Advances in Bioscience and Biotechnology, 2012, 3, 788-804 ABB
http://dx.doi.org/10.4236/abb.2012.326099 Published Online October 2012 (http://www.SciRP.org/journal/abb/)
Nitric oxide-factor, which regulates proliferation and
apoptosis in the adult brain of Amur sturgeon Acipenser
schrenckii*
Е. V. Pushchina1, D. K. Obukhov2
1A. V. Zhyrmunskii Institute of Marine Biology, Far Eastern Division, Russian Academy of Sciences, Vladivostok, Russia
2St. Petersburg State University, St. Petersburg, Russia
Received 29 August 2012; revised 30 September 2012; accepted 4 October 2012
ABSTRACT
The distribution of proliferative zones, NO-producing
cells and apoptosis areas in the medulla oblongata,
cerebellum, optic tectum, thalamus and hypothala-
mus of Amur sturgeon Acipenser schrenckii was in-
vestigated, using techniques of immunoperoxidase
staining of proliferating cell nuclear antigen (PCNA),
neuronal nitric oxide synthase and TUNEL-labeling
of fragmented DNA. It has been established, that in
the sturgeon brain NO can act both as a cytotoxic
proapoptogenic factor, and as a factor, which stimu-
lates cell proliferation. The presence of NO-producing
elements in somato- and viscerosensory areas of me-
dulla oblongata, tectum, cerebellum and thalamus
suppose, that in these brain areas NO constitutes
apoptogenic factor, which induces the cells death in a
territory of postmitotic neuroblasts, renders control-
ling effect on development and differentiating of che-
mosensory, visual, motor and hypophysotropic brain
areas in postnatal ontogenesis. Maximal proliferating
activity and high concentration of NO-ergic cells were
revealed in external layers, adjoining to the medullar,
cerebellar and tectum membranes, that allow to sup-
pose NO participation in postnatal morphogenesis of
these brain structures as a factor, which regulates cell
proliferation. In sensory centers (tectum and nuclei of
the V, VII, and X nerves), significantly varying ratios
of intensities of proliferation and apoptosis were
found; this is indicative of dissimilar rates of growth
and differentiation in visual and chemosensory cen-
ters of the sturgeon brain. Presence of NO-producing
elements in the PCNA-immunola-beling and TUNEL-
labeling brain areas allow to consider NO as a factor,
which balances processes of proliferation and apop-
tosis in the sturgeon brain.
Keywords: Adult Neurogenesis; Nitric Oxide; Apoptosis;
Sturgeon; Neotenia; Development of Sensory Systems
1. INTRODUCTION
It is now generally accepted, that in the teleost brain the
neurons permanently are occurring in the different brain
areas, and promoting their growth in postembryonic pe-
riod [1]. In some studies on different fish species, it was
found that the corresponding proliferative centers are
localized along the entire rostro-caudal brain axis [1,2].
The peculiarities of postnatal proliferation and manifest-
tations of apoptosis in the cerebellum were, in particular,
examined in two fish species, a gymnotiform fish, Ap-
teronotus leptorhynchus [3], and a cyprinoid fish, Danio
rerio [1,4]. In whole, it should be recognized, that the
mechanisms of postembryonic morphogenesis in the
brain of fish remains little studied. Sturgeons form one of
the most ancient groups of vertebrates; they are the most
primitive evolutionary branch of the Actynopterygii.
Evolution of sturgeons was, to a considerable extent,
realized in a paedomorphosis mode characterized by
relative slowdown of ontogenesis of organs and their
systems. In the teleost fishes brain neuronal nitric oxide
synthase was found in neurons and fibers, prevailing
practically in all brain areas and in a spinal cord [5-10].
There is some, but not yet rigorous, evidence, that
NADPH-diaphorase in the teleost fishes brain was local-
ized in different types of neurons [5,8-10], glial cells:
astrocyte and oligodendrocyte [11], and also in endo-
theliocyte [7] and tanicyte [12]. Thus, NO-producing
systems in the teleost fish brain involve heterogeneous in
morphological and functional aspects, population of
neurons and glial cells. Presence of constitutive nitric
oxide synthase in projection areas of majority sensory
nerves in medulla oblongata and integrative brain centers
show direct evidence of NO participation in the sense
process regulation in low vertebrates brain. However up
*This work was supported by Grant of Far Eastern Branch of Russian
Academy of Sciences № 12-III-А-06-095. Conflicts of interests: None
declared.

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 789
to date it is unknown, what influence NO render to the
sensory system development in postembryonic onto-
genesis. The results of experimental and morphological
studies in teleost fishes have shown, that in CNS neurons
NO participate in different functional mechanisms,
which are connected with regeneration [13], differentia-
tion [14,15], cluster communication between neurons
and glial cells [16], neuromorphogenesis [13,17], neuro-
transmission and cellular metabolism [18]. High content
of NO in different types of the teleost brain cells differ
substantially the teleost fishes from all other vertebrates,
especially from the mammals, and may be connected
with producing and death of large number neurons in
postembryonic period of ontogenesis. Today the infor-
mation about influence of NO-producing system on pos-
tembryonic development of the sturgeon’s brain, on in-
terrelations between embryonic and definitive parts in
the structure of their CNS, and on relations between
neotenic organization and differential development of the
brain is absent.
The aim of this study is to find out the distribution of
NO-producing sites in different brain areas and in addi-
tion, we attempted to define possible influences of NO
on the processes of adult neurogenesis and apoptotic cell
death in the brain of the Amur sturgeon Acipenser
schrenckii.
2. MATERIALS AND METHODS
2.1. Fishes Material
In our study, we used nine 3-year-old individuals of the
Amur sturgeon Acipenser schrenckii; body mass 900 to
1.100 g, length 55 to 60 cm. Fishes were obtained in
2011 from the Ryazanov experimental production fish
hatchery. Fishes were anesthetized in a cuvette with
0.1% solution of tricaine methanesulfonate (MS-222;
Sigma, USA) for 10 - 15 min. The intracranial cavity of
the immobilized fish was perfused, using a syringe, with
a 4% solution of paraformaldehyde based on 0.1 М
phosphate buffer (рН 7.2).
2.2. PCNA Immunohistochemistry
To study the proliferative activity of cerebral cells, we
used an indirect avidin-biotin-peroxidase (ABC tech-
nique) immunohistochemical staining of the proliferating
cell nuclear antigen (PCNA). The fish brain fixed using
the above technique was divided into several blocks, the
myelencephalon at the level of nuclei of the V, VII, and
X nerves, the cerebellum, a rostral part of the brainstem
at the level of the isthmus, the thalamus with tectum
hemispheres, and internal lobes of the hypothalamus.
The obtained samples were embedded in paraffin ac-
cording to a conventional technique [19]. Serial frontal
slices (20 μm thick) prepared using a microtome were
mounted on polylysine-covered glass slides. The material
was deparaffined; to increase the membrane permeability,
we used thermal treatment of the obtained preparations
for 45 min at 95˚С with the target retrieval solution
(DAKO, USA). After cooling-down to room temperature,
glass slides were rinsed in distilled water. To remove
manifestations of nonspecific peroxidase activity, the
slices were incubated in 1% solution of hydrogen perox-
ide for 5 min at 37˚С and washed out three times in 0.1
М phosphate buffer (pH 7.2). After these procedures, the
slices were incubated with primary monoclonal mouse
antibodies against PCNA (DAKO, USA) for 20 min un-
der the same temperature conditions and washed out
three times in 0.1 M phosphate buffer. The slices were
incubated with secondary biotin-conjugated mouse anti-
bodies against rabbit immunoglobulins (LSAB 2 System,
HRP; DAKO, USA) for 20 min at 37˚С and then washed
out three times of 0.1 M phosphate buffer (pH 7.2). Then,
slices were incubated with the streptavidin visualization
system (LSAB 2 System, HRP) under the same condi-
tions (20 min, 37˚С) and washed out in 0.1 M phosphate
buffer. Reaction products were detected using diamino-
benzidine (DAB) dissolved (to a concentration of 0.5
mg/ml) in the phosphate buffer; 1- or 2-ml aliquots were
prepared. Before application, hydrogen peroxide based
on the phosphate buffer (0.1 M) was added to aliquots to
the final concentration (0.03%). The obtained prepara-
tions were kept for 5 - 7 min in a thermostat at 37˚С, to
reach a clear visualization of the used marker (process
was controlled under a microscope). Glass slides were
washed out in distilled water; the slices were subjected to
final staining with hematoxylin (Lilly-Mayer) for 30 sec,
washed out with running water for 15 - 20 min, dehy-
drated according to a standard technique, and embedded
in balsam.
2.3. TUNEL-Labeling
To reveal apoptotic cells in the sturgeon brain, we used a
technique for immunoperoxidase labeling of fragmented
DNA chains, TUNEL. After 2-h-long fixation in 4%
solution of paraformaldehyde based on 0.1 M phosphate
buffer (рН 7.2), dissected parts of the sturgeon brain
were washed out for 24 h in 0.1. M phosphate buffer.
Then, these samples were put in 30% solution of sucrose
based on phosphate buffer (0.1 М) for cryoprotection and
kept in this solution up to full immersion. Frontal and
horizontal slices (20 μm thick) were prepared using a
freezing microtome. To identify TUNEL-positive struc-
tures, we used a immunoperoxidase identification system,
ApopTag Peroxidase in Situ Apoptosis Detection Kit
(Chemicon International Inc., USA). For blocking en-
dogenous peroxidase, the slices were incubated in 1%

