Download Calcium Transients in the Garter Snake Vomeronasal Organ

J Neurophysiol
87: 1449 –1472, 2002; 10.1152/jn.00651.2001.
Calcium Transients in the Garter Snake Vomeronasal Organ
ANGEL R. CINELLI,1 DALTON WANG,2 PING CHEN,2 WEIMIN LIU,1 AND MIMI HALPERN1
Department of Anatomy and Cell Biology and 2Department of Biochemistry, State University of New York Downstate
Medical Center, Brooklyn, New York 11203
1
Received 6 August 2001; accepted in final form 19 October 2001
The vomeronasal (VN) system of garter snakes is particularly well developed and is known to be critical for several
species-specific behaviors including prey detection (Burghardt
and Pruitt 1975; Graves and Duvall 1985; Halpern 1982, 1992;
Halpern and Frumin 1979; Halpern and Kubie 1994; Kahmann
1932; Kubie and Halpern 1979; Naulleau 1965; Wilde 1938).
The initial step in prey detection involves the interaction between a prey-derived chemoattractive ligand and specific receptors on the dendritic surfaces of bipolar neurons of the VN
sensory epithelium. We have isolated and purified prey extracts
and used them as selective chemoattractive stimuli. Among the
compounds isolated are EW20 (Wang et al. 1988), a 20-kDa
thiol-containing protein, EW3 (Wang et al. 1992, 1993), a
low-molecular-weight chemoattractant [both derived from
earthworm wash (EW)], and ES20 (Jiang et al. 1990), a 20-kDa
glycoprotein derived from electric shock-induced earthworm
secretion (ESS). ES20 specifically binds to the VN sensory
epithelium in a saturable and reversible manner with a Kd of
⬃0.3 ␮M (Jiang et al. 1990) and does not bind specifically to
other organs, including brain and main olfactory epithelium. G
proteins (Go, Gi, and Gs) have been immunologically detected
in the VN sensory epithelium of garter snakes and ES20
receptors probably are coupled to these G proteins (Luo et al.
1994).
Functional studies have demonstrated that ESS and ES20
evoke depolarizing currents in VN neurons and increase unit
activity in the accessory olfactory bulb (AOB) mitral cells, the
targets of the axons of receptor neurons of the VN epithelium
(Jiang et al. 1990; Luo et al. 1994; Taniguchi et al. 1998,
2000). Binding of ES20 to VN receptors also results in increased levels of inositol 1,4,5-trisphosphate (IP3), suggesting
that this signal cascade pathway may be involved in chemosensory transduction. In contrast, ES20 significantly reduces
basal levels of cAMP as well as GTP␥S- or forskolin-induced
high levels of cAMP (Luo et al. 1994). Nevertheless, an
adenylate cyclase, ACVN, has been cloned from a garter snake
VN epithelial library, which shows high homology to AC type
VI (Liu et al. 1998) and is sensitive to Ca2⫹ regulation (Wang
et al. 1997). Thus within the VN sensory epithelium components of two second-messenger systems, ACVN and phospholipase C (PLC), exist.
In VN neurons, the reversal potential induced by dialysis of
IP3 or its analogue 3-deoxy-3-fluoro IP3 mimics the reversal
currents generated by the chemoattractive stimuli (Taniguchi et
al. 2000). IP3, which is known to mobilize intracellularly
sequestered Ca2⫹ via the IP3 receptor (IP3R), causes elevations
of cytosolic Ca2⫹, which plays multiple functional roles in
neurons including signal transduction (Berridge 1993; Clapham 1995). However, these events in VN neurons remain to be
elucidated.
Taking advantage in the garter snake of the presence of
specific prey extracts as selective chemoattractant stimuli
(ESS), in the present study, we disclose mechanisms triggering
stimulus-induced cytosolic Ca2⫹ transients associated with
chemosensory transduction. Optical recordings were performed in slices from VN neurons selectively loaded with the
fluorescent Ca2⫹ indicator Calcium Green-1 from their axonal
terminals in the AOB by retrograde transport. We report here
that chemoattractants produce initially transient IP3-related ac-
Address for reprint requests: M. Halpern, Dept. of Anatomy and Cell
Biology, Box 5, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203 (E-mail: [email protected]).
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.
INTRODUCTION
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Cinelli, Angel R., Dalton Wang, Ping Chen, Weimin Liu, and
Mimi Halpern. Calcium transients in the garter snake vomeronasal
organ. J Neurophysiol 87: 1449 –1472, 2002; 10.1152/jn.00651.2001.
The signaling cascade involved in chemosensory transduction in the
VN organ is incompletely understood. In snakes, the response to
nonvolatile prey chemicals is mediated by the vomeronasal (VN)
system. Using optical techniques and fluorescent Ca2⫹ indicators, we
found that prey-derived chemoattractants produce initially a transient
cytosolic accumulation of [Ca2⫹]i in the dendritic regions of VN
neurons via two pathways: Ca2⫹ release from IP3-sensitive intracellular stores and, to a lesser extent, Ca2⫹ influx through the plasma
membrane. Both components seem to be dependent on IP3 production.
Chemoattractants evoke a short-latency Ca2⫹ elevation even in the
absence of extracellular Ca2⫹, suggesting that in snake VN neurons,
Ca2⫹ release from intracellular stores is independent of a preceding
Ca2⫹ influx, and both components are activated in parallel during
early stages of chemosensory transduction. Once the response develops in apical dendritic segments, other mechanisms can also contribute to the amplification and modulation of these chemoattractantmediated cytosolic Ca2⫹ transients. In regions close to the cell bodies
of the VN neurons, the activation of voltage-sensitive Ca2⫹ channels
and a Ca2⫹-induced Ca2⫹ release from intracellular ryanodine-sensitive stores secondarily boost initial cytosolic Ca2⫹ elevations increasing their magnitude and durations. Return of intracellular Ca2⫹ to
prestimulation levels appears to involve a Ca2⫹ extrusion mediated by
a Na⫹/Ca2⫹ exchanger mechanism that probably plays an important
role in limiting the magnitude and duration of the stimulation-induced
Ca2⫹ transients.
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
cumulation of [Ca2⫹]i in dendritic regions via two pathways: a
Ca2⫹ release from IP3-sensitive intracellular stores and, to a
lesser extent, a Ca2⫹ influx through the plasma membrane in
dendritic regions. Although other mechanisms can secondarily
participate in the modulation of these chemoattractant-induced
cytosolic Ca2⫹ transients, cAMP seems not to have an important role in the generation these transient Ca2⫹ responses.
METHODS
Animals
Adult garter snakes, Thamnophis sirtalis, of both sexes were obtained from commercial suppliers. They had all resided in the laboratory for ⱖ2 months prior to use in the experiments described here.
VN bipolar neurons were loaded with Calcium Green 1 by means
of retrograde axonal transport of dye placed in the accessory olfactory
bulb (AOB). Snakes were anesthetized with a subcutaneous injection
of methohexital sodium (0.5 mg/g body wt; Eli Lilly, Indianapolis,
IN). A hole above the AOB was drilled through the skull to expose the
AOB bilaterally, and Calcium Green 1 was applied either as crystals
or a 5-␮l injection of Calcium Green solution injected deep into the
glomerular layer. The snakes were allowed to recover for 3– 4 days
prior to the evaluation of Ca2⫹-related fluorescence signals.
Preparation of VN sensory epithelial tissue slices
The methods are essentially similar to those described previously
(Taniguchi et al. 1995, 1996, 2000). In brief, snakes were lightly
anesthetized with methohexital sodium prior to decapitation. The
vomeronasal neuroepithelium was dissected from the head after carefully removing the bony capsule and mushroom body, mounted onto
a carrot block, and cut into slices, ⬃240 ␮m thick, with a vibrating
slicer (Vibratome 3000, Technical Products International, St. Louis,
MO) in snake Ringer solution. This solution consisted of (in mM) 119
NaCl, 4.1 KCl, 2.5 CaCl2, 1.5 MgCl2, 15 glucose, 5 Na-pyruvate, and
10 HEPES (pH 7.4). The tissue slice was then mounted onto a small
plastic petri dish.
Preparation of ESS
The methods are the same as described earlier by Jiang et al.
(1990). Secretions were obtained by passing an electric current from
a 9-V battery (20 6-s bursts with an intershock interval of 30 s)
through the worms. A yellowish mucus-like secretion was collected
and centrifuged, and the supernatant was dialyzed against distilled
water.
Preparation of VN sensory epithelial homogenate
Essentially we followed a standard method as previously reported
(Luo et al. 1994). Dissected VN epithelia were homogenized in cold
buffer (20 mM Tris/HCl, pH 8, 1 mM PMSF, 80 ␮g/ml DTT, 0.5
␮g/ml antipain, 1 ␮g/ml leupeptin, 1 ␮g/ml aprotinin, 0.6 ␮g/ml
chymostatin, and 0.6 ␮g/ml pepstatin) and centrifuged at 500 g for 5
min. The supernatant was recovered and referred to as homogenate.
Monitoring intracellular Ca2⫹ changes
Video imaging of fluorescent changes was used to
monitor calcium responses. The general plan of the optical setup is
based on standard methods (Cinneli and Salzberg 1991, 1992; Cinelli
OPTICAL SETUP.
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CALCIUM IMAGING TECHNIQUES. Fluorescence measurements of
Ca2⫹ levels were performed following standard protocols. Data are
reported as fractional changes over background fluorescence levels
(F/Fo). Standard procedures for background subtraction and calibrations were used for calibration with solutions of known dye concentration (Tsien et al. 1985). After the experiments, in situ calibrations
were performed. Cells were permeabilized with Ca2⫹ ionophores
(ionomycin) or membrane solvents (digitonin or saponin). Fmax and
Fmin were determined in Ringer solution (1 mM Ca2⫹) to saturate the
Ca2⫹indicator and then by subsequently bathing the cells in low-Ca2⫹
Ringer solution supplemented with 5 mM EGTA. Calcium Green
yielded increases in fluorescence signals proportional to Ca2⫹ bound;
these levels were directly related to levels of [Ca2⫹]i. Other terms of
the equation were assessed by in situ calibration (see following text).
IMAGE PROCESSING. Images were digitized and stored in real time
using a frame grabber board in a Pentium IBM-compatible computer
system. Final images were analyzed by applying various digital filterings or convolution algorithms (Cinelli 1998, 2000; Cinelli and
Salzberg 1991, 1992; Cinelli et al. 1995). High spatial resolution
during image acquisition was necessary to preserve the image details
in fine dendrites even when low band-pass spatial filters were used.
Background experiments indicated that low band-pass spatial filters
were often required to suppress pixel noise from the detector or the
image intensifier. High spatial resolutions were also necessary for the
application of deconvolution techniques after low band-pass spatial
filters. Deconvolution techniques were used for improving focus resolution and obtaining better resolution for visualizing particular cellular compartments such as the dendritic terminals of vomeronasal
neurons. Temporal plots of Ca2⫹ transients were obtained from averaged values over 8 ⫻ 8 pixel kernels. In the figures, changes over
time are illustrated by pseudocolors resulting from subtracting basal
levels of [Ca2⫹]i from those obtained after experimental manipulation.
