1Department of Anatomy and Neurobiology and Program in Neuroscience, University of Maryland, Baltimore, Maryland 21201; and 2Section of Neurobiology and 3Department of Neurosurgery, Yale University, New Haven, Connecticut 06520
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ABSTRACT |
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Zufall, Frank, Trese Leinders-Zufall, and Charles A. Greer. Amplification of Odor-Induced Ca2+ Transients by Store-Operated Ca2+ Release and Its Role in Olfactory Signal Transduction. J. Neurophysiol. 83: 501-512, 2000. A critical role of Ca2+ in vertebrate olfactory receptor neurons (ORNs) is to couple odor-induced excitation to intracellular feedback pathways that are responsible for the regulation of the sensitivity of the sense of smell, but the role of intracellular Ca2+ stores in this process remains unclear. Using confocal Ca2+ imaging and perforated patch recording, we show that salamander ORNs contain a releasable pool of Ca2+ that can be discharged at rest by the SERCA inhibitor thapsigargin and the ryanodine receptor agonist caffeine. The Ca2+ stores are spatially restricted; emptying produces compartmentalized Ca2+ release and capacitative-like Ca2+ entry in the dendrite and soma but not in the cilia, the site of odor transduction. We deplete the stores to show that odor stimulation causes store-dependent Ca2+ mobilization. This odor-induced Ca2+ release does not seem to be necessary for generation of an immediate electrophysiological response, nor does it contribute significantly to the Ca2+ transients in the olfactory cilia. Rather, it is important for amplifying the magnitude and duration of Ca2+ transients in the dendrite and soma and is thus necessary for the spread of an odor-induced Ca2+ wave from the cilia to the soma. We show that this amplification process depends on Ca2+-induced Ca2+ release. The results indicate that stimulation of ORNs with odorants can produce Ca2+ mobilization from intracellular stores without an immediate effect on the receptor potential. Odor-induced, store-dependent Ca2+ mobilization may be part of a feedback pathway by which information is transferred from the distal dendrite of an ORN to its soma.
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INTRODUCTION |
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Olfactory receptor neurons (ORNs) are the
chemoreceptive cells that convert odor molecules into electrical
membrane signals, a process known as odor transduction. It is clear
that two ubiquitous second messengers, cAMP and
Ca2+, play pivotal roles in odor transduction.
cAMP is the primary second messenger produced in the olfactory cilia
when odor molecules bind to olfactory receptors, thus causing the
activation of a G-protein-coupled adenylyl cyclase/cAMP
second-messenger cascade leading to the opening of cAMP-gated cation
channels (CNG channels) (for review, see Ache and Zhainazarov
1995; Reed 1992
; Restrepo et al.
1996
). The substantial Ca2+ permeability
of the CNG channels (Dzeja et al. 1999
; Frings et al. 1995
) then causes a rapid Ca2+
increase in the lumen of the olfactory cilia (Leinders-Zufall et
al. 1997
, 1998
). This Ca2+
signal controls both excitation and adaptation. By activating Ca2+-dependent Cl
channels conducting a depolarizing Cl
current,
it serves to increase the gain of transduction (Kleene 1993
; Kleene and Gesteland 1991
;
Kurahashi and Yau 1993
; Lowe and Gold
1993
; Reuter et al. 1998
). The
Ca2+ rise also mediates adaptation by modulation
of CNG channel activity (Kurahashi and Menini 1997
;
Liu et al. 1994
) and
Ca2+/calmodulin kinase II-dependent attenuation
of adenylyl cyclase (Leinders-Zufall et al. 1999
;
Wei et al. 1998
).
Because of the crucial role of Ca2+ in olfactory
signal transduction, it is necessary to develop a detailed
understanding of the mechanisms underlying Ca2+
regulation and homeostasis. Here, we investigate the contribution of
intracellular Ca2+ stores. This study was
motivated, in part, by our previous finding that brief focal odor
stimulation of olfactory cilia produced a wavelike
Ca2+ elevation beginning in the apical cilia and
spreading through the dendrite toward the soma, generating a global
Ca2+ rise in the entire ORN
(Leinders-Zufall et al. 1997, 1998
).
Unlike the monotonic decay of the Ca2+ transients
in the cilia, Ca2+ elevations in the dendrite and
soma often had a more complex appearence and lasted up to 10 times
longer than the ciliary signals, leading us to hypothesize that they
may depend on secondary Ca2+ mobilization.
Two other compelling reasons make the study of
Ca2+ stores in ORNs necessary. First, odorants
sometimes also cause formation of InsP3
(Boekhoff et al. 1990; Ronnett et al.
1993
). It has been proposed that InsP3
mediates a second transduction mechanism by opening
InsP3-gated channels (Boekhoff et al.
1990
; Schild et al. 1995
), but this proposal is
at odds with experiments suggesting that odor transduction is mediated
solely by the cAMP pathway (Belluscio et al. 1998
;
Brunet et al. 1996
). Intracellular
Ca2+ stores could provide an alternative target
for odor-induced InsP3. Second, imaging
intracellular Ca2+ is increasingly
used as a method to visualize odor-induced activity, construct
sensitivity profiles of individual ORNs, and relate this information to
the structure of odor receptor genes (Bozza and Kauer
1998
; Malnic et al. 1999
; Rawson et al.
1997
; Sato et al. 1994
; Tareilus et al.
1995
; Touhara et al. 1999
). If these data are to
be interpreted correctly, more information on the precise mechanisms by
which Ca2+ is generated in ORN compartments is
required. As our results show, odor-induced mobilization of
Ca2+ from intracellular stores
needs to be taken into account.