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 790
solution of hydrogen peroxide for 3 min and then washed
out two times for 5 min in phosphate buffer. The slices
were covered with a smoothing buffer (75 μl) and kept
for 10 sec at room temperature. Then, the slices were
slightly dried, subjected to the action of TdT enzyme (55
μl/5 cm2), incubated in a humid chamber for 1 h at 37˚С,
and immersed in a stop buffer for 10 min. The slices
were washed out in phosphate buffer at room tempera-
ture (three times for 1 min with changing of the solution),
again dried, covered with antidioxygenin conjugate (65
μl/5 cm2), and incubated in a humid chamber for 30 min.
To detect the reaction products, cerebral slices were in-
cubated in the substrate for identification of peroxidase
(VIP Substrate Kit; Vector Labs, USA) with control of
the development of color under a microscope, washed
out in three changes of phosphate buffer, and mounted
on glass slides. The cell nuclei were subjected to final
staining with methyl green according to the technique of
Brasher [19]. The preparations obtained were dewatered
using a conventional technique and embedded in balsam.
2.4. nNOS Immunohistochemistry
To reveal localization of NO-producing neurons and fi-
bers in the sturgeon brain, we used a technique of indi-
rect streptavidinbiotin immunohistochemical labeling of
NOS. The slices were incubated with primary polyclonal
rabbit antibodies against NOS (IСN Biomedicals, USA;
dilution 1:5000) at 4˚С for 24 h. After three washings out
in phosphate buffer, the slices were incubated with sec-
ondary biotin-conjugated goat antibodies against rabbit
immunoglobulins (Biomedicals, Germany) at room tem-
perature for 2 h. The material was washed out three times
in phosphate buffer. Then, the slices were incubated in
the presence of the streptavidinperoxidase complex
(Biomedicals, Germany) at room temperature for 2 h and
again washed out three times in phosphate buffer. Im-
munohistochemical reactions were visualized using a
standard avidinbiotin system (ABC; Vectastain Elite
АВС Kit; Vector Laboratories, USA). To identify the
reaction products, the slices were incubated in a substrate
for detection of peroxidase (VIP Substrate Kit; Vector
Laboratories, USA); the process of staining was con-
trolled under a microscope. Then, the slices were washed
out in three changes of phosphate buffer, mounted on
slides, dehydrated using a standard technique, and em-
bedded in balsam. To estimate the specificity of the im-
munohistochemical reaction, we used a technique of
negative control. The sturgeon brain slices were incu-
bated in a medium containing 1% nonimmune horse se-
rum (instead of primary antibodies) for 48 h, and then all
procedures were performed as was described above. In
all control experiments, the immunopositivity in the
studied cells was absent.
2.5. Statistic Analysis
Morphometric processing was performed using an in-
verted-stage microscope, Axiovert 200 M, equipped with
a module, ApoTome, and digital cameras, Axio Cam
MRM and Axio Cam HRC (Carl Zeiss, Germany). The
measurements were performed at ×400 magnification in
five randomly chosen fields of vision for each studied
region. The proliferation index (PI) and apoptosis index
(AI) were calculated per 1 mm2 of the section using the
following formulas:
of the PCNApositive nuclei
PI100% and
totalof the nuclei
of TUNELpositive fragments
AI 100%.
totalof nuclei
n
n
n
n




Parametric comparison (Student’s t-test) was used for
estimation of the intergroup differences. The data ob-
tained were processed using Statistica and Excel soft-
ware. Numerical data are presented below as means ±
s.e.m.
3. RESULTS
3.1. Labeling of PCNA
In the myelencephalon we studied regions of the V, VII,
and X nerves nuclei; on the external surface of the
myelencephalon, typical thickenings (hypertrophied lobes
of the above nerves) corresponding to these nuclei.
Within the territory of these lobes, we examined periven-
tricular, subventricular, and deeper regions of the mye-
lencephalon, namely the interfascicular region, medial
reticular formation (MRF), and perinuclear parts (adja-
cent to the nuclei) localized far from the opening of the
cerebral ventricle, as well as cells of the somatovisceral
longitudinal column forming the nuclei of the V, VII,
and X nerves.
In the periventricular myelencephalon region, PCNA-ir
cells formed a cellular layer corresponding, by its mor-
phological peculiarities, to the neuroepithelium (Figure
1(A)). This layer included morphologically heterogene-
ous cells; among such units, we classified three main
types. The morphological parameters of PCNA-ir cells in
the periventricular region of the myelencephalon are
mentioned in Table 1. Within this region, we found
small practically round units with mean large/small di-
ameters of 7.8/7.0 μm, as well as oval (11/7.5 μm), and
elongated (16/7.2 μm) units. Therefore, all the labeled
PCNA-ir neurons in this region differed from each other
only in the values of the large diameters of the somata,
while small diameters were nearly the same. The PI for
the periventricular zone of the myelencephalon was, on
average, 21.0% ± 1.3% (Table 1). In the subventricular
region, mainly in caudal regions of the myelencephalon,

