Department of Neurology, UCLA School of Medicine, Los Angeles, California 90095-1769
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ABSTRACT |
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Costantin, James L. and Andrew C. Charles. Spontaneous Action Potentials Initiate Rhythmic Intercellular Calcium Waves in Immortalized Hypothalamic (GT1-1) Neurons. J. Neurophysiol. 82: 429-435, 1999. GT1-1 cells exhibit spontaneous action potentials and transient increases in intracellular calcium concentration ([Ca2+]i) that occur in individual cells and as spatially propagated intercellular Ca2+ waves. In this study, simultaneous cell-attached patch-clamp recording of action currents (indicative of action potentials) and fluorescence imaging of [Ca2+]i revealed that Ca2+ transients in GT1-1 cells were preceded by a single action current or a burst of action currents. Action currents preceded Ca2+ transients in a similar pattern regardless of whether the Ca2+ transients were limited to the individual cell or occurred as part of an intercellular Ca2+ wave. Both the action currents and Ca2+ transients were abolished by 1 µM tetrodotoxin. Removal of extracellular Ca2+ abolished all spontaneous Ca2+ transients without inhibiting the firing of action currents. Nimodipine, which blocks L-type Ca2+ currents in GT1-1 cells, also abolished all spontaneous Ca2+ signaling. Delivery of small voltage steps to the patch pipette in the cell-attached configuration elicited action currents the latency to firing of which decreased with increasing amplitude of the voltage step. These results indicate that spontaneous intercellular Ca2+ waves are generated by a propagated depolarization, the firing of action potentials in individual cells, and the resulting influx of Ca2+ through L-type Ca2+ channels. These patterns of spontaneous activity may be important in driving the pulsatile release of GnRH from networks of cells.
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INTRODUCTION |
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Rhythmic release of hormones is a fundamental
property of the endocrine reproductive axis. The rhythmic, pulsatile
release of luteinizing hormone (LH) from the anterior pituitary is
driven by the pulsatile release of gonadotropin releasing hormone
(GnRH) from neurons located in the hypothalamus (Clarke and
Cummins 1982; Knobil 1980
). Multiple lines of
evidence indicate that the ability to release GnRH in a pulsatile
fashion is an intrinsic property of GnRH neurons. Hypothalamic explants
and primary cell cultures derived from the hypothalamus have been shown
to release GnRH in a pulsatile manner (Bourguignon et al.
1993
; Krsmanovic et al. 1992
). However, the
mechanisms of GnRH release by individual cells and the processes by
which the activity of a network of neurons is coordinated to produce a
pulsatile pattern of GnRH release remain poorly understood.
GnRH neurons in vivo exhibit changes in spontaneous
electrophysiological activity that are correlated with pulsatile
hormone release. LH pulses have been correlated with increases in
multiunit activity (MUA volleys) recorded in the medial basal
hypothalamus of the rat (Kawakami et al. 1982), rhesus
monkey (Knobil 1988
; Wilson et al. 1984
),
and goat (Mori et al. 1991
). Examination of the single
unit component of the MUA volley suggests that the increased activity
is a result of an increase in the firing rate of individual neurons
rather than the recruitment of new bursting neurons (Cardenas et
al. 1993
).
The development of immortalized cell lines of GnRH releasing neurons
has provided the opportunity to study the electrophysiological activity
of GnRH neurons in a reliable and accessible in vitro preparation. The
GT1 cell lines are immortalized GnRH neuronal lines derived by
genetically targeted tumorigenesis in transgenic mice (Mellon et
al. 1990). GT1 cells are highly differentiated neurons both
morphologically and functionally (Wetsel 1995
), and they
release GnRH in a pulsatile manner with a 25- to 35-min interpulse interval (Martinez de la Escalera et al. 1992
;
Wetsel et al. 1992
). Multiple clones of the GT1 line
were established, including the GT1-1 and GT1-7 lines. Both GT1-1
and GT1-7 cells exhibit spontaneous action potentials and
Ca2+ transients in individual cells
(Charles and Hales 1995
; Hales et al.
1994
). In addition, the GT1-1 line (but not the GT1-7 line) shows waves of increased
[Ca2+]i that are
propagated across fields of hundreds of cells (Charles et al.
1996
). The existence of intercellular
Ca2+ waves in GT1-1 cells, the property of dye
coupling between these cells, and the levels of expression of the gap
junction protein connexin26 suggest that the Ca2+
waves are propagated primarily via gap junctions (Charles et al.
