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.

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.

The vomeronasal cavity in adult humans.

We observed the surface of the anterior part of the nasal septum of living subjects using an endoscope. In approximately 13% of 1842 patients without pathology of the septum, the vomeronasal pit was clearly observed on each side of the septum, and in 26% it was observed only on one side. The remaining observations indicated either the presence of putative pits or no visible evidence of a pit. However, repetitive observations on 764 subjects depicted changes over time, from nothing visible to well-defined pits and vice versa. Based on 130 subjects observed at least four times, we estimate that approximately 73% of the population exhibits at least one clearly defined pit on some days. By computer tomography, the vomeronasal cavities were located at the base of the most anterior part of the nasal septum. Histological studies indicated that the vomeronasal cavities consisted of a pit generally connected to a duct extending in a posterior direction under the nasal mucosa. Many glands were present around the duct, which contained mucus. There was no sign of the pumping elements found in other mammalian species. Most cells in the vomeronasal epithelium expressed keratin, a protein not expressed by olfactory neurons. Vomeronasal epithelial cells were not stained by an antibody against the olfactory marker protein, a protein expressed in vomeronasal receptor neurons of other mammals. Moreover, an antibody against protein S100, expressed in Schwann cells, failed to reveal the existence of vomeronasal nerve bundles that would indicate a neural connection with the brain. Positive staining was obtained with the same antibodies on specimens of human olfactory epithelium. The lack of neurons and vomeronasal nerve bundles, together with the results of other studies, suggests that the vomeronasal epithelium, unlike in other mammals, is not a sensory organ in adult humans.

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.

Optics of the developing fish eye: comparisons of Matthiessen's ratio and the focal length of the lens in the black bream Acanthopagrus butcheri (Sparidae, Teleostei).

Matthiessen's ratio (distance from centre of lens to retina:lens radius) was measured in developing black bream, Acanthopagrus butcheri (Sparidae, Teleostei). The value decreased over the first 10 days post-hatch from 3.6 to 2.3 along the nasal and from four to 2.6 along temporal axis. Coincidentally, there was a decrease in the focal ratio of the lens (focal length:lens radius). Morphologically, the accommodatory retractor lentis muscle appeared to become functional between 10-12 days post-hatch. The results suggest that a higher focal ratio compensates for the relatively high Matthiessen's ratio brought about by constraints of small eye size during early development. Combined with differences in axial length, this provides a means for larval fish to focus images from different distances prior to the ability to accommodate.

Structure and function of the vomeronasal organ.

Many animals use their vomeronasal organs to gain direct and specific contact with chemical cues released by congeners and in biological fluids. These cues provide information about the physiological status of the emitter and facilitate or regulate social interactions such as sexual relationships. The present review gives a short description of the discovery of the vomeronasal organ and the pivotal findings of Jacobson. The distribution of the organ and its anatomy in some vertebrates are described. The mechanisms for stimulus entry and egress are discussed, and the findings that led to the appreciation of the vomeronasal organ in mammals as a main chemosensory organ for pheromones mediating reproductive status and inducing sexual behaviour are reported. The anatomical, biochemical and functional properties of the receptor neurones are described.

Scanning electron microscopy and gramicidin patch clamp recordings of microvillous receptor neurons dissociated from the rat vomeronasal organ.

Vomeronasal organs from female rats were dissociated and isolated microvillous receptor neurons were studied. The isolated receptor neurons kept the typical bipolar shape which they have in situ as observed by scanning electron microscopy. We applied the perforated patch-clamp technique using the cation-selective ionophore gramicidin on freshly isolated and well differentiated receptor neurons. The mean resting potential was -58+/-14 mV (n=39). The contribution of the sodium pump current to the resting potential was demonstrated by lowering the K+ concentration in the bath or by application of 100 microM dihydro-ouabain. The input resistance was in the range of 1-6 GOmega and depolarizing current pulses of a few pA were sufficient to trigger overshooting action potentials. In voltage clamp conditions a fast transient sodium inward current and a sustained outward potassium current were activated by membrane depolarization. These observations indicate that freshly isolated vomeronasal receptor neurons of rats can be recorded, using gramicidin, with little modification of the intracellular content. Their electrophysiological properties are very similar to those observed in situ. Four out of eight female vomeronasal receptor cells were depolarized by diluted rat male urine.

