Immunocytochemical identification of primary olfactory afferents in rainbow trout

Abstract

We have used a combination of techniques to analyze the primary olfactory projection in trout: anterograde tract tracing with horseradish peroxidase (HRP) and immunocytochemistry with antisera to olfactory marker protein (OMP) and to keyhole limpet hemocyanin (KLH). HRP labeling and the OMP antiserum revealed a subset of ciliated receptor neurons with a wide dendrite that lacked the protruding knob found on other receptor neurons. The organization of the primary olfactory axons was clearly revealed by antisera to KLH, which reacted with no other neurons. When visualized with anti-KLH, fascicles of olfactory axons penetrated the basal lamina of the olfactory rosette at scattered sites and converged to form the olfactory nerve. Fascicles within the olfactory nerve traveled parallel to the long axis of the nerve until resorted by extensive intermixing as they entered the olfactory bulb. Within the olfactory bulb, most axons terminated in nine discrete terminal fields in the glomerular layer; however, a few olfactory nerve axons projected into the ventral medial telencephalon. Fascicles supplying each terminal field in the glomerular layer followed distinctive trajectories within the olfactory nerve layer. Axons ending in two terminal fields made brush-like terminations rather than the glomerular terminations characteristic of the remaining seven fields. After unilateral olfactory nerve transection, returning olfactory axons reestablished the normal pattern of terminal fields within 14 weeks. It is likely that the organization of afferents in the trout olfactory bulb is similarly well regulated during normal receptor cell replacement.

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THE JOURNAL OF COMPARATIVE NEUROLOGY 324:575-589 (1992)
Immunocytochemical Identification of
Primary Olfactory Afferents in
Rainbow Trout
DAVID R. RIDDLE AND BRUCE OAKLEY
Neuroscience Program and Department of Biology, The University of Michigan, Ann Arbor,
Michigan 48109
ABSTRACT
We have used a combination of techniques to analyze the primary olfactory projection in
trout: anterograde tract tracing with horseradish peroxidase (HRP) and immunocytochemistry
with antisera to olfactory marker protein (OMP) and to keyhole limpet hemocyanin (KLH).
HRP labeling and the OMP antiserum revealed a subset of ciliated receptor neurons with a wide
dendrite that lacked the protruding knob found on other receptor neurons. The organization of
the primary olfactory axons was clearly revealed by antisera to KLH, which reacted with no
other neurons. When visualized with anti-KLH, fascicles of olfactory axons penetrated the basal
lamina of the olfactory rosette at scattered sites and converged to form the olfactory nerve.
Fascicles within the olfactory nerve traveled parallel to the long axis of the nerve until resorted
by extensive intermixing as they entered the olfactory bulb. Within the olfactory bulb, most
axons terminated in nine discrete terminal fields in the glomerular layer; however, a few
olfactory nerve axons projected into the ventral medial telencephalon. Fascicles supplying each
terminal field in the glomerular layer followed distinctive trajectories within the olfactory nerve
layer. Axons ending in two terminal fields made brush-like terminations rather than the
glomerular terminations characteristic of the remaining seven fields. After unilateral olfactory
nerve transection, returning olfactory axons reestablished the normal pattern of terminal fields
within 14 weeks. It is likely that the organization of afferents in the trout olfactory bulb is
similarly well regulated during normal receptor cell replacement.
o 1992 Wiley-Liss, Inc.
