Calcium Entry through Cyclic Nucleotide-Gated Channels in Individual Cilia of Olfactory Receptor Cells: Spatiotemporal Dynamics (original) (raw)

Articles

Journal of Neuroscience 1 June 1997, 17 (11) 4136-4148; https://doi.org/10.1523/JNEUROSCI.17-11-04136.1997

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Abstract

Transient elevations of intracellular Ca2+ play an important role in regulating the sensitivity of olfactory transduction, but such elevations have not been demonstrated in the olfactory cilia, which are the site of primary odor transduction. To begin to understand Ca2+ signaling in olfactory cilia, we used high-resolution imaging techniques to study the Ca2+ transients that occur in salamander olfactory receptor neurons (ORNs) as a result of cyclic nucleotide-gated (CNG) channel activation. To visualize ciliary Ca2+ signals, we loaded ORNs with the Ca2+indicator dye Fluo-3 AM and measured fluorescence with a laser scanning confocal microscope. Application of the phosphodiesterase inhibitor IBMX increased fluorescence in the cilia and other neuronal compartments; the ciliary signal occurred first and was more transient. This signal could be abolished by lowering external Ca2+ or by applying LY83583, a potent blocker of CNG channels, indicating that Ca2+ entry through CNG channels was the primary source of fluorescence increases. Direct activation of CNG channels with low levels of 8-Br-cGMP (1 μm) led to tonic Ca2+signals that were restricted locally to the cilia and the dendritic knob. Elevated external K+, which depolarizes cell membranes, increased fluorescence signals in the cell body and dendrite but failed to increase ciliary Ca2+ fluorescence. The results demonstrate the existence and spatiotemporal properties of Ca2+ transients in individual olfactory cilia and implicate CNG channels as a major pathway for Ca2+ entry into ORN cilia during odor transduction.

Signal transduction in vertebrate olfactory receptor neurons (ORNs) begins in the olfactory cilia when odor molecules bind to specific receptors and initiate a cascade of events leading to rapid cAMP formation, followed by the opening of Ca2+-permeable cyclic nucleotide-gated (CNG) cation channels (for review, see Lancet, 1986; Reed, 1992; Breer, 1993; Zufall et al., 1994). There is also evidence for an inositol 1,4,5-trisphosphate (IP3) pathway causing elevation of intracellular Ca2+ ([Ca2+]i) (cf.Restrepo et al., 1996), but its precise role in odor transduction remains unclear (see Brunet et al., 1996).

[Ca2+]i plays important roles in regulating the sensitivity of olfactory transduction as part of the process underlying odor adaptation. An early phase of odor adaptation is triggered by cAMP-dependent Ca2+ entry. Removal of extracellular Ca2+ results in a less transient cAMP-induced whole-cell current and eliminates the phasic–tonic behavior of odor currents during maintained stimulation (Kurahashi, 1990; Kurahashi and Shibuya, 1990; Zufall et al., 1991b; Kurahashi and Menini, 1997). Ca2+-dependent feedback regulation also can be initiated by the CO/cGMP signaling system (Verma et al., 1993; Ingi and Ronnett, 1995; Leinders-Zufall et al., 1995a), which leads, via persistent activation of CNG channels by cGMP, to the induction of a long-lasting form of odor adaptation (Leinders-Zufall et al., 1996; Zufall and Leinders-Zufall, 1997). Collectively, these results reveal complex effects of [Ca2+]i in ORNs. A complete understanding of the role of [Ca2+]irequires, therefore, detailed knowledge about the temporal and spatial properties of Ca2+ changes caused by stimuli.

Several laboratories have applied Ca2+ imaging to address this problem and to monitor stimulus-induced events in ORNs. These studies detected elevations in [Ca2+]iresulting from stimulation with single odor ligands or odor mixtures (Restrepo et al., 1990, 1993a,b; Restrepo and Boyle, 1991; Sato et al., 1991, 1994; Nakamura et al., 1994; Tareilus et al., 1995), forskolin (Sato et al., 1991; Restrepo et al., 1993b), or dialysis of ORNs with cAMP (Nakamura et al., 1994) or IP3 (Nakamura et al., 1994;Schild et al., 1995). Despite these abundant data, studies to date have not addressed Ca2+ signals in the cilia, the site of odor transduction. Thus, interpretation of the Ca2+ gradients seen in the dendrite and soma happened in the absence of any information about the events occurring at the transduction site. Additional mechanisms may regulate [Ca2+]i in cellular compartments other than the cilia; microdomains of Ca2+ gradients may exist and function with relative independence. Therefore, the goal of the current study was to fill this gap in our knowledge by obtaining Ca2+ images of olfactory cilia. Using micropulse stimulation and high-resolution confocal microscopy, we detect Ca2+ transients in individual cilia resulting from Ca2+ entry through activated CNG channels, thus showing, for the first time, a spatial sequence of Ca2+ signaling in ORNs.

MATERIALS AND METHODS

Preparation and solutions. Olfactory receptor neurons (ORNs) were freshly dissociated from the nasal epithelium of adult land-phase tiger salamanders (Ambystoma tigrinum), as previously described (Firestein et al., 1991b; Zufall et al., 1991b;Leinders-Zufall et al., 1995a), without the use of enzymes during the dissociation procedure. The epithelial sheets were cut carefully into small pieces (<1 mm3) and gently passaged in normal external solution (see below) several times through the fire-polished tip of a Pasteur pipette. Suspended cells were collected with a pipette, and aliquots of ∼200 μl were placed in a recording chamber on glass coverslips that had been coated previously with 0.1% laminin and 0.01% poly-l-lysine. This method was sufficient to ensure that the cells and their normally motile cilia became firmly attached to the coverslips (see Anderson and Hamilton, 1987). All cells used in this study were clearly identifiable as ORNs by their characteristic morphology, having a single thick dendrite with a knob-like swelling from which emanated 6–15 cilia.