RESULTS
Retrogradely labeled VN neurons
Using slices of snake VN sensory epithelium, changes in cytosolic Ca2⫹ associated with chemosensory transduction were
studied in VN neurons loaded with the Ca2⫹ indicator, Ca2⫹
Green 1. VN neurons were labeled by retrograde transport of this
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Loading VN bipolar neurons with Calcium Green 1 using
retrograde axonal transport from the accessory olfactory
bulb
et al. 1995). Essentially the system consists of an upright epi-illumination microscope (Nikon Epiphot) with a video camera (MV-1070;
Marshal Electronics) in the photographic port. Light from a 150-W
xenon lamp (Optic Quip 1600) is collimated and rendered quasimonochromatic by one of several interference filters, focused by
means of a quartz-UV-grade condenser (Nikon), and reflected to the
preparation by a dichroic mirror. The wavelength for the excitation
and emission filters and the dichroic mirror were selected according to
the excitation and emission spectra of Calcium Green 1. To improve
collection efficiency, fluorescent light from the cells was collected by
high numerical aperture (n.a.) water-immersion objectives (⫻20 or
⫻40; Fluo; Nikon), which formed a real image on the CCD sensor of
the video camera located in the image plane of the microscope. To
further improve the sensitivity of this analog camera, image exposures
were extended to increase light integration in the CCD sensor wells
(Cinelli 1998). When fast acquisition rates were needed, an image
intensifier was used to improve the sensitivity of the camera. Fluorescence emission usually remained constant during the experiments.
To assure stability of the recordings and to avoid photobleaching
effects, the excitation light levels were reduced by neutral density
filters until the fluorescence intensity remained constant for 200 s of
illumination. No significant levels of autofluorescence were observed
in VN neurons, and the drugs, at the concentrations used, did not
affect or quench fluorescence levels.
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
dye from their axonal terminals in the AOB. This method allowed
the selective staining solely of mature VN neurons, which are the
only cell elements in the vomeronasal organ (VNO) that send their
axons to the AOB. Transport of the dye from the injection sites to
VN neurons was usually obtained within 72– 84 h. Using this
approach, we observed that not all VN neurons in a slice preparation were found to be labeled with the fluorescence indicator
(Fig. 1A), a condition that probably arose from the rather localized
application of the dye in the AOB. An important advantage of this
system for obtaining selective retrograde staining is that the VNO
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and the AOB are in different and separated tissue compartments.
This condition prevents the undesirable diffusion of the dye to the
VNO and the nonspecific staining of other cell types. We consistently observed in the slices that only mature VN neurons were
labeled with the fluorescence indicator (Fig. 1, A and B); no other
cellular elements, such as sustentacular cells or immature VN
neurons, were labeled. Therefore all of the Ca2⫹ responses evaluated in this study arose exclusively from fluorescence changes in
VN neurons sending their axons to the AOB.
In most of our recordings, the use of nonconfocal optics in
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2⫹
FIG. 1. Ca
transients in retrogradely labeled snake vomeronasal (VN) neurons. A shows a video image illustrating the
selective staining of VN neurons with Ca2⫹ Green after retrograde transport of this dye from their axonal terminals in the accessory
olfactory bulb (AOB). Observe the labeling in the cell bodies of mature VN neurons (red arrows) located in the intermediate region
of the sensory epithelium. No other cellular elements, such as sustentacular cells or immature VN neurons were labeled. L, lumen;
D, dendritic region; S, somata region; BL, basal lamina. Horizontal bar ⫽ 20 ␮m. B: a schematic diagram showing the laminar
organization of cellular elements in the snake VNO. As shown in A, the cell bodies of mature VN neurons (R) are located
exclusively in the middle epithelial laminae with dendrites projecting toward the epithelial lumen. Adjacent to the basal lamina are
basal cells (B); immature VN neurons (N) are located apical to the basal cells, and sustentacular cells (S) are located close to the
luminal surface, intermixed with the dendrites of VN neurons. C: the increase in [Ca2⫹]i recorded in the 1st, 3rd, 5th, and 7th frames
after elecrtic shock-induced earthworm secretion (ESS, 2.0 mg protein/ml) application. Records taken from a 16 video sequence
(1 frame/s; 120-ms image exposure) showing the spatial distribution of Ca2⫹ responses. Abbreviations as in A. Optical signals were
obtained from single runs, and are coded as follow: green: 5–10%; yellow: 11–15%; orange: 16 –20%; red: ⬎20%. In situ
calibration performed after membrane permeabilization indicates that a 20% optical signal roughly corresponds to changes in
cytosolic Ca2⫹ levels on the order of 500 nM. D: the time course of the increase in [Ca2⫹]i over baseline levels following ESS
application from the entire sequence (16 frames) as determined from 3 different sites (8 ⫻ 8 pixels) shown in the first image in
C (a– c). The 1st point in the plots corresponds to basal cytosolic values and the 2nd point to the first response obtained 1 s after
stimulus application. Arrows indicate the times corresponding to the 4 images illustrated in C.
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
Resting Ca2⫹ levels
Changes in cytosolic Ca2⫹ levels were determined with
standardized optical-imaging techniques (Cinelli and Salzberg
1991, 1992; Cinelli et al. 1995). Using Calcium Green 1, single
wavelength emission measurements of changes in fluorescence
intensity represented estimates of [Ca2⫹]i variations. According to “in situ” calibrations performed at the end of the experiments (see METHODS), baseline [Ca2⫹]i was equivalent to
60.2 ⫾ 15.4 (SD) nM (n ⫽ 12), and cytosolic Ca2⫹ response
peaks were in the range of 104 –740 nM elevations above
resting levels, proportional to the intensity of stimulation.
Experiments usually lasted 6 – 8 h. During this period, [Ca2⫹]i
baseline levels remained steady (usually ⬍70 nM), and no
deterioration of the preparations was observed as judged by the
similarity of the Ca2⫹ responses obtained throughout the experimental sessions.
Characteristics of chemoattractant-induced Ca2⫹ responses
To determine whether VN stimulants evoke cytosolic Ca2⫹
changes associated with excitatory responses, VN epithelial
slices (240 –300 ␮m) were exposed to prey chemoattractant
ESS while [Ca2⫹]i was measured. As illustrated in Fig. 1C,
Ca2⫹ transients in VN neurons elicited by the bath application
of ESS ligands consisted of sharp elevations in cytosolic Ca2⫹
J Neurophysiol • VOL
levels that usually reached a peak amplitude within 1 s.
Response peaks were followed by a brief plateau and then
a more prolonged decay phase in which cytosolic Ca2⫹
levels gradually declined to baseline levels within 16 –
32 s.
Ca2⫹ transients evoked by ESS ligands exhibited a rather
widespread distribution. Figure 1, C and D, shows the spatial
distribution and time course of typical Ca2⫹ transients evoked
by bath application of ESS ligands (2.2 mg/ml). In general,
patterns of activity displayed a scattered appearance with a
heterogeneous organization in which it was possible to find
nonuniform foci of activity distributed in multiple epithelial
regions separated by silent sectors. Within each lamina there
were important variations in the amplitude and time course of
ESS responses, suggesting a different degree of activity among
stimulated VN neurons. Usually Ca2⫹ signals from sectors
showing the largest amplitudes exhibited the longest durations
and lowest thresholds. Following successive stimuli at any
given concentration of ESS, the overall organization of these
Ca2⫹ responses among different epithelial sectors was rather
constant. We interpret this finding as indicating that this heterogeneous pattern represents cumulative responses from dissimilar VN neurons responding in different degrees to the ESS
ligands.
ESS stimuli evoked clear dose-dependent Ca2⫹ transients in
the concentration range of 0.5– 4.5 mg/ml protein. Figure 2,
A–C, shows typical changes in kinetics and spatial distributions
of Ca2⫹ responses following ESS stimuli applied at different
concentrations. The concentration threshold for eliciting detectable Ca2⫹ increases with ESS in our slice preparation was
between 0.8 and 1.2 mg/ml protein. These Ca2⫹-dependent
fluorescence signals usually exhibited half-saturating peak responses at concentrations on the order of 7 mg/ml protein. In
this study, most records were obtained with stimuli in the range
of 2.2– 4.4 mg/ml protein, and these concentrations are similar
to those that evoke clear electrophysiological responses in the
VN epithelium (Taniguchi et al. 1998). As a control for the
specificity of ESS ligands in eliciting Ca2⫹ responses associated to chemosensory transduction mechanisms, we evaluated
the effect of actin which is a behaviorally inactive compound
(unpublished observations). In contrast to the clear responses
evoked by ESS, the application of actin to the bath solution
evoked no detectable Ca2⫹ changes (Fig. 2D) even at relatively
high concentrations (4 – 6 mg/ml).
Once the stimulus threshold was reached, Ca2⫹ transients
elicited by ESS exhibited relatively sharp response onsets with
latency rise times in the range of 500 –750 ms according to
temporal plots obtained from video image sequences acquired
at 250 ms/image (data not shown in the figures). Sharp response onsets were also observed even when ESS stimuli were
applied at the lowest concentration (0.75 mg/ml, Fig. 2A, plot).
Higher ESS concentrations evoked Ca2⫹ signals that exhibited
similar response profiles but larger amplitudes and longer
durations. As shown in Fig. 2, A–C, the applications of increasing ESS concentrations evoked a proportional increase in the
amplitude and duration of Ca2⫹ transients in all epithelial
regions. There were also important changes in the spatial
distribution of these responses as ESS concentration increased.
At low concentrations (e.g., 1 mg/ml), there was a limited
number of activated sites that were located almost entirely in
dendritic regions. As ESS stimulus concentration increased,
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a rather thick slice preparation (240 –300 ␮m) prevented us
from obtaining, with certainty, Ca2⫹ signals at single-cell
resolution. In fact, most of our optical recordings represented
population responses from clusters of VN neurons. Despite this
limitation, it was possible to study the spatial distribution of
Ca2⫹ signals in different segments of VN neurons because the
garter snake VNO exhibits a distinctive lamination pattern of
cellular elements (Fig. 1B). In contrast to the less clear laminar
organization of cellular elements found in other species (e.g.,
Halpern et al. 1995), the cell bodies of mature VN neurons are
located exclusively in the middle epithelial laminae. Just above
the basal lamina there are unstained cell elements corresponding to basal cells and immature VN neurons. In the present
study, the latter were not stained because their axons had not
reached the AOB. Sustentacular cells and the dendrites of VN
neurons are located in the apical epithelial regions close to the
luminal surface. As shown in Fig. 1B, these sustentacular cells
were also unstained in our slices because first they lack projections to the AOB and second no diffusion of the dye
occurred from the AOB to the VNO. Thus despite the lack of
single-cell resolution in our recordings, the lamination pattern
of the snake VNO and the specific staining of only VN neurons
allowed us to define the source of the optical signals at least in
three cellular compartments: the cell body region, the dendritic
shaft region, and the apical dendritic region of VN neurons
adjacent to the luminal surface. Consequently, as seen in all
figure labels, Ca2⫹ responses recorded toward the base and in
the middle of the epithelium corresponded to transients predominantly generated in or close to the cell bodies, optical
signals from the upper VN epithelial sectors related primarily
to Ca2⫹ transients from dendritic shafts, and fluorescence
changes adjacent to the VN lumen corresponded to Ca2⫹
responses arising from the most apical segments of VN dendrites.
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
three major effects were observed in the spatial distribution of
these responses. First, Ca2⫹ transients spread from dendritic
locations to the cell body region of VN neurons where magnitudes and duration comparable to those found in dendritic
regions were attained. Second, we observed a relative enlargement of individual foci of activity, involving adjacent epithelial
sectors. Finally, new discontinuous foci of activity appeared in
previously silent epithelial regions (Fig. 2, B and C). These
new sites of activity did not overlap with other active sectors,
suggesting the recruitment of a different set of active VN
neurons. These results suggest that the newly active VN neurons were probably responding to distinct chemical cues
present in ESS ligands that exhibited different response thresholds.
