Drugs Affecting Phospholipase C-Mediated Signal Transduction Block the Olfactory Cyclic Nucleotide-Gated Current of Adult Zebrafish
Li Ma and
William C. Michel
Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah 84108
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ABSTRACT |
Ma, Li and William C. Michel. Drugs affecting phospholipaseC-mediated signal transduction block the olfactory cyclic nucleotide-gated current of adult zebrafish. J. Neurophysiol. 79: 1183-1192, 1998. Amino acid and bile salt odorants are detected by zebrafish with relatively independent odorant receptors, but the transduction cascade(s) subsequently activated by these odorants remains unknown. Electro-olfactogram recording methods were used to determine the effects of two drugs, reported to affect phospholipase C (PLC)/inositol tripohsphate (IP3)-mediated olfactory transduction in other vertebrate species, on amino acid and bile salt-evoked responses. At the appropriate concentrations, either an IP3-gated channel blocker, ruthenium red (0.01-0.1 µM), or a PLC inhibitor, neomycin (50 µM), reduced amino-acid-evoked responses to a significantly greater extent than bile salt-evoked responses. Excised patch recording techniques were used to measure the affects of these drugs on second-messenger-activated currents. Ruthenium red and neomycin are both effective blockers of the olfactory cyclic nucleotide-gated (CNG) current. Both drugs blocked the CNG channel in a voltage-dependent and reversible manner. No IP3-activated currents could be recorded. The differential effects of ruthenium red and neomycin on odor-evoked responses suggest the activation of multiple transduction cascades. The nonspecific actions of these drugs on odor-activated transduction pathways and our inability to record an IP3-activated current do not permit the conclusion that zebrafish, like other fish species, use a PLC/IP3-mediated transduction cascade in the detection of odorants.
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INTRODUCTION |
Olfactory transduction is initiated when membrane-associated odorant receptors, located on the cilia of olfactory receptor neurons (ORN), bind an appropriate odorant. Results obtained using a variety of experimental approaches suggest that one of several distinct transduction cascades may be activated upon ligand binding (Ache and Zhainazarov 1995
; Dionne and Dubin 1994
). The most thoroughly studied transduction pathways are GTP dependent and use either cyclic nucleotides (Buck 1996
; Zagotta and Siegelbaum 1996
; Zufall et al. 1994
) or inositol trisphosphate (IP3) (Bruch 1996
; Honda et al. 1995
; Restrepo et al. 1996
) as second messengers. A general strategy often used to associate an odor-evoked response with a specific transduction cascade requires that one or more drugs specifically perturb both odor and second-messenger-activated responses. A problem with this approach is that the drugs affecting olfactory transduction are frequently not specific (Kleene 1994
). For example, the olfactory cyclic nucleotide-gated (CNG) channel can be blocked by divalent cations (Frings et al. 1992
; Zufall and Firestein 1993
), calcium/calmodulin antagonists (W-7, trifluoperazine) (Kleene 1994
), soluble guanylate cyclase inhibitors [6-anilino-5,8-quinolinedione] (Leinders-Zufall and Zufall 1995
), and epithelial sodium (amiloride) and calcium channel blockers (diltiazem) (Frings et al. 1992
). The olfactory IP3-activated current also is blocked by divalent cations (Restrepo et al. 1992
) and by ruthenium red (Restrepo et al. 1990
), a drug that interacts with many calcium-binding proteins (Amann and Maggi 1991
) and blocks voltage-gated calcium channels (Hamilton and Lundy 1995
). Because few studies have exhaustively screened drug effects on all components of odor-activated transduction pathways and because drug effects may vary across species, it is essential to experimentally confirm that drug effects described in one species account for perturbed odor-evoked responses in other species.
The zebrafish is emerging as an increasingly popular model for studies of the organization of the olfactory system (Baier and Korsching 1994
; Baier et al. 1994
; Byrd and Brunjes 1995
; Friedrich and Korsching 1997
; Weth et al. 1996
) and its development (Barth et al. 1996
; Byrd et al. 1996
; Hansen and Zeiske 1993
; Vogt et al. 1997
). Functional properties of the zebrafish olfactory system have received less attention. Zebrafish respond behaviorally (Algranati and Perlmutter 1981
; Bloom and Perlmutter 1977
; Dill 1974
; Steele et al. 1990
, 1991
; Van Den Hurk and Lambert 1983
) and electrophysiologically (Michel and Lubomudrov 1995
) to most of the common water-soluble odorants detected by other fish species (Hara 1992
). Most of the amino acid and bile salt odorants tested appear to interact with at least partially independent odorant receptors (Michel and Derbidge 1997
) but the transduction cascades subsequently activated remain unknown. Odorants stimulate phospholipase C (PLC) activity and inositol trisphosphate production in the channel catfish (Restrepo et al. 1990
, 1993
) and Atlantic salmon (Lo et al. 1994
). Components of the CNG pathway are present in these fish species [and the zebrafish (Barth et al. 1996
)], but at least in the catfish, odorants do not stimulate rapid cyclic nucleotide synthesis (Restrepo et al. 1993
). Toward understanding the nature of transduction cascade(s) present in zebrafish ORNs, we conducted two experiments. In the first experiment, electro-olfactogram (EOG) methods were used to determine if drugs affecting olfactory transduction in other vertebrates affect the amino acid and bile salt-evoked responses of zebrafish. The two drugs tested, ruthenium red and neomycin, are reported to block IP3-gated channel (Honda et al. 1995
; Restrepo et al. 1990
) and PLC (Slivka and Insel 1988
) activity, respectively. To explore the mechanistic basis of any drug effects, a second series of experiments examined the actions these drugs on olfactory second-messenger-activated channel activity recorded from excised membrane patches of dissociated olfactory receptor neurons.

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| FIG. 1.
Block of odor-evoked responses by ruthenium red is dose dependent. A: Mean ± SE cysteine (Cys; 100 µM) and taurocholic acid (TCA; 1 µM) evoked responses recorded in the presence of 0.01 (n = 4), 0.1(n = 13), 1 (n = 4), and 10 (Cys, n = 2; TCA, n = 4) µM ruthenium red. Data for each odor in the ruthenium red background are normalized to their respective responses in normal artificial fresh water (AFW) recorded before ruthenium red exposure. TCA-evoked responses are significantly greater than the Cys-evoked response in the presence of 10 and 100 nM ruthenium red [2-way analysis of variance (ANOVA), Fisher's least significant difference (LSD) test, P < 0.05]. B: ruthenium red (0.1 µM) reduced the responses to several amino acids to a significantly greater extent than the responses to the bile salts tested (1-way ANOVA, Fisher's LSD test P 0.05). In B, each odorant was tested on 3 fish. Average responses to cysteine and TCA were 0.97 ± 0.59 mV and 1.72 ± 0.94 mV, respectively (n = 13). Arg, L-arginine; Cys, L-cysteine; Glu, L-glutamate; Met,L-methionine; TCDCA, taurochenodeoxycholic acid; TCA, taurocholic acid.
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METHODS |
Animal care and maintenance
Zebrafish (Danio rerio) were purchased from a commercial supplier (Steve Lambourne), housed in recirculating 10-20 gal aquaria equipped with under-gravel and charcoal filtration, and fed a commercially available flake food diet (Tetramin).
Electro-olfactogram methods
EOG responses were obtained using methods as previously described (Michel and Lubomudrov 1995
). Fish were immobilized with an intramuscular injection of gallamine triethiodide (Flaxedil; 60 µg/g body wt) and secured to a silicone elastomer (Sylgard) recording chamber. The olfactory organ immediately was provided with a continuous flow of artificial freshwater (AFW; see Solutions for composition), and the gills were irrigated with a flow of ~5 ml/min of AFW containing the general anesthetic 3-aminobenzoic acid ethyl ester (MS-222; 20 mg/l in AFW). The level of anesthesia was monitored by observation of reflexive movement of the gills and eyes and increased if required. Anesthesia was not provided before immobilization to minimize the loss of afferent sensory activity associated with topical application of anesthetic (Spath and Schweickert 1977
). While under general anesthesia, the small flap of epithelium covering the olfactory organ was removed surgically to facilitate positioning of the recording electrode. The recording electrode (3 M KCl in 1% agar, 5-10 µM tip diam) was positioned between olfactory lamellae near the midline raphe in a location that maximized the response to 100 µM cysteine (Cys). An identical electrode positioned on the head served as the differential electrode and a silver/silver chloride ground electrode positioned beneath the fish in the AFW bath completed the electrical circuit. Responses to olfactory stimuli were amplified (2,000-5,000 times gain) and filtered (2 kHz) by a low-noise differential DC amplifier and displayed on an oscilloscope. The amplifiedsignal also was digitized (100 Hz), stored, and displayed usingAxotape software (Axon Instruments). Each digital record contained ~8 s of prestimulation and 32 s of poststimulation time. Preparations were rejected if 100 µM Cys did not elicit a response >250 µV.
In situ odorant delivery
Methods for odorant delivery have been described previously (Michel and Lubomudrov 1995
). Briefly, the olfactory epithelium was bathed by a carrier flow of AFW (or drugs prepared in AFW) that was supplied by gravity-flow from polyethylene bottles, selected by a six-way valve and regulated to 3 ml/min with a flowmeter. Olfactory stimuli (50 µl) were introduced into the carrier flow of AFW through a rotary loop injector (Rheodyne). The bolus of odor reached a peak concentration of ~84% of the loaded concentration by 8 s after injection and decayed to the baseline concentration after ca. 12 s (Michel and Lubomudrov 1995
). The concentrations reported have not been corrected for dilution. To determine the effects of ruthenium red and neomycin on olfactory sensitivity, odor-evoked responses measured in the presence of these drugs were normalized to responses obtained in normal AFW before drug exposure. Odors tested in a drug background were diluted with the appropriate background solution.
Dissociation of the olfactory epithelium
Fish were killed by decapitation and the olfactory rosettes were dissected into ice-cold Ringer (see Table 1). After rinsing, the rosettes were held in fresh, oxygenated Ringer at 4°C until use. To dissociate ORNs, a rosette was transferred into divalent-free Ringer containing L-cysteine-activated (1.25 mg/ml) papain (0.25mg/ml) for 15-30 min. The rosette was rinsed in divalent-free Ringer, placed onto a concanavalin A-coated glass coverslip containing a single drop of divalent-free Ringer, and dissociated by teasing with fine insect pins or by trituration through fire-polished glass pipettes. The cells were allowed to settle and attach to the coverslip for
10 min before use.

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| FIG. 2.
Phospholipase C inhibitor, neomycin (50 µM), reduces the responses to amino acid odorants to a significantly greater extent than the responses to the bile salt odorants (1-way ANOVA, Fisher's LSD testP 0.05). Amino acid odorants were tested at a concentration of 100 µM, and bile salt odorants were tested at a concentration of either 1 µM(TCA) or 10 µM (all others). Data from 5 preparations are plotted as means ± SE. Gln, L-glutamine; GCA, glycocholic acid; His, L-histidine; LCA, lithocholic acid; Lys, L-lysine.
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Excised patch recording techniques
Coverslips containing dissociated ORNs were placed in a recording chamber situated on the stage of a Nikon MM1 upright microscope and viewed through a ×40 water immersion lens with an Optizoom image inverter/variable magnifier (×0.8-2) and ×10 eye pieces. Recordings were obtained with borosilicate glass patch electrodes (>1 µm tip diam) filled with the appropriate pipette solution and positioned on the soma or dendrite of an isolated ORN under visual control. Signals were amplified with a commercial patch clamp amplifier (Axopatch 200A), displayed on an analog oscilloscope, and stored digitally using pClamp software (Axon Instruments). Recordings were filtered at 1 kHz and digitized at 5 kHz. All reported voltages refer to the intracellular surface of the membrane relative to the extracellular surface.
