Odorant responsiveness of squid olfactory receptor neurons.

In the olfactory organ of the squid, Lolliguncula brevis there are five morphological types of olfactory receptor neurons (ORNs). Previous work to characterize odor sensitivity of squid ORNs was performed on only two of the five types in dissociated primary cell cultures. Here, we sought to establish the odorant responsiveness of all five types. We exposed live squid or intact olfactory organs to excitatory odors plus the activity marker, agmatine (AGB), an arginine derivative that enters cells through nonselective cation channels. An antibody against AGB was used to identify odorant-activated neurons. We were able to determine the ORN types of AGB-labeled cells based on their location in the epithelium, morphology and immunolabeling by a set of metabolites: arginine, aspartate, glutamate, glycine, and glutathione. Of 389 neurons identified from metabolite-labeled tissue, 3% were type 1, 32% type 2, 33% type 3, 15% type 4, and 17% type 5. Each ORN type had different odorant specificity with type 3 cells showing the highest percentages of odorant-stimulated AGB labeling. Type 1 cells were rare and none of the identified type 1 cells responded to the tested odorants, which included glutamate, alanine and AGB. Glutamate is a behaviorally attractive odorant and elicited AGB labeling in types 2 and 3. Glutamate-activated AGB labeling was significantly reduced in the presence of the adenylate cyclase inhibitor, SQ22536 (80 microM). These data suggest that the five ORN types differ in their relative abundance and odor responsiveness and that the adenylate cyclase pathway is involved in squid olfactory transduction.

Cross-species comparison of metabolite profiles in chemosensory epithelia: an indication of metabolite roles in chemosensory cells.

Comparative studies of chemosensory systems in vertebrates and invertebrates have greatly enhanced our understanding of anatomical and physiological constraints of chemical detection. Immunohistochemical comparisons of chemosensory systems are difficult to make across species due to limited cross-reactivity of mammalian-based antibodies. Immunostaining chemosensory tissues with glutaraldehyde-based antibodies generated against small metabolites in combination with hierarchical cluster analyses provide a novel approach for identifying and classifying cell types regardless of species. We used this "metabolite profiling" technique to determine whether metabolite profiles can be used to identify cell classes within and across different species including mouse, zebrafish, lobster and squid. Within a species, metabolite profiles for distinct cell classes were generally consistent. We found several metabolite-based cell classifications that mirrored function or receptor protein-based classifications. Although profiles of all six metabolites differed across species, we found that specific metabolites were associated with certain cell types. For example, elevated levels of glutathione were characteristic of nonsensory cells from vertebrates, suggesting an antioxidative role in non-neuronal cells in sensory tissues. Collectively, we found significantly different metabolite profiles for distinct cell populations in chemosensory tissue within all of the species studied. Based on their roles in other systems or cells, we discuss the roles of L-arginine, L-aspartate, L-glutamate, glycine, glutathione, and taurine within chemosensory epithelia.

Cholinergic innervation of the zebrafish olfactory bulb.

A number of fish species receive forebrain cholinergic input but two recent reports failed to find evidence of cholinergic cell bodies or fibers in the olfactory bulbs (OBs) of zebrafish. In the current study we sought to confirm these findings by examining the OBs of adult zebrafish for choline acetyltransferase (ChAT) immunoreactivity. We observed a diffuse network of varicose ChAT-positive fibers associated with the nervus terminalis ganglion innervating the mitral cell/glomerular layer (MC/GL). The highest density of these fibers occurred in the anterior region of the bulb. The cellular targets of this cholinergic input were identified by exposing isolated OBs to acetylcholine receptor (AChR) agonists in the presence of agmatine (AGB), a cationic probe that permeates some active ion channels. Nicotine (50 microM) significantly increased the activity-dependent labeling of mitral cells and juxtaglomerular cells but not of tyrosine hydroxlase-positive dopaminergic neurons (TH(+) cells) compared to control preparations. The nAChR antagonist mecamylamine, an alpha7-nAChR subunit-specific antagonist, calcium-free artificial cerebrospinal fluid, or a cocktail of ionotropic glutamate receptor (iGluR) antagonists each blocked nicotine-stimulated labeling, suggesting that AGB does not enter the labeled neurons through activated nAChRs but rather through activated iGluRs following ACh-stimulated glutamate release. Deafferentation of OBs did not eliminate nicotine-stimulated labeling, suggesting that cholinergic input is primarily acting on bulbar neurons. These findings confirm the presence of a functioning cholinergic system in the zebrafish OB.

Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica.

Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela, this fluid is a mixture of two secretions from two separate glands, usually co-released: ink, a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study, we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an L-amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela, which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapin's major amino acid substrates--L-lysine and L-arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml(-1), or 30 micromol ml(-1), which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l(-1) and lysine is present in ink at <1 mmol l(-1) but in opaline at 65 mmol l(-1). Our previous results showed that both lysine and arginine mediate escapin's bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapin's antimicrobial effects require concentrations of lysine and/or arginine >1 mmol l(-1), our data lead us to conclude that lysine in opaline is the primary natural substrate for escapin in ink. Furthermore, packaging of the enzyme escapin and its substrate lysine into two separate glands and their co-release and mixing at the time of predatory attack allows for the generation of bioactive defensive compounds from innocuous precursors at the precise time they are needed. Whether lysine and/or arginine are substrates for escapin's antipredatory functions remains to be determined.

Morphological and biochemical heterogeneity in facial and vagal nerve innervated taste buds of the channel catfish, Ictalurus punctatus.

In catfish, the facial nerve innervates taste buds distributed over the entire body including the barbels, while the glossopharyngeal and vagal nerves innervate oropharyngeal taste buds. Facial nerve innervated taste buds (FITBs) are thought to be involved in food detection and localization, while glossopharyngeal and vagal nerve innervated taste buds (VITBs) evaluate the palatability of food prior to ingestion. Physiological studies indicate that both oral and extra-oral taste buds detect sapid substances such as amino acids and nucleotides, but the facial taste system is more sensitive to some of these substances. The anatomical, molecular, and/or physiological mechanisms underlying the functional differences in these two gustatory pathways remain to be identified. In the current investigation we compare the basic morphological features of FITBs and VITBs and the distribution of the following metabolites: gamma-aminobutyric acid (GABA), glutamate, aspartate, alanine, taurine, and glutathione. Vagal innervated taste buds are significantly longer and narrower than FITBs, with fewer taste cells and a smaller nerve plexus. Each of the metabolites examined was heterogeneously distributed in taste cells with notably more GABA positive cells present in the VITBs. Patterns of metabolite colocalization suggest the presence of several taste cell subtypes. The morphological and metabolite differences noted between FITBs and VITBs provide a potential anatomical basis for the previously noted differences in physiological sensitivity.

Assessment of neuronal maturation and acquisition of functional competence in the developing zebrafish olfactory system.

Olfactory coding at the level of the olfactory bulb is thought to depend upon an ensemble response of mitral cells receiving input from chemotopically-organized projections of olfactory sensory neurons and regulated by lateral inhibitory circuits. Immunocytochemical methods are described to metabolically classify neurons in the developing zebrafish olfactory system based on the relative concentrations of taurine, glutamate, GABA (and potentially other small biogenic amines) and a small guanidium-based cation, agmatine, which labels NMDA-sensitive cells by permeating through active ionotropic glutamate receptor channels. Using metabolic profiling in conjunction with activity dependent labeling we demonstrate that neuronal differentiation in the developing olfactory bulb, as assessed by acquisition of a mature neurochemical profile, and sensitivity to an ionotropic glutamate receptor agonist, NMDA, occurs during the second day of development. This experimental approach is likely to be useful in studies concerned with the development of glutamatergic signaling pathways.

Odor-stimulated glutamatergic neurotransmission in the zebrafish olfactory bulb.

The role of glutamate as a neurotransmitter in the zebrafish olfactory bulb (OB) was established by examining neuronal activation following 1). glutamate receptor agonist stimulation of isolated olfactory bulbs and 2). odorant stimulation of intact fish. Four groups of neurons (mitral cells, projection neurons; granule cells, juxtaglomerular cells, and tyrosine hydroxylase-containing cells; interneurons) were identified on the basis of cell size, cell location, ionotropic glutamate receptor (iGluR) agonist/odorant sensitivity, and glutamate, gamma-aminobutyric acid (GABA), and tyrosine hydroxylase immunoreactivity. Immunoreactive glutamate levels were highest in olfactory sensory neurons (OSNs) and mitral cells, the putative glutamatergic neurons. The sensitivity of bulbar neurons to iGluR agonists and odorants was established using a cationic channel permeant probe, agmatine (AGB). Agmatine that permeated agonist- or odor-activated iGluRs was fixed in place with glutaraldehyde and detected immunohistochemically. N-methyl-D-aspartic acid (NMDA) and alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainic acid (KA) iGluR agonists and odorants (glutamine, taurocholic acid) stimulated activity-dependent labeling of bulbar neurons, which was blocked with a mixture of the iGluR antagonists, D-2-amino-5-phosphono-valeric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The AMPA/KA antagonist CNQX completely blocked glutamine-stimulated AGB labeling of granule cells and tyrosine hydroxylase-containing cells, suggesting that, in these cell types, AMPA/KA receptor activation is essential for NMDA receptor activation. However, blocking AMPA/KA receptor activity failed to eliminate AGB labeling of mitral cells or juxtaglomerular cells. Collectively, these findings indicate that glutamate is the primary excitatory neurotransmitter in the zebrafish OB and that iGluR subtypes function heterogeneously in the bulbar neurons.

Amino acid odorants stimulate microvillar sensory neurons.

The olfactory epithelium (OE) of zebrafish is populated with ciliated and microvillar olfactory sensory neurons (OSNs). Whether distinct classes of odorants specifically activate either of these unique populations of OSNs is unknown. Previously we demonstrated that zebrafish OSNs could be labeled in an activity-dependent fashion by amino acid but not bile acid odorants. To determine which sensory neuron type was stimulated by amino acid odorants, we labeled OSNs using the ion channel permeant probe agmatine (AGB) and analyzed its distribution with conventional light- and electron-microscope immunocytochemical techniques. Approximately 7% of the sensory epithelium was labeled by AGB exposure alone. Following stimulation with one of the eight amino acids tested, the proportion of labeled epithelium increased from 9% for histidine to 19% for alanine; amino acid stimulated increases in labeling of 2-12% over control labeling. Only histidine failed to stimulate a significant increase in the proportion of labeled OSNs compared to control preparations. Most amino acid sensitive OSNs were located superficially in the epithelium and immuno-electron microscopy demonstrated that the labeled OSNs were predominantly microvillar. Large numbers of nanogold particles (20-60 per 1.5 microm(2)) were associated with microvillar olfactory sensory neurons (MSNs), while few such particles (<15 per 1.5 microm(2)) were observed over ciliated olfactory sensory neurons (CSNs), supporting cells (SCs) and areas without tissue, such as the lumen above the OE. Collectively, these findings indicate that microvillar sensory neurons are capable of detecting amino acid odorants.

CHEMICAL MEDIATION OF APPETITIVE FEEDING IN A MARINE DECAPOD CRUSTACEAN: THE IMPORTANCE OF SUPPRESSION AND SYNERGISM.

The California spiny lobster, Panulirus interruptus, failed to exhibit appetitive feeding or locomotion in response to a low molecular weight fraction (< 1000 daltons) prepared from a sea water extract of muscle from abalone, a natural prey. This lack of response was caused by chemical suppressants, rather than by lack of stimulatory compounds. Excitatory responses were induced by single, low molecular weight compounds, but these responses were inhibited by suppressants which occur naturally in the muscle fraction. Amino and organic acids were found highly stimulatory to lobsters, but nucleotides and sugars were not. A mixture of monocarboxylic amino acids and dicarboxylic organic acids was much more effective in elliciting behavior than either of the constituents tested alone, at the same overall concentration. Mixtures which combined either ammonium or urea with amino or organic acids significantly reduced behavioral activity caused by these latter substances. Results indicate that tests of single chemicals cannot always reliably predict the stimulatory properties of solutions, combining even as few as two or more compounds. The stimulatory properties of complex odorants, including prey extracts, are best assessed by fractionating and then combining and testing the fractions in bioassays of factorial design.