Heterogeneous distribution of taste cells in facial and vagal nerve-innervated taste buds.

Input from the three gustatory nerves of vertebrates is used to evaluate the nutritional quality of food. In some species, these cranial nerves are modified to accomplish additional specific functions. For example, the facial nerve innervated taste buds distributed over the body surface of catfish aid food search. Physiological studies indicate that this extra-oral taste pathway is more sensitive to amino acids than either the glossopharyngeal or vagal systems of the oral cavity. The current investigation seeks to determine if differences in taste cell subtypes might contribute to the observed differences in sensitivity. The distributions of five low molecular weight metabolites, L-alanine, L-aspartate, L-glutamate, GABA, taurine and the tripeptide glutathione, were examined in 2118 individual taste cells innervated by either the facial or vagal nerve of the channel catfish, Ictalurus punctatus. The metabolite profiles of these cells were determined immunocytochemically and subjected to a k-means clustering algorithm. Fifteen cell classes with quantitatively different patterns of metabolite co-localization were identified. All but one small class of two cells were found in both facial and vagal nerve-innervated taste buds. Four classes (9% of the total cells) had high, two classes (17%) had intermediate and the remaining nine classes (74%) had low levels of GABA immunoreactivity. While the functional significance of differences in metabolite profile remains to be determined, taste cell classes were not uniformly distributed across vagal and facial nerve innervated taste buds and may provide an anatomical basis for previously reported differences in gustatory sensitivity.

Functional units of a compound nose: aesthetasc sensilla house similar populations of olfactory receptor neurons on the crustacean antennule.

The lateral flagellum of the antennule of the spiny lobster Panulirus argus houses more than 1,000 morphologically similar olfactory sensilla, called aesthetascs. By using a high-resolution activity labeling technique that depends on entry of agmatine into olfactory receptor neurons (ORNs) through cation channels during odor stimulation, we examined the distribution of different functional types of ORNs within and across mature aesthetascs. A significant number of ORNs in mature aesthetascs are labeled with agmatine during stimulation by single odorants, including adenosine-5'-monophosphate, ammonium chloride, cysteine, glycine, proline, and taurine. The percentage of ORNs per aesthetasc that was agmatine labeled during odor stimulation averaged 0.5-1.6% for single compounds and 4.6% for a 33-component mimic of oyster tissue. For most antennules and antennular regions studied, the percentage of agmatine-labeled ORNs by stimulation with single or complex odorants was statistically homogeneous across most or all aesthetascs. The extent of heterogeneity among mature aesthetascs was correlated with their age: extensive heterogeneity was observed only in the distal part of the flagellum containing the oldest aesthetascs and their ORNs. Thus, it appears that over most of the length of the aesthetasc-bearing region of the lateral flagellum, different and distinct functional types of aesthetascs do not exist. Rather, aesthetascs appear to be repetitive morphological and functional units in olfactory coding. However, because odor sensitivity of ORNs can change with the age of an aesthetasc, some development-related functional heterogeneity exists among aesthetascs.

Pharmacological characterization of ionotropic glutamate receptors in the zebrafish olfactory bulb.

The distribution of N-methyl-D-aspartate- (NMDA) and kainic acid- (KA) sensitive ionotropic glutamate receptors (iGluR) in the zebrafish olfactory bulb was assessed using an activity-dependent labeling method. Olfactory bulbs were incubated with an ion channel permeant probe, agmatine (AGB), and iGluR agonists in vitro, and the labeled neurons containing AGB were visualized immunocytochemically. Preparations exposed to 250 microM KA in the presence of a NMDA receptor antagonist (D-2-amino-5-phosphono-valeric acid) and an alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist (sym 2206), revealed KA receptor-mediated labeling of approximately 60-70% of mitral cells, juxtaglomerular cells, tyrosine hydroxylase-positive cells and granule cells. A higher proportion of ventral olfactory bulb neurons were KA-sensitive. Application of 333 microM NMDA in the presence of an AMPA/KA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) resulted in NMDA receptor-mediated labeling of almost all neurons. The concentrations eliciting 50% of the maximal response (effective concentration: EC(50)s) for NMDA-stimulated labeling of different cell types were not significantly different and ranged from 148 microM to 162 microM. These results suggest that while NMDA receptors with similar binding affinities are widely distributed in the neurons of the zebrafish olfactory bulb, KA receptors are heterogeneously expressed among these cells and may serve unique roles in different regions of the olfactory bulb.

