Long-Term Recordings of Networks of Immortalized GnRH Neurons Reveal Episodic Patterns of Electrical Activity

Craig S. Nunemaker, R. Anthony DeFazio, Michael E. Geusz, Erik D. Herzog, Gilbert R. Pitts, and Suzanne M. Moenter

Departments of Internal Medicine, Cell Biology, and Biology and National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22908


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nunemaker, Craig S., R. Anthony DeFazio, Michael E. Geusz, Erik D. Herzog, Gilbert R. Pitts, and Suzanne M. Moenter. Long-Term Recordings of Networks of Immortalized GnRH Neurons Reveal Episodic Patterns of Electrical Activity. J. Neurophysiol. 86: 86-93, 2001. The CNS controls reproduction through pulsatile secretion of gonadotropin-releasing hormone (GnRH). Episodic increases in the firing rate of unidentified hypothalamic neurons have been associated with downstream markers of GnRH secretion. Whether this episodic electrical activity is intrinsic to GnRH neurons, intrinsic to other "pulse generator" neurons that drive GnRH neurons, or a combination of these is unknown. To determine if GnRH neurons display episodic firing patterns in isolation from other cell types, immortalized GnRH neurons (GT1-7 cells) were cultured on multiple microelectrode arrays. Long-term, multi-site recordings of GT1-7 cells revealed repeated episodes of increased firing rate with an interval of 24.8 ± 1.3 (SE) min that were completely eliminated by tetrodotoxin, a sodium channel blocker. This pattern was comprised of active units that fired independently as well as coincidentally, suggesting the overall pattern of electrical activity in GT1-7 cells emerges as a network property. The A-type potassium-channel antagonist 4-aminopyridine (1 mM) increased both firing rate and GnRH secretion, demonstrating the presence of A-type currents in these cells and supporting the hypothesis that electrical activity is associated with GnRH release. Physiologically relevant episodic firing patterns are thus an intrinsic property of immortalized GnRH neurons and appear to be associated with secretion. The finding that overall activity is derived from the sum of multiple independent active units within a network may have important implications for the genesis of the GnRH secretory pattern that is delivered to the target organ. Specifically, these data suggest not every GnRH neuron participates in each secretory pulse and provide a possible mechanism for the variations in GnRH-pulse amplitude observed in vivo.


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INTRODUCTION
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Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central control of reproduction. In males and throughout most of the female reproductive cycle, GnRH is released in discrete pulses at intervals ranging from ~30 min to a few hours. Pulses of GnRH initiate pulsatile secretion of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Clarke and Cummings 1982; Levine and Duffy 1988; Levine et al. 1985; Moenter et al. 1991) from the anterior pituitary gland. This intermittent GnRH signal is crucial to reproductive success. In the absence of an episodic GnRH signal, synthesis and secretion of the gonadotropins are suppressed (Belchetz et al. 1978; Haisenleder et al. 1991), leading ultimately to infertility (Marshall et al. 1972). Moreover, changes in GnRH pulse frequency throughout the ovulatory cycle determine changes in the relative amounts of LH to FSH release (Wildt et al. 1981); these changes are prerequisite for proper follicular development (Marshall and Griffin 1993). The neural mechanisms underlying this critical episodic pattern, commonly referred to as the GnRH pulse generator, are not well understood at the cellular or network level.

