Journal of Fish Biology (2017) 91, 473–489

doi:10.1111/jfb.13352, available online at wileyonlinelibrary.com

PSA-NCAM expression in the teleost optic tectum is related

to ecological niche and use of vision in finding food
I. Labak*, V. Pavić*, M. Zjalić*, S. Blažetić*, B. Viljetić†, E. Merdić*
and M. Heffer‡§
*Department of Biology, J. J. Strossmayer in Osijek, Ulica cara Hadrijana 8/A, 31000
Osijek, Croatia, †Department of Medical Chemistry, Biochemistry and Clinical Chemistry,
J. J. Strossmayer in Osijek, Faculty of Medicine, Ulica cara Hadrijana 10, 31000 Osijek,
Croatia and ‡Department of Medical Biology and Genetics, J. J. Strossmayer in Osijek,
Faculty of Medicine, Ulica cara Hadrijana 10, 31000, Osijek, Croatia

(Received 27 February 2017, Accepted 15 May 2017)

In this study, tangential migration and neuronal connectivity organization were analysed in the
optic tectum of seven different teleosts through the expression of polysialylated neural cell adhesion
molecule (PSA-NCAM) in response to ecological niche and use of vision. Reduced PSA-NCAM
expression in rainbow trout Oncorhynchus mykiss optic tectum occurred in efferent layers, while
in pike Esox lucius and zebrafish Danio rerio it occurred in afferent and efferent layers. Zander
Sander lucioperca and European eel Anguilla anguilla had very low PSA-NCAM expression in all
tectal layers except in the stratum marginale. Common carp Cyprinus carpio and wels catfish Silurus
glanis had the same intensity of PSA-NCAM expression in all tectal layers. The optic tectum of
all studied fishes was also a site of tangential migration with sustained PSA-NCAM and c-series
ganglioside expression. Anti-c-series ganglioside immunoreactivity was observed in all tectal layers
of all analysed fishes, even in layers where PSA-NCAM expression was reduced. Since the optic
tectum is indispensable for visually guided prey capture, stabilization of synaptic contact and decrease
of neurogenesis and tangential migration in the visual map are an expected adjustment to ecological
niche. The authors hypothesize that this stabilization would probably be achieved by down-regulation
of PSA-NCAM rather than c-series of ganglioside.
© 2017 The Fisheries Society of the British Isles

Key words: brain; ecological niche; optic tectum; PSA-NCAM; teleost; visual sense.

INTRODUCTION
There is a wide variety of brain morphologies among teleosts that is correlated with
their behaviour and niches they inhabit (Ito et al., 2007). Adult brain proliferation and
neurogenesis are modulated by diverse signals and related to growth of the entire body
and sensory systems (Kaslin et al., 2008). The fish brain has an enormous potential for
continuous production of new neurons throughout adulthood and in teleosts a high level
of neurogenesis is sustained in almost the entire brain (Zupanc et al., 2005; Grandel
et al., 2006).
The optic tectum and retina grow continuously throughout a fish’s life (Marcus
et al., 1999) and relative size of the adult tectum is correlated with an increased

§Author to whom correspondence should be addressed. Tel.: +38 931 505615; email: mheffer@mefos.hr

473
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474 I . L A B A K E T A L.

reliance on vision, characteristics of primary habitat and lifestyle in general (Yopak

& Lisney, 2012). The teleost’s optic tectum is a neatly laminated structure (Ebbesson
& Vanegas, 1976) consisting of paired lobes that form the roof of the mesencephalon
(Butler & Hodos, 2005). It receives input from retinofugal fibres that arise from the
retinal ganglion cells (Yopak & Lisney, 2012) and creates abundant topographically
organized connections (Springer & Gaffney, 1981). The tectal region is also the site
of integration of visual and non-visual information, e.g. those from the somatosen-
sory and octavo-lateral system (Butler & Hodos, 2005), which come from other
sensory modalities and are also organized in a topographical manner. Processing
of multi-sensory information depends on synaptic inputs and connectivity between
different types of neurons (Vanegas et al., 1974) organized in seven layers; from
the deep stratum periventriculare to the superficial stratum marginale (Nieuwenhuys
et al., 1998). The animal cortex represents a remarkable degree of plasticity based on
sensory and environmental stimulation during life (Kiss et al., 2001).
In the present study, plasticity in the optic tectum in relation to vision was inves-
tigated in teleost species that occupy different ecological niches (based on feeding
habits) and differ in their use of vision in the food-finding process. A molecule that
contributes to plasticity is polysialylated neural-cell adhesion molecule (PSA-NCAM)
(Gascon et al., 2007). The NCAM molecule establishes cell–cell adhesion through
homophilic interactions of its extracellular domains (Bonfanti, 2006) and the large
negatively charged PSA chain reduces NCAM-NCAM interactions and therefore
interferes with cell adhesion, allowing dynamic changes in membrane contacts (Kiss
& Rougon, 1997). PSA-NCAM is associated with cell migration, synaptogenesis
and neurite outgrowth (Rutishauser, 2008), while poorly sialylated forms of NCAM
stabilize cell–cell contact (Rougon, 1993). Reorganization in the brain (stabilization
and destabilization of neuronal connectivity) is associated with the balance of NCAM
and PSA-NCAM. The presence of NCAM means firm adhesion and stabilization
of membrane contact, while PSA-NCAM contributes to loose contact and plasticity
(Gascon et al., 2007).
Based on the adaptive diverse brain morphology of teleosts and a high level of
neurogenesis, tangential migration within the optic tectum of all analysed fishes was
expected. PSA-NCAM is used as a marker of tangentially migrating cells (Rousselot
et al., 1995; Doetsch & Scharff, 2001) but expression of these molecules may also
indicate a permanent change in cell interactions and facilitated plasticity in the
structure and function of the tectum (Rutishauser & Landmesser, 1996). Based on
this, the organizations of neuronal connectivity in the optic tectum of seven different
teleosts were analysed through the expression of PSA-NCAM in response to different
ecological niches, as well as difference in using vision to capture prey. It was expected
that adjustments to ecological niches would be reflected in optic tectum, number of
optic nerve fibres and the presence of myelinated axons.