High-resolution functional labeling of vertebrate and invertebrate olfactory receptor neurons using agmatine, a channel-permeant cation.

Methods are described for odor-stimulated labeling of olfactory receptor neurons (ORNs) of the freshwater zebrafish Danio rerio and the marine spiny lobster Panulirus argus. Permeation of a cationic molecule, 1-amino-4-guanidobutane ( = agmatine, AGB), through ion channels following odor stimulation, and its detection by an anti-AGB antibody, allow labeling of odor-stimulated ORNs. Parameters adjusted to optimize activity-dependent labeling included labeling medium ionic composition, stimulation times, and AGB concentration. For lobsters, 7% of ORNs were labeled by a complex odor, oyster mixture, under optimal conditions, which was stimulation for 5 s per min for 60 min with 20 mM AGB in artificial seawater with reduced sodium and calcium concentrations. AGB was a weak odorant for lobsters; it elicited only a small electrophysiological response from ORNs and labeled < 1% of the ORNs during stimulation with AGB in the absence of odors. For the zebrafish, stimulation for 10 s per min for 10 min with 5 mM AGB plus odorant (L-glutamine) in fish Ringer's solution was the optimal labeling condition, resulting in labeling of 17% of the olfactory epithelial area. Approximately 6% of the olfactory epithelium was labeled during stimulation with a control stimulus, AGB alone. This labeling by AGB alone suggests it is an olfactory stimulus for zebrafish; a conclusion supported by electrophysiological recordings. We used electrophysiological assays and channel blockers to examine, for each species, potential ion channels for entry of AGB into ORNs. These results show that AGB can be used as an activity-dependent label for chemoreceptor neurons of diverse phyla living in a range of environmental conditions.

Evidence of distinct amino acid and bile salt receptors in the olfactory system of the zebrafish, Danio rerio.

The molecular receptors for amino acid and bile salt odorants of adult zebrafish, Danio rerio, were partially characterized using electro-olfactogram recording methods. Each of the 14 odorants tested interact with partially independent odorant receptor(s). Based on shared patterns of adaptation, cluster analysis identified two very dissimilar groups of odorants. The first group comprised the 8 amino acids with subgroups of basic, acidic and neutral amino acids. The second group comprised the 6 bile salts with subgroups of non-conjugated, taurine- and glycine-conjugated bile salts. Comparison of the general patterns of partial adaptation of amino acid and bile salt odorants suggests fundamental differences in their odorant receptors. Presumably, the differences in the extent of partial adaptation are due to differences in the transduction cascades activated or the distribution of odorant receptors on individual olfactory receptor neurons.

Sustained primary culture of lobster (Panulirus argus) olfactory receptor neurons.

Appropriate conditions were developed for primary sustained culture of olfactory neurons of the spiny lobster Panulirus argus. Neurons were cultured in a modified Liebowitz L15 media supplemented with Panulirus salts, basic minimal essential (BME) vitamins, L-glutamine, low dextrose, and either fetal calf serum (FCS) or lobster haemolymph. Neurite outgrowth and cell viability was strongly affected by choice of adherent substratum, presence of serum, and length of animal captivity. Neither nerve growth factor 7s (NGF-7s), HEPES, nor preconditioned media from the target organ, the olfactory lobe, had any gross effect on either longevity or neurite outgrowth. Five morphologically distinct neuronal cell types (8-16 mum soma diameter) could be defined based on their number and type of processes. All of these cells were electrically excitable (N = 50), and many (56%) produced either inward or outward currents in response to stimulation with single odors. The proportion of cells responding to odors increased (80%) when 10 cells were sequentially presented with a series of 3-5 odors. The finding that cultured cells maintain responsiveness to odors yet are morphologically more compact than their counterparts in situ, argues for the prospect of using these dissociated cultured olfactory receptor neurons to study signal transduction in olfaction.

Isolation and characterization of the laure olfactory behavioral mutant in the zebrafish, Danio rerio.