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METHODS |
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Preparation of isolated ORNs
Isolated ORNs were prepared following protocols described
previously (Leinders-Zufall et al. 1997). Briefly, ORNs
were acutely dissociated from the nasal epithelium of adult land-phase
tiger salamanders without the use of enzymes. To avoid movement
artifacts, suspended cells were placed in a laminar flow chamber on
glass coverslips that had been previously coated with 0.01%
poly-L-lysine and 0.1% laminin to immobilize the neurons
and their normally motile cilia on the substrate. Only those cilia that
did not change their position during the course of an experiment were
included in the analysis. ORNs were continuously superfused with
physiological Ringer solution containing (in mM) 115 NaCl, 2.5 KCl, 1.0 CaCl2, 1.5 MgCl2, 4.5 HEPES, and 4.5 Na-HEPES, pH 7.6, adjusted to 240 mOsm. To avoid a
contribution to the measured Ca2+ signals from
regenerative action potential discharges, 4 µM tetrodotoxin (TTX) was
added to this solution in all Ca2+ imaging
experiments. TTX was also present in the Ringer solution during the
electrophysiological experiments with the exception of the receptor
potential recordings of Fig. 7. All measurements were carried out at
room temperature.
Calcium imaging
Imaging techniques were essentially as described earlier
(Leinders-Zufall et al. 1997, 1998
). ORNs
(n = 57) were loaded with the
Ca2+ indicator fluo-3 AM (18 µM; Molecular
Probes, Eugene, OR). A laser scanning confocal system (Bio-RAD MRC-600,
Hercules, CA) attached to an Olympus IMT-2 inverted microscope was
employed to visualize Ca2+-mediated fluorescence
changes. A 60×, 1.4 numeric aperture oil immersion objective (Nikon)
was used; images were additionally magnified three to four times using
the confocal's electronic zoom setting. Time-series images were made
by collecting 64 × 64 pixel fluorescence images at a rate ranging
from 0.1 to 3 Hz, depending on the experiment. For the higher spatial
resolution required in some experiments, four individual frames
(768 × 512 pixels/frame) were averaged together, using the Kalman
filter function of the confocal system. Data are presented in arbitrary fluorescence units or as relative changes in fluorescence intensity normalized to baseline fluorescence (
F/F). For the
quantification of Ca2+ signals, regions of
interest were outlined and the average pixel values in these regions
were measured. Usually, these regions corresponded to entire ORN
compartments such as soma, dendrite, knob, and individual cilia, as
schematized in Fig. 1A. We
also tested whether equivalent results were obtained when smaller areas that showed some degree of inhomogeneity in fluorescence level were
taken as representative region (see, for example, Fig. 1, B
and G). In no case did the results obtained by these two
approaches differ qualitatively, and none of the conclusions depend
critically on the small quantitative differences seen (compare for
example Fig. 1, F and G).
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Electrophysiological recording and drug application
For all current-clamp and voltage-clamp recordings
(n = 46), we employed the perforated patch-clamp
technique with amphotericin B (Leinders-Zufall et al.
1995). This approach ensures the least possible disturbance of
the internal milieu of the neurons and avoids artificial
Ca2+ buffering of the intracellular compartments.
The patch pipettes were filled with the following solution (in mM):
17.7 KCl, 105.3 KOH, 82.3 methanesulfonic acid, 5.0 EGTA, and 10 HEPES,
pH, 7.5 (KOH), adjusted to 224 mOsm. In some experiments
K+ was replaced by Cs+.
Current and voltage recording, stimulation sequences, data acquisition, and on-line analysis were controlled by an EPC-9 patch-clamp amplifier in combination with Pulse software (HEKA Electronic,
Lambrecht/Pfalz, Germany) and a Macintosh computer. Currents were
filtered at 300 Hz (
3 dB, 8-pole low-pass Bessel). The indifferent
electrode consisted of a Ag-AgCl wire connected to the bath solution
via an agar bridge. All data reported here have been corrected for junction potentials.
Thapsigargin (Research Biochemicals International, Natick, MA) was initially dissolved in ethanol to give a 2-mM stock solution. The agent was diluted to the final concentration immediately before use, sonicated, and applied by bath perfusion. Final concentrations of thapsigargin solutions contained 0.01% (vol/vol) ethanol. In control experiments (n = 4) we found that this concentration of ethanol alone had no effect on intracellular Ca2+ levels. Other stimuli such as the odorant cineole (eucalyptol, 1,3,3-trimethyl-2-oxabicyclo[2,2,2]-octane), the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), or the ryanodine receptor agonist caffeine were ejected from multibarrel glass pipettes that were placed within 5-10 µm from the cilia or the cell soma. Stimulus pipettes were located downstream from the cells to avoid prestimulation. Unless otherwise stated, all chemicals were obtained from Sigma (St. Louis, MO). Our basic paradigm to deplete intracellular Ca2+ stores was to add 200 nM thapsigargin to the bath solution for 5 min. This treatment resulted, over the course of 10-25 min after the initial application of the drug, in Ca2+ accumulation followed by restoration of the Ca2+ signal to prestimulation levels (see Figs. 1 and 3).
Data analysis
For off-line analysis, eight-bit confocal image files were transferred to a Macintosh G3 microcomputer and analyzed with customized NIH Image 1.61 software. Additional data analyses and calculations were performed using Igor Pro software (WaveMetrics, Lake Oswego, OR). Unless otherwise stated, data are expressed as means ± SD. Statistical tests were performed with SuperAnova 1.1 (Abacus Software, Berkeley, CA). Fisher's LSD (least significant difference) test was used as a post hoc comparison of the ANOVA. Composite images were prepared using Adobe Photoshop 3.0 and printed on a Fujix Pictography 3000 color printer.
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RESULTS |
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Spatial segregation of store-operated Ca2+ release in ORN compartments
To test for the existence of functional
Ca2+ stores in ORNs, we applied thapsigargin, a
potent and irreversible inhibitor of sarcoplasmic-endoplasmic reticulum
Ca2+-ATPases (SERCAs) that mediate the uptake of
cytosolic Ca2+ into endoplasmic reticulum stores
(Pozzan et al. 1994; Thastrup et al.