Anoxic depression of light-evoked potentials in retina and optic tectum of crucian carp.

The crucian carp is an exceptionally anoxia-tolerant vertebrate. For the brain, with its very high rate of ATP use, depression of energy use is likely to be an important strategy for anoxic survival. This study shows that the light-evoked response of the retina and the corresponding evoked potential in optic tectum decrease in amplitude by 69 and 75%, respectively, during 38 min of anoxia, and by about 90% after 1 h in anoxia. Both responses were restored upon reoxygenation. The length of light exposure (5 s or 100 ms) did not affect the degree of anoxic depression. These results are the first to show an anoxia-induced depression of central nervous system (CNS) activity in vivo in this species, and indicate that the crucian carp temporarily turns off its visual sense in order to reduce neural energy use during anoxic condition.

Functional role of receptor neurons in encoding olfactory information.

In the present review we have considered the properties of the olfactory receptor neurons and discuss the strategy these cells use to perform their signaling task. Special emphasis is laid on the mechanisms for setting the membrane potential at rest and the mechanisms that the cell can use to respond with action potentials to significant stimuli only. We demonstrate that the firing properties of the receptor neurons depend upon the initial level of the membrane potential. We present the idea that the olfactory glomerulus can function as a unit in olfactory processing. In this perspective the olfactory receptor neuron is a subunit of the olfactory glomerulus.

Direct influence of the sodium pump on the membrane potential of vomeronasal chemoreceptor neurones in frog.

1. Whole-cell measurements were made from microvillous receptor neurones isolated from the frog vomeronasal organ. We examined the mechanisms that determined the value of the resting membrane potential. 2. Cells recorded in Ringer solution containing 4 mM K+ showed a resting membrane potential of -88 +/- 20 mV (mean +/- 1 S.D., n = 56). Sixty-six per cent of the cells had stable resting potentials more negative than the calculated equilibrium potentials for K+ (EK, -82 mV) indicating the presence of a hyperpolarizing outward pump current. 3. Cells recorded with an intracellular solution containing Na+ instead of K+, to set EK at 0 mV, presented stable membrane potentials in the range -65 to -119 mV when bathed in a normal Ringer solution. 4. Ouabain, a specific inhibitor of the Na+,K(+)-ATPase, blocked the outward sodium pump current (Ip) and depolarized the membrane. 5. The sodium pump current, measured as the current blocked by 0.5 mM dihydro-ouabain, was linearly related to the membrane potential in the range -60 to -120 mV. The reversal potential measured with a calculated free energy of ATP hydrolysis of -36.2 kJ mol-1 was estimated to be -143 mV. 6. Reduction of the external K+ concentration to 0 mM depolarized the membrane to less than -40 mV. Voltage-clamp observations in this condition indicated a reduction of Ip. Ouabain added to the bath reduced the blocking effect of low external K+. The addition of external K+ activated Ip and induced a rapid hyperpolarization of the cell membrane. 7. At membrane potentials more negative than -80 mV, an inward rectifying depolarizing current characterized as Ih was activated. When Ih was blocked by 5 mM external Cs+ the resting membrane potential increased. 8. These data indicate that the membrane potential of the vomeronasal receptor neurones is not generated by a passive diffusion of K+ ions but by the hyperpolarizing current created by the Na+,K(+)-ATPase. We propose that the resting potential is set by a balance between Ip and Ih. The physiological implications of these mechanisms for setting the resting potential are discussed.

[The vomeronasal organ--a rediscovered sensory organ]. / Vomeronasale--et gjenoppdaget sanseorgan.

The authors review the function and anatomy of the vomeronasal organ (organ of Jacobson), a chemosensory organ situated in the nose. In mammals, stimulation of the vomeronasal organ induces sexual behaviour and changes the hormonal status of males and females. Impairment of the vomeronasal organ in rats disrupts both the females' ultrasound calling during oestrus and their maternal behaviour. Stimulation of the vomeronasal organ promotes sexual maturation of juvenile females and induces abortion and production of pheromones. Earlier on, the vomeronasal organ in man was believed to be present only at the foetal stage; in 1991 its presence was reestablished in adults. Electrophysiological studies show that the vomeronasal organ of man is stimulated by steroids found in human skin. The physiological properties of the receptor cells of the vomeronasal organ are different from those of the olfactory organ.