To initiate a genetic analysis of olfactory development and function in the zebrafish, Danio rerio, we developed a behavioral genetic screen for mutations affecting the olfactory sensory system. First, we characterized olfactory responses of wild-type zebrafish to various odors. We found that 3-day-old juvenile zebrafish reacted to the amino acid L-cysteine with an aversive behavioral response. We isolated one mutant, laure (lre), which showed no aversive behavioral response to L-cysteine at 3 days of development, and carried out a preliminary characterization of this mutant's defects. We found that lre mutant fish were also defective in their response to L-serine and L-alanine, but not to taurocholic acid, as young adults. In addition, lre mutant fish had significantly fewer primary olfactory sensory neurons than normal, and the axons of these neurons did not form the characteristic axon termination pattern in the developing olfactory bulb. Nevertheless, the olfactory epithelium of lre mutant fish showed normal or near normal electrophysiological responses to several odorants. Our data suggest that the behavioral defects observed in the lre mutant result from the disruption of the developing olfactory sensory neurons and their axonal connections within the olfactory bulb. The isolation of the lre mutant shows that our behavior-based screen represents a viable approach for carrying out a genetic dissection of olfactory behaviors in this vertebrate model system.

Cyclic nucleotide-gated channel activation is not required for activity-dependent labeling of zebrafish olfactory receptor neurons by amino acids.

The olfactory epithelium of fish is heterogeneous both with respect to the types of receptor cells (ORNs) present and the families of odorant receptors expressed in these cells. As a consequence of this diversity, the transduction cascade(s) activated by odorants has yet to be unambiguously established. In the current study, electrophysiological and activity-dependent labeling techniques were used to assess the role of the cyclic nucleotide-gated channel in zebrafish olfactory transduction. Both amino acid and bile salt odorants elicited robust electrophysiological responses, however, activity-dependent labeling of ORNs could be stimulated only by the amino acid odorants. An adenylate cyclase (AC) activator (forskolin) and a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) also elicited robust electrophysiological responses; generally larger than the responses elicited by either the amino acid or bile salt odorants. However, neither forskolin alone or a mixture of forskolin and IBMX stimulated activity-dependent labeling. Bathing the olfactory epithelium with forskolin, which presumably increased the intracellular concentration of cAMP, reduced the responses to bile salt odorants to a significantly greater extent than amino acid odorants. Collectively, these findings suggest that the transduction of amino acid input does not rely primarily on cyclic nucleotide-gated (CNG) channel activation and that CNG channel activation may be required for the transduction of bile salt input.

Cyclic nucleotides mediate an odor-evoked potassium conductance in lobster olfactory receptor cells.

Odors activate at least two distinct transduction pathways in lobster olfactory receptor cells that, respectively, excite and inhibit the cell. Data presented suggest that odors selectively activate the inhibitory conductance through the second messenger cAMP. Not all cells support both odor-evoked excitatory and inhibitory conductances; in the current investigation, about 50% of the cells tested were inhibited by odors. In the majority of cells that, as a group, support an inhibitory response to odor stimulation, activation of adenylate cyclase with forskolin or inhibition of phosphodiesterase activity with 3-isobutyl-1-methylxanthine (IBMX) elicits an outward current with a time course similar to that of odor-evoked outward currents. The membrane-permeant cyclic nucleotide analogs 8-Br-cAMP and 8-Br-cGMP have a similar effect. Forskolin and IBMX enhance the magnitude of odor-evoked outward currents when the drug and the odor are copresented to the cell. In contrast, these same drugs have little or no effect on cells that, as a group, fail to support an inhibitory response to odor stimulation. This study provides the first direct evidence implicating cAMP in olfactory transduction in an invertebrate and contrasts with similar studies in vertebrates that have implicated cAMP as a second messenger mediating excitation.

Specificity and sensitivity of the olfactory organ of the zebrafish, Danio rerio.