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 791
Table 1. Morphometric characteristics of PCNA-, nNOS- and TUNEL-immunopositive (ip) elements and the proliferation and
apoptosis indices (PI and AI, Respectively; М ± s.e.m.) in the brain of the Amur sturgeon Acipenser schrenckii.
Brain subdivisions Dimensions of PCNA-ip
cells, μm PI, % Dimensions of TUNEL-ip elements, μm AI, % Dimensions of nNOS-ip cells, μm
Myelencephalon
Periventricular region
7.8 ± 0.4/7.0 ± 1.1
11.1 ± 0.8/7.5 ± 1.4
14.5 ± 1.5/7.2 ± 1.0
21 ± 1.3 6.4 ± 0.3/5.9 ± 0.3
8.9 ± 0.5/6.2 ± 0.4 9 ± 0.2
7.6 ± 0.7/7.1 ± 1.3
9.4 ± 0.3/8.5 ± 0.2
12.8 ± 1.0/7.1 ± 0.8
Subventricular region 9.0 ± 0.9/7.3 ± 0.9
11.0 ± 0.6/7.3 ± 0.9 3.3 ± 0.2 7.7 ± 0.5/6.9 ± 0.2 9.1 ± 0.6/7.3 ± 1.2 6 ± 0.2
7.9 ± 0.8/7.3 ± 0.4
9.6 ± 0.3/7.4.1 ± 1.3
10.9 ± 0.8/7.3 ± 0.7
Deep layers of the
myelencephalon
7.9 ± 1.1/5.7 ± 0.3
11.5 ± 1.4/9.1 ± 2.3 8.5 ± 0.2 - -
9.5 ± 0.4/9.4 ± 0.2
11.7 ± 1.0/8.6 ± 1.4
14.1 ± 0.6/12.1 ± 0.8
Cells of the medial reticular
formation (MRF) at the
isthmus level
- -
31.9 ± 2.6/28.6 ± 3.1
17.5 ± 4.4/12.3 ± 1.5
8.3 ± 1.8/5.1 ± 0.6
8.9 ± 0.3
28.3 ± 4.1/22.3 ± 5.32
0.5 ± 2.5/15.5 ± 2.6
16.6 ± 2.3/12.1 ± 3.1
MRF at the level of the
nucleus of the V nerve - -
15.7 ± 1.4/11.7 ± 1.5
9.0 ± 2.3/6.5 ± 0.6
6.2 ± 0.2/5.0 ± 0.6
10.6 ± 0.4
16.8 ± 2.7/12.3 ± 1.4
9.6 ± 1.4/7.2 ± 0.4
6.1 ± 0.2/5.4 ± 0.5
Interfascicular cells - -
30.9 ± 3.3/15.5 ± 2.1
22.4 ± 1.3/11.0 ± 1.2
12.3 ± 1.4/8.7 ± 1.5
9 ± 0.4
36.2 ± 7.3/24.4 ± 6.3
28.7 ± 4.5/22.7 ± 5.4
16.6 ± 3.2/13.2 ± 2.7
Cells of the somatosensory
column - - 8.5 ± 1.2/5.6 ± 0.4 3.4 ± 0.3
39.3 ± 4.3/28.1 ± 2.3
48.3 ± 6.5/28.7 ± 3.6
55.6 ± 8.2/43.4 ± 5.7
Cells of the external zone
6.7 ± 0.2/5.7 ± 0.9
8.0 ± 0.7/6.1 ± 0.9
11.3 ± 0.2/8.7 ± 1.3
4.5 ± 0.3 - -
6.8 ± 0.3/6.0 ± 0.9
8.1 ± 0.7/6.0 ± 0.7
10.6 ± 1.2/7.3 ± 2.5
Cerebellum
Molecular layer 8.0 ± 0.9/6.3 ± 0.9
10.3 ± 0.6/7.8 ± 0.9 9.9 ± 0.7 6.7 ± 2.2/5.3 ± 2.0 9.9 ± 1.3/7.0 ± 1.2 12.5 ± 0.9 6.7 ± 1.4/5.3 ± 0.9 8.8 ± 0.7/6.8 ± 0.7
Ganglionary layer - - 12.5 ± 1/10.2 ± 0.5 6 ± 0.5 14.8 ± 0.8/11.2 ± 0.3
17.5 ± 1.3/8.9 ± 0.6
Granular layer 7.8 ± 0.4/6.5 ± 1.4
12.1 ± 1.8/8.2 ± 1.4 15.8 ± 0.6
22.8 ± 1.2/17.8 ± 1
16.0 ± 0.8/10.8 ± 0.5
11.9 ± 0.6/9.2 ± 0.4
8.8 ± 0.9/7.2 ± 1.5
0.77 ± 0.1 6.8 ± 0.8/5.5 ± 1.0
Tectum
Periventricular layer
7.8 ± 0.4/7.0 ± 1.1
10.8 ± 0.9/7.4 ± 1.2
14.3 ± 1.2/7.3 ± 1.0
12.1 ± 1.4
3.6 ± 0.4/2.3 ± 0.2
6.0 ± 0.7/4.7 ± 0.5
7.9 ± 0.8/7.1 ± 0.3
2 ± 0.3 6.8 ± 0.4/5.8 ± 1.1
9.2 ± 0.6/7.1 ± 1.1
Marginal layer 6.7 ± 0.2/5.7 ± 0.9
8.0 ± 0.7/6.1 ± 0.9 17.8 ± 1.5 6.9 ± 1.3/5.7 ± 0.6 15 ± 1.6 6.4 ± 0.5/5.9 ± 0.7 8.2 ± 0.3/6.3 ± 0.5
Optic layer 7.2 ± 0.2/6.8 ± 0.4 5.4 ± 0.3 6.8 ± 0.6/5.4 ± 0.2 1.5 ± 0.2 -
Central gray layer 10.3 ± 0.6/7.8 ± 0.9
12.1 ± 0.8/8.2 ± 1.4 5.6 ± 0.3 7.8 ± 0.5/7.3 ± 0.2 9.9 ± 1.3/7.0 ± 1.2 8 ± 0.4 17.3 ± 1.2/7.8 ± 2.0 11.1 ± 0.8/7.2 ± 0.7
External cellular/fibrous
layer
16.5 ± 1.5/7.2 ± 1.0
12.1 ± 0.8/8.2 ± 1.4 6.3 ± 0.4 7.4 ± 0.3/7.0 ± 0.2 9 ± 0.5
14.3 ± 1.2/6.2 ± 1.2
10.1 ± 1.2/7.8 ± 1.1
7.2 ± 0.8/5.9 ± 0.3
Inner fibrous layer 10.3 ± 0.6/7.8 ± 0.9
8.0 ± 0.7/6.1 ± 0.9 8.1 ± 0.3 7.5 ± 0.3/7.1 ± 0.3 20 ± 2.3 9.7 ± 0.4/6.2 ± 0.9 7.2 ± 0.5/5.3 ± 0.4

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 792
Continued
Thalamus
Periventricular region 7.8 ± 0.4/7.0 ± 1.1
14.5 ± 1.5/7.3 ± 1.0 12 ± 1.1 6.4 ± 0.1/5.6 ± 0.2 7.6 ± 0.3 14.7 ± 1.5/7.3 ± 1.0 8.1 ± 0.5/7.3 ± 0.4
Subventricular region 9.0 ± 0.9/7.3 ± 0.9 4.3 ± 0.5 6.7 ± 0.1/5.6 ± 0.2 2.8 ± 0.5 8.7 ± 0.6/7.3 ± 0.5
10.0 ± 0.4/7.3 ± 0.3
Dorsal thalamic nuclei 9.1 ± 1.3/7.7 ± 0.5
10.1 ± 0.2/7.3 ± 0.6 7.6 ± 0.4
8.5 ± 0.9/6.5 ± 0.8
11.5 ± 0.3/7.9 ± 2.1
13.4 ± 0.2/8.7 ± 2.1
13 ± 1.3 9.1 ± 1.3/7.7 ± 0.5
12.4 ± 0.7/7.8 ± 0.6
Hypothalamus
Periventricular region 7.6 ± 0.2/7.2 ± 0.8
14.5 ± 1.5/7.3 ± 1.0 9.6 ± 0.6 6.3 ± 0.2/5.9 ± 0.3 3 ± 0.4 8.1 ± 0.8/7.2 ± 0.5 15.5 ± 1.5/7.3 ± 1.0
Subventricular region 9.1 ± 0.7/7.4 ± 0.9 6.6 ± 0.3 7.1 ± 0.2/5.8 ± 0.2 6 ± 0.3 8.7 ± 0.6/7.3 ± 0.5
11.0 ± 0.6/6.9 ± 0.6
Lateral hypothalamus 9.2 ± 0.9/7.3 ± 0.9
12.4 ± 0.7/8.8 ± 1.4 4 ± 0.5
14.8 ± 0.7/11.2 ± 3.0
19.1 ± 1.3/13.2 ± 1.2
28.4 ± 2.5/14.5 ± 1.9
18 ± 2.0 9.9 ± 0.6/7.2 ± 0.6
13.4 ± 0.4/8.2 ± 1.1
Ventral hypothalamus 9.1 ± 0.7/7.4 ± 0.9
11.8 ± 0.8/8.1 ± 1.4 4.2 ± 0.4 11.5 ± 0.3/7.9 ± 2.1 13.3 ± 0.4/10.2 ± 2.0 6.8 ± 0.3 9.4 ± 0.6/7.8 ± 0.3 12.8 ± 0.5/7.6 ± 1.4
Footnotes. Dimensions of ip elements are presented as mean values of their greater and smaller diameters separated by a slash. In deep layers of the myelen-
cephalon, PCNA-ip cells were found in the MRF at levels of the isthmus, nuclei of the V, VII, and X nerves, and interfascicular region.
we observed two types of PCNA-ip cells (Table 1). In
the rostral part of the brainstem, the PIs in the subven-
tricular and caudal zones were 3.3% and 14.3%, respect-
tively. In the projection region of the trigeminal nerve,
PCNA immunolocalization was observed in cells of the
periventricular zone adjacent to the nucleus of V nerve,
as well as in single cells of the perinuclear zone (Figure
1(b)). For these regions, the PI values were 6.3 and 9.3%,
respectively (Figure 2(A)). In the region of projection of
the facial nerve, PCNA immunopositivity was also ob-
served mostly in the periventricular zone; however, large
weakly labeled PCNA cells prosessing processes were
observed in the nucleus per se. These units did not be-
long to the population of periventricular neuroblasts
(Figure 1(C)). The PI values for the nucleus of the facial
nerve and for the adjacent perinuclear region were 9.6
and 5.6%, respectively (Figure 2(A)). Most PCNA-ip
cells localized in deep layers of the myelencephalon
were classified as belonging to different regions of the
reticular formation, to the rostral part (at the level of the
isthmus and nucleus of the trigeminal nerve) and caudal
one (at the level of projection of the vagus nerve) (Table
1). For these zones in the rostral and caudal regions of
the myelencephalon, the PI values were 8.5% and 15.6%,
respectively. In the region of projection of the X nerve,
the overwhelming majority of PCNA-ip cells were lo-
calized periventricularly, and the PI was very high
(23.7%). In the subventricular region, the number of
PCNA-ir cells was considerably smaller (PI 4.9%), while
this index was 5.7% in the deep layer (Figure 2(B)). In
the sturgeon myelencephalon, PCNA-ip elements were
also observed near the external surface adjacent to the
pia matter (Figure 1(D)). These immunopositive cells
were divided into three types; among them, two cell
types were also common for other regions, while one
type was formed by smaller units (Table 1).
In the cerebellum, PCNA-ip cells were found in the
granular and molecular layers. In the dorsal part of the
cerebellar body, the maximum number of small round
and oval PCNA-ip cells was observed (Table 1). The PI
values for granular-layer cells of the caudal and ventral
parts of the cerebellar body were 15.6% and 7.6%, re-
spectively. In different regions of the cerebellar body, the
values of this index of proliferative activity (PI) varied
(Table 1). In the rostral part, this index for cells local-
ized in the granular layer was, on the whole, lower (in
the dorsal and ventral regions, 9.6% and 7.6%, respect-
tively). In various parts of the molecular layer of the
cerebellar body, the number of PCNA-ip cells also dif-
fered from each other (Table 1). In the rostral part, the PI
was 10%, while in the caudal part this index varied from
8.3% to 7.6%. In the cerebellar valve, the majority of
PCNA-ip cells were localized in the periventricular layer
close to the opening of the intertectal ventricle (Figure
1(E)). In the molecular layer of the valve, we found
small PCNA-ip cells; the PI for these cells was 8.3%. In
the central part of the valve, large weakly labeled PCNA-
ip cells (Figure 1(E)) that could not be classified as
typical PCNA-ip units were also observed.
In the tectum, the number of labeled PCNA cells
demonstrated clearly pronounced differences. Such dis-
similarities were clear, on the one hand, in rostral and