Characteristics of different cytosolic Ca2⫹ transients in VN
neurons
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could be activated following an initial Ca2⫹ influx mediated
through VSCC, we evaluated the characteristics of Ca2⫹ transients elicited by high-[K⫹]o depolarization following the depletion of internal Ca2⫹ stores. Depletion of intracellular stores
were obtained either using thapsigargin or ryanodine. Thapsigargin depletes intracellular Ca2⫹ store because it is a potent
inhibitor of the intracellular Ca2⫹ pump and prevents Ca2⫹
reuptake into these pools following spontaneous or stimulusinduced depletions. As a consequence of this action, thapsigargin was used to determine the effect obtained after the depletion of all intracellular Ca2⫹ pools. Under our experimental
conditions, we found that the action of thapsigargin (1 ␮M for
10 –15 min) in depleting internal Ca2⫹ stores was predominantly stimulus dependent because the spontaneous Ca2⫹ depletion from these stores was rather low. As a consequence, the
effect of thapsigargin was assessed following repetitive stimulation (see following text). Under these conditions, we observed that thapsigargin could shorten the time course and
reduce the magnitude of K⫹-elicited Ca2⫹ signals, but this
reduction was observed exclusively during the late phase of the
response (n ⫽ 3; data not shown). Thus this result indicates
that Ca2⫹ release enhanced the decay phase of VSCC-mediated
signals.
To further determine the involvement of ryanodine-sensitive
pools in this effect, we used ryanodine instead of thapsigargin
to selectively deplete these stores. Ryanodine acts as a selective antagonist of one of the two types of Ca2⫹ stores by
forcing their release channels into a permanently opened state.
For this purpose, we applied ryanodine (10 ␮M) to the bath
solution in the presence of high concentrations of caffeine (25
mM) for 10 –15 min. In all cases (n ⫽ 18), this protocol
completely depleted ryanodine-sensitive Ca2⫹ stores as judged
by the suppression of caffeine-elicited responses (as in other
cell systems, we also found that in snake VN neurons Ca2⫹
transients evoked by caffeine depend exclusively on a Ca2⫹
release from ryanodine-sensitive stores; see following text).
As illustrated in Fig. 4C, middle, following ryanodine treatment, K⫹-elicited Ca2⫹ signals exhibited a shortened time
course and a reduction in magnitude during the late phases of
the response. These changes were observed in both dendritic
and somatic regions but were more obvious in regions adjacent
to the cell bodies of VN neurons. At this level, Ca2⫹ transients
elicited by K⫹ depolarizations exhibited a reduction during the
decay phase equivalent to 20 –35% of control responses (n ⫽
5). In no case was the initial onset or the peak amplitude of
these responses affected. Thus K⫹ depolarization appears to
elicit an initial Ca2⫹ influx through the activation of VSCC that
in turn triggers a secondary Ca2⫹ release from ryanodinesensitive internal stores, potentiating and prolonging these responses. Therefore both thapsigargin and ryanodine actions
indicate the presence of a CICR mechanism enhancing the late
phases of the Ca2⫹ signals mediated through the activation of
VSCC.
CHARACTERISTICS OF CYTOSOLIC CA2⫹ ELEVATIONS ELICITED BY
RELEASE FROM INTRACELLULAR STORES. To determine the
characteristics of the Ca2⫹ transients in snake VN neurons that
depend on Ca2⫹ release from internal stores, we evaluated the
action of caffeine. By readily crossing the plasma membrane in
different cell systems, caffeine evokes strong Ca2⫹ release,
which depends exclusively on its reversible binding to intra-
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To elucidate possible mechanisms involved in the generation
of ESS responses, we first established the general characteristics of different types of Ca2⫹ transients in VN neurons. Figure
3 compares the spatial distribution and kinetics of different
types of Ca2⫹ signals evoked in snake VN neurons. Both ESS
and caffeine (2–5 mM) applications gave rise to prolonged
Ca2⫹ signals, which lasted 20 –50 s depending on the stimulus
concentrations. These two responses, however, differed in their
spatial distribution and onset characteristics. In contrast with
ESS responses, caffeine signals were found adjacent to the cell
body region, and the pattern of activation was more uniform
across different regions. Caffeine signals also exhibited a
slower rise time with a more progressive build-up, a characteristic that was evident especially at low concentrations (⬍2.5
mM; Fig. 3C). High [K⫹]o elicited Ca2⫹ transients that were
relatively rapid in onset and brief in duration compared with
other types of Ca2⫹ signals found in snake VN neurons (Fig.
3B). They were found predominantly in the cell body region
and, as with caffeine-induced responses, were practically absent in apical dendritic segments.
ROLE OF VOLTAGE-SENSITIVE CA2⫹ CHANNELS. To evaluate the
role of voltage-sensitive Ca2⫹ channels (VSCC) in the generation of cytosolic Ca2⫹ changes, we determined the properties
of Ca2⫹ responses elicited by high KCl (100 mM; Fig. 3B).
The short duration and monotonic decay of these signals occurred even when high [K⫹]o was still present in the bath. This
finding suggests a relatively rapid inactivation of VSCC in
snake VN neurons. The total duration of these cytosolic Ca2⫹
changes was in the range of 3.5–5.5 s, lasting an average of
4.52 ⫾ 1.87 (SD) s (n ⫽ 7). Thus the time course of these
transients were considerably shorter than those elicited by ESS.
Response latencies were also shorter, usually shorter than the
fastest time resolution tested in this study (250 ms/image).
Response peaks attained fluorescent fractional changes equivalent to 25–32%, values that corresponded to [Ca2⫹]i elevation
on the order of 175–290 nM. These Ca2⫹ signals were reversibly suppressed when extracellular Ca2⫹ was removed from the
medium (Fig. 4A). They were also completely blocked after the
application of common VSCC blockers such as cadmium
(Cd2⫹; Fig. 4B) or cobalt (Co2⫹; 50 –100 ␮M; data not shown),
confirming that they resulted primarily from a Ca2⫹ influx
through VSCC.
To determine whether a Ca2⫹-induced Ca2⫹ release (CICR)
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cellular ryanodine receptors (Usachev and Thayer 1999; Usachev et al. 1993). We found that relatively low doses of
caffeine (2.5 mM) could trigger strong Ca2⫹ transients in snake
VN neurons (Fig. 3C). Similar to ESS responses, the amplitude
and duration of these signals were dose dependent. Ca2⫹
transients evoked by 2.5 mM caffeine had peak amplitudes
similar to responses evoked by 2.2 mg/ml ESS (Fig. 3, A and
C). Under these conditions, optical signals exhibited fluorescence increases of 28.5 ⫾ 4.7% (⌬F/Fo; mean ⫾ SD; n ⫽ 8)
that corresponded to [Ca2⫹]i elevations in the range of 190 –
200 nM. But in contrast to the ESS responses, Ca2⫹ transients
elicited by caffeine attained their largest amplitudes in the cell
body region of VN neurons. As we mentioned before, this
distribution was similar to the responses elicited by high-[K⫹]o
applications.
Caffeine responses exhibited moderate rise times, followed
by a plateau, and then a prolonged declining phase in which
[Ca2⫹]i slowly returned to baseline (Figs. 3C and 5A). In
contrast to ESS transients, the rise times of caffeine signals
were highly dependent on stimulus concentration. At low concentrations, they exhibited a slow onset, which progressively
became sharper at higher concentrations.
Ca2⫹ signals elicited by caffeine appear to be generated
entirely by Ca2⫹ release from internal pools. In all cases
2⫹
FIG. 2. General characteristics of Ca
transients evoked by ESS ligands in garter snake VN neurons. A–C: patterns of activity
show 8 consecutive records from 16 frame sequences illustrating [Ca2⫹]i over baseline levels, just before (1st frame) and following
(remaining 7 frames) the application of ESS stimuli at 3 different concentrations (l, 2, and 3.3 mg protein/ml). Note that the luminal
surface of the epithelium (L) is to the right, the basal lamina (BL) to the left. D: a similar sequence but with records obtained
following the application of actin (3 mg/ml), which was used as a control and produced no detectable Ca2⫹ changes. Observe that
in B and C, the increases in odorant concentrations evoked larger and more widespread responses over different regions of the
vomeronasal sensory epithelium. Plots on the right illustrate the corresponding time course of Ca2⫹ signals from the whole
sequence of images (16) at the 3 sites shown in A, 1st image (a– c are indicated by —, - - -, and 䡠 䡠 䡠 , respectively). In all cases,
[Ca2⫹]i returns to baseline levels after 12–16 s, but higher concentrations produce longer response durations. Optical signals were
obtained from single runs (1 frame/s, 120-ms image exposure). The 1st 2 points in the plots correspond to basal cytosolic values
and the 3rd point to the 1st response obtained 1 s after stimulus application. Cytosolic Ca2⫹ levels were coded in pseudocolors as
in Fig. 1C and overlapped on a fluorescent image of labeled VN neurons. — in D ⫽ 75 ␮m.
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FIG. 3. Spatiotemporal characteristics of
Ca2⫹ signals evoked by ESS (A), high K⫹ (B),
and caffeine (C) in VN neurons. Patterns of
activity in A–C correspond to 8 consecutive
records from 16 frame sequences reflecting
cytosolic Ca2⫹ elevations following stimulus
applications. Right: plots correspond to the
time course of Ca2⫹ signals from the whole
sequence at 3 different epithelial sites shown in
A, first image (a– c). Observe in all the responses the presence of broad patterns of activity spread throughout different epithelial
sectors. Responses, however, had different spatial distributions. Note in A that not all retrogradely labeled receptor cells respond to ESS
stimulation with increased Ca2⫹ signals. ESS
signals reached their maximum in multiple, but
confined, sectors in VN dendritic regions close
to the epithelial lumen. In contrast, high-K⫹
and caffeine responses are found predominantly in proximity of the cell body region, a
location where they attained their maximum
peak values and longest durations. Observe
also the differences in response kinetics; ESS
and caffeine evoked Ca2⫹ signals having a
long time course in comparison with the brief
transient elevations evoked by high-K⫹ depolarization. They also exhibited more complex
profiles during their decaying phases that contrast with the rather rapid monotonic decay of
K⫹-elicited Ca2⫹ signals. ESS and high-K⫹
responses exhibited signals with sharp rise
times, while Ca2⫹ responses evoked by caffeine have a slower onset, especially in small
amplitude responses (see trace a). Optical signals were obtained from single runs (1 frame/s;
120-ms image exposure) and superimposed on
a fluorescent image of the VN slice. The 1st
point in the plots corresponds to basal cytosolic
values and the 2nd point to the 1st response
obtained 1 s after stimulus application. Cytosolic Ca2⫹ levels were coded as described in
Fig. 1C. Abbreviations in Fig. 1A. Horizontal
bar in C ⫽ 25 ␮m.
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2⫹
FIG. 4. Properties of Ca
transients in snake VN neurons evoked by KCl (100 mM)-induced membrane depolarization. Plots
a and b correspond to optical signals at 2 different sites in the cell body region of VN neurons. A: the effect of removing external
Ca2⫹ from the medium on the Ca2⫹ transients; left: control responses; middle: the suppression of all Ca2⫹ signals following the
removal of Ca2⫹ from the medium (0 [Ca2⫹]o supplemented with 5 mM EGTA); and right: recovery in normal Ringer solution.
B: the action of cadmium, a general VSCC blocker, on the Ca2⫹ signals elicited by K⫹ depolarization. Left: Ca2⫹ signals under
control conditions; middle: complete suppression of Ca2⫹ signals following the application of Cd2⫹ (50 ␮M); and right: recovery
in normal Ringer solution. C: the effect of depleting ryanodine-sensitive internal Ca2⫹ stores on K⫹-induced Ca2⫹ transients. Left:
K⫹-induced Ca2⫹ signals under control conditions; middle: K⫹-induced Ca2⫹ signals obtained after the depletion of ryanodine
stores. The depletion of ryanodine-sensitive stores shortens the duration of Ca2⫹ transients by selectively reducing the amplitude
of the responses during their late decaying phases. Right: illustration of the lack of recovery of the late decaying response phase
even after repetitive washings in normal Ringer solution, indicating an irreversible effect of ryanodine. Plots of Ca2⫹ signals were
taken from single runs, 16 image sequences (1 frame/s; 120-ms image exposure). The 1st point in the plots corresponds to basal
cytosolic values and the 2nd point to the first response obtained 1 s after stimulus application.
studied (n ⫽ 7), the absence of Ca2⫹ from the medium (0
[Ca2⫹]o, supplemented with 5 mM EGTA) did not affect the
characteristics of caffeine responses evaluated immediately
J Neurophysiol • VOL
after the Ca2⫹ removal. Under these conditions, Ca2⫹ responses exhibited profiles, time courses (14.8 ⫾ 2.3 s), and
peak magnitudes (29.5 ⫾ 3.7 ⌬F/Fo) similar to controls (Fig.