Episodic changes in the rate of action potential firing by hypothalamic neurons appear to comprise part of the mechanism that gives rise to pulsatile GnRH release. Rhythmic volleys of hypothalamic electrical activity in primates, goats, and rats were invariably coincident with pulsatile LH release (Hiruma and Kimura 1995; Tanaka et al. 1995; Wilson et al. 1984), but the neurosecretory phenotype(s) of the episodically active neurons could not be determined in these studies. Questions thus remain whether or not the observed rhythmic electrical activity was endogenous to GnRH neurons and related to GnRH secretion. In marked contrast to the episodic hypothalamic electrical activity associated with LH release in animal models, whole cell recordings of GT1-7 cells, an immortalized, tumor-derived murine GnRH cell line (Mellon et al. 1990), displayed nearly continuous activity or patterns on a vastly shorter time scale than that of hormone release intrinsic to this cell line (Besecke et al. 1994; Charles and Hales 1995; Costantin and Charles 1999; Krsmanovic et al. 1992, 1993; Martinez de la Escalera et al. 1992; Pitts et al. 2001; Van Goor et al. 1999; Wetzel et al. 1992). One interpretation of these findings is that the episodic firing pattern arises outside of the GnRH neural network. However, the short duration of the recordings in these studies (<30 min) precluded observation of episodic changes in firing rate on the same time scale as pulsatile hormone release from this cell line, i.e., approximately half hourly (Besecke et al. 1994; Krsmanovic 1993; Martinez de la Escalera et al. 1992; Pitts et al. 2001; Wetzel et al. 1992). In the present study, long-term extracellular recordings were used to monitor multiple GT1-7 cells simultaneously for several hours, revealing an episodic pattern of firing rate with a frequency consistent with pulsatile GnRH release (Besecke et al. 1994; Krsmanovic et al. 1993; Martinez de la Escalera et al. 1992; Pitts et al. 2001; Wetzel et al. 1992).


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METHODS
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Cells

All cell culture reagents were obtained from Life Technologies (Grand Island, NY) unless noted. GT1-7 cells (passage <27), generously provided by Dr. Richard Weiner (University of California at San Francisco), were maintained in DMEM-F12 supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum in a humidified 37°C-5% CO2 atmosphere. At 70-80% confluence, cells were harvested with trypsin and 1-2 × 105 cells were plated onto multimicroelectrode plates (MMEPs, Dr. Guenter Gross, University of North Texas). MMEPs were prepared for cell culture by flame heating with a butane torch to render the surface hydrophilic. Matrigel (Collaborative Research, Bedford, MA), diluted 1:10 in PBS, was applied and then immediately aspirated to form a thin coating, which was allowed to gel at 37°C for 60 min before plating cells. Cells were grown on MMEPs for 1.5-2 days in the preceding medium and then switched to DMEM-F12 containing the defined supplement B-27 plus 100 IU/ml penicillin and 100 µg/ml streptomycin for 2.5-4 days to 85-95% confluence; total culture duration before recording was 4-6 days. Cultures were incubated 1-2 h before recording in either Locke's buffered salt solution [n = 3, containing (in mM) 154 NaCl, 5.6 KCl, 2.2 CaCl2, 1 MgCl2, 10 glucose, 2 HEPES, 6 NaHCO3, 0.05% BSA, and 0.02 bacitracin, pH 7.4] or modified Kreb's Ringer solution (n = 4, 140 NaCl, 4.7 KCl, 2.6 CaCl2, 0.6 MgCl2, 10 glucose, 10 HEPES, 0.05% BSA, and 0.02 bacitracin, pH 7.4). For pattern analysis, cells were recorded for 4.5-13.5 h in either static or perfusion conditions. Although the baseline firing rate was generally lower in Locke's, similar episodic patterns were obtained under all conditions; results were thus combined for pattern analysis. For drug treatment, MMEP cultures were perfused first with Kreb's, followed by a 10-min treatment of one of the following diluted in Kreb's: [4-aminopyridine (4-AP), 1 mM, n = 6; bicuculline, 20 µM, n = 4; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 20 µM, n = 4]; duration of tetrodotoxin (TTX, 500 nM, n = 6) treatment was 5 min.

Electrical recordings

MMEPs contain an array of transparent indium-tin-oxide leads terminating in 64 gold-plated electrodes (1-3 M impedance, ~10 µm in diameter) arranged in a 600 × 600 µm grid consisting of 4 rows 200 µm apart and 16 columns 40 µm apart (Gross et al. 1977, 1993). Cyberamp AI 401 10 × gain low-noise differential preamplifiers (Axon Instruments, Foster City, CA) were used to record signals from up to eight electrode pairs (1 preamplifier per pair) at various positions on the MMEP. The output from each preamplifier (channel output) represented the difference in signal between the two electrodes of each pair. The potential difference between the electrode pairs was further amplified by the Cyberamp AI 380 (Axon Instruments) at 2,000-5,000 gain, notch-filtered at 60 Hz, band-pass filtered at 300-3,000 Hz, and then digitized at 12-bit resolution and a 15-kHz sampling rate. Total gain was 20,000 to 50,000. Array Acquire software (Thomas Breeden, University of Virginia, Charlottesville, VA) was used to acquire extracellular potential from each amplifier channel on an IBM-compatible personal computer (Herzog et al. 1997, 1998). When the extracellular potential exceeded a preset threshold value of 25-50% greater than noise (estimated at ±20-35 µV), maximum and minimum voltages were recorded along with their respective times of occurrence; each voltage excursion beyond threshold was counted as a single spike. All detected spikes were collected in 2-min bins throughout the recording for analysis of firing rate. Spikes of similar signature in polarity and size were grouped together as one "active unit" and separated from spike waveforms of differing signature. The term active unit refers to the cell or group of cells producing a characteristic waveform; since as many as four cells were observed covering a single electrode, the number of cells comprising an active unit cannot be determined at this level of analysis. In most cases, each amplifier channel recorded only a single active unit, although on occasion two or more units could be distinguished on a single channel based on either direction of deflection or size (see Fig. 1). A second program, Wave Stability (Thomas Breeden, University of Virginia) was used to record the waveform shape of individual signals. This software cycled through each amplifier channel for a 20-s sampling period once every 3 min throughout the recording session. Control recordings were performed with MMEPs devoid of cells but perfused with recording solution and showed no action potentials or episodic activity.

Data analysis

The CLUSTER7 computer algorithm (Veldhuis and Johnson 1986) identified episodes of increased electrical activity within each data stream using a configuration that kept false positives under 5%. The minimum amplitude for episode identification was varied, depending on the baseline activity of each amplifier channel. Interval between episodes was calculated for each active unit that exhibited two or more episodes of increased firing rate. Approximately 50% of MMEPs (n = 7) showed episodic activity on three to six channels, a percentage that is comparable to the percentage of GT1 cultures exhibiting pulsatile GnRH release in our lab (Pitts and Moenter, unpublished observations). All active units on a MMEP were summed and termed composite activity; inter-episodic intervals were determined as in the preceding text. The hypothesis that episodes were coincident among multiple active units was tested using the statistical software HYPERGEO (Veldhuis et al. 1991), a probability function that performs co-variate analysis for several data sets. Coincidence was defined as peak activity that occurred in the same or adjacent 2-min bins. For pharmacological treatments, ANOVA, blocked for plate effects, was used to compare average firing rates 10 min before and 10 min after drug addition. All data are presented as means ± SE, and statistical significance was set at P < 0.05.

GnRH measurement

Due to low number of cells grown on MMEPs, GnRH secretion from these cultures was undetectable unless samples were collected at a minimum of 10-min intervals. This low rate of sampling did not provide the resolution required to examine episodic GnRH secretion but was adequate to study the response to pharmacological challenge. GnRH was thus measured during the 10 min preceding, 10 min during, and 10 min after application of the potassium channel blocker 4-AP in perfusion (1 mM, n = 6 MMEPs). GnRH levels were determined using a slight modification of a previously described radioimmunoassay (Nett et al. 1973). Briefly, primary antibody (R1245) was incubated for 24 h with 100 µl sample, tracer was added with incubation continuing an additional 24 h, and separation of bound and free tracer was achieved by ethanol precipitation. All samples were assayed in duplicate in a single assay; the limit of sensitivity was 0.14 ± 0.04 pg/tube and the intra-assay variation was 9.0%. Hormone values were compared using a paired one-tailed t-test assuming unequal variances.


