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*ESTRADIOL
Endocrinology Vol. 143, No. 6 2284-2292
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Estradiol-Sensitive Afferents Modulate Long-Term Episodic Firing Patterns of GnRH Neurons

Craig S. Nunemaker, R. Anthony DeFazio and Suzanne M. Moenter

Departments of Internal Medicine and Cell Biology and National Science Foundation Center for Biological Timing, 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, Jefferson Park Avenue, University of Virginia, Charlottesville, Virginia 22908. E-mail: . smm4n{at}virginia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH neurons comprise the final common pathway of an estrogen-sensitive pattern generator controlling fertility. To determine estradiol effects on GnRH neuron firing patterns, adult transgenic mice were ovariectomized (OVX), and half were treated with estradiol (OVX+E). One week later targeted single-unit extracellular recordings were made from GnRH neurons identified by green fluorescent protein expression. Estradiol markedly affected GnRH neuron firing patterns, increasing the percentage and duration of time these cells were quiescent (<=1 action current/min). Estradiol increased the interval between episodes of increased firing rate determined by Cluster analysis of recordings more than 45 min (OVX+E 38.8 ± 7.2 min, OVX 16.7 ± 2.1 min, n = 6 each). Possible mechanisms of estradiol modulation were examined by simultaneously blocking ionotropic secretion of {gamma}-aminobutyric acid and glutamatergic receptors. This treatment had no effect on cells from OVX mice (n = 10), indicating episodic firing of GnRH neurons is not driven by activation of these receptors. Receptor blockade eliminated estradiol effects on GnRH neurons in the midventral preoptic area (n = 7) but not elsewhere (n = 7). Individual GnRH neurons thus display episodic firing patterns at intervals previously reported for secretory pulses. Estradiol modulates episode frequency to exert feedback control; in a substantial subset of GnRH neurons, estradiol feedback is enforced via GABAergic and/or glutamatergic afferents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTRADIOL TARGETS MANY FUNCTIONS of the central nervous system, including cognition, cell survival, behavior, and reproduction (1). The GnRH neurosecretory system provides a unique model for examining central estradiol actions because steroid feedback effects on this system are well studied at the whole-animal level. GnRH neurons play a pivotal role in reproduction by forming the final common pathway for the central control of fertility. GnRH release is distinctly patterned, with pulses of secretion separated by prolonged periods (minutes to hours) of little detectable hormone release (2, 3, 4). GnRH initiates secretion of the gonadotropins LH and FSH (5, 6). The pulsatile nature of GnRH release is crucial for maintenance of reproduction; continuous elevation of GnRH suppresses gonadotropin synthesis and secretion (7), causing infertility. Modulation of the frequency of GnRH release is equally critical as GnRH pulse frequency is a primary determinant of differential pituitary responses. Specifically, higher-frequency pulses favor LH release, whereas lower-frequency pulses favor FSH (7). This change in the relative levels of gonadotropin is critical to proper ovarian follicular maturation (8).

Estradiol is an important feedback regulator of GnRH release. LH pulses and volleys of hypothalamic electrical activity, two markers of GnRH secretion, and GnRH release itself are modulated by estradiol (4, 9, 10, 11, 12, 13, 14). Debate continues, however, over the sites and mechanisms of estradiol action on GnRH release. Specifically, does estradiol act directly on GnRH neurons or through estrogen-sensitive afferents? Historically, no evidence for estrogen accumulation (15) or receptor expression (16, 17) was found in GnRH neurons, suggesting estradiol feedback was conveyed to GnRH neurons via estrogen-sensitive afferents (18). Recent studies using more sensitive methods indicate GnRH neurons express ERß, suggesting direct action is also possible (19, 20). Both direct and transsynaptic mechanisms may cooperate in the regulation of GnRH release.

