Sustaining olfaction at low salinities: evidence for a paracellular route of ion movement from the hemolymph to the sensillar lymph in the olfactory sensilla of the blue crab Callinectes sapidus.

Evidence reported previously suggests that in low-salinity conditions the integrity of the olfactory dendrites of the blue crab is sustained by a diffusion-generated ionic microenvironment within the aesthetascs. Diffusion of ions from the hemolymph to the sensillar lymph is proposed to maintain this microenvironment. In this study, using lanthanum as an electron-dense marker of extracellular fluid space, we find morphological evidence for paracellular continuity between the hemolymph and the sensillar lymph. Lanthanum penetrates extracellular fluid spaces within the aesthetascs when antennules are either perfused or bathed externally with solutions containing lanthanum nitrate. This was found in both freshwater- and seawater-acclimated animals. Evidence for ion diffusion from the aesthetascs was obtained using self-referencing, ion-selective microelectrodes. Both Ca2+ and K+ exhibit outwardly directed flux gradients associated with the aesthetasc tuft in low-salinity conditions. These findings are consistent with the concept that ion diffusion from the hemolymph to the sensillar lymph generates an ionic/osmotic microenvironment within the aesthetascs at low salinities.

Sustaining olfaction at low salinities: mapping ion flux associated with the olfactory sensilla of the blue crab Callinectes sapidus.

To test the hypothesis of a diffusion-generated, ionic/osmotic microenvironment within the olfactory sensilla (aesthetascs), flux gradients of Ca(2+) and K(+) associated with the external surfaces of these sensilla were spatially mapped using self-referencing, ion-selective microelectrodes. Blue crabs (Callinectes sapidus) acclimated to low-salinity conditions (15% sea water and fresh water) showed a net efflux of ions from the aesthetascs. The region of maximum flux associated with each aesthetasc conformed to that predicted from structural data and corresponded to the permeable region of the cuticle separating the olfactory dendrites from the external environment. Estimates of net flux from the entire tuft of aesthetascs for both Ca(2+) and K(+) fell within the predicted range on the basis of comparisons with (22)Na(+) flux measured previously and assuming a passive diffusion model of ion movement from the hemolymph to the sensillar lymph and, ultimately, to the external environment. The maximum concentrations of these ions measured deep within the tuft are discussed in the light of a potential across the aesthetascs that may limit ion efflux at low salinities.

Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4, 5-trisphosphate receptor in lobster olfactory receptor neurons.

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.

Stimulants of Feeding Behavior in Fish: Analyses of Tissues of Diverse Marine Organisms.

Analyses of the free amino acids, quaternary amines, guanido compounds, nucleotides, nucleosides, and organic acids in extracts of tissues from 10 species of marine teleost fishes and 20 species of invertebrates are reported. With multidimensional scaling techniques, the relative concentrations of the above chemicals in fishes, molluscs, and crustaceans are shown to cluster into separate taxon-specific groups. The greatest differences are between the fishes and the two groups of invertebrates. Similarities are more evident between the molluscs and crustaceans where eight of the nine most abundant substances are identical: i.e., betaine, taurine, trimethylamine oxide, glycine, alanine, proline, homarine, and arginine. The major tissue components in the fishes and invertebrates are correlated with compounds previously shown to stimulate feeding behavior in 35 species of fish. Glycine and alanine are major tissue components and are also the two most frequently cited feeding stimulants in the 35 species. Molluscs and crustaceans each contain high concentrations of five of the most frequently cited stimulants (glycine, alanine, proline, arginine, and betaine); these substances all occur in much lower concentrations in fish. Some minor tissue components, such as tryptophan, phenylalanine, aspartic acid, valine, and uridine 5`-monophosphate, are, however, important feeding stimulants for some fish species. Stimulants for herbivores and carnivores are often different. Several major feeding stimulants are substances that serve as "compensatory solutes," stabilizing enzymes and structural proteins.

Sulfide as a Chemical Stimulus for Deep-Sea Hydrothermal Vent Shrimp.

