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The Journal of Neuroscience, September 15, 2000, 20(18):6773-6780
Dependence of Transient and Residual Calcium Dynamics on
Action-Potential Patterning during Neuropeptide Secretion
Martin
Muschol1 and
B. M.
Salzberg1, 2
Departments of 1 Neuroscience and
2 Physiology, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104-6074
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ABSTRACT |
Secretion of the neuropeptide arginine vasopressin (AVP) from the
neurohypophysis is optimized by short phasic bursts of action potentials with a mean intraburst frequency around 10 Hz. Several hypotheses, most prominently action-potential broadening and buildup of
residual calcium, have been proposed to explain this frequency dependence of AVP release. However, how either of these mechanisms would optimize release at any given frequency remains an open question.
We have addressed this issue by correlating the frequency-dependence of
intraterminal calcium dynamics and AVP release during action-potential stimulation.
By monitoring the intraterminal calcium changes with low-affinity
indicator dyes and millisecond time resolution, the signal could be
dissected into three separate components: rapid Ca2+
rises ( [Ca2+]tr) related to
action-potential depolarization, Ca2+ extrusion
and/or uptake, and a gradual increase in residual calcium ([Ca2+]res) throughout the
stimulus train. Action-potential stimulation modulated all three
components in a manner dependent on both the stimulation frequency and
number of stimuli. Overall, the cumulative [Ca2+]tr amplitude initially
increased with fStim and then rapidly deteriorated, with a maximum around
fStim  5 Hz. Residual calcium levels,
in contrast, increased monotonically with stimulation frequency.
Simultaneously with the calcium measurements we determined the amount
of AVP release evoked by each stimulus train. Hormone release increased
with fStim beyond the peak in
[Ca2+]tr amplitudes, reaching its
maximum between 5 and 10 Hz before returning to its 1 Hz level. Thus,
AVP release responds to the temporal patterning of stimulation, is
sensitive to both [Ca2+]tr and
[Ca2+]res, and is optimized
at a frequency intermediate between the frequency-dependent maxima in
[Ca2+]tr and
[Ca2+]res.
Key words:
calcium dynamics; excitation-secretion coupling; arginine
vasopressin; exocytosis; neurohypophysis; action potential; residual
calcium
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INTRODUCTION |
Secretion of the peptide hormones
arginine vasopressin (AVP) and oxytocin (OXT) from the mammalian
posterior pituitary is differentially regulated by the pattern of
action-potential activity that evokes release (Poulain and Wakerley,
1982 ; Cazalis et al., 1985; Gainer et al., 1986; Bicknell, 1988). AVP
secretion is optimized by phasic bursts of action potentials, whereas
optimal OXT release is associated with sustained high-frequency
stimulation. Frequency-dependent action-potential broadening is thought
to be one of the mechanisms involved in this pattern-dependent
regulation of secretion (Gainer et al., 1986; Bourque, 1990; Jackson et
al., 1991). Buildup of residual calcium levels during bursts of action
potentials is considered another contributing factor (Jackson et al.,
1991; Stuenkel and Nordmann, 1993; Stuenkel, 1994). In addition,
P/Q-type Ca2+-channels are
expressed in AVP terminals, but not OXT terminals, and are believed to
play a direct role in AVP secretion in the rat (Wang et al., 1997).
The purpose of our present study was twofold. First, we aimed to
resolve the relative changes of
[Ca2+]i
within a given stimulation train, i.e., on a millisecond time scale. This time resolution would enable us to track relative changes
in the near-membrane level of intraterminal
Ca2+, as well as in the kinetics of the
Ca2+ extrusion and/or uptake process, and
to follow the amplitude and kinetics of residual calcium levels during
trains of action potentials. Furthermore, for a meaningful comparison
of stimulation-induced [Ca2+]i changes,
on the one hand, and the corresponding amount of hormone secretion, on
the other hand, we wanted to measure both quantities simultaneously
from the same preparation. To accomplish these goals, we used the
low-affinity calcium indicator dye Mag-Fluo-4, which improved the
temporal resolution of the
[Ca2+]i records
from ~1 sec (Jackson et al., 1991; Stuenkel and Nordmann, 1993;
Stuenkel, 1994) to 1 msec. Concurrently, we applied a quantitative enzyme immunoassay (EIA) to sample aliquots withdrawn from the bathing
saline immediately after a given stimulation train to assess the amount
of evoked hormone secretion.
The improved temporal resolution of the
[Ca2+]i
measurements permitted us to isolate different components of
[Ca2+]i and to
determine their distinct patterns of modulation within a given train of
action potentials. We demonstrate that these patterns vary with both
the frequency of stimulation and the number of action potentials within
the train. We also show that AVP release is shaped by the detailed and
distinct dependence of transient and residual calcium on the temporal
patterning of stimulation.
Portions of this work have been published previously in abstract form
(Muschol et al., 1999; Muschol and Salzberg, 2000).
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MATERIALS AND METHODS |
Preparation and apparatus. Details of the preparation
and apparatus have been described previously (Salzberg et al., 1983, 1985, 2000a; Gainer et al., 1986; Obaid et al., 1989). Briefly, 30- to
60-d-old CD-1 female mice (Jackson Laboratories, Bar Harbor, ME) were
sacrificed using CO2 asphyxiation followed by
decapitation. The neurohypophysis, together with the pars intermedia
and a short segment of the infundibular stalk, was separated from the
anterior pituitary. The preparation was mounted in an optical recording chamber and perfused with oxygenated (95% O2,
5% CO2) physiological saline containing (in
mM): NaCl 155, KCl 5.6, CaCl2 2.2, MgCl2 1, glucose
10, HEPES 20, pH 7.4, at a rate of 300 µl/min. Trains of action
potentials were generated in the nerve terminals of the neurohypophysis
by field depolarization of their axons at the level of the infundibular
stalk with a pair of Teflon-insulated platinum (90%)-iridium (10%)
wires. A Master-8 pulse generator (A.M.P.I., Jerusalem, Israel),
driving two ISO-Flex stimulus isolators (A.M.P.I.), provided the
bipolar pulses of 500 µsec duration that were used for field
depolarization. All experiments were performed at room temperature
(23 ± 2°C). All chemicals used were from Sigma (St. Louis, MO)
unless indicated otherwise.
