Mixed input to olfactory glomeruli from two subsets of ciliated sensory neurons does not impede relay neuron specificity in the crucian carp.

The consensus view of olfactory processing is that the axons of receptor-specific primary olfactory sensory neurons (OSNs) converge to a small subset of glomeruli, thus preserving the odour identity before the olfactory information is processed in higher brain centres. In the present study, we show that two different subsets of ciliated OSNs with different odorant specificities converge to the same glomeruli. In order to stain different ciliated OSNs in the crucian carp Carassius carassius we used two different chemical odorants, a bile salt and a purported alarm substance, together with fluorescent dextrans. The dye is transported within the axons and stains glomeruli in the olfactory bulb. Interestingly, the axons from the ciliated OSNs co-converge to the same glomeruli. Despite intermingled innervation of glomeruli, axons and terminal fields from the two different subsets of ciliated OSNs remained mono-coloured. By 4-6 days after staining, the dye was transported trans-synaptically to separately stained axons of relay neurons. These findings demonstrate that specificity of the primary neurons is retained in the olfactory pathways despite mixed innervation of the olfactory glomeruli. The results are discussed in relation to the emerging concepts about non-mammalian glomeruli.

Ocean acidification impairs olfactory discrimination and homing ability of a marine fish.

The persistence of most coastal marine species depends on larvae finding suitable adult habitat at the end of an offshore dispersive stage that can last weeks or months. We tested the effects that ocean acidification from elevated levels of atmospheric carbon dioxide (CO(2)) could have on the ability of larvae to detect olfactory cues from adult habitats. Larval clownfish reared in control seawater (pH 8.15) discriminated between a range of cues that could help them locate reef habitat and suitable settlement sites. This discriminatory ability was disrupted when larvae were reared in conditions simulating CO(2)-induced ocean acidification. Larvae became strongly attracted to olfactory stimuli they normally avoided when reared at levels of ocean pH that could occur ca. 2100 (pH 7.8) and they no longer responded to any olfactory cues when reared at pH levels (pH 7.6) that might be attained later next century on a business-as-usual carbon-dioxide emissions trajectory. If acidification continues unabated, the impairment of sensory ability will reduce population sustainability of many marine species, with potentially profound consequences for marine diversity.

Visualizing a set of olfactory sensory neurons responding to a bile salt.

In the present study, we exposed the olfactory epithelia of crucian carp, Carassius carassius, and brown trout, Salmo trutta, to dextran coupled with Alexa dyes together with odorants. Dye uptake was severely reduced after pre-exposure to nocodazole, an inhibitor of microtubule polymerization that impairs endocytosis, supporting the hypothesis that odour-activated olfactory receptor molecules undergo endocytosis. Application of the bile acid taurolithocholate, a potent and specific odorant for fish, resulted in the labelling of a sparse (less than 3%) cell population with the typical morphology of ciliated sensory neurons (CSNs) - long dendrites and cell somata deep in the sensory epithelium. The dye was distributed throughout the sensory neuron, also revealing axons and target glomeruli. Stained axons redistribute at the entrance of the olfactory bulb and terminate in two small target areas, a dorsal and a medial one. These results are consistent with the notion that taurolithocholate is detected specifically by a few ciliated sensory neurons. Application of the olfactory epithelium of brown trout to bile acid stained cells with the appearance of CSNs. Application of an alarm agonist, hypxanthine-3-N-oxide, to crucian carp olfactory organ caused staining of another set of sensory neurons. Furthermore, our results show that odour-induced uptake of a dye can serve to identify the subtype of olfactory sensory neurons responding to a particular odorant, and to pinpoint the target regions of these neurons in the olfactory bulb as a first step to elucidating the neuronal network responding to a particular odour.

The functional organization of the fish olfactory system.

Recent developments in the functional anatomy and physiology of the fish olfactory system reveal three parallel pathways from the sensory epithelium, via the olfactory bulb to the telencephalon. There are three morphological types of sensory neurones spread in a seemingly overlapping arrangement in the olfactory epithelium. The axons of each type of sensory neurones converge to a specific region of the olfactory bulb and connect to separate sets of relay neurones. The axons of these relay neurones leave in three bundles to the telencephalon. Each bundle conveys specific information that elicits sets of characteristic behaviour in response to odours involved in essential life processes in the fish. One pathway is tuned to social cues, another to sex pheromones, and the third to food odours.

Avoidance behavior and brain monoamines in fish.