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2⫹
FIG. 5. Properties of Ca
signals evoked by caffeine in snake VN neurons. A: the characteristics of caffeine responses obtained
following the removal of Ca2⫹ from the medium. Left: corresponds to control responses obtained from a 4 ⫻ 4 pixel area in the
layer of VN neuron somata. Middle: plots of Ca2⫹ signals elicted by caffeine (10 mM) following the removal of Ca2⫹ from the
medium (0 [Ca2⫹]o with 5 mM EGTA). Although no significant change was observed in the initial response (—), successive
caffeine applications evoked a use-dependent gradual reduction in consecutive Ca2⫹ signals, as shown in responses obtained after
4 and 8 caffeine applications (- - - and 䡠 䡠 䡠 , respectively). Right: the recovery of this Ca2⫹ transient after restitution of normal
[Ca2⫹]o (2–3 min). B: the effect of depleting internal Ca2⫹ stores with thapsigargin on caffeine-elicited Ca2⫹ signals. Left: a control
response; middle: traces correspond to the progressive reduction in these Ca2⫹ responses following 2, 4, and 8 caffeine stimulations
after incubation of the slice with thapsigargin (1 ␮M, 15 min). Observe that on the 8th trial, no detectable signals were obtained,
and this suppression was not reversed following repetitive rinses with normal Ringer solution (right). C: Ca2⫹ signals evoked by
caffeine following the depletion of ryanodine-sensitive internal stores. Left: corresponds to a control signal obtained before the
application of ryanodine (20 ␮M, 15 min). Middle: the absence of caffeine-induced Ca2⫹ transients after cytosolic Ca2⫹ levels
returned to basal levels following ryanodine treatment (see further details in text). Similar to thapsigargin effects, the ryanodine
suppression of optical signals did not recover after rinsing the preparation in normal Ringer solution. Tissue slices in B and C were
tested in normal snake Ringer solution. Plots correspond to Ca2⫹ signals from the cell body region of VN neurons taken from a
single 16 image sequence (1 frame/s; 120-ms image exposure). The 1st point in the plots corresponds to basal cytosolic values and
the 2nd point to the 1st response obtained 1 s after stimulus application.
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
Role of the IP3 signaling cascade in the generation of Ca2⫹
transients
Previous studies have suggested that phosphoinositide turnover leading to IP3 formation may constitute the second-messenger system mediating chemosensory transduction in the VN
system (Holy et al. 2000; Kroner et al. 1996; Luo et al. 1994;
Sasaki et al. 1999; Wekesa and Anholt 1997). Thus we were
interested in determining whether the activation of this pathway could give rise to cytosolic Ca2⫹ changes similar to those
elicited by ESS ligands. In different cell systems, bradykinin
(BK) stimulates phosphoinositide turnover, which, in turn,
among other actions, can increase production of IP3 (Kirischuk
et al. 1995; Seymour-Laurent and Barish 1995; Verkhratsky
and Kettenmann 1996). Thus it has been used rather extensively to evaluate the role of IP3 in intracellular Ca2⫹ signaling
(see Berridge 1993, 1998). To determine the effect of BK in
snake VNO and compare it to ESS ligands, we evaluated the
J Neurophysiol • VOL
actions of BK and ESS stimuli on IP3 levels in homogenates of
the snake VN sensory epithelium. IP3 production was measured following protocols previously reported (Luo et al.
1994). VN homogenates (50 ␮g protein) were incubated either
with ESS (13 ␮g) or BK (300 nM) in a reaction solution (500
␮l) of 25 nM Tris-acetate, pH 7.6, 5 mM MgAc2, 0.5 mM
ATP, 1 mM DTT, 0.01 mM GTP, 0.1 mM CaCl2, and 0.1
mg/ml BSA). As a control, distilled water was used instead of
ESS or BK. In all cases the incubation time was 1 min. Under
these experimental conditions, ESS evoked IP3 levels equivalent to 210 ⫾ 10 (SD) pmol/mg proteins (n ⫽ 4) while BK
evoked IP3 levels equivalent to 255 ⫾ 5.0 pmol/mg proteins
(n ⫽ 4). These IP3 increases differed significantly from those
obtained under control conditions [75 ⫾ 15 (SD) pmol/mg
proteins; n ⫽ 4], suggesting that both ESS and BK induce
similar phosphoinositide turnover leading to IP3 formation. To
further determine whether these increases elicited by BK correspond to IP3 changes arising from VN neurons, IP3 levels
were determined also in homogenates from VN preparations
previously deafferented from the AOB. These VN preparations
lack mature VN neurons because the sectioning of their axons
induce their degeneration. In contrast with the intact VNO, in
deafferented homogenates, BK and ESS incubations exhibited
IP3 levels that did not differ significantly from those found
under control conditions. Thus these data indicate that both
ESS and BK increase IP3, and these increases take place
predominantly in mature snake VN neurons.
Ca2⫹ signals evoked by BK applications also exhibited
strong similarities with the Ca2⫹ transients elicited by ESS
ligands. As shown in Fig. 6, BK stimuli evoked Ca2⫹ transients
that consisted of an initial sharp [Ca2⫹]i rise followed by a
brief plateau and then a prolonged declining phase in which
cytosolic Ca2⫹ levels slowly returned to basal values, with a
halftime in the range of 9 –14 s, depending on the stimulus
concentration. BK responses were also dose dependent in the
concentration range tested in this study (50 –300 nM) with
amplitudes and durations directly proportional to stimulus intensity. Application of 200 nM of BK evoked peak fluorescent
signals attaining fractional changes (⌬F/Fo) equivalent to 25–
40%. These values roughly corresponded to elevation in
[Ca2⫹]i on the order of 150 –390 nM. Under these conditions,
Ca2⫹ signals exhibited a time course in the range of 12–16 s.
Like ESS responses, BK signals exhibited relatively sharp rise
times even at low concentrations (Fig. 6B), characteristics that
differed from the Ca2⫹ responses elicited by caffeine (Fig. 3C).
Ca2⫹ transients elicited by ESS and BK also shared a similar
spatial distribution with responses distributed in somatic as
well as in dendritic sectors (Fig. 6, A and C). This presence of
Ca2⫹ signals in dendritic regions, even those adjacent to the
luminal surface, differed from the localization of caffeine
responses found largely in the cell body region of VN neurons.
Despite their similarities, we found some differences between the overall distribution of BK and ESS elicited Ca2⫹
signals. Unlike ESS responses, BK patterns of activity were
more uniformly distributed across different sectors of the epithelium, and lacked the multifocal appearance characteristic of
ESS responses. As illustrated in Fig. 6, even at relatively low
concentrations, BK-evoked responses were homogeneously
distributed over relatively large epithelial regions. This more
uniform distribution probably reflects a lack of selectivity by
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5A, middle, 1st trace). However, we consistently found that
successive applications of caffeine in Ca2⫹-free medium
evoked Ca2⫹ transients that exhibited a progressive reduction,
first in their amplitude and then in their duration (n ⫽ 7; Fig.
5A, middle, - - - and 䡠 䡠 䡠 ). The most likely explanation for this
result is that these reductions are a consequence of the inability
of intracellular Ca2⫹ stores to be fully refilled in Ca2⫹-free
medium (Usachev and Thayer 1999). This interpretation is
corroborated by the finding that there was a complete recovery
of caffeine responses when normal [Ca2⫹]o was restored to the
medium (Fig. 5A, right).
Further confirmation about the role of intracellular Ca2⫹
release in the generation of caffeine responses was obtained by
evaluating these signals after thapsigargin or ryanodine treatment. Both thapsigargin (1 ␮M; Fig. 5B) and ryanodine (20
␮M; Fig. 5C) suppressed caffeine-induced Ca2⫹ transients but
with some differences in their actions. VN slices preincubated
with thapsigargin (10 –15 min) evoked no major changes in
basal cytosolic Ca2⫹ levels, and initial caffeine applications
elicited Ca2⫹ signals that exhibited only a slight reduction in
their amplitude and duration. Under these conditions, however,
successive caffeine applications evoked a progressive reduction in magnitude and duration of Ca2⫹ transients that led, after
8 –15 trials, to a complete response suppression (Fig. 5A, series
of traces in the middle plot). No major change was observed
using different intervals between trials (n ⫽ 5). In contrast, the
magnitude of these reductions was use-dependent because it
was related to the magnitude and duration of the previous
caffeine responses. Altogether this evidence suggests that the
reduction and eventual suppression of caffeine responses were
caused by the gradual and irreversible depletion of internal
Ca2⫹ stores.
Ryanodine depletion also suppressed caffeine-elicited Ca2⫹
signals (Fig. 5C). But in contrast to the gradual effects observed with thapsigargin, the protocol used here to deplete
ryanodine-sensitive stores (see preceding text) evoked a complete suppression of all caffeine responses from the first trial
(Fig. 5C, middle and right, respectively). This irreversible
suppression of caffeine responses following ryanodine treatment not only confirms that these Ca2⫹ transients arise entirely
from internal Ca2⫹ release but further indicates that this release
depends primarily on ryanodine-sensitive pools.
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
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BK in the activation of VN neurons that have different chemosensory specificities.
Source of Ca2⫹ signals related to IP3 production
To further characterize the sources of the Ca2⫹ responses
evoked by ESS and BK stimuli, we determined whether these
responses depend on a Ca2⫹ release from intracellular stores or
a Ca2⫹ influx through the plasma membrane. According to our
present results (see preceding text), both BK and ESS stimuli
appeared to trigger an IP3 increase in VN neurons, and this
J Neurophysiol • VOL
molecule can elicit important cytosolic Ca2⫹ elevations that, in
different cell systems, depend entirely on a Ca2⫹ release from
internal pools (for review, see Berridge 1998). On the other
hand, IP3 can also evoke cytosolic Ca2⫹ increases through a
plasma membrane Ca2⫹ influx via an IP3-activated cation
conductance as has been demonstrated in some invertebrate
chemosensory systems (Munger et al. 2000; see also Schild
and Restrepo 1998 for review).
BK RESPONSES IN CA2⫹-FREE MEDIUM. To determine whether
BK responses in snake VN neurons depend on Ca2⫹ entry
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2⫹
FIG. 6. Comparison of the spatiotemporal characteristics of Ca
signals evoked by ESS and the IP3 agonist bradykinin (BK).
A–C: the spatial distribution of Ca2⫹ signals during the peak amplitudes (left) and the decaying phase of these responses (right, 8th
frame). Observe that the Ca2⫹ signals elicited by BK (A and B) were distributed more evenly among different epithelial sectors than
those evoked by ESS (C). Right: plots correspond to the time course of the Ca2⫹ signals obtained at 3 different locations shown
in the 1st image in A. Optical signals were obtained from single runs of 16 image sequences (1 frame/s; 120-ms image exposure).
The 1st point in the plots corresponds to basal cytosolic values and the 2nd point to the 1st response obtained 1 s after stimulus
application. Cytosolic Ca2⫹ levels were coded as in Fig. 1C. Abbreviations as in Fig. 1. Horizontal bar in C ⫽ 25 ␮m.