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 793
Figure 1. Immunolocalisation of proliferating cell nuclear an-
tigen (PCNA) in the Amur sturgeon Acipenser schrenckii brain.
(A) Cross section of dorsal parts of the myelencephalon at the
caudal level. Triangular red arrows indicate PCNA-ip cells in
the periventricular (PVZ) and subventricular (SVZ) zones and
at the level of the medial reticular formation (MRF); (B) Sec-
tion through the nucleus of the trigeminal (V) nerve (region of
the nucleus (NV) is outlined by a rectangle); triangular green
arrows indicate PCNA-ip cells, black arrows indicate immu-
nonegative cells; (C) Section through the nucleus of the facial
(NVII) nerve (outlined by a rectangle); blue arrows indicate
processes of immunonegative cells of the nucleus; two small
figured arrows indicate two daughter cells possessing a com-
mon process; (D) Fragment of the myelencephalon external
zone (EZ) adjacent to the cerebral covers; (E) Section of the
valve of the cerebellum; green arrows indicate large slightly
labeled PCNA-ip cells; (F) Section through the tectum; (G)
Section at the level of the ventral thalamus, green arrow indi-
cate immunonegative cell in subventricular area. Designation:
IV-fourth ventricle; PNZ-perinuclear zone; MoL-Molecular
layer; PVL-periventricular, IFL-inner fibrous, CCL-central
cellular, ECFL-external cellular/fibrous, OL-optic, and ML-
marginal layers; VH-ventral halamus, Inf-infundibulum. Scale:
(A), (B), (D), (E)—100 µm, (C), (G)—50 µm, (F)—200 µm.
caudal parts of this structure and, on the other hand, in
medial and lateral zones (Figure 3). Most PCNA-ip cells
were localized in the tectal PVL (Figure 1(F)) where we
found all patterns of cells typical of the periventricular
zone of the myelencephalon (Table 1). In the medial
region, the relative number of proliferating cells within
the PVL was more than two times greater than in the
(A)
(B)
Figure 2. Intensity of the processes of proliferation and apop-
tosis in different parts of the myelencephalon of the Amur
sturgeon Acipenser schrenckii. Data are shown as M ± m. Ab-
scissa axis: (A) In the nuclei of trigeminal and facial nerves
(NV and NVII, respectively) and perinuclear zones adjacent to
these nuclei (PNZ V and PNZ VII, respectively); (B) In the
lobe of the X nerve. PVZ, SVZ, and DZ-periventricular, sub-
ventricular, and deep zones, respectively. Ordinate axis-Prolif-
eration index, PI (blue columns) and apoptosis index, AI (pink
columns), %.
lateral region; the PI values in these zones were 17.6%
and 7.0%, respectively (Figure 3). Highly immunoposi-
tive cells were found near the external border of the mar-
ginal layer (ML). Rather high PI values in the lateral and
medial zones were nearly the same (17.0% and 18.6%,
respectively; Figures 1(F) and 3). In the optic layer (OL),
the number of stained PCNA-ip cells was smaller. In the
medial and lateral parts, the PI values were 4.3% and
6.6%, respectively. In the central cellular layer (CCL)
and external cellular/fibrous layer (ECFL), the number of
PCNA-ip cells in the lateral zone appreciably exceeded
that in the medial parts (Figure 3). Therefore, mor-
phometric analysis showed that the majority of PCNA-ip
cells in the medial part of the tectum are concentrated in
the PVL and ML, while their numbers were relatively

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 794
Figure 3. Intensity of proliferative activity in the tectum of the
Amur sturgeon Acipenser schrenckii brain. Data are shown as
M ± m. Abscissa axis, PCNA activity in the layers of the optic
tectum: PVL-periventricular, IFL-inner fibrous, CCL-central
cellular, ECFL-external cellular/fibrous, OL-optic, and ML-
marginal layers. Ordinate axis: Proliferation index (PI), %.
Green and red columns correspond to the PI values in the me-
dial and lateral zones of the tectum, respectively.
small in the CCL, ECFL, and OL (Figure 3). In the lat-
eral zone, most PCNA-ip cells were found in the ML,
while in the PVL their number was smaller. At the same
time, a considerable proportion of the PCNA-ip cells
were localized in the deep layer of the tectum.
In the thalamus, most PCNA-ip cells were localized in
the periventricular region (mean PI value, 12% ± 1.1%)
and deep layers (Figure 1(G)). On the territory of the
dorsal thalamic nuclei adjacent to the Meynert’s retroflex
bundle, we also found weakly labeled PCNA-ip cells.
The PI value for this region was 7.6% ± 0.4% (Table 1).
In the subventricular region, we found relatively rare
PCNA-ip cells (PI 4.3 ± 0.5%) and rather numerous im-
munonegative units (Figure 1(G)). In the hypothalamus,
the relative number of PCNA-ip cells in the periven-
tricular region was greater (PI 9.6% ± 0.6%) than that in
the subventricular region (PI 6.6% ± 0.3%). In deep lay-
ers of the lateral and ventral hypothalamus, the normal-
ized number of PCNA-ip cells was 4.0% to 4.2% (Table
1).
3.2. TUNEL Labeling
In the sturgeon myelencephalon, TUNEL-labeled ele-
ments were indentified in the periventricular and sub-
ventricular zones, longitudinal somatovisceral neuronal
column, and reticular formation (Figures 4(A) and (B)).
TUNEL-positive structures in this cerebral part demon-
strated different stages of the apoptotic process. Among
the corresponding neurons, we identified both cells with
fragmented chromatin and units at later stages of the
above-mentioned process (Figure 4(C)). In the vicinity
of large reticular formation neurons containing clearly
distinguishable fragmented TUNEL-labeled chromatin,
we frequently observed numerous glial cells whose nu-
clei were stained with methyl green (Figure 4(B) and
(C)). Morphometric parameters of apoptotic elements in
different regions of the sturgeon myelencephalon are
shown in Table 1.
In the periventricular region of the sturgeon myelin-
cephalon, at the level of the projection of the trigeminal
nerve nucleus, we found apoptotic bodies with mean
diameters of 6.4/5.9 μm, which were localized among
intact cells of the periventricular region. The AI values in
the periventricular region of the myelencephalon were
8.9% to 9.0% (Table 1). In the subventricular zone, the
total proportion of TUNEL-positive cells was smaller
(6.0%; Table 1). In this zone, we found apoptotic bodies
with dimensions of 7.6/6.9 μm (Table 1). In the MRF,
neurons in earlier apoptotic stages were observed;
TUNEL-labeled chromatin was fragmented and looked
like small granules. These cells were gathered in small
groups (Figure 4(C)). The AI values in the isthmus re-
gion of the reticular formation at the level of the
trigeminal nerve and in cells of the interfascicular region
differed insignificantly from each other (8.9%, 10.6%,
and 9%, respectively; Table 1). A high relative density
Figure 4. TUNEL-containing elements in the myelencephalon
of the Amur sturgeon Acipenser schrenckii (cell nuclei are
additionally stained with methyl green, by Brasher). (A) Cross
section through the viscerosensory column of neurons; red
triangle arrows indicate apoptotic bodies; (B) Longitudinal
section at the level of the reticular formation; neurons of the
reticular formation, which contain fragmented TUNEL-labeled
chromatin (shown by blue arrow) and are surrounded by glial
cells (shown by small figured arrows); (C) Fragment containing
neurons with TUNEL-labeled fragments of DNA (shown by
triangle blue arrows) is outlined by a square in B and are sur-
rounded by glial cells (shown by black arrows); (D) Longitu-
dinal section at the level of the facial nerve (outlined by a rec-
tangle). VSC-viscerosensory column, and MLF-medial longi-
tudinal fascicle. Other designations are the same as in Figure 1.
Scale: (A), (B), (D)—100 µm, (C)—50 µm.