Membrane patches were excised from the soma or dendrite of acutely dissociated ORNs in the inside-out configuration. The use of low [Ca2+] solutions and a brief air exposure often resulted in successful inside-out patches. The presence of one or many second-messenger-activated channels was confirmed by exposing the intracellular face of the patch to either adenosine 3
,5
-cyclic monophosphate (cAMP) or IP3 and noting either single- or multichannel activity. Only excised patches estimated to have five or more second-messenger-activated channels were used in this study. Since excised patches frequently vesiculated, no attempt was made to determine the proportion of patches containing second-messenger-activated currents. The magnitude of the second-messenger-activated current was measured from the average of four replicate voltage ramps from
60 to +60 mV (duration = 30 ms). The intracellular surface of the excised patch was exposed to cAMP for 3-5 s before initiating the ramp protocol. Leak currents, measured using an identical ramp protocol in the absence of second messenger, were subtracted from the second-messenger-activated current before calculating the slope conductance. The slope conductance was calculated from a linear regression of the average of the leak-corrected current over the voltage range of
30 to +30 mV. Plots of the slope conductance versus second-messenger concentration were fit with the Hill equation to calculate the K1/2 and Hill coefficient for second messenger binding. Bi-ionic methods were used to measure the potassium/sodium (K+/Na+) permeability ratio (Frings et al. 1992
). Second messengers and drugs were applied to excised patches using a flow microswitching device (Warner Instruments, Hamden, CT). Unless otherwise indicated, all data are reported as means ± SE.

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| FIG. 3.
Application of adenosine 3 ,5 -cyclic monophosphate (cAMP) to the intracellular surface of membrane patches excised from the dendrite or soma of many zebrafish olfactory receptor neurons (ORNs) activates a dose-dependent cyclic nucleotide-gated (CNG) current. A: CNG current in 1 inside-out patch from the soma is activated by low micromolar concentrations of cAMP and saturates at cAMP concentrations ~10 µM. Patch was stimulated with 0.4, 0.8, 1.6, 2, 3, 5, 10, 20, 30, and 40 µM cAMP). In this and all subsequent patches, the CNG-specific current was calculated by subtracting the "leak" currents (measured in the absence of cAMP; inset shows an example from another patch) from the total currents measured in the presence of cAMP. B: plot of normalized slope conductances of the data shown in A were fit with the Hill equation to yield a K1/2 for cAMP activation and Hill coefficient for this patch of 2.4 µM and 2.1, respectively. Slope conductance for each trace was normalized to the highest slope conductance. Data was obtained from inside-out patches in symmetrical NaCl solutions.
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| FIG. 4.
Pharmacology of the zebrafish olfactory CNG channel is similar to other vertebrate olfactory CNG channels. Two calcium/calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7; A) and trifluoperazine (TFP; B), blocked the CNG current in a dose-dependent fashion. A partial block was evident in the presence of low micromolar concentrations of these drugs. Amiloride (C) and diltiazem (D) also blocked the CNG channel but required higher concentrations. Block by all of these drugs was completely reversible (not shown for W7 and TFP). Data plotted are representative of data obtained from 3 to 5 different patches for each drug. CNG current was activated with 40 µM cAMP in all patches. Data was obtained from inside-out patches in symmetrical NaCl solutions.
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Solutions
The compositions of all solutions are listed in Table 1. Stock solutions of IP3 and cAMP were prepared in distilled water and stored at
20°C. Stock solutions of ruthenium red, (+)-cis-diltiazem, and amiloride were prepared in the NaCl bath solution and stored at 4°C. Stock solutions of N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) and trifluoperazine were prepared in ethanol and stored at 4°C. 6-anilino-5,8-quinolinedione(LY-83583) was obtained from Calbiochem (San Diego, CA), and a 10 mM stock solution, prepared in dimethyl sulfoxide, was stored at 4°C. All other chemicals were obtained from Sigma Chemical (St. Louis, MO).
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RESULTS |
Effects of ruthenium red and neomycin on odor-evoked responses
Odor-evoked responses were affected significantly by both of the drugs tested. Submicromolar ruthenium red significantly reduced the response to the amino acid L-cysteine without affecting the response to the bile salt taurocholic acid. Ten and 100 nM ruthenium red reduced the response to 100 µM Cys by ca. 50 and 80%, respectively (Fig. 1A). In contrast, responses elicited by 1 µM taurocholic acid (TCA) were unaffected by 10 nM ruthenium red and reduced by <15% by 100 nM ruthenium red. When the ruthenium red concentration was increased to either 1 or 10 µM, the Cys-evoked response was eliminated and TCA-evoked responses were attenuated significantly. Cys-evoked responses recovered slower than TCA-evoked responses regardless of the ruthenium red concentrations tested (data not shown). At 10 µM ruthenium red, the response to cysteine did not recover within 30 min. The responses to three other amino acids, thought to interact with odorant receptors independent of the receptor(s) mediating the cysteine response (Michel and Derbidge 1997
), also were attenuated significantly by 100 nM ruthenium red (Fig. 1B).
Odorants stimulate the olfactory PLC activity of several fish species (Boyle et al. 1987
; Har Lo et al. 1993
; Huque and Bruch 1986
; Restrepo et al. 1993
). If PLC activation is essential to odor transduction, then drugs blocking PLC activity also should block odor-evoked responses. The PLC inhibitor, neomycin (50 µM) reduced amino acid-evoked responses to a significantly [2-way analysis of variance (ANOVA), Fisher's least significant difference (LSD) test, P < 0.05] greater extent than bile salt-evoked responses (Fig. 2). The responses elicited by the eight amino acids tested were reduced by >80-90%, whereas the responses to the four bile salts tested were only reduced by <20% to ~50%.