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Episodic electrical activity

GT1-7 cells recorded simultaneously at several locations within a culture exhibited episodic patterns of increased firing rate. Examples of typical waveforms counted as spikes from three different MMEPs are shown in Fig. 1. Each graph in panels (A-C) displays all spikes detected during one 20-s sampling window. Unambiguous waveform spikes were detectable above the noise threshold. These waveforms are consistent with previous reports of extracellular recordings using planar electrode arrays (Gross et al. 1985). More than one waveform was sometimes detectable on a single amplifier channel based either on direction of deflection (Fig. 1B) or size (Fig. 1C). These likely reflect firing of different active units; the term "active unit" refers to a cell or group of cells producing a characteristic waveform. Although the examples reveal that distinct active units can be monitored, in most cases, only one active unit per amplifier channel fired repeatedly during the recording (e.g., Fig. 1A).



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Fig. 1. Representative examples of action potential waveforms recorded and counted as spikes by the analysis software. A-C: spikes detected during one 20-s sampling window. Groups of similar traces were vertically offset for clarity; baseline voltage for all traces was near 0 mV, and dashed horizontal lines indicate the positive and negative thresholds for spike detection. A: traces of 4 action potentials recorded from 1 electrode. The unitary amplitude and shape of the voltage traces (superimposed at the time they crossed the positive threshold) indicates they are from a single active unit defined as a cell or group of cells producing a characteristic waveform. In most cases, only 1 active unit was observed on each channel. Occasionally, two units were discriminated on a channel as shown by the distinct waveforms in B and C. B: an example of 2 active units with different polarity. C: an example of 2 active units with different waveform amplitude. D: example of spike frequency of a single unit displayed over time. Wave Analysis software detected and counted every instance of the waveform signature in C indicated by the box to generate a plot of spike frequency for the duration of the recording for this single unit. Similar plots were generated for each unique active unit recorded.

Waveforms of individual active units were counted to generate plots of firing rate over time, as shown in Fig. 1D. Firing rate composites, shown in Fig. 2, were obtained by adding the firing rates of all active units on each MMEP; these composites thus represent the total recorded activity from each culture. GT1-7 cultures displayed striking episodic increases in firing rate. The inter-episode interval was consistent among all seven MMEPs (24.8 ± 1.3 min, range 20.4-30.3 min) and corresponded well with the observed inter-pulse intervals reported for GnRH secretion from GT1 cell lines (Besecke et al. 1994; Krsmanovic 1993; Martinez de la Escalera et al. 1992; Pitts et al. 2001; Wetzel et al. 1992). All firing was completely and reversibly eliminated by application of the sodium-channel blocker tetrodotoxin as shown in Fig. 3, demonstrating the electrical signals were generated by sodium-dependent action potentials. These data suggest rhythmic generation of action potentials at a period physiologically relevant to secretion is an endogenous property of GT1-7 cells.



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Fig. 2. Four examples of composite electrical activity from separate multimicroelectrode plate (MMEP) cultures. All individual active units from each of 4 MMEPs were summed to create the 4 composite patterns shown. *, indicate episodes of significant changes in firing rate detected by CLUSTER7. Inter-episodic interval (mean ± SE) for the composite firing pattern of each MMEP culture shown here: A, 24.9 ± 2.9 min (4 active units); B, 26.0 ± 3.6 min (3 active units); C, 27.3 ± 3.5 min (4 active units); and D, 20.9 ± 3.3 min (5 active units). Similar patterns were observed in cells recorded with either Locke's (A and B) or Kreb's (C and D) buffer. Note that active unit firing patterns summed to produce each composite (A-D) were recorded from different channels representing different locations within each of the 4 individual MMEP cultures illustrated. On each of these 4 MMEP cultures, each amplifier channel recorded only a single active unit with 1 exception: 2 of the 5 units comprising the composite shown in D were observed on the same channel. The 2 waveform signatures from this channel are shown in Fig. 1C.



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Fig. 3. The sodium channel blocker tetrodotoxin (TTX, 500 nM) eliminated all firing. A: firing pattern of a representative active unit before, during (solid bar), and after (dashed line) 5-min treatment with TTX. B: mean ± SE firing rate of all active units recorded (n = 20 units on 6 MMEPs) before (0.33 ± 0.06 Hz), during (0.00 ± 0.00 Hz), and after (0.17 ± 0.09 Hz) TTX treatment (*P < 0.001).