Voltage-dependent currents in microvillar receptor cells of the frog vomeronasal organ.

Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 microns) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was -61 +/- 13 mV (mean +/- SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of -60 to -86 mV, with almost no spontaneous action potential. The input resistance was in the G omega range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2-10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near -45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near -75 mV. An outward K+ current was blocked by internal Cs+ ions or by external tetraethylammonium or Ba2+ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd2+ ions. A voltage-dependent Ca2+ current was observed in an iso-osmotic BaCl2 solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.

Behaviour of dogs during olfactory tracking.

The ability to detect the direction of a track is of vital importance to animals of prey and is retained in many modern breeds of dogs. To study this ability, four trained German shepherd tracking dogs, equipped with head microphones to transmit sniffing activity, were video-monitored after being brought at right angles to a track where the position of each footprint was known. Three phases could be recognized in the dogs' behaviour: (1) an initial searching phase, during which the dog tried to find the track, (2) a deciding phase, during which it tried to determine the direction of the track and (3) a tracking phase, in which it followed the track. During ten tests on 20-min-old tracks on grass, and ten tests on 3-min-old tracks on concrete, the dogs always followed the track in the correct direction (i.e. in the direction the track was leading). During the deciding phase the dogs moved at half the speed and their periods of sniffing lasted three times as long as during the other two phases. The deciding phase lasted 3-5 s, while the dogs sniffed at 2-5 footprints. The dogs' ability to determine track direction in this time must rely on accurate methods of sampling air and a remarkable sensitivity for certain substances.

The effect of Cu (II) on the electro-olfactogram (EOG) of the Atlantic salmon (Salmo salar L) in artificial freshwater of varying inorganic carbon concentrations.

The effect of inorganic copper species was studied by recording the receptor potential, electro-olfactogram (EOG), from the olfactory epithelium of Atlantic salmon (Salmo salar L). In a series of experiments, the olfactory organ was irrigated with aqueous copper solutions with concentrations of the free cupric ion (Cu2+) ranging from 0.2 to 9.7 microM. The diverse copper species were created by varying the amount of bicarbonate (NaHCO3) in artificial freshwater solutions of equal total copper concentrations. In general, these copper solutions induced a slow depolarization of the baseline followed by a hyperpolarization. The amplitudes of these variations in baseline potentials increased with increasing concentrations of Cu2+ ion, i.e., decreasing concentrations of NaHCO3. Stimulating the olfactory epithelium with L-alanine during the copper exposure evoked atypical EOG responses. The amplitudes and form of the EOGs changed drastically with increasing Cu2+ concentrations, with significant correlation between the reduction in EOG amplitudes and the Cu2+ concentration. The results indicate that among the copper species tested the toxic effect is caused mainly by the dissolved Cu2+ ion. The results also suggest that the Cu2+ ion exerts its toxic effects on the transduction mechanisms of the olfactory receptor cells. The different EOG profiles obtained in response to varying Cu2+ concentrations indicate that this ion affects the transduction mechanisms at different stages.

The central connections of the olfactory bulbs in cod, Gadus morhua L.

The central projections of the "classical" olfactory system of the cod, Gadus morhua were examined with horseradish peroxidase and cobalt tracing techniques. Label was applied to the olfactory bulb or selectively to central stumps of sectioned individual olfactory tract bundlets. The olfactory bulb projects bilaterally to restricted areas of the dorsolateral, ventromedial and basolateral telencephalon, anterior commissural and preoptic areas, habenular nuclei, dorsal thalamus and to the nucleus posterior tuberis in the diencephalon. An interbulbar connection courses in the medial olfactory tract (MOT). Contralateral projections were less pronounced than on the ipsilateral side. More specifically, the lateral olfactory tract (LOT) projects ipsilaterally to the telencephalon into the Dlv, Dc, Vs and Dp areas. The lateral bundlet of the medial olfactory tract (IMOT) terminates in the Dlv and Dc areas. The medial bundlet of the medial olfactory tract (mMOT) terminates in Vv and Vd. The fused lMOT and mMOT project to the caudal telencephalon in the Vs and Dp. Neurons projecting to the olfactory bulb were located bilaterally in the telencephalon. The majority of the bulbopetal fibers course via the lateral part of the MOT; a few neurons also project to the bulb through the other bundlets of the olfactory tract. The results are compared with previous studies on the olfactory projections of other teleost species and discussed with respect to the reported functional differentiation of the olfactory system in teleosts.