Fluorescence measurements of calcium changes. The
neurohypophysis was incubated for 90 min in a physiological saline
containing 5 µM of the AM form of the low-affinity
(KD,Ca = 22 µM) calcium indicator dye Mag-Fluo-4 (Molecular
Probes, Eugene, OR), 0.9% dimethyl sulfoxide, and 0.1% Pluronic F-127
(BASF Wyandotte, Wyandotte, MI). All fluorescence measurements were
performed on an upright UEM Microscope (Zeiss, Jena, Germany) using a
Nikon 10× Fluor objective (NA = 0.5). Light from a 300 W tungsten
halogen lamp was passed through a 470 ± 20 nm excitation filter
(Zeiss). Dye fluorescence was monitored at  > 515 nm (Schott
Optical Glass Co., Duryea, PA) with a single large-area photodiode
(PV-444, Perkin Elmer Optoelectronics, Vaudreuil, Canada). The
photocurrent was converted into a voltage signal using a custom-built
two-stage sample-and-hold amplifier (Cellular and Molecular Physiology
Electronics Shop, Yale University School of Medicine, New Haven, CT).
The sample-and-hold circuitry enabled us to measure and subtract the resting fluorescence, F0, before
second-stage amplification of the small, stimulation-induced
fluorescence changes, F, superimposed on the fluorescence
background F0. The output from the
second-stage amplifier was filtered at 1 kHz with a LPF-8 low-pass
eight-pole Bessel filter (Warner Instrument Corp.ration, Hamden, CT)
and acquired at 16-bit resolution with an AT-MIO-16XE-50 data
acquisition board (National Instruments, Austin, TX) running under the
control of LabView Software (National Instruments). The fluorescence
changes, F, were corrected for dye bleaching by
subtracting a stimulation-free reference record collected immediately
after each measurement. Total measurement duration for the fluorescence
records varied from 45 sec at 1 Hz down to 6 sec for all stimulation
frequencies above 10 Hz. In a typical experiment, the tissue was
allowed to rest for 15 min between stimulation trains. Trapping of
calcium ionophores (A23187, ionomycin) in the outer layers of the
neurohypophysis prevented us from using them to convert the
F/F0 values into absolute changes in
[Ca2+]i.
Enzyme immunoassay of AVP release. The amount of AVP
released during a given stimulus train was determined using a
solid-phase EIA Assay kit (Assay Designs, Ann Arbor, MI). To counteract
the proteolytic activity in the tissue, the protease inhibitors
phenylmethanesulfonyl fluoride (0.1 mM; Fluka Chemical
Corp., Ronkonkoma, NY), leupeptin (2 µg/ml), and aprotinin (5.2 × 10 3
TIU/ml) were added to the physiological saline. Immediately
before stimulation, superfusion flow through the recording chamber was halted. After a 5 min waiting period, 350 µl of the perfusate surrounding the tissue was collected and kept at 4°C. A 96-well EIA
kit was used to analyze the AVP content in the samples. At the end of
the second incubation period, sample absorbance at = 405 nm
was determined with a Titertek Multiskan MCC/340 plate reader (Flow
Laboratories, McLean, VA). Absorbance readings were converted into
absolute AVP concentrations by interpolation from the absorbance of
eight AVP calibration standards fitted to a four-parameter logistic
equation [for details, see Chan (1987)].
Data analysis. Fluorescence data were processed using the
IGOR data analysis software (Wavemetrics, Lake Oswego, OR). We used a
seven-point binomial smoothing algorithm to reduce noise in the
F/F0 traces. This
considerably improved the reliability of subsequent peak-detection
procedures without affecting the time course or amplitude of the
F/F0 changes.
Furthermore, the amplitudes of the first stimulation-induced peak in
F/F0 from all records within one experiment were matched to each other. As can be seen in
Figure 1B, these amplitudes were already within 10%
of one another before normalization. Values for
F/F0 before and at the peak immediately after each stimulus were extracted using a custom macro. These values were used to determine the amplitudes of the transient Ca2+ rise,
[Ca2+]tr, and
the change in residual calcium,
[Ca2+]res.
Potential artifacts: relative fluorescence change,
F/F0, and intraterminal
Ca2+-dynamics. We were concerned about
excluding contributions to or distortions of
F/F0 from sources other
than stimulation-induced calcium changes inside the nerve terminals.
Dye signals from AM-loaded preparations might represent a superposition
of [Ca2+]i
changes from several cellular components within the preparation. The
neurohypophysis (pars nervosa) comprises millions of nerve terminals
and secretory swellings (Herring bodies), and these are the most
abundant structure in this tissue, accounting for some 99% of the
excitable membrane in rat (Nordmann, 1977). The only other cellular
structures with significant total volume are pituicytes. Because
pituicytes apparently lack voltage-gated calcium channels, we do not
expect these glial elements to contribute to stimulation-induced
changes of F/F0.
