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INSERM U-469, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 34094 Montpellier Cedex 5, France; and the Department of Anatomy and Neurobiology, University of Vermont College of Medicine (J.F.), Burlington, Vermont 05405
Address all correspondence and requests for reprints to: Dr. Patrice Mollard, INSERM U-469, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France. E-mail: mollard{at}u469.montp.inserm.fr
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Abstract |
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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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The apparent requirement for cell to cell associations in the intact tissue to retain certain aspects of calcium signaling patterns (13, 14, 15) prompted us to examine calcium signaling in a preparation that preserves both the architecture of the pituitary glandular parenchyma and the occurrence of spontaneous [Ca2+]i firing (4). Recently, we reported that a large proportion of endocrine cells in acute slices from female guinea pigs exhibited in situ spontaneous [Ca2+]i transients due to electrical activity, which fired either asynchronously in cells of the same field or synchronously in neighboring cells probably coupled by gap junctions (16). However, the limited length of optical [Ca2+]i recordings (30 sec to 2 min) did not allow us to discriminate distinct spiking patterns (16). Here, the improvement of fluorescent [Ca2+]i recordings over long time scales together with numerical methods for pattern analysis has enabled us to identify a special processing of [Ca2+]i transients infrequently observed in isolated single cells. This pattern occurred spontaneously in about 40% of endocrine cells in slices prepared from male as well as cycling and lactating female rats. It was rhythmic and bistable in nature. Each cycle period was characterized by a burst during which [Ca2+]i spike frequency was relatively high and constant and an interburst interval during which [Ca2+]i spikes occurred randomly or were absent. The remainder of recordings from the endocrine cells demonstrated either sporadic [Ca2+]i transients or were silent. We also discovered that the ability of cells to fire rhythmic [Ca2+]i bursts could be regulated by hypothalamic factors. The secretagogue TRH 1) induced a bursting mode in cells that were previously either silent or randomly active and 2) finely adjusted the timing of burst generation in bursting cells without altering the average intraburst frequency of [Ca2+]i transients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Confocal cytosolic calcium imaging
Before recordings, slices were transferred to a chamber attached
to the stage of an upright microscope (Axioskop-FS, Carl Zeiss, Le Pecq, France) and continuously superfused with
Ringer’s saline at 32 C. Cells were viewed with a x63 (0.9 numerical
aperture) water immersion objective lens (Carl Zeiss) and
then exposed intermittently to 15 µM Oregon Green 488
BAPTA-1 acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR; 3 sec every 20 sec for 30 min)
delivered onto a cell field via a blunt micropipette (4). To measure in
real-time the averaged changes in Oregon Green 488 BAPTA-1 emission in
endocrine cells, a fast-scanning confocal microscope (Noran Odyssey XL,
Noran Instruments, Inc., Middleton, WI) was used (16). The bright over
time tool of the software package was applied to live images (120
images/sec with averaging every four frames). A 100-µm slit was used
for [Ca2+]i signals,
giving bright images with a 3.1-µm axial resolution. Acquired data
were processed for analysis with InterVision 1.5.1 (Noran) and Igor Pro
3.13 (Wavemetrics, Inc., Lake Oswego, OR) software. The effects of both
dye leak and photobleaching due to laser illumination were attenuated
by correcting the basal level over the recording time. The baseline of
each recording was fitted according the equation F(t) = A +
Be-Ct, where A, B, and C are parameters, F is
the fluorescence level, and t is the time. The corrected level Fc was
then obtained using the equation Fc(t) = F(t) -
Be-Ct. The correction, however, did not
completely overcome the apparent decay of burst amplitudes that
occurred during long lasting optical recordings. Because Oregon Green
488 BAPTA-1 is a single wavelength dye, its emission is a function of
both intracellular Ca2+ and dye concentrations.
[Ca2+]i changes were,
therefore, expressed as the F/Fmin ratio, where Fmin was the minimum
fluorescence intensity measured after off-line correction of the basal
level.
Burst analysis
Direct determination, during optical recordings, of the
positions of bursts was occasionally problematic because it was
difficult to accurately determine the limits of the bursts when
[Ca2+]i transients
occurred randomly between two bursts. Therefore, to analyze our
recordings as accurately as possible, we performed a frequency analysis
of the profiles of
[Ca2+]i spiking (17, 18, 19).