1990
; Thomas and Hanley 1994
; Treiman et
al. 1998
). To visualize changes in the intracellular
Ca2+ concentration, ORNs were loaded with the
Ca2+ indicator dye fluo-3 AM, and confocal
fluorescence imaging was done as described previously
(Leinders-Zufall et al. 1997
, 1998
). Figure 1 illustrates the effect of bath-applied thapsigargin (200 nM)
on Ca2+ fluorescence in the various cellular
compartments of an ORN. At rest before stimulation (Fig.
1B), fluorescence intensity was generally rather low except
for domains of higher fluorescence in discrete spots at the rim of the
nucleus and at proximal and distal portions of the dendrite. The
distribution of these fluorescence spots resembled the distribution of
cellular organelles such as endoplasmic reticulum, mitochondria, and
variegated and multivesicular bodies (Simmons et al.
1981
). The soma contains a large elliptically shaped nucleus
that allows only a thin rim of surrounding cytoplasm on the lateral and
basal aspects of the soma. The light and dark areas within the soma
reflect the nuclear "checkerboard" pattern of chromatin typical for
mature salamander ORNs (Simmons et al. 1981
). Consistent
with our previous results, resting Ca2+
fluorescence within single cilia was below detection level in most
ORNs. Thapsigargin induced a substantial long-lasting elevation in
intracellular Ca2+ (Fig.
1C). This thapsigargin-evoked Ca2+
rise overlapped spatially with the distribution of endoplasmic reticulum, occurring in the knob, dendrite, and soma, but not in the
olfactory cilia. This effect of thapsigargin was highly robust and
reproducible in virtually every ORN tested (soma:
F/F = 26.4 ± 12.3%, mean ± SD;
dendrite:
F/F = 19.9 ± 8.9%; knob:
F/F = 9.0 ± 6.8%; n = 17).
The elevated Ca2+ recovered to near baseline
levels over the course of 10 min after superfusing the ORN with Ringer
solution (Fig. 1D).
Given that thapsigargin acts as an irreversible SERCA inhibitor, a likely explanation for this observation is that the Ca2+ stores were emptied at this point and Ca2+ clearance took place. This notion was supported further in other experiments in which we applied thapsigargin for a prolonged time (200 nM for 30 min, not shown). Despite the continued presence of thapsigargin in the bath solution, the Ca2+ elevation always recovered to baseline levels within 10-25 min after the initial application of thapsigargin (n = 9). Together with additional experiments shown in Fig. 3, this suggests that the recovery of the Ca2+ signal as seen in Fig. 1D does not reflect reversibility of the thapsigargin-induced effect but instead is evidence for an almost complete depletion of the thapsigargin-sensitive Ca2+ pools.
The thapsigargin-induced fluorescence changes were plotted as a function of time (Fig. 1, E-G). In Fig. 1, E and F, spatially averaged pixel values were obtained from entire ORN compartments as outlined in Fig. 1A. No measurable signal was detected in the cilia of this cell (Fig. 1E), or in cilia from any other ORN exposed to thapsigargin (n = 29). Within the soma, dendrite, and knob the thapsigargin-induced Ca2+ rise occurred approximately simultaneously (Fig. 1F). The onset of this signal was initiated within 30 s and reached a maximum by ~3-4 min after drug application (Fig. 1F). Figure 1G shows the thapsigargin-induced Ca2+ rise analyzed in four smaller regions within the cell soma corresponding to dark and light areas (regions are outlined in Fig. 1B). There was no qualitative difference in the thapsigargin-induced effect between these smaller areas and the entire soma. When we applied a higher dose of thapsigargin (400 nM), intracellular Ca2+ arose much faster, but cell death occurred regularly during the course of these experiments, making the use of higher thapsigargin concentrations impracticable.
To strengthen further the evidence that store-operated
Ca2+ release is compartmentalized in ORNs, we
utilized caffeine to induce Ca2+ release.
Caffeine is known as a stimulator of neuronal ryanodine receptors that
act as intracellular Ca2+ release channels
(Garaschuk et al. 1997; McPherson et al.
1991
; cf. Taylor and Broad 1998
). When a pulse
of caffeine (10 mM) was focally applied onto the soma, dendrite and
cilia of an ORN, there was a transient rise in
Ca2+ in all cellular compartments except for the
cilia (n = 14; Fig. 2,
A-C). The Ca2+ transients rose to a
peak within 2 s and decayed back to baseline within 13-42 s after
termination of the stimulus. They reached fluorescence changes of
F/F = 23.1 ± 5.3%, n = 5 (analyzed in the dendritic compartment), which are in a similar range
as those induced by thapsigargin. When the duration of the caffeine
pulses was increased, there was no further increase in the measured
Ca2+ signals, indicating that the responses were
close to their maximum (data not shown). Another known action of
caffeine, inhibition of phosphodiesterase (PDE), was almost certainly
not involved in the caffeine effect. PDE is known to be highly
concentrated in the ORN cilia, and application of the PDE inhibitor
IBMX leads to robust ciliary Ca2+ elevations
(Leinders-Zufall et al. 1997
). The absence of such Ca2+ increases after caffeine stimulation argues
against a significant inhibition of olfactory PDE by the caffeine
stimuli used here.