Organisms dependent on deep-sea hydrothermal vents for their existence face extinction when their vents expire, unless they can establish populations on neighboring vents or on new vent sites. Propagules, including larvae and motile adults, are readily dispersed broadly by seafloor currents, but how they recognize active hydrothermal sites is problematical. Compelling evidence that vent organisms can find and colonize hydrothermal sites has been provided by a series of observations on the East Pacific Rise (1). New hydrothermal vents created there following a volcanic eruption on the seafloor in March 1991 were colonized by sessile invertebrates in less than one year. On the Mid-Atlantic Ridge, shrimp that normally cluster on sulfide surfaces have been observed to swim directly back to the surfaces when displaced from them. How do vent animals locate new or existing vents? Passive transport by currents (2) or active swimming without guidance by some physical cue is not likely to result in success (3). Chemicals present in hydrothermal fluids have been proposed as attractants. We provide the first evidence of a chemosensory response in a vent invertebrate to sulfides, which are prevalent in vent fluids and provide the energy,for chemosynthetic primary production at vents.

Modulation of behavior by biogenic amines and peptides in the blue crab, Callinectes sapidus.

Using the blue crab Callinectes sapidus as a model system, we have investigated the effects of potential neuromodulators on freely behaving animals. Of interest is the modulatory effect of a number of drugs on three rhythmic behaviors of the blue crab: courtship display (CD) of the male crab, sideways swimming and backward swimming. The drugs tested were proctolin, dopamine, octopamine, serotonin, and norepinephrine. Injection of each drug elicited a unique posture or combination of limb movements. These experiments showed two results pertinent to CD behavior: A posture identical to the CD posture was displayed after dopamine injection; and rhythmic leg waving similar to CD was evoked by proctolin. An unusual combination of flexion and extension of all limbs and movements of some limbs occurred after serotonin injection. Injection of octopamine led to a posture antagonistic to CD posture. The effects of these drugs were concentration- and time-dependent. Injection of dopamine, octopamine, or serotonin produced effects that were seasonally-dependent, and the influence of proctolin proved to be dependent on developmental stage. Quantitative analysis of leg waving movements after proctolin injection allowed for comparison of these movements to naturally-occurring behavior.

cDNA clones from the olfactory organ of the spiny lobster encode a protein related to eukaryotic glutamine synthetase.

We report here the nucleotide (nt) sequence of clones from a lobster olfactory organ cDNA library encoding a protein homologous to glutamine synthetase (GS) from eukaryotes. The cDNA for the lobster putative GS is 2045 bp in length, and includes a 5'-untranslated region 55 nt in length, a 1083-nt open reading frame, and a 907-nt 3'-untranslated region. The encoded protein shows 65, 64, and 63% identity with the reported GS sequences of chicken, human and fruit fly, respectively.

Ecto-ATPase/phosphatase activity in the olfactory sensilla of the spiny lobster, Panulirus argus: localization and characterization.

Electrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has chemoreceptors that are selectively excited by adenine nucleotides in seawater. Biochemical studies have revealed that these same nucleotides can be rapidly dephosphorylated by ectoenzymes associated with the olfactory sensilla (aesthetascs). In this study the distribution of ecto-ATPase/phosphatase activity within aesthetascs was determined cytochemically and the nature of the adenine-nucleotide dephosphorylating activity was dissected biochemically. Cytochemically, the distribution of ATP-dephosphorylating activity was similar to that shown previously for AMP and beta-glycerol phosphate; i.e., cerium phosphate reaction product was specifically localized to the transitional zone where the sensory dendrites develop cilia and branch to form the outer dendritic segments. Unlike the dephosphorylation of AMP and beta-glycerol phosphate, Mg2+ or Ca2+ was required for ecto-ATPase/phosphatase activity. Biochemical measures of both AMP- and ATP-dephosphorylating activity within aesthetascs corroborated the cytochemical evidence that these activities are localized to the transitional zone. A major portion of the AMP dephosphorylation (about 67%) derives from nonspecific alkaline phosphatase activity that is insensitive to levamisole and L-bromotetramisole. In contrast, nonspecific phosphatase activity accounted for a much smaller part of the ATP dephosphorylation (about 15%). Ectoenzymatic activity in the transitional zone may be an important means of removing excitatory/inhibitory nucleotides from this region.

Ectonucleotidase activities associated with the olfactory organ of the spiny lobster.