First, the plots of F/Fmin ratio reflecting the
[Ca2+]i changes were
transformed to exclude the contribution of the baseline noise. The
plots of [Ca2+]i were
differentiated so that the baseline fluctuated around 0.0 (see Fig. 2B
). Second, the time of occurrence of each sharp deflection above 0
and above a threshold (corresponding largely to the onset of each
[Ca2+]i transient) was
represented by a vertical line. Bursts in which several
[Ca2+]i transients
occurred were, therefore, represented by groups of closely spaced
parallel lines (raster plots). The frequency of
[Ca2+]i transients
measured using the interline intervals was then plotted as a function
of time. Third, values above a threshold, determined as one fifth of
the maximal frequency during the recording, helped identify the
boundaries of individual bursts. Only bursts that consisted of a
minimum of three separated
[Ca2+]i transients were
included in the analysis as an individual burst. Two parameters were
then used as reliable indicators of bursting behavior (8, 20): 1) the
duty cycle, defined as the proportion of one cycle period (equal to a
burst duration plus an interburst interval), in which the
[Ca2+]i bursting is on;
and 2) the bursting activity quotient (Q; the ratio between the total
duration of bursts and the duration of the recording), which
corresponds to the average duty cycle.
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Test substances
Drugs were pressure ejected from an extracellular micropipette
(tip diameter, 2–4 µm), the tip of which was positioned in the
vicinity of the recorded cells. The concentrations reported were those
in the pressure pipette. Puffer applications of Ringer’s saline had no
effect on [Ca2+]i
activity (n = 12). TRH, tetrodotoxin (TTX; a
Na+ channel blocker), nickel chloride
(Ni2+; a low threshold Ca2+
channel blocker), and Lucifer Yellow were all purchased from
Sigma (St. Louis, MO). To obtain a
Ca2+-free solution, CaCl2
was omitted from and 5 mM EGTA was added to Ringer’s
saline.
Statistics
When the spike frequency was analyzed within bursts, the bursts
were divided into five equal parts, leading to five successive groups
of [Ca2+]i transients.
Differences between mean spike frequencies were assessed by using a
paired ANOVA. Analysis of cell distribution was carried out by using a
2 test. Differences with P <
0.05 were considered significant. Numerical data are expressed as the
mean ± SD. All curve fits were performed
with the analysis menu of Igor Pro 3.13.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A–C). Recordings from cells not exhibiting a bursting mode were either
silent or randomly active (Fig. 1D). A number of variations were
encountered in the patterns of the
[Ca2+]i bursts during the
optical recordings. In the vast majority of bursting cells (called
bursters), both [Ca2+]i
spike trains and interburst intervals were irregular in length (range,
15–240 sec; Fig. 1A; n = 201). A few
[Ca2+]i spikes were also
detected during the interburst intervals. On rare occasions bursts were
identified with nearly identical epochs of oscillatory activity that
recurred on a regular basis (Fig. 1B; n = 33). Both regular and
irregular patterned
[Ca2+]i bursts were
blocked by external Ca2+ removal (in the presence
of 5 mM EGTA; 11 of 11 cells; Fig. 1C) and markedly
suppressed by either 50 µM Ni2+ (14
of 16 cells) or 5 µM TTX (8 of 15 cells; data not
shown).
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) were differentiated, and peak values
above a threshold were represented by raster plots (Fig. 2B). The spike
frequency was then plotted as a function of time (Fig. 2C). No marked
acceleration in spike frequency was noticeable within bursts
(P > 0.5). Frequency analysis also permitted us to
identify burst boundaries during optical recordings (Fig. 2C, values
above a threshold denoted by the dashed horizontal line; see
also Materials and Methods).
Distribution of spontaneously bursting cells in the anterior
pituitary
How representative is the
[Ca2+]i burst pattern in
the anterior pituitary tissue? Based on the criteria for burst
identification described above, we observed that a large proportion of
endocrine cells spontaneously fired bursts of
[Ca2+]i transients in
male, cycling female, and lactating rats (Fig. 3). More strikingly, three endocrine cell
types (lactotrophs, somatotrophs, and corticotrophs), which together
represent the majority of endocrine cells within the glandular
parenchyma (21), displayed
[Ca2+]i bursts (Fig. 4A). The remaining cells were either
randomly active or silent (Fig. 4B). In these three cell types, both
burst and interburst durations were variable in length (up to several
minutes). We do not preclude that the remainder of the cell types
present in the adenohypophysis (gonadotrophs and thyrotrophs) display
endogenous [Ca2+]i
bursts; we have not attempted to record from these cells because their
density is low within the gland (22).