Correlation between firing rate and secretion

To determine if increased firing rate correlates with increased secretion, GT1-7 cells were treated with the A-type potassium-channel antagonist 4-AP (1 mM). This pharmacological manipulation is expected to increase excitability by broadening action potentials and increasing input resistance (Kita et al. 1985). Figure 4A illustrates the effect of 4-AP on the firing pattern of a representative active unit. On average, 4-AP increased the firing rate threefold from 0.14 ± 0.05 to 0.47 ± 0.16 Hz (P < 0.05, n = 21 of 25 active units monitored on 6 MMEPs, Fig. 4B). Simultaneous measurement of GnRH release from these MMEP cultures revealed a doubling of GnRH secretion (1.04 ± 0.21 to 2.07 ± 0.15 pg/ml, n = 6 cultures, P < 0.05, Fig. 4C), demonstrating a correlation between increased firing rate and hormone release.



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Fig. 4. Potassium channel blockade increases firing rate and gonadotropin-releasing hormone (GnRH) secretion. A: firing activity of a representative active unit before, during (dashed line) and after (solid bar) 10-min treatment with 4-aminopyridine (4-AP, 1 mM). B: mean ± SE firing rate of all active units recorded (n = 25 units on 6 MMEPs) before, during, and after treatment. C: GnRH secretion from the same MMEPs during the 10-min intervals before, during, and after treatment with 4-AP (*P < 0.05). Note that washout was incomplete in the 10 min immediately following treatment due to perfusion dynamics.

Independently active units generate both coincident and noncoincident episodic changes in firing rate

The firing patterns of individual units recorded from different regions of a culture were further examined to study how the overall pattern of episodic activity of a culture was generated. Due to the distance between electrodes, each active unit was considered a unique cell or group of cells (with the one exception mentioned in the preceding text in which two units from the same electrode were identified). Episodes were considered coincident among two or more active units if their peaks occurred within ±2 min of one another. Using this criterion, 65% of episodes in composite patterns were coincident between two active units. Of these, 50% were coincident among three or more active units. For example, in Fig. 5 all four peaks in firing rate identified in Fig. 5B coincided with peaks in Fig. 5, A and C. The probability of such coincident increases in firing rate occurring by chance was calculated for each MMEP using HYPERGEO. For the example shown (Fig. 5), the probability that the four peaks among three data sets were coincident due to chance was P < 6.7 × 10-12 (range for all MMEPs: P < 0.025 to P < 1 × 10-26). These data imply GT1 cells are actively coordinating firing.



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Fig. 5. Coordinated activity between areas of the culture gives rise to episodic changes in network activity. Three active units (A-C) from different locations on the same MMEP, demonstrate both coincident and independent firing. *, episodes of significant changes in firing rate detected by CLUSTER7. Four coincident peaks among all 3 active units were detected at 115, 140, 155, and 210 min. Additional coincident peaks were observed with units 1 and 3 at 30, 60, and 200 min that were absent with unit 2. D: sum of active units 1-3.

To investigate possible mechanisms of cell-cell communication, glutamatergic AMPA/kainate receptors and GABAA receptors were tested for effects on firing rate using their respective antagonists, CNQX and bicuculline. Although these agents block the primary means of synaptic communication in the hypothalamus (Decavel and van den Pol 1990; van den Pol et al. 1990), they had no effect on firing rate of GT1-7 cells on MMEPs (CNQX n = 19 active units on 4 MMEPs, average firing rate: 0.23 ± 0.06 to 0.24 ± 0.08 Hz with CNQX; bicuculline n = 14 active units on 4 MMEPs, average firing rate 0.14 ± 0.06 to 0.30 ± 0.15 with bicuculline). Although there was a tendency toward increased activity in the presence of bicuculline, this was not statistically significant as responses were variable, with some units increasing firing, some decreasing and some remaining unchanged. The role of GnRH peptide as a possible communication mechanism was not examined, as the GT1 cells used in these studies do not express message for known GnRH receptor when evaluated by nested RT-PCR (S. J. Susalka and S. M. Moenter, unpublished observations).