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Together, the results of Figs. 1 and 2 indicate that salamander ORNs contain functional Ca2+ stores in their knob, dendrite, and soma. The fact that Ca2+ increases were readily induced by thapsigargin or caffeine before the activation of any Ca2+ entry pathway provides evidence that even at rest the stores contain a releasable pool of Ca2+. Because thapsigargin acts as an uptake inhibitor, the data suggest that Ca2+ stores in ORNs continuously leak Ca2+ and that this loss is counterbalanced by active uptake (sequestration) of Ca2+ by the SERCA pumps into the stores. The fact that we have selectively increased Ca2+ in the dendritic knob but not in the cilia by releasing it from Ca2+ stores in the knob shows that this Ca2+ remains in the knob and does not spread into the cilia. Ca2+ release from thapsigargin-sensitive stores is therefore unlikely to be significantly involved in the primary odor transduction process or in its Ca2+-dependent feedback regulation through modulation of enzymatic cascades present in the olfactory cilia.
Thapsigargin discharges Ca2+ from caffeine-sensitive stores
To test that the source of the thapsigargin-induced
Ca2+ increase was indeed intracellular, we
lowered the extracellular Ca2+ concentration to
0.6 µM, which is sufficient to eliminate odor- and cyclic
nucleotide-induced Ca2+ responses in these cells
(Leinders-Zufall et al. 1997, 1998
). Under these conditions thapsigargin still elicited a substantial increase in Ca2+ with no obvious difference in
the spatial distribution of the signal compared with normal
extracellular Ca2+ concentrations (Fig.
3A). However, there was a
strong modification in the temporal behavior of the
thapsigargin-induced Ca2+ rise in that the
response became more transient, now recovering back to baseline within
3-4 min during the continuous presence of the drug (Fig.
3A; n = 7). This shift from a sustained to a more transient response following removal of extracellular
Ca2+ is consistent with previous results in a
number of nonexcitable cell types and is sometimes interpreted as
evidence for the existence of capacitative Ca2+
entry (Putney 1986
; cf. Thomas and Hanley
1994
). In the "capacitative entry" model the initial
Ca2+ transient induced by thapsigargin is caused
by release from intracellular stores, whereas the sustained
Ca2+ elevation reflects
Ca2+ entry caused by the activation of
store-operated Ca2+ entry channels located in the
plasma membrane, but it is unclear whether such capacitative
Ca2+ entry occurs in neurons (see
Garaschuk et al. 1997
). The results of Fig.
3A may suggest that this is the case in ORNs, although more
specific testing will be required for a final assessment of this
possibility.
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Figure 3B illustrates an experiment in which an ORN was stimulated by two successive thapsigargin applications (200 nM) that were interrupted by a 10-min washing period. Although the first stimulus induced a Ca2+ rise that gradually recovered back to baseline over the course of ~12 min, the second application was unable to elicit any detectable Ca2+ increase (n = 3). This provides further evidence that a one-time treatment with 200 nM thapsigargin was sufficient to discharge the thapsigargin-sensitive Ca2+ pools to a large degree if not completely. Therefore this stimulus was utilized in all subsequent experiments for discharging Ca2+ stores.
To investigate the relation between thapsigargin- and caffeine-depleted stores, an ORN was stimulated with a 5-s pulse of caffeine (10 mM) as described in Fig. 2, and the resulting Ca2+ transients were analyzed in the knob (Fig. 3C) and dendrite (Fig. 3D). The experiment was done with low external Ca2+ concentration (0.6 µM) to avoid release-activated Ca2+ entry. After the caffeine test pulse, thapsigargin was applied, causing store depletion. A second caffeine stimulus was now unable to induce a Ca2+ transient in the knob (n = 4; Fig. 3C). This indicates that caffeine and thapsigargin target the same set of Ca2+ pools within the knob. In the dendrite, however, the situation was more complicated in that the caffeine-evoked Ca2+ transient was reduced (by 3- to 5-fold) but not completely eliminated after thapsigargin treatment (n = 4; Fig. 3D).
That thapsigargin and caffeine target the same set of
Ca2+ stores is further suggested by the results
of Fig. 3E in which we examined the role of
thapsigargin-dependent Ca2+ uptake on refilling
of caffeine-sensitive stores. Multiple consecutive caffeine pulses
delivered at a rate of 0.1 Hz induced an initial large response
followed by much smaller Ca2+ transients,
suggesting considerable depletion of Ca2+ stores
(Fig. 3E). When the same protocol was repeated 6.6 min later, the initial large Ca2+ transient was fully
recovered, indicating that the stores underwent spontaneous refilling.
However, when this protocol was repeated after treatment of the cell
with thapsigargin (200 nM), the initial large
Ca2+ transient failed to recover, whereas there
was very little effect on the smaller Ca2+
transients seen with repetitive caffeine stimulation (n = 5). These findings indicate that refilling of caffeine-sensitive
stores in ORNs is mediated by SERCA pumps. The data demonstrate that thapsigargin and caffeine overlap, at least partially, in their actions, thus targeting the same set of Ca2+
stores. There may be a second type of caffeine-sensitive
Ca2+ pool in these cells that is resistant or
less sensitive to thapsigargin treatment (for comparison see
Thomas and Hanley 1994), although a nonuniform internal
thapsigargin concentration cannot be ruled out entirely.
Ca2+ stores control the waveform of odor-induced Ca2+ transients in the dendrite and soma but not in the cilia
What is the precise role of thapsigargin-sensitive
Ca2+ stores in Ca2+
signaling of ORNs? The endoplasmic reticulum may serve 1) as an intracellular source for Ca2+
being involved in Ca2+ mobilization and
Ca2+-induced Ca2+ release
following cellular stimulation, or 2) it may mediate rapid
sequestration and uptake to govern clearance of
Ca2+ after an odor stimulus (Miller
1991; Simpson et al. 1995
). To distinguish
between these possibilities, we emptied Ca2+
stores with thapsigargin and examined the effect of this treatment on
stimulus-induced Ca2+ responses in the ORN compartments.
