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.

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.

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)

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.

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.

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.

Stereoselective detection of amino acids by lobster olfactory receptor neurons.

1. Biochemical and electrophysiological assays were used to test the hypothesis that the olfactory system of the Caribbean spiny lobster, Panulirus argus, contains populations of chemosensory receptors that are differentially sensitive to the L- and D-stereoisomers of the amino acid alanine. 2. Independent binding sites for L-alanine (dissociation constant (KD) of 6.6 microM and maximum binding (Bmax) of 16.8 fmole/microgram protein) and for D-alanine (KD of 21.6 microM and Bmax of 17.8 fmole/microgram protein) were characterized biochemically. The interaction of ligand with each binding site is rapid, reversible and saturable with respect to both time and concentration. 3. Based on a difference of at least 20% in the relative sensitivity of an olfactory receptor cell to alanine enantiomers, 44% and 34% of the 77 neurons tested were classified as L-alanine and D-alanine sensitive, respectively. The relative sensitivity to alanine enantiomers was independent of the concentration tested. Stereoselective receptors are likely for 17 of 20 other amino acids tested. 4. The congruence of biochemical and electrophysiological results leads to the conclusion that the lobster's responses to D- and L-alanine are mediated by receptors specific for each stereoisomer and that the receptors are differentially distributed among receptor cells.

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.

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)

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.