1. The specificity and sensitivity of the olfactory organ of adult zebrafish, Danio rerio, to selected amino acid, bile acid, and steroid odorants were characterized using the electro-olfactogram recording technique. The olfactory organ was responsive to 28 of the 29 odorants tested. 2. All of the 100 microM amino acid and bile acid stimulants elicited a negative-going response that was significantly greater than the response to the artificial fresh-water control. The general pattern of relative stimulatory effectiveness established for the amino acid stimuli was neutral amino acids > basic amino acids > acidic amino acids > imino acids. The general pattern of relative stimulatory effectiveness of 100 microM bile acid stimuli was taurine-conjugated bile acids > glycine-conjugated bile acids approximately non-conjugated bile acids. The responses to the most stimulatory bile acid odorants were up to 40% larger than the responses to the most stimulatory amino acid odorants. 3. The response threshold for cysteine and taurocholic acid, the most stimulatory of the amino acid and bile acid stimuli tested, was approximately 10(-8) M. Females are significantly more sensitive to these odorants than males.

Odor-evoked inhibition in primary olfactory receptor neurons.

Odors can inhibit as well as excite lobster olfactory receptor cells. Inhibitory components of an odor mixture act within the normal, first 500 ms odor sampling interval of the animal to reduce the peak magnitude and increase the latency of the net excitatory receptor potential in a concentration-dependent manner. The intracellular effects are reflected in the propagated output of the cell. The results argue that inhibitory odor input is functional in olfaction by potentially serving to increase the diversity of the neuronal patterns that are thought to be the basis of odor discrimination.

Mechanisms of afterhyperpolarization in lobster olfactory receptor neurons.

In lobster olfactory receptor neurons (ORNs), depolarizing responses to odorants and current injection are accompanied by the development of an afterhyperpolarization (AHP) that likely contributes to spike-frequency adaptation and that persists for several seconds after termination of the response. A portion of the AHP can be blocked by extracellular application of 5 mM CsCl. At this concentration, CsCl specifically blocks the hyperpolarization-activated cation current (Ih) in lobster ORNs. This current is likely to be active at rest, where it provides a constant, depolarizing influence. Further depolarization deactivates Ih, thus allowing the cell to be briefly hyperpolarized when that depolarizing influence is removed, thus generating an AHP. Reactivation of Ih would terminate the AHP. The component of the AHP that could not be blocked by Cs+ (the Cs(+)-insensitive AHP) was accompanied by decreased input resistance, suggesting that this component is generated by increased conductance to an ion with an equilibrium potential more negative than the resting potential. The Cs(+)-insensitive AHP in current clamp and the underlying current in voltage clamp displayed a reversal potential of approximately -75 mV. Both EK and ECl are predicted to be in this range. Similar results were obtained with the use of a high Cl- pipette solution, although that shifted ECl from -72 mV to -13 mV. However, when EK was shifted to more positive or negative values, the reversal potential also shifted accordingly. A role for the Ca(2+)-mediated K+ current in generating the Cs(+)-independent AHP was explored by testing cells in current and voltage clamp while blocking IK(Ca) with Cs+/Co(2+)-saline. In some cells, the Cs(+)-independent AHP and its underlying current could be completely and reversibly blocked by Cs+/Co2+ saline, whereas in other cells some fraction of it remained. This indicates that the Cs(+)-independent AHP results from two K+ currents, one that requires an influx of extracellular Ca2+ and one that does not. Collectively, these findings indicate that AHPs result from three phenomena that occur when lobster ORNs are depolarized: 1) inactivation of the hyperpolarization-activated cation current, 2) activation of a Ca(2+)-mediated K+ current, and 3) activation of a K+ current that does not require influx of extracellular Ca2+. Roles of these processes in modulating the output of lobster ORNs are discussed.

A hyperpolarization-activated cation conductance in lobster olfactory receptor neurons.