Some active units appeared not to participate in every episode identified in composite activity profiles, a phenomenon that may be relevant to the mechanisms underlying the production of the GnRH secretory pattern. For example, peaks in firing rate at ~30 and 60 min occurred in the active units illustrated in Fig. 5, A and C, that were absent in B. Of the total episodes detected in composites, an average of 35% (range 12-65%) were generated by a single active unit; examples of such independent activity were observed on all seven MMEPs. These units can only be regarded as independent with regard to the area of the culture being monitored. That is, these units could be coordinated with active units outside of the region being observed. On occasion, different active units within a culture exhibited very different patterns (Fig. 6, A and B). The sum of these units produced a composite activity pattern with intervals between episodes that were more consistent than those of the individual active units (Fig. 6C).



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Fig. 6. Records from 2 active units on the same MMEP (A and B) display independent activity. *, episodes of significant change in firing rate detected by CLUSTER7. C: sum of active units in A and B. Inter-episode intervals: A, 59.0 ± 26.6 min; B, 53.6 ± 18.5 min; and C, 33.5 ± 4.4 min (mean ± SE). Note increased regularity of episodes and decreased inter-peak interval for the summed versus individual active unit data.


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The extracellular recordings of the present study reveal GT1-7 cells produce episodic increases in firing rate, suggesting episodic activity is intrinsic to the GnRH neural network. This supports and extends previous reports that used the frequency of pulsatile GnRH release by GT1 cells as a measure of episodic GnRH-pulse generator activity (Besecke et al. 1994; Krsmanovic 1993; Martinez de la Escalera et al. 1992; Pitts et al. 2001; Wetzel et al. 1992). The similarity of the interval between the increases in firing rate monitored in this study and the release of GnRH in previous reports is consistent with the hypothesis that increased firing rate is coupled to secretion as has been demonstrated in other neuroendocrine systems (Cazalis et al. 1985; Dutton and Dyball 1979; Summerlee and Lincoln 1981). Both coincident and independent firing among cells contributed to the overall pattern of activity, an observation consistent with the hypothesis that the overall pattern of the GnRH-pulse generator may emerge as a network property.

Whether or not increases in firing rate are associated with pulses of hormone release is a persistent question. Due to limitations of GnRH assay sensitivity, we were unable to detect spontaneous secretion at intervals under 10 min from the small number of cells grown on MMEPs; this did not provide the necessary time resolution for pulse detection. We thus cannot definitively conclude that these episodes of electrical activity underlie GnRH release. The interval between electrical episodes in the present study is essentially identical to the intervals between secretory pulses previously reported for this cell line when techniques compatible with greater numbers of cells were utilized (Besecke et al. 1994; Krsmanovic 1993; Martinez de la Escalera et al. 1992; Pitts et al. 2001; Wetzel et al. 1992). This is consistent with the hypothesis that these events are causally linked.

This link is strengthened by the present observation that both firing rate and GnRH secretion simultaneously increased in response to the potassium-channel blocker 4-AP. This implies an A-type potassium current has a role in determining membrane excitability in GT1 cells and perhaps in GnRH neurons. In various model systems, 4-AP has been shown to depolarize membranes (Kita et al. 1985), enhance synaptic input (Schoppa and Westbrook 1999), broaden action potentials associated with Ca2+ influx (Rogawski and Barker 1983), and increase the number of transmitter quanta released by single presynaptic nerve stimulation (Lundh 1978). Such effects are consistent with the observed increases in both firing rate and secretion. Moreover, TTX, which eliminated electrical activity in the present study, has previously been reported to decrease GnRH release (Mellon et al. 1990). These data agree with other studies reporting that depolarization of cell membranes increased both Ca2+ oscillations (Charles and Hales 1995) and GnRH secretion (Krsmanovic et al. 1992; Mellon et al. 1990) in GT1 cells and cultured GnRH neurons (Keen et al. 2000; Terasawa et al. 1999).