Gonadotropin releasing hormone (GnRH)--a novel olfactory stimulant in fish.

The aim of this study was to elucidate the putative role of the gonadotropin releasing hormone (GnRH or LHRH) as an olfactory stimulant in fish. We report for the first time extreme sensitivity of the olfactory organ in the rainbow trout (Oncorhynchus mykiss) to GnRH. Recordings of the electroolfactogram (EOG) showed an electrophysiological response to 10(-16) M GnRH four times the amplitude of the response to a fresh water control stimulus. By stimulating the olfactory epithelium with several GnRH analogs and fragments of the decapeptide, the biologically active region of GnRH could be partly elucidated. The response profile of GnRH differed from that of the positive control odorant L-alanine, suggesting that separate receptors or receptor cells are involved. We propose that this potent odorant may act as a reproductive pheromone in fish.

Differential effects of mercurial compounds on the electroolfactogram (EOG) of salmon (Salmo salar L.).

The effects on the salmon (Salmo salar L.) electroolfactogram (EOG) of the two mercurials, mercuric chloride (HgCl2) and methylmercuric chloride (CH3HgCl), were studied. The EOG responses were evoked by stimulating the olfactory epithelium with 340 microM L-alanine for 10 sec every second minute during a 1-hr period. Each EOG response consisted of an initial peak component followed by a sustained component with an amplitude about 40% below the peak value. Three experimental series, each comprising six fish, were carried out. In the first series, the rosette was irrigated solely with artificial "fresh water." In the two other series, a 5-min exposure to mercury (HgCl2 or CH3HgCl, at 10(-5) M) was included after 10 min and a 15-min exposure after 45 min. The mercuric ion (Hg2+) eliminated the peak response within 2 min and suppressed the sustained response to about 35%. During the subsequent irrigation with mercury-free fresh water, both EOG components regained about 50% of their initial amplitudes. In contrast, methylmercury induced a steady and parallel decline of both the peak and the sustained responses, which were not reversed by rinsing the epithelium with fresh water. The results of this study demonstrate the vulnerability of the olfactory receptor function in fish to mercury exposure. Also, they show the very different effects of inorganic and organic mercurials upon the EOG.

Histochemical demonstration of mercury in the olfactory system of salmon (Salmo salar L.) following treatments with dietary methylmercuric chloride and dissolved mercuric chloride.

The deposition of organic and inorganic mercury compounds was studied histochemically in the salmon (Salmo salar L.) olfactory system. One group of salmon was given fodder pellets containing methylmercuric chloride (CH3HgCl, 99 micrograms Hg/g) for 4 weeks. Other groups of fish were exposed to dissolved mercuric chloride (HgCl2, 270 micrograms Hg/liter) for 2, 6, and 12 hr, respectively. In both series of experiments, the radioisotope 203Hg was included in order to determine the accumulation of mercury in the olfactory system. Gamma-spectrometry showed that both mercury compounds accumulated in the olfactory rosettes and their nerves. Tissue sections from the rosettes and olfactory nerves were subjected to autometallographic silver enhancement, thereby rendering mercury deposits visible for light and electron microscopy. Microscopic analysis demonstrated an intense and comprehensive Hg deposition in the axons and Schwann cells of both methylmercury- and inorganic mercury-exposed fish. On the other hand, the two mercury compounds showed different staining patterns in the sensory epithelium. The silver grains evoked by methylmercury were localized predominantly in lysosome-like inclusions within the receptor cells, while those produced by HgCl2 exposure were situated mainly along the borders of neighboring cells. The present findings that organic and inorganic mercury compounds were deposited in the olfactory system along its whole length, from the receptor cell apices to the brain, support the electrophysiological results presented elsewhere (Baatrup et al., 1990, Ecotoxicol. Environ. Safety 20, 269-276).