Cells were held in a laminar flow chamber and superfused continuously at a rate of ∼100 μl/sec with physiological Ringer’s solution containing (in mm): NaCl 115, KCl 2.5, CaCl21.0, MgCl2 1.5, HEPES 4.5, and Na-HEPES 4.5, pH 7.6, adjusted to 240 mOsm. To avoid a contribution to the measured Ca2+ signals from regenerative action potential discharges, we added 4 μm tetrodotoxin (TTX) to this solution in all experiments, a dose that fully blocks currents through Na+channels in adult salamander ORNs (our unpublished observations). To reduce the external Ca2+ concentrations (as used for some experiments in Fig. 5), we buffered the bath solution with EGTA to give a free Ca2+ concentration of ≤1 μm. An estimate for this value was calculated from the stability constants, giving 0.6 μm free Ca2+ (Leinders-Zufall et al., 1996). Chemicals were applied either in the bath or by micropipette pressure ejection, as described previously (Leinders-Zufall et al., 1996). In the latter case, different solutions were ejected from multibarrel glass pipettes that were placed within 5–10 μm from the cilia or, as in one type of experiment, from the cell soma. Stimulus pipettes were located downstream from the cells to avoid prestimulation. Under these conditions the solution switching time was 30–40 msec, as measured by the electrical response to elevated K+ solutions (Leinders-Zufall et al., 1996).

Fig. 5.

Fig. 5.

Evidence that IBMX-stimulated fluorescence increases in the cilia depend on Ca2+ entry through activated CNG channels. A, Phase contrast image of an ORN showing the dendritic knob and several cilia. Scale bar, 1 μm.B, High-resolution image (gray scale) of the same ORN indicating the fluorescence intensity at rest, with normal (1 mm) Ca2+ in the extracellular bath solution. C, After an IBMX pulse the cilia show enhanced fluorescence intensity and are clearly resolvable.D, The same IBMX pulse fails to increase significantly the ciliary fluorescence levels in lowered external Ca2+(≤1 μm). E, The IBMX-induced fluorescence increase recovers after normal Ca2+ levels are restored in the bath solution. F, Analysis of the time courses of IBMX-induced fluorescence increases in one cilium under various conditions of external Ca2+ concentration, the same experiment as in B–E; sampling rate, 3 Hz.G, Effect of the CNG channel blocker LY83583 (40 μm) on IBMX-stimulated fluorescence changes. The IBMX-induced signal is abolished nearly fully in the presence of LY83583. The effect of LY83583 can be reversed after wash-out of the drug.

ORN membrane potentials were analyzed by patch-clamping the neurons and using the current-clamp mode of an EPC-9 patch-clamp amplifier, as previously described (Leinders-Zufall et al., 1995a). The perforated patch-clamp technique with amphotericin B was used for these measurements (Leinders-Zufall et al., 1995a, 1996).

IBMX (3-isobutyl-1-methylxanthine) and LY83583 [6-(phenylamino)-5,8-quinolinedione; Research Biochemicals International, Natick, MA] were prepared in DMSO. Dilutions were made fresh with final DMSO concentrations of <0.1% (v/v ) and were delivered directly to individual cells via the multibarrel pipette. This concentration of DMSO has been shown not to alter the electrophysiological characteristics of salamander ORNs (Leinders-Zufall et al., 1995a). 8-Br-cGMP was dissolved in Ringer’s solution. Unless otherwise stated, all chemicals were obtained from Sigma (St. Louis, MO).

Dye loading and calcium-imaging techniques. Intracellular Ca2+ was monitored with Fluo-3 AM (Molecular Probes, Eugene, OR), a membrane-permeant long wavelength fluorescence indicator, using techniques previously described (Leinders-Zufall et al., 1994). Fluo-3 AM was dissolved in a solution of DMSO and 20% Pluronic F-127 (Molecular Probes), which then was added to normal Ringer’s solution, sonicated briefly, and added to the experimental chamber to give a final concentration of 18 μm Fluo-3 AM, 0.08% Pluronic F-127, and 0.4% DMSO. Cells were incubated in the dye solution for 60 min at room temperature and then perfused with dye-free Ringer’s solution for 10 min before beginning the experiments. A laser scanning confocal system (Bio-Rad MRC-600, Hercules, CA) attached to an Olympus IMT-2 inverted microscope was used to visualize Ca2+-mediated fluorescence in the ORNs. The confocal system was equipped with a krypton–argon ion laser. A 60×, 1.4 numeric aperture objective (Nikon) was used; images additionally were magnified 3–4× with the electronic zoom setting of the confocal microscope, which scanned a smaller area, using the same number of pixels in the image frame. The excitation illumination was 488 nm; emitted fluorescence was collected with a long-pass filter for wavelengths ≥515 nm. The confocal optical section was ∼1.5 μm thick (estimated by the tilt–reflector method; Bio-Rad Technical Bulletin 101). Each ORN was imaged individually, with the optical section adjusted to show the cell body, dendrite, and cilia, if possible. High-resolution scans (768 × 512 pixels/frame) were made either in standard confocal mode or, for phase contrast images, using a Bio-Rad transmitted light detector. Time series images were made by collecting 64 × 64 pixel fluorescence images at a rate of either 1 or 3 sec−1. All scanning head settings except gain (i.e., black level, detector aperture, and laser neutral density filter) were kept the same over the series of experiments; the gain usually was adjusted slightly, but only once, at the beginning of the experiment during acquisition of the baseline image. High-resolution images of baseline fluorescence were taken for subsequent comparison with the time series images, and several images were acquired at the beginning of each time series before experimental solutions were applied. Eight-bit confocal image files were transferred to a Macintosh Quadra 800 microcomputer and analyzed with National Institutes of Health Image 1.59 software. Regions of interest corresponding to different cell compartments were outlined, and the average pixel values in these regions were measured. Data are presented in arbitrary fluorescence units or as relative changes in fluorescence intensity (Δ_F_) normalized to baseline fluorescence (F).