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
To further characterize the component of BK responses that was independent
of Ca2⫹ release, we evaluated these signals after the depletion
intracellular Ca2⫹ stores. As with Ca2⫹ transients evoked by
caffeine, the preincubation of VN slices with thapsigargin
(1 ␮M for 10 –15 min) did not affect initial BK responses to a
large extent (Fig. 8B). Subsequent BK applications, however,
evoked a progressive decrease affecting both the amplitude and
duration of these Ca2⫹ signals. These reductions were observed equally in all epithelial regions and depended on the
stimulus concentration and the number of previous stimulations. There was, however, some difference in the evolution of
these changes as repetitive BK stimuli were delivered. In
regions corresponding to the cell bodies of VN neurons, cuBK RESPONSES AFTER DEPLETING CA2⫹ STORES.
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2⫹
FIG. 7. Characteristics of Ca
signals evoked in apical dendritic regions of
VN neurons by BK in the absence of [Ca2⫹]o. A: responses to BK (300 nM)
obtained in normal Ringer solution (control) and immediately following removal of Ca2⫹ from the bath {0 [Ca2⫹]o with 2 mM EGTA (1st trial)}.
Observe the rapid reduction in the peak amplitude of the Ca2⫹ signal that
occurred without delay after removing extracellular Ca2⫹. B: the effect of
repetitive BK applications in Ca2⫹-free medium. After their initial reduction,
Ca2⫹ responses in dendritic regions maintained rather constant characteristics
for a few runs (2–5) and progressively decreased following subsequent BK
applications. This cumulative decrease gradually affected both the amplitude
and duration of Ca2⫹ responses until all cytosolic Ca2⫹ changes become
undetectable (usually after 20 runs using this stimulus concentration; bottom
trace). C: full recovery shown of BK-elicited Ca2⫹ signals after the VN slices
were returned to normal Ringer solution for 5 min. All Ca2⫹ signals were taken
from single runs of 16 image sequences (1 frame/s; 120-ms image exposure).
The 1st point in the plots corresponds to basal cytosolic values and the 2nd
point to the 1st response obtained 1 s after stimulus application.
mulative reduction progressed until all Ca2⫹ responses elicited
by BK were abolished. Depending on the stimulus concentration, this occurred usually after 8 –20 consecutive applications
of BK. In apical dendritic regions, however, responses failed to
be completely suppressed. Instead, the reduction progressed
until it revealed a small component that remained insensitive to
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through the plasma membrane or a release from internal pools,
we first evaluated these responses in [Ca2⫹]o-free medium. In
general, the absence of extracellular Ca2⫹ did not abolish BK
responses, suggesting that these signals depend largely on a
Ca2⫹ release from internal stores. In all the slices evaluated
(n ⫽ 6), applications of BK (100 –200 nM) in 0 [Ca2⫹]o
medium (supplemented with 2 mM EGTA) evoked Ca2⫹ signals that exhibited similar characteristics and kinetics to control responses. Under these conditions, most Ca2⫹ transients
preserved their relatively sharp onsets, magnitudes, and durations.
Although in most epithelial regions there was no modification, Ca2⫹-free conditions affected BK signals in apical dendritic regions close to the luminal surface of the epithelium. In
this sector, we consistently observed that immediately after the
removal of Ca2⫹ from the bath there was a sudden but constant
reduction in the magnitude of BK responses equivalent to a
decrease of 10 –18%. These decreases were observed in the
amplitude at the peak of the responses as well as during their
early decay phase. These changes are illustrated in Fig. 7A,
which shows BK signals recorded in apical epithelial regions
before (—) and after (- - -) the removal of Ca2⫹ from the
medium. Full response recovery was always obtained without
delay after the restitution of normal [Ca2⫹]o in the bath. This
evidence indicates that Ca2⫹ signals elicited by BK stimuli in
these apical dendritic segments of VN neurons has a component generated by a Ca2⫹ influx through the plasma membrane.
In this region, however, there is still a response remaining that
is unaffected by removal of Ca2⫹ from the medium. This
component, present in Ca2⫹-free medium, appears to be largely
dependent on Ca2⫹ release from intracellular stores as is the
case for BK-evoked signals obtained in more basal regions.
In contrast with the initial, sudden reduction found in apical
dendritic regions, a different type of response decrease was
found in Ca2⫹-free medium following repetitive BK stimulations (trials 8 –20). These changes were observed in all epithelial regions, including the apical dendritic locations, and consisted of a progressive and cumulative decrease in response
magnitude and duration following subsequent applications of
BK (6 –20; Fig. 7B). The degree of reduction in these Ca2⫹
signals was dependent on the number and the concentration of
previous BK applications. As was the case for Ca2⫹ transients
elicited by repetitive applications of caffeine when the VN
slices were in Ca2⫹-free medium, these reductions probably
arise from the progressive inability of intracellular Ca2⫹ stores
to be replenished in 0 [Ca2⫹]o.
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
1461
thapsigargin (n ⫽ 7) even after multiple BK applications
(12–25 trials). This component attained its largest amplitude in
locations adjacent to the epithelial luminal surface with a
magnitude equivalent to 15–24% of the control responses and
a duration of ⬃4 – 6 s. The real amplitude and duration of these
Ca2⫹ transients, however, could be underestimated in our
recordings because it appears to be generated in a highly
localized dendritic region that cannot be independently assessed due to the population nature of our optical signals.
Figure 8A illustrates the characteristics of BK responses
recorded in apical dendritic regions after the depletion of
internal Ca2⫹ stores by thapsigargin. As seen in the middle
plots, BK applications in thapsigargin-treated slices evoked a
reduction of Ca2⫹ signals, and after the sixth trial, it is possible
to distinguish the emergence of a thapsigargin-resistant component, which maintained a rather constant magnitude and
J Neurophysiol • VOL
duration during subsequent stimulus applications (12–20 trials;
middle and right). Under this condition, caffeine applications
failed to evoke any detectable [Ca2⫹]i changes in responses
recorded in the soma region (data not shown), indicating that
this dendritic component does not depend on a partial depletion
of intracellular Ca2⫹ stores. In contrast, the removal of Ca2⫹
from the medium reversibly abolished this thapsigargin-resistant component. Thus altogether this evidence indicates that
this component is not generated by an internal Ca2⫹ release but
instead is dependent on a Ca2⫹ influx through the plasma
membrane. Confirming this notion, the magnitude and duration
of this component matches the initial reductions in control BK
responses observed in the same region when Ca2⫹ was removed from the medium (Fig. 7A).
In contrast with thapsigargin, ryanodine-induced Ca2⫹ depletion did not greatly affect the characteristics of Ca2⫹ tran-
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2⫹
FIG. 8. Ca
signals recorded in apical dendritic regions of VN neurons evoked by BK following depletion of internal Ca2⫹
stores. A: the effects of depleting internal Ca2⫹ stores on responses following the preincubation of a VN slice with thapsigargin
(1 ␮M). Left: Ca2⫹ signals under control conditions. Middle: traces illustrate Ca2⫹ responses evoked by multiple BK stimuli tested
15 min after the application of thapsigargin; note the reduction, but not total suppression, of these Ca2⫹ signals. Right: the
thapsigargin-resistant portion of the original BK response remained constant after 20 trials. B: Ca2⫹ signals in dendritic and somatic
regions evoked by BK after the depletion of ryanodine-sensitive stores. Left: BK responses under control conditions. Middle: Ca2⫹
signals obtained after the depletion of ryanodine stores (see protocol in the text). Observe that the Ca2⫹ signal from dendritic
locations shows no detectable changes, while that from the somata region exhibits a significant reduction during its decaying phase.
Right: after ryanodine treatment there was no detectable Ca2⫹ signal evoked by caffeine stimulation, corroborating the full
depletion of ryanodine-sensitive stores under this condition. Ca2⫹ signals obtained from single runs of 16 image sequences
(1 frame/s; 120-ms image exposure). The 1st point in the plots corresponds to basal cytosolic values and the 2nd point to the 1st
response obtained 1 s after stimulus application.
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
ESS RESPONSES IN CA2⫹-FREE MEDIUM. The mechanisms involved in the generation of Ca2⫹ signals elicited by ESS
ligands were analyzed using protocols similar to those used
with BK stimulation. First, we evaluated whether Ca2⫹ transients evoked by ESS ligands depend on Ca2⫹ influx or reflect
a release from internal stores. As with BK signals we found
that the absence of Ca2⫹ in the medium (0 [Ca2⫹]o, supplemented with 5 mM EGTA) did not suppress ESS responses.
Immediately after the removal of Ca2⫹ from the medium, Ca2⫹
signals in most epithelial locations exhibited kinetics and characteristics similar to control responses. Ca2⫹ signals obtained
in apical dendritic regions adjacent to the epithelial lumen,
however, exhibited a slight reduction in their peak amplitudes
of ⬃12–20% (Fig. 9B). As observed with reductions in BK
responses, the late phase of these responses was less affected.
These changes were also fully reversible.
In contrast with these local and immediate effects, repetitive
ESS stimuli under Ca2⫹-free conditions evoked a different type
of response reduction. As with the effects found in BK signals,
numerous ESS applications (8 –20 trials) in Ca2⫹-free medium
evoked a progressive decrease in Ca2⫹ signals in all epithelial
regions. These reductions were use dependent because they
were related to the number and concentration of preceding
stimulations and affected both the size and duration of the
J Neurophysiol • VOL
2⫹
FIG. 9. Characteristics of Ca
signals in apical dendritic regions of snake
VN neurons evoked by ESS ligand in the absence of [Ca2⫹]o. A: traces
correspond to Ca2⫹ responses elicited by ESS (2.2 mg/ml) under control
conditions and immediately after the removal of Ca2⫹ from the bath (0 [Ca2⫹]o
with 2 mM EGTA). Observe the immediate reduction in the peak amplitude
and initial decaying phase of the response. B: superimposed traces illustrate
decremental Ca2⫹ signals evoked by successive ESS applications in VN slices
maintained in Ca2⫹-free medium. Like BK responses illustrated in Fig. 7,
following the initial decrease, successive ESS applications evoke a progressive
response reduction that affected both the amplitude and duration of Ca2⫹
signals, until these cytosolic Ca2⫹ elevations become undetectable (usually
after 16 –20 runs using this stimulus concentration; bottom trace). C: full
recovery of ESS-evoked Ca2⫹ signals after the VN slices were returned to
normal Ringer solution for 5 min. Ca2⫹ signals were obtained from single runs
of 16 image sequences (1 frame/s; 120-ms image exposure). The 1st point in
the plots corresponds to basal cytosolic values and the second point to the 1st
response obtained 1 s after stimulus application.
Ca2⫹ signals until they disappeared. Figure 9B illustrates the
progressive decay of ESS responses following repeated stimulation in Ca2⫹-free medium. This type of change usually
appeared after the fourth ESS application, and following the
eighth stimulus, Ca2⫹ signals started to exhibit major reductions in peak amplitude (70 – 85%) and duration. At this stage,
Ca2⫹ transients also exhibited slower rise times and longer
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sients elicited by BK stimuli. After Ca2⫹ depletion from ryanodine stores (application of 10 ␮M ryanodine with high
concentrations of caffeine, 25 mM), Ca2⫹ transients elicited by
BK applications were affected significantly only in the cell
body region of the epithelium (Fig. 8B, middle, - - -). But even
changes in this region were limited because they consisted of
only slight reductions in peak amplitudes (equivalent to 12–
18%) and a minor shortening in duration (in the range of 30%)
of these responses. Figure 8B illustrates typical Ca2⫹ signals
evoked in dendritic (—) and somatic regions (- - -) before (left)
and after (middle) the depletion of ryanodine-sensitive stores.
Practically no change is observed in the signals evoked by BK
in dendritic regions (a), and the Ca2⫹ signals from the somatic
regions (b) exhibited only a slight reduction in magnitude,
which occurred predominantly during its decay phase. Under
these condition, however, caffeine applications (2–5 mM)
evoked no detectable Ca2⫹ signals (Fig. 8B, right), indicating
that the lack of major changes in BK responses was not caused
by an incomplete depletion of ryanodine stores. Therefore
these data suggest that BK responses (especially in dendritic
regions) depend largely on a Ca2⫹ release from nonryanodinesensitive pools, probably IP3-sensitive stores.