The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobster's olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5'-ectonucleotidase, ecto-ADPase, and ecto-ATPase activities of this olfactory system showed that each activity was characterized by Michaelis-Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 microM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5'-ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto-ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.

The role of perireceptor events in chemosensory processes.

In a review of vertebrate olfaction, Getchell et al. coined the term 'perireceptor events' to denote processes, ancillary to both receptor activation and transduction, that influence the entry, exit or residence time of odorant molecules in the receptor environment. The present review describes recent advances in our understanding of perireceptor events and shows that these processes are integral components of chemical sensing systems of organisms as diverse as bacteria, slime molds, yeast, insects, crustaceans and mammals. Moreover, it emphasizes that perireceptor processes are essential components of chemical sensing systems, rather than simply interesting adjuncts to the 'main events' of receptor activation and transduction.

The Efflux of Amino Acids from the Olfactory Organ of the Spiny Lobster: Biochemical Measurements and Physiological Effects.

The amino acids taurine and glycine are odorants that activate specific chemosensory cells in the olfactory sensilla (aesthetascs) of the spiny lobster, Panulirus argus. We show that the aesthetascs themselves contain large intracellular concentrations of taurine (≈2 mM) and glycine (≈ 85 mM); these concentrations are more than 10,000-fold greater than the response thresholds of the chemosensory cells. A net efflux of at least five amino acids occurs when the olfactory organ is immersed in amino acid-free seawater. With taurine and glycine, efflux continues until an apparent equilibrium is reached between the sensilla and the external medium; for taurine the equilibrium with seawater occurs at ≈12 to 28 nM, and for glycine at ≈100 to 500 nM. Aesthetascs may achieve these equilibria within 300 ms. Hence, even during the brief interval between consecutive flicks of the antennule, olfactory receptors are exposed to a background of odorants escaping from intracellular stores. Electrophysiological studies show that both the spontaneous and evoked activities of taurine-sensitive chemosensory cells are markedly affected by a taurine background simulating that measured in the efflux studies. Uptake systems may participate in establishing the equilibria between sensilla and seawater since (1) the net efflux of amino acids increases in sodium-free seawater; and (2) guanidinoethane sulfonate, a competitor for taurine uptake, selectively increases net taurine efflux. Effluxes from an olfactory organ may contribute noise to the chemosensory process; alternatively, background substances could contribute functionally by affecting membrane proteins.

ATP-sensitive chemoreceptors: antagonism by other nucleotides and the potential implications of ectonucleotidase activity.

As measured by extracellular single-cell recording, the responses to adenosine triphosphate (ATP) by ATP-sensitive chemoreceptors (ATP cells) on the olfactory organ of the spiny lobster are markedly suppressed by adenosine diphosphate (ADP), adenosine monophosphate (AMP) and to a lesser extent, adenosine, when each is presented in binary mixture with ATP. In the presence of ADP, the dose-response function for ATP exhibits an apparent parallel displacement to the right suggesting that this antagonism may occur via competition at the ATP receptor. Structure-activity relationships reveal that the structural requirements for antagonism by diphosphate analogs of ADP bear little relationship to the requirements for the agonistic activity of corresponding triphosphate analogs. Under Mg2+-free conditions, the desensitization of ATP cells tends to be delayed resulting in enhanced responses to ATP. Desensitization does not appear to be related to the generation of the antagonist, ADP, from ATP via ecto-ATPase activity. The results of this study suggest that the responses of ATP cells to the ATP contained in natural stimulus (odor) mixtures can be tempered by the suppressive interactions of other nucleotides in the mixtures. Furthermore, these interactions may be mitigated and/or intensified by the actions of sensillar ectonucleotidases.

Biochemistry of an olfactory purinergic system: dephosphorylation of excitatory nucleotides and uptake of adenosine.

The olfactory organ of the spiny lobster, Panulirus argus, is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of [3H]-adenosine is saturable with increasing concentration, linear with time for up to 3 h, sodium dependent, insensitive to moderate pH changes and has a Km of 7.1 microM and a Vmax of 5.2 fmol/sensillum/min (573 fmol/micrograms of protein/min). Double-label experiments show that sensilla dephosphorylate nucleotides extracellularly; 3H from adenine-labeled AMP or ATP is internalized, whereas 32P from phosphate-labeled nucleotides is not. The dephosphorylation of AMP is very rapid; 3H from AMP is internalized at the same rate as 3H from adenosine. Sensillar 5'-ectonucleotidase activity is inhibited by ADP and the ADP analog alpha, beta-methylene ADP. Collectively, these results indicate that the enzymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.

