333
The fine structure of the olfactory surface of teleostean
fishes
By L. H. BANNISTER
(From the Department of Biology, Guy's Hospital Medical School, London)
With 4 plates
Summary
The olfactory receptors of Phoxinus are of at least two distinct types: those with
ciliated tips, and those with rod-shaped tips to their dendrites. Both Phoxinus and
Gasterosteus possess ciliated receptors with 4 to 6 cilia arranged in a ring on each
dendritic tip. The cilia resemble non-sensory motile cilia in most respects. They are,
however, shorter, and lack 'arms' on the outer ring of double fibrils. They also lack
striated rootlets, but possess basal feet, which point towards the centre of the ring of
cilia. The rod-tipped receptors contain three or more longitudinally oriented bundles
of fibres. Microtubules are present in receptor dendrites, in supporting cells, and in
ciliated non-sensory cells. Their mean diameter in dendrites is 180 A.
The different forms of receptor, and their function, are discussed.
Introduction
ALTHOUGH the structure of the olfactory organ of teleostean fishes has been
studied by a number of workers, (Dogiel, 1887; Jagodowski, 1901; Burne,
1909; Hopkins, 1926; Pipping, 1926; Liermann, 1933; Laibach, 1937; Holl,
1965; and others), the nature of the olfactory receptor endings themselves
is poorly understood. Laibach (1937) examined these endings in the eel
Anguilla, Trujillo-Cenoz (1961) studied them in two species of cyprinodont
fish with methods including the use of electron microscopy, and Holl (1965)
investigated them in a wide range of teleosts. All three of these authors found
the receptor endings to be similar to those of all other vertebrates so far
examined. Their results, however, conflict with the earlier findings of Jagodowski (1901) for the pike Esox, and of Hopkins (1926) for the marine scup
Stenesthes. Both of these authors reported a single long apical process on each
olfactory receptor instead of the usual group of cilia.
The aim of the present study was to throw light on this problem, and to
examine the olfactory surface in detail. A comparison was made between its
condition in two teleosts: the minnow Phoxinus phoxinus, a member of the
Cyprinidae, which is a relatively unspecialized teleost family showing several
primitive actinopterygian features, and the three-spined stickleback Gasterosteus aculeatus, which belongs to the Gasterosteidae, a relatively advanced
teleost group (Bertin and Arambourg, 1958).
Phoxinus, like all the Cyprinidae, has a well-developed olfactory organ, and
may be classed with that group of fishes which depends upon ciliary action
[Quart. J. micr. Sci., Vol. 106, pt. 4, pp. 333-42, 1965.]
334
Bannister—Olfactory surface of teleosts
and the movement of the fish relative to the surrounding water for the
ventilation of the olfactory cavity (Pipping, 1926). This is the primitive
condition, found in all the less advanced Actinopterygii. There are two
external nares on each side, placed close together; water is drawn through the
anterior and expelled through the posterior aperture, passing across the
olfactory mucosa in the process. This current is created by the large numbers
of cilia which surround the mucosa, and by groups of cilia on the mucosa
itself. The olfactory epithelium is thrown into a variable number of folds,
the lamellae, which project far into the lumen of the olfactory organ.
Gasterosteus forms a useful contrast in being a microsmatic species having
a different method of olfactory ventilation. The olfactory cavity is small and
quite devoid of motile cilia. There is only one nasal aperture on either side,
through which water is inhaled and expelled by pressure changes brought
about by normal respiratory movements, transmitted from the pharynx. The
olfactory epithelium is again thrown into a number of folds, but these are of
small dimensions.
Materials and methods
Small (3 to 5 cm) specimens of Phoxinus and Gasterosteus were used in this
study. Olfactory organs for electron miscroscopy were carefully excised from
freshly killed fish, and fixed in the 1% osmium fixative (pH 7-4) employed
by Robertson, Bodenheimer, and Stage (1963). After fixation for 4 h at
o° to 2° C, the pieces of tissue were dehydrated in an ethanol series, and
embedded in Araldite. Sections were cut with a glass knife mounted in
a Huxley ultramicrotome, and most were stained for 1 h in 1% potassium
permanganate, differentiating in 0-5% citric acid. All micrographs shown in
this paper are of sections stained in this manner. Some pieces of tissue were
also stained in block with phosphotungstic acid.
