Alarm reaction in the crucian carp is mediated by the medial bundle of the medial olfactory tract

Experiments were performed to determine which bundles of the olfactory tracts were essential for mediating alarm reaction in crucian carp (Carassius carassius L.). The fish were maintained in physiological saline after surgery to preserve the remaining tracts and postoperative inspections revealed the functionality of the intact tracts. Operations on the tracts were performed symmetrically on both sides. Sham-operated and non-operated fish showed the typical alarm behaviour of fast swimming to the bottom, dashing movements and aggregation when exposed to skin extract which contain alarm substance. Fish with only the medial bundle of the medial olfactory tract intact also displayed the alarm behaviour upon exposure; however, these fish did not react to the amino acid, L-alanine with either feeding response or alarm reaction. Crucian carp which had the medial bundle of the medial olfactory tract cut, leaving both the lateral bundle of the medial olfactory tract and the lateral olfactory tract intact, did not display any alarm reaction to skin extract; however, these fish reacted to exposure to L-alanine with feeding behaviour. There were statistically significant differences between the behaviour scores for the fish subject to different treatments. The present study demonstrates that the medial bundle of the medial olfactory tract appears to be both necessary and sufficient for mediation of the alarm reaction. The results also show that the sensory neurons which respond to alarm substance terminate and make synaptic connections with the secondary neurons that make up the medial bundle of the medial olfactory tract; thereby demonstrating the specificity of the spatial aspect of olfactory processing. The results are discussed with respect to the spatial aspect of organization within the olfactory system, the pattern of generalization across orders of fish, and the functional implications of the spatial arrangement of information transmission between the peripheral olfactory organ and the brain.     

Is feeding behaviour in crucian carp mediated by the lateral olfactory tract?

Experiments were performed to investigate which bundle of the olfactory tract was essential for mediating feeding behaviour in crucian carp. Fish were divided in three groups: control fish, fish with only the lateral olfactory tracts (LOTs) intact and fish with the LOTs cut. The fish were maintained in physiological saline after surgery to preserve the remaining tracts and postoperative inspections revealed the functional status of the remaining tracts. With the injection of food odour into the aquaria the scores for various feeding behaviours--biting, snapping, mouth openings and vertical posture--were not significantly different between those of the control fish and the fish with the LOT intact. Those fish that had the LOT cut but the medial and lateral parts of the medial olfactory tract (mMOT, lMOT) intact had significantly lower feeding-related scores than the other two groups of fish. The results of the present study indicate that the LOT is necessary to maintain the full qualitative and quantitative extent of feeding behaviour in crucian carp.     

Does the lateral bundle of the medial olfactory tract mediate reproductive behavior in male crucian carp?

The olfactory tract in crucian carp (Carassius carassius) is divided into three distinct bundles: the lateral tract (LOT) and the lateral (lMOT) and medial (mMOT) bundles of the medial tract. The LOT has been shown to mediate information associated with feeding behavior, whereas the mMOT mediates information associated with alarm response. The role of the medial olfactory tract (lMOT and mMOT) in reproductive behavior is still under debate. In the present experiment, male reproductive behavior towards prostaglandin-injected females was investigated before and after cutting off the different olfactory tract bundles, to determine which of the tract bundles is essential for mediating reproductive behavior in male crucian carp. The fish were maintained in physiological saline before and after surgery to preserve the remaining tract bundles. Operations were performed symmetrically on both sides and post-operative inspections revealed the functionality of the intact tracts. Sham-operated males and males with only the lMOT intact showed typical reproductive behavior, with following of the female and inspections of the female anal papilla. However, males in which the lMOT was cut, leaving both the mMOT and the LOT intact, showed reduced reproductive behavior. Our results suggest that the lMOT mediates reproductive behavior in male crucian carp.     