Furthermore, the considerably larger volume of pituicytes (275 µm3) versus nerve endings (2-14
µm3) (Nordmann, 1977), and their
correspondingly smaller surface to volume ratio, should result in
substantially higher dye concentrations inside AM-loaded nerve
terminals than inside pituicytes. That expectation was confirmed by the
dramatically lower resting fluorescence observed after AM loading of
the large cells in the neighboring pars intermedia (>20
µM diameter) (Bourque, 1990). This difference in dye concentration will significantly enhance the contributions of
nerve terminals to F/F0
signals. Another possible concern is the heterogeneity of the nerve
terminal population itself, which contains both AVP- and
OXT-secreting terminals. Although different types and distributions of
Ca channels have been identified pharmacologically (Wang et al., 1997),
patch-clamp measurements showed nerve terminals to be homogeneous in
their Ca-channel properties (Branchaw et al., 1997). Calcium
measurements from isolated nerve terminals (Stuenkel, 1994; Fisher and
Fernandez, 1999) or in tissue slices (Jackson et al., 1991) showed
calcium kinetics to be insensitive to terminal peptide identity.
Uptake of AM-loaded dyes into subcellular compartments presents another
potential source of F/F0
distortions (Almers and Neher, 1985). Indeed, loading of Fura-2 into
the subcellular compartments of the rat neurohypophysis has been
reported (Troadec et al., 1998). If compartmentalization occurs with
Mag-Fluo-4, it does not appear to contribute to the stimulation-induced
fluorescence changes. In Figure 1B we have
superimposed F/F0 data
from the first action potential at each stimulation frequency. Figure
1C illustrates the corresponding values of the resting
fluorescence F0 for each of these
measurements. Note that the
F/F0 amplitudes remain
constant throughout the experiment, whereas loss of dye from the tissue
typically resulted in a reduction in
F0 by a factor of 2 or more. Given
this dye loss, the relative proportion of cytosolic versus
compartmentalized dye is bound to change over time. If
compartmentalized dye contributed to
F/F0, this dye redistribution in turn would alter
F/F0 amplitudes. This
was not observed.
Dye properties such as binding kinetics, dye saturation, or sensitivity
to Mg2+ can distort both the time course
and the relative amplitudes of
F/F0 measurements
(Baylor and Hollingworth, 1988; Helmchen et al., 1997). Mag-Fluo-4 is a
low-affinity calcium indicator (KD,Ca = 22 µM) (Haugland, 1996) with fast on/off
rates. Because we estimate the maximal dye concentration in the
terminals at 100-200 µM, distortions of the
time course of F/F0 are
not expected. In fact, the intrinsically fast kinetics of this
indicator dye compared with its high-affinity variants (i.e., Fluo-4 or
Fluo-3) enabled us to detect the rapidly changing components of
[Ca2+]i.
Distortions of F/F0
amplitudes resulting from dye saturation are also negligible. For
typical resting levels of intraterminal [Ca2+]i of
100-300 nM (Jackson et al., 1991; Stuenkel,
1994), the resting fluorescence F0 of
Mag-Fluo-4 will be close to its zero-calcium value,
Ffree, whereas the dynamic range,
(Fbound  Ffree)/Ffree, of Mag-Fluo-4 exceeds a factor of 25 in our microscope measurement system. The maximal value of
F/F0 during measurements
did not exceed 15%, well within the compass of linear dye response. In addition, F/F0
amplitudes obtained with KCl depolarization ranged from 50 to 200%
without showing signs of dye saturation (data not shown).
Finally, to evaluate potential Mg2+
contributions to the measurements, we repeated
F/F0 measurement with
two additional low-affinity Ca2+
indicators, Magnesium Green and Mag-Fura-2. The relative Ca/Mg sensitivities among these three dyes differ by as much as a factor of
4, with Mag-Fluo-4 being the least Mg2+
sensitive. Nevertheless, all three dyes yielded identical
F/F0 traces (results not
shown). We therefore presume that Mg2+
changes make no discernable contribution to
F/F0 on the time scale
of our measurements, in agreement with observations on nerve terminals
in the Calyx of Held (Helmchen et al., 1997). On the basis of all of
these arguments, we conclude that the observed fluorescence changes,
F/F0, reported below are
linear and that they provide a reliable, if uncalibrated,
representation of intraterminal calcium changes.
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RESULTS |
Intraterminal calcium changes ([Ca]i)
during trains of action potentials
Figure 1 presents a summary of
intraterminal Ca2+ changes,
[Ca2+]i, in the
neurohypophysis resulting from stimulation with trains of action
potentials (NStim = 40) at the
indicated frequencies. For any given train,
[Ca2+]i can be
separated into three distinct phases. Each action potential triggers a
rapid ( 15 msec), transient calcium rise,
[Ca2+]tr.
Between stimuli, a slower decay phase,
[Ca2+]dec,
related to Ca2+ extrusion and/or uptake,
prevails. In addition, residual calcium, [Ca2+]res,
accumulates as a result of incomplete Ca2+
removal during the interstimulus period.
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Figure 1.
Frequency dependence of intraterminal calcium
changes. A, Superposition of the fluorescence changes,
F/F0, recorded with
the low-affinity calcium indicator dye Mag-Fluo-4/AM
(KD, Ca = 22 µM) in the
mouse neurohypophysis during trains of 40 action potentials at the
indicated frequencies. The fluorescence data were low-pass-filtered at
1 kHz and corrected for dye bleaching by subtraction of a
reference trace without stimulation. The data in A were
also passed through a seven-point smoothing algorithm, and their final
amplitudes were matched to the amplitude of the first
[Ca2+]tr of the control trace
at 15 Hz. In the inset, the complete time course of the
truncated 1 Hz fluorescence trace is shown. B, Initial
[Ca2+]tr amplitudes from the data
in Figure 1 before matching their peaks. Even without peak matching,
F/F0 values are within
10% of one another. In contrast, the corresponding values for
the resting fluorescence F0 (not in
chronological order) changed nearly twofold during the course of these
recordings, as shown in C.
