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Neuroscience Letters 472 (2010) 65–67

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

G protein alpha subunits in the olfactory epithelium of the holocephalan fish

Chimaera monstrosa
Sara Ferrando a,∗ , Lorenzo Gallus a , Chiara Gambardella a,b , Marino Vacchi c , Grazia Tagliafierro a
a
LIBiOM, Department of Biology, University of Genoa, Viale Benedetto XV 5, I-16132 Genoa, Italy
b
Department of Animal Biology and Marine Ecology, University of Messina, Salita Sperone 31, I-98166 Messina, Italy
c
ISPRA, c/o National Antarctic Museum, University of Genoa, Viale Benedetto XV 5, I-16132 Genoa, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Receptor neurons in the olfactory and vomeronasal epithelia of vertebrates have dendritic specialization
Received 20 November 2009 that is correlated to the receptor gene family they express and the G protein coupled with that receptor (in
Received in revised form 22 January 2010 particular the G protein alpha subunit). There are not very many data in the literature about the morpho-
Accepted 26 January 2010
logical and molecular features of the olfactory epithelium of Chondrichthyes. In this work, the presence
and distribution of different types of G protein alpha subunits (G␣o , G␣q and G␣olf ) were investigated
Keywords:
in the olfactory epithelium of the holocephalan Chimaera monstrosa using immunohistochemistry. Only
Olfactory epithelium
G␣o -like immunoreactivity was detected in the microvillous receptor neurons and in numerous axon
G␣o
Cartilaginous fish
bundles of the fila olfactoria. These preliminary data are in agreement with studies showing G protein
Holocephalan alpha subunits in elasmobranchs and support the data present in the literature about putative odorant
receptor families in the sequenced genome of the holocephalan Callorhinchus milii.
© 2010 Elsevier Ireland Ltd. All rights reserved.

The olfactory and vomeronasal epithelia of vertebrates contain
receptor neurons (RNs), whose dendritic specializations seem to be
correlated with the receptor family expressed in that neuron. Olfac-
tory receptors (ORs) are expressed in the ciliated RNs, while type 1
and type 2 vomeronasal receptors (V1Rs and V2Rs) are expressed
in the microvillous RNs [4,14,17–19]. Each receptor family has a
distinct G protein coupled alpha subunit, so the expression of the G
protein alpha subunits can also be correlated to the dendritic spe-
cialization (cilia or microvilli) of the RNs [1,3,13,17–19,21]. There
are some differences in this system between fish and tetrapods. In
tetrapods, the olfactory and the vomeronasal epithelia are actually
two distinct epithelia, while in fish there is only one epithe-
lium, called the olfactory epithelium (OE), which has features of
both the olfactory and vomeronasal systems [17–19]. Moreover, in
tetrapods, V1Rs is usually well represented, while in fish, the two
more common receptor families are the ORs and the V2Rs, with
the V1Rs only represented by a few genes [11,23]. The morphology
and ultrastructure of the OE of Chondrichthyes have been stud-
ied in different species of elasmobranchs [6,7,9,25,26] and in one
holocephalan [20], allowing researchers to observe epithelial cili-
ated cells and the microvillous RNs, and highlighting the absence
of the otherwise ubiquitous ciliated RNs. A third type of RN, spe-
cific to fish, the crypt neuron, has been described in elasmobranchs
[6,7]. The molecular features of the OE of Chondrichthyes are largely
∗ Corresponding author. Tel.: +39 010 3537015; fax: +39 010 3538047.
E-mail address: sara.ferrando@unige.it (S. Ferrando).
unknown. A study on the distribution of G proteins has been per-
formed in the OE of the elasmobranch Scyliorhinus canicula [8],
but there are no data about the presence and distribution of the
receptor families. Genes of the odorant receptor families have been
investigated in the partially sequenced genome of the holocephalan
Callorhinchus milii [5], as it is the only known genome in Chon-
drichthyes, and the V2R family seems the only odorant receptor
family broadly represented (with 32 genes found) [12,22]. Only
one functional OR gene and two functional V1R genes have been
found in the genome of C. milii [12,22] and there are no data in the
literature regarding the site of expression of these putative genes.
These fragmentary data offer a picture of a very particular olfac-
tory system in cartilaginous fishes, both from a structural and
molecular point of view. On the other hand, holocephalans and elas-
mobranchs diverged very early along the cartilaginous fish branch
and any evidence seen in one group could be not representative of
the other. To understand if this very peculiar kind of olfactory sys-
tem is general to the whole group of Chondrichthyes, all features
need to be verified in both elasmobranchs and holocephalans.
The aim of the present study was to investigate the presence of
different G protein alpha subunits in the OE of Chimaera monstrosa
by immunohistochemical methods. To identify the presence or
absence of the ORs, we used an antibody against the typical G
protein alpha subunit coupled to the ORs in the olfactory system
of all vertebrates, G␣olf [17–19]. As the V2Rs seem to be the only
receptor family well represented in holocephalans, we assessed
the presence of the two G protein alpha subunits usually coupled
to the V2Rs in fish: the G␣o and G␣q [17–19]. The rabbit fish,

