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Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425
Address all correspondence and requests for reprints to: Dr. L. Stephen Frawley, Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425. E-mail: frawleys{at}musc.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important advance in our understanding of the cellular basis for GnRH pulsatility was the finding that GnRH release from populations of GT1 clones, immortalized mouse GnRH neurons (5), was episodic (6, 7, 8, 9, 10) with an interpulse interval comparable to that observed for primary cultures from castrated rodents (11). From this and other work, several groups have speculated that the pulse generator must reside at the level of either the neuronal network (6, 12, 13) or the individual cell (7, 8, 9, 10). In the former scenario, it can be envisaged that individual cells might harbor the ability to release GnRH, but that this would lack an episodic dynamic in the absence of network connectivity. In the latter scenario, individual neurons might release GnRH in a pulsatile fashion, independent of their network connectivity. Unfortunately, there is currently little basis to distinguish between these possibilities because the available evidence for secretion has been derived entirely from cell populations that provide limited insight into the contribution of single cells to secretory dynamics.
Recently, Betz and colleagues adapted the use of FM1–43 for monitoring granule to plasma membrane fusion in living cells during exocytosis (14, 15). Upon fusion, the granule membranes become exposed to and are subsequently labeled by this fluorescent dye present in the culture medium. Much of this newly incorporated dye is internalized during the endocytotic vesicle formation that follows exocytosis. Thus, this dye provides an exceptional marker for membrane changes associated with the secretory process. In previous work we adapted this technique for use with individual GT1–7 cells (16). Our results demonstrated clearly that FM1–43 uptake was increased after treatment with GnRH stimulatory secretagogues and inhibited with exposure to an inhibitory secretagogue. These findings indicated that many of the changes in FM1–43 fluorescence reflect membrane changes associated with the secretory process in individual cells. In the current study we further confirmed that this technique can be used as a marker of exocytosis in single cells and determined the impact of cell-cell communication on GT1–7 pulsatile activity. This strategy enabled us to address the compelling issue of whether the pulse generator resides in the GT1–7 neuronal network or the individual neuron.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monitoring of FM1–43 fluorescence
A mixture of medium 199 and DMEM/F-12 (1:1) supplemented with 1
mM HEPES and 1% streptomycin/penicillin was used as the
imaging medium. After three rinses with this medium, cells were exposed
to the fluorescent dye FM1–43
[N-(3-triethylammoniopropyl)-4-(p-dibutylaminostyryl
piridinium dibromide, 2 µM; Molecular Probes, Inc., Eugene, OR] for 30 min at 37 C. The coverslips
bearing the cells were then mounted in Sykes-Moore chambers (Bellco
Glass, Inc., Vineland, NJ). The chambers were next placed on a
temperature-controlled stage of a fluorescence microscope (Axiovert,
Carl Zeiss, Jena, Germany) fitted with a Carl Zeiss Fluar x40 oil immersion objective (numerical
aperture = 0.9), and a field of cells was selected. Then cells
were epi illuminated at 490 nm for 100–500 msec every 5 min for 2–25
h. Image acquisition was controlled using Metafluor PC-software
(Universal Imaging Corp., West Chester, PA), and fluorescence emissions
were captured using a CCD camera (C-4880–80, Hamamatsu,
Hamamatsu City, Japan) running in 10-bit mode. The images acquired were
transferred to off-line storage for analysis at a later time. Regions
of the same field devoid of cells were selected for continuous
monitoring of the background throughout each experiment. Inasmuch as
the illumination intensity was reduced to less than 5% by placing
filters in the light path, and the absorption spectrum of FM1–43 (490
nm) approached visible light, the phototoxic effects that prolonged
exposure to light can induce were minimized. Treatments with nimodipine
(0.1 µM in imaging medium) and octanol (20
µM in imaging medium) were performed 90 and 30
min before image acquisition, respectively. Nimodipine was maintained
in the imaging medium throughout each experiment.
Evaluation of gap junction functionality
GT1–7 cells were plated onto Matrigel-coated, 25-mm coverslips
that were photoengraved with an alpha-numeric grid pattern. These
cultures were then incubated for 2–4 days or 5–7 days, after which
selected cells (1–20/field) were microinjected with a dye mixture
consisting of 1.25% Lucifer Yellow CH (LY; lithium salt;
Mr, 457 Da; Molecular Probes, Inc.)
and 1.25% rhodamine B-dextran (Mr, 10,000 Da;
Molecular Probes, Inc.) in water. After excitation at 480
nm (for LY) and 550 nm (for rhodamine), microinjected cells were
identified by their fluorescent emissions at both 528 nm (for LY) and
590 nm (for rhodamine). Gap junction functionality was estimated as the
ratio (R) of the number of cells showing LY fluorescence to the number
of cells exhibiting both LY and rhodamine fluorescence minus 1 [R
= (LY/LY and rhodamine) - 1]. The rationale for subtracting 1
was to allow the degree of functionality to equal zero in instances
where no intercellular transit of dye occurred.
