Chem. Senses 27: 395-398,
2002
© Oxford University Press 2002
SHORT COMMUNICATION |
The Alarm Reaction in Crucian Carp is Mediated by Olfactory Neurons with Long Dendrites
Division of General Physiology, Department of Biology, PO Box 1051, University of Oslo, N-0316 Oslo, Norway
Correspondence to be sent to: Kjell B. Døving, Division of General Physiology, Department of Biology, PO Box 1051, University of Oslo, N-0316 Oslo, Norway. e-mail: kjelld{at}bio.uio.no
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To date, three morphological types of olfactory receptor neurons (ORNs) have been described in the fish olfactory epithelium: those with tall, intermediate and short dendrites (Ichikawa and Ueda, 1977
; Thommesen,
1983; Hansen et al.,
1997; Hansen and Finger,
2000). In the catfish, Ictalurus punctatus, application
of the lipophilic neural tracer DiI to restricted regions of the olfactory
bulb has shown that ORNs can be morphologically divided into different
categories, depending on the localization of the soma and the length of the
dendrite (Morita and Finger,
1998). The appearance of ORNs of these categories in the olfactory
epithelium depends on which specific part of the olfactory bulb is labelled.
In a previous study (Hamdani et
al., 2001a), we observed that the sensory neurons with
intermediate dendrites and microvilli were labelled concomitantly with the
labelling of the lateral olfactory tract (LOT) axons. As we also observed that
the LOT mediates feeding behaviour (Hamdani
et al., 2001b), we concluded that ORNs with intermediate
dendrites and microvilli mediate feeding behaviour in the crucian carp.
Given the exquisite relationship between morphology and behaviour in the
feeding behaviour neurons, we undertook the present study to explore the
possibility that a particular morphological type of sensory neuron connects to
the medial bundle of the medial olfactory tract (mMOT) and participates in the
alarm reaction (Hamdani et al.,
2000). The lipophilic neural tracer DiI (1,
1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)
was applied to the synaptic region in the medial olfactory bulb of the crucian
carp. In the cases where we observed a staining only of the axons of the mMOT
and not in other parts of the tract, the majority of sensory neurons stained
had long dendrites.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The procedures followed in this study were the same as those used in our previous study (Hamdani et al., 2001a
). Crucian carp, Carassius carassius L., were caught
in a small lake in the outskirts of Oslo, Norway. They were transported to the
aquarium facilities at the Department of Biology. The aquaria had free-flowing
de-chlorinated city water provisions and the fish were fed ad libitum
three times a week. Six fish (21-32 g) were netted from the aquaria and anaesthetized with benzocaine (45 mg/l). After exposure sufficient for lethality, each fish was placed in a holding apparatus and were perfused transcardially with 4% buffered paraformaldehyde (phosphate buffer 0.1 M, pH 7.4). The cranial bones just above the olfactory bulbs and tracts were removed and the mesenchymal tissue in the brain case was aspirated and the meninges around the olfactory bulbs were removed by fine forceps. The heads were then cut at a level corresponding to the most anterior portion of the opercula and were placed in fixative (paraformaldehyde). After 2 days, the skull preparations were placed under a dissection microscope. Small crystals of DiI (Molecular Probes, Eugene, OR, USA) were inserted by a sharp needle into discrete caudal areas in the medial part of the olfactory bulb in situ. The olfactory systems from both sides of each fish were used, giving a total of 12 preparations. After application of the dye, the brain cavity was filled by a 2% agar—agar solution to prevent migration of the crystal away from the site of application. These preparations were placed into buffered paraformaldehyde and kept at room temperature for 6 weeks to permit diffusion of the dye. After this time period, the olfactory epithelium, the olfactory nerve, the olfactory bulb and a part of the olfactory tract on each side was dissected out as a single unit. The preparations where then embedded in 12% gelatin solution and placed into separate casting moulds. The blocks were fixed in 4% paraformaldehyde at 4°C for a minimum of 2 days and cut at 50 µm sections on a Vibratome. Sections obtained were inspected with fluorescence (550 nm excitation, 565 nm emission) on an Olympus microscope (BX50WI) and photographed by an Olympus digital camera (DP50) to show the distribution of the labelled neurons within the lamella.
To visualize the distribution of soma of all cells in the olfactory
epithelium, a DNA probe, the nuclear stain Bisbenzimid (H33258, Reidel de Haen
AG, Sultze, Hanover, Germany), was applied to two slices of the histological
preparations used for DiI. In a segment of a lamella, all nuclei in the
different layers were counted. We should note that for this part of the
present study, we used data from our previous study
(Hamdani et al.,
2001a).
For all Vibratome sections of the olfactory rosette, the position of the cell body of each stained sensory neuron was coarsely categorized by the location of its nucleus within the epithelium. The sensory epithelium was divided into five equal layers from the surface to the basal lamina; layer 1 being the uppermost layer and layer 5 closest to the basal membrane (see Figure 1). Thus, the position of each cell soma was assigned to a particular layer.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fluorescent probe for DNA
The sensory epithelium is pseudostratified
(Farbman, 1992) and cell somas
are found in all depths from the surface to the basal lamina. To observe the
distribution of all cell bodies, a DNA probe was applied to two preparations
of lamellae and cell somas were apparent at various depths. Counting all
nuclei in a limited region of a lamella revealed 426 stained cell somas. The
position of each soma was carried out by coarsely dividing the epithelium in
five layers (Figure 1). As
described, layer 1 is localized at the epithelium surface and layer 5 is
localized at the basal lamina. The distribution revealed 
20% of the cells
in each of the five layers (Figure
3).