MATERIALS AND METHODS

E X P E R I M E N TA L A N I M A L S , E T H I C S A N D T I S S U E
COLLECTION
Nine zebrafish Danio rerio (Hamilton 1822), 4–5 cm in total body length (LT ), raised under
standard laboratory conditions in the Department of Zoology, Faculty of Science, University

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of Zagreb, Croatia, were used in the experiment. Twenty-one common carp Cyprinus carpio
L. 1758, 38–42 cm LT , six zander Sander lucioperca (L. 1758), 54–59 cm LT , six pike Esox
lucius L. 1758, 75–79 cm LT and six wels catfish Silurus glanis L. 1758, 96–101 cm LT ,
were used from polycultural C. carpio artificial lakes and 21 rainbow trout Oncorhynchus
mykiss (Walbaum 1792), 55–60 cm LT and 12 European eels Anguilla anguilla (L. 1758),
67–71 cm LT , were used from monocultural artificial lakes. All fishes from artificial lakes
were collected in collaboration with the Faculty of Agriculture, University of Osijek, Croa-
tia. In the artificial lakes, the environmental conditions (soil, water, temperature, pH, light,
turbidity) simulated natural habitat and supported good survival and optimum growth of
fishes. Turbidity and water colour were measured in the lakes as they influence visual stim-
uli. Turbidity was between 40 and 70 cm in all lakes. Water colour was light-green in the
polyculture artificial lake and clear or pale in the monoculture lakes and for the D. rerio held
under standard laboratory conditions. All fishes used were adults, determined by their size
and reproductive ability. Fishes were killed by anaesthetic overdose with clove oil (Aromara;
www.aromara.hr) and decapitated. For immunohistochemical analysis, dissected brains were
immersed and fixed in 4% paraformaldehyde for 48 h, cryoprotected in up to 20% sucrose,
frozen and stored at –80∘ C, while brains used for ganglioside extraction and Western blot
were immediately frozen in liquid nitrogen. All animal experiments were conducted in the
Laboratory of Neurobiology at the Faculty of Medicine in Osijek. The original research
reported here was performed under guidelines established by the European Council (E.U.,
2010). The study was approved by the Ethical Committee of the Faculty of Medicine in Osijek
(2158/61-02-145/2-06).

B R O M O U R I D I N E L A B E L L I N G A N A LY S I S
Twenty milligram of 5-bromo-2-deoxyuridine (BrdU) solution in 1X phosphate-buffered
saline (PBS) was intraperitoneally injected per kilogram of body mass in D. rerio, O. mykiss
and C. carpio. Three individuals from each species were used in this experiment. Four hours
after the injection, the fishes were sacrificed and the brains were dissected according to the
tissue collection protocol above, followed by immunohistochemical and histological staining
methods.

I M M U N O H I S T O C H E M I C A L A N A LY S I S
Free-floating immunohistochemistry was performed on 35 μm thick serial coronal sections
cut on a cryostat (Leica; http://www.leicabiosystems.com/) in all seven fish species studied,
using three individuals per species. This method was also performed on the brains labelled with
BrdU. All steps were done at 4∘ C. First, endogenous peroxidase was blocked in 1% hydrogen
peroxide/PBS for 30 min, followed by blocking unspecific binding of antibodies using 1%
bovine serum albumin (Sigma-Aldric; www.sigmaaldrich.com) and 5% goat serum in 0·1 M
PBS (Gibco; www.thermofisher.com) for 2 h. Next, sections were incubated with primary
antibody. The following primary antibodies were used: anti-PSA-NCAM (Chemicon; www
.merckmillipore.com) diluted 1:1000, anti-NeuN (Chemicon) diluted 1:2000, anti-SMI312
(Stenberg Monoclonals; www.novusbio.com) diluted 1:10 000, anti-Glial fibrillary acidic
protein (GFAP) (DAKO; www.1degreebio.org) diluted 1:4000, anti-BrdU (Roche; www.roche
.com) diluted 1:200 and anti-Q211 (kind gift of H. Rösner, Stuttgart, Germany) diluted 1:16.
All antibodies were prepared in a blocking solution and sections were incubated overnight at
4∘ C, except anti-BrdU, which was prepared in a blocking solution with Triton and incubated
for 48 h at room temperature.
After incubation with primary antibody, sections were incubated for 4 h at 4∘ C with
secondary antibody [(biotinylated goat anti-mouse immunoglobulin G (IgG) or biotiny-
lated goat anti-mouse immunoglobulin M (IgM) antibody] (Jackson Immunoresearch; www
.jacksonimmuno.com) diluted 1:500 in blocking buffer. The sections were transferred in
tertiary complex, Vector Elite kit (Vector Laboratories; www.vectorlabs.com) prepared by
the manufacturer’s protocol and incubated for 2 h at 4∘ C. A 3,3′ -Diaminobenzidine (DAB)
substrate kit (Vector Laboratories) was used for detection. Each of the steps after primary
antibody incubation were separated by washes (3 × 10 min each) in 1 × PBS. After detection,