The selective reduction of amino acid-evoked, but not of bile salt-evoked, responses by submicromolar ruthenium red and by neomycin is consistent with the presence of a PLC/IP3 transduction cascade activated by amino acids but not by bile salts. However, the specific effects of ruthenium red and neomycin on any potential odorant-activated transduction cascades in zebrafish are unknown. Consequently, in the second phase of this investigation, we tested the effects of these drugs on second-messenger-activated currents in excised patch recordings.
Second-messenger-activated currents in excised patch recordings
Second-messenger-activated currents were identified by application of cAMP or IP3 to the intracellular surface of membrane patches excised from the soma or dendrites of isolated ORNs and recording currents during voltage clamp protocols. A cAMP-activated current was recorded routinely, but we failed to observe an IP3-activated current in excised patches from >40 zebrafish ORNs, including eight patches that were sensitive to cAMP (data not shown). Our failure to identify an IP3-activated current might have been due to an inappropriate calcium concentration, the use of patches generally excised from somatic membrane, or the absence of these channels in zebrafish ORNs. The effects of ruthenium red and neomycin on the CNG current are considered in the following sections.
General properties of zebrafish olfactory cng channel
Low micromolar concentrations of cAMP activated the zebrafish olfactory CNG channel (Fig. 3). The averageK1/2 for cAMP was 3.0 ± 0.6 µM (n = 10, range 0.94-6.6 µM). A Hill coefficient of 2.8 ± 0.5 (n = 10) indicates cooperativity of cAMP binding. At concentrations of cAMP sufficient to elicit a saturating response, the average slope conductance was 1.2 ± 1.2 nS, (n = 102, range, 0.09-6.3 nS). The calculated K+/Na+ permeability ratio of 0.9 ± 0.01 (n = 5) indicates that the zebrafish olfactory CNG channel is a relatively nonspecific cation channel. Shifting EC1 from 0 to
60 mV changed the reversal potential of the CNG current by only 1 mV (n = 3 patches; data not shown).
Before determining the effects of ruthenium red and neomycin on the zebrafish olfactory CNG current, the basic pharmacology of the zebrafish CNG current was compared with other vertebrate olfactory CNG currents. Four of five drugs previously reported to block other vertebrate olfactory CNG channels reversibly blocked the zebrafish olfactory CNG channel (Fig. 4, Table 2). At 1-10 µM, the calcium/calmodulin antagonists W-7 and trifluoperazine partially blocked the zebrafish olfactory CNG channel. At 100 µM, either calcium/calmodulin antagonist effectively blocked the CNG current (Fig. 4, A and B). Amiloride and diltiazem partially blocked the zebrafish olfactory CNG channel at millimolar concentrations (Fig. 4, C and D) but were ineffective at concentrations <200 µM (not shown). LY-83583 (10 µM) blocked the tiger salamander olfactory CNG channel (Leinders-Zufall and Zufall 1995
) but failed to block the zebrafish olfactory CNG channel when concentrations ranging from 10 to 80 µM were applied to either the intracellular or extracellular surface of the membrane (Table 2). The pharmacological data indicates that the zebrafish olfactory CNG channel has properties similar to other vertebrate CNG channels.
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TABLE 2.
Effects of drugs reported to block other vertebrate olfactory CNG conductances on the zebrafish olfactory CNG conductance
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Effect of ruthenium red on the zebrafish olfactory CNG current
To determine if ruthenium red affected the CNG current, we coapplied 40 µM cAMP and one of several concentrations of ruthenium red to the intracellular surface of the excised membrane patch (Fig. 5, A, C, and D). The lowest concentration of ruthenium red tested (100 nM) blocked ~25% of the outward component of the CNG current. At an intermediate concentration of ruthenium red (1 µM), nearly 90% of the outward component was blocked. At the highest ruthenium red concentrations (10 and 100 µM), the block of the outward component of the CNG current was generally complete. In some patches, at the intermediate and higher ruthenium red concentrations, a negative slope conductance at positive membrane potentials was observed, indicating the ruthenium red block is voltage dependent. The inward component of the CNG current also was blocked but only ruthenium red concentrations
10-100 µM (Fig. 5D). The block of the inward component of the CNG current was also voltage dependent. To determine if extracellular ruthenium red blocked the CNG current, we included ruthenium red in the recording pipette solution. Although the block developed slowly, extracellular ruthenium red also affected the CNG current (Fig. 5B). Initially, only the outward component of the CNG current was partially blocked by ruthenium red (the block was most notable at positive transmembrane potentials). After 30 min, both inward and outward components of the CNG current were blocked by ruthenium red.

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| FIG. 5.
Intracellular ruthenium red blocks the CNG channel at submicromolar concentrations. A: ramp currents recorded in the presence of cAMP and increasing concentrations of ruthenium red reveal that the block is dose-dependent and affects both inward and outward components of the CNG current. B: extracellular ruthenium red (10 µM ruthenium red in the recording pipette) blocked the CNG channel in excised inside-out patches. Block developed slowly; within 5 min the CNG current was blocked at more positive transmembrane potentials (more than +30 mV). In this patch, the outward CNG current was blocked completely by 12 min. Block of the inward component developed more slowly but was essentially complete within 30 min. Different intracellular ruthenium red concentrations are required to block inward (C; n = 4) and outward (D; n = 4) components of the CNG current. In C and D, the inward and outward currents for each patch were normalized to the cAMP-activated currents measured in the absence of ruthenium red at 60 and +60 mV, respectively. CNG current was activated with 40 µM cAMP. Data was obtained from inside-out patches in symmetrical NaCl solutions.