The effects and mechanisms of estradiol feedback on long-term (i.e. minutes to hours) GnRH neuron activity have not been examined directly at the GnRH neuron itself. Recently developed transgenic mice have permitted positive identification of living GnRH neurons through green fluorescent protein (GFP) expression (21, 22). We targeted GFP-identified GnRH neurons for noninvasive single-unit extracellular recordings to monitor firing patterns for up to 31/2 h. Long-duration recordings were necessary to allow detection of changes in long-term firing patterns that occur on the time scale of GnRH release. Using this approach, we examined the modulatory effects of estradiol on GnRH neuron firing pattern and the role synaptic mechanisms, specifically those activating ionotropic GABAergic and glutamatergic receptors, play in estradiol modulation of firing patterns.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
GnRH neurons were recorded from adult female, GnRH-GFP mice (22). Our goal was to examine the effects of estradiol on the firing pattern of GnRH neurons in isolation from other ovarian factors. For this reason we chose to compare GnRH neurons from mice that were ovariectomized (OVX) with those that were OVX and treated with estradiol (OVX+E). A time point for recording of 1 wk plus or minus 2 d post ovariectomy was chosen as a compromise between allowing sufficient time for endocrine adjustment to ovariectomy and avoiding side effects of long-term steroid withdrawal. Mice were anesthetized with Metofane (Janssen Pharmaceuticals, Ontario, Canada), bilaterally OVX, and were either implanted with a SILASTIC (Dow Corning Co., Midland, MI) capsule containing 0.625 µg estradiol (OVX+E, n = 13 mice) or not treated further (OVX, n = 15 mice). estradiol was given only to the animal and was not present in any of the recording solutions. Endocrine status was confirmed by measurements of uterine weight and LH levels on the day of the experiment. All animals were held on a 14-h light, 10-h dark cycle with lights off at 1600 h. Experiments were begun in the late afternoon near lights off, with recordings extending into evening. The Animal Care and Use Committee of the University of Virginia approved all procedures used in these experiments.

Brain slice preparation
All reagents were purchased from Sigma (St. Louis, MO). All solutions were bubbled with 95% O2/5% CO2 mixture throughout the experiments and for at least 30 min before exposure to tissue. Mice were decapitated and the brain was rapidly removed and placed in ice-cold high-sucrose saline solution containing mM 250 sucrose, 3.5 KCl, 26 NaHCO3, 10 glucose, 1.3 NaHPO4, 1.2 MgSO4, 3.8 MgCl2. Coronal 200-µm brain slices were cut with a vibratome (Ted Pella, Inc., Redding, CA) and then incubated for 30 min at room temperature in a solution of 50% high-sucrose saline and 50% normal saline (NS) solution, containing mM 125 NaCl, 3.5 KCl, 26 NaHCO3, 10 glucose, 1.3 NaHPO4, 1.2 MgSO4, 3.8 MgCl2, 2.5 CaCl2. Slices were then transferred to a solution of 100% NS solution at 30–32 C for at least 90 min before recording.

Electrophysiology
Targeted single-unit extracellular recordings were chosen as the approach for this study because this method allows recording from an identified neuron with minimal impact on the behavior of that neuron. Only one cell per slice was recorded; typically one to two slices per animal were used. During recording, slices were placed in a recording chamber continuously superfused at 5–6 ml/min with oxygenated NS solution kept at 30–32 C with an inline-heating unit (Warner Instruments, Hamden, CT). Cells were observed with an Olympus Corp. BX50WI upright fluorescent microscope equipped with infrared differential interference contrast (Opelco, Dulles, VA). GnRH neurons were identified by brief illumination (15–45 sec) at 470 nm to visualize the GFP signal. Cells with bipolar morphology were preferentially targeted in each slice. Recording pipettes (1–3 M{Omega}) were made from capillary glass (type 7052, OD/ID 1.65/1.1 mm, World Precision Instruments, Sarasota, FL) using a two-stage pipette puller (Narashige, Japan). Patch pipettes were filled with NS solution or normal HEPES buffered solution containing in mM 150 NaCl, 10 HEPES, 10 glucose, 2.5 CaCl2, 1.3 MgCl2, 3.5 KCl. No difference in firing was observed between these two pipette solutions. Pipettes were targeted to GnRH neurons using an MP-285 micromanipulator (Sutter Instruments, Novato, CA). Slight positive pressure was applied to the pipette before entering the NS solution bath and maintained until reaching a few microns from the target cell. Pressure was released and the pipette was moved next to the GnRH neuron. Initial seal resistances ranged from 4 to 18 M{Omega} and either remained stable or increased slowly over time up to as high as 31 M{Omega}. Gentle negative pressure was applied if amplitude of action currents was less than 50 pA; if slice movement was noted during the recording, the pipette was repositioned slightly to compensate. The location of each GnRH neuron studied was mapped on sketches of coronal sections obtained from a mouse brain atlas (23).