1996
).
To further investigate the mechanisms of intrinsic electrophysiological activity of GT1-1 cells and the processes by which this activity is coordinated in networks of cells, we have examined the spontaneous activity of GT1-1 cells with simultaneous cell-attached patch-clamp recording of a single cell and video imaging of [Ca2+]i from the entire field of cells containing the patched cell.
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METHODS |
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Electrophysiological recording
CELL-ATTACHED PATCH-CLAMP RECORDING.
Action currents were measured in the cell-attached configuration
(Hamill et al. 1981) as a capacitive current that
charges the membrane (Charles and Hales 1995
;
Fenwick et al. 1982
; Peters et al. 1989
).
The advantage of measuring action currents in the cell-attached
configuration, as compared with whole cell recording of action
potentials, is that the intracellular contents of a cell are not
disturbed while action currents are monitored. The records are
displayed as they appeared on the oscilloscope, i.e., patch outward
ionic current in the cell-attached configuration is plotted as a
negative (downward) current. The patch is voltage clamped to 0 mV
relative to the bath. The capacitive current that is measured when the
cell fires an action potential appears as a brief spike in the downward
direction. The bath and pipette solution consisted of (in mM) 137 NaCl,
2.8 KCl, 2.0 MgCl2, 1.0 CaCl2, 10 glucose, and 10 HEPES (pH 7.2 with
NaOH); nominally Ca2+-free solution was the same
except 1.0 mM CaCl2 was not added. Axon
instruments software (Axoscope and pClamp 7) and a Digidata 1200 A/D
board were used to record the action currents onto a Pentium PC.
WHOLE CELL PATCH-CLAMP RECORDING.
Ca2+ channels were recorded using a bath solution
containing (in mM): 115 NaCl, 5.4 CsCl, 10.8 BaCl2, 1.0 MgCl2, 10 glucose, and 10 HEPES plus 0.5 µM TTX (pH 7.2 adjusted with NaOH),
and a pipette solution containing (in mM): 130 CsCl, 10 EGTA, 3 MgATP, and 10 HEPES (pH 7.2 adjusted with CsOH). The leak subtraction voltage
protocol used was P/N = 4 (Bezanilla and Armstrong
1977
).
VIDEO FLUORESCENCE IMAGING.
Changes in [Ca2+]i were
measured after loading the cells in the bath solution with 5 µM fura2
(Calbiochem) for 30-40 min. We measured
[Ca2+]i with a
fluorescence imaging system and analysis methods described previously
(Charles et al. 1991). Some sequences of fura2
fluorescence were recorded to videotape and subsequently digitized
using the Axon Imaging Workbench software and Digidata 2000 A/D board
(Axon Instruments). The change in fura2 fluorescence measured with
380-nm excitation (
F) is shown in pixel intensity values
(0-255 scale). The bath solution used for video fluorescence
experiments was the same as that used for cell-attached patch-clamp recording.
CORRELATING VIDEO IMAGING WITH ELECTROPHYSIOLOGY. Synchronization of the cell-attached electrophysiological experiments with the fluorescence microscopy was achieved by simultaneously placing an audio tag on the videotape and a digital tag on the Axoscope file. The tags were aligned with respect to time by the frame number on the video tape and by their position on the Axoscope file. A single-frame animation video cassette recorder (Sanyo GVR-S950) was used. The Society of Motion Picture and Television Engineers (SMPTE) method of identifying individual frames was used. The SMPTE time code was "striped" onto audio channel 2 using the VCR and the audio tag on channel 1 was assigned a frame number. Thus individual tags on the Axoscope file could be correlated with a frame number on the videotape.
CELL CULTURE. GT1-1 cells were a generous gift of Dr. Richard Weiner. Cells were maintained in culture in DMEM/F12 media (Mediatech, Cat No. 10-092) supplemented with 5% fetal bovine serum, 5% horse serum, 100 IU/ml penicillin and 100 µg/ml streptomycin in 252 cm flasks. Cells were grown to ~60-80% confluency and then passaged or transferred onto poly-D-lysine-coated glass coverslips, on which they were grown for 3-10 days to a confluence of ~60-80% before experimentation.