Functional and morphological regeneration of olfactory tracts and subtracts in goldfish

Goldfish are ideal vertebrates for the study of regeneration within the central nervous system. The present behavioural and neuroanatomical investigations after bilateral transection of the entire olfactory tracts of either lateral or medial subtracts have been designed (1) to examine the relationship between morphological changes and changes in the perception of spontaneously preferred chemosensory stimuli, (2) to investigate the animals' ability to qualitatively discriminate amino acids in olfactory concentrations (below taste threshold, 10^-6–10^-8 M), one of which had been rewarded preoperatively (specific regeneration), and (3) to examine the discriminative ability for amino acids at concentrations above taste threshold (> 10^-5 M) in intact sham-operated, and in operated specimens at various time intervals before functional regeneration. Within 10–14 days after bilateral transection of the lateral olfactory tracts, specific regeneration was observed. After bilateral transection of the medial olfactory tracts, no immediate behavioural change was recorded for 1 week. Thereafter, goldfish behaviour became unstable and dropped to the chance level for 3–4 weeks. Subsequent to this time the goldfish returned to the preoperative level. Following bilateral crushing of the olfactory tracts and after total tractotomy, a specific regeneration was observed after 4 weeks and 6–8 weeks, respectively, post op. HRP studies showed that after bilateral lesioning a qualitative reinnervation of the respective nuclei within the forebrain by the medial and lateral olfactory subtracts was evident.Abbreviations FB funnel biting - FO funnel orientation - HRP horseradish peroxidase - LOT lateral olfactory tract - MOT medial olfactory tract     

The role of olfaction in the behavioural and physiological responses to conspecific skin extract in Brycon cephalus

A behavioural alarm reaction was shown by 78% of the non-operated juvenile matrinxã Brycon cephalus exposed to a conspecific skin extract, whereas the other 22% did not change their behaviour. The main reaction (72% of those reacting) consisted of a brief initial phase (2 min) of dashing or very rapid swimming followed by a long-lasting period of immobility (up to 30 min). The second most frequent behaviour (22% of the reacting fish) was immobility, with the animals becoming motionless immediately after exposure to the conspecific skin extract, for periods ranging from 5 to 30 min. Only one animal slowed its swimming activity. The responses, evaluated by the number of times the animal crossed the grid in the rear of the aquarium, were significantly lower when compared to the baseline swimming activity. Histological examination of matrinxã epidermis revealed the existence of club cells. Complete sectioning of the olfactory tracts as well as bilateral sections of the olfactory sub-tracts (medial or lateral), stopped the alarm reaction, confirming the importance of olfaction in the ability to recognize the chemical stimulus. Despite the behavioural responses, no variation was detected in blood glucose or cortisol levels, a result that is discussed in terms of the intensity of the disturbance caused by the alarm substance.     

Olfactory bulb axonal outgrowth is inhibited by draxin

Olfactory bulb (OB) projection neurons receive sensory input from olfactory receptor neurons and precisely relay it through their axons to the olfactory cortex. Thus, olfactory bulb axonal tracts play an important role in relaying information to the higher order of olfactory structures in the brain. Several classes of axon guidance molecules influence the pathfinding of the olfactory bulb axons. Draxin, a recently identified novel class of repulsive axon guidance protein, is essential for the formation of forebrain commissures and can mediate repulsion of diverse classes of neurons from chickens and mice. In this study, we have investigated the draxin expression pattern in the mouse telencephalon and its guidance functions for OB axonal projection to the telencephalon. We have found that draxin is expressed in the neocortex and septum at E13 and E17.5 when OB projection neurons form the lateral olfactory tract (LOT) rostrocaudally along the ventrolateral side of the telencephalon. Draxin inhibits axonal outgrowth from olfactory bulb explants in vitro and draxin-binding activity in the LOT axons in vivo is detected. The LOT develops normally in draxin−/− mice despite subtle defasciculation in the tract of these mutants. These results suggest that draxin functions as an inhibitory guidance cue for OB axons and indicate its contribution to the formation of the LOT.     

