Calcium entry through cyclic nucleotide-gated channels in individual cilia of olfactory receptor cells: spatiotemporal dynamics - PubMed (original) (raw)
Calcium entry through cyclic nucleotide-gated channels in individual cilia of olfactory receptor cells: spatiotemporal dynamics
T Leinders-Zufall et al. J Neurosci. 1997.
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 microM) 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.
Figures
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 m
m
) 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 μ
m
). 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 μ
m
) 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.
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.
Fig. 2.
Effect of a 1 min administration of IBMX (500 μ
m
) 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).
Fig. 3.
Time course of IBMX-stimulated fluorescence increases (500 μ
m
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).
Fig. 4.
Spatiotemporal analysis of Ca2+fluorescence stimulated by a brief pulse of IBMX (500 μ
m
applied 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.
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 μ
m
8-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 μ
m
) analyzed in the four main compartments of the neurons. Low levels of 8-Br-cGMP (0.5–1 μ
m
) 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 μ
m
). The following increases in fluorescence were measured: 0.5 μ
m
8-Br-cGMP, 7.5 ± 8.7% (knob), 0.75 ± 1.5% (dendrite), 0% (soma); 1 μ
m
8-Br-cGMP, 18.7 ± 8.9% (cilium), 17.3 ± 12.5% (knob), 1.3 ± 2.5% (dendrite), 0.5 ± 1.0% (soma); 100 μ
m
8-Br-cGMP, 29.3 ± 21.5% (cilium), 58.7 ± 27.8% (knob), 86.5 ± 25.4% (dendrite), 68.8 ± 22.8% (soma).
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 m
m
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 m
m
8-Br-cGMP plus 20 μ
m
LY83583. 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).
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 m
m
KCl 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 m
m
KCl 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 m
m
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.
References
- Ahmad I, Leinders-Zufall T, Kocsis JD, Shepherd GM, Zufall F, Barnstable CJ. Retinal ganglion cells express a cGMP-gated cation conductance activatable by nitric oxide donors. Neuron. 1994;12:155–165. - PubMed
- Anderson PAV, Hamilton KA. Intracellular recordings from isolated salamander olfactory receptor neurons. Neuroscience. 1987;21:167–173. - PubMed
- Anholt RRH, Rivers AM. Olfactory transduction: cross-talk between second-messenger systems. Biochemistry. 1990;29:4049–4054. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous