Departments of Internal Medicine, Cell Biology, and Biology and National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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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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RESULTS |
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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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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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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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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 × 1012 (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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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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DISCUSSION |
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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.
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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.
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FOOTNOTES |
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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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REFERENCES |
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