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It is instructive to compare the overall shape of the observed
[Ca2+]i traces
with the predictions from a simple one-compartment model of
Ca2+ kinetics during repetitive
stimulation (Regehr et al., 1994). This model accounts for periodic
Ca2+ influx during stimulation trains,
Ca2+ buffering by endogenous buffers and
Ca2+ indicators, and a
Ca2+ extrusion process linearly
proportional to
[Ca2+]i, the free
calcium concentration in the terminal. For a typical nerve terminal of
radius r 1-3 µm (Nordmann, 1977; Sattelle, 1988), and
presuming an intraterminal diffusion coefficient of 6 × 106
cm2/sec (Blaustein and Hodgkin, 1969), the
time for spatial Ca2+ gradients to
dissipate can be estimated as  Diff r2/6DCa 2.5 msec (Crank, 1975). The neglect of diffusion effects is
therefore a good approximation for the experimental situation with
which we are concerned. The model readily reproduces the overall shape
of the [Ca2+]i
traces. In particular, it predicts that for any given stimulation frequency, fStim, residual calcium
levels will increase until they reach a steady-state plateau at which
the Ca2+-dependent extrusion/uptake rate
between stimuli exactly balances the Ca2+
rise during stimulation. The amplitude of the residual calcium plateau
is expected to increase linearly with
fStim, because the time-averaged
Ca2+ influx is presumed constant. The
model therefore provides an important reference point against which to
compare the experimentally observed Ca2+
dynamics. In the remainder of this section we will explore the effects
of various stimulation parameters on the transient
Ca2+ rises
([Ca2+]tr),
Ca2+ extrusion/uptake
([Ca2+]dec),
and residual calcium changes
([Ca2+]res), as
well as their impact on AVP release.
Facilitation and depression of
[Ca2+]tr amplitudes during trains
of action potentials
In Figure 2 we have plotted
[Ca2+]tr
amplitudes versus the stimulus number,
nStim, from the data in Figure 1. Both
stimulation frequency, fStim, and
total number of stimuli, NStim, affect
[Ca2+]tr
amplitudes. At fStim = 1 Hz, a barely
perceptible facilitation of
[Ca2+]tr
amplitudes occurs within the first few stimuli. As stimulation extends
beyond 10 stimuli,
[Ca2+]tr
amplitudes begin to deteriorate, declining to 80% of their initial
value at the end of a 40 stimulus train. At
fStim = 3 Hz, facilitation of early
[Ca]tr amplitudes is dramatic, with the
amplitude of the fifth response >60% greater then the initial response. Again, as stimulation continues,
[Ca2+]tr
deteriorates within the train. With increasing stimulation frequency,
facilitation does not persist beyond the first few stimuli: e.g., at 15 Hz, [Ca2+]tr
returns to its initial amplitude within <10 stimuli. Above 15 Hz,
depression of
[Ca2+]tr
dominates throughout the stimulation train.
[Ca2+]tr
depression progresses further with
fStim, until at 40 Hz the [Ca2+]tr
amplitude at the end of 40 stimuli has deteriorated to only 20% of its
value at the onset of the stimulus train.
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Figure 2.
Amplitude variation of the transient
Ca2+ rise. Shown are fractional changes in the
amplitude of the transient Ca2+ rise,
[Ca2+]tr, during a train of
40 action potentials at the indicated stimulation frequencies ( , 1 Hz;  , all other frequencies), normalized to the first transient of
each train. Three different patterns of facilitation and depression
emerge. At low frequencies
(fStim 1 Hz),
[Ca2+]tr amplitudes are essentially
constant throughout the train (). At moderate frequencies (1 Hz < fStim 15 Hz),
[Ca2+]tr amplitudes facilitate
during the initial stimuli but deteriorate later in the train. Finally,
at higher frequencies, depression of
[Ca2+]tr amplitudes dominates
throughout the train, resulting in
[Ca2+]tr amplitudes deteriorating
to <20% of their initial values at the end of 40 stimuli.
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Action-potential stimulation therefore produces three distinct patterns
of Ca2+ rises as a function of frequency.
At or below fStim = 1 Hz,
[Ca2+]tr
amplitudes are nearly constant, with mild depression developing slowly
during extended stimulation. Between 1 and 10 Hz, early facilitation of
[Ca2+]tr
amplitudes dominates throughout the stimulation train. Beginning at
~10 Hz, early facilitation is eroded by subsequent depression. Overall, depression develops at progressively smaller
nStim and at an increasingly rapid
rate as fStim increases. Eventually, [Ca2+]tr
amplitudes deteriorate monotonically throughout the train.