Calibration procedures. We estimated [Ca2+]i in ORNs in some experiments by using the calibration procedures of Kao et al. (1989) (also see Thomas and Delaville, 1991). Accordingly, the following equation was used to determine [Ca2+]i at rest:Embedded Image Equation 1in which F is the measured baseline fluorescence intensity and _K_d is the Ca2+-Fluo-3 dissociation constant (400 nm). _F_minis the fluorescence intensity in the absence of Ca2+, and_F_max is the fluorescence intensity of the Ca2+-saturated dye. Both parameters are calculated as average pixel values determined independently for each region of interest. _F_max is calculated as (_F_Mn − _F_bkg)/0.2 +_F_bkg, in which _F_Mn is the fluorescence intensity with ionomycin (10 μm) and MnCl2 (2 mm), and _F_bkgis the fluorescence intensity with ionomycin (10 μm) and MnCl2 (2 mm) after lysis with 12 μm digitonin. As detailed in Kao et al. (1989), we checked this procedure by measuring the fluorescence intensity during ionomycin treatment in 1 mm external Ca2+before addition of MnCl2 and found that this value approached the calculated _F_max. We also tested for a potential contribution of extracellular indicator dye that might have been present even after washing by adding 0.1 mmMnCl2, which was chelated after 20 sec with 0.25 mm diethylenetriaminepentaacetic acid (DTPA). There was no change in fluorescence, and thus a correction for extracellular dye was not necessary.

Data analysis. Data analyses and calculations were performed with Igor Pro software (WaveMetrics, Lake Oswego, OR) running on a Macintosh computer. Via this program user-defined functions in combination with an iterative Levenberg–Marquardt nonlinear, least-squares fitting routine were applied to the data. Unless otherwise stated, data are expressed as mean ± SD and number of cells (n). Statistical tests were performed with SuperAnova 1.1 (Abacus Software, Berkeley, CA). Fisher’s LSD (least significant difference) test was used as a post hoc comparison of the ANOVA. Composite images were prepared with Adobe Photoshop 3.0 and printed on a Codonics NP 1600 color printer.

RESULTS

Visualization of olfactory cilia

To visualize changes in [Ca2+]i in single olfactory cilia, we loaded ORNs with Fluo-3 AM (18 μm) and observed them with a confocal microscope. We chose Fluo-3 as the Ca2+ indicator for this study because, among presently available indicators, it provides large optical signals per molecule and thus allows for the detection of Ca2+transients in small cytoplasmic volumes (Minta et al., 1989). The data reported here were obtained from 115 individual ORNs, all of which had retained their cilia and showed the typical ORN morphology. Imaging of these neurons, in particular of the ciliary compartment, relies on the ability to immobilize the cells and the cilia on the substrate (see Materials and Methods). Figure 1 shows representative examples of ORNs as phase contrast and silhouette images to illustrate, after attachment and dye loading, the basic morphology and the four main compartments of the cells, including the soma, a single thick dendrite containing a knob-like swelling (termed olfactory knob) from which emanated half a dozen or more cilia (Fig.1A–E). ORNs tended to retract their dendrites so that the overall dendritic length was reduced. The morphology of the cilia, which are 200–300 nm in diameter (Simmons et al., 1981), was similar to that seen in earlier studies (Firestein et al., 1991b). Only those cells in which the majority of the cilia did not change their position during an experiment were included in the analysis. Individual cilia that moved during the course of an experiment were excluded from the analysis. Cells with longer dendrites were chosen for those experiments in which we were interested mainly in the spatiotemporal relation of Ca2+ fluorescence between dendrite and soma. However, there were no significant differences among the various morphological cell types with respect to their Ca2+signaling.

Fig. 1.

Fig. 1.

A–E, Morphology of acutely dissociated salamander olfactory receptor neurons after attachment and dye loading. Characteristic ORNs are shown as phase contrast and silhouette images. All studied neurons had intact cilia and a clearly identifiable dendritic knob. F, Example of the fluorescence intensity at rest (gray scale image) showing characteristic domains of higher fluorescence in discrete spots at the rim of the nucleus and at proximal and distal regions of the dendrite. The cilia are not detectable. Scale bars, 10 μm.

A fluorescence image of a representative ORN at resting conditions is shown in Figure 1F. Fluorescence intensity at rest was generally rather low. In many ORNs, however, domains of higher fluorescence were detectable in discrete spots at the rim of the large nucleus and at proximal and distal portions of the dendrite (see also Fig. 2B, for comparison). These fluorescence spots corresponded to locations where cellular organelles such as variegated and multivesicular bodies, mitochondria, and endoplasmic reticulum have been described in ORNs (Simmons et al., 1981). It is unclear whether higher fluorescence in these locations represents dye accumulation in organelles or whether it is attributable to higher Ca2+ levels. Consistent with the very low resting fluorescence of Fluo-3, significant fluorescence signals associated with single cilia usually could not be detected in ORNs without stimulation of the cyclic nucleotide pathways (Fig.1F; see also Fig. 5). In ∼15% of the cells, however, single cilia could be resolved faintly at rest. An example is seen in Figure 2B. Using the procedures described above (see Materials and Methods), we estimated the intracellular free Ca2+ concentration at rest to be 40.1 ± 9.1 nm (n = 4) in the cilia and 57.5 ± 33.3 nm (n = 4) in the dendritic knob.

Fig. 2.

Fig. 2.

Effect of a 1 min administration of IBMX (500 μm) on the Ca2+ fluorescence of an ORN.A, phase contrast image. Scale bar, 5 μm.B–D, Fluorescence images in pseudocolor scale taken at rest in the absence of IBMX (B), at peak fluorescence in the presence of IBMX (C), and after wash-out of IBMX and recovery of fluorescence intensity to near baseline levels (49 sec after the end of the IBMX stimulus; D). Images were generated by averaging four individual frames together, using the Kalman filter function of the confocal system. E, Time series images of the same ORN acquired during the first 32 sec of stimulation with IBMX and after recovery of the fluorescence signal (93 sec). Sampling rate, 3 Hz. Images are labeled in seconds, starting from an arbitrary zero time point; the stimulus was triggered at 2.5 sec. The earliest increases in Ca2+fluorescence took place in the cilia and the olfactory knob (arrow, 5 sec), followed by changes in the dendrite and cell body (frames 7 sec–32 sec).

Ca2+ transients induced by IBMX: spatiotemporal pattern

To investigate the Ca2+ signals associated with activation of odor-sensitive CNG channels, we first stimulated the ORNs with the phosphodiesterase inhibitor IBMX. Previous experiments have shown that this treatment results in activation of the same transduction channels as odor stimulation (Firestein et al., 1991b;Lowe and Gold, 1993a), presumably via elevation of cAMP levels because of the high basal activity of olfactory adenylyl cyclase (see Lowe and Gold, 1993a). Because the CNG channels are highly permeable to Ca2+ (Zufall and Firestein, 1993; Frings et al., 1995), IBMX stimulation should increase [Ca2+]i in the ORNs. Averaged fluorescence images (pseudocolor scale) are depicted in Figure 2B–D under three different conditions: (1) at rest before stimulation (Fig. 2B), (2) at peak fluorescence during a 1 min administration of IBMX (500 μm) (Fig. 2C), and (3) after wash-out of IBMX and recovery of the fluorescence intensity to near-baseline levels (Fig. 2D). The presence of IBMX caused a prominent, reversible increase in fluorescence across the entire cell. Particularly evident is the fluorescence signal associated with the characteristic “checkerboard” chromatin pattern of salamander ORN nuclei (Simmons et al., 1981), which can be observed in the soma region showing enhanced intensity during IBMX stimulation. A time series of images of the same experiment is shown in Figure 2E. Images were acquired before (first image, 0 sec), during (second to fifth image, 5–32 sec), and after recovery from stimulation with IBMX (last image, 93 sec). Note that the earliest IBMX-stimulated fluorescence increases took place in the cilia and the knob (see arrow in Fig. 2E,5 sec), followed by changes in the dendrite and soma (see images, 5–32 sec, Fig. 2E). Similar results were obtained in 15 other cells.

After stimulation of the cells, fluorescence increases in individual cilia could be resolved clearly. Figure 3_A_illustrates a high-resolution image taken at peak fluorescence showing the distal dendrite, knob, and several cilia of the same ORN as depicted in Figure 2. A quantitative comparison of the time course of the fluorescence signals was obtained from the outlined regions of three different cilia (Fig. 3B, areas 1–3) and a control region outside the cell (area 4). The analysis (Fig. 3C) revealed that the presence of IBMX (500 μm) resulted in a significant increase in Ca2+ fluorescence within these three regions but did not induce a measurable increase in the control region. Despite the continuous presence of IBMX the time course of the fluorescence increase was transient. The IBMX stimulus produced very similar time courses of Ca2+ fluorescence in the three different cilia. The signal reached its peak within 3.85–4.88 sec. The decay phase of the traces was fit with single exponential functions, giving time constants (τoff) ranging from 5.68 to 11.81 sec. The relative increase in fluorescence in the three cilia was similar, ranging from 41 to 58%. This suggests that the calculated fluorescence changes represented the expected cellular response caused by stimulation with IBMX. Because the ciliary membrane does not contain voltage-activated ion channels (see below), IBMX-induced increases in fluorescence in this compartment theoretically should be attributable entirely to CNG channel activation, even under the nonvoltage-clamp conditions used here. Evidence for this view is given by the close match of the time courses of the signals of Figure 3C with the ionic currents through CNG channels during continuous intracellular perfusion by cyclic nucleotides (Kurahashi, 1990; Firestein et al., 1991a; Leinders-Zufall et al., 1995a). Together with the fact that olfactory CNG channels are strongly permeable to Ca2+, these results thus suggest that the increase in fluorescence in the cilia resulted from Ca2+ influx through activated CNG channels.

Fig. 3.

Fig. 3.

Time course of IBMX-stimulated fluorescence increases (500 μm IBMX) analyzed in different cilia of the same ORN as that depicted in Figure 2. A, High-resolution confocal image (gray scale) taken at peak fluorescence intensity. Individual cilia are clearly identifiable. Scale bar, 2 μm. B, Same image as in_A_ but with outlined regions of interest indicating where average fluorescence intensity was measured.C, Plot of the time course of fluorescence increases stimulated by IBMX in the four different regions. The control area (4) that lacks cilia did not show any significant fluorescence change. Recovery of the signals was fit with single exponential functions, as indicated by the continuous lines, giving decay time constants of 7.4 sec (region_1_), 5.7 sec (region 2), and 11.8 sec (region 3).

To compare the spatiotemporal pattern of IBMX-induced fluorescence changes in the various cellular compartments in more detail, we next stimulated ORNs with brief pulses (1 sec) of IBMX (500 μm) (Lowe and Gold, 1993a). Stimuli were applied locally such that only the cilia came in contact with the test solution, but not the rest of the ORN, as done in previous electrophysiological experiments (Lowe and Gold, 1991). This technique has not yet been applied to the analysis of Ca2+ signals in ORNs. This treatment was sufficient to elicit marked fluorescence increases in all four major compartments of a given ORN (n = 19). A characteristic example is depicted in Figure4A, and results from several experiments are summarized in Table 1. The IBMX stimulus evoked a brief Ca2+ transient in a given cilium with a time to peak of 3 sec. The decay time course could be well fit by a monoexponential function yielding a time constant τoff = 2.8 sec (Fig. 4A,B). The time course of the fluorescence signal measured in the knob was qualitatively similar although slightly slower (see comparison in Table1). In contrast, the fluorescence increases in the dendrite and the soma resulting from the IBMX pulse showed very different temporal behavior. Most notably, they were sustained for a longer period before recovering back to baseline, often for >60 sec (Fig.4A,B). The Ca2+fluorescence in the dendrite displayed the slowest recovery of the four different compartments (n = 6) (see Table 1). Its recovery time course displayed complicated kinetics (Fig.4A), suggesting that several distinct mechanisms contributed to the overall fluorescence increase in the dendrite. To compare the onset and offset rates of the Ca2+ fluorescence in the different compartments in more detail, we rescaled signals to give identical peak amplitudes and plotted them at two different time scales (Fig. 4B,C). There was a clear temporal relation among the fluorescence increases in the various compartments. IBMX-stimulated Ca2+ fluorescence was detected first in the cilia and, with a slight delay, also in the knob. There was, however, a marked delay between the ciliary signal and the fluorescence change in the dendrite and soma. Interestingly, the rise of the signals in dendrite and soma occurred at a time when the ciliary response already was decaying. These results are fully consistent with previous evidence indicating that the cilia are the primary site of olfactory signal transduction and that they contain the highest density of CNG channels (cf. Zufall et al., 1994). These data also suggest that the fluorescence increases monitored in the dendrite and the soma probably reflect the activity of additional Ca2+ signaling mechanisms secondary to the primary transduction events because of CNG channel activation.