An R.C.A. EMU-3 electron microscope, operating at 50 kV, was employed
in making observations. This instrument was calibrated with a carbon replica
of an optical grating, giving a 2% limit of confidence at highest magnifications.
Thick sections for light microscopy were taken from Araldite-embedded
tissue, and viewed under the -j^ in. objective of a phase-contrast microscope.
Paraffin sections were also cut, and were stained with haematoxylin and eosin,
toluidine blue, and silver proteinate. The osmium-fixed and Aralditeembedded tissue was, however, by far the best for observing the finer details
of the olfactory surface, especially when stained with potassium permanganate.
The lengths of olfactory and non-olfactory cilia were determined in such
material.
Preparations to show the form of individual receptor cells were made by
a double rapid Golgi method, as follows. Freshly killed fish were decapitated,
and the lower jaw and cranial roofing bones removed. The remaining pieces
of tissue were placed in a solution containing 1% osmium tetroxide and
2-5% potassium dichromate, and kept in the dark for 3 days. They were then
washed in 2 changes of 0-75% silver nitrate, being kept in the final solution
Bannister—Olfactory surface of teleosts
335
for 48 h in the dark. The specimens were returned to the original osmicdichromate solution for 2 days, and treated once more with silver nitrate for
a further 4 days. At the end of this period they were washed for 6 h in several
changes of 70% ethyl aclohol, dehydrated, and embedded rapidly in lowviscosity nitrocellulose. Sections were cut at 120 to 140 ft on a sledge microtome, and mounted in neutral balsam without a coverslip.
The lengths of cilia and 'rods' were measured from camera lucida drawings,
using phase-contrast equipment with a T ^ in. objective. With electron micrographs, the dimensions of relatively large structures were determined from
photographic enlargements of X 16,000. Smaller structures, such as microtubules, were measured with a travelling microscope on negative plates taken
at a magnification of X 50,000. In this latter case, care was taken that only
transverse sections of tubules in sharp focus were measured.
Observations
Light-microscope studies of the olfactory epithelium have been made by
several authors in a number of species of teleost, as mentioned in the introduction, and its histology will therefore be considered only briefly. A diagram
of the arrangement of the structures at the olfactory surface, as seen in
Phoxinus, is shown in fig. 1. The mucosa itself consists of a simple columnar
epithelium resting on a basal membrane and supported by connective tissue.
As in all vertebrates, it comprises the olfactory receptor cells, supporting
cells, and basal cells. Gasterosteus lacks the normal ciliated epithelial cells
present in Phoxinus (fig. 1), which there cover the tips of the lamellae, and
are scattered in groups over the whole surface. Mucous gland cells are found
in association with these ciliated epithelial cells.
The individual receptor cells were found to be similar to those of other
vertebrates in general appearance (fig. 2). As noted by several authors (see
Discussion), there is a great variation in the shape of the cells and in the
relative lengths of dendrites within a given olfactory organ (figs. 3 and 4).
Another type of variation is in the form of the free endings of the dendrites.
These may possess a flower-like array of cilia, which is the usual pattern in
vertebrates, or they may simply project from the epithelial surface as tapering
rods. I have seen the latter type of ending, which appears not to have been
described before, in Golgi preparations, in Araldite and in paraffin sections;
the electron-microscope appearance will be described later in this paper.
The electron-microscope studies of the olfactory epithelium will now be
reported. As there is little difference from the condition found by TrujilloCen6z (1961) in the deeper parts of the epithelium these observations will
largely concern the structures at or near its surface (fig. 1). The surface is
formed by the free ends of the supporting cells and of the receptors. The
latter endings are sometimes situated in shallow depressions of the surface,
and sometimes are raised above it. The outer surface of the epithelium,
including the cilia, is covered by an electron-dense layer some 200 A thick.