Chondroitin fragments are odorants that trigger fear behavior in fish

The ability to detect and avoid predators is essential to survival. Various animals, from sea urchins to damselfly larvae, use injury of conspecifics to infer the presence of predators. In many fish, skin damage causes the release of chemicals that elicit escape and fear in members of the shoal. The chemical nature of the alarm substance ("Schreckstoff" in German), the neural circuits mediating the complex response, and the evolutionary origins of a signal with little obvious benefit to the sender, are unresolved. To address these questions, we use biochemical fractionation to molecularly characterize Schreckstoff. Although hypoxanthine-3 N-oxide has been proposed to be the alarm substance, it has not been reliably detected in the skin and there may be other active components. We show that the alarm substance is a mixture that includes the glycosaminoglycan (GAG) chondroitin. Purified chondroitins trigger fear responses. Like skin extract, chondroitins activate the mediodorsal posterior olfactory bulb, a region innervated by crypt neurons that has a unique projection to the habenula. These findings establish GAGs as a new class of odorants in fish, which trigger alarm behavior possibly via a specialized circuit.     

Anti-Predator behaviour of naïve compared experienced juvenile roach

Juvenile roach Rutilus rutilus without any previous contact with predatory fishes reacted to the presence of alarm substances more intensively than fish with previous experience. Among several types of behavioural response observed, some, such as hiding, were not registered in experienced fish. The most pronounced difference between the two juvenile fish categories was manifested in feeding. Naïve fish used more time for feeding than experienced ones. The experiment showed that alarm substance originating from small and young and medium roach induced a stronger reaction than the substance extracted from large and oldéê fish.     

Chemosensory mediation of food searching in the Buenos Aires tetra <Emphasis Type="Italic">Hyphessobrycon anisitsi</Emphasis> (Characidae)

Food searching behavior of the Buenos Aires tetra Hyphessobrycon anisitsi has been investigated before and after experimentally induced anosmia combined with visual deprivation. The threshold concentration of a water extract of familiar food (chironomid larvae) for fish with intact olfaction was 10^–6 g/L. Induced anosmia led to complete loss of the search reaction to food odor. The chemosensory behavioral response to the water extract of food used at high concentrations (10^-1-10^-2 g/L) was not restored in fish after 1.5, 5, or 13.5 months of anosmia. The obtained results show that the external sense of taste is absent or extremely poorly developed in the Buenos Aires tetra.     

Alarm reaction and alert state inGambusia Affinis (Pisces,Poeciliidae) in response to chemical stimuli from injured conspecifics

We studied the behavior of the Poeciliid fishGambusia affinis after the introduction of 3 substances into their tank: a homogenization ofGambusia affinis, a homogenization of the Anabantid fishBetta splendens, and a blank made of distilled water. The response of the fish was measured as a change in their spatial distribution in the tank after the introduction of the substance. Two sizes of fish were used, and theGambusia homogenization produced clear alarm reactions in both, the fish fleeing to the bottom of the tank. This is one of a few examples available of recognition of alarm substances in non-ostariophysian fish. In addition, we found that the small fishes that had recently been exposed to the alarm substance stayed in an ‘alert state’, in which they had an increased sensitivity to mechanical and visual fright stimuli.     

The sensory basis of olfactory search behavior in banded kokopu (<Emphasis Type="Italic">Galaxias fasciatus</Emphasis>)

The sensory basis of olfactory search behavior was investigated in the banded kokopu, Galaxias fasciatus, using a flow tank. In the presence of a 2 cm s(-1) current flow, banded kokopu use both water current and chemical information to locate a food odor source. The superficial neuromasts of the lateral line system mediate the rheotactic component of the odor search. A physical block of one olfactory nostril did not affect the olfactory search strategy employed by banded kokopu in still water or in the presence of a current flow. Thus, there is no evidence that banded kokopu perform a bilateral comparison of the olfactory stimulus during their odor search. Previously, olfaction and gustation have been the only sensory systems shown to directly mediate orientation and movement towards odor sources in fish. The use of hydrodynamic cues by fish in location of an olfactory source has been previously proposed, but without direct experimental identification of the sensory systems employed. This study identifies the contributing roles of both olfactory and hydrodynamic sensory systems to the olfactory search repertoire of fish.     

Projection of sensory neurons with microvilli to the lateral olfactory tract indicates their participation in feeding behaviour in crucian carp.