To evaluate the physiological significance of the observed facilitation
and depression of
[Ca2+]tr, we
need to consider the factors that might contribute to [Ca2+]tr:
Ca2+ influx,
Ca2+ mobilization, and
Ca2+ extrusion and uptake, as well as
Ca2+ buffering by the endogenous
Ca2+ buffers and the
Ca2+ indicator dye. As we have argued in
Materials and Methods, distortion of the intrinsic
Ca2+ dynamics resulting from dye
saturation, slow dye kinetics, or competition with the intrinsic
Ca2+ buffers is negligible. Furthermore,
Ca2+ binding by both dye and buffer and
diffusion of Ca2+ in these small terminals
are fast enough that the Ca2+-indicator
dye is always in quasi-equilibrium with
[Ca2+]i. Studies
on isolated nerve terminals have shown that >99% of the
Ca2+ influx is rapidly bound to endogenous
Ca2+ buffer(s), thereby dramatically
limiting the amplitude of
[Ca2+]tr
(Stuenkel, 1994). Saturation of the endogenous
Ca2+ buffer therefore might produce the
apparent facilitation of
[Ca2+]tr. The
linear relationship between ICa and
[Ca2+]i observed
in these nerve terminals using Fura-2 (Stuenkel, 1994, his Fig. 2)
suggests that Ca2+ influx does not
saturate the endogenous buffer under these conditions. Furthermore,
numerical calculations, using the above-mentioned one-compartment model
(Regehr et al., 1994; Tank et al., 1995), indicate that buffer
saturation results in a sigmoidal shape for [Ca2+]res, which
was not observed in our experiments. Given the slow time course of
Ca2+ extrusion and/or uptake (see below),
these factors are also unlikely to affect the time course or amplitudes
of
[Ca2+]tr.
This leaves changes in Ca2+ influx and,
potentially, Ca2+ mobilization as the
relevant sources for the observed changes in
[Ca2+]tr
amplitudes. The role of Ca2+ influx in
excitation-secretion coupling is well established (Douglas, 1963;
Douglas and Poisner, 1964). Release of
Ca2+ from intracellular stores is tightly
coupled to secretion in many other secretory cells (Tse and Tse, 1999).
Mobilization of Ca2+ from isolated
granules themselves, for example, has been reported in pancreatic
acinar (Gerasimenko et al., 1996) and chromaffin cells (Yoo and
Albanesi, 1990). We have recently localized both Type 2 Ryanodine and
Type 1 InsP3 Ca2+
release channels on the dense-core secretory granules in the mouse
neurohypophysis (Salzberg et al., 2000b). Together with reports of high
Ca2+ concentrations in these granules
(Thirion et al., 1997), the possibility of
Ca2+ mobilization from secretory granules
(or other intraterminal stores) cannot be excluded. However, because
there is as yet no direct evidence for
Ca2+ release from intraterminal
compartments in the neurohypophysis, we will treat the terms
"Ca2+ influx" and
"[Ca2+]tr"
as equivalent.
Ca2+ extrusion or uptake
Figure 3 shows the
Ca2+ decay
([Ca2+]dec)
immediately after the last
[Ca2+]tr rise,
on a linear (Fig. 3A) and a logarithmic (Fig. 3B)
time scale. As is apparent from Figure 3A,
[Ca2+]dec
displays a bimodal decay pattern: an initial rapid decay switches over
to a very slow decay as
[Ca2+]i approaches
resting levels. The initial rapid decay phase, in turn, contains two
distinct components. Figure 3D summarizes the results from
double-exponential fits to this initial decay phase. The two components
of the rapid decay phase display a significantly different dependence
on residual calcium levels. The time constant 1fast of the
faster component increases from a low of ~25 msec to a maximum of 200 msec at the highest level of residual calcium. This increase is
directly reflected in Figure 3B in the increasing delay
before the onset of the steep decay. Any single
Ca2+-sensitive
Ca2+ extrusion/uptake mechanism would be
expected to increase its macroscopic rate of
Ca2+ extrusion with increasing
Ca2+ load. The observed decrease,
therefore, might result from a transition between two distinct
extrusion mechanisms with separate times scales of
1fast(min) and
1fast(max),
respectively.
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Figure 3.
Decay of intraterminal calcium levels after
stimulation. Shown is the decay of
[Ca2+]i at the end of stimulation with
40 action potentials at the indicated fStim
on linear (A) and logarithmic
(B) time scales. The y-axis scale
in B is identical to the one in A. The
solid lines in A represent the raw data
points. In B the raw data from A were
resampled uniformly on a logarithmic time scale. The open
circles in B are the results from
double-exponential fits through the data. C, Changes in
the shape of the decay phase of the 1st ( ), 5th, and 15th
(- - -) [Ca2+]i response
during action-potential stimulation at
fStim = 15 Hz. D,
Dependence of the amplitudes (open symbols) and time
constants (closed symbols) of the double-exponential
fits in B on stimulation frequency ( , constant
background;  ,  , first and , , second fast
component).
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In contrast, the time constant of the second fast component,
2fast, remains
fairly constant at 400 ± 50 msec. The slow decay is not well
resolved in this record and, for the purpose of the double-exponential
fit shown in Figure 3, was set equal to a constant background.
Quantitative measurement of the slow time constant was hampered by its
sensitivity to small errors in the bleaching correction (see Materials
and Methods). Nevertheless, from extended F/F0 recordings we
estimate the slow time constant to be slow = 15-25 sec, i.e., 50-fold slower than the initial decay. Inspection of
the raw data in Figure 3A reveals clearly that the relative contributions of the three decay components of
[Ca2+]i vary with
the level of residual calcium. Although the amplitudes of the slow and
the second fast component both saturate around fStim = 10 Hz, the amplitude and time
constant of the first fast component increase essentially monotonically
with stimulation frequency.
The presence of at least three different decay components with both
amplitudes and decay rates dependent on the level of residual calcium
adds another level of complexity to the temporal coding of
[Ca2+]i transients
during stimulation trains. The dominant role of the slow decay
component at low frequencies, for example, provides the terminals with
a "long-term" memory for their stimulation history even after
brief, low-frequency stimulation. Similarly, the changing amplitudes
and time constants of the fast decay components modulate the kinetics
of [Ca2+]i
decay, particularly within the first few stimuli of a given train of
action potentials. This is highlighted in Figure 3C, which
shows a superposition of the 1st, 5th, and 15th response in a 15 Hz
stimulus train. Initially,
[Ca2+]dec
between stimuli is clearly bimodal, whereas it becomes essentially single exponential as
1fast increases
beyond the interstimulus period. Without definitive pharmacological
data, however, any specific assignment of different Ca2+ decay components to particular
extrusion/uptake mechanisms must remain tentative.