This covering is shown in section surrounding a cilium in fig. 15. Extending
336
Bannister—Olfactory surface of teleosts
down from the epithelial surface at the boundaries of all cells, are junctional
complexes of the type described by Farquhar and Palade (1963). These
consist of three units: a distinct tight junction (figs. 5 and 18), or zonula
occludens; a rather, indistinct adhering band, or zonula adhaerens; and a
desmosome (figs. 18 and 19), or macula adhaerens. Farquhar and Palade (1963)
non-sensory ciliated epithelial cell
ciliated receptor
rod-shaped
receptor ending
supporting cell
receptor with microvilli
FIG. 1. Diagrammatic reconstruction of the olfactory surface of Pkoxinus, based on
a series of sections cut parallel to its plane.
found two types of desmosome in mammalian epithelium, distinguishable by
their relation to the adjoining tonofibrils, in that the latter either abut at right
angles, or run parallel. In the olfactory epithelia studied here, I have found
the first type to be present only between adjacent non-sensory (that is, supporting and ciliated) cells (fig. 18). The second type of desmosome lies only at the
junction of supporting cells and receptor dendrites (fig. 19). In this last case,
tonofibrils are present in large numbers only on the side of the supporting
cell, although a few have been seen in the dendrites.
FIGS. 2 to 8. Olfactory epithelium of Phoxinus.
FIGS, 2 to 4. Golgi preparations of ciliated receptors. The axon infig.3 is not in the plane
of focus. FIG. 5. Section cut vertical to the surface, showing various cell types. FIG. 6. Section as in fig. 5, but through a receptor possessing microvilli. FIG. 7. Section cut slightly
obliquely to the surface. FIG. 8. Section as in fig. 7, showing the microvilli of a receptor.
a, axon of an olfactory receptor; c, cilium; ce, centriole; cep, ciliated non-sensory cell;
cr, ciliated receptor cell; d, dendrite of olfactory receptor; m, mitochondrion; mr, receptor
with microvilli; mv, microvillus; rr, rod-shaped receptor ending; rt, rootlet of cilium;
sc, supporting cell; z, zonula occludens.
FIGS. 2-8
L. H. BANNISTER
FIGS.
9-17
L. H. BANNISTER
Bannister—Olfactory surface of teleosts
337
The receptor cells
These cells are typical vertebrate olfactory receptors consisting of bipolar
neurons with thin axons and thicker, but tapering dendrites, the tips of which
are usually slightly swollen and form the receptor surface. Internally, all
dendrites contain longitudinally oriented microtubules, or neurotubules
(figs. 13, 19, and 25). The presence of these within a cell process of dendritic
dimensions is taken as an indication of that cell being a receptor.
The diameters of 32 microtubules, representing 5 ciliated receptors in
a 3 cm specimen of Gasterosteus, were carefully measured, giving a mean
value of 183 A i i o / 5 A (standard deviation), that is about 180 A.
Among the microtubules are scattered longitudinally oriented mitochondria,
and vesicles varying in size and electron density (fig. 21).