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RESULTS |
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Simultaneous measurements of electrical activity and [Ca2+]i in GT1-1 cells revealed that each increase in [Ca2+]i was preceded by a single action current or a burst of action currents (n = 6 different coverslips). The patterns of action currents observed with simultaneous measurements of [Ca2+]i were consistent with independent measurements of action currents (n = 42) and [Ca2+]i (n = 33). A typical example of a continuous recording of action currents (spikes) measured using the cell-attached patch-clamp technique (Fig. 1A) is shown with the corresponding [Ca2+]i measurements obtained using fluorescence microscopy (Fig. 1B). The Ca2+ oscillations followed the action currents after a variable latency. A small percentage of action currents were not associated with an increase in [Ca2+]i (Fig. 1Ae). There was considerable variability in the duration of action current bursts and in the number of spikes per burst (Fig. 1C). In most cases, subsequent bursts of action currents occurred only after [Ca2+]i had recovered fully from the increase associated with the previous burst.
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Groups of 5-50 cells within each field of 100-200 GT1-1 cells showed repetitive intercellular Ca2+ waves with consistent sites of initiation and patterns of propagation (Fig. 2). Ca2+ waves within an individual group were in some cases confined to that group, in some cases propagated to an adjoining group, and in some cases initiated by a wave originating in an adjoining group. GT1-1 cells appeared to have groups of cells that behaved as "initiators" and groups of cells ("followers") that exhibited Ca2+ oscillations only as part of an intercellular wave propagated from neighboring cells. Action currents preceded the increase in [Ca2+]i in all experiments regardless of whether the cell being measured was an initiator or a follower. The data in Fig. 1 are from a cell within a group of initiator cells, whereas the data in Fig. 2C are from a follower cell. The Ca2+ waves propagated to varying degrees away from the sites of initiation. An extensive wave is shown in Fig. 2A (a-f); the wave initiated in the top right corner of the field and propagated to the left and down, involving almost the entire field of cells. The position of the on-cell recording pipette is seen in a bright-field image (Fig. 2B). The acquisition time of each individual frame (a-f) is indicated on the tracing of measured electrical activity (Fig. 2C). Note that the burst of action currents occurs before the increase in [Ca2+]i seen near the pipette. A more limited wave that initiated in the same group of cells but propagated to fewer cells is shown in Fig. 2D. The temporal pattern of the extensive and limited intercellular waves was consistent throughout the 30-min recording. The limited waves occurred with a regular pattern having a mean interwave interval of 18 ± 0.25 (SE) s. The extensive waves appeared less frequently with an interwave interval of 24 ± 2 s. A raster plot from the same field of cells during a 5.5-min time period is shown in Fig. 2E. Each row of the plot corresponds to the cell indicated by the black line. Increases in [Ca2+]i within individual cells are seen as bright bands within each row. The more extensive waves can be seen as bright vertical bands that traverse the entire plot while the more limited waves do not. The extensive waves appeared to cluster in groups of two to four waves separated by a longer time interval between clusters. Cells within sites of initiation and cells acting as followers were seen consistently in all experiments (n = 39).
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The measured action currents in GT1-1 neurons could be due to voltage-gated Na+ channels, Ca2+ channels, or both. Bath application of the potent voltage-gated Na+ channel blocker TTX completely abolished the action currents (Fig. 3A; n = 8 cells) and Ca2+ waves (Fig. 3B; n = 10 coverslips). The L-type Ca2+ channel blocker nimodipine blocked the Ca2+ waves (Fig. 3C) but not the action currents (data not shown; n = 4). The action currents did not require the influx of Ca2+; they continued to fire after the removal of extracellular Ca2+ (Fig. 4A; n = 7 cells). Removal of extracellular Ca2+ caused a cessation in the Ca2+ waves that was reversible after a few minutes (Fig. 4B; n = 7 coverslips). These results indicate that the action currents are generated by Na+ action potentials and that these action potentials are required to produce repetitive Ca2+ transients through the activation of voltage-gated Ca2+ channels.