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To investigate the recovery from ruthenium red block, ruthenium red was applied to the intracellular surface of an excised patch and 40 µM cAMP was applied in ruthenium red until a stable level of block was achieved. To start the recovery period, ruthenium red was removed and cAMP was tested alone. At each concentration of ruthenium red tested, the magnitude of the CNG current was measured before, in the presence of, and at several recovery intervals after the removal of ruthenium red (Fig. 6). The data plotted are representative of data obtained from at least three patches at each concentration. In general, recovery from ruthenium red block was relatively rapid and complete. Recoveries from 100 nM and 1 µM ruthenium red were complete in <5 min. Recoveries from 10 and 100 µM ruthenium red exposure were incomplete. Nearly 80% of the outward current recovered in <5 min, but no further recovery was measured even after 30 min.

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| FIG. 6.
Time course of recovery from ruthenium red application was investigated at 4 ruthenium red concentrations (A-D). In all patches, the ruthenium red block of the CNG current was allowed to completely develop (plotted) before returning the patch to cAMP alone and measuring recovery over time. Recovery was rapid and complete at lower ruthenium red concentrations ( 1 µM). At higher ruthenium red concentrations ( 10 µM), the recovery was rapid and largely complete, but a portion of the CNG current did not recover. Data from 4 different inside-out patches are plotted in A-D. Each patch was tested with the concentration of ruthenium red indicated in the figure. CNG current was activated with 40 µM cAMP. Data was obtained from inside-out patches in symmetrical NaCl solutions.
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Effect of neomycin on the zebrafish olfactory CNG current
Application of neomycin to the intracellular surface of the membrane partially blocked the olfactory CNG current. The block was dose dependent (Fig. 7A). Neomycin (50-200 µM) blocked ~50% of the outward current at +60 mV. The effect of neomycin concentration on the average CNG currents of three excised patches is illustrated in Fig. 7B. Extracellular neomycin (included in the pipette solution,n = 3 patches) blocked only a small portion (<10%) of the peak outward current (Fig. 7C). The most pronounced neomycin block was observed when it was applied to both sides of the membrane patch (Fig. 7D). In three patches with 50 µM neomycin bathing the intracellular surface and 50-100 µM neomycin bathing the extracellular surface, nearly all of the outward component and ~50% of the inward component of the CNG current was blocked.

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| FIG. 7.
Phospholipase C (PLC) inhibitor neomycin blocked the CNG channel. A: intracellular neomycin blocked the CNG current in a dose-dependent manner. Membrane patches became very unstable when exposed to concentrations of neomycin >200 µM. B: average ± SE block of the outward component of the CNG current, measured in 3 excised patches, is plotted as a function of intracellular neomycin concentration. C: extracellular neomycin (100 µM) weakly blocks the outward component of the CNG current (neomycin included in the recording pipette). D: neomycin nearly completely blocked the outward component of the CNG current when present on both sides of the membrane. In all patches, the CNG current was activated with 40 µM cAMP. Data was obtained from inside-out patches in symmetrical NaCl solutions.
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DISCUSSION |
Two significant findings are reported in this study. First, drugs, previously reported to block phospholipase C activity (neomycin) and IP3-gated channel activity (ruthenium red), differentially affected odor-evoked responses of the zebrafish olfactory epithelium. Both drugs reduced the responses evoked by amino acids to a significantly greater extent than the responses evoked by bile salts. Submicromolar concentrations of ruthenium red (10-1,000 nM) selectively blocked amino acid evoked responses, 10 µM ruthenium red blocked all odor-evoked responses. Fifty micromolar neomycin selectively blocked amino acid-evoked responses. This neomycin concentration is 20- to 200-fold lower than generally used to perturb PLC activity. Collectively, these results are consistent with the interpretation that a PLC/IP3 transduction cascade mediates amino acid-evoked responses. The second significant finding of this study was that ruthenium red and neomycin both blocked the zebrafish olfactory CNG current. The later finding renders any conclusion about the transduction pathway(s) used by amino acid and bile salt odorants premature. To our knowledge, we are the first to directly examine the actions of these drugs on the olfactory CNG channel.
Ruthenium red is an organometallic dye that reportedly affects the function of many calcium-binding proteins (Amann and Maggi 1991
), blocks voltage-gated calcium channel activity (Hamilton and Lundy 1995
) and nociceptor function (Dray et al. 1990
), and associates with negatively charged phospholipids (Voekler and Smejtek 1996
). In olfaction, ruthenium red originally was used to block norleucine-evoked responses of the channel catfish olfactory epithelium as well as IP3-activated channel activity of enriched catfish olfactory cilia membranes incorporated into lipid bilayers (Restrepo et al. 1990
). Ruthenium red has similar affects on the olfactory IP3-activated currents/channels of other vertebrate (Honda et al. 1995
; Okada et al. 1994
; Restrepo et al. 1990
, 1992
) and invertebrate (Fadool and Ache 1992
) species. The olfactory CNG channel must be added to the list of cellular substrates affected by ruthenium red.
The block of the zebrafish olfactory CNG current by ruthenium red was dose dependent and effective at submicromolar concentrations. One hundred-fold lower concentrations of ruthenium red were required block the outward component of the CNG current. Extracellular ruthenium red also blocked the outward component of the CNG current to a greater extent than the inward component. Collectively, these findings suggest that the primary site of ruthenium red action is on the intracellular surface. Ruthenium red association with negatively charged phospholipid residues (Voekler and Smejtek 1996
) near the CNG channel presumably would reduce the local monovalent cation concentration, resulting in a block similar to that produced during intracellular acidification (Frings et al. 1992
). If the adsorption of ruthenium red to these membrane phospholipids is strong, ruthenium red levels might be expected to persist even after ruthenium red application is stopped. The slow and incomplete recovery of both odor responsiveness and CNG currents after exposure to the higher ruthenium red concentrations might reflect its adsorption to phospholipids.