Buildup and decay of residual calcium levels
Figure 4A depicts
the net increase in residual calcium during stimulation by plotting the
F/F0 values at the foot
of each individual
[Ca2+]tr within
a train. The initial mismatch between Ca2+
influx during stimulation and the rate of
Ca2+ extrusion between action potentials
effects this net increase in
[Ca2+]res. For any
given stimulus train,
[Ca2+]res
increases until the Ca2+ extrusion rate
begins to match the time-averaged Ca2+
influx. This behavior is in overall agreement with a one-compartment model for
[Ca2+]i
kinetics that predicts a single-exponential rise in
[Ca2+]res (Regehr
et al., 1994; Tank et al., 1995). With increasing stimulation
frequency, however, single-exponential fits only conform to
progressively shorter segments of the rising phase of residual calcium
and overestimate its plateau amplitudes (Fig. 4B,
solid lines). This deviation from the model behavior is
caused by the rapid depression of calcium influx during continuous
stimulation in this frequency range.
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Figure 4.
Changes in residual calcium during stimulation.
Shown is the increase of residual calcium,
[Ca2+]res, in the nerve
terminals of the neurohypophysis as a function of
(A) time and (B) the
stimulus number, nStim, during a
train of 40 action potentials at the indicated
fStim. The open circles
represent the values of indicator fluorescence changes
(F/F0) immediately
before a transient Ca2+ rise. In B,
the spacing between data points () at different
fStim represents an increment in time of
t = (fStim)1
sec, respectively. The solid lines represent
single-exponential fits through the data. With increasing
fStim, the range of data points
conforming to this single-exponential rise rapidly decreases.
C, Initial rise times () of
[Ca2+]res obtained from the
single-exponential fits. The open squares represent the
values of the plateau amplitudes of
[Ca2+]res. Because the
single-exponential fits did not reproduce plateau amplitudes properly,
their values were instead set equal to
[Ca2+]res at
nStim = 25. The solid
lines in C are only guides to the eye.
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|
Figure 4C shows both the plateau amplitude () and rise
time () of
[Ca2+]res versus
fStim. Several features are
noteworthy. First, the plateau amplitude does not pass through the
zero-Ca2+ origin but appears to have a
finite offset. This offset is caused by the presence of the slow decay
component, which keeps
[Ca2+]res elevated
for many tens of seconds after a stimulation train is completed. The
corresponding low-frequency component of
Ca2+ extrusion is not resolved on the
frequency scale of our experiments. Second, the saturation of
[Ca2+]res at
higher frequencies arises from the pronounced depression of
[Ca2+]tr during
action-potential stimulation. In contrast, fixed
[Ca2+]tr
amplitudes would result in a strictly linear increase of the [Ca2+]res plateau
with frequency, as observed in the crayfish neuromuscular junction
[Tank et al. (1995), their Fig. 3B]. Finally, the rise time of residual calcium is a highly nonlinear function of stimulation frequency because, apparently, increasingly effective extrusion pathways are activated during the rapid
Ca2+ rise at high-frequency stimulation.
Frequency dependence of AVP secretion
Figure 5A summarizes the
dependence of AVP release on fStim.
The aliquots used for the EIA analysis were
obtained from the same preparation, immediately after collection of the
F/F0 data for a given
frequency. Although the absolute amount of AVP release varied somewhat
from one assay to the other, the relative changes in AVP release with
fStim were readily reproducible. Even
at the lowest fStim of 1 Hz, AVP
secretion was elevated well above basal release. Secretion further
increased to a broad peak value around fStim = 5-10 Hz and then declined
again. At fStim = 40 Hz, release had
declined to the levels elicited with 1 Hz stimulation. Because we do
not have a millisecond time-resolved measure of release, we constructed
a time-averaged measure for the frequency dependence of the transient
and residual components of
[Ca2+]i
instead. Figure 5B displays the cumulative amplitudes,
 [Ca2+]tr and
[Ca2+]res,
i.e., the amplitude of each component summed over the stimulus train,
as a function of fStim. Comparison of
the frequency dependence of release with that of
[Ca2+]tr and
[Ca2+]res
permits several conclusions. Clearly, Ca2+
influx during action-potential stimulation alone is sufficient to
induce secretion in this preparation, as has been established for
nearly 40 years (Douglas, 1963; Douglas and Poisner, 1964). More
interestingly, the pattern of action-potential firing plays an active
role in enhancing release through facilitation of
Ca2+ rises at low-stimulation frequencies.
This is particularly apparent during the steep increase in AVP release
from 1 to 5 Hz, which correlates with the pronounced
[Ca2+]tr
facilitation in this frequency range. On the other hand, the peak in
AVP secretion does not coincide with the peak in
[Ca2+]tr.
Furthermore, for fStim beyond 15 Hz,
[Ca2+]tr drops
well below its 1 Hz level, whereas release remains at or above the 1 Hz
level out to fStim = 40 Hz. These
results indicate how the increase in
[Ca2+]res can
sustain elevated release levels despite a substantial reduction in
Ca2+ influx.
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Figure 5.
AVP release and cumulative Ca2+
changes. A, Basal release () and changes in AVP
secretion () from the neurohypophysis in response to a train of 40 action potentials at the indicated fStim.