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sections were mounted onto gelatin-coated histological slides and coverslips attached using
VectaMount (Vector Laboratories). Control sections of each fish were performed in the same
way as described above, but with the omission of primary antibody from the experiment.
Instead, control sections were incubated in a blocking buffer. Immunohistochemically stained
sections were photographed using a digital camera (Olympus; www.getolympus.com) on a
Zeiss Axioskop 2 MOT microscope (Zeiss; www.micro-shop.zeiss.com). All sections were
imaged at ×1000 magnification, except for BrdU in the O. mykiss sections, which were imaged
at ×2000 magnification.
PSA-NCAM immunoreactivity, as a crucial antibody in the study, was analysed in the optic
tectum layers of each fish species by IMAGE J software (NIH; www.imagej.en.softonic.com)
using a scale ranging from 0 (the greatest staining intensity) to 255 (no staining) (Moreira
et al., 2014). Several layers were quantified: stratum album central and stratum periventriculare
(sac + spv) combined; stratum griseum centrale inner plexiform layer and stratum griseum cen-
trale deeper layer (sgc-ip + sgc); stratum opticum and stratum fibrosum et griseum superficiale
(so + sfgs). Stratum marginale (sm) was analysed singly. Tectal layers were named according
to (Nieuwenhuys et al., 1998) nomenclature. PSA-NCAM stained sections were normalized
against equivalent regions from the negative control sections and compared between the layers
of the same fish species.

H I S T O L O G I C A L S TA I N I N G M E T H O D S
To confirm that BrdU labelled only stem cells, counterstaining with methylene blue which
marks all other types of cells except stem cells was done on the brains from fishes injected
with BrdU. Sections were mounted on microscope slides, air dried and incubated for 5 min at
room temperature in methylene blue solution (Sigma Aldrich; https://www.sigmaaldrich.com/
european-export.html) (0·005% in water) after which slides were washed in distilled water
and air dried. For histological staining, optic nerves from all seven analysed fishes, three from
each species, were paraffin embedded and cut at 7 μm with a microtome (Leica). After cutting,
sections were deparaffinized in xylene, rehydrated in ethanol and stained with toluidine blue
for 2 min. The excess of dye was removed by washing three times in distilled water. Stained
optic nerves were left to air dry and then they were covered with VectaMount covering medium.
Nerve cross sections were calculated using an ellipse formula. Each optic nerve was imaged
at ×1000 magnification. The number of nerve fibres was manually counted and the number of
nerve fibres was recalculated as the number of nerve fibres per square millimetre of optic nerve.

OPTIC TECTUM MEASUREMENTS

Optic tectum volume measurement and nerve fibres analyses were performed on all seven
studied fish species, three individuals from each. Volume fractions of the optic tectum were
determined by a modified water displacement method in a 2-ml graduated cylinder.

G A N G L I O S I D E E X T R AC T I O N
Ganglioside extraction was performed according to the modified protocol by (Schnaar, 1994).
Fresh frozen brain samples of whole brain and separated optic tectum from O. mykiss, C. car-
pio and A. anguilla, three individuals per each species for each analysis, were homogenized
in three volumes of water using an ice-cold homogenizer. After homogenization, eight vol-
umes of methanol was added and mixed well. All further steps were carried out at room tem-
perature. Next, four volumes of chloroform were added and centrifuged for 15 min at 805g.
The supernatant was collected and a 0·173 volume of water (based on supernatant volume)
was added, mixed and centrifuged again under the same conditions. The upper phase contain-
ing extracted gangliosides was separated and applied to the thin-layer chromatography plates
(Sigma-Aldrich) and developed in a solvent system chloroform–methanol–0·25% KCl–2·5 M
NH4 OH (60:42:10·5:0·5). After development, gangliosides were visualized by Svennerholm’s
reagent. Plates were scanned and gangliosides were quantified as previously described (Schnaar
& Needham, 1994) using Image J software.