Key words: axons, regeneration, olfactory marker protein, olfaction, fish
In studies of teleostean olfactory systems, several investi-
gators have described the concentric arrangement of cell
types and resulting lamination of the olfactory bulb
(Johnston, 1898, '02; Sheldon, '12; Holmgren, '20; Droo-
gleever-Furtuyn, '61; Story, '64; Ichikawa, '76; Bass, '81a;
Oka, '83). The laminar organization of teleost olfactory
bulbs is less pronounced but generally comparable to that
in mammals (reviewed in Allison, '53; Nieuwenhuys, '67;
Andres, '70; Alonso et al., '89a). Studies using degenera-
tion, axonal tracing, and electrophysiological techniques
have demonstrated that the efferent axons arising from the
medial and lateral regions of the olfactory bulb project
primarily through the medial and lateral olfactory tracts,
respectively, to partially segregated central projections
(Scalia and Ebbesson, '71; Ito, '73; Braford and Northcutt,
'74; Finger, '75; Satou et al., '79; Dubois-Dauphin et al.,
'80; Bass, '81b). This division into medial and lateral
pathways may reflect the segregation of odor information
associated with reproduction and feeding (Doving and
Selset, '80; Stacey and Kyle, '83; Sorensen et al., '88; Satou,
'90). Given the possibility of such functional segregation of
the second order projections in the olfactory system, it is
important to understand how the primary olfactory axons
are organized and distributed within the glomerular layer
of the olfactory bulb.
Recent anterograde and retrograde mapping experiments
in trout have demonstrated that each point in the glomeru-
lar layer receives information from the entire olfactory
rosette (Riddle and Oakley, '91). Here we extend our
studies of the organization of the primary olfactory projec-
tion in trout, using tract tracing and immunocytochemical
labeling to demonstrate major morphological subdivisions
within that projection, which may reflect the segregation of
axons into functional groups within the olfactory bulb.
Olfactory receptor neurons were exposed to HRP applied to
the mucosal surface in order to anterogradely label their
projections to the olfactory bulb. In other trout, the distri-
Accepted June 26,1992
Address reprint requests to Dawd R. Riddle whose present address is
Department of Neurohioloa, Duke University Medical Center, Box 3209,
Durham, NC 27710.
10 1992 WILEY-LISS, INC.

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576
bution of olfactory axons was characterized by immunocy-
tochemistry. Neuroanatomical investigations of mamma-
lian and amphibian olfactory systems have benefitted from
the use of several antibodies and lectins that react with
olfactory receptor neurons (Allen and Akeson, '85a,b; Fujita
et al., '85; Hempstead and Morgan, '85a,b; Mori et al., '85;
Schwob and Gottlieb, '86; Key and Giorgi, '86a,b; Morgan,
'88; Barber, '89; Shinoda et al., '89). The most useful
markers permit one to trace the trajectory of an identified
group of neurons from source to termination in normal
tissue. One of the antisera employed in the present study,
anti-olfactory marker protein (anti-OMP), has been shown
to bind to olfactory receptor neurons in a variety of
vertebrates (Margolis, '80; Chuah and Zheng, '87; Baker et
al., '89). In fish, OMP-like immunoreactivity (OMP-IR) has
been detected previously by radioimmunoassay (Margolis,
'80); the present study is the first immunocytochemical
demonstration of OMP-IR in fish. In addition to the
antiserum against OMP, we used antisera to keyhole limpet
hemocyanin (anti-KLH) as a novel marker of olfactory
receptor neurons.
As well as characterizing the organization of the primary
olfactory projection in trout, this study provides new infor-
mation regarding two other issues of interest to those
studying the chemical senses of fishes-the
multiple receptor cell types and of a direct projection of
primary olfactory afferents into the telencephalon. A num-
ber of investigators have demonstrated that there are at
least two distinct types of olfactory receptor neurons in
teleosts (reviewed in Yamamoto, '82, see also Rhein et al.,
'81; Zielinski and Hara, '88) and in mammals (Rowley et al.,
'89). One cell type bears a distinct olfactory knob with long
cilia. The other has a less prominent apical knob bearing
numerous microvilli. It has been suggested that in salmonid
fishes (Salmo alpinus L., Salvelinus fontinalis, and Onco-
rhynchus mykiss), the two cell types represent functionally
distinct classes (Thommesson, '82, '83), that microvillous
cells respond to amino acids and ciliated cells respond to bile
salts. Other investigators, however, have been unable to
find such a correlation in the catfish (Ictalurus punctatus)
between morphology and functional specificity (Erickson
and Caprio, '84). Even as the functional significance of
these two cells types remains unresolved, other studies
have provided evidence for yet a third class of receptor cells.