Data analysis
Inasmuch as FM1–43 was maintained in the imaging medium for as
long as the data were collected, the fluorescence signal tended to
increase continuously. Increments in fluorescence are manifested in the
fluorescent profile as stair-step increases in total signal. To
demonstrate that these stepwise increases were actually reflective of
exocytosis, we studied FM1–43 incorporation in cells in which fusion
of granules to the cell membrane was blocked by cytoplasmic
microinjection of 1 mg/ml mouse monoclonal antibody to SNAP-25 (SMI 81,
Sternberger Monoclonals, Inc., Lutherville, MD). After loading with
FM1–43, microinjected cells were identified by their fluorescent
emission at 590 nm (for rhodamine dextran, which was coinjected with
the antibody (1:1), and FM1–43 incorporation was monitored as
described above. Cells injected with a nonspecific antibody (1 mg/ml
mouse IgG1 
; MOPC 21, Sigma-Aldrich Corp., St. Louis,
MO) that presents the same isotype as that of SMI 81 and
rhodamine-dextran (1:1) were used as a control.
The stair-step increases in FM1–43 uptake were better resolved by transforming our data to a differential over time (dy/dt; rate of dye uptake), which yielded profiles with identifiable upstrokes and downstrokes that could be analyzed subsequently by the PULSAR program [developed by Drs. Merriam, Kozuch, and Wachter, NICHHD, (Bethesda, MD), and University of California (Berkeley, CA)]. PULSAR enabled analysis of various pulsatile parameters, the most relevant of which were frequency and duration of peaks. Pulse amplitude was calculated from the original datasets as the difference between the areas under the curve defined by the three points following and preceding a pulse. Rises greater than 5% were considered significant deflections in FM1–43 incorporation (only 0.8% of pulses resolved by PC PULSAR analysis were rises smaller than 5% and were not considered significant pulses). To avoid false positives, we did not consider single point peaks as pulses in any of the FM1–43 profiles analyzed.
To quantify the degree of synchronization among cells within neuronal clusters, we compared the extent of synchronization with that expected if pulse coincidence were random. We used an algorithm model that consisted of a number of numeric channels corresponding to the number of cells in any given experiment. Each channel in the random model was comprised of a numeric series corresponding to the number of 5-min points in the related experimental dataset. Each time point in the random model was assigned a value based on the probability that a pulse would occur at that moment. Thus, the dataset generated for the random model paralleled that observed for the neurons, except that pulse concurrence was determined by random coincidence. This random model provided a measure of the extent to which random coincidence could be expected to occur during an experiment. Therefore, the predicted probability that more than 50% of the cells would display a pulse simultaneously was low and approached zero. We took this 50% to be a cut-off point for discrimination between random and nonrandom synchronization. The calculated index of synchronization (IOS) of pulses was, therefore, the ratio of pulse coincidence observed in more than 50% of cells to that predicted by the random model. Accordingly, a value of 1 indicated that true synchronization was low, because pulse coincidence in the experimental dataset was not different from that expected for random concurrence. The effect of time in culture on synchronization of secretory pulses was evaluated by comparing IOSs for neurons cultured less than 5 days with those cultured for at least 5 days. Under both culture conditions, clusters of neurons exhibiting more than 80% confluence were selected for study.
Statistical analysis
Statistical differences in frequency, duration, and amplitude of
peaks in the absence or presence of nimodipine or octanol, and
diffusion of LY toward neighboring cells in the absence or presence of
octanol were assessed by one-way ANOVA, followed by the Newman-Keuls
multiple comparison test. The time dependence of synchronization and
the effects of nimodipine on this phenomenon were evaluated by one-way
ANOVA followed by the Mann-Whitney U test. The effect of octanol on
synchronization was assessed by one-way ANOVA, followed by unpaired
Student’s t test. All differences were considered
statistically significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a representative
example of results obtained during long-term monitoring of FM1–43
uptake in individual GT1–7 neurons. Regardless of the degree to which
individual neurons contacted one another, each cell exhibited stepwise
fluctuations of FM1–43 uptake or pulses. These fluctuations, which are
illustrated more clearly in an expanded portion of the example (Fig. 1B), differed considerably from neuron to neuron. Indeed, these
differences were manifested as changes in both frequency and amplitude
of pulses. However, on the average, these cells displayed features of
pulsatility (frequency, 1.4 ± 0.1 pulses/h; duration of peaks,
17.3 ± 0.6 min) comparable to those reported recently for entire
cultures of perifused GT1–7 neurons (17). To further
evaluate whether these stair-step increases in dye uptake were
associated with exocytosis, we compared fluorescence profiles of
normally secreting cells to those that had been injected with SMI 81,
an antibody shown to arrest exocytosis in synaptosomal preparations
(18). As illustrated (Fig. 1C), the frequency of stepwise
increases in FM1–43 incorporation was strongly inhibited, with only an
occasional pulse observed (0.17 ± 0.06 pulses/h; n = 4; 16
cells). Cells injected with the control antibody (1.2 ± 0.1
pulses/h; n = 3; 16 cells) were indistinguishable from noninjected
controls (see above) and from temporally coincident noninjected
controls (1.1 ± 0.1 pulses/h; n = 9; 12 cells). These
results demonstrate that stair-steps increases in membrane fluorescence
actually reflect endocytosis immediately following exocytotic
bursts.
