Barbara S. Zielinski

Gustatory responses of the rainbow trout (Salmo gairdneri) palate to amino acids and derivatives

Summary1.Gustatory responses to amino acids and derivatives obtained from the palatine nerve (VIIth cranial nerve) of rainbow trout (Salmogairdneri) were studied.2.The response to an amino acid was characterized by its fast-adapting, phasic nature that returned to baseline within 5 s during continuous stimulation.3.Complete recovery of the response took place 120 s after stimulation when tested with two identical stimuli given successively (Figs. 2, 3).4.Among common amino acids tested, onlyL-isomers of proline, hydroxyproline, alanine, leucine, and phenylalanine were stimulatory (Table 1), demonstrating that the facial taste system is more narrowly-tuned to amino acid stimuli than the olfactory system. The arginine derivative,L-α-amino-β-guanidinopropionic acid (L-AGPA) andL-argininic acid, and betaine were also effective.5.Concentration-response relations, when plotted semi-logarithmically, were generally sigmoidal, saturating at higher concentrations (>1 mM) (Fig. 5). The threshold concentration forL-proline, the most effective amino acid tested, was estimated to be 50 nM. The thresholds for other stimulatory chemicals ranged between 1 and 100 μM.6.L-AGPA, though having higher threshold (10 μM), could induce responses three times the magnitude of that ofL-proline at 1 mM.7.The stimulatory activity of heterocyclic-imino acids having 4-, 5-, and 6-membered rings and also of thioproline and hydroxyproline suggests that the receptor recognizes primarily the imino acid region (Fig. 6).8.Taste responses to amino acids were independent of pH, except that arginine analogues were only active at basic pHs (Fig. 7 and Table 3). larginine was active only at pHs higher than 8.5. The palatal chemoreceptors were stimulated by waters with pHs below 7.0, suggesting the existence of receptors for pH and/or CO2 (Fig. 7).

Male Sea Lampreys, Petromyzon marinus L., Excrete a Sex Pheromone from Gill Epithelia

Abstract During the period when they are producing sperm, male sea lampreys (Petromyzon marinus L.) release a sex pheromone 7α, 12α, 24-trihydroxy-5α-cholan-3-one-24-sulfate (3 keto-petromyzonol sulfate, 3ketoPZS) that induces search and preference behaviors in ovulating females. In this study, we conducted a series of experiments to demonstrate that release of this pheromone into water takes place exclusively through the gills. In a behavioral maze, water conditioned with the anterior region of spermiating males induced an increase of search and preference behaviors in ovulating females. Similar behavior was not elicited by water conditioned by the posterior region. The anterior region washings and whole-body washings from spermiating males also elicited large and virtually identical electro-olfactogram responses from female sea lampreys, while the posterior washings produced negligible responses. Further, mass spectrometry and immunoassay confirmed that virtually all the 3ketoPZS released into water was through the gills. Immunocytochemistry revealed some gill epithelial cells and hepatocytes from spermiating males contained dense immunoreactive 3ketoPZS, but not those from prespermiating males. These results demonstrate that 3ketoPZS is released through the gill epithelia and suggest that this pheromone or its precursor may be produced in the liver.

The Morphology of the Olfactory Epithelium in Larval, Juvenile and Upstream Migrant Stages of the Sea Lamprey, Petromyzon marinus

The structure of the olfactory epithelium in the larval, juvenile and upstream migrant stages of the sea lamprey, Petromyzon marinus, was investigated by light microscopy and by scanning and transmission electron microscopy. Ciliated olfactory receptor cells (ORC) were present in all stages. In larval specimens, the number of ORC was 20 +/- 8 ORC per 100 microns length of olfactory epithelial surface. In juveniles and in upstream migrants the ORC density dropped to 9 +/- 2 and 6 +/- 2 ORC/100 microns, respectively. Sustentacular cells were microvillar in the smallest larval stage (with a body length of 15 mm) and ciliated in larger larvae and later life stages. The morphological characteristics of the olfactory mucosa suggest that the sea lampreys capacity for use of the olfactory system extends into the larval stage, and that there are specific changes associated with metamorphosis.