Horseradish peroxidase (HRP) applied to the olfactory
nerve or bulb of goldfish (Carassius auratus) and carp (I.
punctatus) labeled ciliated cells with a wide dendrite termi-
nating at the surface of the epithelium (Type I1 ciliar cells,
Muller and Marc, '84). We present additional evidence from
both HRP and immunocytochemical studies that supports
the existence of Type I1 ciliar receptor cells in trout,
suggesting that at least three different olfactory receptor
cell types cell types may be present in teleosts.
In addition to finding several morphological types of
olfactory receptor neurons, these studies provide further
evidence that a small subset of olfactory axons terminates
caudal to the olfactory bulb. Each marker of olfactory
receptor neurons used in this study also labeled olfactory
nerve axons that projected through the olfactory bulb into
the ventral-medial telencephalon.
presence of
D.R. RIDDLE AND B. OAKLEY
MATERIALS AND METHODS
Rainbow trout (Oncorhynchus mykiss), 18-30 cm long,
were obtained from Spring Valley Trout Farm (Dexter, MI)
and maintained in 75 gallon aquaria at 14-16°C. Trout
were anesthetized for surgery with tricaine methane sul-
fonate (MS-222,100 mgil of aquarium water), wrapped in a
wet towel, packed in ice and immobilized in a polystyrene
holder. A constant stream of chilled aquarium water contain-
ing the anesthetic (MS-22, 80 mg/l) flowed across the gills.
One olfactory mucosa in each of two normal fish was
extensively labeled with horseradish peroxidase (HRP,
Sigma type VI) so that the distribution of HRP-labeled
olfactory axons could be examined. A pledget of gelfoam
soaked in 5% HRP was inserted through the anterior naris
and placed against the olfactory rosette. The ipsilateral
nares were then sealed with Parafilm and cyanoacrylate
glue. Three weeks later the fish were re-anesthetized and
perfused sequentially with heparinized physiological saline,
fixative (1% paraformaldehyde and 1.25% glutaraldehyde in
phosphate buffer), and 10% sucrose in phosphate buffer.
Cryostat sections of the telencephalon and attached olfac-
tory bulbs, the olfactory nerves, and the olfactory mucosae
were thaw-mounted on gelatin-coated slides and HRP was
visualized by means of tetramethyl benzidine (TMB, Mesu-
lam, '82). Alternate sections of the mucosa were treated
with diaminobenzidine (DAB), according to the intensifica-
tion method of Adams ('81). The soma1 depth was deter-
mined for HRP-filled cells in the material treated with
DAB. For a labeled cell that appeared in its entirety in a
single section, the distance from the epithelial surface to
the center of the nucleus was measured with a calibrated
eyepiece reticle.
Seven normal fish were anesthetized with MS-222 and
perfused for immunocytochemistry with heparinized physi-
ological saline followed by fixative (6% HgCl,, 1% sodium
acetate, 0.1% glutaraldehyde). Six other normal trout were
perfused with alternative fixatives (Bouin's fluid, 4%
paraformaldehyde, or 70% ethanolil09 acetic acid).
All tissue samples were dehydrated through graded alco-
hol, cleared in xylene and embedded in paraffin. Sections
were cut at 7-10 pm and mounted on gelatin-coated slides.
Following rehydration, the HgClz-fixed tissue was treated
with alcoholic iodine followed by 5% sodium thiosulfate to
remove residual mercury. All tissue was preincubated in
0.3% Hz02 to block endogenous peroxidase activity, fol-
lowed by 0.5% normal serum.
Sections of olfactory mucosae and bulbs fixed with Bouin's
fluid, ethanoliacetic acid, or HgCl, were incubated with
antiserum to olfactory marker protein (anti-OMP, gift of F.