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The voltage-gated Ca2+ currents present in GT1-1
cells were characterized in terms of their sensitivity to voltage and
Ca2+ channel toxins. Ca2+
channels are subdivided broadly based on the degree of depolarization required to open them. Low-voltage-activated (LVA)
Ca2+ channels are opened by small depolarizations
from the holding potential; high-voltage-activated (HVA)
Ca2+ channels require depolarizations near 40
mV and above. HVA channels are subdivided further based on their
sensitivity to peptide toxins and dihydropyridines (DHPs)
(Birnbaumer et al. 1994
). L-type HVA channels are
blocked by DHP antagonists (such as nimodipine) and enhanced by DHP
agonists. N-type HVA channels are blocked by the cone snail toxin
-conotoxin GVIA (
-CTx GVIA). P-type HVA channels are blocked by
the funnel web spider toxin
-agatoxin IVA (
-Aga IVA). The
currents through voltage-gated Ca2+ channels were
measured in GT1-1 cells using Ba2+ as the charge
carrier (Fig. 5). Currents elicited by
voltage steps from the holding potential (
90 mV) to depolarized
potentials are shown in Fig. 5A. The corresponding
current-voltage (I-V) curve of peak inward currents is
plotted (Fig. 5B). The I-V relationship is
typical of HVA channels; the peak of the curve is near 10 mV and
currents activate near
40 mV. There is an additional shoulder of
current near
50 mV that may indicate that there is a component of LVA
channels in these cells (Nowycky et al. 1985
). The
inward Ca2+ current was blocked completely by 200 µM Cd2+ (Fig. 5, C and
D). The HVA current was not an N- or P-type current as the
addition of
-CTx GVIA or
-Aga IVA had no effect on inward currents Fig. 5C (n = 5, both toxins). The
time course of the blockade of peak currents by 200 µM
Cd2+ as well as the lack of effect of N- and
P-type channel blockers is shown in Fig. 5D. Nimodipine
reduced the peak HVA currents by 0.70 ± 0.15 (n = 6) when compared with control currents (Fig. 5E). The time
course of the current reduction by 50 µM nimodipine is shown in Fig.
5F. These results indicate that the HVA channels present in
GT1-1 cells are predominantly L-type. There is, however, a residual
component of HVA current that remains after the addition of nimodipine.
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The measured cell-attached current waveform in GT1-1 cells consisted of at least two components; the Na+ action currents and a sustained negative current of varied duration. The sustained current occurred near the end of a burst of action currents, and in some cases did not occur at all (Fig. 6). Action currents within a burst typically showed a progressively declining amplitude. Small spikes suggestive of truncated action currents (Figs. 1C, 6A, and 7) often were superimposed on the sustained negative current. Figure 6A shows three bursts of action currents with the sustained current, whereas a fourth burst had no sustained current. Recordings from two different cells (Fig. 6, B and C) illustrate further the temporal relationship between the two components as well as some of the variability seen in the duration of the sustained current.
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The duration of action current bursts and the amplitude and duration of their accompanying sustained negative current were correlated with the amplitude and duration of Ca2+ transients (Fig. 7). Single action currents were often not associated with increases in [Ca2+]i. Longer trains of action currents with larger sustained negative currents were associated with longer and higher-amplitude Ca2+ transients.
Induction of action current firing in bovine chromaffin cells has been
shown to occur with positive voltage pulses delivered to the pipette
(Fenwick et al. 1982). The positive pulse in the pipette, while hyperpolarizing the patch itself, depolarizes the cell
as a whole (to a lesser extent). A subset of GT1-1 cells (<10%) was
capable of firing action currents in response to very small voltage
pulses delivered to the pipette. A previously quiescent cell that fired
action currents in response to small depolarizing pulses is shown in
Fig. 8. An increase in the magnitude of
the pulse delivered to the pipette caused an increase in the
probability of action current firing (Fig. 8A) as well as a
decrease in the latency to firing (Fig. 8B).
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DISCUSSION |
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GT1-1 cells exhibit intrinsic activity characterized by
spontaneous action potentials and increases in
[Ca2+]i that are either
limited to individual cells or propagated to neighboring cells as an
intercellular Ca2+ wave. The results presented
here show that each increase in
[Ca2+]i in an individual
cell is preceded by a burst of action potentials. The ionic mechanisms
involved in the individual cell Ca2+ oscillations
of GT1-7 and GT1-1 cells appear to be the same (Bosma 1993; Charles and Hales 1995
). In both clones of
the GT1 line, TTX blocks the action currents and the
Ca2+ oscillations. Nimodipine or removal of
extracellular Ca2+ eliminates the
Ca2+ oscillations but not the spontaneous action
currents. Ca2+ oscillations continue after
depletion of intracellular stores with thapsigargin, and agents that
cause Ca2+-induced Ca2+
release have no effect on Ca2+ signaling
(Charles and Hales 1995
). These results are consistent with a model previously proposed in which a spontaneous membrane depolarization leads to the firing of
Na+-dependent action potentials, further cell
depolarization, and an influx of Ca2+ through
voltage-gated Ca2+ channels (Charles and
Hales 1995
).