The crucian carp performs a typical avoidance behavior when exposed to olfactory cues from injured skin of conspecifics. They swim rapidly to the bottom and hide in available material. This work examines the effects of skin extract exposure and availability of hiding material on this behavior, and concomitant changes in brain monoaminergic activity in crucian carp. Individual fish were exposed to skin extract in aquaria with or without hiding material. Exposure to skin extract resulted in the expected avoidance behavior consisting of rapid movement towards the bottom of the aquarium. This lasted for 1-2 min. Activity then decreased below the level observed before exposure, suggesting a "freezing" type of avoidance behavior. This behavior was independent of availability of hiding material. Brain dopaminergic activity increased in telencephalon and decreased in the brain stem following skin extract exposure, again independent of availability of hiding material. However, fish kept in aquaria without hiding material showed an elevation of serotonergic activity in the brain stem and the optic tectum compared to fish with available hiding material. Absence of hiding material increased serotonergic activity also without exposure to skin extract. In aquaria with hiding material, the fish stirred up a cloud of fine sediments and showed a more pronounced decrease in locomotor activity in agreement with this being a more efficient freezing or immobile avoidance behavior. These results show that basic components of avoidance behavior and related brain changes are present in the fish brain, in accordance with the common phylogenetic roots of avoidance behavior in all vertebrates.

The effect of sublethal concentrations of the water-soluble fraction of crude oil on the chemosensory function of Caspian roach, Rutilus caspicus (YAKOVLEV, 1870).

The water-soluble fraction of crude oil is a complex and toxic mixture of hydrocarbons. Because aquatic organisms directly encounter it, the water-soluble fraction plays an important role in the toxicity of crude oil in aquatic environments. To determine whether fish are attracted to or avoid the water-soluble fraction, Caspian roaches (Rutilus caspicus) were exposed to different concentrations of the water-soluble fraction in a choice maze apparatus. The results showed that Caspian roaches can detect and avoid 2 mg/L of the water-soluble fraction. To study the effect of the water-soluble fraction on the olfactory function of fish, Caspian roaches were exposed to 3.2 mg/L and 16 mg/L of the water-soluble fraction for 96 h; afterward, exposed fish encountered food extract in a choice maze apparatus. The present study showed that the water-soluble fraction significantly impairs the olfactory function of roaches. To investigate the effect of olfactory system dysfunction on the feeding behavior of fish, Caspian roaches were exposed to 3.2 mg/L and 16 mg/L of the water-soluble fraction. After 4 d, 8 d, and 12 d of exposure, the feeding behavior toward the food extract was tested. The results showed that both 3.2 mg/L and 16 mg/L of the water-soluble fraction suppress the feeding behavior of Caspian roaches. The present study demonstrates that sublethal concentrations of crude oil's water-soluble fraction impair the olfactory function of fish and consequently suppress the feeding behavior.

Physiological and behavioural responses to noxious stimuli in the Atlantic cod (Gadus morhua).

In the present study, our aim was to compare physiological and behavioural responses to different noxious stimuli to those of a standardized innocuous stimulus, to possibly identify aversive responses indicative of injury detection in a commercially important marine teleost fish, the Atlantic cod. Individual fish were administered with a noxious stimulus to the lip under short-term general anaesthesia (MS-222). The noxious treatments included injection of 0.1% or 2% acetic acid, 0.005% or 0.1% capsaicin, or piercing the lip with a commercial fishing hook. Counts of opercular beat rate (OBR) at 10, 30, 60, 90 and 120 min and observations of behaviour at 30 and 90 min post-treatment were compared with pre-treatment values and with control fish injected with physiological saline, an innocuous stimulus. Circulatory levels of physiological stress indicators were determined in all fish at 120 minutes post-treatment. All treatments evoked temporarily increased OBR that returned to pre-treatment levels at 60 minutes (saline, 0.005% capsaicin, hook), 90 minutes (0.1% acetic acid, 0.1% capsaicin), or 120 minutes (2% acetic acid), but with no significant differences from the control group at any time point. Fish treated with 0.1% and 2% acetic acid and 0.1% capsaicin displayed increased hovering close to the bottom of the aquaria and fish given 2% acetic acid and 0.1% capsaicin also displayed a reduced use of shelter. The only effect seen in hooked fish was brief episodes of lateral head shaking which were not seen pre-treatment or in the other groups, possibly reflecting a resiliency to tissue damage in the mouth area related to the tough nature of the Atlantic cod diet. There were no differences between groups in circulatory stress indicators two hours after treatment. This study provides novel data on behavioural indicators that could be used to assess potentially aversive events in Atlantic cod.