The receptor cells fall into 3 categories, distinguished by the form of their
distal tips. These will now be considered. The first category comprises the
ciliated olfactory receptors (see particularly figs. 1, 9 to 17, and 25), each of
which possesses a ring of 4 to 6 cilia upon its convex distal end. The diameters
of 11 of these distal dendritic tips were measured in a 3 7 cm specimen of
Phoxinus, giving a mean of i*56j&±o-24/x. The olfactory cilia are shorter
than the neighbouring non-sensory cilia, in Phoxinus. In a 3 7 cm fish, for
example, 50 cilia of each type were measured, giving a mean length of 5-0 ^ i
O"9/x for olfactory cilia, against 12-0/x±2'6p for non-olfactory ones. The
diameter of the olfactory cilia (o-34/x=|=oiO42^, from 31 measurements in
a 3 7 cm specimen of Phoxinus) is, however, much the same as that of the
non-sensory type. In most respects, the internal structure of the olfactory
cilia resembles that of the non-sensory ones (figs. 14 to 16). The usual 9 pairs
of outer fibrils and the 2 central ones are present; fine strands connect the
outer fibrils with the central pair, and with the ciliary membrane. The basal
body, too, has its usual appearance and bears a striated basal foot pointing
towards the centre of the ring of cilia (figs. 11, 13, and 25). Some of the
dendritic microtubules are found connected to the basal body and basal foot
(fig. 25). The olfactory cilia differ from the non-sensory ones in two important
respects: 'arms' (Gibbons and Grimstone, i960)are absent from the peripheral
fibrils of the cilia (fig. 15), although they are found in neighbouring nonsensory cilia in Phoxinus, and there are no striated rootlets attached to the
Fics. 9, 10, and n . Transverse sections through ciliated receptor endings at three successive levels (Phoxinus). FIGS. 9 and 10 show six cilia, and fig. 11 has five. T h e cilia in fig. 9
marked X do not belong to this group (determined from serial sections).
FIGS. 12 and 13. Oblique sections through ciliated receptor tips {Phoxinus).
FIGS. 14 and 15. Transverse sections through olfactory cilia at two levels, showing the
normal cilium structure (Gasterosteus). 'Arms' are missing on the outer fibrils in fig. 15.
F I G . 16 is the basal body of a cilium with a basal foot (Gasterosteus).
F I G . 17. An atypical cilium from Gasterosteus. Figs. I 4 t o 17 are all at the same magnification.
b, basal body of cilium; bf, basal foot of cilium; c, cilium; cf, central fibrils of cilium; cm, cell
membrane; h, hillock on tip of dendrite; mt, microtubule; nw, microvillus; o, outer electrondense sheath of olfactory epithelium; pf, peripheral fibrils of cilium; sc, supporting cell;
v, vesicle.
2421.4
A3 2
338
Bannister—Olfactory surface of teleosts
basal bodies. A unit membrane of the normal type forms the ciliary membrane,
but often its outer lamella stains only indistinctly, and merges with the
electron-dense covering of the epithelium, as shown in figs. 14, 15 and 17.
While the foregoing description applies largely to both species of fish, it is
noticeable that in Gasterosteus there is a significant number of atypical cilia,
whose fibrils differ from the usual 'nine-plus-two' pattern. One of these is
pictured in fig. 17.
The second type of receptor ending bears a variable number of microvilli
instead of cilia (figs. 1, 6, and 8). In other ways, however, it closely resembles
the ciliated form, and possesses a number of randomly oriented centrioles
buried at various depths in the cytoplasm of the dendrite (fig. 6). The presence
of these structures is strong evidence that this type of cell is an immature form
of the ciliated receptor (see the description of developing ciliated cells by
Sotelo and Trujillo-Cenoz, 1958).
The third type of receptor ending I found only in Phoxinus, and appears
to be unlike any olfactory structure that has been described before in vertebrates. This type bears neither cilia nor microvilli, but extends simply as
a naked rod from the epithelial surface (figs. 1, 20, 21 to 23). The diameter of
this structure at the level of its emergence from the surrounding cells is
approximately the same as that of the ciliated receptors (fig. 20), but it tapers
gradually to end bluntly in a single or double point. The mean length of such
endings in a 3-7 cm specimen of Phoxinus was found to be approximately 4/z,
although it is possible that they are longer than this in life, and are damaged
during preparation for sectioning.
Internally, this type of receptor resembles the ciliated type in the presence
of longitudinally arranged microtubules and mitochondria, and of small
vesicles. There are, however, in addition to these, 3 or more bundles of
closely packed fibres (figs. 20, 22 to 24) which extend from a position deep
in the dendrite into its naked tip (fig. 23). Each constituent fibre is about 50 A
thick. The rod-shaped endings seem to be present only in some parts of the
epithelium, and where present are much less frequent than the ciliated endings,
the ratio being about 1:10.