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 795
of glial cells has significant impact on the total AI values
within the sturgeon myelencephalon. Results of meas-
urements of morphometric indices of apoptotic bodies
showed that a significant number of cases where we
identified apoptosis in the brainstem MRF and isthmus
zone belong just to the glial cells. In the region of the
nuclei of the V nerve, we found heterogeneous TUNEL-
positive elements. Among such elements, we verified
neurons with fragmented chromatin and apoptotic bodies
of different sizes (Table 1). In this zone, the relative
number of these elements (AI) was 12.7%, while the AI
in the perinuclear zone was 10.6% (Figure 2(A)). In the
projection of the nucleus of the VII nerve, the number of
TUNEL-positive elements was smaller (Figure 4(D)). In
this zone, the AI was 5.6% vs 6.7% in the perinuclear
zone (Figure 2(A)). At the level of the trigeminal nerve
outlet, we observed in the MRF TUNEL-labeled mid-
sized neurons, apoptotic bodies similar to those observed
in the isthmus region, and also an additional type of
small dense bodies absent in the isthmus region (Table
1). In the interfascicular region, the parameters of
TUNEL-positive elements corresponded, in general, to
those observed in the isthmus region. Therefore, in the
reticular formation of the sturgeon brain, a population of
TUNEL-positive elements demonstrated noticeable mor-
phological heterogeneity that probably correlated with
the functional one. In the visceral motor column, we
found TUNEL-positive structures localized between the
motoneurons (Figure 4(A)). Labeled chromatin was lo-
calized in small neurons and also in glial cells; groups of
apoptotic bodies (final stage of cell degradation) were
frequently observed. In this region of the myelencepha-
lon, the AI was 3.4% (Table 1).
In the cerebellum, TUNEL-positive elements were
found in the molecular, granular, and ganglionary layers
(Figure 5(A)). The pattern of apoptotic manifestations in
different parts of the cerebellar body is shown in Figure
6. In the dorsal, central, and lateral regions of the cere-
bellar body, the number of apoptotic elements was ap-
preciably different in the above layers. The maximum
number of TUNEL-labeled cells was found in the mo-
lecular layer of the dorsal part of the cerebellar body
(Figures 5(A) and 6). In the molecular layer, we ob-
served morphologically heterogeneous labeled structures
of two types, small (6.7/5.3 μm) and larger (9.9/7.0 μm;
Table 1). The AI for the cells of the dorsal region of the
molecular layer was 18%. In the lateral part, the relative
number of cells with apoptotic manifestations was
smaller (AI 12%), while in the central part we detected
the minimum number of such elements (AI 7.5%). In the
ganglionary layer of the central and lateral parts of the
cerebellar body, we found TUNEL-labeled structures
whose sizes were 12.5/10.2 μm (Table 1). The AI values
for cells of the ganglionary layer of the central and lat-
eral parts were 14% and 4%, respectively (Figure 6). In
the dorsal part of the cerebellar body, TUNEL-labeled
elements in the ganglionary layer were absent. Accord-
ing to the characteristics of TUNEL-positive elements,
Figure 5. TUNEL-labeled elements in the cerebellum, tectum
and thalamus of the Amur sturgeon Acipenser schrenckii brain.
(A) Longitudinal section through the central part of the cere-
bellar body; red arrows indicate TUNEL-positive structures in
different layers of the body, blue arrows–the Purkinje cells; (B)
Section through the medial zone of the tectum; apoptotic bodies
shown by figured arrows; (C) Horizontal section through the
central thalamic nucleus (CThN), delineated by rectangle, con-
taining apoptotic elements, shown by red figured arrows, the
vessels shown by blue figured arrows; (D) Apoptotic bodies
(compact or irregular-shaped grained; shown by white arrows)
localized among gliocytes and neurons (shown by small red
figured and black arrows, respectively) in the central thalamic
nucleus. Other designations are the same as in Figure 1. Scale:
(A), (B), (D)—100 µm, (C)—50 µm.
Figure 6. Intensity of the apoptotic process in different regions
of the cerebellar body of the Amur sturgeon. Vertical scale-
Apoptosis index (AI), %. CR, DR, and LR-Central, dorsal, and
lateral regions of the cerebellar body. Yellow, blue, and pink
columns correspond to the values of the AI in the granular,
ganglionary, and molecular cerebellar layers, respectively.

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 796
the granular layer was characterized by maximum mor-
phological heterogeneity; among such elements, we clas-
sified four main types (Table 1). Another peculiarity of
the granular layer of the sturgeon cerebellum was the
rather high density of cells localized in this layer. This
was noticeably reflected on the AI values for this region;
in its dorsal, central, and lateral parts, the A. Is were
1.2%, 0.45%, and 0.7%, respectively (Figure 6).
In the tectum, TUNEL-labeled structures were ob-
served in all layers (Figure 5(B)). Among such elements,
apoptotic bodies (6.0/4.7 μm) localized in the CCL and
ECFL were most frequently observed (Table 1). Smaller
bodies (3.6/2.3 μm) were found in the inner fibrous layer
(IFL) and ECFL, while comparatively large labeled ele-
ments (8.0/7.1 μm) were identified in the CCL and ECFL.
In the PL, the concentration of TUNEL-positive struc-
tures was low (AI was equal to 2%). In contrast, we
found the maximum number of apoptotic structures in
the IFL; in this layer, the AI reached its maximum (20%).
In the CCL and ECFL, the number of apoptotic elements
was moderate; the AI values were 8% and 9%, respec-
tively (Table 1). In the OL, the number of TUNEL-
positive elements was the lowest (AI was 1.5%), while in
the ML we observed 15.0% of cells with manifestations
of apoptosis (Table 1).
In the thalamus, we identified four types of TUNEL-
positive structures (Figure 5(C)). Smaller elements
(6.4/5.6 μm) were observed in the subventricular region
and lateral thalamus. These elements were, probably,
products of apoptotic degeneration of glial cells. Larger
sites (8.5/6.5 μm) were found in the dorsal thalamic
zones at a distance from the periventricular region. In the
region of the central thalamic nuclei, the largest (11/7.9
and 13.4/8.4 μm) TUNEL-labeled apoptotic bodies (com-
pact or irregular-shaped grained ones) were present (Fig-
ure 5(C)). It is obvious that such bodies are indicative of
the late stages of apoptotic degradation of cells in the
central thalamic nuclei (Figure 5(D)). In the lateral
thalamus, we observed large cell conglomerates with
apoptotic manifestations (diameter 31.5/25.8 μm) to-
gether with single apoptotic bodies. The AI in the stur-
geon thalamus was 7.6%.
In the hypothalamus, small TUNEL-positive apoptotic
bodies (6.3/5.9 μm) were present in the periventricular
and subventricular regions. In periventricular, subven-
tricular, and lateral hypothalamus, the AI values were,
respectively, 3%, 6%, and 18%. Larger elements (8.6/6.5
μm) were localized in deep layers of the lateral and ven-
tral thalamus. The sturgeon hypothalamus was charac-
terized by a very low density of glial cells compared with
that in the thalamus and brainstem. In the lateral hypo-
thalamus, we found TUNEL-positive conglomerates con-
sisting of several cells (dimensions of these structures are
shown in Table 1), which were localized near the exter-
nal surface.
3.3. Immunolocalisation nNOS
In the sturgeon myelencephalon nNOS-labeled elements
were indentified in the periventricular and subventricular
areas, deep brain areas and cells, which are located near
the external brain surface, adjoining to the brain mater as
well as to heterogeneous neuron population of medial
and median reticular formation, glial cells and neurons of
somatovisceral medullar column (Figures 7(A)-(D)). In
periventricular area the heterogeneous populations of
nNOS-ip cells are arranged on a border with IV ventricle,
forming a laminated layer (Figure 7(A)). In subventricu-
lar area the number of nNOS-ip cells much less, but they
are presented by morphologically heterogeneous popula-
tion (Figure 7(A), Table 1). The majority of the soma-
tovisceral medullar neurons express nNOS too, while
immunopositivity was revealed in large multipolar cells
(obviously motoneurons) as well as in not so large neu-
rons (Figure 7(A)). In deep layers of medulla the nNOS-
ip cells were revealed in a content of median and medial
reticular formation (Figures 7(B) and (C)). The distribu-
tion density of small nNOS-ip cells in brainstem was
sufficiently big, but the number of large reticulospinal
neurons was comparatively little. Near the brain external
surface, bordering with the brain membranes and adjoin-
ing areas, the heterogeneous nNOS-ip cells owing high
enzyme activity level were revealed (Figure 7(D)).
In cerebellum the nNOS-immunopositivity was re-
vealed in the cells of molecular, granular and ganglionic
layers (Figure 7(E)). In molecular layer the small inten-
sively nNOS-labeled cells of two types were revealed
(Table 1), which were located solely or in small groups
(Figure 7(E)). In ganglionic layer two types of nNOS-ip
cells, the oval 14/11 and spindle-shaped 17/9 (Figure
7(E)), forming the Purkinje’s cells and eurydendroid
cells population, accordingly, were revealed. In granular
layer a vast majority of nNOS-ip cells was presented by
small round and oval neurons, sizing 7/6 (Figure 7(E),
Table 1).
In tectum nNOS-ip cells in all layers, except optical
one, were revealed (Figure 7(F)). In marginal layer the
fibers and two types of cells, the small 6/6 and bigger 8/6,
were intensively labeled (Table 1, Figure 7(F)). In
ECFL nNOS was demonstrated in three types of large
cells (Table 1, Figure 7(F)). In CCL two types of
nNOS-ip cells, spindle-shaped and oval, were detected
(Figure 7(E)). In PL and IFL two types of small moder-
ately labeled nNOS-ip cells were determined (Table 1,
Figure 7(F)).
In thalamus nNOS-ip cells in periventricular and sub-
ventricular areas were verified (Table 1). In periventricu-
lar area large number of elongated highly immunopositive