Furthermore, the relative lack of action of ryanodine on
these BK-induced Ca2⫹ signals indicates that at least in dendritic regions a functional separation between ryanodine- and
nonryanodine-sensitive Ca2⫹ pools probably exists. Assuming
that this interpretation is correct, the reduction observed in
somatic regions after the depletion of ryanodine-sensitive
stores might be secondary and reflect an impairment in CICR
mechanisms, which perhaps participate in amplifying BK responses. This interpretation is consistent with the observation
that the effect is predominantly in the decaying portion of these
responses. This hypothesis is also consistent with the reduction
in the Ca2⫹ signals evoked by K⫹ depolarization observed
following the depletion of ryanodine-sensitive stores (see preceding text; Fig. 4C).
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
peak latencies. A complete recovery of ESS responses was
observed after 5–10 min incubation in normal [Ca2⫹]o (Fig.
9C). As with similar use-dependent reductions found in caffeine and BK responses, this progressive response decrease
following repetitive ESS stimulation in Ca2⫹-free medium
probably reflects the inability of intracellular Ca2⫹stores to be
replenished.
ESS RESPONSES AFTER THE DEPLETION OF CA2⫹ STORES. To
confirm the characteristics of the components dependent and
independent of internal Ca2⫹ release, ESS responses were
evaluated after the depletion of these intracellular stores. As
with BK signals, following the application of ESS ligands, VN
slices preincubated with thapsigargin (1 ␮M for 10 –15 min)
1463
evoked Ca2⫹ signals that exhibited a gradual reduction in
amplitude and duration, consistent with a progressive depletion
of internal Ca2⫹ stores (Fig. 10A). In the cell body region, ESS
responses were completely suppressed by multiple ESS applications (8 –10 trials), indicating that at this level these responses depend entirely on Ca2⫹ release from internal stores.
In apical dendritic regions, however, the depletion induced by
thapsigargin failed to completely suppress all Ca2⫹ signals,
even following numerous applications of ESS ligands (⬎20
trials). As with BK responses, there was a thapsigargin-resistant component that was revealed after the disappearance of the
overlapping ESS response dependent on intracellular Ca2⫹
release (Fig. 10A). Fig. 10A, right, shows that this ESS-evoked
J Neurophysiol • VOL
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2⫹
FIG. 10. Spatiotemporal properties of Ca
signals in VN neurons evoked by ESS after
the depletion of internal Ca2⫹ stores. A: the
effects on ESS responses of depleting internal
Ca2⫹ stores with thapsigargin (1 ␮M, 15
min). Left: the image and the plot correspond
to Ca2⫹ signals under control conditions.
Middle: traces illustrate the use-dependent reduction in the Ca2⫹ signals evoked by successive applications of ESS after thapsigargin
treatment (15 min). The top image of the right
panel illustrates the distribution of Ca2⫹ signals in a thapsigargin-treated slice after 10
ESS stimulations, and the lower plot (right
panel) shows the presence of this component
from the apical dendritic region even after 20
trials. Top images correspond to the distribution of activity at the signal peaks, and lower
plots illustrate the time course of Ca2⫹ signals
as recorded from the apical dendritic region
(E in the 1st image). B: the characteristics of
ESS responses after the depletion of ryanodine-sensitive stores. Left: the image and the
plots correspond to Ca2⫹ signals under control conditions; right: image and plots correspond to responses obtained after the depletion of ryanodine stores (20 ␮M ryanodine;
see protocol in the text). The top images illustrate the spatial distribution of responses at
their peaks, while the lower plots show the
time course and amplitude of Ca2⫹ signals
from somatic (a) and dendritic (b) regions.
Observe that Ca2⫹ signals in dendritic locations (- - -) show no detectable changes, while
the amplitude and duration of the response
from the somata region (—) was affected
predominantly during its late phase. Optical
signals were obtained from single runs
(1 frame/s; 120-ms image exposure). The 1st
point in the plots corresponds to basal cytosolic values and the 2nd point to the 1st response obtained 1 s after stimulus application.
Cytosolic Ca2⫹ levels were coded in pseudocolors as in Fig. 1C. Abbreviations as in Fig.
1. Horizontal bar in B ⫽ 25 ␮m.
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
Possible role of cAMP in responses to ESS
In the main olfactory system, odor transduction and associated Ca2⫹ transients rely on the activation of a cAMP pathway.
The binding of odorants to olfactory receptors causes a Gprotein activation of adenylate cyclase, which, via cAMP,
activates a cyclic nucleotide-gated (CNG) channel. The opening of this channel generates an inward transduction current
carried by Na⫹ and Ca2⫹, and this influx is largely responsible
for the Ca2⫹ transients elicited during odor stimulation. To
determine whether similar CNG channel activity participates in
a Ca2⫹ influx in snake VN neurons, we evaluated ESS responses after the application of the specific CNG channel
blockers LY 83583 and L-cis-diltiazem (LCD), which are
known to abolish Ca2⫹-related odor responses in olfactory
receptor neurons (ORNs) (Kolesnikov et al. 1990; LeindersZufall et al. 1997, 1998). As illustrated in Fig. 11, preincubation of VN slices with either LY 83583 (80 ␮M; B) or LCD (40
␮M; C) for 10 –15 min did not affect the general spatial
distribution (Fig. 11, B and C) or the kinetics (Fig. 11E) of
Ca2⫹ transient elicited by ESS ligands. The amplitudes, latenJ Neurophysiol • VOL
cies, and time courses of ESS responses remained practically
unchanged after the application of either of these CNG channel
blockers (Fig. 11E). A similar absence of effect was observed
in all the slices evaluated (n ⫽ 5). Furthermore, no changes
were observed even in the dendritic regions adjacent to the
luminal surface of the epithelium where we found a component
arising from Ca2⫹ influx.
To eliminate the possibility that in VN neurons ESS responses are mediated by CNG channels that are insensitive to
the blockers tested or by the participation of an unknown
cAMP-related mechanisms, we evaluated the effects of forskolin on cytosolic Ca2⫹ changes in these cells. As in other cell
systems, forskolin appears to act as an adenylate cyclase (AC)
activator directly inducing an increase in cAMP levels in VN
neurons (Luo et al. 1994; Wang et al. 1997). Despite this
action, we found that the addition of forskolin (10 ␮M) to the
bath evoked no detectable [Ca2⫹]i changes, at least in the time
frame in which Ca2⫹ responses were analyzed in this study
(n ⫽ 5; Fig. 10F). Thus altogether these results indicate that
cAMP mechanisms do not directly participate in mediating the
Ca2⫹ transients that occur during the early phases of chemosensory transduction in snake VN neurons.
Role of Na⫹/Ca2⫹ exchanger in ESS responses
We also investigated whether the Ca2⫹ influx observed
during ESS response was dependent on a reverse mode of
operation of the Na⫹/Ca2⫹ exchanger. In different cell systems, the operation of the Na⫹/Ca2⫹ exchanger is fully reversible and can mediate either a Ca2⫹ influx or efflux depending
on the ionic gradients present across the plasma membrane
(DiPolo and Beauge 1987, 1999). Thus it is conceivable that
during VN chemosensory transduction an increase in cytosolic
Na⫹ could activate the Na⫹/Ca2⫹ exchanger in its reverse
mode forcing Ca2⫹ into the cell as has been reported recently
in squid (Danaceau and Lucero 2000).
To characterize the functional role of the Na⫹/Ca2⫹ exchanger in VN neurons, we evaluated ESS responses under
conditions that inhibit its activity. Because monovalent cations
cannot substitute for Na⫹ on the Na⫹/Ca2⫹ exchanger (Dipolo
and Beauge 1987, 1999; Fiero et al. 1998), we blocked the
exchanger by replacing external Na⫹ with Li⫹ or choline.
After the substitution of Na⫹ by equimolar concentrations of
Li⫹ or choline, we observed no permanent changes in basal
[Ca2⫹]i. In some cases (n ⫽ 3), however, immediately after the
substitution of [Na⫹]o we observed increases in cytosolic Ca2⫹
that returned to normal values within 1–2 min. We interpret
these Ca2⫹ elevations as reflecting a brief Ca2⫹ influx through
the exchanger operating in reverse mode following a sharp
depletion of [Na⫹]i.
During ESS responses, however, we found that the Na⫹/Ca2⫹
exchanger operates in the forward mode, mediating instead a
Ca2⫹ efflux. In all the cases studied (n ⫽ 6), we found that the
blockade of exchanger activity following the replacement of Na⫹
by Li⫹ enhanced ESS responses. Under this condition, Ca2⫹
signals exhibited slight increases in their peak amplitude, and
major increases in their duration and magnitude. As seen in Fig.
12B, the changes in amplitudes were more prominent during the
late phases of ESS responses. These Ca2⫹ signals exhibited longer
plateaus and slower decay rates, characteristics that increased the
total duration of these transients by 35–50%. Because Li⫹ may
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component persisted after numerous stimulus applications (20
trials). This component was, however, reversibly suppressed in
Ca2⫹-free medium, supporting the notion that it arises directly
from Ca2⫹ influx through the plasma membrane. This remaining ESS response exhibited its largest amplitudes in locations
just adjacent to the luminal surface of the epithelium (Fig. 10A,
right), similar to the BK-induced response. In this location, the
response attained a magnitude equivalent to 15–20% of the full
response obtained before the thapsigargin-induced Ca2⫹ depletion. However, as we previously mentioned for the similar
component in BK signals, the actual size of this Ca2⫹ influx
could be underestimated in our population optical recordings
due to the difficulty of accurately determining cytosolic Ca2⫹
levels from localized regions.
These data indicate that Ca2⫹ transients associated with
chemosensory transduction in VN neurons depend on two
different mechanisms: a widespread component generated by
Ca2⫹ release from intracellular stores and a more restricted
component that depends on Ca2⫹ influx in apical dendritic
regions. In addition, the strong similarities between the kinetics
and properties of BK and ESS responses suggest that both
Ca2⫹ transients are closely interrelated, probably sharing similar mechanisms linked to PLC activation and IP3 production.
Because we found previously a functional separation between
ryanodine- and nonryanodine-sensitive stores, we determined next
whether cytosolic Ca2⫹ elevation evoked by ESS stimuli depends
on Ca2⫹ release from nonryanodine (IP3-sensitive) stores. For this
purpose, we evaluated the presence and characteristics of ESS
responses after the depletion of ryanodine-sensitive stores. As
with BK responses, no major changes in the Ca2⫹ signals elicited
by ESS ligands were observed following the depletion of ryanodine-sensitive stores. The depletion of ryanodine stores only affected ESS signals in the cell body region of VN neurons (Fig.
10B, a). Consistent with similar effects observed in Ca2⫹ transients elicited by K⫹ depolarization (Fig. 4C) and BK stimuli
(Fig. 8B), it is likely that these effects depend on an impairment in
CICR mechanisms that we have interpreted previously to be
mediated predominantly by Ca2⫹ release from ryanodine-sensitive pools.
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
1465
interfere with IP3 mechanisms (Worley et al. 1988) and ESS
responses seem to rely on this pathway, we also tested the effect
of blocking the exchanger by choline substitution (n ⫽ 3). Consistent with our previous results, the replacement of Na⫹ by
choline yielded similar effects to those obtained using Li⫹. As
illustrated in Fig. 12C, under this condition, ESS responses also
exhibited larger amplitudes and longer durations, and these
changes resulted mainly from the slower decay rate of the cytosolic Ca2⫹ elevations elicited by ESS ligands. Thus both Li⫹ and
choline substitution confirm that during VN transduction the Na⫹/
Ca2⫹ exchanger participates in the clearance of Ca2⫹ overloads.