Margolis, Roche Institute). The tissue was incubated in
normal rabbit serum (NRS) followed by the OMP antise-
rum diluted 1:200 in NRS. Antibody binding was visualized
by means of a biotinylated rabbit anti-goat secondary
antibody (Sigma, St. Louis, MO), avidin-biotin-peroxidase
complex (ABC, Vector Labs, Burlingame, CA) and DAB
(Sigma).
In addition to the OMP antiserum, four commercially
available polyclonal antisera were tested for reactivity in
the olfactory system of the trout. Three of the antisera were
raised against neuropeptides conjugated to KLH: luteiniz-
ing hormone-releasing hormone (anti-KLHi LHRH, Incstar,
Stillwater, MN), substance P (anti-KLHiSP, Incstar), and
vasoactive intestinal peptide (anti-KLHiVIP, Amersham,
Arlington Heights, IL)). The three antisera were originally
obtained to investigate the presence of the neuropeptides in
the brain of the trout. After observing that the three
antisera produced identical patterns of labeling and that
the labeling could not be blocked by preadsorption with the

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MARKERFOROLFACTORYRECEPTORAXONSINTROUT
577
neuropeptides (see Results), we were led to suspect that the
observed reactivity actually resulted from antibodies against
the KLH to which the peptides had been conjugated. This
was confirmed by the elimination of the reactivity of the
three antisera by preadsorption with KLH and also by using
an antiserum against unconjugated KLH (U.S. Biochem,
Cleveland, OH). The antisera against KLH and KLH
conjugates were used at final dilutions from 1:1,000 to
1:10,000 in normal goat serum (NGS). Antibody binding
was detected with biotinylated goat anti-rabbit IgG (Sig-
ma), ABC, and DAB. Immunocytochemical controls in-
cluded substitution of the primary antisera with NGS,
substitution of the secondary antibody or ABC with buffer,
preadsorption of the antisera to peptide conjugates with the
appropriate neuropeptide (Sigma, 10-100 pgiml antibody
solution), and preadsorption of each antiserum with KLH
(U.S. Biochem., 10 pgiml antibody solution).
In addition to our studies of normal trout, we lesioned the
olfactory system in 18 trout in order to verify the identity of
the neuronal elements labeled by anti-KLH and to compare
the organization of the normal and regenerated systems.
The fish were anesthetized as described above and a small
hole was drilled in the midline of the skull at the level of the
posterior margin of the orbit. The hole was enlarged with
fine rongeurs to expose the olfactory bulbs and posterior
portion of the olfactory nerves. In 9 of 18 trout, one
olfactory nerve was transected approximately 1 mm ante-
rior to the olfactory bulb and the cut ends of the nerve
reapposed. In seven other trout, the olfactory bulb was
resected and removed along with the posterior portion of
the olfactory nerve. Gelfoam controlled occasional bleeding
and was used to fill the hole in the skull. The incision was
closed with 5-0 nylon sutures (Ethicon). Finally, in each of
the two remaining trout, one olfactory rosette was exposed
and extirpated with microscissors. The flow of fresh aquar-
ium water across the gills revived the fish and restored
vigorous opercular movement. After 2 to 14 weeks of
recovery, the fish were re-anesthetized with MS-222 and
perfused with the HgClz fixative as described above. Sec-
tions from both the lesioned and contralateral sides of the
olfactory system of experimental fish were processed simul-
taneously so that immunoreactivity could be compared in
lesioned and control tissue processed under identical condi-
tions.
Following immunocytochemical labeling, we made cam-
era lucida drawings of the glomerular layer from every
fourth section of one olfactory bulb from each of four
normal trout. Using a digitizing tablet and a microcom-
puter, we determined the total volume of the glomerular
layer, the volume of each of the nine terminal fields, and the
percentage of the total glomerular layer volume in each
terminal field (See Table 1 footnote). In addition, for two
fish in which the olfactory nerve had been transected 9-14
weeks prior, two-dimensional representations of the size
and position of the normal and regenerated terminal fields
were prepared and analyzed with a digitizing tablet and
computer (See Fig. 10 legend).