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W E S T E R N B L OT
Fresh frozen brains and separated optic tectum, from C. carpio and O. mykiss, three indi-
viduals per species for each analysis, were homogenized in protein extraction reagent, T-PER
tissue protein extraction reagent (Thermo Fisher; www.thermofisher.com) and centrifuged
for 20 min at 45 730g at 4∘ C. Protein samples (10 μg) were separated by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 7·5% polyacrylamide gel and
transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, www.sigmaaldrich
.com) in transfer buffer Blot Transfer Buffer (20×) (Thermo Fisher). Blots were blocked in
1 × PBS containing 5% non-fat dried milk and 0·1% Tween-20 for 2 h at room temperature and
incubated, one in anti-PSA-NCAM (Chemicon; www.merckmillipore.com) diluted 1:1000 and
the other in anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam; www.abcam
.com) overnight at 4∘ C. After incubation with primary antibody, blots were incubated for 4 h
at room temperature with secondary antibody (biotinylated goat anti-mouse IgG or biotinylated
goat anti-mouse IgM antibody) (Jackson Immunoresearch lab) diluted 1:500. A Vector Elite
kit was used as tertiary complex with 2 h incubation at room temperature followed by detection
using a DAB substrate kit (Vector Laboratories). Each of the steps after primary antibody
incubation was separated by washes (3 × 10 min each) in 1 × PBS. One membrane after transfer
blot was incubated with sialidase (44 U ml−1 ) (Sigma-Aldrich) in acetate buffer pH 5·5 for 48 h
at room temperature. After incubation the blot was incubated in anti-PSA-NCAM following
the procedure described previously.

S TAT I S T I C A L M E T H O D S
After checking for normality with the Shapiro-Wilk test, Mann-Whitney U-tests were used to
compare staining in the different tectal layers of each fish. Statistical significance was defined
as P < 0·05 with Bonferroni correction because of multiple comparisons. Statistical tests were
performed using the statistical software package Statistica 12 (www.statsoft.com).

RESULTS

I M M U N O H I S T O C H E M I S T RY
Organization of neuronal connectivity in response to ecological niche and use of
vision was analysed through the expression of PSA-NCAM. In the O. mykiss optic
tectum, PSA-NCAM expression decreased from the higher to the lower layers with
the weakest staining in the sac + spv [Figs 1 and 2(a)]. In those layers PSA-NCAM
expression was almost the same as control staining and they were significantly dif-
ferent from sgc-ip + sgc (Mann-Whitney U = 8·50, n1 = n2 = 9, P < 0·05), sfgs + so
(U = 9·00, n1 = n2 = 9, P < 0·05) and sm (U = 3·00, n1 = n2 = 9, P < 0·05) layers.
In the D. rerio optic tectum, the lowest PSA-NCAM expression was observed in
sfgs + so [Figs 1 and 2(b)]. There was a statistically significant difference between these
layers and sm (U = 3·00, n1 = n2 = 9, P < 0·05), sgc-ip + sgc (U = 9·50, n1 = n2 = 9,
P < 0·05) and sac + spv (U = 17, n1 = n2 = 9, P < 0·05) layers. Similar to D. rerio, in
E. lucius the lowest PSA-NCAM expression was also in sfgs + so [Figs 1 and 2(c)].
Statistically significant differences occurred between these layers and sm (U = 9·00,
n1 = n2 = 9, P < 0·05), sgc-ip + sgc (U = 0·00, n1 = n2 = 9, P < 0·05) and sac + spv
(U = 0·00, n1 = n2 = 9, P < 0·05).
Cyprinus carpio had the highest PSA-NCAM expression in sm [Figs 2(d) and
3]. A statistically significant difference was present only between sm and sac + spv
layers (U = 4·00, n1 = n2 = 9, P < 0·05). Silurus glanis had intensive expression of

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478 I . L A B A K E T A L.

(a) Ctrl PSA-NCAM Q211 GFAP NeuN SMI 312

sm sm
Oncorhynchus mykiss
so so
sfgs sfgs

sgc-ip sgc-ip

sgc sgc

sac sac
spv spv
200 μm

(b)
sm sm
Danio rerio
so so
sfgs sfgs

sgc-ip sgc-ip

sgc sgc

sac sac
spv spv

50 μm

(c) sm sm
Esox lucius so so
sfgs sfgs

sgc-ip sgc-ip

sgc sgc

sac sac
spv spv
200 μm

Fig. 1. Coronal sections of optic tectum stained with antibodies to polysialylated neural cell adhesion molecule
(PSA-NCAM), Q211, glial fibrillary acidic protein (GFAP), NeuN and SMI312 in (a) Oncorhynchus mykiss,
(b) Danio rerio and (c) Esox lucius. spv, stratum periventriculare; sac, stratum album centrale; sgc, stratum
griseum centrale deeper layer; sgc-ip, stratum griseum centrale inner plexiform layer; sfgs, stratum fibrosum
et griseum superficiale; so, stratum opticum; sm, stratum marginale.

PSA-NCAM in all layers [Figs 2(g) and 3], but there was no statistically significant
difference between layers.
Sander lucioperca had the highest expression of PSA-NCAM in sm and very
weak, almost none, in all other layers [Figs 2(e) and 4]. The sm showed differences
with sgc-ip + sgc (U = 4·00, n1 = n2 = 9, P < 0·05) and sac + spv layers (U = 3·00,
n1 = n2 = 9, P < 0·05). Sfgs + so was significantly different from sgc-ip + sgc
(U = 0·00, n1 = n2 = 9, P < 0·05) and sac + spv layers (U = 0·00, n1 = n2 = 9, P < 0·05).