DISCUSSION
General characteristics of optical signals in snake VN
neurons
In this study we have shown the presence of major cytosolic
Ca2⫹ transients associated with chemosensory transduction in the
J Neurophysiol • VOL
VNO of the garter snake. The combined use of optical techniques
with the selective labeling of VN neurons by retrograde transport
of the Ca2⫹ indicator made it possible for us to assess the characteristics and sources of cytosolic Ca2⫹ elevations elicited by a
well-defined chemoattractant exclusively in VN neuron. In our
slice preparation, the relatively low resolution of the images in
relation to the image blur caused by the lack of confocal optics
(Cinelli 2000) prevented single-cell resolution of fluorescence
signals in most cases. Nevertheless, two important experimental
conditions have facilitated the analysis of Ca2⫹ responses in snake
VN neurons. First, fluorescence signals in this study arose exclusively from VN neurons because only these cells were retrogradely labeled with the Ca2⫹ indicator from the AOB. Therefore
no optical signals could arise from cell types other than mature
VN neurons whose axons project to the AOB. Another advantage
in the snake VNO is its laminar organization. As illustrated in Fig.
1B, the cell bodies of mature VN neurons are located in the
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2⫹
FIG. 11. Contribution of the cAMP signaling pathway to the generation of ESS-elicited Ca
transients. Cyclic nucleotide-gated
(CNG) channel blockers evoked no detectable effects either in the spatial distribution nor the time courses of Ca2⫹ signals evoked
by ESS. Top: images show the spatial distribution of Ca2⫹ signals elicited by ESS ligands (2.2 mg/ml) under control conditions
(A) and after preincubation of the VN slices (10 min) with the CNG channel blockers LY83583 (40 ␮M; B) and L-cis-diltiazem
(LCD, 20 ␮M; C). Patterns correspond to the amplitude peaks of the Ca2⫹ responses plots illustrated in D and E. No detectable
changes were observed either in the spatial distribution or the time course of ESS responses following incubation with CNG
blockers. F: plot corresponds to cytosolic Ca2⫹ levels during the application of forskolin (5 ␮M) to the bath; no measurable changes
in [Ca2⫹]i were observed. Optical signals were obtained from single runs (1 frame/s; 120-ms image exposure). The 1st point in the
plots corresponds to basal cytosolic values and the 2nd point to the 1st response obtained 1 s after stimulus application. Cytosolic
Ca2⫹ levels were coded in pseudocolors as in Fig. 1C. Abbreviations as in Fig. 1. Horizontal bar in C ⫽ 40 ␮m.
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
intermediate region of the sensory epithelium with dendrites projecting apically, toward the epithelial lumen. Thus optical signals
recorded at intermediate epithelial levels represented Ca2⫹
changes in the cell body regions, while signals toward the luminal
epithelial surface corresponded to Ca2⫹ responses from VN dendrites.
In general we found that the average basal [Ca2⫹]i values in
snake VN neurons were in reasonable agreement with those
found in other neurons, including ORNs. The cytosolic Ca2⫹
elevations elicited by the chemoattractant ESS also exhibited
characteristics and kinetics similar to the Ca2⫹ responses
evoked in rat VN neurons (Leinders-Zufall et al. 2000) and
ORNs (Leinders-Zufall et al. 1997, 1998; Restrepo and Boyle
1991; Restrepo et al. 1990, 1993; Sato et al. 1991; Tareilus et
al. 1995) during chemosensory transduction.
J Neurophysiol • VOL
Spatial distribution of Ca2⫹ transient elicited by ESS ligands
An interesting finding was that Ca2⫹ signals elicited by ESS
ligands in snake VN neurons exhibited a rather broad distribution with optical signals arising from an apparently large
population of VN neurons. These Ca2⫹ transients, however,
were not uniformly distributed and exhibited dissimilar thresholds, characteristics that suggest the presence of heterogeneous
responses in different VN neurons. We also observed a recruitment of new VN neurons that became active as ESS concentration increased. But even at the highest concentration tested
not all VN neurons responded to ESS stimuli, suggesting a
certain degree of selectivity in the response characteristics of
these neurons. Corroborating this notion, the heterogeneous
response characteristics among different VN neurons con-
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⫹
2⫹
FIG. 12. Contribution of the Na /Ca
exchanger in the generation of ESS-elicited Ca2⫹ transients. Left: images and the
corresponding time-course plots (right) show the spatial distribution of ESS-elicited Ca2⫹ signals in normal Ringer solution (A) and
after replacing Na⫹ with Li⫹ (B) or with choline (C) in the bath to block Na⫹/Ca2⫹ exchanger activity. Images correspond to frames
taken during the peak and late phases of the responses (frames 2nd and 8th, respectively). Under both conditions that block
Na⫹/Ca2⫹ exchanger activity, there was a significant increase in response magnitude during the late phases of these responses that
was also accompanied by a slight increase in peak magnitude. Optical signals were obtained from single runs (1 frame/s; 120-ms
image exposure). The 1st point in the plots corresponds to basal cytosolic values and the 2nd point to the 1st response obtained
1 s after stimulus application. Cytosolic Ca2⫹ levels were coded in pseudocolors as in Fig. 1. Abbreviations as in Fig. 1.
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
Mechanisms involved in Ca2⫹ transients in VN neurons
Our results indicate that in snake VN neurons, Ca2⫹ responses elicited by ESS ligands depend on two different mechanisms: a Ca2⫹ influx through the plasma membrane and a
Ca2⫹ release from intracellular stores. We can assume that
these two components correspond to cytosolic Ca2⫹ elevations
exclusively from VN neurons because only these cellular elements were stained and contribute to the optical response
evaluated here (see preceding text). The component generated
by a Ca2⫹ influx was found after depleting intracellular stores
with thapsigargin and exclusively observed in the apical dendritic region of VN neurons. This distribution is interesting
because it suggests that this component occurs at locations in
close proximity to the sites where VN transduction takes place
and could be related to the activation of a primary transduction
current. The component dependent on Ca2⫹ release from internal stores displayed a more widespread distribution with
responses spreading to different cellular segments. Nevertheless, it seems likely that this component also starts in apical
dendritic regions because there it exhibited its lowest threshold. If this is the case, it is interesting that both Ca2⫹ influx and
Ca2⫹ release seem to coexist in similar dendritic domains.
Within the limitation imposed by the time resolution of our
recordings, both components seem to appear almost simultaJ Neurophysiol • VOL
neously because we were unable to detect any difference either
in their latencies or their rise times. It is unlikely that this
characteristic results from the “in vitro” conditions used here
because ESS ligands should only stimulate VN neurons
through the activation of VN receptors. Thus even in our slice
preparation, only responses associated with VN transduction
should give rise to these Ca2⫹ signals.
We also found these two components to be relatively independent because the suppression of one of them did not abolish
the other. In fact, the depletion of internal stores by thapsigargin abolishes most Ca2⫹ transients elicited by ESS ligands
except those arising from a Ca2⫹ influx. On the other hand, this
Ca2⫹ influx was reversibly abolished when Ca2⫹ was removed
from the medium, but this condition did not suppress the Ca2⫹
signals elicited from Ca2⫹ release. Therefore we cannot consider either of these two components to be secondary to the
other. Instead it is likely that both components constitute
primary responses that appear to be triggered in parallel by a
common mechanism associated with the initial stages of VN
signal transduction.
BK applications evoked Ca2⫹ transients that exhibited kinetics and properties similar to the Ca2⫹ signals evoked by
ESS ligands. The main difference observed was in relation to
their spatial distribution. In contrast to the heterogeneous appearance of ESS responses, Ca2⫹ signals elicited by BK stimuli were distributed more uniformly among different VN neurons. This characteristic probably reflects the lack of selectivity
of the BK stimulus in activating different VN neurons. But
similar to ESS responses, Ca2⫹ signals elicited by BK also
exhibited two components, one arising from Ca2⫹ influx and
the other from Ca2⫹ release. The component arising from Ca2⫹
influx was also found exclusively in apical dendritic regions
close to the epithelial lumen. This is an interesting finding
because in our “in vitro” slice preparation, BK stimuli directly
applied to the bath probably activate BK receptors from all
cellular segments because it is believed that in general these
receptors are broadly distributed in neurons (Koizumi et al.
1999). Despite this putative global effect, however, we observed in VN neurons only a Ca2⫹ influx in apical dendritic
segments, indicating that this component probably exists only
at this cellular level. This evidence provides further support for
the notion that it might represent the opening of a conductance
that is normally activated by VN transduction. In addition, as
with ESS signals, these two components also appear to be
relatively independent because the suppression of one did not
abolish the presence of the other. Finally, Ca2⫹ release in
response to both ESS and BK was largely unaffected by the
selective depletion of ryanodine-sensitive stores (see following
text). This finding indicates that this Ca2⫹ release depends
predominantly on nonryanodine-sensitive stores, probably
those stores activated by IP3 (Berridge 1998). Only the Ca2⫹
responses from the cell body seem to be affected by the
depletion of ryanodine stores but with reductions that appear to
be secondary because only the late phases of these Ca2⫹
transients are affected.
Taken together with the evidence obtained in the biochemical assay indicating that both ESS and BK stimuli evoked
similar IP3 elevations, these results lead to the interpretation
that the Ca2⫹ transients elicited by ESS ligands depend primarily on mechanisms linked to IP3 production. Consistent
with this interpretation, previous functional studies have sug-
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trasted with the more uniform signals evoked by other stimuli
such as caffeine, high-[K⫹]o depolarization and BK.
The broad and heterogeneous responses elicited by ESS
stimuli in snake VN neurons contrast with the more restricted
patterns found in mouse VN neurons following stimulation
with single chemicals (Leinders-Zufall et al. 2000). In this
case, Ca2⫹ transients were found only in limited VN neurons,
the number of which remained constant at higher stimulus
concentrations. It is likely that the differences observed in the
distribution of Ca2⫹ signals in these two preparations may arise
as a consequence of the different types of stimuli employed.
Natural compounds such as ESS ligands have a heterogeneous
molecular structure that probably comprises multiple chemical
cues. Thus it is likely that each of these cues could activate
different sets of VN neurons having distinct molecular receptive ranges. In contrast, pure single chemicals such as those
tested in the mouse VNO probably activate a more selective
and uniform set of VN neurons. Supporting this hypothesis,
recent electrophysiological studies in rat VNO have shown that
complex pheromonal stimulants found in urine elicit broad
activation of large numbers of VN neurons (Holy et al. 2000).
Moreover, similar to our findings in snake VN neurons, higher
concentrations of this natural stimulus also seem to increase the
number of active VN neurons. In general, in both systems it is
conceivable that the simultaneous detection of multiple cues by
a large number of VN neurons may reflect a distributed coding
strategy that is useful for the detection of complex chemoattractants. In the garter snake, such a strategy would be useful
in identifying a large number of chemical cues associated with
different types of prey. A similar combinatorial code strategy
has been proposed as the basis for odor recognition in the main
olfactory system (Cinelli et al. 1995; Friedrich and Korsching
1997; Rubin and Katz 1999) and certain complex pheromonal
compounds in invertebrates (for review, see Sorensen et al.
1998).
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A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
FIG. 13. Summary of the different mechanisms postulated to participate in
the generation of Ca2⫹ transients during chemosensory transduction in snake
VN neurons. Our results support the existence of 2 levels of organization in the
generation of ligand-induced Ca2⫹ signals. First, ESS activates selective
receptors that appear to trigger IP3 production and mediate an increase in
cytosolic Ca2⫹ levels through 2 different mechanisms: a Ca2⫹ release from
internal stores, and a Ca2⫹ influx through a putative IP3-gated conductance in
the plasma membrane. These initial Ca2⫹ elevations appear to be activated in
parallel and subsequently amplified through ⱖ2 other secondary mechanisms.
One includes a further Ca2⫹ release through a Ca2⫹-induced Ca2⫹ release
mechanism mediated by functionally independent ryanodine-sensitive pools.