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 797
Figure 7. Immunolocalisation neuronal nitric oxide synthase
(nNOS) in the brain of Amur sturgeon Acipenser schrenckii. (A)
Cross section of dorsal parts of the myelencephalon, triangular
black arrows indicate nNOS-ip cells in the periventricular zone,
blue arrows indicate ip neurons in the somatovisceral column;
black vertical arrows indicate small ip cells of deep area; (B)
Immunopositive neurons (shown by black arrows) in the me-
dian and medial in (C) reticular formation, (D) Fragment of the
myelencephalon external zone adjacent to the cerebral covers;
(E) Cross section through the central part of the cerebellar body;
black vertical arrows indicate nNOS-positive structures in mo-
lecular and granular layers of the body, blue arrows–the Pur-
kinje cells, green arrow–eurydendroid neuron; (F) Cross sec-
tion through the medial part of the optic tectum; arrows indi-
cate nNOS-positive cells in different layers; (G) Horizontal
section through the hypothalamus. Other designations are the
same as in Figures 1 and 4. Scale: (A)-(D), (F), (G)—100 µm,
(E)—50 µm.
cells was founded. In dorsal thalamic area the oval and
elongated nNOS-ip cells were revealed (Table 1). In
periventricular area of hypothalamus the number of
nNOS-ip cells was very high (Figure 7(G)), the most of
them have had spindle-shaped form and they were or-
ganized as neuroepithelium. In subventricular area im-
munopositive cells were not so numerous and have rep-
resented two types of cells (Table 1). In deep layers of
lateral and ventral hypothalamus the number of nNOS-ip
cells was reduced, but the labeled cells were bigger in
comparison with those in periventricular and subven-
tricular areas (Figure 7(G)).
4. DISCUSSION
The results of experimental and morphological investiga-
tions on teleost fishes have shown, that in the CNS neu-
rons NO is participating in different functional mecha-
nisms, connected with regeneration [13], differentiation
[14,15], cluster communications between neurons and
glial cells [16], neuromorphogenesis [13,17], neuro-
transmission and cell metabolism [18]. The results of
immunohistochemical and morphometric analysis of the
nNOS-, PCNA and TUNEL-positive elements in three-
years-old sturgeon brain have shown the availability of
large amount of immunopositive cells, but ratio between
proliferation and apoptosis in the different sturgeon brain
parts were differed substantially, what may be connected
with unequal rate of growth and differentiation of the
different brain regions.
Taking into account the known fact, that proliferative
processes in matrix brain areas are not restricted by em-
bryonic period, but continue during postembryonic
stages of development; the special role in these procesess
is given in low vertebrates, especially in teleost fishes, to
nitric oxide [20,21]. In the brain of adult zebrafish,
nNOS mRNA-expressing populations are closely associ-
ated with the proliferation zones [22], that generate new
cells throughout life [23]. The results of present study
demonstrate the nNOS presence in periventricular areas,
which contain ependymal cells, subventricular area, deep
brain layers and also external zone, adjoining to the brain
coat in adult specimen of sturgeon. In the same brain
areas the PCNA-ip and ТUNEL-labeled structures were
identified, what witness the NO participation both in
processes postembryonic cells proliferation and in apop-
tosis. One proposed mechanism for the effects of NO in
developmental processes is a suppressive influence on
DNA synthesis, whereby NO acts a negative regulator on
precursor cells and thereby affects the balance of cell
proliferation, differentiation and apoptosis [13,24]. In
mammals, the presence of NO-producing sustems and
NO-mediated action in developmental processes of the
CNS have preferentially been studied during early post-
natal stages [25]. It has been shown, that in early postna-
tal period the development continue in ependymal cells
of third ventricle in rats [14], central canal of spinal cord
in mouse [26], and in lateral ventricle in rabbit [27]. The
main sources of NO-mediated reactions, including those
in neurogenesis and neuronal plasticity, are nNOS or
similar isoforms of enzyme in different species [24,25],
including in restricted brain areas with ongoing neuro-
genesis and neuronal plasticity in adult mammals [28,
29]. The involvement of NO in postnatal neurogenesis
was found in different vertebrates [30]. In lower verte-
brates such as teleosts, NO has been emphasized to play
a versatile role in the developmental of the CNS during

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 798
both embryonic and post-embrionic life stages [20,21].
Two main neurogenesis sites have been identified in the
adult mammalian brain. They include the subventricular
zone (SVZ), and the subgranular zone (SGZ) of dentate
gyrus (DG). The process of neurogenesis is composed of
following main steps, which include precursor prolifera-
tion, migration; differentiation, integration and survival.
It has been demonstrated, that SVZ of mammals is sur-
rounded by nNOS positive neurons [31] and cells ex-
pressing nNOS, also have been identified in neuronal
precursors in DG [28]. These findings suggest nNOS
might take part in neurogenesis regulation. In morpho-
logical study on mammals the adult mouse brain have
been shown, that nitric oxide (NO), a signaling molecule
in the nervous system with a role in embryonic neuro-
genesis, may be produced in the proximity of the pro-
genitor cells in the adult brain, as a prerequisite to pro-
posing a functional role for NO in adult neurogenesis
[29]. Proliferating and immature precursor cells were
identified by immunohistochemistry for bromodeoxyu-
ridine (BrdU) and nitrergic neurons by either NADPH-
diaphorase staining or immunohistochemical detection of
neuronal NO synthase. Nitrergic neurons with long vari-
cose processes were found in the SVZ, intermingled with
chains of cells containing BrdU. Within the olfactory
bulb, many small cells in the granular layer and around
the glomeruli expressed either BrdU or nNOS and, in
some cases, both markers [29]. Colocalization was also
found in a few isolated cells at a certain distance from
the neurogenesis areas. The anatomical disposition
shown indicates that NO may be released close enough
to the neuronal progenitors to allow a functional influ-
ence of this messenger in adult neurogenesis.
The participation of nitric oxide in the postembryonal
proliferation process in different brain areas of sturgeon
confirm the results of correlation analysis of the nNOS-
and PCNA-ip cells parameters in periventricular, sub-
ventricular and deep medullar areas (Table 2). It has
been shown, that correlation coefficients (CC) between
parameters of nNOS- and PCNA-ip cells in periven-
tricular area constitute 0.9; in subventricular area—0.8;
and in deep medullar layers 0.9 (in Table 2, the CC val-
ues are shown by bold letters). The analysis of the data
obtained for this cerebral structure shows the presence of
relationships between NO-ergic and proliferative cells. In
present time it is established, that in vertebrates NO con-
stitutes a signal molecule acting not only during embry-
onic neurogenesis, but in postembryonic period too [29].
The results of our investigation confirm this resume and
allow to suggest the nNOS participation in postembry-
onic morphogenesis of the sturgeon brain.
Studies in mammals have shown that during embry-
onic development apoptosis leads to elimination of
young cells, which, after arrival at the target site, have
failed to make proper connections with other neurons
and to receive adequate amounts of specific survival
factors produced by cells in the target area [1]. Although
not yet examined in the context of adult neurogenesis, it
is possible that a similar mechanism regulates the num-
ber of cells born in the adult brain. The investigations of
cellular and molecular mechanisms of apoptosis have
shown that these processes are caused by the free radi-
cals accumulation, especially of the NO-metabolites.
They caused the glutamate exitotoxicity development,
which, in turn, leads to oxidative stress [12,32]. The re-
sults of the nNOS immunolocalisation investigation have
shown that, NO-producing systems include the morpho-
logically and functionally heterogeneous neurons and
glial cells. Our data are in accordance with results in dif-
ferent teleost species: Oreochromis niloticus [9], goldfish
[6,7] and Danio rerio [22]. It has been shown, that NO in
normal conditions in the teleost fishes brain is present in
various types of neurons [5,8-10], as well as in astrocytes
and endotheliocytes [7], oligodendrocytes [11], and
tanicytes [12], which are widespread practically in all
areas of brain and spinal cord. It is known, that excessive
NO production in the neuron cytoplasm stimulates the
local formation of superoxide ions, which form the first
component of cytotoxic effect [33]. The zone of super-
oxide anion toxicity is determined by the diffusion con-
stant in the brain tissue. NO has been shown [34] to be
able to diffuse to some 130 μm, what exceeds considera-
bly the average size of cellular somata, but quite conform
for the intercellular transmission. This may bring to for-
mation of peroxynitrite in adjacent cells. Toxic oxidants
disturb the connections between components of cell
membranes and cytoplasmic proteins, triggering the
mechanisms of apoptosis [35]. The molecular properties
of NO prevent it from being deposited in organelles and
synaptic terminals, though it serves ideal condition for
spatial signaling between neurons [24]. The results of our
investigation demonstrate, that NO-ergic production in
the brain cells of sturgeon is sufficiently high, so NO may
easily penetrate through nuclear membrane and influence
autocrinically a genetic apparatus of neuboring neurons
and glial cells, causing their mass mortality. This con-
firms the results of immunolocalisation TUNEL- and
nNOS-ip cells in different parts of sturgeon brain. NO is
capable to diffuse on substantial distances, securing the
mechanisms of apoptotic death in whole conglomerates
of neuronal cells, which we revealed in sturgeon in
thalamus, cerebellum and lateral hypothalamus. High
coefficients in apoptotic and NO-ergic elements were
shown in investigation of TUNEL-labeled and nNOS-ip
cells in different brain areas. Thus, in the isthmus area
CC have constituted 0.8; in central thalamic nucleus—0.8;
caudal brain stem area—0.9; (in Table 3 CC values are
shown by bold letters). This allow to suggest the NO