A Novel Neural Substrate for the Transformation of Olfactory Inputs into Motor Output

Anatomical and physiological experiments in the lamprey reveal the neural circuit involved in transforming olfactory inputs into motor outputs, which was previously unknown in a vertebrate.

Olfactory sensory neurons in the sea lamprey display polymorphisms

The sea lamprey (Petromyzon marinus) is an ancient jawless fish phyletically removed from modern (teleost) fishes. It is an excellent organism in the study of olfaction due to its accessible olfactory pathway, which is susceptible to manipulation, and its important location in the evolution of vertebrates. There are many similarities in the olfactory systems of all fishes, and they also share characteristics with the olfactory system of mammals. Teleost fishes lack the distinctive vomeronasal organ of mammals; rather all odours are processed initially by olfactory sensory neurons (OSNs) of three morphotypes within the olfactory epithelium. We sought to identify olfactory sensory neuron polymorphisms in the sea lamprey. Using retrograde tracing with dyes injected into the olfactory bulb, we identified three morphotypes which are highly similar to those found in teleosts. This study provides the first evidence of morphotypes in the sea lamprey peripheral olfactory organ, and indicates that olfactory sensory neuron polymorphism may be a trait highly conserved throughout vertebrate evolution.

Glomerular Territories in the Olfactory Bulb from the Larval Stage of the Sea Lamprey Petromyzon marinus

The goal of this study was to investigate the spatial organization of olfactory glomeruli and of substances relevant to olfactory sensory neuron activity in the developing agnathan, the sea lamprey Petromyzon marinus. A 45‐kD protein immunoreactive to Golf, a cAMP‐dependent olfactory G protein, was present in the ciliary fraction of sea lamprey olfactory epithelium and in olfactory sensory neurons of larval and adult sea lampreys. This result implies that Golf expression was present during early vertebrate evolution or evolved in parallel in gnathostome and agnathostome vertebrates. Serial sectioning of the olfactory bulb revealed a consistent pattern of olfactory glomeruli stained by GS1B4 lectin and by anterograde labeling with fluorescent dextran. These glomerular territories included the dorsal cluster, dorsal ring, anterior plexus, lateral chain, medial glomeruli, ventral ring, and ventral cluster. The dorsal, anterior, lateral, and ventral glomeruli contained olfactory sensory axon terminals that were Golf‐immunoreactive. However, a specific subset, the medial glomeruli, did not display this immunoreactivity. Olfactory glomeruli in the dorsal hemisphere of the olfactory bulb, the dorsal cluster, dorsal ring, anterior plexus, lateral chain, and medial glomeruli, were seen adjacent to 5HT‐immunoreactive fibers. However, glomeruli in the ventral hemisphere, the ventral ring, and ventral cluster did not display this association. The presence of specific glomerular territories and discrete glomerular subsets with substances relevant to olfactory sensory neuron activity suggest a spatial organization of information flow in the lamprey olfactory pathway. J. Comp. Neurol. 465:27–37, 2003.

Nitric oxide synthase in the olfactory mucosa of the larval sea lamprey (Petromyzon marinus)

The use of nitric oxide, a product of enzymatic reduction of L‐arginine by nitric oxide synthase, as a modulator of processes within the olfactory mucosa was investigated in larval sea lampreys, extant fish of ancient vertebrate origin. In the present study, we demonstrated that the sea lamprey olfactory mucosa is specifically sensitive to L‐arginine, that the L‐arginine responses are inhibited by an inhibitor of nitric oxide synthase, No‐nitro‐L‐arginine, and that nitric oxide synthase is present in olfactory receptor cells, sustentacular cells, and basal cells. Electron microscopic examination using NADPH‐diaphorase histochemistry revealed intense labeling within secretory vesicles of sustentacular cells and in proximity to mitochondria within olfactory receptor cell dendrites and sustentacular cells. At the base of the olfactory epithelium, NADPH‐diaphorase staining was intense in the perinuclear cytoplasm of a subpopulation of basal cells, moderate in sustentacular cell foot processes, and scattered in olfactory receptor cell axons. Throughout axons in the olfactory epithelium and the lamina propria, labeling predominated in axonal profiles with mitochondria. These physiological and ultrastructural studies imply that in sea lamprey larvae, nitric oxide modulates peri‐receptor events of L‐arginine chemostimulation, olfactory receptor cell axonal activity, and developmental processes.