Neomycin is an aminoglycoside antibiotic that reportedly affects many cellular substrates. Neomycin blocks phospholipase C activity by binding to the enzyme's substrate, phosphoinositol bisphosphate (PIP2) (Slivka and Insel 1988
).A variety of ion channel types, including skeletal muscleL-type Ca2+ channels, Ca2+-activated K+ channels, and mechanosensitive channels are blocked by neomycin and by other aminoglycoside antibiotics (Haws et al. 1996
; Winegar et al. 1996
). In isolated rat ORNs, 200 µM neomycin blocked the [Ca2+]i increase elicited by an IP3-activating odor mixture, presumably by inhibiting PLC activity (Tareilus et al. 1995
), but also appears to have partially blocked the response to the cAMP-activating odor mixture. Our finding that neomycin blocks olfactory CNG current suggests that the partial block of the response to the cAMP-activating odor mixture previously noted may have been a result of a direct action of neomycin upon the CNG channel. Although higher concentrations of neomycin frequently are used to perturb PLC function, we found that excised patches became unstable at concentrations of neomycin >200 µM. Because neomycin only partially blocks the CNG current and is known to have actions on other potential substrates involved in olfactory transduction, particularly PLC activity, we did not further characterize its actions on the CNG current.
Our findings that both ruthenium red and neomycin block the olfactory CNG current are not likely to be due to properties unique to the zebrafish CNG channel. Biophysical and pharmacological properties of the olfactory CNG channel of zebrafish are similar, in most respects, to the olfactory CNG channels of other vertebrates. In a low calcium environment (ca. 10 nM), the K1/2 of 3.0 µM for cAMP activation of zebrafish CNG channels is similar to K1/2 values reported for the toad [3.4 µM (Nakamura and Gold 1987
)], channel catfish [2.5 µM (Bruch and Teeter 1990
; Goulding et al. 1992
)], frog [3.4-4 µM (Frings et al. 1992
)], rat [2.5 µM (Frings et al. 1992
)], and the tiger salamander [20 µM (Zufall et al. 1991
)]. The Hill coefficient of 2.8 measured for the zebrafish CNG channel is slightly larger than values reported for other olfactory CNG channels (range, 1.4-2.1), but is consistent with the general idea of cooperative cyclic nucleotide binding to a heterotetrameric channel (Zagotta and Siegelbaum 1996
). The K+/Na+ permeability ratio of 0.9 falls within the range of previously reported values (Frings et al. 1992
). Pharmacological properties of the zebrafish olfactory CNG channel are also similar to other olfactory CNG channels. Four of five drugs previously used to block vertebrate olfactory CNG channels were effective blockers of the zebrafish olfactory CNG channel. Low micromolar concentrations of the calcium/calmodulin antagonists W-7 and trifluoperazine blocked the zebrafish (current report) and bullfrog (Kleene 1994
) olfactory CNG channels. As reported for other olfactory CNG channels (Zagottaand Siegelbaum 1996), relatively high concentrations of(+)-cis-diltiazem (1 mM) were required to partially block the zebrafish olfactory CNG channels. Although shown to be a relatively potent blocker of the olfactory CNG current in the tiger salamander (Leinders-Zufall and Zufall 1995
), LY-83583 (10-80 µM) did not block the zebrafish olfactory CNG channel from either the intracellular or extracellular surface of the membrane.
Multiple odor-activated transduction cascades have been confirmed in several species (Ache and Zhainazarov 1995
; Dionne and Dubin 1994
) and are indicated in the channel catfish (Miyamoto et al. 1992
). The issue of whether multiple odor-activated transduction cascades are present in zebrafish has yet to be adequately resolved. If amino acid and bile salt sensitivity is segregated among the ciliated and microvillar receptor cells in zebrafish like pheromone and amino acid sensitivity appears to be in goldfish (Zippel et al. 1997
), then differences in CNG channel microenvironment in these cell type might differentially affect the blocking efficacy of these drugs. On the surface, however, the selective reduction of amino acid-evoked responses, but not of bile salt-evoked responses, by both ruthenium red (at lower concentrations) and neomycin suggests the presence of multiple transduction cascades. Because concentrations of either drug significantly attenuating amino acid-evoked responses had very little affect on the CNG current, it is possible that an as-yet unidentified component involved in the transduction of amino acid odorants by zebrafish is affected primarily by these drugs. For example, at low ruthenium red concentrations the block of amino acid-evoked responses might be due to drug interactions with an anionic binding site present on amino acid receptors but not on bile salt receptors. At higher concentrations, ruthenium red might eliminate all odor-evoked responses through its action on the CNG current. Alternately, the block of amino acid-evoked responses observed at lower drug concentrations might be due to an action of these drugs on an independent transduction cascade, perhaps mediated by PLC and an IP3-gated current. Our failure to identify an IP3-activated current may have been due to any number of factors, including recording from the soma rather than cilia or an inappropriate calcium concentration. Because bile salt-evoked EOG responses and CNG currents are both blocked 10 µM ruthenium red but only slightly affected by 50 µM neomycin, it is perhaps likely that bile salts activate a CNG transduction cascade.
 |
ACKNOWLEDGEMENTS |
We thank D. Derbidge, M. Pearson, and A. Reed for assistance with electro-olfactogram recordings. We thank Dr. Mary Lucero for reviewing an earlier version of the manuscript.
This work was supported by National Institute of Deafness and Other Communications Disorders Grant DC-01418.
 |
FOOTNOTES |
Address for reprint requests: W. C. Michel, Dept. of Physiology, University of Utah School of Medicine, 410 Chipeta Way, Salt Lake City, UT 84108.
Received 9 September 1997; accepted in final form 20 November 1997 .
 |
REFERENCES |
-
ACHE, B. W.,
ZHAINAZAROV, A.
Dual second-messenger pathways in olfactory transduction.
Curr. Opin. Neurobiol.
5: 461-466, 1995.[Medline]
-
ALGRANATI, F. D.,
PERLMUTTER, A.
Attraction of zebrafish, Brachydanio rerio, to isolated and partially purified chromatographic fractions.
Environ. Biol. Fish
6: 31-38, 1981.
-
AMANN, R.,
MAGGI, C. A.
Ruthenium red as a capsaicin antagonist.
Life Sci.
49: 849-856, 1991.[Medline]
-
BAIER, H.,
KORSCHING, S.