The duration of recordings lasted 20–220 min, with mean duration for each treatment group of approximately 60 min. Because of the prolonged interval between secretory pulses, it was anticipated that GnRH neurons might be electrically quiescent for long periods. Shorter-term whole-cell recordings of GFP-identified GnRH neurons also indicated sustained periods of quiescence (21, 24). To resolve whether a long period of quiescence was owing to inactivity of the cell or to having lost the recording, 10–15 mM KCl was added to the bath if no activity was observed for 30 min. If the cell fired in response to this treatment, the data set was truncated for purposes of analysis at the moment before KCl stimulation. If the cell did not respond to KCl, the data set was truncated for analysis at the last minute containing spontaneous action currents.

Drug treatments
To begin to examine the role of synaptic transmission in mediating estradiol effects, long-term targeted extracellular recordings were made from additional GnRH neurons in the presence of ionotropic GABAergic and glutamatergic receptor antagonists (OVX block, n = 10 cells from six mice; OVX+E block, n = 14 cells from five mice). These receptor types were chosen because they are the target of the primary means for synaptic transmission in the hypothalamus (25, 26). Slices were preincubated (15–30 min) and then recorded in NS solution containing 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM 2-amino-5-phosphonovaleric acid (APV) to block ionotropic glutamatergic transmission and 20 µM bicuculline to block ionotropic GABAergic transmission. Acute effects of this receptor antagonist mixture were also assessed in cells from OVX mice by switching between control saline and saline containing 20 µM bicuculline, 50 µM APV, and 20 µM CNQX during the recording (switch after 45 min, n = 5).

Data collection
Single-unit extracellular recordings were made using an EPC-7 or EPC-8 amplifier (HEKA, Germany) with Igor Pro software (Instrutech, Port Washingtion, NY) running on a G4 Macintosh computer (Apple Computer, Cupertino, CA) to acquire data. Recordings were made in voltage-clamp mode with a holding potential of 0 mV, filtering at 10 kHz, and digitized with an ITC-18 acquisition interface (Instrutech). Action currents (events), the membrane currents associated with action potential firing, were detected using Pulse Control Event Tracker software (Instrutech). Events were identified using the following Event Tracker settings: 10 µsec sampling interval, -50 to -150 pA threshold (depending on noise levels and initial action current amplitude), 1 point over threshold to trigger. Noise typically ranged from 20 to 25 pA peak to peak. For each event, the time of the event and 10 msec centered on the event were digitized and stored to a data file. To eliminate artifacts, we designed a program to detect spurious events using a minimum event width of 0.05 msec at half-peak amplitude. Using custom programs written for Igor Pro (Instrutech), events (action currents) were counted and binned at 1-min intervals to identify changes in long-term firing patterns. Using Excel (Microsoft Corp., Redmond, WA), binned data were evaluated for several criteria as detailed below.

Data analysis
Our goal was to examine long-term firing patterns under various conditions, looking for episodes of increased firing rate (referred to as episodes hereafter). This requires recordings of sufficient duration for multiple episodes to occur; the duration of observation would naturally increase as episodes become further apart. We were successful in obtaining 27 recordings long enough to detect episodes of increased firing rate using the Cluster 7 algorithm (27, 28). Two of these were arrhythmic as described below so that the total number of cells analyzed by Cluster was 25 (n = 6 cells each OVX, OVX+E, OVX+E block, n = 7 OVX block). Cluster 7 compares clusters of points by pooled t testing to look for nadirs and peaks over time. Using peak and nadir clusters of 4 and 6 points, respectively, Cluster 7 identified episodes and calculated the interepisode intervals for recordings with multiple identified episodes. Because it requires a string of data flanking a peak for positive identification, Cluster 7 often missed episodes very near the beginning or end of data streams; obvious episodes in these positions that could not be identified by Cluster 7 analysis were identified by eye (e.g. see first and last episode in Fig. 4AGo, lower panel). Some recordings did not display two or more episodes for one of two reasons. First, some recordings were of insufficient duration for more than one episode to occur given the average interepisode intervals (n = 25 total, n = 8 OVX, n = 8 OVX+E, n = 3 OVX block, n = 6 OVX+E block). Second, two recordings that were of sufficient duration did not exhibit identifiable changes in firing rate; one was continuously active and one showed very sparse activity. These 27 recordings were analyzed using alternative methods discussed below.



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  		Figure 4. Blocking ionotropic GABAergic and glutamatergic receptors did not significantly alter firing patterns either acutely or in population comparisons of GnRH neurons from OVX mice. A, Representative examples of firing patterns are shown for two cells recorded in receptor antagonists for the duration of the experiment. B, A representative example of the firing pattern of a cell recorded in NS solution for 45 min and then recorded in NS solution containing 20 µM bicuculline, 20 µM CNQX, and 50 µM APV (blockade). Data are plotted as mean firing rate at 1-min intervals. Vertical lines at the top of each graph illustrate timing of individual action currents detected. Horizontal bars indicate duration of blockade treatment. Asterisks (*) indicate episodes of increased firing rate detected by Cluster 7.

 
Rather than discard this sizable database of 27 recordings that could not be analyzed with the Cluster algorithm, we analyzed these records for the following parameters: mean frequency, percent quiescence, and duration of quiescence. Mean firing rate was determined by dividing the total number of events detected by the duration of the recording. Quiescence was defined as 1-min bins containing 1 or fewer events/min. The percent of total bins that were quiescent and the duration of consecutive quiescent bins (a surrogate measure of the long-term firing pattern) was determined for each recording. Altering the quiescence threshold to 0 or 2 events/min did not change interpretation of the data. For comparison, the 25 rhythmic recordings that were long enough for Cluster analysis were also analyzed using these alternative criteria; Cluster analysis agreed with the less formal measures, validating the use of the latter for extracting information from the shorter-duration recordings. In total, n = 14 cells each OVX, OVX+E, OVX+E block, and n = 10 for OVX block, for a total of 52 cells were analyzed.

Comparison of percent quiescence was performed using a Wilcoxon rank-sum test. Mean frequency, interepisode interval, and quiescence duration were compared by one-way ANOVA followed by post hoc analysis with both Fisher’s protected least significant difference and Student-Newman-Keuls for pair-wise comparisons when appropriate.

LH immunoassay
Trunk blood was collected from each mouse at the time of brain slice preparation. Serum LH concentration was determined by a modified supersensitive two-site sandwich immunoassay described previously (29, 30). Mouse LH reference preparation (AFP-5306A) provided by the National Hormone and Pituitary Program was used as standard. The assay sensitivity was 0.07 ng/ml; intra- and interassay coefficients of variation were 7.7% and 14%, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LH levels and uterine weights reflect estradiol action
Estradiol is known to modulate episodic GnRH release and alter reproductive status (18). In this study, we investigated the effects of estradiol treatment on the firing patterns of individual GnRH neurons in OVX mice. To confirm endocrine status after surgery and implantation, measurements of LH levels and uterine weights were made. LH levels were significantly higher (OVX 3.6 ± 0.6 ng/ml, OVX+E 0.1 ± 0.0 ng/ml), and uterine weights were significantly lower (OVX 22.2 ± 2.2 mg, OVX+E 118.8 ± 9.7 mg) among OVX animals, compared with OVX+E animals (P < 0.05), as expected because of loss of estradiol action.

The firing pattern of GnRH neurons differs from that of non-GnRH neurons
Using a targeted single-unit recording technique, GnRH neuron firing patterns were recorded for up to 3.5 h. GnRH neurons had a mean firing rate (total events/duration of recording) of 0.20–0.45 Hz that did not differ among the four treatment groups (P > 0.25). There was a high degree of variability in firing rate in GnRH neurons because firing occurred in episodes separated by quiescent periods (Fig. 1AGo). Tetrodotoxin (500 nM) eliminated all activity in GnRH neurons (n = 5), indicating the detected events were because of sodium-dependent action potentials (data not shown). The episodic firing pattern observed in GnRH neurons was markedly different from that of magnocellular neurons in the paraventricular nucleus of the hypothalamus (Fig. 1BGo), in which firing rate was relatively constant (2–3 Hz, n = 5 cells), consistent with previous reports (31). Of interest, the action current waveforms of GnRH neurons (Fig. 1CGo) and non-GnRH neurons (Fig. 1DGo) also differed qualitatively.