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Setting the bursting pattern by TRH
As a number of cellular functions are dynamically orchestrated
when a bolus of hypothalamic factors invades the glandular parenchyma,
one would expect the bursting pattern to change upon the episodic
arrival of a hypothalamic stimulus. To explore this, we investigated
whether the secretagogue TRH modifies or even triggers patterned bursts
in the anterior pituitary tissue. There are several lines of evidence
that TRH promotes the firing of
[Ca2+]i transients in
isolated lactotrophs and thyrotrophs (23) and perhaps in other cell
types (24). We, therefore, performed these experiments using acute
pituitary slices from lactating female rats, in which the density of
lactotrophs is high (25). Baseline
[Ca2+]i activity was
recorded for several minutes before the effects of a brief exposure to
TRH (1 nM, 30 sec) were examined. As most spontaneous
[Ca2+]i bursts were
irregular in nature, we measured the bursting activity quotient (Q)
both before and after TRH application. During the control periods, Q
was nulled in both silent and randomly active cells (Fig. 5, Aa and Ab, respectively); however, it
was distributed over a wide range (14–98%) in endogenous bursters
(Fig. 5, Ac–Ae).
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, Aa and
Ab). Second, TRH reset the pacing in endogenous bursters. The effects
of TRH depended on the bursting activity level of the cell before
stimulation (Fig. 5, Ac–e and B). In cells showing an initial low Q
value (<50%), TRH caused a 2- to 3-fold increase in the Q value (Fig. 5Ac). When cells displayed an intermediate Q level (50–80%) before
TRH, TRH finely readjusted their rhythmic pattern (Fig. 5Ad), whereas
in highly active cells (Q >80%), TRH caused a decrease in the Q level
(Fig. 5Ae). Another important finding was the unimodal distribution of
Q values around a peak level of 67% in responsive cells regardless of
whether they were bursting before TRH stimulation (Fig. 5C). Even
though an acceleration of spike frequency was observed at the beginning
of the first burst in some agonist-stimulated cells (17 of 63 cells),
TRH had no effect on the average intraburst
[Ca2+]i spike frequency
when examined over long time scales (Fig. 5D).
As more than 50% of endocrine cells are lactotrophs in the lactating
rat anterior pituitary, we investigated whether TRH modulated the
information processing of
[Ca2+]i transients in
lactotrophs. To conduct this experiment, the PRL content of cells was
postidentified by immunofluorescence (anti-PRL guinea pig antibody).
Eighty-six percent of cells (33 of 39) that displayed a fine adjustment
of [Ca2+]i bursts in
response to 1 nM TRH were lactotrophs (Fig. 6). The slices were also processed for
TSH immunodetection (anti-TSH rabbit antibody). None of the bursters
recorded was immunoreactive for TSH (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Long lasting optical recordings with the Ca2+ dye