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 799
Table 2. Correlation coefficients for relations between PCNA and nNOS-ip parameters of neurons in medulla oblongata of the Amur
sturgeon Acipenser schrenckii.
PVZ
(NO)а
PVZ
(NO)б
PVZ
(PCNA)а
PVZ
(PCNA)б
SVZ
(NO)а
SVZ
(NO)б
SVZ
(PCNA)а
SVZ
(PCNA)б
DZ
(NO)а
DZ
(NO)б
DZ
(PCNA)а
DZ
(PCNA)б
PVZ
(NO)а 1
PVZ
(NO)b −0.02 1
PVZ
(PCNA)а 0.92 −0.21 1
PVZ
(PCNA)b −0.06 −0.30 −0.003 1
SVZ
(NO)а 0.95 −0.27 0.94 0.08 1
SVZ
(NO)b 0.48 0.11 0.37 −0.01 0.43 1
SVZ
(PCNA)а 0.86 −0.10 0.90 0.17 0.89 0.21 1
SVZ
(PCNA)b 0.31 0.11 0.47 −0.05 0.29 −0.34 0.65 1
DZ
(NO)а 0.91 −0.18 0.93 0.16 0.93 0.61 0.86 0.31 1
DZ
(NO)b 0.34 0.01 0.49 0.25 0.31 0.26 0.29 0.15 0.48 1
DZ
(PCNA)а 0.96 −0.06 0.93 −0.01 0.96 0.53 0.88 0.32 0.93 0.31 1
DZ
(PCNA)b 0.68 0.15 0.54 0.23 0.67 0.68 0.67 0.13 0.73 −0.01 0.76 1
Footnotes: Meaningful correlation coefficients values are shown by bold letters, explanation see text. Designations: PVZ-periventricular; SVZ-subventricular;
DZ-deep zone of medulla oblongata, a and b-maximum length (large diametr) and maximum width (small diametr) of the cell cection.
Table 3. Correlation coefficients for relations between TUNEL-labeled and nNOS-ip parameters of neurons in the isthmus, central
thalamic nuclei and caudal part of medulla oblongata of the Amur sturgeon Acipenser schrenckii.
IA (TUNEL) IA (NO) CTN (TUNEL) CTN (NO) CMO (TUNEL) CMO (NO)
IA (TUNEL) 1
IA (NO) 0.87 1
CTN (TUNEL) 0.77 0.76 1
CTN (NO) 0.57 0.66 0.80 1
CMO (TUNEL) 0.65 0.85 0.77 0.50 1
CMO (NO) 0.81 0.71 0.90 0.52 0.95 1
Footnotes: Meaningful correlation coefficients values are shown by bold letters, explanation see text. Designations: IA-isthmus area, CTN-central thalamic
nucleus, CMO-caudal part of medulla oblongata.
participation in the cell death control in postnatal onto-
genesis in the amur sturgeon brain.
Thereby, the results of investigation show, that in the
sturgeon brain NO may act as a cytotoxic proapopto-
genic factor, as well as a factor stimulating proliferation.
Cytotoxic and neuroprotective effects may regarded as
interconnected elements of one action. Supposing, that
redundant NO formation potentiate apoptosis mecha-
nisms in the zones of postmitotic neuroblasts localization,
but the factors, which decrease NO production, may be

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 800
regarded as compensatory. NO, which has proapoptotic
actions, is actively involved in the mechanisms of neu-
rogenesis. Taken together, these findings suggest that
apoptosis is used as a mechanism which regulates the
number of young cells after they have reached their tar-
get areas. A similar observation has been made in the
teleostean retina during postembryonic development.
Apoptotic cell death occurs predominantly in areas
where the new cells differentiate and become integrated
into visual circuits [36].
4.1. Proliferation and Apoptosis in
Postembrionic Ontogenesis of Sturgeon
According to the modern concept, cell proliferation, cell
differentiation, and apoptosis in the fish brain within the
postembryonic period and also in adult individuals
should be considered an integral set of interrelated func-
tional phenomena [37]. The development of “newborn”
cells in the adult brain of teleost fishes is characterized
by a few specificities, either remain near the region
where they developed or can migrate (during 1 to 2
weeks from the moment of their formation) from the
proliferation zone (where they were formed) to the spe-
cific target regions [38]. In young (2-month-old) stur-
geons, active proliferation of the cells was observed in
the periventricular regions of the telencephalic, preoptic,
and thalamic brain subdivisions [38]. Within this stage of
development, an extremely high proliferation potential in
the sturgeon brain also results from the formation of
secondary foci of cell proliferation (secondary matrix
zones of the brain). In 3-ears-old sturgeons, a high pro-
liferative activity is preserved in the periventricular zone
of different cerebral subdivisions, and the intensity of
proliferation significantly exceeds that of apoptosis.
These phenomena are more expressed in the caudal divi-
sions of the myelencephalon; this fact allows us to con-
sider this region as the main center of postembryonic
neurogenesis. In contrast, the number of cells subjected
to apoptosis either exceeds the number of proliferating
units (in the myelencephalon) or is nearly equal to the
above index (in the hypothalamus and thalamus) in the
subventricular zone of the sturgeon brain. Studies carried
out on fishes, e.g., on Apterono tus , showed that about
50% of “adult-born” brain cells are eliminated according
to the apoptotic scenario during several weeks from the
moment of their formation and migration to deep cere-
bral layers [39]. The remaining 50% of the cells can re-
main in a resting state during the entire period of life of
the fish or can differentiate into neurons or gliocytes [39].
In deep layers of the sturgeon myelencephalon, we have
found small PCNA-ip cells, which were probably glio-
cytes.
In contrast, the distribution of apoptotic and NO-ergic
elements was observed in functionally heterogeneous
cell populations (neurons of the reticular formation, nu-
clei of the craniocerebral nerves, and perinuclear regions
of V, VII, and X nerves), as well as in gliocytes. Such
distribution of apoptotic phenomena in adult fishes is
typical for the postmitotic differentiating cerebral zones
[2]. The presence of NO in V, VII, X craniocerebral nu-
clei and adjoining reticular formation, earlier was re-
vealed in other species of adult teleost fishes [5,7-10,40],
but the presence of NO-producing elements in the
somato- and viscerosensor medullar areas may mean,
that in these areas NO is acting as apoptogenic factor,
which induces cellular death in areas of the postmitotic
neuroblasts localization and render the controlling effect
on development and differentiation of chemosensory
areas in postembryonic development. We consider the
presence of highly immunoreactive cells in the external
(adjacent to brain coats) layers a special feature of the
above-mentioned age period. Such PCNA- and nNOS-
labeled cells were found in the myelencephalon, cere-
bellum, and tectum. Our data agree with observations
made on other fish species within the period of postnatal
ontogenesis [1,2]. Recent studies on fishes demonstrated
that numerous centers of proliferation are localized along
the entire rostro-caudal axis of the brain. However, the
patterns of postnatal proliferation were investigated in
detail only in three fish species, namely in the stickle-
back (Gasterosteus aculeatus, Perciformes), a gymnoti-
form electric fish (knifefish), Apteronotus leptorhynchus,
and a cyprinid fish, danio (Danio rerio) [37].
In the sturgeon cerebellum, a considerable prolifera-
tive potential was found in the cells localized in the mo-
lecular layer. Alongside with intense proliferation in this
layer, we also observed here clearly pronounced apop-
tosis. Investigation of nNOS immunolocalisation have
shown, that in molecular and granular layers in sturgeon
brain the nNOS-ip cells are presented by morphologi-
cally homogeneous type of neurons, morphometric char-
acteristic of which are in accordance with such of apop-
totic bodies, revealed in the same zones. Thus, we may
prognosticate, that NO in sturgeon cerebellum serve as
proapoptogenic factor, regulating death of postmitotical
cells of molecular and granular layers in sturgeon. The
NO-ergic activity identification in projection neurons of
ganglionic cerebellum layer in Purkinje and euryden-
droid cells confirm the data on other species of fishes [5,
6,41]. Nitric oxide, synthesized in Purkinje cells and in
somas of eurydendroid neurons, may act as a volume
neurotransmitter to the significant distance in the cere-
bellum, by extracerebellar projections forming, which
arise from eurydendroid neurons [42]. The presence of
TUNEL-positive elements, corresponding parameters of
gandlionic cells, confirm the participation of NO as a
factor, regulating quantity of Purkinje cells and euryden-