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  		Figure 1. Firing patterns and waveforms from GnRH neurons differ from other hypothalamic neurons. Representative examples of firing patterns from a GnRH neuron (A) and a magnocellular neuron from the paraventricular nucleus of the hypothalamus (B). Data are plotted as mean firing rate at 1-min intervals for a 30-min segment of the recording. Vertical lines at the top of each graph illustrate timing of individual action currents detected during the 1-min expanded segment. Individual action current waveforms of GnRH neurons (C) and magnocellular neurons (D) were also qualitatively different. The horizontal dashed line indicates the baseline trace location. Scale bars indicate action current amplitude and time scale of the corresponding panel.

 
Firing patterns in OVX vs. OVX+E
Because mean firing rate did not differ among groups and it is the pattern of GnRH release that is critical to reproductive function, we investigated the effects of estradiol on the patterning of action currents in GnRH neurons. Firing patterns were examined in three time domains: long-term (>20 min), short-term (<1 min), and over 4 msec to compare action current waveforms. Long-term firing patterns recorded from neurons in the OVX group displayed higher baseline activity (Fig. 2AGo, top), whereas for the OVX+E group, firing patterns typically consisted of longer periods of quiescence (Fig. 2BGo, top). Short-term firing patterns in both OVX and OVX+E states typically consisted of brief firing bursts separated by a few seconds (Fig. 2Go, middle). Action current waveforms (Fig. 2Go, bottom) were qualitatively similar in both OVX and OVX+E states. The remainder of this report deals with estradiol actions on the long-term firing patterns because these occur on an interval consistent with secretory pulses from these cells; short-term patterns will be considered in a separate report.



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  		Figure 2. Representative firing patterns of GnRH neurons from OVX (A) and OVX+E (B) mice. Records are shown on three time scales: 30 min (top), 30 sec (middle), and 4 msec (bottom). The boxed portions of long-term patterns (top) are expanded to show short-term firing patterns (middle). Individual action current waveforms indicated by the arrows on the middle trace are expanded below (bottom). The horizontal dashed lines indicate the baseline trace location in each section. Scale bars indicate action current amplitude and time scale.

 
One striking difference in the long-term firing pattern of GnRH neurons was the extended periods of minimal activity induced by estradiol. We quantified this in all recordings with two measures: percent quiescence (defined as percent of 1-min bins containing <= 1 event/min) and duration of quiescence (defined as number of consecutive 1-min bins containing <= 1 event/min). Quantitatively, a significantly greater percentage of quiescence was observed in cells from OVX+E mice (49% ± 6%, n = 14) than those from OVX mice (18% ± 5%, n = 14, P < 0.002). Further, the duration of quiescence was greater (P < 0.01) for cells from OVX+E mice (6.9 ± 1.4 min) than those from OVX mice (1.7 ± 0.4 min), suggesting that estradiol may reorganize GnRH neuron firing patterns.

Recordings longer than 45 min were further analyzed with the Cluster 7 algorithm to determine if significant episodes of increased firing rate occurred in either group. Figure 3Go shows representative examples of firing patterns from both groups with episodes detected by Cluster 7 marked with an asterisk. Interepisode interval was greater for cells from OVX+E mice (38.8 ± 7.2 min), compared with cells from OVX mice (16.7 ± 2.1 min, P < 0.02). Together these findings suggest estradiol-induced alterations in GnRH secretion are reflected directly at the GnRH neuron as changes in firing pattern.



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  		Figure 3. Estradiol lengthens the interval between episodes of increased firing rate. Representative examples of firing patterns are shown for two GnRH neurons from OVX (A) and two from OVX+E (B) mice. Data are plotted as mean firing rate at 1-min intervals. Vertical lines at the top of each graph illustrate timing of individual action currents detected. Asterisks (*) indicate episodes of increased firing rate detected by Cluster 7.

 
Blockade of ionotropic GABAergic and glutamatergic transmission does not significantly alter long-term firing patterns in GnRH neurons from OVX mice
To determine whether the effects of estradiol on firing patterns were mediated by ionotropic GABAergic and/or glutamatergic communication, recordings were made in the presence of the {gamma}-aminobutyric acid A (GABAA) receptor antagonist bicuculline, the N-methyl-D-aspartate receptor antagonist APV and the {alpha}-amino-3-hydroxy-S-methylisoxazale-4-proprionic acid receptor antagonist CNQX. In GnRH neurons from OVX control mice, there was no effect of the receptor antagonist cocktail on any measure of firing activity (i.e. interepisode interval, quiescence, or mean firing rate, Fig. 4Go, see Figs. 6Go and 7Go for summary, n = 10). This indicates activity was not generated or significantly modulated by ionotropic GABAergic or glutamatergic communication in our slice preparation. This finding held true for all measures described above and whether recordings were made following preincubation in the receptor antagonist cocktail (Fig. 4AGo) or after acute receptor blockade (Fig. 4BGo).