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 D I S T R I B U T I O N O F P S A- N C A M I N T E L E O S T T E C T U M 479

(a) *
(b)
300 * *
* * *
250
200
150
100
50
0
(d)
(c) * * *
250 *

200

150

100
Relative staining intensity

50

0
(e)
* (f) *
300 * * * * *
*
250
200
150
100
50
0
sm so+sfgs sgc-ip + sgc sac + spv
(g)
250

200

150

100

50

0
sm so+sfgs sgc-ip + sgc sac + spv

Fig. 2. Mean ± s.e. relative immunoreactive staining intensity of polysialylated neural cell adhesion molecule
(PSA-NCAM) ( ) in (a) Oncorhynchus mykiss, (b) Danio rerio, (c) Esox lucius, (d) Cyprinus carpio, (e)
Sander lucioperca, (f) Anguilla anguilla and (g) Silurus glanis. , control; 0, greatest intensity; 255, no
staining; *, significant difference between samples (P < 0·05). spv, stratum periventriculare; sac, stratum
album centrale; sgc, stratum griseum centrale deeper layer; sgc-ip, stratum griseum centrale inner plexiform
layer; sfgs, stratum fibrosum et griseum superficiale; so, stratum opticum; sm, stratum marginale.

Also, there was a significant difference between sgc-ip + sgc and sac + spv layers
(U = 16·50, n1 = n2 = 9, P < 0·05). Similar to S. lucioperca, A. anguilla also had
the highest expression of PSA-NCAM in sm and very weak, almost none, in all
others layers [Figs 2(f) and 4]. There was a statistically significant difference
between PSA-NCAM expression in sm and sfgs + so (U = 0·00, n1 = n2 = 9, P < 0·05),

© 2017 The Fisheries Society of the British Isles, Journal of Fish Biology 2017, 91, 473–489
480 I . L A B A K E T A L.

Ctrl PSA-NCAM Q211 GFAP NeuN SMI 312

(a) Cyprinus carpio sm
sm
so so
sfgs sfgs

sgc-ip sgc-ip

sgc sgc

sac sac
spv spv

(b) Silurus glanis

sm sm
so so
sfgs sfgs

sgc-ip sgc-ip

sgc sgc

sac sac
spv spv

Fig. 3. Coronal sections of optic tectum stained with antibodies to polysialylated neural cell adhesion molecule
(PSA-NCAM)-NCAM, Q211, glial fibrillary acidic protein (GFAP), NeuN and SMI312 in (a) Cyprinus
carpio and (b) Silurus glanis. spv, stratum periventriculare; sac, stratum album centrale; sgc, stratum gri-
seum centrale deeper layer; sgc-ip, stratum griseum centrale inner plexiform layer; sfgs, stratum fibrosum
et griseum superficiale; so, stratum opticum; sm, stratum marginale.

sgc-ip + sgc (U = 1·00; n1 = n2 = 9, P < 0·05) and sac + spv (U = 0·00, n1 = n2 = 9,

P < 0·05) layers.
Tangential migration was detected with the monoclonal antibody PSA-NCAM which
recognizes polysialic epitope bound to the extracellular domain of the NCAM molecule
(glycoprotein) and with monoclonal antibody Q211 which recognizes three sialic acids
on the inner galactose (c-pathway of gangliosides) (Letinić et al., 1998). Monoclonal
antibody GFAP was used as a marker of a glial supportive system (glial fibrillary
acidic protein, marker of radial glial cells). For detection of proliferating cells mono-
clonal antibody BrdU was used. For establishing mature neurons, monoclonal antibody
NeuN (neuronal nuclear antigen) was used. Monoclonal antibody SMI312 was used as
a marker of phosphorylated neurofilaments typical for myelinated axons.
In the O. mykiss optic tectum, lipid epitope of sialic acid (positive Q211 immunore-
activity) was equally distributed as protein epitope (positive PSA-NCAM expression).
Radial glia cells were detected only in sm where PSA-NCAM expression was the most
intense. In that layer, mature neurons and myelinated axons were not detected. Mature
neurons were abundant in spv and sparsely arranged in the other layers (except in sm).
Myelinated axons colocalized with mature neurons and were most densely packed in
the spv (Fig. 1). In this layer positive proliferating cells were also detected (Fig. 5).

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 D I S T R I B U T I O N O F P S A- N C A M I N T E L E O S T T E C T U M 481

(a) Ctrl PSA-NCAM Q211 GFAP NeuN SMI 312

sm
Anguilla anguilla so sm
sfgs so
sfgs

sgc-ip sgc-ip

sgc sgc

sac sac
spv spv

(b) sm
Sander lucioperca sm
so so
sfgs sfgs

sgc-ip sgc-ip

sgc sgc

sac sac
spv spv

Fig. 4. Coronal sections of optic tectum stained with antibodies to polysialylated neural cell adhesion molecule
(PSA-NCAM), Q211, glial fibrillary acidic protein (GFAP), NeuN and SMI312 in (a) Anguilla anguilla
and (b) Sander lucioperca. spv, stratum periventriculare; sac, stratum album centrale; sgc, stratum griseum
centrale deeper layer; sgc-ip, stratum griseum centrale inner plexiform layer; sfgs, stratum fibrosum et
griseum superficiale; so, stratum opticum; sm, stratum marginale.