Silver nanoparticles disrupt olfaction in Crucian carp (Carassius carassius) and Eurasian perch (Perca fluviatilis).

The present study investigates the effect of silver nanoparticles on olfaction in Crucian carp (Carassius carassius) and Eurasian perch (Perca fluviatilis). The electro-olfactogram (EOG) signal was recorded by stimulating the olfactory epithelium with pulses of the odorant L-alanine during the pre-exposure, silver exposure and recovery periods, respectively. The nanosilver suspension concentrations applied were 0.00, 0.45 and 45 µg L⁻¹, respectively. Secondly, to compare the toxicity of silver nanoparticles with silver ions, perch were exposed to ionic silver. During exposure to nanosilver suspension, the olfactory epithelium rapidly hyperpolarized, which was not found after exposure to silver ion solution. Exposure to 0.45 µg L⁻¹ nanosilver suspension led to enhanced EOG responses, whereas exposure to 45 µg L⁻¹ silver nanoparticle suspension and silver ion solution resulted in suppressed EOG signals. The EOG signals partly recovered in silver-free water. The silver nanoparticle olfactory toxicity is believed to be a combination of silver particles and released silver ions.

The alarm reaction in fishes--odorants, modulations of responses, neural pathways.

In 1938, von Frisch discovered the alarm reaction ("schreck reaktion") in fishes. He found that substances stored in the skin are released upon injury and detected by the olfactory organ. Several agonists that induce alarm reactions have been identified since then, but the true nature of the alarm substances remains veiled. Behavioral experiments have demonstrated that skin extract of conspecifics is more potent than that of heterospecifics. During development, the alarm reaction is seen in the larval stages in several cyprinid species; the threshold for eliciting the reaction decreases with age. Further, in crucian carp the alarm reaction was shown to be suppressed in the majority of females during the spawning period. The anatomy of the olfactory organ of this species is advantageous for studying underlying mechanisms of olfactory-induced behaviors. Crucian carp have been applied as a model organism in our lab for identification of the neural pathway of the alarm reaction, mainly in primary and secondary olfactory neurons. In the olfactory bulb, there is a distinct region that responds specifically to skin extract. The neural basis for discrimination between extracts of skin from different species has been investigated, suggesting that identification of conspecifics may be based on activation of specific subsets of neurons.

Ligand-specific induction of endocytosis in taste receptor cells.

We demonstrate a ligand-specific induction of endocytosis in cells of juvenile brown trout taste buds. The process is fast, massive and selective, as only a few cells in each taste buds are stained by exposure of the oral cavity to the taste stimulant l-cysteine together with a dye at 20 degrees C. Low temperature (+2 degrees C) and disruption of microtubules with nocodazole caused a substantial reduction in the number of taste cells stained, indicating endocytic uptake of dye and transport towards the cell soma in vesicles. As endocytosis is evoked by the presence of ligands, it is most likely that the stained cells are the so-called receptor cells, which have taste receptors and the molecular machinery for downstream processing. The number of stained taste cells and taste buds containing stained taste cells increased with the concentration of l-cysteine. Control experiments with different dyes revealed great variability in the ability to induce staining on their own. In particular, Texas Red dextran was efficient and stained many cells within each taste bud. Behavioural experiments demonstrated that Texas Red dextran is a deterrent taste substance for brown trout. In fish first exposed to the stimulant l-cysteine plus a dye and subsequently to a deterrent, either Texas Red, or glycine, the majority of stained cells were found in separate taste receptor cells, indicating that the majority of taste receptors for stimulants and deterrents are expressed in separate taste buds. These results also strengthen the assumption that the stained cells take part in the initiation of taste processes that are related to perception. The functional implication of the induced endocytosis is discussed.

Single unit responses to skin odorants from conspecifics and heterospecifics in the olfactory bulb of crucian carp Carassius carassius.

Injured fish skin leaks alarm substances that induce the fright reaction upon olfactory detection. The skin also contains a multitude of other odorants traditionally related to other behaviors, but to what extent they are detected upon injury is unknown. We have performed single unit recordings in the olfactory bulb (OB) of crucian carp while exposing the olfactory epithelium to skin extracts from conspecifics and three other species of the carp family, common carp, tench and bream. The aims were to investigate whether neural activity may be induced by different types of skin odorants and how well the odorants from injured conspecifics are distinguished from other species. The OB of crucian carp shows a clear chemotopy as units located in different regions respond to either food-related odorants, to pheromones or to alarm odorants respectively. Units in all regions responded to skin extracts, which indicate the detection of odorants usually involved in reproduction and feeding, in addition to the alarm substances. Among OB units responding to only one of the skin extracts, most were sensitive to conspecific skin extract. Furthermore, pair-wise comparisons showed that the discrimination between conspecific skin extract and skin extract from another species was in general better than the discrimination between skin extracts from two heterospecifics. The findings suggest that identification of injured fishes may be based on different groups of odorants and that the crucian carp olfactory system discriminates well between odorants from conspecifics and those from other fish species.