Projections from the accessory olfactory organ into the medial region of the olfactory bulb in the sea lamprey (Petromyzon marinus): a novel vertebrate sensory structure?

Although four different primary olfactory pathways have been described in tetrapod vertebrates, polymorphic olfactory sensory neurons comingle in the olfactory epithelium and project axons into separate bulbar regions in teleost fish. However, spatially segregated neurons may exist in the peripheral olfactory organ of lampreys, extant representatives of ancestral jawless vertebrates. In lampreys, the caudoventral portion of the peripheral olfactory organ contains tubular diverticula, named the accessory olfactory organ (AOO). Short, ciliated AOO cells were retrogradely labelled following application of biocytin or carbocyanine dyes to the medial region of the olfactory bulb. Tracer application to eight radial locations within the layer of glomeruli with mitral cells, of the olfactory bulb, showed that AOO projections were restricted to the medial region of the olfactory bulb. The outer boundary of the AOO projection extended to the ventromedial region of glomerular neuropil in 43% of the specimens. The olfactory sensory neurons in the main olfactory epithelium projected to glomerular neuropil throughout the olfactory bulb, including sparse projections to the medial region of the olfactory bulb. This study shows that these AOO neurons and their projections in the medial region of the olfactory bulb are anatomically distinct regions of the primary olfactory pathway in the sea lamprey. J. Comp. Neurol. 516:105–116, 2009.

Behavioural responses of female round gobies ( Neogobius melanostomus ) to putative steroidal pheromones

Although reproductive (RF) and non-reproductive (NRF) female round goby are attracted to washings of conspecific reproductive males and RF, respectively, behavioural responses of females to synthesized steroids has not been studied. We tested attraction and avoidance of RF and NRF to different blends of steroids previously shown to be either produced by reproductive males and/or detectable at a concentration of at least 10 –8 M by the olfactory epithelium of RF. Three blends of steroids were used, including: etiocholanolone (3 α -hydroxy-5 β -androstan-17-one), 11-oxo-etiocholanolone (3 α -hydroxy-5 β -androstane-11,17-dione), androstenedione, 11 β -hydroxy-androstenedione, and 11-ketotestosterone (‘free’ steroid blend); etiocholanolone glucuronide, etiocholanolone sulfate, 11-oxo-etiocholanolone glucuronide and 11-oxo-etiocholanolone sulfate (‘conjugated’ steroid blend); and all nine steroids together (‘total’ steroid blend). NRF were attracted to the free steroid blend and avoided the conjugated blend. RF did not reveal any significant bias to the steroid blends, but there was a tendency for RF to prefer conjugated steroids and avoid free steroids. Because there was no significant attraction by RF to particular blends of synthesized steroids tested, other compounds (sex attractants) not yet identified from the male round goby are likely responsible for initiating courtship and/or spawning behaviours in reproductive females.

Immunohistochemical localization of glutathione S-transferase pi in rainbow trout olfactory receptor neurons

In the fish olfactory system, glutathione S-transferases (GST) which detoxify electrophilic substances and participate in reactions of lipophilic compounds, may be active in the biotransformation of odorants and xenobiotics. In this study GST activity in the rainbow trout olfactory mucosa was high (477.6 +/- 218 nmol/min per mg protein). The GST pi class was demonstrated by Western immunoblot analysis and localized by immunofluorescence to the dendritic and perinuclear regions of olfactory receptor neurons; areas previously shown to contain elevated glutathione. The presence of GST and glutathione in fish olfactory receptor neurons suggests that these cells utilize the glutathione pathway.