Fig. 4.

Fig. 4.

Spatiotemporal analysis of Ca2+fluorescence stimulated by a brief pulse of IBMX (500 μmapplied for 1 sec). The stimulus was directed exclusively at the cilia.A, Comparison of the time courses of fluorescence increases evoked by a brief IBMX pulse. The signal in any given cilium shows the highest rate of activation and recovery. In contrast, fluorescence increases in the dendrite and soma remained sustained for tens of seconds after a brief stimulus (compare Table 1). Note that the magnitude of relative changes in fluorescence in a given cilium cannot be compared directly with those in the knob or other parts of the ORN, because the diameter of a single cilium was much smaller than the optical section of the confocal microscope. B, C, For a better comparison of the time courses, fluorescence signals from_A_ are rescaled to give the same peak amplitudes and are plotted at two different time scales. Sampling rate, 1 Hz.

Table 1.

Analysis in various cellular compartments of temporal properties of fluorescence increases resulting from a 1 sec pulse of IBMX

IBMX-stimulated fluorescence increase depends on Ca2+entry through CNG channels

To investigate further the precise source of the ciliary fluorescence increases, we performed two additional tests. To examine whether the fluorescence change depended on Ca2+ entry or whether it was caused by intracellular release of Ca2+, we measured the IBMX responses in the presence of lowered extracellular Ca2+ concentrations. Figure 5_A–E_illustrates an example in which IBMX-stimulated Ca2+fluorescence was measured under two different conditions of external Ca2+ concentration, with physiological levels of 1 mm Ca2+ and with Ca2+ lowered to ≤1 μm (Leinders-Zufall et al., 1996). The temporal analysis of the same experiment is depicted in Figure 5F. At resting conditions the baseline fluorescence in this ORN was very low so that the cilia were barely detectable (Fig. 5B). Stimulation with IBMX in the presence of 1 mmCa2+ caused a marked increase in Ca2+fluorescence of the cilia (17.2%) with the characteristic transient time course (Fig. 5C,F). However, under conditions in which the external Ca2+ was lowered, the same IBMX stimulus failed to induce any measurable fluorescence increase (Fig.5D,F). Full recovery of the original IBMX response occurred after restoring the Ca2+level in the bath solution (Fig.5E,F) (n = 5). This experiment indicates that Ca2+ entry was essential for the generation of IBMX-stimulated fluorescence signals in the cilia. We also found no detectable fluorescence change in other ORN compartments under lowered external Ca2+ (data not shown), which further underscores the notion that Ca2+ entry is the primary Ca2+ source for the generation of the measured signals.

Second, we tested whether the ciliary fluorescence changes depended on Ca2+ permeation through CNG channels by using a pharmacological blocker for CNG channels, LY83583 (Leinders-Zufall and Zufall, 1995). LY83583 can be applied from the external side, shows rapid and complete recovery from its blockade of CNG channels on wash-out, and appears to block ionic flux through CNG channels by binding to the open channel pore (Leinders-Zufall and Zufall, 1995). Consistent with these results, the IBMX-stimulated fluorescence increase was abolished nearly fully in the presence of LY83583 (40 μm; n = 3) (Fig. 5G). The effect of LY83583 was reversible on wash-out of the drug (Fig.5G). Collectively, these two experimental approaches provide strong evidence to conclude that the measured increases in fluorescence in the cilia were caused by Ca2+ entry through CNG channel activation.

Fluorescence changes in response to 8-Br-cGMP

Native olfactory CNG channels are known not only to be gated by cAMP but also by cGMP (Nakamura and Gold, 1987; Zufall et al., 1991a;Frings et al., 1992). Given that cGMP mediates the induction of a long-lasting form of odor adaptation (Leinders-Zufall et al., 1996;Zufall and Leinders-Zufall, 1997), interest has focused on the mechanisms by which cGMP elevation leads to olfactory adaptation. Because the build-up of cGMP in ORNs is slow, reaches only low micromolar levels, and can outlast the primary cAMP-mediated odor response for minutes (Breer et al., 1992; Verma et al., 1993;Leinders-Zufall et al., 1995a), endogenous odor-stimulated cGMP formation results in currents through CNG channels with small amplitudes but minute-long time courses (Leinders-Zufall et al., 1996;Zufall and Leinders-Zufall, 1997). We have hypothesized that ionic flux through these persistent currents causes significant Ca2+accumulation in olfactory cilia as part of Ca2+-dependent feedback regulation. To test this idea, we exposed ORNs to low amounts of 8-Br-cGMP (1 μm) (Fig.6A,B). This low cGMP concentration was sufficient to produce significant fluorescence increases in olfactory cilia. The cGMP-stimulated fluorescence increase developed on a slow time course, reaching a sustained plateau within ∼2 min. More importantly, the increase in fluorescence was confined locally to the cilia and the knob (n = 5) (Fig. 6A,B). With a delay of ∼40 sec a much smaller increase in fluorescence could be detected in the dendritic compartment, whereas a fluorescence change in the soma was barely detectable. 8-Br-cGMP-stimulated fluorescence increases were blocked reversibly by LY83583 (20 μm) (data not shown), consistent with CNG channel activation (Leinders-Zufall and Zufall, 1995). Stronger stimulation with 8-Br-cGMP (100 μm) also promoted marked fluorescence increases in all parts of the ORN (Fig.6B). These data indicate that steep Ca2+gradients between single cilia and the dendrite and soma, lasting several minutes, developed on weak stimulation with cGMP. Thus, cGMP concentrations known to occur in ORNs after brief odor stimuli could serve to maintain elevated Ca2+ levels in olfactory cilia for time spans an order of magnitude longer than the cAMP-mediated responses.