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  		Figure 6. Summary of the effects of the ionotropic GABAergic and glutamatergic receptor antagonist mixture on the firing patterns of GnRH neurons from OVX and OVX+E mice. Mean values for interepisode interval (A), percent quiescence (B), and duration of quiescence (C). Unfilled bars represent values from untreated cells, and filled bars indicate values from cells treated with the receptor antagonist mixture (block). *, P < 0.05 vs. OVX and OVX block values. #, P < 0.05 vs. OVX+E block values. The number of recordings is indicated above each bar.

 


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  		Figure 7. Subdivision of data reveals two populations of GnRH neurons from OVX+E mice with regard to response to ionotropic GABAergic and glutamatergic receptor antagonists. A, Distribution of percent quiescence values in descending order for cells from OVX (unfilled circle), OVX block (filled circle), OVX+E (unfilled triangle), and OVX+E block (filled triangle) mice. Data are normalized to the number of observations per group. B, Bar graphs display mean values for percent quiescence above the median and below the median. Unfilled bars represent values from untreated cells, and filled bars indicate values from cells treated with receptor antagonist mixture (block). C, D, Display duration of quiescence data in the same manner as described for percent quiescence. *, P < 0.05 vs. OVX and OVX block values. #, P < 0.05 vs. OVX+E block values. The number of recordings is indicated above each bar in B and D. Note different ordinate scales within B and D.

 
Blockade reverses the effects of estradiol in a subset ofGnRH neurons
In contrast to findings in OVX mice, ionotropic GABAergic and glutamatergic receptor blockade reversed the effect of estradiol in half of GnRH neurons as detailed below (Fig. 5Go). Cells that responded are referred to as "responders," those that did not as "nonresponders." All but one of the cells that was recorded for sufficient duration for Cluster analysis (n = 5 of 6) fell into the responder group. As a consequence, the estradiol-mediated increase of interepisode interval as determined by Cluster analysis was reversed by the receptor antagonist cocktail (Fig. 6AGo).



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  		Figure 5. Blockade reverses estradiol effects in a substantial population of GnRH neurons from OVX+E mice. Representative examples of firing patterns are shown for two cells from OVX+E mice that responded (A) to receptor antagonists (20 µM bicuculline, 20 µM CNQX, and 50 µM APV, blockade) and two cells that did not respond (B) to receptor antagonists based on measures of quiescence (see Fig. 6Go). Data are plotted as mean firing rate at 1-min intervals. Vertical lines at the top of each graph illustrate timing of individual action currents detected. Horizontal bars indicate duration of blockade treatment. Asterisks (*) indicate episodes of increased firing rate detected by Cluster 7.

 
Interestingly, analysis of the complete OVX+E data set, including recordings too short for Cluster analysis, revealed no significant differences between cells recorded in control solution and those recorded in the receptor antagonist mixture (block) in mean percent quiescence (Fig. 6BGo) or duration of quiescence (Fig. 6CGo). Although no differences were observed in these measures for the complete data set, cells from OVX+E mice recorded in the receptor antagonist mixture fell into two groups, as mentioned above, with regard to the quiescence measures examined. We thus examined the distribution of data and compared values above the median to those below the median for mean percent quiescence and quiescence duration. The distribution of percent quiescence reveals the two groups, responders and nonresponders, within the OVX+E block group (Fig. 7AGo, closed triangles). Specifically, values above the median of the OVX+E block group were similar to those from the untreated OVX+E group (Fig. 7AGo, open triangles), whereas values below the median of the OVX+E block group were similar to those from the OVX and OVX block groups (Fig. 7Go, circles, summary in Fig. 7BGo). A similar pattern is seen when duration of quiescence was examined in this manner (Fig. 7Go, C and D). Percentage quiescence rank order matched that of duration of quiescence, showing the same cells demonstrated both of these trends.