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), whereas a strong coordinated
relationship was evident in cultures maintained for at least 5 days, as
evidenced by the intermittent, temporal overlap of pulses (Fig. 2B).
Quantification of this relationship was achieved by comparing the IOS
for the two groups. As shown in Fig. 2C, the IOS for cells maintained
less than 5 days approached 1 (0.9 ± 0.1; n = 5
experiments), indicating that the degree of synchronization was not
different from that predicted by a random model. In contrast, the IOS
for cells cultured 5 days or more was 8.5 ± 2.2 (n = 7
experiments) or more than 8-fold higher than that expected to occur
randomly. This difference in the IOS reveals that synchronized
pulsatility is achieved only after several days in culture and
indicates strongly that maturation of neuronal network communication is
needed to attain this synchronized functional state. This effect on
synchronization was not an artifact of cell density, because we
selected fields in which the degrees of confluence (Fig. 2, D and E)
were similar (80.5 ± 4.5% vs. 89.6 ± 3.7% in
cells cultured for <5 and >5 days, respectively; P >
0.05).
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). These results demonstrate clearly
that the influx of Ca2+ through L-type channels
is not only involved in intrinsic pulsatility, but is also required for
synchronization of these pulses.
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, we found that diffusion of dye in
nonsynchronized (<5 days in culture) cells was limited, as evidenced
by the fact that each LY-staining cell was also stained with rhodamine.
Because rhodamine is conjugated to dextran and cannot pass through gap
junctions, this costaining would indicate that LY is present only in
the cells originally injected. In contrast, many cells in synchronized
cultures (>5 days in culture) were stained with LY (Fig. 4B, arrowheads), but not rhodamine, revealing that diffusion had
occurred (Fig. 4B). Interestingly, treatment of these synchronized
cultures with octanol (20 µM), which is known
to specifically block junctional conductance (25),
severely reduced the transfer of dye (Fig. 4C). These relationships are
illustrated more quantitatively in Fig. 4D. Notably, the degree of gap
junction functionality was about 4-fold greater for synchronized
(cultured for >5 days) vs. nonsynchronized (cultured for
<5 days) cells, and treatment with octanol abolished this difference.
These results demonstrate that octanol can inhibit functionality of gap
junctions in GT1–7 neurons. Additionally, the ability of octanol to
inhibit gap junction communication was accompanied by an inhibition of
synchronization among cells. The IOS found after administration of
octanol was about 2-fold lower than that exhibited by GT1–7 cells
under control conditions (Fig. 4E). Thus, it appears that functional
gap junctions are necessary for cell-cell communication and that this
form of communication underlies the synchronization of pulsatility in
GT1–7 cell populations. It must be emphasized, however, that the
ability of an individual cell to pulse is not dependent on gap
junctions. This is evidenced by the observation that cells in the
absence or presence of octanol exhibited similar frequency (1.8 ±
0.1 vs. 1.9 ± 0.2 pulses/h, respectively;
P > 0.05), amplitude (723.8 ± 269.7
vs. 764.2 ± 183.5 arbitrary units,
respectively; P > 0.05) and duration of pulses
(18.9 ± 1.7 vs. 17.6 ± 2.1 min, respectively;
P > 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial results made it very difficult to understand the manner in which these individual units could function in harmony until we observed that this individual neuronal activity came to be coordinately regulated after a fixed time in culture. Because this coordination did not appear to be a function of cell density, we reasoned that it reflected a maturational or differentiational process required for establishment of a functional neuronal network. Possible mechanisms that have been associated with GnRH cell function and could provide the necessary degree of signal concurrence required for synchronization include paracrine messengers, synaptic connections, and communication through gap junctions (for review, see Ref. 31). Because of identification of functional gap junctions or proteins related to these communication structures in GT1–7 cells (12, 13, 22) and the ability of gap junctions to transmit a coordinating signal among adjacent cells in a fast and effective manner, we directed our efforts to explore the participation of gap junctional communication in the maintenance of coordinated exocytosis among GT1–7 cells.