Fig. 6.

Fig. 6.

Differential effects of weak and strong stimulation with 8-Br-cGMP on Ca2+ fluorescence in ORNs.A, Temporal analysis of the effect of 1 μm8-Br-cGMP on fluorescence levels in various cellular compartments. The rising phase of the ciliary response can be fit with a single exponential function with a time constant of τon = 67 sec. Note that there is a slow, cumulative increase in Ca2+fluorescence in a given cilium, reaching a plateau after ∼2 min.B, Plot showing the increase in Ca2+fluorescence resulting from various concentrations of 8-Br-cGMP (0.5, 1, and 100 μm) analyzed in the four main compartments of the neurons. Low levels of 8-Br-cGMP (0.5–1 μm) produced spatially heterogeneous fluorescence changes that were locally restricted to the cilia and the knob. Large fluorescence increases could be detected in all neuronal compartments after strong stimulation with 8-Br-cGMP (100 μm). The following increases in fluorescence were measured: 0.5 μm 8-Br-cGMP, 7.5 ± 8.7% (knob), 0.75 ± 1.5% (dendrite), 0% (soma); 1 μm 8-Br-cGMP, 18.7 ± 8.9% (cilium), 17.3 ± 12.5% (knob), 1.3 ± 2.5% (dendrite), 0.5 ± 1.0% (soma); 100 μm 8-Br-cGMP, 29.3 ± 21.5% (cilium), 58.7 ± 27.8% (knob), 86.5 ± 25.4% (dendrite), 68.8 ± 22.8% (soma).

Are there domains of higher fluorescence in various parts of individual cilia?

Previous ultrastructural experiments have suggested that the distal cilia are the main site of olfactory signal transduction, containing the molecular components of the cyclic nucleotide cascade (Menco et al., 1992; Menco, 1994). We therefore examined whether the fluorescence increase resulting from CNG channel activation showed a heterogeneous distribution in individual cilia, displaying higher intensity at distal cilia as compared with more proximal regions (Fig.7). Potential problems in this analysis could result from the fact that the diameter of a given cilium is not constant over its length (Menco, 1994) and that the optical section of the confocal microscope used here was larger than the ciliary diameter. Thus, differences in fluorescence intensity over the length of a given cilium simply could reflect differences in the path length and the number of dye molecules. To avoid this problem, we measured the relative decrease in cGMP-induced fluorescence (100 μm 8-Br-cGMP) that occurred on blocking the CNG channels by LY83583 (20 μm) (Fig. 7A,B). For the analysis the cilia were divided into proximal, medial, and distal regions (Fig.7C), and the relative changes in fluorescence intensity in these outlined areas were compared (Fig. 7D). The LY83583-mediated decline in fluorescence intensity was quite constant in different regions of a given cilium, showing no significant differences (Fig. 7D). There was also very little difference among individual cilia (Fig. 7D). Thus, at the level of resolution obtainable, there was no evidence in single cilia for a heterogeneous pattern of fluorescence changes with respect to CNG channel activation, consistent with conclusions from previous electrophysiological recordings (Lowe and Gold, 1991).

Fig. 7.

Fig. 7.

Lack of evidence for the existence of subdomains of higher fluorescence within individual cilia of an ORN.A, High-resolution image (4 averages) in pseudocolor scale taken in the presence of 1 mm 8-Br-cGMP showing strong fluorescence intensity in the distal dendrite, knob, and eight individual cilia. Scale bar, 1 μm. B, Same ORN in the presence of 1 mm 8-Br-cGMP plus 20 μmLY83583. As a result of CNG channel blockade by LY83583, the fluorescence intensity is decreased markedly but recovers back to high levels on wash-out of the blocker (data not shown). C, Drawing illustrating the ciliary regions in which fluorescence intensity was measured. Each cilium was subdivided into proximal (gray), medial (white), and distal (black) areas. D, Plot of the relative decrease in fluorescence levels mediated by LY83583 in various cilia (indicated by the numbers 1–8) and in subciliary parts (proximal, medial, and distal, as indicated in C). There is no significant difference in fluorescence changes in the various regions (LSD, p = 0.42–0.71).

Fluorescence changes in response to membrane depolarization by elevated K+

The existence of marked fluorescence increases in olfactory cilia generated by activation of CNG channels raised the question of the extent to which these changes were mediated by depolarization of the ORN and, in particular, whether subsequent Ca2+ entry through voltage-gated Ca2+ channels could account for the detected signals. To discriminate between these possibilities, we used micropulse stimulation with elevated external KCl solutions and measured the differential fluorescence changes in different cellular compartments.

The first aim was to stimulate the neurons with a 120 mmKCl pulse (1 sec), which is sufficient to drive the ORN membrane potential to values positive of 0 mV. This treatment failed to elicit any detectable fluorescence increase if the stimulus was directed exclusively at the cilia (Fig. 8A) (n = 6). This result reinforces the notion that the spatial distribution of ion channels in the ORNs is highly asymmetric and that the cilia lack significant voltage-activated membrane channels (cf. Zufall et al., 1994). When the soma of the ORNs was stimulated with a 120 mm KCl pulse, a marked and rapid increase in Ca2+ fluorescence was detected in the soma compartment (32 ± 21%) (Fig. 8B). The fluorescence signal reached its peak within 2.7 ± 0.8 sec (n = 7) and decayed on an exponential time course with a time constant of 16.6 ± 11.1 sec (n = 7). This signal was most likely attributable to activation of high voltage-gated Ca2+channels, followed by Ca2+ entry (Schild et al., 1994). Temporally similar but smaller fluorescence changes were detectable simultaneously in the dendrite and knob (Fig. 8B). Notably, there was no detectable fluorescence increase in the cilia (n = 7) (Fig. 8B). These data rule out the possibility that the ciliary fluorescence changes caused by activation of CNG channels resulted from backpropagation of Ca2+ from the dendritic compartment and that activation of voltage-dependent Ca2+ channels contributed to the measured fluorescence changes in the olfactory cilia.