Chemoreceptors of crustaceans: similarities to receptors for neuroactive substances in internal tissues.

A description is given of crustacean chemosensory systems and the neurophysiological procedures used to study them. Their response properties and tuning characteristics are discussed. A review is then provided of specific crustacean chemoreceptors that are stimulated selectively by either purine nucleotides, taurine, glutamate, or glycine, all of which have neuroactive properties in internal tissues. Two distinctly different types of purinergic chemoreceptors occur on the antennules of the spiny lobster. P1-like chemoreceptors have a potency sequence of AMP greater than ADP greater than ATP greater than adenosine and show a strict structural requirement for the ribose phosphate moiety. P2-like chemoreceptors have a potency sequence of ATP greater than ADP greater than AMP or adenosine and show a broad sensitivity to nucleotide triphosphates with modifications in both the purine and ribose phosphate moieties. Sensilla containing the dendrites of chemosensory neurons also possess an ectonucleotidase(s) that inactivates excitatory nucleotides to yield adenosine which is subsequently internalized by a sensillar uptake system. Narrowly tuned taurinergic chemoreceptors are present on both the antennules and legs of lobsters. Although taurine itself is the most effective stimulant, the taurine analogs hypotaurine and beta-alanine are also very excitatory. Structure-activity studies indicate these chemoreceptors have marked similarities to taurine-sensitive systems in internal tissues of vertebrates. By contrast, comparative studies of glutamatergic chemoreceptors on the legs of lobsters indicate response spectra different from those of the glutamate receptors in lobster neuromuscular junctions and the three classes of excitatory amino acid receptors identified internally in vertebrates. Crustacean chemoreceptors for glycine, ecdysteroids, and pyridine are also described. The hypothesis that receptors for internal neuroactive agents may have originally evolved as external chemoreceptors of primitive aquatic organisms is discussed.

Amino acid suppression of taurine-sensitive chemosensory neurons.

Single unit recordings from chemoreceptors on the antennule of the spiny lobster revealed a population of taurine-sensitive cells whose response is suppressed when taurine is presented in mixture with certain amino acids. A synthetic mixture of 21 amino acids plus betaine, which mimics the composition of a natural food stimulus (crab muscle tissue) and itself contains taurine, totally and reversibly blocked the taurine response of this group of receptor cells. An analysis of the contribution to this suppression by the six major components (based on concentration) in the mixture revealed partial or complete inhibitory activity by five of the compounds. In a sample group of the inhibited cells, mean percent suppression of the taurine response was 99% for glycine and L-arginine, 98% for L-glutamine, 60% for L-alanine and 43% for L-proline. Both glycine and alanine in binary mixture with taurine caused a right-shift in the concentration-response function for taurine, suggesting a competitive mechanism of suppression. pA2 values determined from these data yielded 4.17 for glycine and 3.55 for alanine. These results suggest that the processing of chemical information in quality and/or intensity coding of natural stimulus mixtures can be tempered by interactions of the components at the receptor-cell level, and possibly at the receptor-sites themselves.

Characterization of a sex pheromone in the blue crab,Callinectes sapidus: Crustecdysone studies.

The molting hormone/sex pheromone hypothesis of Kittredge and Takahashi (1972) and Kittredge et al. (1971) was explored inC. sapidus. Two concentrations of crustecdysone (5 × 10(-5) M and 5 × 10(-6) M) were presented to male crabs in a bioassay system in which courtship behavior was monitored. The results demonstrate that crustecdysone does not stimulate courtship in this species. The physical properties of crustecdysone were also compared to those of the partially purified sex pheromone derived from pubescent females. Using HPLC and mass spectral analysis, no correspondence of crustecdysone with the bioactive material could be shown. These results, in conjunction with the findings of others, do not support an evolutionary relationship between the molting hormone and sex pheromone communication in the Crustacea.