The supporting cells
These are polygonal columnar epithelial cells, and lie in the same relation
to the receptors as found by de Lorenzo (1957) in the rabbit: they lie between
or envelop the receptors so that no two of the latter lie in juxtaposition
(figs. 1, 5, 7, and 18). Their surfaces are usually slightly convex, and bear
FIG. 18. Section cut parallel to plane of surface, showing electron-dense vesicles in the
supporting cells, desmosomes, and zonulae occludentes {Gasterosteus).
FIG. 19. Enlarged portion of fig. 18, showing the type of desmosomal junction between
receptor and supporting cells.
FIG. 20. Section in the same plane as fig. 18, showing the dendrites of rod-tipped and
ciliated receptors (Phoxinus).
cr, ciliated receptor; dm, desmosome; mt, microtubule; rr, rod-tipped receptor; sc, supporting
cell; t, tonofibrils; v, vesicle; z, zonula occludens.
FIGS. 18-20
L. H. BANNISTER
FIGS. 21-24
L. H. BANNISTER
Bannister—Olfactory
surface of teleosts
339
a small number of irregular microvilli. The cytoplasm immediately beneath
the free surface is poorly provided with endoplasmic reticulum, but carries
many vesicles and large canals. In Gasterostew the vesicles are particularly
numerous and electron-dense (fig. 18). In this position also, in both species of
fish, a small number of microtubules are present, having dimensions similar
to those of the ciliated receptors. Internal to this zone is a layer containing
tonofibrils which run parallel to the epithelial surface, and connect with the
desmosomes (figs. 18 and 19), as mentioned already in the general remarks
on the olfactory surface. Tonofibrils and desmosomes are also found deeper
in the cytoplasm, but there they do not form a distinct stratum. An endoplasmic reticulum and mitochondria are present, as usual, in the deeper parts
of the cell.
Ciliated non-sensory cells
These cells, which are not found in Gasterosteus, are typical of normal ciliated epithelial cells in their fine structure (figs. 1 and 5). Each possesses a
large number of long cilia with basal feet, striated rootlets, microtubules
connected to the ciliary bases, and a zone of mitochondria beneath. It is
noteworthy that each cilium carries only one striated rootlet, which runs at an
angle towards the mitochondrial zone and bears on its undersurface a deep
groove.
Discussion
In the present study it has been shown that in Phoxinus there are at least
two types of olfactory receptor ending, one ciliated, and the other rod-shaped.
The latter type may correspond to the single olfactory process of Jagodowski
(1901) and Hopkins (1926), although it appears to be too short to do so exactly.
Its absence in Gasterosteus may indicate that it is a primitive teleost feature,
but a much wider survey of teleost olfactory organs is necessary to confirm
this viewpoint. The majority of endings in the two species studied here, as
found in a variety of other teleost species by Laibach (1937), Trujillo-Cenoz
(1961), and Holl (1965), however, conform to the pattern found in other
groups of vertebrates (Allison, 1953; de Lorenzo, 1957, 1963).
The significance of there being more than one type of ending in one species
is not clear. Many authors have described differences in the appearance of
the complete receptor cells within the olfactory organs of various vertebrates
(Dogiel, 1887; Miiller, 1955; Neuhaus, 1955; Le Gros Clark, 1956; Vinnikov,
FIG. 21. Section in a plane vertical to the surface, showing rod-shaped and ciliated receptor
endings (Phoxinus).
FIG. 22. Higher magnification of rod-shaped ending in fig. 21.
FIG. 23. Transverse section near the tip of a rod-shaped ending with three bundles of
fibres.
Fie. 24. Vertical section through the dendrite of a rod-tipped receptor, showing fibrous
bundle and vesicles.
cr, ciliated receptor; fb, bundle of fibrous material; rr, rod-shaped receptor ending; sc,
supporting cell; v, vesicle.