The hormone levels were measured with an EIA. Aliquots from the
recording chamber were collected for analysis immediately after
the measurement of the stimulation-induced changes in
[Ca2+]i. B, Relative
changes in the cumulative amplitudes of the transient
Ca2+ rise
([Ca2+]tr, ) during
the action potential, and the cumulative residual calcium increase
( [Ca2+]res, )
throughout the stimulus train versus fStim.
Data in B are normalized to their respective peak
values. Solid lines in A and
B are guides to the eye. The error bars reflect the SDs
from six different experiments.
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|
Effect of burst stimulation on
[Ca2+]i and AVP secretion
We used a burst-stimulation protocol to explore the facilitation,
depression, and recovery behavior of
[Ca2+]tr. This
protocol has the additional virtue that short stimulation bursts will
reduce the net amplitude of
[Ca2+]res and
permit us to examine its relevance to AVP release. The preparation was
stimulated with a continuous train of 40 stimuli at 5 Hz (reference) or
shorter bursts of 4 × 10, 8 × 5, or 20 × 2 stimuli,
each at an intraburst frequency of 5 Hz, and separated by interburst
intervals of 4, 2 or 1 sec, respectively. We chose 5 Hz as intraburst
frequency because it was close to the optimal frequency for AVP release
and displayed strong
[Ca2+]tr
facilitation during continuous stimulation. Figure
6A-C shows the [Ca2+]tr
amplitudes for these four stimulation protocols. From these traces it
is apparent that facilitation and depression of
[Ca2+]tr
operate as separate mechanisms. For example, each one of the 10 stimuli
bursts in Figure 6A facilitates to a comparable
degree, indicating that facilitation does not persist beyond the 4 sec burst separation. For the shorter burst separations of 2 sec (Fig. 6B, five stimuli/burst) and 1 sec (Fig.
6C, two stimuli/burst), facilitation sometimes followed the
reference curve for one or two bursts before falling off in subsequent
bursts (data not shown). At the same time, the amplitude of the first
Ca2+ transient in each subsequent burst
follows the pattern of depression established by the reference record.
Clearly, long-term depression persists beyond the burst separation but
does not interfere with the facilitation within each burst.
[Ca2+]tr
amplitudes for burst stimulation drop by ~10-20% from their value
during continuous stimulation (Fig. 6D, white
bars). AVP release, however, is reduced by nearly 40% (Fig.
6D, gray bars). The substantial difference
between the reduction in
[Ca2+]tr and
the decline in AVP secretion using burst stimulation suggests that the
reduced plateau level of
[Ca2+]res is the
main reason for the disproportionate deterioration in AVP
secretion.
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|
Figure 6.
Effect of burst stimulation on transient
Ca2+ rise and AVP release. Shown are
[Ca2+]tr amplitudes during
stimulation of the neurohypophysis with trains of action-potential
bursts at 5 Hz. The bursts () consisted of either
(A) 4 × 10, (B)
8 × 5, or (C) 20 × 2 stimuli at 5 Hz,
separated by interburst intervals of 4, 2, and 1 sec, respectively. The
reference trace () in Figure
6A-C represents the initial,
continuous action-potential train of 40 stimuli at 5 Hz. The
solid lines through the data are guides to the eye.
D, Changes in cumulative
[Ca2+]tr amplitudes (white
bars) and AVP release (gray bars) for the
data in A-C with respect to the values observed with
the first reference trace. Values for a second reference trace at the
end of the experiments and for the basal release of AVP are shown as
well. Results are normalized to the values obtained with the first
reference stimulus train.
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|
| |
DISCUSSION |
At first sight, the shape of intraterminal
Ca2+ changes conforms well to the
predictions of a simplified one-compartment model of
Ca2+ dynamics (Regehr et al., 1994; Tank
et al., 1995), which presumes fixed Ca2+
influx and a single Ca2+ extrusion
mechanism. In particular, the model clearly embodies the essential
features underlying the development of a steady-state plateau of
residual calcium and the increase of the plateau amplitude with
stimulation frequency.
On closer inspection, however, a more complex picture emerges. Most
importantly, trains of action potential at different frequencies cause
dramatically different patterns of Ca2+
influx. Amplitudes of Ca2+ influx can
undergo pronounced facilitation and depression in the course of a
single action-potential train. Dependent not only on
fStim but also on
NStim, the net
Ca2+ influx can be enhanced or depressed.
To our knowledge, this is the first time concurrent facilitation and
depression of Ca2+ influx during
action-potential stimulation has been described in the neurohypophysis.
In addition,
[Ca2+]i decay
occurs on at least three distinct, widely separated time scales. Both
of these factors conspire to alter the rise times, decay times, and
plateau amplitudes of residual calcium. Thus, residual calcium itself
is intricately and inextricably coupled to the shifting balance between
Ca2+ influx and extrusion, and the
underlying pattern of action-potential stimulation. This raises the
question of the physiological origin(s) of the observed changes in
Ca2+ influx and
[Ca2+]i decay.
Changes in Ca2+ influx
Changes in the action-potential shape have long been postulated as
an important factor contributing to the frequency dependence of release
(Gainer et al., 1986; Bourque, 1990; Jackson et al., 1991).
Conceivably, changes in action-potential shape might be sufficient to
produce both facilitation and depression of
[Ca2+]tr. For
example, broadening of the action potential (Gainer et al., 1986;
Bourque, 1990) might enhance the influx of
Ca2+ early in a train, whereas the
decrease in action-potential amplitude (Salzberg et al., 1985; Bourque,
1990) would result in depression. On the other hand, the rapid onset of
facilitation of Ca2+ influx does not agree
well with the gradual development of action-potential broadening
[Bourque (1990), their Fig. 9].
Inactivation of Ca2+ channels might be
another factor altering Ca2+ influx.