In D. rerio and E. lucius, lipid epitope of sialic acid was present in all tectal layers,
even in the layers in which the PSA-NCAM expression was reduced (Fig. 1). In D.
rerio glial cells were present in sm and spv. At the same time, mature neurons were
abundant in spv and sparsely, but very rarely, distributed in other layers (except in sm).
Thick myelinated axons were present in all layers (except in sm) (Fig. 1). The spv layer
was positive for proliferating cells (Fig. 5). In E. lucius glial cells were visible in spv,
where mature neurons were also abundant. In the other layers, there were no mature
neurons. Thin myelinated axons were distributed from spv to sgc-ip (Fig. 1).
In C. carpio and S. glanis optic tectum, lipid epitope of sialic acid was equally dis-
tributed as protein epitope and the glia cells were vertically extended through the entire
tectum. Mature neurons were abundant in spv and sparsely arranged to sgc. Thin myeli-
nated axons were distributed in all layers (except in sm) (Fig. 3). In the spv layer
positive proliferating cells were determined (Fig. 5). In S. glanis, glia cells were present
in sm and spv. Mature neurons were very rarely arranged in all layers except in sm.
Myelinated axons were not detected (Fig. 3).
In the optic tectum of S. lucioperca and A. anguilla, protein epitope of sialic acid was
very reduced but lipid epitope was present in all tectal layers (Fig. 4). In S. lucioperca,

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482 I . L A B A K E T A L.

Cyprinus carpio Oncorhynchus mykiss Danio rerio

Methylene blue / BrdU

Fig. 5. Immunolabelling of BrdU proliferating cells in stratum periventriculare of (a) Cyprinus carpio, (b)
Oncorhynchus mykiss and (c) Danio rerio.

glia cells were present in the spv layer which was also abundant in mature neurons and
thin myelinated axons. In A. anguilla, glia cells vertically extended through the entire
tectum and mature neurons were abundant in the spv layer. Myelinated axons were not
detected (Fig. 4).

O P T I C T E C T U M V O L U M E A N D N U M B E R O F N E RV E F I B R E S
The ratio of the tectum volume in relation to the whole brain was calculated for all
fishes. The tectum of the O. mykiss and D. rerio took up half the volume of the whole
brain, while the volume of the tectum in the other fishes took up less: E. Lucius 38%, S.
glanis 35%, A. anguilla 32% and S. lucioperca 24%. Cyprinus carpio had the smallest
tectum (20%) in relation to the whole brain. The number of fibres in the optic tectum
was not proportional to the tectum volume. Sander lucioperca had the highest number
of fibres and D. rerio had the smallest number of fibres (Table I).

G A N G L I O S I D E E X T R AC T I O N
Ganglioside extraction, followed by thin layer chromatography (TLC), was per-
formed on the brain of O. mykiss, C. carpio and A. anguilla. The whole brain and
optic tectum were analysed separately for each fish. Chromatography was used to
detect and separate gangliosides, especially c-pathway of gangliosides. Oncorhynchus
mykiss and C. carpio had more c-pathways of gangliosides in the optic tectum com-
pared with the whole brain, but differences between proportion of the c-pathways of
gangliosides in the optic tectum in relation to the whole brain were smaller than in

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 D I S T R I B U T I O N O F P S A- N C A M I N T E L E O S T T E C T U M 483

Table I. Whole brain and optic tectum (OT) volume, mass and number of fibres in optic nerve
of seven teleost species

Danio Oncorhynchus Sander Esox Cyprinus Silurus Anguilla

rerio mykiss lucioperca lucius carpio glanis anguilla
Mean brain volume 20 216.67 136·6 450 966.67 323.33 65
(μl)
Mean OT volume 10 100 33·33 175 200 116.6 21·66
(μl)
Mean brain mass (g) 0·0295 213 0·205 0·48 933 0·33 0·066
Mean OT mass (g) 0·0028 0·0633 0·042 0·16 173 73 0·0176
Numbers of fibres 5917 11 228 18 703 16 071 10 901 8331 6696
per mm2 of optic
nerve*
Numbers of fibres in 1866 25 825 55 175 65 947 28 452 2027 2370
optic nerve
*Smaller fibres may have been missed using light microscopy

O. mykiss. Cyprinuns carpio, as well as A. anguilla, had almost the same proportion
of c-pathways of ganglioside in the tectum and the whole brain. Anguilla anguilla had
the most c-pathways of ganglioside in the optic tectum (Fig. 6).

W E S T E R N B L O T A N D N E U R A M I N I D A S E T R E AT M E N T
Western blot analyses of PSA-NCAM of C. carpio and O. mykiss showed that
PSA-NCAM was expressed in the whole brain and in the optic tectum with the same
intensity. To confirm expression of PSA-NCAM, blots were treated with sialidase, an
enzyme that cleaves sialic acid from PSA-NCAM. After sialidase treatment there was
no positive staining on the blot, which confirmed that the antibody recognizes only the
polysialylated form of NCAM (Fig. 7).

DISCUSSION
Tangential migration (PSA-NCAM and c-series ganglioside expression) occurred in
all tectal layers of all studied fishes. Certain layers of the optic tectum of fishes that are
visual predators, such as O. mykiss and E. lucius (Bogut et al., 2006; Randall, 2014),
showed a reduced PSA-NCAM expression in certain layers. The same phenomenon
was observed in D. rerio optic tectum. In S. lucioperca and A. anguilla, which actively
feed at dusk and during the night (Deelder, 1984; Bogut et al., 2006), low PSA-NCAM
expression was detected in the whole optic tectum. In the optic tectum of C. carpio and
S. glanis, which feed on benthic organisms and have barbels (Bogut et al., 2006), the
same intensity of PSA-NCAM expression was observed in all tectal layers. A previous
study reported correlation of PSA expression with visual stimuli and the ability of
reorganization of retinotectal axons (Williams et al., 1996). According to the results,
reduced PSA-NCAM expression could be related to the use of vision in prey capturing.
Most of the cell bodies (NeuN expression) in all fishes were in the stratum periven-
triculare, while myelinated axons (SMI312 expression) were arranged in upper tectal

© 2017 The Fisheries Society of the British Isles, Journal of Fish Biology 2017, 91, 473–489
484 I . L A B A K E T A L.