In vivo studies have made similar indirect links between electrical activity recorded in the hypothalamus and GnRH secretory patterns by demonstrating volleys of multi-unit electrical activity are invariably associated with secretion of LH (Hiruma and Kimura 1995; Tanaka et al. 1995; Wilson et al. 1984). Related studies in cultured olfactory placode GnRH neurons from monkeys suggest these cells coordinate rapid calcium oscillations at an interval consistent with secretion (Terasawa et al. 1999). A very recent report from this latter model system suggest only those calcium oscillations that are coordinated among many GnRH neurons are associated with the uptake of the lipophilic dye FM 1-43, a marker of exocytosis (Keen and Terasawa 2000). Both the pattern of electrical activity observed in vivo and the interval between coordinated calcium oscillations in primary cultures are similar to that shown in the present study, in which GnRH-derived GT1 cells were the only cell type present. A possible interpretation of these combined results is that networks of GnRH neurons in vivo are endogenously rhythmic and thus produce a significant component, if not all, of the hypothalamic electrical activity associated with luteinizing hormone release.

MMEPs are well suited to study endogenous rhythmic patterns in GT1 cells because this noninvasive technique allows for long-term recordings necessary to observe episodic patterns of electrical activity. These results differ, however, from most previous electrophysiological studies of GT1 cells in which no episodic changes in firing rate consistent with the pattern of hormone release were observed (Charles and Hales 1995; Costantin and Charles 1999; Krsmanovic et al. 1992; Van Goor et al. 1999). Due to the relative brevity of these patch-clamp recordings (several minutes vs. several hours on MMEPs in the current study), changes in firing pattern over this duration would have been difficult to detect. The one report of long-term patch-clamp recordings of these cells suggested that two GT1-7 cells were active briefly at 20- to 30-min intervals, consistent with the present observations (Bosma 1993).

Because GT1 cells are tumor-derived, it is important to consider the role cell cycle activity may play in the generation of the episodic activity observed. In this regard, the doubling time of GT1 cells indicates a cell cycle of ~48 h, roughly two orders of magnitude greater than the interspike interval observed in the present study, suggesting these rhythmic events are not causally linked. Moreover, whole cell recordings of nondividing GFP-expressing GnRH neurons in brain slices demonstrated bursts of activity flanked by periods of quiescence of <= 30 min (Suter et al. 2000), consistent with the interval between episodes observed in this study.

MMEPs are also well suited to study network interactions because they simultaneously record firing rate at multiple locations. Thus unlike secretory studies that describe the integrated output of the entire culture, MMEPs can unmask, to some extent, how individual cells or groups of cells behave with regard to others. Since the locations of active units were known for many recordings, attempts were made to determine if patterns of activity emerged and spread from a single location. In most cases, episodes began robustly and coincidentally at all participating locations, suggesting close coordination throughout the recording area. This conclusion is limited to the resolution of data collection, in this case 2 min. In this regard, a previous study that monitored intracellular calcium levels every second also failed to identify consistent foci of activity in the GT1-7 cell line (Charles and Hales 1995). In the present study, when a single unit did precede others during one episode, the same unit did not persistently lead in initiating subsequent episodes nor did any units consistently lag. There was also no indication that the likelihood of firing depended on location. Together, these results tentatively suggest that there was not a single specific pacemaker that dictated the activity of the culture.

Although it is difficult to conclude regarding focal points for activity generation, a more discernable aspect of the observed patterns was the number of active units that contributed to them. Specifically, independent episodes recorded on a single amplifier channel were frequently the only contributors to an increase in firing rate in the composite pattern. If activity within the area of the electrode array on the MMEP (<0.1% of the culture area) is representative of cell participation rates throughout the culture and electrical activity is indeed correlated with hormone release as hypothesized, then some secretory pulses may involve more cells than others. In vivo, the GnRH pulse generator is characterized by changes in both frequency and amplitude (Levine and Duffy 1988; Moenter et al. 1991), but the cellular bases for these changes are unknown. Although speculative, selective participation observed presently in GT1 cells may suggest that a change in the number of GnRH neurons firing is one possible mechanism for modulation of pulse amplitude. This hypothesis remains to be tested.