Seasonal variations in olfactory sensory neurons--fish sensitivity to sex pheromones explained?

Olfactory sensory neurons of vertebrates regenerate throughout the life of the animal. In fishes, crypt cells are a type of olfactory sensory neurons thought to respond to sex pheromones. Here, we demonstrate that the number of crypt cells in the olfactory epithelium of the crucian carp varies dramatically throughout the year. During winter, few crypt cells are observed at any location within the sensory epithelium. In spring, the majority of crypt cells are located deep in the epithelium not yet exposed to the environment. However, during the summer spawning season, crypt cells are positioned at the epithelial surface. These findings may explain previous studies demonstrating a relationship between circulating androgen and olfactory sensitivity to sex pheromones.

Effects of antalarmin, a CRF receptor 1 antagonist, on fright reaction and endocrine stress response in crucian carp (Carassius carassius).

The corticotrophin-releasing factor (CRF) receptors show striking homogeneity throughout the vertebrate subphylum. In mammals, the CRF(1) receptor (CRFR(1)) plays an important role in mediating behavioral and endocrine responses to fear and stress. The specific roles of this receptor subtype in fear and stress reactions in non-mammalian vertebrates are largely unknown. Crucian carp displays the olfactory-mediated fright reaction, a stereotypic behavioral response to waterborne cues from damaged skin of conspecifics. This reaction shows several similarities to basic components of avoidance behavior in mammals. In the present study, we applied the non-peptide CRFR(1) antagonist, antalarmin, to crucian carp 1 h before exposure to conspecific skin extract. This treatment resulted in a suppression of the fright reaction. After skin extract exposure, antalarmin treatment also lead to lower plasma cortisol values, as compared to vehicle treatment. This suppression of the behavioral fright reaction and the stress induced rise in plasma cortisol in crucian carp suggests that the functions of the CRFR(1) are conserved by evolution.

Female crucian carp, Carassius carassius, lose predator avoidance behavior when getting ready to mate.

In predator-prey interactions, the prey often have to compromise fitness-related behaviors such as feeding, courting, and territorial defense in order to avoid predators. In these trade-off situations, some behaviors have priority over others. These priorities are not rigid, and may be context-dependent; for instance, many animals show increased risk-taking during courtship behavior by paying less attention to potential predators. We investigated whether the fright reaction, a stereotypical avoidance response to olfactory cues from injured conspecifics, may be affected by reproductive status in a teleost fish, the crucian carp. We demonstrate that among individuals not responding to alarm substances with a fright reaction, the majority were ovulated or spermiated. In females, mean plasma concentrations of 17beta-estradiol and testosterone, gonadal steroids known to decrease during the later stages of sexual maturation, were lower in the individuals not responding with a fright reaction compared to those responding. In males, there were no differences between responsive and non-responsive individuals in mean plasma levels of androgens (testosterone and 11-ketotestosterone) involved in spermatogenesis and male sexual behavior. As the fright reaction in crucian carp consists of behavior incompatible with spawning behavior, we hypothesize that this short-term suppression of the alarm response has evolved so that spawning can occur uninterrupted.

Inflammatory obstruction of the olfactory clefts and olfactory loss in humans: a new syndrome?

The first step in the olfactory perception is the activation by odorants of sensory neurones in the olfactory epithelium. In humans, this sensory epithelium is located at 2 narrow passages, the olfactory clefts, at the upper part of the nasal cavities. Little is known about the physiology of these clefts. We examined, in 34 patients, the impact of obstructed clefts upon detection and postlearning identification of 5 odorants. The location and extension of the obstructions were assessed using endoscopy, CT scans, and MRI. The inflammatory obstruction was usually bilateral, extending anteroposteriorly, and confined to the clefts, with no sign of obstruction or any inflammatory disease in the rest of the nasal cavities and sinuses. When tested with 5 odorants, these patients showed greatly impaired olfaction compared with a group of 73 normosmic subjects. The majority of these 34 patients had sensory deficits equivalent to that found in another group of 41 congenital anosmic patients, where inspection with MRI indicated the lack of olfactory bulbs. This study demonstrates that the olfactory clefts, in human, function as an entity that is different from other regions of the nasal cavity and is the target for local inflammatory events that are apparently not responding to corticoid and antibiotic treatments.