The differences revealed by the arbitrary division into two groups around the median are of interest with regard to the anatomical location of cells in each group. Cells from OVX+E animals that were responsive to the receptor antagonists were predominantly located in the midventral preoptic area (5 of 7, Fig. 8Go). The only designated unresponsive cell located within this area was on the cusp between values from both OVX and OVX+E animals. Other unresponsive cells were widely distributed but typically located outside the midventral preoptic area (6 of 7, Fig. 8Go). These data suggest that communication of estradiol actions to GnRH neurons may occur through phenotypically different mediators, depending on the location of specific subpopulations of GnRH neurons.



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  		Figure 8. Response to receptor antagonists correlates with location of GnRH neurons. Sketches of five overlapping coronal mouse brain slices, displayed rostral to caudal. GnRH location data from all recordings (n = 14 slices) are compiled on these sketches. The majority of neurons responsive to ionotropic GABAergic and glutamatergic receptor blockade (filled circles, n = 7) were located in the midventral preoptic area (center). GnRH neurons that did not respond to this treatment (marked by X, n = 7) were distributed throughout the recording region. Brain slice sketches adapted from Ref. 23 . ac, Anterior commissure; cc, corpus callosum; lv, lateral ventricle; oc, optic chiasm; III, third ventricle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To demonstrate estradiol effects on electrophysiological correlates of GnRH-pulse generator function, the present experiments used targeted single-unit extracellular recording in acute brain slice preparations, a technique that does not disturb the intracellular milieu. GFP-expressing GnRH neurons displayed repeated episodes of increased firing rate separated by quiescent periods of varying lengths. Estradiol increased the interval between episodes on the long-term time scale predicted for GnRH release. This estradiol feedback signal also reduced LH release, likely caused in part by reduced frequency of GnRH secretion. Blockade of ionotropic GABAergic and glutamatergic transmission reversed estradiol effects in half of GnRH neurons examined. This result supports the hypothesis that estradiol feedback is mediated by afferent network systems involving these transmitters in a substantial population of GnRH neurons.

The present findings are consistent with several reports of the effect of estradiol on indirect measures of GnRH-pulse generator activity in rodents. For example, estradiol reduces frequency of volleys of hypothalamic electrical activity associated with LH release (32). Consistent with this, sharp electrode recordings of GnRH neurons in guinea pigs demonstrated acute inhibition of firing by bath application of estradiol (33); further work in this model implied activation of an inward-rectifying potassium channel in this inhibition (34). Previous studies using whole-cell current clamp to examine GnRH neurons directly have demonstrated that GnRH-GFP neurons in acute slice preparations are capable of firing spontaneous action potentials (21) or single episodes of increased firing flanked by periods of quiescence (24). The present data extend these findings by revealing, for the first time, not only repeated bouts of activity from individual GnRH neurons that occur with an interval relevant to GnRH release but also in vivo estradiol modulation of these episodic firing patterns in a manner consistent with estradiol-induced changes in LH release. This study also has implications from a technical standpoint. Specifically, reproductive changes elicited through use of an in vivo animal model (OVX vs. OVX+E) produced changes in the GnRH neurosecretory system that could be documented and studied in acutely prepared brain slices in the absence of estradiol.

Possible mechanisms of estradiol modulation were examined to determine if estradiol-sensitive afferents were involved in mediating these effects. The choice to investigate ionotropic GABAergic and glutamatergic neurotransmitter systems in this regard was based on several factors. First, these are the dominant excitatory and inhibitory neurotransmitters in the hypothalamus (25, 26), so these transmitter systems are likely candidates to be used by interneurons within local circuits interacting with GnRH neurons or perhaps between GnRH neurons themselves. With regard to the latter, GnRH neurons possess small clear vesicles indicative of classical neurotransmitters (35) and GT1 cells (36) and prenatal GnRH neurons (37) have been shown to synthesize GABA. Second, GABA and glutamate receptors have been identified in GnRH neurons (18, 21, 38). Third, estrogen-sensitive neurons located near or projecting to the region of GnRH neurons in the preoptic area and hypothalamus synthesize and secrete GABA (39) or glutamate (40, 41). Moreover, estradiol has been shown to increase the density of synaptic contacts in the hippocampus (42) as well as to enhance the postsynaptic response following activation of ionotropic receptors (43). Fourth, indirect evidence from whole-animal studies suggests both GABA and glutamate affect LH release (18, 44). Finally, because we wished to determine whether estradiol feedback was transmitted indirectly to GnRH neurons, we chose to block simultaneously GABAergic and glutamatergic receptors. Simultaneous blockade should eliminate dominant forms of neural communication, consistent with this goal, as well as minimize difficulties in interpretation that arise because of interactions between these systems in the complex networks present in the brain slice.