0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2010.01.059
 Author's personal copy

66 S. Ferrando et al. / Neuroscience Letters 472 (2010) 65–67

C. monstrosa, is the only holocephalan living in the Mediterranean.
It is a deep sea fish that feeds mainly on bottom-living inverte-
brates [10]. Two adult female specimens of C. monstrosa (standard
length 62 and 59 cm) were collected in the Ligurian Sea (NW
Mediterranean Sea) by bottom trawlers. The estimated age of the
samples was 18–20 years, calculated using the von Bertalanffy
growth equation using the parameters given by Calis et al. [2].
The fish were brought on board lifeless and rapidly dissected to
collect the olfactory organs. Time of death was estimated to be
about an hour before fixation. The olfactory organs were fixed in
4% paraformaldehyde in 0.1 M phosphate buffered solution (PBS,
pH 7.4) at 4 ◦ C, embedded in Paraplast (Bio-Optica, Milano, Italy)
and sectioned in 5 ␮m thick sections. Histological observations
were performed by hematoxylin–eosin (Bio-Optica, Milano Italy).
Immunohistochemical reactions were carried out to highlight
three different G protein alpha subunits using the following
polyclonal antisera from Santa Cruz Biotechnology Inc. (CA,
USA): anti-G␣o (Cat. sc-387), anti-G␣q (Cat. sc-393) and anti-G␣olf
(Cat. Sc-383), all raised against mammalian proteins. The secondary
antibodies used were Alexa 488 conjugated anti-rabbit (1:800 in
PBS, Molecular Probes, Invitrogen Corporation, Carlsbad, CA, USA)
or, alternatively peroxidase conjugated anti-rabbit (Sigma, USA).
Only the anti-G␣o antiserum gave a positive result, so its specificity
was tested by neutralizing this primary antibody with its antigen,
the G␣o blocking peptide (Santa Cruz Biotechnology, Inc., Cat. No.
sc-387P). The mixture of the antiserum with its blocking peptide
(five-fold by weight as suggested by the manufacturer) was left to
react for 24 h at 4 ◦ C before use. Negative controls were performed
by omitting the primary antiserum. Positive controls were per-
formed using tissues of teleost and mammals; in particular, the OE
of Dicentrarchus labrax, which has been shown to express all three
proteins, was used to test the three antisera [17–19]. In addition,
rat testis was used as a positive control for the antibody against
G␣olf [24].
Sections were examined by a BX60 Olympus light and epi-
fluorescence microscope and visualized with an Olympus CCD
Color-ViewII Camera (Olympus, Tokyo Japan) with analySIS soft-
ware (Soft Imaging System GmbH, Germany).