1. The current underlying inward rectification in lobster olfactory receptor neurons was investigated with the use of whole-cell patch-clamp techniques. Inward rectification could most likely result from an inwardly rectifying potassium conductance or a hyperpolarization-activated cation conductance. To distinguish between these possibilities, the current underlying inward rectification was examined with respect to its sensitivity to extracellular Cs+ and Ba2+, time course of activation, and reversal potential. 2. In current clamp, injection of negative current led to a hyperpolarization followed by a partial return (sag) toward the initial holding potential. The rate and magnitude of the sag depended on the magnitude of the hyperpolarizing current with larger currents leading to larger, faster depolarizing sags. In voltage clamp, hyperpolarizing steps elicited a slowly activating, noninactivating inward current clamp. Both the sag and the slow inward current were blocked reversibly by extracellular application of 5 mM CsCl but were unaffected by 2 mM BaCl2. 3. The rate of inward current activation was best approximated by a single exponential function with time constants that were voltage dependent, ranging from 7.8 s at -69 mV to 248 ms at -114 mV. 4. Cells normally exhibited an average input resistance of 0.99 G omega over the range of -69 to -114 mV. With the hyperpolarization-activated inward current blocked by 5 mM CsCl, the average input resistance increased to 2.12 G omega over the same range. 5. Analysis of tail currents revealed that the average predicted reversal potential of the hyperpolarization-activated inward current was 1.7 mV and was not affected significantly by a shift in ECl.(ABSTRACT TRUNCATED AT 400 WORDS)

Drugs affecting phospholipase C-mediated signal transduction block the olfactory cyclic nucleotide-gated current of adult zebrafish.

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 microM), or a PLC inhibitor, neomycin (50 microM), 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.

Physiological evidence for the discrimination of L-arginine from structural analogues by the zebrafish olfactory system.

Although it is generally assumed that fish are capable of discriminating amino acid odorants on the basis of differences in side-chain structure, less is known about their ability to discriminate amino acids with modifications to alpha-carboxyl and alpha-amino groups. In this study, the ability of the zebrafish olfactory system to detect and presumably discriminate analogues of the basic amino acid Arg was assessed, by using cross-adaptation and activity-dependent labeling techniques. Electrophysiological recordings established that esterification (L-arginine methyl ester; AME) or deletion (agmatine or amino-4-guanidobutane; AGB) of the alpha-carboxyl group yielded odorants more potent than Arg, whereas deletion of the alpha-amino group (L-argininic acid; AA) yielded a less potent analogue. In cross-adaptation experiments, no test-competitor odorant combination yielded complete cross-adaptation, suggesting the detection of these Arg analogues by multiple odorant receptors (ORs) with partially nonoverlapping specificities. Activity-dependent immunocytochemical labeling of olfactory receptor neurons supported this conclusion. AGB, an ion-channel-permeant probe (and odorant), labeled 4.9 +/- 0.4% (n = 24) of sensory epithelium, whereas the addition of Arg, 1-ethylguanidine sulfate, L-alpha-amino-beta-guanidinopropionate, or AME to AGB resulted in a significant elevation of labeling (8-14%). This study provides evidence that the olfactory system has the potential to discriminate among amino acid odorants with modified alpha-carboxyl and alpha-amino groups.

Inhibition of lobster olfactory receptor cells by an odor-activated potassium conductance.

1. Whole cell current-clamp recordings show that odors not only depolarize but may also hyperpolarize lobster olfactory receptor cells. Odor-evoked hyperpolarizations occurred in 36% of 178 receptor cells examined. Cell-attached recordings of action potentials followed by current-clamp recordings in the same cell indicate that depolarizing and hyperpolarizing responses were associated with increases (excitation) and decreases (inhibition) in action potential frequency, respectively. Since odorants that hyperpolarized one receptor cell depolarized other cells and since individual cells may be both excited and inhibited, the inhibitory and excitatory nature of the response must be conferred by the odorant-receptor and transduction processes expressed by the receptor cell. 2. The input resistance dropped from 1.73 G omega at rest to 1.45 G omega during odor-evoked hyperpolarization, and the membrane time constant correspondingly decreased from 114 to 61 ms. The increased conductance persisted throughout the stimulation period (5 s). 3. Shifting the K+ reversal to a more negative potential by lowering the [K+]o from 14 to 2.8 mM increased the magnitude of hyperpolarization. The hyperpolarization could be reversibly blocked by dendritic treatment with 5-10 mM 4-aminopyridine (4-AP) or 10 mM cesium ion, but not by 10 mM tetraethylammonium (TEA). 4. Substituting 80% of the [Cl-]o with NO3- increased the amplitude of the hyperpolarization. Based on a calculated equilibrium potential of -32 mV for chloride, an increase in chloride conductance in a low [Cl-]o environment should have decreased the magnitude of the response. Presumably the change in [Cl-]o acts through the dendritic steady-state chloride conductance to shift the membrane potential further from the reversal potential for K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Odor sensitivity of cultured lobster olfactory receptor neurons is not dependent on process formation.