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 801
droid neurons in postnatal development.
4.2. The Sensory System Development
In contrast to mammals in which central divisions of
sensory systems are completely formed and correspond
strictly to the number of sensory receptors at the moment
of birth and/or immediately after this event, sensory pro-
jections in the fish brain continue their growth and de-
velopment during the entire life. Such a peculiarity of the
fishes is related to the fact that the CNS organization
must adapt to a significant permanent increase in the size
of the body and, correspondingly, to a rise in the volume
of incoming sensory information. Identification of nNOS
in projections areas of the somato- and viscerosensory
nuclei of the myelencephalon and tectum opticum of the
sturgeon confirmed the hypothesis about participation
NO in controlling of postnatal development of sensory
systems. Our findings agree with the published data on
intensification of differential growth in primary sensory
regions in the lobe of the nucl. vagus of the carp, as well
as in the Danio retina and tectum, compared with other
cerebral regions [43,44]. In the retina and tectum of birds
and reptiles, the intensity of neurogenesis decreases sig-
nificantly within the postembryonic period, up to com-
plete cessation of this process [45]. Up to now, it remains
unknown whether all types of neurons develop and are
integrated into the corresponding networks of the grow-
ing brain of fishes. It seems probable that some initial
level of organization of neuronal networks in fishes is
already pre-formed at the moment of their hatching, and
only some types of neurons continue their formation and
integration into existing networks during the later period
of life.
It is believed that the weak ability for substitution or
development of new neurons in the mammalian brain is
related to the limited ability of such cells in animals of
this class to be integrated into mature neuronal networks
[46]. It is hypothesized that neurons formed de novo in
adult animals are distinguished by a higher plasticity
compared with that of preexisting cells [47,48]. This
viewpoint agrees well with our findings on the sturgeon
and allows us to suppose that postembryonic neurogene-
sis correlates with coordinated growth of the sensory
systems and sensory structures of the brain. Therefore,
this phenomenon can open possibilities for the process-
ing of new ontogenetic experience. The intensity of pro-
liferative activity of cells in the medial zone of the tec-
tum opticum of the sturgeon brain exceeds that in the
lateral zone. This allows us to hypothesize that the
growth of the tectum is asymmetric; this structure is
enlarged mostly at the expense of the medial zone. In
contrast to the activity of PCNA within early postnatal
development stages, the maximum respective activity in
the tectum of 3-year-old sturgeons is observed in the ML.
In this part, in contrast to the myelencephalon and cere-
bellum, we found cell populations with PCNA-, TUNEL
and NO-production. This findings allow us to suppose
the presence of NO-dependent mechanism of regulation
in tectum opticu m postnatal morphogenesis. Proliferating
cells were also observed in deep layers of the tectum, and
the intensities of proliferation in such layers of the me-
dial and lateral zones differed from each other signifi-
cantly. The maximum concentration of apoptotic ele-
ments was observed within the IFL corresponding to the
zone of localization of postmitotic neuroblasts, which
left the proliferative cycle in the PVL.
In the sturgeon myelencephalon, the chemosensory
region corresponding to projections of the facial and
vagus nerves was characterized by increased prolifera-
tive activity; the intensity of the latter exceeded the level
of apoptosis in these regions. High values of the AI in
the perinuclear regions of the trigeminal, facial, and
vagus nerves in the sturgeon brainstem allow us to con-
sider these zones as postmitotic differentiating regions
typical of late stages of neurogenesis. Such a conclusion
agrees with the data obtained on the Apteronotus [37]. It
is likely that excessive neuroblasts formed in the periven-
tricular zone and migrating to the zones of differentiation
are eliminated in these regions of the sturgeon brain ac-
cording to the apoptosis scenario. Incorporation of new
cells into the neuronal networks existing earlier in the
sensory regions is directly related, first of all, to an in-
crease in the size of the brain in the course of growth of
the fish. However, it should be taken into account that
fishes, immediately after hatching, possess relatively
well preformed sensory and motor systems making pos-
sible rather rapid training for complex behavioral habits,
e.g., active catching of food and avoidance of predators.
This indicates that some parts of the CNS of fishes,
which are responsible for information processing and
realization of functional needs of the organism necessary
within a certain life period, begin to function before
hatching. The later postembryonic growth can be con-
sidered a process of delayed development related to the
maintenance of the functions necessary in future, e.g., for
the formation of zoosocial communication or sexual be-
havior. Therefore, our conclusion that some parts of the
sturgeon brain remain, in fact, in the neotenic state over a
rather long postembryonic period seems to be quite logi-
cal. This hypothesis explains high indices of proliferative
activity in some brain regions in cartilaginous ganoid
fishes.
5. CONCLUSIONS
Therefore, our findings allow us to conclude that the
processes of proliferation and apoptosis in the brain of

E. V. Pushchina, D. K. Obukhov / Advances in Bioscience and Biotechnology 3 (2012) 788-804 802
3-year-old sturgeons remain rather intense, and the ratios
between these processes in different brain structures are
rather dissimilar.
Nitric oxide in the Amur sturgeon brain may play a
role of cytotoxic proapoptogenic factor and a factor,
which stimulate cell proliferation. The availability of
NO-producting elements in somato- and viscerosensory
medullar areas, tectum, cerebellum, thalamus and hypo-
thalamus of Amur sturgeon permits to suppose that in
these areas NO constitutes a proapoptogenic factor, in-
ducing the programmed cell death, occurring in the areas
of the postmitotic neuroblasts localization, causing regu-
lating impact on development and differentiation of
chemosensory, visual, motor and hypophysotrophic brain
areas in postnatal development. The maximal prolifera-
tion activity and high concentration of NO-ergic cells
were revealed in external layers, adjacent to the subpial
medullar, cerebellar and tectal zones, what allows to
suppose participation of NO in postembryonic neuro-
genesis in these brain structures as a factor, which regu-
late cells proliferation. The highest concentration of pro-
liferating cells was revealed in the medullar periven-
tricular area, that allows to regard this area as a major
brain region of postembryonic neurogenesis. The differ-
ent ratios between proliferation and the programmed cell
death indices were revealed in sensor brain centers: tec-
tum and projections of V, VII and X cranial nuclei, wit-
nessing the presence of different growth and differentia-
tion speed, taking place in visual and chemosensory cen-
ters of the Amur sturgeon brain.
6. ETHICAL APPROVAL
The experiments were approved by the Animal Ethics Committee of A.
V. Zhirmunskii Institute of Marine Biology, Far Eastern Branch of
Russian Academy of Sciences Vladivostok, Russia.
7. AUTHOR STATEMENTS
The manuscript is original, has not been submitted to or is not under
consideration by another publication, has not been previously published
in any language or any form, including electronic, and contains no
disclosure of confidential information or authorship/patent application
disputations.
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