Functional significance of the co-localization of taste buds and teeth in the pharyngeal jaws of the largemouth bass, Micropterus salmoides.

Studies of feeding behavior in the largemouth bass, Micropterus salmoides, revealed that live goldfish or artificial food balls are ingested in three discrete steps: inhalation of the food into the oral cavity, passage through the pharyngeal cavity, and swallowing. Food balls with or without a feeding stimulant were inhaled with equal frequency; thus, vision was clearly the major sense affecting inhalation. However, food balls with defined concentrations of a feeding stimulant were swallowed in a dose-dependent manner, whereas food balls without a feeding stimulant were promptly expelled. Thus, gustation played a major role in stimulating swallowing. Videotaped observations of feeding behavior suggested that both food processing and gustation occur in the pharynx and take place before the swallowing of either goldfish or food balls. The well-developed pharyngeal jaws of largemouth bass consist of six major pads of caniform teeth in the upper pharynx and two pads in the lower pharynx. Scanning electron microscopy showed that taste buds were abundant around most of these pharyngeal teeth. Histological sections prepared from all pharyngeal pads revealed that both elevated and flattened taste buds occur with the teeth. The morphology of these taste buds was typical of that described in other teleosts. Neuronal profiles, visualized with an HNK-1 monoclonal antibody, were observed entering each taste bud. The antibody also selectively stained a group of one to four putative sensory cells in each taste bud and the distal processes of these cells in the receptor area. The co-localization of teeth and taste buds on the pharyngeal jaws indicates that food processing and gustation both occur there, and that together these processes determine whether a potential food item is swallowed.

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.

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.

Purinergic modulation in the brain of the spiny lobster.

Earlier studies identified purinergic chemoreceptors in the olfactory organ of the spiny lobster, Panulirus argus. In this study, electrophysiological experiments demonstrate that purinergic substances can modulate both the spontaneous activity and the evoked responses of neurons within the brain of this animal. Perfusion of the brain with 100 microM adenosine 5'-monophosphate (AMP) modulated the spontaneous activity of 71% of the brain interneurons that were monitored. AMP also modulated the electrically or chemically evoked activity of 25% of the monitored interneurons. The effects were dose-dependent (down to 1 microM) and reversible. The modulatory effects of adenosine were similar to those of AMP, and were antagonized by the adenosine receptor antagonist 1,3-dipropyl-8-p-sulfophenylxanthine. The modulation by AMP or adenosine was depressive in most but not all neurons, as is the case with purinergic effects in the brain of vertebrates. We believe this is the first demonstration of modulatory effects of purinergic substances in the nervous system of any invertebrate.

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.

Chemically stimulated feeding behavior in marine animals : Importance of chemical mixtures and involvement of mixture interactions.

A review is provided of the chemical components in tissue extracts that elicit feeding behavior in marine fish and crustaceans. For most species, the major stimulants of feeding behavior in excitatory extracts are an assemblage of common metabolites of low molecular weight including amino acids, quaternary ammonium compounds, nucleosides and nucleotides, and organic acids. It is often mixtures of substances rather than individual components that account for the stimulatory capacity of a natural extract. Recent studies using a shrimp,Palaemonetes pugio, are described in which behavioral bioassays were conducted with complex synthetic mixtures formulated on the basis of the composition of four tissue extracts. These results indicate that synergistic interactions occur among the mixture components. The neural mechanisms whereby marine crustaceans receive and code information about chemical mixtures are also reviewed. Narrowly tuned receptor cells, excited only by particular components of food extracts such as specific amino acids, nucleotides, quaternary ammonium compounds, and ammonium ions, are common in lobsters and could transmit information about mixtures as a labeled-line code. However, since physiological recordings indicate that most higher-level neurons in the brain each transmit information about many components of mixtures, rather than about a single component, it is suggested that information about a complex food odor is transmitted as an across-fiber pattern, instead of a labeled-line code. Electrophysiological recordings of responses of peripheral and central neurons of lobsters to odor mixtures and their components reveal that suppressive interactions occur, rather than the synergistic interactions noted earlier in the behavioral studies. Possible reasons for these differences are discussed. Evidence from the behavioral study indicates that the "direction" of a mixture interaction can be concentration-dependent and the synergism may occur at low mixture concentrations, while suppression may occur at high concentrations.

ANTENNULAR CHEMOSENSITIVITY IN THE SPINY LOBSTER, PANULIRUS ARGUS: COMPARATIVE TESTS OF HIGH AND LOW MOLECULAR WEIGHT STIMULANTS.

Antennular chemoreceptors in the spiny lobster, P. argus, were surveyed electrophysiologically for responsiveness to natural stimuli of different molecular weights to gain further insight into the stimulatory role of macromolecules. Extracts and body fluids from eight potential food organisms were prepared and tested both before and after fractionation by ultrafiltration. Data presented verify chemosensitivity of lateral antennular filaments and show that for all extract types. the components of low molecular weight (< ca. 10,000) were significantly more stimulatory than the components of higher molecular weight; and stimulus values of low molecular weight fractions did not differ significantly from those of the unfractionated extracts.