell types.
The production of an episodic GnRH signal and the switch from episodic to surge mode in females are critical to fertility. The present study, in combination with previous work, provides strong support for the hypothesis that autoregulation of GnRH neurons by their peptide product is a key step in the cycle of events that comprises the GnRH pulse and surge generator. How this autocrine regulation is influenced by development and stage of the female reproductive cycle and the downstream signaling mechanisms engaged by the receptor are exciting questions for future research.
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Acknowledgments |
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Footnotes |
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Abbreviations: GAP, GnRH-associated peptide; GnRHR-1, type I GnRH receptor; GFP, green fluorescent protein; NS, normal saline.
Received May 6, 2003.
Accepted for publication October 10, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid-evidence for an autocrine regulation. Eur J Neurosci 10:1145–1152[CrossRef][Medline]
-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons. Mol Endocrinol 16:2872–2891This article has been cited by other articles:
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  P. Chen and S. M. Moenter GABAergic Transmission to Gonadotropin-Releasing Hormone (GnRH) Neurons Is Regulated by GnRH in a Concentration-Dependent Manner Engaging Multiple Signaling Pathways J. Neurosci., August 5, 2009; 29(31): 9809 - 9818. [Abstract] [Full Text] [PDF]
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 J. C. Gill, B. Wadas, P. Chen, W. Portillo, A. Reyna, E. Jorgensen, S. Mani, G. A. Schwarting, S. M. Moenter, S. Tobet, et al. The Gonadotropin-Releasing Hormone (GnRH) Neuronal Population Is Normal in Size and Distribution in GnRH-Deficient and GnRH Receptor-Mutant Hypogonadal Mice Endocrinology, September 1, 2008; 149(9): 4596 - 4604. [Abstract] [Full Text] [PDF]
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D. Gonzalez-Martinez, C. De Mees, Q. Douhard, C. Szpirer, and J. Bakker Absence of Gonadotropin-Releasing Hormone 1 and Kiss1 Activation in {alpha}-Fetoprotein Knockout Mice: Prenatal Estrogens Defeminize the Potential to Show Preovulatory Luteinizing Hormone Surges Endocrinology, May 1, 2008; 149(5): 2333 - 2340. [Abstract] [Full Text] [PDF]
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C. Xu, T. A. Roepke, C. Zhang, O. K. Ronnekleiv, and M. J. Kelly Gonadotropin-Releasing Hormone (GnRH) Activates the M-Current in GnRH Neurons: An Autoregulatory Negative Feedback Mechanism? Endocrinology, May 1, 2008; 149(5): 2459 - 2466. [Abstract] [Full Text] [PDF]
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Z. Chu and S. M. Moenter Physiologic Regulation of a Tetrodotoxin-Sensitive Sodium Influx That Mediates a Slow Afterdepolarization Potential in Gonadotropin-Releasing Hormone Neurons: Possible Implications for the Central Regulation of Fertility. J. Neurosci., November 15, 2006; 26(46): 11961 - 11973. [Abstract] [Full Text] [PDF]
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 C. De Mees, J.-F. Laes, J. Bakker, J. Smitz, B. Hennuy, P. Van Vooren, P. Gabant, J. Szpirer, and C. Szpirer Alpha-Fetoprotein Controls Female Fertility and Prenatal Development of the Gonadotropin-Releasing Hormone Pathway through an Antiestrogenic Action. Mol. Cell. Biol., March 1, 2006; 26(5): 2012 - 2018. [Abstract] [Full Text] [PDF]
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Z. Chu and S. M. Moenter Endogenous Activation of Metabotropic Glutamate Receptors Modulates GABAergic Transmission to Gonadotropin-Releasing Hormone Neurons and Alters Their Firing Rate: A Possible Local Feedback Circuit J. Neurosci., June 15, 2005; 25(24): 5740 - 5749. [Abstract] [Full Text] [PDF]
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 G S Harrison, M E Wierman, T M Nett, and L M Glode Gonadotropin-releasing hormone and its receptor in normal and malignant cells Endocr. Relat. Cancer, December 1, 2004; 11(4): 725 - 748. [Abstract] [Full Text] [PDF]
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 A. J. Martinez-Fuentes, L. Hu, L. Z. Krsmanovic, and K. J. Catt Gonadotropin-Releasing Hormone (GnRH) Receptor Expression and Membrane Signaling in Early Embryonic GnRH Neurons: Role in Pulsatile Neurosecretion Mol. Endocrinol., July 1, 2004; 18(7): 1808 - 1817. [Abstract] [Full Text] [PDF]
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