(a) 40·0
35·0

30·0
Ganglioside fraction %

25·0

20·0

15·0

10·0

5·0

0·0
c-serie GT1b X1 GD1b GD1a GD2 GD3 GM2 X2 GM3 GM4 X3

(b) X3
GM4

GM3
X2
GM2
GM1
GD3
GD2
GD1a
GD1b
X1
GT1b

c-serie

St Owb Oot Cwb Cot Awb Aot

Fig. 6. Results of ganglioside extraction from the whole brain of Oncorhynchus mykiss ( , Owb), Cyprinus car-
pio ( , Cwb) and Anguilla Anguilla ( , Awb) and separated optic tectum O. mykiss ( , Oot), Cyprinus
carpio ( , Cot) and Anguilla anguilla ( , Aot). (a) Quantification of specific gangliosides based on thin layer
chromatography (TLC) and (b) TLC of extracted gangliosides. X1, unknown ganglioside 1; X2, unknown
ganglioside 2; X3, unknown ganglioside 3; St, gangliosides standard.

layers. This correlates with previous research where the optic tectum is divided into
two main areas: the stratum periventriculare, with cell bodies of most tectal neurons
and synaptic neuropil area (Bene et al., 2011) and the stratum opticum, a layer of
bundles of the optic nerve terminals (Corbo et al., 2012). Retinal axons target the
stratum opticum and the stratum fibrosum et griseum superficiale and make gluta-
matergic synapses with the dendrites of neurons located in the stratum periventriculare,
thereby receiving visual information (Kinoshita et al., 2005; Kinoshita & Ito, 2006).
Non-polysialylated NCAM contributes to the stabilization of membrane contact (Gas-
con et al., 2007). According to this, reduced PSA-NCAM expression in the O. mykiss,
D. rerio, E. lucius, S. lucioperca and A. anguilla indicate stabilization of membrane
interactions, i.e. undisturbed contact between retinal axons and dendrites from tectum
based on the use of vision.

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 D I S T R I B U T I O N O F P S A- N C A M I N T E L E O S T T E C T U M 485

(a) (b) (c) (d)

170 kD
PSA-NCAM

GAPDH 37 kD

Fig. 7. Western blot for polysialylated neural cell adhesion molecule (PSA-NCAM) of Cyprinus carpio (a) whole
brain and (b) optic tectum and Oncorhynchus mykiss (c) whole brain and (d) optic tectum. +, blot incubated
with sialidase; –, blot incubated without sialidase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Oncorhynchus mykiss has a larger optic tectum (50% volume fraction) and thicker
myelinated axons than E. lucius and S. lucioperca, which have more nerve fibres. The
presence of a large number of fibres and myelinated axons leads to the conclusion that
for these fishes, vision could be their primary sense. The optic tectum is indispensable
for visually guided prey capture (Gahtan et al., 2005), which means that for these fishes,
reduced PSA-NCAM expression in the optic tectum is necessary.
Danio rerio is a fish that has barbells, but its PSA-NCAM expression is similar to
fishes that do not have barbels and use day feeding. The relation of the optic tectum to
the whole brain is similar to O. mykiss which confirms a correlation between relative
size of the tectum and increased use of vision (Yopak & Lisney, 2012). Compared with
all analysed fishes, D. rerio had the smallest number of fibres in the optic nerve and very
thick myelinated axons. All this leads to the conclusion that during feeding, D. rerio
relies basically on vision, which could be the cause of reduced PSA-NCAM expression
in the optic tectum.
As with S. lucioperca, A. anguilla feeds on benthic organisms at night and had a
reduced PSA-NCAM expression in the optic tectum. Anguilla anguilla did not have
myelinated axons and had the lowest number of optic nerve fibres from all the anal-
ysed fishes without barbels. The tectum is also the site of integration of non-visual
information, e.g. from the somatosensory and octavo-lateral system (Butler & Hodos,
2005). These results indicate that A. anguilla do not rely on vision during prey capture.
Cyprinus carpio and S. glanis feed on benthic organisms and possess barbels. Cypri-
nus carpio had the highest PSA-NCAM expression of all analysed fishes and the same
expression of PSA-NCAM in all layers, similar to S. glanis. The relation between
C. carpio optic tectum size and the whole brain was smaller than in S. glanis. Cyprinus
carpio had more nerve fibres than S. glanis and unlike S. glanis had myelinated axons.
These results indicate that C. carpio uses both vision and barbels in detecting prey.
Silurus glanis has small eyes, related to its nocturnal activities (Bogut et al., 2006). The
optic tectum of blind fishes and fishes with a rudimentary sense of vision comprises of
seven layers but the layers related to creating connections with the retina are missing
or are significantly reduced (Voneida & Fish, 1984). Based on high stabilization of
membrane interactions in the optic tectum, the results confirmed that for C. carpio and

© 2017 The Fisheries Society of the British Isles, Journal of Fish Biology 2017, 91, 473–489
486 I . L A B A K E T A L.