For these reasons, we hypothesized estradiol-induced effects on GnRH neuron firing patterns were conveyed through a pathway involving GABAergic and/or glutamatergic neurons. Approximately half of the neurons from estradiol-treated mice responded to blockade of ionotropic GABAergic and glutamatergic receptors by reverting to OVX-like long-term firing patterns. This suggests that estradiol effects are mediated by activation of these receptors in a sizable subpopulation of GnRH neurons and supports previous descriptions of functional heterogeneity among GnRH neurons (45, 46). Interestingly, GnRH neurons from OVX+E mice that responded to blockade were located within or just outside the midventral preoptic area. In contrast, nonresponsive GnRH neurons were typically located outside this area, indicating a possible correlation between anatomical location and use of GABA and/or glutamate acting through ionotropic receptors as mediators of estradiol effects.

In addition to anatomical location, there are other possible explanations for a lack of response to ionotropic receptor blockade in half of the GnRH neurons examined from OVX+E mice. First, slice preparation could damage local network circuitry, possibly affecting some GnRH neurons more than others, depending on location or innervation. In this regard, functional synapses have been demonstrated in the absence of the presynaptic cell body (47), but it is important to point out that indirect action through multiple interneurons could be compromised in slice preparations. Second, recent evidence supports the possibility of direct effects of estradiol acting through the ß isoform of the estrogen receptor on GnRH neurons (19, 20). Direct action of estradiol on GnRH neurons could explain why some cells did not respond to blockade of ionotropic GABAergic and glutamatergic receptors because interneurons would not be required for direct effects. Third, estradiol effects could be mediated by afferents that communicate through other neurotransmitters such as norepinephrine (18), NPY (48, 49), or opioid peptides (50).

Another intriguing observation arising from these studies was that blockade of ionotropic GABAergic and glutamatergic transmission had no significant impact on the long-term firing patterns of GnRH neurons from OVX mice. Eliminating both steroid feedback by ovariectomy and specific transmitted effects by receptor blockade permits speculation about the origin of GnRH neuron rhythmicity. Continued episodic firing from these cells following receptor blockade is consistent with the hypothesis that individual GnRH neurons contain the oscillatory mechanisms necessary for inherent pulse generation. The notion that GnRH neurons are intrinsically rhythmic is supported by work on immortalized GnRH neurons, GT1 cells (51). Cultures of GT1 cells exhibit pulsatile secretory patterns (52, 53, 54). Studies of patterns in membrane recycling associated with exocytosis (55) and action potential firing (28) have extended this observation to the level of individual or small clusters of GT1 cells. Recent work on primary cultures of GnRH neurons examining action potential firing (56) or oscillations in intracellular calcium (57) concur with these findings in GT1 cells.

The hypothesis that long-term changes in firing pattern associated with GnRH release emerge from an oscillator intrinsic to GnRH neurons fits well with our current observations that estradiol feedback alters neuronal networks that influence GnRH firing patterns. We propose the role of estradiol feedback through these networks is to adjust the pattern of GnRH neuron activity, rather than the overall firing rate. In this way, estradiol can regulate the interval of GnRH release as needed to drive shifts in fertility, without affecting the fundamental oscillatory mechanism. The data have implications at the mechanistic level for shifts among different reproductive states that are driven by changes in estradiol feedback such as parts of the reproductive cycle (4, 58), seasonal transitions (59), and the attainment of puberty (60).


   Acknowledgments
 
We thank Ms. Valerie Long for performing LH immunoassays; Dr. Martin Straume for statistical advice; and Glenn Harris, Shannon Sullivan, and Pei-San Tsai for editorial comments.


   Footnotes
 
This work was supported by NIH Grants HD-34860 and HD-41469 (to S.M.M.), the NICHD/NIH through cooperative agreement U54HD28934 as part of the specialized cooperative centers program in reproduction research, and the NSF Center for Biological Timing.

Abbreviations: APV, 2-Amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; GABA, {gamma}-aminobutyric acid; GFP, green fluorescent protein; NS, normal saline; OVX, ovariectomized; OVX + E, ovariectomized mice treated with estradiol.

Received January 30, 2002.

Accepted for publication February 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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