34°
Bannister—Olfactory surface of teleosts
1956; Holl, 1965). Also, Le Gros Clark (1957) suggested that such differences
might form a basis for peripheral olfactory discrimination; that is, structural
and staining differences may imply physiological ones, different receptors
'mitochondrion
FIG. 25. Diagrammatic reconstruction of the distal tip of a ciliated receptor,
cut away to show internal structure. The dotted line indicates the level of
the junction with the neighbouring supporting cells.
being particularly sensitive to particular odours. Some of these differences,
such as the gross form of the cell body and dendrite, appear to be rather
superficial. The wide variation of the latter (figs. 2, 3, and 4) would seem to
be just that expected of bipolar cells packed tightly with supporting cells so
as to give a maximum number of sensory endings per unit area of epithelial
Bannister—Olfactory surface of teleosts
341
surface. The receptor with a rod-shaped ending does, however, constitute
a possible case of a specific chemoreceptor, in view of its peculiar internal
structure. On the other hand, it may merely represent another method of
increasing the area of the sensory surface. A ciliated ending with a diameter
of 1-56/1*, possessing 4 cilia each 5 ^ in length and 0-34^ in diameter, would
have an area of approximately 23 JU,2, 90% of this being accounted for by the
cilia. A rod 4/x long, with the same diameter as the tip of a ciliated receptor
dendrite, would have about the same area. An additional possibility is that the
rod-shaped endings, since they project further into the lumen of the olfactory
organ, could receive chemical stimuli not affecting the ciliated receptors,
especially when strong water currents depress the cilia of the latter into the
mucus overlying the epithelium. Lastly, it may be argued that this type of
cell is innervated by the trigeminal nerve, and that it constitutes a gustatory
receptor. In Golgi preparations, however, its axon is seen to run with those
of other receptors as bundles of the olfactory nerve; I have also examined
several series of Golgi, and silver proteinate-stained preparations, and have
failed to find any branches of the trigeminal entering the olfactory mucosa.
The receptor with the rod-shaped ending must therefore be considered for
the present as olfactory in function.
Structures similar to cilia have been described in many sensory cells,
from photoreceptors on the one hand, to the locust ear and crab chordotonal
scolopale organ (Gray, i960; Whitear, 1962) on the other; such cilia often
lack the central pair of fibrils. The olfactory receptors found here contain the
full quota of fibrils, including the central pair. Therefore, the question arises
whether the olfactory cilium is capable of movement. Slow, random movements have been described in saline-flooded olfactory cilia in amphibians
(Schultze, 1856, 1862; Hopkins, 1926), reptiles (Hopkins, 1926), and in
mammalian receptors in tissue culture (Pomerat, cited by Le Gros Clark,
1957), suggesting that this may be the case in all vertebrates. If so, the absence
both of 'arms' on the outer ring of ciliary fibrils, and of a striated rootlet,
reported here, may be of great significance. The 'arms' may be essential
for the rapid, effective beating of normal motile cilia, but not for the slow,
intermittent movements of olfactory ones. Indeed, in view of the strong water
currents obtaining in the olfactory cavity, it is possible that the olfactory cilia
only move as a response to deformation by the ambient current. The full
complement of fibrils, and other normal components of the ciliary apparatus
may, then, be merely a means of keeping the cilia erect and fully presented
to the olfactory current, which would otherwise depress them against the
epithelial surface and lower their efficiency as chemoreceptors.
Microtubules of approximately 180 A diameter, found in the present study
in receptor cell dendrites, supporting cells, and non-sensory ciliated cells,
correspond to the 200A microtubules reported in dendrites by Palay (1956)
in ciliated epithelium in Anodonta by Gibbons (1961), and in similar positions
by others. Their relation to the ciliary basal bodies in both sensory and nonsensory cells suggests that in these two cases, at least, they are homologous.
342
Bannister—Olfactory surface of teleosts
I wish to express my thanks to Professor G. E. H. Foxon for reading and criticizing
my manuscript, to Professor R. Warwick for the technical facilities of the Anatomy
Department, to Mr. G. Stevens for his tuition in electron-microscope techniques, and
to the National Spastics Society for the use of the electron microscope housed in
Guy's Hospital Medical School.
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