S. glanis vision is not a primary sense for detecting prey, unlike in species that are
visual predators or that actively feed at dusk and night and do not possess barbels.
Previous studies have shown that fishes have a high level of neurogenesis in almost
the entire brain (Zupanc & Horschke, 1995; Zikopoulos et al., 2000; Ekström et al.,
2001). The majority of new cells in the adult fish brain are generated in the cerebellum
(Hinsch & Zupanc, 2007), while a minority are produced in other regions (Zupanc,
2008), including the optic tectum. Continuous neurogenesis in the periventricular grey
zone (PGZ) of the optic tectum has been reported in adult teleosts such as D. rerio
(Zupanc et al., 2005). In the present study, the results showed positive proliferating
cells (BrdU) in the stratum periventriculare of the optic tectum of O. mykiss, D. rerio
and C. carpio. According to some authors (Marcus et al., 1999; Zupanc et al., 2005;
Grandel et al., 2006), proliferating cells exist in this layer of the adult D. rerio optic
tectum. Cyprinus carpio had more proliferating cells than O. mykiss and D. rerio, which
could be correlated with higher PSA-NCAM expression in the C. carpio optic tectum.
Recent studies have shown that juvenile Atlantic salmon Salmo salar L. 1758 that
spent 8 weeks in an enriched environment had increased plasticity in the telencephalon,
meaning that within the brain, enrichment affects neurogenesis and synaptic plasticity
(Salvanes et al., 2013). Both O. mykiss and C. carpio in the present study came from
artificial lakes, but C. carpio lived in a richer environment than O. mykiss. Danio rerio,
as well as O. mykiss, lived in a less enriched environment (cultivated in an aquarium)
and had the same number of proliferating cells as O. mykiss (Fig. 5).
The expression of c-series gangliosides in fishes is a general feature of the nervous
tissue, over the whole life time (Greis & Rösner, 1990). In the optic tectum of all fishes,
c-series of gangliosides is present (Q211 positive), which means active axonal growth
and cell migrations (Letinić et al., 1998). PSA-NCAM expression also promotes axon
growth and fasciculation (Cremer et al., 2000) and migration (Yoshida et al., 1999).
Adult teleosts have abundant radial glial cells in the brain (Xing et al., 2014). Sev-
eral studies report that the regeneration capacity in adult teleosts is associated with
the presence of radial glia (Diotel et al., 2010; Kroehne et al., 2011). In the present
study, all fishes had GFAP-positive glial cells. The optic tectum of all analysed fishes
already had differentiated neurons (NeuN) and plasticity occurred in terms of axonal
sprouting and cell migration. Glia cells vertically extended through the entire tectum
only in C. carpio and A. anguilla. Therefore, ganglioside extraction was performed
only in C. carpio, A. anguilla and O. mykiss to compare the proportion of c-series
of gangliosides with species that did not have vertical extension through the tectum.
Also, positive SMI312 immunoreactivity means a presence of projecting axons and
represents the use of vision in detecting prey. Cyprinus carpio with thin myelinated
axons and A. anguilla without myelinated axons had a higher proportion of c-series
gangliosides in the optic tectum than O. mykiss (Fig. 6), where myelinated axons were
thicker and GFAP was present only in a higher tectal layer. A higher proportion of
c-series gangliosides in the C. carpio and A. anguilla optic tectum may also be associ-
ated with an enriched environment which leads to certain adjustments to environmental
conditions.
In this comparative study of differences in tangential migration and neuronal con-
nectivity in the optic tectum between various fish species in relation to their ecological
niche and use of vision, a whole animal control is missing because only completely
blind fish with a developed optic tectum could be used. In the future projects, the focus
will be on developing control fish models for visual studies. To compensate for the lack

© 2017 The Fisheries Society of the British Isles, Journal of Fish Biology 2017, 91, 473–489
 D I S T R I B U T I O N O F P S A- N C A M I N T E L E O S T T E C T U M 487

of a whole-animal control, a wide range of fish species that differ in their use of vision
was studied, including typical models, as well as unusual species.
In conclusion, in the present study, PSA-NCAM expression in the teleost optic tec-
tum was observed through a dual function: organization of neuronal connections and
tangential migration. Lipid epitope of sialic acid was present in all tectal layers of all
analysed fishes, even in layers where protein epitope was reduced. Therefore, tangential
migration occurs in the optic tectum, but in layers where stabilizations of neuronal con-
nectivity are necessary, PSA-NCAM expression was reduced. These results confirmed
that there was adjustment to environmental conditions and the use of vision, manifested
through stabilization of membrane contact rather than absence of tangential migration.

This work was supported by the Croatian Ministry of Science, Education and Sports
grant ‘Lipid rafts and glycoconjugates in development and regeneration of CNS’ (no.
219-0061194-2158; https://www.grid.ac/institutes/grid.425828.1). The authors wish to thank
I. Bogut, P. Odvorčić, R. Ozimec, A. Štambuk and M. Balog who provided fish specimens
during research field work and who greatly assisted this research.

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