Olfactory glomeruli in the zebrafish form an invariant pattern and are identifiable across animals.
J. Neurosci.
14: 219-230, 1994.[Abstract]
-
BAIER, H.,
ROTTER, S.,
KORSCHING, S.
Connectional topography in the zebrafish olfactory system: random positions but regular spacing of sensory neurons projecting to an individual glomerulus.
Proc. Natl. Acad. Sci. USA
91: 11646-11650, 1994.[Abstract/Free Full Text]
-
BARTH, A. L.,
JUSTICE, N. J.,
NGAI, J.
Asynchronous onset of odorant receptor expression in the developing zebrafish olfactory system.
Neuron
16: 23-34, 1996.[Medline]
-
BLOOM, H. D.,
PERLMUTTER, A. A
sexual aggregating pheromone system in the zebrafish, Brachydanio rerio (Hamilton-Buchanan).
J. Exp. Zool.
199: 215-226, 1977.[Medline]
-
BOYLE, A. G.,
PARK, Y. S.,
HUQUE, T.,
BRUCH, R. C.
Properties of phospholipase C in isolated olfactory cilia from the channel catfish (Ictalurus punctatus).
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
88: 767-775, 1987.
-
BRUCH, R. C.
Phosphoinositide second messengers in olfaction.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
113: 451-459, 1996.[Medline]
-
BRUCH, R. C.,
TEETER, J. H.
Cyclic AMP links amino acid chemoreceptors to ion channels in olfactory cilia.
Chem. Senses
15: 419-430, 1990.
-
BUCK, L. B.
Information coding in the vertebrate olfactory system.
Annu. Rev. Neurosci.
19: 517-544, 1996.[Medline]
-
BYRD, C. A.,
BRUNJES, P. C.
Organization of the olfactory system in the adult zebrafish: histological, immunohistochemical, and quantitative analysis.
J. Comp. Neurol.
358: 247-259, 1995.[Medline]
-
BYRD, C. A.,
JONES, J. T.,
QUATTRO, J. M.,
ROGERS, M. E.,
BRUNJES, P. C.,
VOGT, R. G.
Ontogeny of odorant receptor gene expression in zebrafish, Danio rerio. J.
Neurobiol.
29: 445-458, 1996.
-
DILL, L. M.
The escape response of the zebra danio (Brachydanio rerio). II. The effect of experience.
Anim. Behav.
22: 723-730, 1974.
-
DIONNE, V. E.,
DUBIN, A. E.
Transduction diversity in olfaction.
J. Exp. Biol.
194: 1-21, 1994.[Abstract/Free Full Text]
-
DRAY, A.,
FORBES, C. A.,
BURGESS, G. M.
Ruthenium red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurones as well as the activation of peripheral nociceptors in vitro.
Neurosci. Lett.
110: 52-59, 1990.[Medline]
-
FADOOL, D. A.,
ACHE, B. W.
Plasma membrane inositol 1,4,5-trisphosphate-activated channels mediate signal transduction in lobster olfactory receptor neurons.
Neuron
9: 907-918, 1992.[Medline]
-
FRIEDRICH, R. W.,
KORSCHING, S. I.
Combinatorial and chemotopic odorant coding in the zebrafish olfactory bulb visualized by optical imaging.
Neuron
18: 737-752, 1997.[Medline]
-
FRINGS, S.,
LYNCH, J. W.,
LINDEMANN, B.
Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.
J. Gen. Physiol.
100: 45-67, 1992.[Abstract]
-
GOULDING, E. H.,
NGAI, J.,
KRAMER, R. H.,
COLICOS, S.,
AXEL, R.,
SIEGELBAUM, S. A.,
CHESS, A.
Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.
Neuron
8: 45-58, 1992.[Medline]
-
HAMILTON, M. G.,
LUNDY, P. M.
Effect of ruthenium red on voltage-sensitive Ca2+ channels.
J. Pharmacol. Exp. Ther.
273: 940-947, 1995.[Abstract]
-
HANSEN, A.,
ZEISKE, E.
Development of the olfactory organ in the zebrafish, Brachydanio rerio. J.
Comp. Neurol.
333: 289-300, 1993.[Medline]
-
HAR LO, Y.,
BRADLEY, T. M.,
RHOADS, D. E.
Stimulation of Ca2+-regulated olfactory phospholipase C by amino acids.
Biochemistry
32: 12358-12362, 1993.[Medline]
-
HARA, T. J.
In: Fish Chemoreception.,
. Amsterdam: Elsevier, 1992
-
HAWS, C. M.,
WINEGAR, B. D.,
LANSMAN, J. B.
Block of single L-type Ca2+ channels in skeletal muscle fibers by aminoglycoside antibiotics.
J. Gen. Physiol.
107: 421-432, 1996.[Abstract]
-
HONDA, E.,
TEETER, J. H.,
RESTREPO, D.
InsP3-gated ion channels in rat olfactory cilia membrane.
Brain Res.
703: 79-85, 1995.[Medline]
-
HUQUE, T.,
BRUCH, R. C.
Odorant-and guanine nucleotide-stimulated phosphoinositide turnover in olfactory cilia.
Biochem. Biophys. Res. Commun.
137: 36-42, 1986.[Medline]
-
KLEENE, S. J.
Inhibition of olfactory cyclic nucleotide-activated current by calmodulin antagonists.
Br. J. Pharmacol.
111: 469-472, 1994.[Abstract]
-
LEINDERS-ZUFALL, T.,
ZUFALL, F.
Block of cyclic nucleotide-gated channels in salamander olfactory receptor neurons by the guanylyl cyclase inhibitor LY83583.
J. Neurophysiol.
74: 2759-2762, 1995.[Abstract/Free Full Text]
-
LO, Y. H.,
BELLIS, S. L.,
CHENG, L.-J.,
PANG, J.,
BRADLEY, T. M.,
RHOADS, D. E.