The cilia of an ORN were stimulated with a brief pulse of the PDE
inhibitor IBMX (300 µM for 1 s), and confocal images were acquired at the peak of the resulting Ca2+
response (Fig. 4, A-C). IBMX
treatment is known to stimulate the odor-sensitive cAMP pathway present
in the cilia of these cells causing CNG channel opening and
Ca2+ entry (Firestein et al.
1991b; Leinders-Zufall et al. 1997
). As reported
previously, IBMX caused the generation of a characteristic Ca2+ wave spreading through the ORN with distinct
spatiotemporal properties starting in the cilia and eventually leading
to a global Ca2+ rise in the dendrite and soma
(Fig. 4B). After recovery of this Ca2+
wave, Ca2+ stores were emptied with thapsigargin
(200 nM for 5 min) until the resulting Ca2+
elevation recovered back to baseline as shown in Fig. 3B.
Following this treatment, a second IBMX pulse of the same strength was
applied that evoked a Ca2+ transient within the
cilia very similar to that of the control measurement. But in contrast
to the control situation, the propagation of the
Ca2+ wave into the dendrite and soma was now
strongly reduced. This can be seen in the confocal image of Fig.
4C in which the cilia exhibited enhanced
Ca2+ fluorescence in response to IBMX but the
fluorescence intensity in the dendrite and soma resembled more that of
the unstimulated cell shown in Fig. 4A. Closely similar
results were obtained in a total of six ORNs (see Table
1 for a more complete analysis of the
data).
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An equivalent effect was observed when the cells were stimulated with odorant (cineole, 1 s, 300 µM) instead of IBMX. Cineole triggered a similar Ca2+ wave as IBMX, starting in the cilia and then propagating toward the soma (Fig. 4D). A characteristic property of this Ca2+ wave is the prolonged Ca2+ elevation in the dendrite and soma, which is distinct from the more transient Ca2+ rises in the cilia and the olfactory knob. Consistent with the results of Fig. 4, A-C, Ca2+ store depletion with thapsigargin caused a 2.5- to 11-fold decrease in the peak amplitude of the transients as well as a 2- to 4-fold shortening of the response duration in the dendrite and soma (n = 4; Fig. 4D). There was no detectable effect of store depletion on the ciliary odor-induced Ca2+ transients (n = 4; Fig. 4D). A moderate degree of response shortening was seen in the olfactory knob (n = 4; Fig. 4D). Data from several independent experiments are summarized in Table 1. We conclude therefore that a substantial portion of the prolonged Ca2+ responses in the dendrite and soma results from Ca2+ release by thapsigargin-sensitive stores. This provides evidence that Ca2+ released from thapsigargin-sensitive pools, at least under the conditions of odor stimulation employed here, amplifies and prolongs incoming Ca2+ signals, thus boosting the magnitude and duration of odor-induced Ca2+ transients in the ORN dendrite and soma giving rise to the propagation of a characteristic Ca2+ wave from the cilia to the soma.
These data provide evidence against a significant contribution of Ca2+ released from thapsigargin-sensitive stores to the ciliary Ca2+ signals elicited by odor stimulation. Together with the results of Figs. 1 and 2, they thus confirm and extend the notion that there is no detectable back spread of Ca2+ from the dendritic knob during the odor response and that each cilium can function as a Ca2+ signaling unit that is relatively independent from Ca2+ changes in other cilia and the dendritic knob.
Depolarization-induced Ca2+ transients depend on the filling state of thapsigargin-sensitive stores: evidence for Ca2+-induced Ca2+ release (CICR)
Having provided evidence that odor stimuli can trigger Ca2+ mobilization from thapsigargin-sensitive stores in the olfactory knob, dendrite, and soma, we next investigated whether Ca2+ entering the ORN through voltage-operated Ca2+ channels (VOCCs) contributes to this effect. To test for the presence of such CICR, we examined whether depolarization-induced Ca2+ transients are affected by Ca2+ store depletion (Fig. 5). If CICR contributes to the depolarization-induced Ca2+ transients, then their magnitude should be diminished after store depletion.
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When the soma of an ORN was stimulated with a 1-s pulse of KCl
(120 mM), a rapid Ca2+ elevation was detected in
the soma (F/F = 44 ± 28%; half recovery time = 21 ± 6 s; n = 9) and in other
cellular compartments (Fig. 5A) but not in the cilia as
shown previously (Leinders-Zufall et al. 1997
).
Following store depletion by thapsigargin (200 nM for 5 min), a second
identical KCl stimulus elicited a Ca2+ response
that was strongly reduced both in peak amplitude and duration as
compared with the control measurement (
F/F = 13 ± 14%; half recovery time = 12.2 ± 7 s;
n = 9; Fig. 5B). As with odor stimulation,
the effect of store depletion on depolarization-induced Ca2+ transients was somewhat variable causing
peak amplitude decreases ranging from 2.5- to 12-fold
(n = 9). A comparable decrease in depolarization-induced Ca2+ transients was never
seen in control experiments without thapsigargin treatment, indicating
that the described effect was not caused by dye bleaching
(n = 4). Thus a large fraction of the
depolarization-induced Ca2+ transients in the
knob, dendrite, and soma of ORNs appears to depend on CICR.
Despite the fact that thapsigargin is widely accepted as a relatively
specific inhibitor of SERCA pumps, previous work has shown that it
sometimes can also act as an inhibitor of voltage-gated Ca2+ channels, at least when used at micromolar
concentrations (Rossier et al. 1993; Shmigol et
al. 1995
; Treiman et al. 1998
). Although we did
not utilize such high doses in the current study, we considered the
possibility that the effect on depolarization-induced
Ca2+ transients as seen in Fig. 5B was
mediated by thapsigargin-induced blockade of VOCCs. VOCCs were
investigated in response to ramp depolarization under voltage clamp
(holding potential,
60 mV) using the perforated patch technique (see
METHODS for details). Voltage-gated
Na+ currents were inhibited by TTX (4 µM), and
K+ outward currents were blocked to a large
degree with Cs+, which was dialyzed from the
patch pipette into the cytoplasm via the pores formed by amphotericin
B. A typical recording example is depicted in Fig. 5C
illustrating the resulting current-voltage (I-V) curve.
Salamander ORNs exhibited relatively small currents through VOCCs with
an activation threshold near
40 mV and a peak amplitude
60 pA
(n = 6), consistent with previous results
(Firestein and Werblin 1987
). These currents were
blocked completely by Cd2+ (500 µM; Fig.
5C). Figure 5D illustrates that thapsigargin had no direct effect on VOCCs (n = 5). Thus
thapsigargin-mediated inhibition of VOCCs is unlikely to account for
the effects documented in Fig. 5B.
Taken together, the results of Figs. 4 and 5 indicate that an
unexpectedly large fraction of the odor-induced
Ca2+ elevations in the knob, dendrite, and soma
(but not in the cilia) of salamander ORNs is mediated by
Ca2+-induced Ca2+ release
from thapsigargin-sensitive stores. We show that CICR is present in
ORNs and that its extent is sufficient to account for the
store-operated amplification of odor-induced Ca2+
transients. The Ca2+ release cannot be explained
by cAMP diffusing from the cilia to the soma: when we lowered the
external Ca2+ concentration to 0.6 µM, which
does not attenuate cAMP formation, there was no
Ca2+ mobilization in any of the ORN compartments
(Leinders-Zufall et al. 1997, 1998
). We
therefore conclude that the prolonged Ca2+
responses seen in the ORN dendrite and soma following a brief pulse of
odorant result primarily from CICR.
Depletion of Ca2+ stores by thapsigargin induces membrane hyperpolarization and activation of a K+ conductance
To begin to assess the relation between store-operated
Ca2+ release and the electrical properties of the
cells, we examined whether Ca2+ depletion by
thapsigargin leads to a change in membrane potential (Fig.
6). Thapsigargin sometimes depolarizes
neurons through activation of cationic conductances (Knox et al.
1996). In salamander ORNs, however, thapsigargin (200 nM)
induced a slow tonic hyperpolarization of
2.2 ± 1.6 mV (from a
resting potential of
60 mV, n = 6) lasting for
7.2 ± 1.3 min. The reversal potential of this hyperpolarization was derived from a plot of the magnitude of the thapsigargin-induced voltage change as a function of the holding potential, yielding Erev =
71 mV (n = 16; regression: P = 0.84; data not shown). The fact
that thapsigargin application was associated with membrane hyperpolarization but not with depolarization rules out that
Ca2+ entry through VOCCs contributed to the
thapsigargin-induced Ca2+ rises documented above.
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Figure 6B illustrates that thapsigargin produced a decrease
in input resistance (monitored through hyperpolarizing current pulses),
from 4.1 ± 0.4 G to 3.3 ± 0.6 G
(n = 6). The onset and recovery of this resistance change paralleled the
general time course of the thapsigargin-induced hyperpolarization,
suggesting that Ca2+ store depletion evoked the
activation of specific ion channels.
To test this notion further, we measured the effect of thapsigargin in
response to stimulation with a depolarizing voltage ramp on whole cell
currents in voltage-clamped ORNs (holding potential, 60 mV). In this
case, unlike the experiment shown in Fig. 5, C and
D, K+ was not replaced with
Cs+ in the intracellular pipette solution. Figure
6C illustrates that, under these ionic conditions,
thapsigargin induced the activation of an outward current (thap,
5 min). Digital subtraction of the two I-V curves
(thap, 5 min
control) revealed a conductance with
outwardly rectifying properties and a characteristic N-shape showing a
local mininum near +50 mV, close to the expected reversal potential for
Ca2+ flux through VOCCs (Fig. 6D;
n = 9). Because this type of conductance is usually
associated with the activation of Ca2+-dependent
K+ channels (Meech and Standen
1975
; Thomas 1984
), the main effect of
thapsigargin-induced Ca2+ store depletion on ORN
membrane properties is a net hyperpolarization caused by the activation
of putative Ca2+-activated
K+ channels. Such a conductance was never seen
when the ORNs were dialyzed with Cs+ (see Fig.
5D). Ca2+-dependent
K+ channels have previously been shown to be
present in the cell body, dendrite, and knob of ORNs (Maue and
Dionne 1987
). In eight of nine experiments, we also observed
activation of a small inward current with an amplitude of
10 pA in
response to thapsigargin (see arrow in Fig. 6D). This
current component was somewhat reminiscent of calcium release-activated
calcium currents (ICRAC) that have been described in a number of nonexcitable cell types (cf.
Parekh and Penner 1997
) and could underlie the effects
observed in Fig. 3A. Detailed investigation of this
thapsigargin-evoked inward current, however, was beyond the scope of
the current study.
Lack of effect of Ca2+ store depletion on odor-induced receptor potential
Thus far our results indicate that odor stimulation causes mobilization of Ca2+ from intracellular stores via CICR and that Ca2+, if discharged by thapsigargin, tends to hyperpolarize the cells through activation of a K+ conductance. This raises the question of whether odor-induced Ca2+ mobilization serves to shape the receptor potential thus modifying chemoelectrical signaling and odor processing in ORNs.
To test this idea, odor-induced receptor potentials were analyzed
before and after store depletion with thapsigargin (Fig. 7). Surprisingly, however, we found that
the waveform of the receptor potential was nearly independent of the
filling state of Ca2+ stores. In the example
shown in Fig. 7A, the cell had a resting potential of 60
mV before thapsigargin application. A 1-s pulse of cineole (300 µM)
induced a typical depolarizing receptor potential to
24 mV lasting
for ~3 s. During the rising phase, a transient burst of action
potential discharges occurred, which was followed by a silent period.