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Dil injection into the medial part of the olfactory bulb
The olfactory bulb has an ellipsoid shape 1.6 mm long and 1.3 mm in
diameter. Of the 12 preparations where the DiI was applied in the medial part
of the bulb, there were only three preparations that showed a selective
staining of the axons in the mMOT. It is important to realize that only in the
three preparations where there was a selective staining of the axons of the
mMOT, was there a selective staining of the ORNs in the olfactory epithelium.
In the other preparations where there was a staining of other bundles of the
olfactory tract, all types of sensory neurons were stained.
Figure 1 shows two typical
sensory neurons with cell bodies in layers 4 and 5. These neurons have long
dendrites and can be further characterized by a thickening of the dendrite a
short distance from the soma. These sensory neurons end in a distinct
olfactory vesicle. Although we could reveal cilia at the olfactory vesicle in
some cases, further studies are needed to ascertain the kind of appendages
associated with these sensory neurons.
In nine preparations, the DiI application was placed so that axons of all three bundles in the olfactory tract were stained and in the olfactory epithelium one could observe all three types of sensory neurons at various depths. These observations strengthen our results because placing the DiI crystals outside the synaptic region would tend to increase the possibilities of staining different types of primary as well as secondary neurons.
Counting the ORNs labelled in the epithelium revealed 1856 in preparation 1, 716 in preparation 2 and 718 in preparation 3 (Figure 3). The stained sensory neurons were found in all lamellae of the olfactory rosette. There was no indication that a particular lamella had more ORNs than others and there was no apparent aggregation of ORNs in any particular region of a lamella. Concomitant with the selective staining of the mMOT, when DiI was applied to the medial region of the bulb, the majority of the cell somas of ORNs occurred in layers 4 and 5 of the olfactory epithelium (Figure 2). These were sensory neurons with long dendrite. As seen from the histogram in Figure 3, between 86 and 98% of the cell somas of the sensory neurons stained were found in layers 4 and 5.
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In summary, our results indicate that ORNs with long dendrites terminate on secondary neurons that have projections to the brain via the medial part of the medial olfactory tract.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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It seems to be a general feature of the olfactory system that primary olfactory neurons expressing a particular receptor, although randomly distributed in domains of the epithelium, project their axons to one or a small number of glomeruli (Ressler et al., 1994
; Vassar et
al., 1994; Mombaerts
et al., 1996). The anterograde staining of the sensory
neurons by application at their terminals in the olfactory bulb in fish
demonstrates that a discrete set of neurons can be visualized
(Morita and Finger, 1998). In
the present study on crucian carp, we have associated the sensory neurons,
which have their cell bodies close to the basal lamina, with the secondary
neurons that form the mMOT. Since this bundle is known to mediate the alarm
reaction, it is conceivable that this type of sensory neuron participates in
the reception of alarm substance. Morphology and function
Our investigations have demonstrated a previously overlooked feature of the
pattern of connections of sensory neurons to the olfactory bulb, permitting us
to suggest that a particular morphological type of sensory neuron may be
allotted to a specific behaviour. In the present study, we show that ORNs with
somas in the deep layer of the olfactory epithelium and possibly equipped with
cilia project to the medial part of the bulb, suggesting that they participate
in an alarm reaction elicited by pheromones. ORNs with somas in the middle
layer of the olfactory epithelium equipped with microvilli project to the
lateral part of the bulb and participate in feeding behaviour. Our findings
are in accordance with physiological data from isolated ORNs of the rainbow
trout showing that ciliated sensory neurons respond to pheromones, and that
microvillous sensory neurons respond to amino acids
(Sato and Suzuki, 2001). We
can, therefore, speculate on the third type of ORNs, i.e. the crypt cells with
short dendrites forming the apical layer. If these crypt cells project to the
lateral bundle of the medial olfactory tract (IMOT) it is possible that they
participate in the reception of sexual pheromones, as this part of the tract
mediates the behavioural patterns related to courtship in goldfish
(Stacey and Kyle, 1983) and
cod (Døving and Selset,
1980). This statement is supported by the presence of all three
types of sensory neurons in our preparations where staining was not confined
to axons of a single olfactory tract.
Sensory neurons with long dendrites and alarm
In previous experiments, we found that the mMOT mediates the alarm
reaction. This finding does not, however, imply that this is the only type of
behaviour reactions mediated by the mMOT. Consequently, the sensory neurons
that terminate on the secondary neurons making up the mMOT may also be devoted
to other types of behaviour induced by pheromones. Interestingly, recent
studies indicate that alarm reaction might not only be confined to
ostariophysi (Schutz, 1956),
but also to other groups of fishes (non-ostario-physian), including gobies
(Smith, 1989;
Smith et al., 1991),
poeciliids (Reed, 1969;
García et al.,
1992; Brown and Godin,
1999), cichlids (Wisenden and
Sargent, 1997) and salmonids
(Brown and Smith, 1997; Mirza
and Chivers, 2000,
2001). Consequently, it is
plausible that the alarm reaction is more common than hitherto believed. Thus,
the present study may imply that the sensory neurons with long dendrites
respond to alarm pheromones.
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Acknowledgments |
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This study was supported by the Research Council of Norway. The authors are grateful to Alexander Kasumyan for comments on this manuscript and to George Alexander for correcting the English language.
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Accepted January 24, 2002
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