RESULTS
The gross anatomy of the primary olfactory system in
trout is similar to that in other salmonids (Pfeiffer, '63;
Yamamoto, '82). The olfactory epithelium is located on the
broad surfaces of 12-16 lamellae that radiate from the base
of each of the bilaterally paired olfactory rosettes. The
edges and tip of each lamella are covered with nonsensory
epithelium, as are secondary folds that separate strips of
olfactory epithelium on each face of the lamellae. The
olfactory nerve, approximately 1 cm long in a 25 cm trout,
emerges from the ventral posterior region of each rosette
and projects to the ipsilateral olfactory bulb. The olfactory
bulbs are seseile, i.e., directly attached to the telencephalon.
The results of the HRP and anti-OMP studies will be
presented first, followed by the investigations of the normal
and regenerated olfactory system using the novel marker of
olfactory receptor neurons, anti-KLH.
HRP labeling
HRP applied to the olfactory epithelium labeled two
distinct morphological classes of cells. Each cell had a fine
basally directed process (arrows in Fig. lA,B). The apical
process of some cells ended in a 1-2 pm diameter knob that
extended slightly above the surface of the epithelium (small
arrowhead in Fig. 1B). The remaining HRP-filled cells had
an apex approximately 4 pm wide that ended flush with the
epithelial surface (large arrowheads in Fig. lA,B). All
HRP-labeled cells fit into these two classes; there were no
labeled cells with wide protruding apices or with narrow flat
apices. The somata of labeled cells with wide apical pro-
cesses were usually deeper in the olfactory epithelium than
those of the cells bearing olfactory knobs. In two animals,
the average somal depth of 280 HRP-filled receptor cells
with dendritic knobs was 26 t 6 pm (mean i_ S.E.M.),
while the mean somal depth of 400 cells with wide, flat
apices was 38 5 8 km.
HRP-filled axons were distributed throughout the olfac-
tory nerve layer and glomerular layer of the ipsilateral
olfactory bulb but were absent from the contralateral
olfactory bulb (Riddle and Oakley, '91). The distribution of
labeled s o n s within the glomerular layer was not continu-
ous; rather the labeled fibers appeared to terminate in
several discrete fields (e.g., Fig. 1C). The pattern of termina-
tions appeared similar in both of the labeled fish. Fascicles
of labeled axons projected through the olfactory bulb and
into the ventral medial telencephalon.
Olfactory marker protein
Cells immunoreactive for the OMP antiserum were un-
evenly distributed in the olfactory epithelium. In some
regions, immunoreactive cells were present every 3-5 pm,
while in other areas, they were absent from stretches of
olfactory epithelium more than 100 pm long. All immunore-
active cells were intensely and similarly labeled. Each
OMP-like immunoreactive cell had a stained axon, un-
stained nucleus, and a darkly stained dendrite that ex-
tended to the surface of the epithelium. The dendrites of a
few immunoreactive cells ended in apical knobs, but most
immunoreactive dendrites had a wide apex approximately 4
pm in diameter, which terminated flush with the epithelial
surface (Fig. 2A).
OMP-like immunoreactivity was also evident in axonal
bundles beneath the olfactory epithelium, in the olfactory
nerve, and in the olfactory nerve layer and glomerular layer
of the olfactory bulb (Fig. 2B). Some immunoreactive axons
projected from the olfactory nerve through the olfactory
bulb and into the ventral medial telencephalon, following
the same path as the forebrain projection labeled by HRP.
No other neurons in the forebrain were immunoreactive.
Omission of the OMP antiserum, the secondary antibody,
or the ABC eliminated labeling. No attempt was made to

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D.R. RIDDLE AND B. OAKLEY
Fig. 1. Horseradish peroxidase (HRP) labeling in the primary
olfactory system. A, B: Several HRP-filled cells arc visible in these
sections through the olfactory epithelium. Two distinct morphological
classes of labeled cells arc evident, one characterized by a narrow apex
topped by an olfactory knob (small arrow), the other characterized by a
wider apex terminating flush with the epithelial surface (arrowheads).