Fig. 1. C. monstrosa (a) OE, hematoxylin-eosin, scale bar 20 ␮m. (b) Olfactory lamellae, G␣o -like immunoperoxidase, scale bar 50 ␮m. (c) Olfactory lamellae, G␣olf -like
immunoperoxidase, scale bar 100 ␮m. (d) Olfactory lamellae, G␣q -like immunoperoxidase, scale bar 100 ␮m. (e) Olfactory lamellae, G␣o -like immunofluorescence, scale
bar 50 ␮m. (f) OE, G␣o -like immunofluorescence, Scale bar 20 ␮m. (d) Olfactory lamellae, G␣o -like immunofluorescence after preincubation of the primary antiserum with
its antigen, scale bar 50 ␮m. Arrow = microvillar layer; arrowhead = dendritic process of the receptor neuron; asterisk = vacuolar region of the ciliary cells; B = basal cell;
BV = blood vessel; C = ciliated cell; E = epithelial thickness; FO = fila olfactoria; N = receptor neuron.
 Author's personal copy
S. Ferrando et al. / Neuroscience Letters 472 (2010) 65–67
The ultrastructure of the OE has been described before [20]. Our
observations confirmed the presence of ciliated epithelial cells with
vacuoles in the apical region. The RNs were recognizable by the
nuclei in the mid-zone of the epithelium, characterized by a promi-
nent nucleolus. Crypt neurons, a type of sensory neurons peculiar to
fish, were not seen. The nuclei of the basal cells were visible close
to the basal lamina (Fig. 1a). The three antibodies gave positive
results on the OE of D. labrax, and the anti-G␣olf showed positive
signal on the rat spermatozoans (data not shown). Immunoreactiv-
ity (ir) for G␣o was detected in the olfactory mucosa of C. Monstrosa,
while no ir for G␣olf and G␣q was seen (Fig. 1b–e). G␣o -like ir
was clearly seen in the layer of microvilli, in the dendritic process,
along the membrane of the RN cell bodies (Fig. 1f) and in the fila
olfactoria in the lamina propria; ir was likely present in all visi-
ble RNs and in all, or the vast majority, of the fila olfactoria in the
sections examined (Fig. 1b and e). The omission of the primary anti-
serum, as well as its preincubation with its antigen, eliminated the
ir (Fig. 1g).
The commercial antisera used in this study are raised against
mammalian G proteins, but they have been successfully used on
different fish species [17–19], and on Chondrichthyes in particu-
lar [8]. Moreover, we obtained positive controls in D. labrax and
rat tissues to confirm that they worked correctly. The peptide used
to obtain this antiserum against G␣o was the 105–124 aa of the
Rattus norvegicus protein [18]. No G␣o sequence is available for
any cartilaginous fish, but the genome of the holocephalan C. milii
has been partly sequenced; thus we performed a search for the
amino acid sequence KMVCDVVSRMEDTEPFSAEL on the protein
blast available on the Elephant Shark Genome Project website [5].
This query gave an alignment with a predicted C. milii peptide of
KMVCDVVSRMEDTEPFSPEL, with a difference of only one amino
acid. The predicted C. milii peptide was used as a query on BLASTP,
and alignment was found with the G␣o of many species, also with
100% identity. Thus, we considered the antiserum reliable for the
detection of G␣o in C. monstrosa.
From our morphological observations, the absence of the crypt
neurons is noteworthy. This neuron type has been shown to be
peculiar to fish [16] and it has also been found in elasmobranchs
[6,7]. As the number of crypt neuron has been shown to vary appre-
ciably in different seasons in some fish species [15], we have to
consider the possibility that the absence of crypt neuron in our
samples, which were both female fish collected in October, is not
representative of a real absence of this cell type in this species.
Research performed on the genome of the holocephalan C. milii
found 32 putative V2Rs, two putative V1R and only one putative OR
[12,22]. The V1R family is often not well represented in fish, and it
seems to be more diffuse in tetrapods [23]. The OR family is usually
well represented in all vertebrates and these receptors are always
coupled to G␣olf [14,15]. Our preliminary results about the absence
of G␣olf -like ir, in agreement with our observations in elasmo-
branchs [8], support the hypothesis about the near absence of ORs
in holocephalans. The V2R family in fish is usually coupled to G␣o
or G␣q [17,18]; our preliminary data suggest the absence of G␣q in
holocephalans, as seen in elasmobranchs. The use of a single anti-
G␣olf antibody and not the immediate fixation of the tissues after
death, does not represent the ideal circumstances of work; different
antibodies, as well as different fixation methods, should be used in
order to acquire more confidence. However all available evidence
supports that the V2R could be the main odorant receptor family
in holocephalans [12,22] and that it is probably coupled to G␣o .
67
Acknowledgement
The authors are grateful to the crew of “Moana 1” fishing vessel
(Port of Genoa) for collecting the specimens.
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