William C. Michel

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, γ‐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 α‐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. J. Comp. Neurol. 454:294–309, 2002.

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. J. Comp. Neurol. 418:270–280, 2000.

Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system

SUMMARY To better understand the full extent of the odorant detection capabilities of fish, we investigated the olfactory sensitivity of zebrafish to a monoamine and several polyamines using electrophysiological and activity-dependent labeling techniques. Electro-olfactogram (EOG) recording methods established the relative stimulatory effectiveness of these odorants as: spermine >> spermidine ≈ agmatine > glutamine > putrescine ≥ cadaverine ≥ histamine > artificial freshwater. The detection threshold for the potent polyamines was approximately 1 μmol l–1. Cross-adaptation experiments suggested that multiple receptors are involved in polyamine detection. Three observations indicated that polyamine signaling may involve a transduction cascade distinct from those used by either amino acids or bile salts. Like bile salts and the adenylate cyclase activator forskolin, but unlike amino acid odorants, polyamines failed to stimulate activity-dependent labeling of olfactory sensory neurons with the cation channel permeant probe agmatine, suggesting a signaling pathway different from that used by amino acid stimuli. Also supporting distinct amino acid and polyamine signaling pathways is the finding that altering phospholipase C activity with the inhibitor U-73122 significantly reduced amino acid-evoked responses, but had little effect on polyamine- (or bile salt-) evoked responses. Altering cyclic nucleotide-mediated signaling by adenylate cyclase activation with forskolin, which significantly reduced responses to bile salts, failed to attenuate polyamine responses, suggesting that polyamines and bile salts do not share a common transduction cascade. Collectively, these findings suggest that polyamines are a new class of olfactory stimuli transduced by a receptor-mediated, second messenger signaling pathway that is distinct from those used by amino acids or bile salts.

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 Ringers 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.

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

SUMMARY 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) escapins 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 μmol 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 escapins bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapins 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 escapins antipredatory functions remains to be determined.

THE SCENT OF DANGER: TETRODOTOXIN (TTX) AS AN OLFACTORY CUE OF PREDATION RISK

Larvae of the California newt (Taricha torosa) exhibit striking predator-avoidance behavior, escaping to refuges in response to a chemical cue from cannibalistic adults. In laboratory flow-tank experiments, stream water collected near free-ranging adults induced hiding responses in 100% of the larvae tested. Solutions prepared by bathing adults (in field and laboratory) also evoked strong hiding behaviors. Insensitive to adult feeding status (fed or starved), and clearly not an excretory product, the chemical cue was released from adult skin (i.e., in swabs of adult backs, sides, and bellies). Tetrodotoxin (TTX) was found in skin swabs of adults and in bathwater at 1 × 10−7 mol/L using reversed-phase high-pressure liquid chromatography (HPLC). Concentrations of 1 × 10−7 to 1 × 10−9 mol/L TTX standard, and equivalent dilutions of bathwater, triggered hiding behaviors in larvae, with no subsequent sublethal toxicity. The presence of TTX-sensitive cells within larval olfactory epithelium was confirmed by beh...

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.

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 μM) 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 α7‐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. J. Comp. Neurol. 504:631–645, 2007.

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 μM). 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. Anat Rec, 291:763‐774, 2008.

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.