Cultured lobster olfactory receptor neurons (ORNs) were surveyed for their odor sensitivity with whole-cell, voltage-clamp recording. The nature of the adequate stimuli, the degree of tuning (response spectra) of the cells, the threshold of sensitivity and the dual polarity of the odor-evoked currents are consistent with chemosensitivity in the cultured ORNs being olfactory. The ability of odors to evoke currents in cultured ORNs that lack processes suggests that lobster ORNs can be induced in vitro to insert all the elements of the transduction cascade in the soma, including those that might normally be confined to processes. This should greatly facilitate analysis of olfactory transduction in these cells.

Chemical induction of feeding in California spiny lobster,Panulirus interruptus (Randall): : Responses to molecular weight fractions of abalone.

Molecular weight fractions of abalone muscle were tested for the ability to induce appetitive feeding and locomotor behavior in the spiny lobster,Panulirus interruptus. Fractions of <1000, 1000-10,000 and >10,000 daltons were isolated by ultrafiltrations and gel chromatography from a seawater extract of abalone muscle. The two lower-molecular-weight fractions (<1000, 1000-10,000) were the least stimulatory of the three fractions tested, and both were ineffective as feeding stimulants. Solutions combining any two of the three isolated fractions produced behavioral activity equal to that caused by whole extract; thus, no single fraction was essential to the stimulatory capacity of abalone. The >1000-dalton fraction was also highly stimulatory, meaning that large and not small molecules were essential in initiating feeding. Finally, a 75% ethanol-insoluble component of the <10,000 fraction was effective, while the ethanol-soluble portion was not. Since the insoluble material consisted predominantly of peptides and polypeptides, it is probable that these molecules act as principal stimulants in abalone muscle.

Single odors differentially stimulate dual second messenger pathways in lobster olfactory receptor cells.

Quench-flow measurements are used to determine the subsecond kinetics of odor-induced changes in second messenger concentrations in lobster olfactory receptor neurons. Individual odors transiently and differentially increase the production of both adenosine cAMP and inositol 1,4,5-trisphosphate (IP3) within 50 msec of odor stimulation. The ability of two different odors to stimulate cAMP and IP3 correlates with the odors' ability to excite and inhibit receptor cells physiologically. These results strengthen the proposition, heretofore based largely on evidence from cultured cells, that dual second messenger pathways mediate excitatory and inhibitory input to lobster olfactory receptor cells.

Voltage- and Ca(2+)-gated currents in zebrafish olfactory receptor neurons.

Voltage- and Ca(2+)-gated currents were recorded from isolated olfactory receptor neurons (ORNs) of the zebrafish Danio rerio using the whole-cell voltage-clamp technique. Zebrafish ORNs had an average capacitance of 0.66 pF and an average apparent input resistance of 8.0 G omega. Depolarizing steps elicited transient inward currents followed by outward currents with transient and sustained components. The transient inward current (INa) was sensitive to 1 mumol l-1 tetrodotoxin, activated between -74mV and -64mV, and reached half-maximal conductance at -28 mV. Its peak amplitude averaged -101pA. Steady-state inactivation of INa was half-maximal at an average test potential of -78mV and recovery from inactivation proceeded with two time constants averaging 23 ms and 532 ms. A sustained, Co(2+)-sensitive current (ICa) activated between -44mV and -34mV and reached a peak amplitude averaging -9pA at -14mV. Outward currents were carried by K+, based on the reversal potentials of tail currents, and consisted of a Ca(2+)-dependent K+ current, a delayed rectifier current (IDR) and a transient K+ current (IA). The Ca(2+)-dependent K+ current (IK(Ca)) activated between -44mV and -34mV, whereas IDR and IA activated between -34mV and -24mV. In summary, zebrafish ORNs possess a complement of gated currents similar but not identical to that of ORNs from other vertebrates and which appears well suited for encoding a graded receptor potential into a train of action potentials.