Specific projection of the sensory crypt cells in the olfactory system in crucian carp, Carassius carassius.

To study the projection of a special type of sensory neuron called crypt cells in the olfactory system in crucian carp, Carassius carassius, we applied the neural tracer 1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in the olfactory bulb (OB). Small crystals of DiI were applied in a small area at the synaptic region at the ventral part of the OB, where a population of secondary neurons specific for sex pheromones has been identified. In those samples (4 out of 24) where only axons in the lateral bundle of the medial olfactory tract were stained, the majority (50-66%) of olfactory sensory neurons stained were crypt cells situated in the peripheral layer of the olfactory epithelium. Because this bundle of the tract mediates reproductive behavior, it is conceivable that crypt cells express olfactory receptors for sex pheromones.

Gender distinction in neural discrimination of sex pheromones in the olfactory bulb of crucian carp, Carassius carassius.

Studies on projection of the sensory neurons onto the olfactory bulb in fish have revealed a clear subdivision into spatially different areas that each responded specifically to different classes of odorants. Amino acids induce activity in the lateral part, bile salts induce activity in the medial part, and alarm substances induce activity in the posterior part of the medial olfactory bulb. In the present study, we demonstrate a new feature of the bulbar chemotopy showing that neurons specifically sensitive to sex pheromones are located in a central part of the ventral olfactory bulb in crucian carp. Extensive single-unit recordings were made from these neurons, stimulating with four sex pheromones, 17,20beta-dihydroxy-4-pregnen-3-one, 17,20beta-dihydroxy-4-pregnen-3-one-20-sulfate, androstenedione, and prostaglandin F(2alpha), known to induce specific reproductive behaviors in males of carp fish. All substances were applied separately to the sensory epithelium at a concentration of 10(-9) M. Of the 297 neurons recorded in males, the majority (236 or 79.5%) responded exclusively to one of the four sex pheromones and thus showed a high specificity. Of the 96 neurons recorded from the olfactory bulb in females, only 1 unit showed such a specific activation. These findings reflect remarkable differences between males and females in the discriminatory power of the olfactory neurons toward these sex pheromones. The gender differences are discussed in relation to behavior studies, expression of olfactory receptors, and the convergence of sensory neurons onto the secondary neurons in the olfactory bulb.

Site fidelity and homing in tropical coral reef cardinalfish: are they using olfactory cues?

A number of tropical coral reef fish hold station and display restricted home ranges. If artificially displaced, they will return to their home site. We questioned if marine fish are using the same mechanisms for home site detection as many freshwater fish, that is, by olfactory sensing of chemical signals deposited on the substrate by conspecific fish. Behavioral experiments were conducted on Lizard Island Research Station, Queensland, Australia, in 2001 and 2002. Five-lined cardinalfish (Cheilodipterus quinquelineatus) were tested in groups with split-branded cardinalfish (Apogon compressus) as a reference species and individually against Apogon leptacanthus as well as conspecifics of another reef site. The group tests showed that both species preferred artificial reef sites that had previously been occupied by conspecifics. Individual C. quinquelineatus preferred scent of conspecifics from their own reef site to that from another site. They also preferred the scent released by artificial reefs previously occupied by conspecifics of their reef site to that of similar reefs previously occupied by conspecifics of another reef site. No discrimination between species from the same reef site was obtained in experiments with individual fish. Our data suggest that cardinalfish are keeping station and are homing by use of conspecific olfactory signals.

The alarm reaction in crucian carp is mediated by olfactory neurons with long dendrites.

In the present study, we applied a lipophilic tracer, Dil (1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), to the synaptic region of the medial olfactory bulb in formaldehyde-fixed preparations from the crucian carp. We observed staining both in the axons of secondary neurons leading to the brain and in the olfactory receptor neurons (ORNs) of the olfactory epithelium. In those preparations, where staining of the tract was restricted to axons of the medial part of the medial olfactory tract, the majority (86-98%) of the somata of the sensory neurons were found in the deep layers of olfactory epithelium. Since the medial bundle of the medial olfactory tract mediates alarm behaviour in the crucian carp, we conclude that the sensory neurons with long dendrites participate in the reception of alarm pheromones.