When activity was observed at multiple locations, episodes were typically coincident. As a result, we were interested in exploring possible mediators of network communication. In vivo, coordination among widely dispersed GnRH neurons is presumed necessary to produce episodic hormone signals large enough to promote the downstream pituitary activity required for reproductive function. Many neurotransmitters, diffusible factors and other mediators have been reported to affect GT1 cells under various culture conditions (Moenter and Weiner 1995). We focused on testing if blockade of the excitatory (glutamate) and inhibitory (GABA) neurotransmitters altered activity in GT1-7 cells for three reasons. First, these are the major excitatory and inhibitory, respectively, neurotransmitters in the hypothalamus (Decavel and van den Pol 1990; van den Pol et al. 1990). Second, these are the only transmitter systems for which there is functional evidence for direct action on GnRH neurons (Spergel et al. 1999), suggesting relevance to in vivo function. Third, GnRH neurons have small clear vesicles that suggest they have a traditional neurotransmitter release product that could be used in network coordination (Jennes et al. 1985). The non-NMDA antagonist CNQX failed to block activity and the GABAA antagonist bicuculline failed to increase firing rate, suggesting GT1-7 cells do not stimulate or inhibit one another, respectively, via these synaptic mechanisms. These data must be interpreted with caution as the phase of the pulse generator could affect results. Further, if activity were a spontaneous product of individual cells, synaptic blockade would not have an effect. Additional studies will be needed to determine the mechanisms involved in pulse propagation.

Of note, the observed synchronization appears to be independent of communication via GnRH peptide, as these GT1-7 cells do not express the known GnRH receptor (S. J. Susalka and S. M. Moenter, unpublished observations). Whether or not GT1 cells express the GnRH receptor appears to vary, but episodic secretion has been reported both in the presence (Krsmanovic et al. 1993) and absence (Pitts et al. 2001) of expression of the GnRH receptor. This suggests GnRH peptide is either not necessary for the generation of coordinated pulses (in GT1 cells) or is redundant with another mechanism for GT1 cells to coordinate activity. The issue of whether GnRH neurons in vivo express the GnRH receptor remains to be resolved, although there is a report that cultured primary GnRH neurons express GnRH receptor peptide after several days in vitro (Krsmanovic et al. 1999).

The episodic patterns of firing rate recorded from GT1-7 cells in the present study display characteristics one would associate with a pulse generator. The ultradian interval of this activity was consistent with previous reports of secretory pulses of GnRH from these cells (Besecke et al. 1994; Krsmanovic 1993; Martinez de la Escalera et al. 1992; Pitts et al. 2001; Wetzel et al. 1992) and induced increases in firing rate correlated with increases in secretion. Activity was often coincident, but on occasion small populations of cells appeared to be the sole contributors to the overall pattern. The emergence of the overall pattern from components at different locations suggests that although individual GnRH neurons are perhaps intrinsically rhythmic, the GnRH-pulse generator emerges as a network property of GnRH neurons.


    ACKNOWLEDGMENTS

We thank Dr. Pei-San Tsai, S. Sullivan, and J. Saunders for editorial suggestions. We also thank Dr. Guenter Gross and his lab for valuable assistance with MMEP culturing techniques, Dr. Edward Blumenthal for MMEP trouble-shooting, Dr. Richard I. Weiner for the GT1 cell lines, A. Walter for technical support, and T. Breeden for software and data interpretation.

This work was supported by National Institute of Child Health and Human Development (NICHD) Grant HD-34860 (S. M. Moenter), the NICHD/National Institutes of Health through cooperative agreement U54HD-28934 as part of the specialized cooperative centers program in reproduction research, and the National Science Foundation Center for Biological Timing.

Present addresses: M. E. Geusz, Dept. of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403; E. D. Herzog, Dept. of Biology, Washington University, St. Louis, MO 63130; G. R. Pitts, Dept. of Physiology and Biophysics, University of Kentucky, Lexington, KY 40536.


    FOOTNOTES

Address for reprint requests: S. M. Moenter, Dept. of Internal Medicine, PO Box 800578, University of Virginia, Charlottesville, VA 22908 (E-mail: smm4n{at}virginia.edu).

Received 21 November 2000; accepted in final form 19 March 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society