After repolarization there was a pronounced afterhyperpolarization
(AHP) lasting for a few seconds. The cell was then treated with
thapsigargin (200 nM for 22 min), and the same odor stimulus was
applied. As shown in Fig. 7, A and B, there was
neither a change in the resting potential nor a significant difference
in the waveform of the odor-induced receptor potential. Closely similar
results were obtained in a total of seven ORNs.
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Overall, these results reveal that pharmacological disruption of
Ca2+ store function has no immediate effect on
the electrophysiological responses to odors, at least under the
conditions of odor stimulation used here, indicating that the receptor
potential and its underlying transduction currents are relatively
independent of the filling state of Ca2+ stores.
Thus, stimulation of ORNs with odorants can produce
Ca2+ mobilization from intracellular stores
without changing significantly the electrical properties of the ORNs.
This situation is reminiscent of a class of synaptic responses
involving Ca2+ release from intracellular stores
in some dendritic spines (Takechi et al. 1998).
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DISCUSSION |
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A combination of confocal imaging and electrophysiological recording provided insight into the role of intracellular Ca2+ stores in odor transduction of ORNs. Several new findings emerge from this work. 1) ORNs contain a releasable pool of Ca2+ that can be discharged at rest applying the SERCA pump inhibitor thapsigargin or the ryanodine receptor agonist caffeine. 2) These Ca2+ stores are distributed differentially within the ORNs; they are present in the soma, dendrite, and knob, but not in the cilia, the site of primary odor transduction. 3) SERCA pumps mediate the refilling of caffeine-sensitive stores. 4) Ca2+ released from thapsigargin-sensitive pools serves to amplify odor-induced Ca2+ transients in the knob, dendrite, and soma, but not in the cilia; this effect underlies Ca2+ wave propagation from the cilia to the soma. 5) The amplification is primarily due to Ca2+-induced Ca2+ release. 6) Thapsigargin-induced Ca2+ release tends to hyperpolarize the ORN membrane potential by stimulating a Ca2+-activated K+ current. 7) Thapsigargin-sensitive stores do not seem to be necessary for generation of an immediate electrophysiological odor response, at least under the experimental conditions described here.
It seems likely that our results will also apply to mammals, although
we have not yet tested whether thapsigargin-sensitive stores exist in
mammalian ORNs. However, there are also differences in
Ca2+ signaling between salamander and mammalian
ORNs. For example, human ORNs frequently appear to respond with a
decrease in Ca2+ to odorant stimulation
(Rawson et al. 1997), whereas such responses were not
observed in salamander (unpublished observations).
It should be noted that a small percentage of ORNs both in amphibians
and rodents sometimes exhibit inhibitory odor responses (Duchamp-Viret et al. 1999; Morales et al.
1997
). Such inhibitory responses were also seen infrequently in
our experiments (unpublished observations). Because
thapsigargin-induced Ca2+ release tended to
hyperpolarize the ORNs, store-dependent Ca2+
release could potentially be involved in these inhibitory odor responses. However, the small percentage of ORNs that exhibited such
responses prevented us from testing this hypothesis directly.
Store-operated Ca2+ release amplifies odor-induced Ca2+ transients via CICR
A key result presented in this communication is that thapsigargin-sensitive Ca2+ stores determine the waveform of odor-induced Ca2+ transients. Following store depletion, the magnitude and duration of odor-induced Ca2+ elevations was strongly reduced relative to the control response (2.5- to 11-fold). This effect occurred only in the dendrite and soma and to a lesser degree in the knob, but not in the cilia. This provides evidence that one function of Ca2+ stores is to serve as an intracellular source for Ca2+ involved in Ca2+ mobilization and release following an odor stimulus. We show that CICR is present in ORNs and that its extent is sufficient to explain the store-dependent amplification of odor-induced Ca2+ transients.
The fact that odor-induced Ca2+ responses in the
soma and dendrite depend critically on the filling state of
Ca2+ stores has two important implications for
experiments in which Ca2+ imaging of the soma is
utilized to construct odor sensitivity profiles of single ORNs
(Bozza and Kauer 1998; Malnic et al.
1999
; Rawson et al. 1997
; Sato et al.
1994
; Tareilus et al. 1995
; Touhara et
al. 1999
). First of all, our results indicate that the
odor-induced Ca2+ rise in the dendrite and cell
body is causally related to the responses in the cilia and therefore
can be used as an assay for imaging odor responsiveness. Thus
Ca2+ imaging at the soma of mammalian ORNs, where
it has not been possible so far to measure intracellular
Ca2+ in the cilia, is a valid method. However,
some caution must be exercised if the magnitude of the cell body
responses is used for generating odor receptor sensitivity profiles,
because the concentration dependence of these responses may not reflect
with fidelity the concentration dependence and sensitivity of the
responses in the cilia. From our experiments, it would appear that odor spectra based on somatic Ca2+ imaging need to be
interpreted in the light of our evidence that they reflect, at least in
part, the filling state of Ca2+ stores. This
filling state may change over the course of an experiment in which an
ORN is challenged with a large number of different odor stimuli. Also,
Ca2+ release imposes a threshold on the measured
responses such that their magnitudes become highly nonlinear with
stimulus strength. Weak odor stimuli that may still be sufficient to
produce ciliary Ca2+ transients may not be
sufficient to induce Ca2+ release at the soma. In
this case, one would underestimate the true sensitivity of an odor
receptor for a given odorant by analyzing the
Ca2+ responses in the cell body.
Evidence for this notion has already been provided: weak activation of
CNG channels produces Ca2+ elevations that are
spatially restricted to the cilia and the knob, whereas strong CNG
channel activation produces Ca2+ rises in all
cellular compartments (Leinders-Zufall et al. 1997
).
Compartmentalization of Ca2+ release mechanisms
We show that Ca2+ can be raised in the knob
by discharging it from Ca2+ stores but that this
Ca2+ remains in the knob and does not spread to
the cilia thus providing evidence for the spatial segregation of
store-operated Ca2+ release in divergent regions
of an ORN. This effect is not due to a diffusion barrier between the
olfactory knob and the cilia because the spread of other substances
such as horseradish peroxidase (Kauer 1981) and
cAMP/cGMP (Firestein et al. 1991a
; Kurahashi 1990
; Leinders-Zufall et al. 1995
) is relatively
unhindered. If cAMP is dialyzed from a patch pipette into the soma of
an ORN, the resulting CNG channel activation in the cilia occurs after only a few hundred milliseconds.
The separation of distinct Ca2+ signaling systems
in spatial domains of an ORN is well-suited for olfactory signaling. It
has been shown that the dynamics of the ciliary
Ca2+ transients determine the rate of odor
adaptation (Leinders-Zufall et al. 1998). Partitioning
of Ca2+ signaling in neuronal compartments
ensures that the more sustained store-dependent
Ca2+ signals in the knob, dendrite, and soma do
not interfere with the signals in the cilia. This suggests that the
Ca2+ signals in the dendrite/soma serve separate
functions than the ciliary Ca2+ transients. In
fact, our finding that store-dependent
Ca2+ does not spread into the cilia
provides evidence that it is not involved significantly in gain control
and adaptation of ciliary enzymatic cascades.
Ca2+ release provides a signal that can spread from the dendrite to the nucleus
The filling state of Ca2+ stores had very
little influence on the odor-induced receptor potential in these
experiments, but more tests will be needed to investigate specifically
whether this is also the case with different conditions of odor
stimulation, e.g., using repeated stimuli of varying durations and
intervals. Nonetheless, this leaves an open question: on the one hand
odor stimuli generate a striking store-dependent
Ca2+ wave propagating from the cilia to the soma;
on the other this effect seems relatively unimportant for the
electrical odor responses that are transmitted to the brain. Given this
evidence, we suggest that store-operated Ca2+
release serves alternative yet unknown functions beyond odor transduction. This notion is consistent with a growing body of evidence
in other cells indicating that propagating Ca2+
waves can provide a molecular signal by which information is transferred from distal parts of a neuron to its nucleus (cf. Berridge 1998). There is increasing evidence that
somatic Ca2+, together with cAMP, has important
roles in nuclear gene activation (Dolmetsch et al. 1997
;
Hardingham et al. 1997
). It will be interesting to test
directly whether store-dependent Ca2+ release is
part of a feedback pathway involved in neuronal gene transcription,
specifically of the odor receptor genes.
Model of Ca2+ sigaling in ORNs: Ca2+ regulation differs between the cilia and the dendrite/soma
In Fig. 8, we have summarized
diagramatically both the experimental results presented here and
results taken from other published studies. Following odor recognition,
Ca2+ signaling begins in the olfactory cilia. The
primary pathway for Ca2+ entry into the cilia are
CNG channels gated by odor-induced cAMP elevations (see
INTRODUCTION). The CNG channels are highly
compartmentalized and exist at high density in the cilia. Thus far, CNG
channels represent the only known Ca2+ entry
pathway into ORN cilia, although InsP3-gated
cation channels have also been proposed to mediate this function (see
INTRODUCTION). If the stimulus strength is sufficient, the
ciliary Ca2+ transients are followed by a
regenerative Ca2+ wave that spreads through the
entire ORN, leading to a global Ca2+ elevation in
the knob, dendrite, and soma. A likely starting signal for this
Ca2+ wave is the passive electrotonic spread of
membrane depolarization initiated by the transduction current in the
cilia. Depolarization leads to Ca2+ entry through
voltage-operated Ca2+ channels into the ORN
dendrite and soma. This Ca2+ signal then triggers
CICR from intracellular stores, most likely through activation of
ryanodine receptors. A posssible role of InsP3
receptors in this process remains to be explored; the synthesis of a
novel membrane-permeant caged InsP3 ester
(Li et al. 1998) opens up new experimental strategies.
Refilling of the stores occurs through thapsigargin-sensitive SERCA
pumps. Store release may also cause activation of a third
Ca2+ entry pathway in the dendrite and soma via
capacitative Ca2+ influx.
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Because Ca2+ is critical for setting the gain and
adaptation level of ORNs, the intracellular Ca2+
concentration must be tightly regulated. The diagram of Fig. 8
indicates some of the mechanisms by which Ca2+
can be extruded from ORNs. There is increasing evidence for the presence of a Na+/Ca2+
exchange mechanism in olfactory cilia (Noé et al.
1997; Reisert and Matthews 1998
) and in the
ORN dendrite (Jung et al. 1994
). A
Ca2+-ATPase has also been isolated from ORNs
(Lo et al. 1994
). We hypothesize that
Ca2+ extrusion can be independently controlled
between cilia and dendrite/soma, given that stimulus-induced
Ca2+ dynamics as well as
Ca2+ entry and storage sites are fundamentally
different between these cellular compartments. It will be interesting
to test whether there is compartmentalization of
Na+/Ca2+ exchanger and
Ca2+-ATPase between cilia and other ORN regions,
in analogy to Ca2+ extrusion in photoreceptors
(Krizaj and Copenhagen 1998
).
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ACKNOWLEDGMENTS |
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We thank G. M. Shepherd for comments on this manuscript.
This work was supported in part by National Institutes of Health Grants NS-37748 to F. Zufall, DC-003773 to T. Leinders-Zufall, and DC-00210 to C. A. Greer.
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FOOTNOTES |
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Address for reprint requests: F. Zufall, Dept. of Anatomy and Neurobiology, University of Maryland, 685 West Baltimore St., Baltimore, MD 21201.
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 7 July 1999; accepted in final form 23 September 1999.
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REFERENCES |
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