Our next series of experiments involved the use of fluorescent dyes to determine the degree of gap junctional communication between GT1–7 cells. Our ability to abolish synchronization of pulsatile activity by treatment with the gap junction inhibitor octanol demonstrates for the first time that this form of cell communication is an absolute requirement for concerted pulsatility in GT1–7 neurons. From a functional point of view, gap junctions are permeable to a variety of different secondary metabolites and intracellular ions, any of which might convey the coordinating signal required for synchronization. Included among these are cAMP (32), Ca2+, inositol trisphosphate (32, 33, 34, 35), Na+, K+, and cADP-ribose (34), but at this time, it is not known which (if any) of these is involved in synchronization of pulsatility in GnRH neurons. A recent intriguing hypothesis (36), which holds that cAMP and associated signaling events comprise an oscillatory timing mechanism in single GT1 neurons, coupled with the capacity of this nucleotide to traverse gap junctions make it a particularly strong candidate to drive both inherent and synchronized pulsatility.
Our current observations also appear to more clearly define the cellular basis for secretory pulsatility. The ability of octanol to abolish synchronization of pulsatile release patterns was in stark contrast to its lack of effect on the episodic pulsatility measured in individual GT1–7 neurons. Remarkably, the frequency, duration, and amplitude of pulsatile episodes in single cells were unaffected. Thus, in addition to defining the central role of gap junctions in synchronization, these observations also provide compelling evidence to support the conclusion that synchronization and pulsatility are to a certain extent mutually exclusive and independent processes. Indeed, the distinct ability of octanol to abolish synchronization without effects on single cell pulsatile secretory activity implies that the former depends entirely on junctional network communication and the latter does not.
Intriguingly, we found that nimodipine, an L-type, voltage-gated Ca2+ channel blocker, exerted differential effects on GT1–7 neurons depending on whether they were cultured sufficiently long to achieve synchronization. In cells cultured for less than 5 days (nonsynchronized cells), nimodipine severely attenuated pulse frequency and amplitude. The ability of nimodipine to reduce these parameters was minimized in cells that were already synchronized, demonstrating a difference in the extent to which L-type calcium channels contributed to secretory pulsatility before and after neurons were fully differentiated. Moreover, the modest partial inhibitory effect of nimodipine on pulsatile activity in single cells paled in comparison with its dramatic inhibition of synchronization. As was the case for the differential effects of octanol described above, this observation reinforces our proposal that inherent pulsatility and synchronization are fundamentally independent processes. The requirement of functional L-type channels for pulse synchronization raises the question of what mechanistic role they play. Inasmuch as we have demonstrated the necessity of gap junctions for synchronization, we must speculate further that action potential propagation could occur via bidirectional monovalent cationic flux through gap junctions. Presumably, the resulting depolarization in adjoining cells would lead to L-type calcium channel opening and further activation of calcium spiking activity. The initiation of L-type calcium channel activity could lead to a synchronized release of GnRH.
In summary, we have demonstrated clearly that the individual cell is the functional unit responsible for elaboration of pulsatility in cultures of GT1–7 neurons. The lack of LY dye transfer or octanol influence on pulsatile activity in nonsynchronized cells coupled with the paucity of evidence for other types of rapid communication between GT1–7 cells indicate that pulsatility within a given neuron does not require cell-cell communication. Moreover, we have also demonstrated that concurrence of secretory pulses among adjacent neurons relies upon the development of gap junctional channels that provide for synchronized communication. Taken together, our results indicate that two components, intrinsic cellular pulsatility and cell-cell communication, are responsible for the elaboration of synchronized secretory pulses from populations of GT1–7 cells. Our findings, when viewed in light of the extensive functional similarities between these clonal cells and hypothalamic GnRH-secreting neurons, lend credence to the possibility that these two components form the cellular basis for the elusive GnRH pulse generator in the hypothalamus.
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Acknowledgments |
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Footnotes |
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Received November 10, 2000.
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) and cells cultured for at least 5 days
(
). These data are expressed as the mean ± SEM of
five and seven independent experiments, respectively. *,
P < 0.05 vs. cells cultured for
less than 5 days. D and E, Phase (upper) and fluorescent
(lower) images of GT1–7 neurons cultured for 3 (D) and
7 (E) days after 30 min of incubation with FM1–43 (x400). Note that
the confluence of cells in both culture conditions is similar.
; n = 3
experiments; 22 regions of interest). *, P < 0.05
vs. cells cultured for less than 5 days. #,
P < 0.05 vs. cells cultured for
more than 5 days. Note that the diffusion of LY to adjacent cells
through gap junctions was more extensive in cells cultured for at least
5 days. The blockade of gap junctions with octanol inhibited the
transfer of dye to adjacent cells. E, The blockade of gap junctions was
also reflected in the synchronization of secretory pulsatility.
Presented here are the IOS values (see Materials and
Methods) of synchronized cells cultured for at least 5 days
(control conditions,