Fig. 8.

Fig. 8.

Effect of membrane depolarization induced by elevated external K+ on fluorescence levels in various cellular compartments. A, Lack of depolarization-induced fluorescence change under conditions in which the 120 mmKCl pulse (1 sec) is directed exclusively at the cilia.B, Rapid fluorescence increases occur in the soma, dendrite, and, to a smaller extent, in the knob if a 120 mmKCl pulse is directed at the cell soma. No signal is detectable in the cilia (the results from only one cilium are plotted). The fluorescence increase in the soma decays with a monoexponential time course yielding a time constant of 15.8 sec. C, Effect of a 20 mm KCl pulse (1 sec) directed at the cell soma. There is no measurable fluorescence change in the cilia and the knob, whereas a very small and delayed signal is detectable in the dendrite and soma.

Previous work has suggested that high voltage-activated Ca2+ channels are the predominant form of Ca2+channels in ORNs and that membrane potentials sufficient to gate these channels probably are reached only during action potential generation (Schild et al., 1994). To evaluate this hypothesis further, we measured the membrane potentials (receptor potentials) resulting from stimulation of the ORNs with saturating concentrations of either IBMX (500 μm), 8-Br-cGMP (100 μm), or the odor ligand cineole (100 μm) by using the current-clamp mode of the patch-clamp amplifier. The perforated patch technique was applied in these experiments (Leinders-Zufall et al., 1995a, 1996), and the cells were held in TTX (4 μm) to avoid impulse discharges. Under these experimental conditions the maximal membrane potential elicited by any of the three stimuli reached −27.1 ± 3.4 mV (n = 9). This indicates that the voltage- and time-dependent conductances in the dendrite and soma of salamander ORNs are adjusted in a manner that ensures that the receptor potential never reaches levels positive to approximately −25 mV. The same extent of depolarization can be generated by a 20 mm KCl pulse (1 sec) directed at the ORN soma (data not shown). The fluorescence changes resulting from a 20 mm KCl stimulus are illustrated in Figure 8C. The results indicate that depolarization of the ORN membrane potential to −25 mV was not sufficient to generate fluorescence changes comparable to those of Figure8B. Except for a small and delayed signal, there was very little detectable fluorescence increase in the dendrite and soma under these conditions of stimulation (n = 3). This is consistent with the hypothesis that impulse generation is required to produce significant Ca2+ entry from activation of voltage-gated Ca2+ channels in these neurons.

From these results we conclude that, even under the nonvoltage-clamp conditions used here, the measured fluorescence increases in the cilia were caused entirely by Ca2+ entry through CNG channels.

DISCUSSION

In this study we have shown the existence and properties of Ca2+ transients in individual olfactory cilia caused by the activation of CNG channels. Several main findings emerge from the current work: (1) the ability to resolve spatially and temporally Ca2+ transients in single olfactory cilia; (2) the demonstration that Ca2+ transients in olfactory cilia are mediated by CNG channels within the cilia and not by backpropagation from the dendrite; (3) the demonstration that Ca2+transients in the dendrite and soma occur with a temporal delay after stimulus-induced events in cilia; (4) the possibility that Ca2+ transients in all of the cilia of individual cells are equivalent, suggesting equipotentiality in odor transduction; and (5) the likelihood that sites of transduction are distributed throughout the cilium, because Ca2+ signals at distal and proximal sites within a single cilium appeared to be similar.

Calcium imaging of salamander olfactory cilia

Confocal laser scanning microscopy proved to be an effective method to detect Ca2+ changes in olfactory cilia. The experiments were facilitated by the use of the isolated salamander ORN preparation (Firestein and Werblin, 1989), which shows robust cellular responses on activation of the cAMP second messenger system and has played a significant role in developing a basic model of the second messenger apparatus mediating the detection of odor ligands (Firestein et al., 1991a,b; Lowe and Gold, 1991, 1993b; Zufall et al., 1991a,b;Leinders-Zufall et al., 1996). The present work reveals that salamander ORNs are also advantageous for the analysis of Ca2+ signals in olfactory cilia. Thus, although previous studies have shown cytoplasmic Ca2+ responses in cell bodies and dendrites of ORNs from rat (Restrepo et al., 1993b; Tareilus et al., 1995), mouse (Sato et al., 1994), man (Restrepo et al., 1993a), catfish (Restrepo et al., 1990; Restrepo and Boyle, 1991), newt (Nakamura et al., 1994), or frog (Sato et al., 1991; Lischka and Schild, 1993), this is the first to demonstrate Ca2+ transients within the ORN cilia.

During the experiments there was neither significant photobleaching nor any evidence of illumination-mediated damage of the cells under our experimental conditions. Fluorescence changes showed full recovery to prestimulation levels. In some cases ORNs were imaged for >30 min and >380 frames. The indicator dye we used (Fluo-3) does not undergo significant shifts in emission or excitation wavelengths on binding Ca2+, precluding ratio imaging. However, calibration by the use of Ca2+ ionophore and heavy metal chelation as outlined by Kao et al. (1989) resulted in an estimate of [Ca2+]i in unstimulated cilia of ∼40 nm, which is consistent with previous work that uses ratiometric methods. Tareilus et al. (1995) described resting values of 40–80 nm [Ca2+]i in the soma and knob of rat ORNs, using the methods of Lipp and Niggli (1993), and similar values were found by others using Fura-2 (Sato et al., 1991;Lischka and Schild, 1993; Restrepo et al., 1993b; Nakamura et al., 1994). The relative fluorescence changes (Δ_F_/F) of ∼20% in the cilia caused by IBMX pulses (see Table 1) then correspond to a change in [Ca2+]i of ∼80 nm, assuming a_K_d value of 400 nm. Saturation would produce a relative increase of ∼110–120%. These high values usually were not reached in our experiments (Table 1), ruling out the possibility that dye saturation posed a significant problem.