Measurements on slices of the rat neurohypophysis demonstrated pronounced voltage-dependent Ca2+ channel
inactivation, with recovery rates from this inhibition noticeably
reduced by intraterminal Ca2+ (Branchaw et
al., 1997). Hence, during low-frequency stimulation, inactivation
should be minimal because the separation between action potentials is
long and [Ca2+]res
rises are slow and small in amplitude. As
fStim increases, depression sets in
earlier and proceeds at a faster rate because action-potential
separation decreases and
[Ca2+]res rises
with increasing rapidity to higher levels. This behavior agrees well
with our observations (Fig. 2). High-frequency depression of
Ca2+ influx could also result from the
buildup of extracellular potassium (Frankenhaeuser and Hodgkin, 1956)
during repetitive stimulation (Salzberg et al., 1985; Leng and Shibuki,
1987). Indeed, its contribution to Ca2+
influx depression might be exaggerated under our stimulation protocol
as a result of the unphysiologically synchronous stimulation of the
axons in the infundibular stalk. Failure of action-potential propagation (Jackson and Zhang, 1995; Obaid and Salzberg, 1996), with a
reduction in the number of activated terminals, is another mechanism
that could contribute to the measured depression of Ca2+ influx with increasing stimulation frequency.
Thus, there are several possible mechanisms for the depression of
Ca2+ influx. The physiological origin of
the observed facilitation of the Ca2+
influx is less obvious. Clearly, this is an important factor in the
frequency-dependent regulation of intraterminal calcium changes and
deserves additional scrutiny.
Ca2+ extrusion and/or uptake
The engagement of different Ca2+
extrusion/uptake pathways with different pump rates and
Ca2+ sensitivities has to be considered an
active part of the cellular control of
Ca2+ dynamics during exocytosis. For
example, an important transient release component of fast synaptic
transmission depends critically on the decay rate of
[Ca2+]res (Chen
and Regehr, 1999). In the neurohypophysis, the most obvious effect of
changes in Ca2+ extrusion/uptake dynamics
is on the buildup and decay of residual Ca2+ levels during repetitive stimulation.
Our attempts to identify the different sources of
Ca2+ extrusion/uptake pharmacologically
have been clouded by uncertainties regarding drug permeance across the
plasma membrane, or by direct drug interference with excitability in
the intact tissue. A comprehensive patch-clamp study on
Ca2+ buffering and removal in isolated
nerve terminals identified mitochondrial (ruthenium red sensitive)
uptake as the dominant pathway of Ca2+
removal during repetitive depolarization, with minor contributions from
Ca2+ ATPases (Stuenkel, 1994).
Mitochondrial Ca2+ uptake during
exocytosis has also been observed in bullfrog presynaptic peptidergic
terminals (Peng, 1998). Secretory granules themselves might contribute
to Ca2+ uptake in nerve terminals as well
(Troadec et al., 1998). These findings would suggest that the slow
extrusion in our data is associated with a plasmalemmal
Ca2+ ATPase, whereas the fast component(s)
reflects high-capacity, low-affinity uptake processes inside the terminals.
AVP release and [Ca2+]i
Several hypotheses, most prominently action-potential broadening
(Gainer et al., 1986; Bourque, 1990; Jackson et al., 1991) and buildup
of residual calcium (Jackson et al., 1991; Stuenkel and Nordmann, 1993;
Stuenkel, 1994), have been proposed to explain the frequency dependence
of AVP release from the neurohypophysis. None of these hypotheses
directly addressed the issue of why release should be optimal at any
specific frequency, rather than simply increasing monotonically with
frequency. The results of our
[Ca2+]i
measurements suggest a compelling solution to this question. AVP
secretion is sensitive to both the Ca2+
rise during action potentials and the amplitude of residual
Ca2+ levels between action potentials.
Action-potential stimulation facilitates
Ca2+ rises at low
fStim but eventually depresses
Ca2+ rises with increasing
fStim. In contrast, residual
Ca2+ increases monotonically throughout
the whole frequency range. Therefore, AVP release should be optimized
at some frequency beyond the maximum in
Ca2+ influx, consistent with the release
data in this and many previous experiments (Cazalis et al., 1985;
Gainer et al., 1986; Bicknell, 1988). The data also suggest a
patterning of action potentials for optimizing AVP release within any
given burst: initial high-frequency stimulation to quickly raise
residual Ca2+, followed by stimulation in
the maximal range of release around 3-10 Hz. This protocol precisely
matches the firing pattern reported for AVP neurons in the hypothalamus
(Poulain and Wakerley, 1982). Finally, the extended interburst
quiescence observed in AVP neurons fits well with the extended recovery
period required to overcome long-term depression of
Ca2+ influx. It remains to be seen how
[Ca2+]tr and
[Ca2+]res
interact and how they couple into various components of the secretory
pathway to accomplish the frequency modulation of release.
| |
FOOTNOTES |
Received April 19, 2000; revised June 14, 2000; accepted June 26, 2000.
This work was supported by United States Public Health Service
Grant NS16824. We are grateful to Dr. E. L. Stuenkel for useful discussions and, in particular, for bringing the EIA assay for AVP
measurements to our attention. We also acknowledge Dr. Meyer Jackson
for helpful comments on this manuscript, Dr. S. Kraner for her help in
implementing the EIA assay, and Dr. J. Lindstrom for making his cold
room and plate reader available. We have also received valuable input
and support from our colleague Dr. A. L. Obaid.
Correspondence should be addressed to Dr. Brian M. Salzberg, Department
of Neuroscience, 234 Stemmler Hall, Philadelphia, PA 19104-6074. E-mail: bmsalzbe{at}mail.med.upenn.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