In the olfactory system of vertebrates, a large number of primary sensory neurons terminate in glomeruli in the olfactory bulb, where they make synapses with a significantly smaller number of secondary neurons. We applied small amounts of a lipophilic neural tracer (Dil) in the glomerular regions of the lateral olfactory bulb in crucian carp, and investigated the centrifugal migration of this stain through the secondary neurons towards the brain and peripherally to the sensory neurons of the olfactory epithelium. In preparations where only the secondary neurons of the lateral olfactory tract (LOT) were stained, the majority (76%) of sensory neurons had cell bodies in the intermediate layer of the olfactory epithelium. Scanning electron microscopy revealed that most of the sensory neurons with cell bodies in the intermediate layers of the olfactory epithelium feature microvilli. Based on observations that the secondary neurons of the LOT mediate feeding behaviour, we feel that there is strong evidence to indicate that the sensory neurons that exhibit microvilli are responsible for mediating the behavioural patterns related to feeding. These results are discussed in relation to physiological experiments on the properties of the sensory neurons and to studies of the innervation pattern of sensory neurons.     

Neurophysiologic detector-a selective and sensitive tool in high-performance liquid chromatography

In the present study neurons from the olfactory system of the fish crucian carp, Carassius carassius L. were used as components in an in-line neurophysiologic detector (NPD) to measure physiological activities following the separation of substances by high-performance liquid chromatography (HPLC). The skin of crucian carp, C. carassius L. contains pheromones that induce an alarm reaction in conspecifics. Extra-cellular recordings were made from neurons situated in the posterior part of the medial region of the olfactory bulb known to mediate this alarm reaction. The nervous activity of these specific neurons in the olfactory bulb of crucian carp was used as an in-line neurophysiologic detector. HPLC was performed with an HP 1100 model equipped with a diode array detector (DAD) and ChemStation software. An adsorbosphere nucleotide-nucleoside 7 microm column was used to separate the substances in the skin extract using artificial pound water (APW) as the mobile phase. UV spectral detection was performed at 214, 254 and 345 nm, and scans (190-400 nm) were collected continuously. This system enabled the selection of peaks in the chromatogram with fish alarm pheromone activity. The neurons in parts of the olfactory system from different aquatic organisms and vertebrates can be used for the detection of species-specific stimuli such as sexual and alarm signals, food odours, and other physiologically significant substances. NPDs clearly offer new and promising options for in-line HPLC as highly selective and sensitive detectors in biological, medical and pharmaceutical research.     

Patterns of olfactory projections in the desert iguana,Dipsosaurus dorsalis

The neural organization of the olfactory system in the desert iguana, Dipsosaurus dorsalis, has been investigated by using the Fink-Heimer technique to trace the efferents of the main and accessory olfactory bulbs, and Golgi preparations to determine the spatial relations between olfactory afferents and neurons in the primary olfactory centers. The accessory olfactory bulb projects to the ipsilateral nucleus sphericus via the accessory olfactory tract. The main olfactory bulb projects to the ipsilateral telen-cephalon via four tracts. The medial olfactory tract projects to the rostral continuation of medial cortex and to the septum. The intermediate olfactory tract projects to the olfactory tubercle and retrobulbar formation. The lateral olfactory tract projects to the rostral part of lateral cortex. The intermediate and lateral olfactory tracts also merge caudally to form the stria medullaris, which crosses the midline in the habenular commissure and distributes fibers to the contralateral hemisphere via two tracts. The lateral corticohabenular tract terminates in the contralateral lateral cortex. The anterior olfactohabenular tract terminates in the contralateral olfactory tubercle, retrobulbar formation and septum. The relation of olfactory afferents to neurons in the medial cortex, lateral cortex, nucleus sphericus, and septum corresponds to a pattern of organization that is typical of many olfactorecipient structures. Such structures are trilaminar, with neurons whose somata are situated in the intermediate layer (layer 2) sending spine-laden dendrites into an outer, molecular layer (layer 1). Olfactory afferents intersect the distal segments of these dendrites. By contrast, other olfactorecipient structures in Dipsoaurus deviate from the familiar pattern. Olfactory afferents intersect somata lying in layer 2 of the retrobulbar formation. Olfactory afferents include some fibers which course perpendicularly to the surface of the olfactory tubercle and extend deep to layer 2.     