The abolition of Ca2+ signaling by TTX shows that the firing of Na+ -dependent action potentials is necessary for Ca2+ oscillations to occur. This finding suggests that depolarizations of the membrane potential that underlie the spontaneous activity in GT1-1 neurons are not sufficient to cause influx of Ca2+ without the firing of Na+ action potentials. A significant implication of this property is that the intercellular communication of a Ca2+ response requires the firing of action potentials in a neighboring cell, thereby creating a threshold for intercellular communication. Additionally, the finding that action potential bursts continue following the removal of extracellular Ca2+, despite the abolition of all measurable Ca2+ signaling, indicates that the spontaneous electrical activity of GT1-1 cells does not require increases in [Ca2+]i.
GT1-1 cells differ from GT1-7 cells in that they are capable of
Ca2+ signaling within groups of cells through
coordinated intercellular Ca2+ waves. Our
previous studies provide evidence that these intercellular Ca2+ waves are propagated through gap junctions
composed of connexin26 (Charles et al. 1996). The
results presented here are consistent with the hypothesis that the
intercellular Ca2+ waves are mediated by a
propagated depolarization rather than by the diffusion of
Ca2+ itself. Ca2+ increases
in individual cells always were preceded by action potentials
if the
diffusion of Ca2+ was responsible for the
propagation of the response, one would expect to see some increases in
[Ca2+]i that were not
preceded by action potentials. This was never observed. The conclusion
that the intercellular Ca2+ waves are propagated
by depolarization also is supported by the observation that individual
cells showed large increases in
[Ca2+]i that in some
cases occurred as part of an intercellular wave and in some cases were
limited to the individual cell.
The Ca2+ waves consistently originated from small groups of cells (initiators) and propagated through the field across cells acting as followers. The initiator cells were frequently near the edge of a field of cells or adjacent to a gap containing no cells. Otherwise there were no distinguishing morphological or electrophysiological characteristic of the cells that acted as initiators. These observations suggest that initiator cells do not represent a distinct subpopulation of cells but rather that they become initiators of intercellular activity because of their position in a field of cells and the amount of input they receive from neighboring cells.
The sustained negative current measured in the cell-attached configuration associated with a burst of action currents was correlated with the occurrence of a significant increase in [Ca2+]i in that cell. At this time, we have not definitively identified the source of the current. The evidence that the sustained current occurs near the end of a burst of Na+ spikes leads us to believe that it is activated when a cell becomes strongly depolarized. There are at least two possible explanations for this current. The first possibility is that the current is due to outward ionic currents passing through voltage-activated K+ channels in the patch of membrane that is delimited by the pipette. A second possibility is that the sustained current is due to a decrease in the resistance of the cell membrane due to the opening of voltage-gated Ca2+ and K+ channels that cause a short-circuiting of the membrane and a deformation of the action current waveform.
The small depolarizing stimulus required to evoke the firing of action potentials indicates that GT1-1 cells have a resting potential that is close to the threshold for action potentials (Fig. 8). In addition, the latency to firing and the probability of action potential firing depends on the strength of the depolarizing input. Because each GT1-1 cell receives input from multiple neighboring cells, different patterns of action potential bursts may result from the integration of these multiple inputs. Different patterns of action potential bursts evoke Ca2+ signals with different amplitudes and durations as shown in Fig. 7. Differences in the patterns of Ca2+ signaling may be important for changes to Ca2+-dependent GnRH release by individual cells.
GT-1 cells have been shown to possess the features of the GnRH pulse
generator: namely rhythmic, pulsatile release of GnRH with a frequency
similar to that measured in rodents (Kawakami et al.
1982; Martinez de la Escalera et al. 1992
). At
this stage, it is not clear how the patterns of electrical and
Ca2+ signaling in GT1-1 cells are related to a
pulsatile pattern of GnRH release. Pulsatile release could be
stimulated by a change in the frequency of spontaneous signaling, a
change in cellular coupling, a change in secretory function, or some
combination of the above. We are presently pursuing simultaneous
measurement of GnRH secretion by GT1-1 cells to identify patterns of
electrical and Ca2+ signaling that are correlated
with pulsatile GnRH release.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grants R29-NS-32283 and P01-NS-02808 to A. C. Charles.
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FOOTNOTES |
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Address reprint requests to J. L. Costantin.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 December 1998; accepted in final form 23 March 1999.
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REFERENCES |
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