Origin of the Ca2+ signals

The results provide strong evidence that Ca2+ entry through open CNG channels was the primary source of IBMX-stimulated fluorescence increases in the ORN cilia. Olfactory CNG channels are strongly permeable for Ca2+ (Zufall and Firestein, 1993;Frings et al., 1995) and are highly enriched in the ciliary compartment and at the olfactory knob of ORNs (Nakamura and Gold, 1987; Kurahashi and Kaneko, 1991), although they also can be found at a lower density in dendritic and somatic portions of the cells, at least under the conditions of acute isolation of the cells (Zufall et al., 1991a). This result predicts any Ca2+ signal associated with the opening of CNG channels to start at the distal end of the cell and, more specifically, in the cilia. The results of Figure 4 demonstrate that this was the case. In addition, ciliary fluorescence changes depended on Ca2+ entry and were abolished reversibly by LY83583 (Fig. 5), a potent blocker of olfactory CNG channels (Leinders-Zufall and Zufall, 1995). Furthermore, the activation time courses of ciliary Ca2+ changes were fully consistent with those of CNG channel responses (Kurahashi, 1990; Firestein et al., 1991a;Leinders-Zufall et al., 1995a). Depolarization of the neuron with high K+ solution was not sufficient to elicit measurable Ca2+ changes in the cilia (Fig. 8), ruling out a contribution of voltage-gated Ca2+ channels. Therefore, our results demonstrate that the fluorescence changes detected in the cilia were caused entirely by the activation of CNG channels.

An interesting result was that the Ca2+ responses in the dendrite and soma displayed much longer time courses than those in the cilia and remained sustained for >1 min, even after brief stimulation with IBMX (Fig. 4). The distinct time course and complex recovery kinetics of these signals indicate that they were generated by additional mechanisms secondary to Ca2+ entry through CNG channels. Membrane depolarization by high K+ solution was not sufficient to generate similar Ca2+ changes (Fig. 8). Although the exact mechanisms underlying these dendritic and somatic responses are unknown, preliminary results suggest that Ca2+ stores may be involved. Treatment of the ORNs with thapsigargin, a Ca2+-ATPase inhibitor that promotes Ca2+ release from intracellular compartments, resulted in shortened dendritic responses although no effects were seen on the ciliary Ca2+ transients (Greer et al., 1996). Collectively, the data of Figures 4 and 8 suggest that olfactory cilia function, at least to some extent, as discrete biochemical compartments with Ca2+ dynamics that are relatively independent from those in the rest of the neuron, a situation not unlike that in other small neuronal compartments such as dendritic spines (cf. Woolf and Greer, 1994; Shepherd, 1996).

Implications for transduction and adaptation mechanisms

The results give added support to the current consensus model of odor detection in which odor receptor activation results in cAMP formation and CNG channel opening, which ultimately leads to Ca2+ entry into the cilia. Detection of Ca2+transients in individual cilia is of particular interest with respect to the mechanisms of sensory adaptation in ORNs. Elevated [Ca2+]i governed by the activity of CNG channels plays important roles in feedback control of the sensitivity of olfactory transduction and is involved in a variety of specific regulatory functions such as desensitization during sustained stimulation (Kurahashi and Shibuya, 1990; Zufall et al., 1991b), rapid termination and recovery of the primary odor response (Kurahashi, 1989), and initiation of long-term adaptation (Zufall and Leinders-Zufall, 1997). Although many of the specific mechanisms and consequences triggered by Ca2+ entry remain to be investigated, it is clear from previous work that elevated [Ca2+]i can influence the activity of several molecular components of the cAMP pathway, including adenylyl cyclase (Sklar et al., 1986; Anholt and Rivers, 1990; Jaworski et al., 1995), CNG channels (Zufall et al., 1991b; Kramer and Siegelbaum, 1992; Chen and Yau, 1994; Lynch and Lindemann, 1994), calmodulin-dependent phosphodiesterase (Borisy et al., 1992; Yan et al., 1995), Ca2+-activated Cl− channels (Kleene, 1993;Kurahashi and Yau, 1993; Lowe and Gold, 1993b; Zhainazarov and Ache, 1995), and Ca2+-activated K+ channels (Morales et al., 1995).

Our finding that weak stimulation of the CNG channels with low levels of cGMP results in significant Ca2+ elevation in the cilia (Fig. 6) fits well with results from previous work indicating that cGMP-dependent Ca2+ entry plays a significant role in a long-lasting form of odor adaptation (Zufall and Leinders-Zufall, 1997). The main difference between cAMP and cGMP-dependent Ca2+ entry seems to be in the time course of the signals. Activation of the cAMP system leads to more rapid and transient Ca2+ elevations, whereas cGMP signals can maintain elevated Ca2+ levels in olfactory cilia for much longer time spans than the cAMP signals. Furthermore, the cAMP-mediated Ca2+signals reach higher levels, compared with the responses to physiological (micromolar) concentrations of cGMP. The result that [Ca2+]i reaches a plateau during sustained stimulation with 1 μm 8-Br-cGMP (Fig. 6) raises the question of how Ca2+ extrusion is being accomplished from olfactory cilia. It is likely that ATP-driven Ca2+ pumps (Lo et al., 1994) and Na+/Ca2+ exchangers (Jung et al., 1994) described in the apical ORN compartments play a role in these processes.

Conclusion

The present results establish the concept that activation of the cAMP and cGMP second messenger pathways leads to significant Ca2+ elevation within the olfactory cilia and that Ca2+ entry through CNG channels plays a predominant role in this process. The protocols developed here should prove useful for analyzing odor-stimulated Ca2+ signals in the cilia; our preliminary results (Zufall et al., 1996) indicate that odor stimulation evokes ciliary Ca2+ signals with spatiotemporal dynamics similar to those obtained in this study by manipulation of the intracellular cyclic nucleotide concentration. Finally, this approach should help to determine the precise functions of CNG channels expressed in other neurons of the nervous system (cf. Ahmad et al., 1994; Leinders-Zufall et al., 1995b; Kingston et al., 1996; Bradley et al., 1997).

Footnotes