The minimum number of neurons in the central olfactory pathway in relation to its function: a retrograde fiber tracing study.

The present study was aimed at determining the functionally essential size of the neuronal population in the central olfactory nervous system. Using conditioned rats who had learnt to avoid repellent (cycloheximide) solution by olfaction, varying degrees of injuries were made to the lateral olfactory tract, a major central olfactory pathway connecting the olfactory bulb to the olfactory cortex. After examining their olfactory ability to discriminate cycloheximide solution from water, intact bulbar projection neurons (mitral cells) with fiber connections to the olfactory cortex were quantified using a retrograde fiber tracing technique. The numbers of retrogradely labeled mitral cells from the rats with normal olfaction ranged between 20 and 92% of the control value, while those numbers from the anosmic rats ranged between 0 and 22%. We conclude that the functionally essential neuronal population is approximately one-fifth of the total in the central olfactory pathway, a presumed threshold value in terms of the ability to avoid cycloheximide solution by olfactory discrimination.     

Chemoreception in chronically anosmiated fish: A phenomenon of compensatory development of the gustatory system

The main results of studying the phenomenon of recovery of sensitivity to food smells after a long olfactory deprivation found in fish are presented. This ability is detected in fish of different taxonomic groups (Chondrostei, Teleostei) having normally external gustatory receptors. At the behavioral level, this effect starts to manifest itself six weeks upon anosmia, and its development apparently ends during the first three to five months in a partial, rather than complete, recovery of sensitivity to food smells. A more prolonged olfactory deprivation (6–14 months) does not cause any further decrease in the threshold concentration of stimulus solutions. The response to other types of chemical signals (alarm pheromone) is not restored. The ability of fish after a prolonged olfactory deprivation to recover sensitivity to food smells is provided by compensatory processes taking place in the external gustatory system. Recovery occurs due to substances that have taste attraction for fish. Species were found in which the food search response in the norm is provided simultaneously by the olfactory and the gustatory systems. The anosmia of such fish leads only to a partial decrease in the efficiency of search behavior. The complementarity of chemosensory systems, their functional interaction, capacity for a compensatory development, and vicariation are considered sensory mechanisms providing the reliability of realization by fish of the most important behavioral patterns, of food, in particular.     

Connections of the olfactory bulb in the piranha (Serrasalmus nattereri)

Summary The connections of the olfactory bulb were studied in the piranha using the Nauta and horseradish-peroxidase methods. Three olfactory tracts project to seven terminal fields in the telencephalon and one in the diencephalon, all of them bilaterally. The contralateral olfactory bulb also receives a small input. All contralateral projections decussate in the anterior commissure and are relatively weak compared to the ipsilateral projections. HRP-containing cells were found in all of the ipsilateral telencephalic aggregates receiving an olfactory tract projection; the contralateral side was free of labeled cell bodies. Although only about one fourth of the entire telencephalon receives a direct olfactory input, the high degree of differentiation of the olfactory system suggests that the piranha depends substantially on the sense of olfaction and that this species may be a good model for further studies on olfactory mechanisms.     

Paradoxical role of C1561T glutamate carboxypeptidase II (GCPII) genetic polymorphism in altering disease susceptibility

Although olfaction could play a crucial role in underwater habitats by allowing fish to sense a variety of nonvolatile chemical signals, the importance of olfaction in species-rich cichlids is still controversial. In particular, examining whether cichlids rely on olfaction for reproduction is of primary interest to understand the mechanisms of speciation. In the present study, we explored the V1R (also known as ora) genes, which are believed to encode reproductive pheromone receptors in fish, in the genomes of Lake Victoria cichlids. By screening a bacterial artificial chromosome library, we identified all six intact V1R genes (V1R1 to V1R6) that have been reported in other teleost fish. Furthermore, RT-PCR and in situ hybridization analyses showed that all of the V1R genes were expressed in the olfactory epithelium, indicating that these receptors are functional in cichlids. These observations indicate that cichlids use V1R-mediated olfaction in some ways for their social behaviors.