Signal transduction for taurocholic acid in the olfactory system of Atlantic salmon.
Chem. Senses
19: 371-380, 1994.[Abstract]
-
MICHEL, W. C.,
DERBIDGE, D. S.
Functional characterization of amino acid and bile salt receptors in the olfactory system of the zebrafish, Danio rerio.
Brain Res.
764: 179-187, 1997.[Medline]
-
MICHEL, W. C.,
LUBOMUDROV, L. M.
Specificity and sensitivity of the olfactory organ of the zebrafish, Danio rerio.
J. Comp. Physiol. [A]
177: 191-199, 1995.[Medline]
-
MIYAMOTO, T.,
RESTREPO, D.,
CRAGOE, E. J. JR,
TEETER, J. H.
IP3- and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish.
J. Membr. Biol.
127: 173-183, 1992.[Medline]
-
NAKAMURA, T.,
GOLD, G. H. A
cyclic nucleotide-gated conductance in olfactory receptor cilia.
Nature
325: 442-444, 1987.[Medline]
-
OKADA, Y.,
TEETER, J. H.,
RESTREPO, D.
Inositol 1,4,5-trisphosphate-gated conductance in isolated rat olfactory neurons.
J. Neurophysiol.
71: 595-602, 1994.[Abstract/Free Full Text]
-
RESTREPO, D., BOEKHOFF, I., AND BREER, H. Rapid kinetic measurements of second messenger formation in olfactory cilia from channel catfish. Am. J. Physiol. 264 (Cell Physiol. 33): C906-C911, 1993.
-
RESTREPO, D.,
MIYAMOTO, T.,
BRYANT, B. P.,
TEETER, J. H.
Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish.
Science
249: 1166-1168, 1990.[Medline]
-
RESTREPO, D.,
TEETER, J. H.,
HONDA, E.,
BOYLE, A. G.,
MARECEK, J. F.,
PRESTWICH, G. D.,
KALINOSKI, D.L.
Evidence for an InsP3-gated channel protein in isolated rat olfactory cilia.
Am. J. Physiol.
263: C667-C673, 1992.[Abstract/Free Full Text]
-
RESTREPO, D.,
TEETER, J. H.,
SCHILD, D.
Second messenger signaling in olfactory transduction.
J. Neurobiol.
30: 37-48, 1996.[Medline]
-
SLIVKA, S. R.,
INSEL, P. A.
Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells.
J. Biol. Chem.
263: 14640-14647, 1988.[Abstract/Free Full Text]
-
SPATH, M.,
SCHWEICKERT, W.
The effect of metacaine (MS-222) on the activity of the efferent and afferent nerves in the teleost lateral-line system.
Naunyn-Schmiedeberg's Arch. Pharmacol.
297: 9-16, 1977.[Medline]
-
STEELE, C. W.,
OWENS, D. W.,
SCARFE, A. D.
Attraction of zebrafish Brachydanio-rerio to alanine and its suppression by copper.
J. Fish Biol.
36: 341-352, 1990.
-
STEELE, C. W.,
SCARFE, A. D.,
OWENS, D. W.
Effects of group size on the responsiveness of zebrafish Brachydanio-rerio Hamilton Buchanan to alanine a chemical attractant.
J. Fish Biol.
38: 553-564, 1991.
-
TAREILUS, E.,
NOÉ, J.,
BREER, H.
Calcium signals in olfactory neurons.
Biochim. Biophys. Acta Mol. Cell Res.
1269: 129-138, 1995.[Medline]
-
VAN DEN HURK, R.,
LAMBERT, J.G.D.
Ovarian steroid glucuronides function as sex pheromones for male zebrafish, Brachydanio rerio.
Can. J. Zool.
61: 2381-2387, 1983.
-
VOEKLER, D.,
SMEJTEK, P.
Adsorption of ruthenium red to phosholipid membranes.
Biophys. J.
70: 818-830, 1996.[Abstract]
-
VOGT, R. G.,
LINDSAY, S. M.,
BYRD, C. A.,
SUN, M.
Spatial patterns of olfactory neurons expressing specific odor receptor genes in 48-hour-old embryos of zebrafish Danio rerio. J.
Exp. Biol.
200: 433-443, 1997.
-
WETH, F.,
NADLER, W.,
KORSCHING, S.
Nested expression domains for odorant receptors in zebrafish olfactory epithelium.
Proc. Natl. Acad. Sci. USA
93: 13321-13326, 1996.[Abstract/Free Full Text]
-
WINEGAR, B. D.,
HAWS, C. M.,
LANSMAN, J. B.
Subconductance block of single mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics.
J. Gen. Physiol.
107: 433-443, 1996.[Abstract]
-
ZAGOTTA, W. N.,
SIEGELBAUM, S. A.
Structure and function of cyclic nucleotide-gated channels.
Annu. Rev. Neurosci.
19: 235-263, 1996.[Medline]
-
ZIPPEL, H. P.,
SORENSEN, P. W.,
HANSEN, A.
High correlation between microvillous olfactory receptor cell abundance and sensitivity to pheromones in olfactory nerve-sectioned goldfish.
J. Comp. Physiol. [A]
180: 39-52, 1997.
-
ZUFALL, F.,
FIRESTEIN, S.
Divalent cations block the cyclic nucleotide-gated channel of olfactory receptor neurons.
J. Neurophysiol.
69: 1758-1768, 1993.[Abstract/Free Full Text]
-
ZUFALL, F.,
FIRESTEIN, S.,
SHEPHERD, G. M.
Analysis of single cyclic nucleotide-gated channels in olfactory receptor cells.
J. Neurosci.
11: 3573-3580, 1991.[Abstract]
-
ZUFALL, F.,
FIRESTEIN, S.,
SHEPHERD, G. M.
Cyclic nucleotide-gated ion channels and sensory transduction in olfactory receptor neurons.
Annu. Rev. Biophys. Biomol. Struct.
23: 577-607, 1994.[Medline]