The other is dependent on Ca2⫹ influx through the activation of voltagesensitive Ca2⫹ channels following membrane depolarization. The 1st stage of
these Ca2⫹ transients occurs predominantly in dendritic regions, while secondary events occur predominantly in the somata region. During these responses, Na⫹/Ca2⫹ exchanger activity contributes in limiting the magnitude
and duration of these cytosolic Ca2⫹ elevations by Ca2⫹ efflux.
J Neurophysiol • VOL
results seem to reveal two levels of organization in the generation of ligand-induced Ca2⫹ signals. First, the binding of
specific ligands to receptors triggers the activation of a secondary signaling mechanism in which IP3 production appears
to be involved. This signaling molecule, in turn, appears to
mediate both an increase in cytosolic Ca2⫹ levels through a
release from internal stores and a Ca2⫹ influx through the
plasma membrane. These initial Ca2⫹ elevations are then amplified through secondary mechanisms localized predominantly in the cell body region. These mechanisms probably
include a Ca2⫹ influx through VSCC activated by membrane
depolarization associated with transduction mechanisms and a
further Ca2⫹ release from ryanodine-sensitive internal pools
through a CICR mechanism. Thus the first stage of these Ca2⫹
transients occurs predominantly in dendritic regions, whereas
secondary events occur predominantly in the somata region.
Finally, the Na⫹/Ca2⫹ exchanger participates in maintaining
basal cytosolic Ca2⫹ by extruding the Ca2⫹ excess resulting
from activation of chemosensory transduction mechanisms. In
accordance with our results, this model does not include the
participation of a cAMP activation of CNG channels in the
generation of the Ca2⫹ transients associated with VN transduction.
Both high [K⫹]o and caffeine evoked Ca2⫹ transients near
the cell body region of VN neuron, but there were important
differences between these Ca2⫹ signals. High [K⫹]o elicited
relatively brief Ca2⫹ signals that depended on the activation of
VSCC because they were suppressed by the removal of Ca2⫹
from the medium and by common VSCC blockers. Caffeine
also evoked major cytosolic Ca2⫹ elevations in the somata
region, but these signals exhibited a more prolonged time
course and depended exclusively on Ca2⫹ release from internal
stores. These caffeine responses also differed from ESS signals
at least in three aspects. First, Ca2⫹ transients evoked by
caffeine exhibited a more gradual rise-time with a progressive
buildup. Second, caffeine signals were absent in apical dendritic regions, and the removal of extracellular Ca2⫹ did not
reduce the magnitude of these responses in any region. Third,
Ca2⫹ signals elicited by caffeine were completely abolished by
depletion of ryanodine-sensitive stores, indicating that these
responses arise entirely from Ca2⫹ release from these pools.
An interesting finding of this study was that depletion of
ryanodine stores did not affect the overall characteristics of
ESS responses. These data indicate that a Ca2⫹ release from
ryanodine-sensitive stores does not constitute the primary
source of the Ca2⫹ elevations associated with chemosensory
transduction. In addition, it suggests that in snake VN neurons
a functional separation exists between IP3 and ryanodinesensitive Ca2⫹ pools, which appear to be unevenly distributed
among different cellular segments. In this regard, we observed
that caffeine responses were found predominantly in the cell
body region, while ESS and BK responses were more broadly
distributed toward dendritic regions. Our data also suggest that
ryanodine-sensitive pools mediate an amplification of Ca2⫹
responses through a CICR mechanism because depletion of
these stores affected the decay phase of the Ca2⫹ transients
evoked either by ESS or high-[K⫹]o depolarization. In rat
ORNs, a similar CICR mechanism also seems to amplify Ca2⫹
signals elicited by odor stimuli (Zufall et al. 2000).
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gests that IP3 is a good candidate as a second messenger in VN
transduction. In the garter snake VN epithelium, exposure of
prey-derived chemoattractants induced dose-dependent IP3 accumulation (Luo et al. 1994), and similar IP3 elevations have
been found in microvillar membranes from prepubertal female
porcine VNO incubated with boar seminal fluid or urine
(Wekesa and Anholt 1997). Aphrodisin, which is a pheromone
excreted by female hamsters, also induces IP3 accumulation in
the VN epithelium of male hamsters (Kroner et al. 1996).
Finally, urinary pheromones evoke IP3 increases in rat VN
epithelium (Sasaki et al. 1999), and the VN neuronal discharges evoked by these urinary pheromones are selectively
blocked by the PLC inhibitor U-73122 (Holy et al. 2000).
Besides the putative IP3-mediated Ca2⫹ responses, we also
found that other mechanisms contribute to the generation of
cytosolic Ca2⫹ elevations elicited by chemoattractants. Figure
13 is a summary diagram of the mechanisms that might participate in the generation and regulation of Ca2⫹ transients in
VN neurons during chemosensory transduction. Basically, our
CHEMOATTRACTANT-INDUCED CALCIUM RELEASE
Component of ESS response dependent on a Ca2⫹ release
Component of ESS response dependent on Ca2⫹ influx
Besides the contribution of Ca2⫹ release, we found, in the
region adjacent to the luminal surface of the VN epithelium, a
component of ESS responses that depends on Ca2⫹ influx. In
our recordings, this component only represents 12–20% of the
total cytosolic Ca2⫹ transients elicited by ESS ligands, but the
magnitude could be underestimated, since our optical signals
represent population responses from several VN neurons together. Under these conditions, stronger neighboring signals
arising from Ca2⫹ release might minimize the real magnitude
of this Ca2⫹ influx, which probably occurs in confined regions
of the microvilli.
It is interesting that this Ca2⫹ influx does not appear to be a
cAMP-mediated mechanism because specific blockers of CNG
channels do not affect, to any measurable degree, the characteristics of this component, nor does the direct application of
forskolin evoke detectable [Ca2⫹]i changes. This interpretation
contrasts with the primary mechanism generating Ca2⫹ responses in ORNs (Leinders-Zufall et al. 1997, 1998) and leads
to the suggestion that in snake VN neurons a cAMP signaling
pathway does not play a fundamental role during the initial
stages of VN signal transduction. This Ca2⫹ influx does not
depend either on the reverse operation of the Na⫹/Ca2⫹ exchanger (DiPolo and Beauge 1987) because we found that its
blockage resulted in an increase in the amplitude and duration
of the Ca2⫹ transients. Thus this exchanger probably participates in restoring basal [Ca2⫹]i as has been reported for other
neurons (Fierro et al. 1998), including ORNs (Jung et al. 1994;
Noe et al. 1997; Reisert and Matthews 1998; Zufall et al.
J Neurophysiol • VOL
2000). Because we found that both ESS and BK responses
seem to depend on IP3 production, it appears likely that this
Ca2⫹ influx depends on activation of an IP3 conductance in VN
dendritic membranes. The existence of IP3-gated cation channels has been proposed in different chemosensory systems, but
unfortunately their role is still unclear (for review, see Schild
and Restrepo 1998). The best available evidence for the existence of these types of channels in chemosensory transduction
is in lobster ORNs, where it has been possible to characterize
an IP3-gated channel coupled to a G protein (Munger et al.
2000). In vertebrates, however, the presence of an IP3 transduction pathway in olfaction is still controversial. Transgenic
mice deficient in either CNG channels or G-protein-coupled
adenylate cyclase type III (Golf) fail to respond to odors
(Belluscio et al. 1999; Brunet et al. 1996), suggesting that IP3
cannot be considered an alternate transduction pathway in this
system.
Among the possible candidates involved in mediating the
Ca2⫹ influx found here are members of the transient receptor
potential (TRP) channels. Some TRP proteins are nonspecific
cation-permeable, store-operated channels (SOC) activated by
the depletion of intracellular Ca2⫹ stores (Boulay et al. 1999;
Minke and Selinger 1996a,b; Vannier et al. 1999; Zhu et al.
1996). However, it has been reported that some members in
this family seem to be activated also by phosphoinositide
turnover but not after store depletion (Okada et al. 1998;
Schaefer et al. 2000). This may involve both functional and
physical interactions between IP3 receptors and TRP channels
(Birnbaumer et al. 2000; Boulay et al. 1999; Kiselyov et al.
1998). In situ hybridization and immunohistochemical studies
(Liman et al. 1999) in rats indicate that the mRNA of a member
of this channel type (rTRP2) is expressed exclusively in the
receptor cells of the vomeronasal sensory epithelium and that
the protein is localized to the microvilli of these neurons.
Interestingly, this location corresponds to the Ca2⫹ influx here.
If similar TRP channels are expressed in snake VN neurons in
this region, it is unlikely that they would be solely SOC
because our results indicate that depletion of intracellular Ca2⫹
stores alone failed to elicit the Ca2⫹ influx observed in this
study.
Still to be resolved are the relationship between these two
components found in responses to ESS and BK as well as the
exact role of these cytosolic Ca2⫹ elevations during VN transduction. It is likely, however, that these cytosolic Ca2⫹ elevations play a significant role in the regulation of VN responses.
In the main olfactory system, cytosolic Ca2⫹ elevations with
similar kinetics play an important role, among other functions,
in odor adaptation. In ORNs, the main mechanism of odor
adaptation depends on an increase in cytosolic Ca2⫹ that activates a calmodulin (CAM)-dependent protein kinase II (CAMkinase II) that, in turn, phosphorylates the adenyl cyclase and
lowers CNG channel activity (Chen and Yau 1994; Kurahashi
and Menini 1997). A similar mechanism is unlikely to occur in
snake VN neurons because we found that Ca2⫹ transients
associated with VN transduction do not depend on the activation of CNG channels. Other mechanisms, however, can still
be triggered by cytosolic Ca2⫹ and act on either the primary
transduction currents or downstream. Interestingly, in Caernorhabditis elegans (Colbert et al. 1997) and Drosophila
(Störtkuhl et al. 1999) mutants defective in some TRP members exhibit impairment in olfactory adaptation. In addition,
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An unexpected finding of this study was the presence of
major Ca2⫹ transients elicited by ESS ligands in the absence of
extracellular Ca2⫹. This result differs from otherwise similar
Ca2⫹ signals associated with chemosensory transduction in
mouse VN neurons (Leinders-Zufall et al. 2000) and in ORNs
(Leinders-Zufall et al. 1997; Restrepo et al. 1990, 1993; Sato et
al. 1991; Schild et al. 1995; Tareilus et al. 1995), which are
completely suppressed in 0 [Ca2⫹]o. It is interesting to note
that Ca2⫹ release from internal Ca2⫹ pools also occurs in
ORNs, but this mechanism appears to be secondary to an initial
Ca2⫹ influx that is mediated through the opening CNG channels (Leinders-Zufall et al. 1998; Zufall et al. 2000).
In snake VN neurons, we found that Ca2⫹ release can be
directly triggered by VN transduction mechanisms, a condition
that may depend on two factors. First, in this system transduction seems to be mediated by phosphoinositide turnover, and it
is known that IP3 elevations in multiple cell systems mediate
major Ca2⫹ mobilization from intracellular pools (for review,
see Berridge 1993, 1998). Second, in snake VN neurons, it
appears that a tight coupling exists between the IP3 elevation
associated with VN signal transduction and the activation of
Ca2⫹ release from intracellular stores. Previous ultrastructural
studies of these cells demonstrated well developed cisternae of
endoplasmic reticulum (ER) extending close to microvillar
processes (Wang and Halpern 1980a,b). Perhaps, the proximity
of these potential Ca2⫹ stores to the microvilli permits efficient
coupling for the activation of Ca2⫹ release during VN signal
transduction.
1469
1470
A. R. CINELLI, D. WANG, P. CHEN, W. LIU, AND M. HALPERN
This work was supported by National Institute on Deafness and Other
Communication Disorders Grant DC-02531 (M. Halpern) and National Science Foundation Grant IBN9905700 (D. Wang).
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