Oregon Green 488 BAPTA-1 allowed us to discern characteristic features
of rhythmic [Ca2+]i
bursts that occurred in acute anterior pituitary slices. The pattern of
the bursting mode was mostly irregular whenever endocrine cells fired
[Ca2+]i transients
spontaneously or in response to TRH. Irregular bursts of an
intracellular message are not unique to the anterior pituitary, as
irregular trains of action potentials have been commonly observed in
different brain areas such as the cortex and hippocampus (6). There are
now several lines of evidence that this variability of patterned bursts
in situ reflects the dynamic nature of cell activities on
short time scales (6, 7, 26). The second striking characteristic of the
bursting mode in endocrine pituitary cells was that the firing rate of
[Ca2+]i transients was
sustained at a high average level (
0.7 Hz) throughout the bursts.
This constant level of
[Ca2+]i spiking activity
during the bursts resembles that of the pacemaker mode widely observed
in isolated endocrine pituitary cells (3). However, in the intact
glandular parenchyma, the
[Ca2+]i spike firing was
episodically reduced (i.e. a drop in spike frequency) or
even interrupted during the interburst periods, which lasted up to
several minutes. The
[Ca2+]i transients were
probably due to Ca2+-dependent action potentials
because they were markedly suppressed upon either external
Ca2+ omission or application of
Na+ and Ca2+ channel
blockers (TTX and Ni2+, respectively) (1, 2, 3). The
mechanisms for generating the trains of action potentials that underlie
the [Ca2+]i bursts may
differ among the cell types. They may involve various pacing currents,
such as low threshold Ca2+ channel currents (27),
pacemaker/bursting (If/Ih) currents (28),
Ca2+-dependent K+ channel
currents (29), and ATP-sensitive inward K+
currents (30).
Although the intrinsic activity of cells seems to influence the occurrence of spontaneous [Ca2+]i bursts, bursting is probably not a fixed property of endocrine cells. Two arguments support this assumption. First, neighboring bursting cells could be synchronized, suggesting that the trigger cell, but not necessarily the follower cell(s), expresses the intrinsic mechanisms for generating [Ca2+]i bursts (16). Second, the ability to fire bursts can be regulated in cells (mainly lactotrophs) from lactating rats in response to TRH. This secretagogue switched on patterned bursts in some cells and harmonized the burst generation on long time scales so that rhythmic [Ca2+]i bursts displayed an average duty cycle that was roughly identical for a large number of endocrine cells scattered throughout the glandular parenchyma. Burst setting by agonists has also been observed in vivo in hypothalamic vasopressin neurons, in which local vasopressin release could establish a special pattern of action potential discharges (19). This raises the possibility that one of the best stimuli for exciting, in a coherent fashion, a large number of peptide-releasing cells in their tissue context is to produce single cell rhythmic bursting patterns of intracellular messages with the same duty cycle.
The present work shows that TRH had no effect on average intraburst [Ca2+]i spike frequency. This is at variance with studies performed on isolated pituitary cells in which a prolonged change in [Ca2+]i spike frequency has been described as the major action of low concentrations of TRH (23, 31). The present results should, therefore, renew interest in the mechanism(s) by which TRH, and perhaps other stimuli (e.g. GH-releasing factor and CRF) (32, 33, 34), act on [Ca2+]i signaling in endocrine cells from the anterior pituitary. It should be noted that although the majority of cells responding were lactotrophs (86%), other cells also displayed rhythmic [Ca2+]i bursts upon agonist stimulation, suggesting an action of TRH (direct receptor stimulation and/or via paracrine interaction) on the bursting behavior of other cell types.
What are the functions of rhythmic [Ca2+]i bursts? Although no information is available yet on the role of [Ca2+]i bursts in the function of endocrine pituitary cells, the high efficiency of bursts of [Ca2+]i spikes to trigger exocytosis in cells releasing peptides and catecholamines from large dense core vesicles has already been demonstrated (9, 10, 11, 12). In addition, regulation of rhythmic [Ca2+]i bursts can optimize the timing and amount of hormone release once they are paced with adequate rest periods (10, 11, 12), such as in lactotrophs upon TRH stimulation in lactating rats. Finally, cell to cell mechanisms (e.g. gap junction-mediated cell synchronization and paracrine factors) (16, 35, 36) might help coordinate the tempo of single cell hormone secretion due to [Ca2+]i bursts throughout the glandular parenchyma (37, 38). Further experiments in which [Ca2+]i monitoring will be combined with single cell detection of exocytosis (39, 40) will be needed to address these questions in acute pituitary slices.
In addition to exocytosis, other Ca2+-dependent functions might also be regulated by rhythmic [Ca2+]i bursts. One candidate would be hormonal gene expression, which may depend on temporal transitions between silent and [Ca2+]i spiking periods (41). It should be noted that the bursting pattern comprises two interdependent parameters of timing information: the cycle period (burst plus interburst period) and the duty cycle (percentage of time within a cycle period a cell takes to fire a burst) (20). The dynamic interplay between both parameters could, therefore, provide a subtle code that the endocrine cell could interpret and transduce into the regulation of one or more functions over long time scales.
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Acknowledgments |
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Footnotes |
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Received August 10, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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