Bar = 10 Fm. C: Horizontal section through one olfactory bulb three
weeks after the ipsilateral olfactory rosette was labeled with HRP
(darkfield). The olfactory nerve enters from the left. At this level along
the dorsal-ventral axis of the olfactory bulb, HRP-labeled axons project
into three discrete terminal fields, anterior medial (small arrowhead),
lateral (large arrowhead) and posterior lateral (small arrow). No
HRP-labeled axons extend into the deeper laminae of the olfactory bulb.
Bar = 250 Fm.

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MARKERFOROLFACTORYRECEPTORAXONSINTROUT
579
Fig. 2. Olfactory marker protein-like immunoreactivity (OMP-IR)
in the olfactory epithelium and olfactory bulb. A: Several immunoreac-
tive cells are visible in the olfactory epithelium. Note the unstained
nucleus (arrowhead) and the broad dendrite (arrow), which extends
apically and terminates flush with the surface of the olfactory epithe-
lium. B: OMP-IR in this horizontal section of the olfactory bulb appears
to be limited to the olfactory nerve layer (ONL) and glomerular layer
(GL). The terminating s o n s form relatively discrete glomeruli similar
to those seen in other vertebrates (arrowheads). Bar in A = 10 pm for A
and 100 pm for B.
block the binding of the OMP antisera by preadsorption,
thus it remains to be determined whether the molecule in
trout recognized by the antisera is the same as mammalian
OMP.
KLH-like immunoreactivity
Specificity of labeling. Antisera against KLH and
against KLH conjugated to neuropeptides reacted intensely
with trout olfactory receptor axons (see Fig. 3A). The four
antisera produced almost identical labeling; anti-KLHiVIP
produced slightly lower background reactivity than other
equidiluted antisera. Reactivity was minimally affected by
variation in fixation, although paraformaldehyde reduced
the background slightly. Omission of the primary antise-
rum, the secondaly antibody, or ABC, eliminated labeling.
Preadsorption of each antiserum to a KLHipeptide conju-
gate with the corresponding peptide (e.g., preadsorption of
anti-KLHiVIP with VIP) did not affect reactivity, even at
Fig. 3. Keyhole limpet hemocyanin-like immunoreactivity (KLH-
IRj in the olfactory bulb. A: Horizontal section from the olfactory bulb
stained with anti-KLHiluteinizing hormone releasing hormone (LHRH),
preadsorbed with LHRH (100 pmiml antiserum). The same pattern of
immunoreactivity was observed with antiserum that was not pread-
sorbed and with each of the four antisera tested. Immunoreactive axons
are limited to the olfactory nerve layer and glomerular layer and project
into discrete terminal fields, such as the anterior medial (arrowhead),
lateral (large arrow) and posterior lateral (small arrow). Note that the
pattern of labeled axons is similar to the distribution of HRP-filled
axons in Figure 1C. B: This section, adjacent to A, is stained with
anti-KLHiLHRH preadsorbed with KLH (10 pmiml). KLH-IR is
completely eliminated. Bar = 250 pm for A and B.
an antigen concentration of 100 p,g/ml of antiserum (Fig.
3A). We were unable to confirm the findings of Alonso et al.
('89b, 'go), who, using anti-VIP from another source,
reported specific VIP-like immunoreactivity in rainbow
trout olfactory nerve and bulb. Preadsorption of each of the
four antisera with KLH (10 yg/ml antiserum) completely
eliminated labeling (Fig. 3B). Since the four antisera pro-
duced identical patterns of reactivity, and since all immu-
noreactivity was eliminated by preadsorption with KLH
while preadsorption with the neuropeptides had no effect,
we concluded that all labeling was due to antibodies against
KLH, and hence, we refer to all reactivity as KLH-like
immunoreactivity (KLH-IR). Characterizing the antigen(s)