Engineering, 2012, 5, 76-79
doi:10.4236/eng.2012.410B020 Published Online October 2012 (
Copyright © 2012 SciRes. ENG
Interaction of Atoms with Grain Surfaces in Steel: Periodic
Dependence of Binding Energy on Atomic Number and
Influence on Wear Resistance*
E. V. Pushchina1, D. K. Obukho v2
1A. V. Zhirmunski Institute of Marine Biology, Far East Division, Russian Academy of Sci ences,
ul. Pal'chevsk ii 17, Vladivostok, 690059 Russia ru
2St. Petersburg State University, Universitetskaya Naberezhnaya 7/9, St. Petersburg, 199034 Russia
Email: puschina@mail
Received 2012
The data of our investigations contribute to understanding of cellular mechanisms of the teleost fishes CNS forming in postembryo-
nic development. The revealed peculiarities of structural and neurochemical organization and description of basic histogenetic
processes (proliferation, migration and neuronal cell differentiation) during the brain forming in fish, which have signs of fetal or-
ganization, widen the existing knowledge about histogenesis of these structures in postembryonic development. It seems conceivable,
that during postembrional development in teleo st fishes so me neurot ransmitters and gaseou s mediators ( NO and H2S) act as factor s,
which initiate and regulate the cellular and the tissues processes of genetic program during the brain development. Materials of this
investi gat ion define a new experimental model for studying of postembrional neurogenesis processes.
Keywords: Teleostei; Postnatal Neurogenesis; Proliferative Cell Nuclear Antigen (PCNA); Neurotransmitter Signaling; Migration;
Tyrosine Hydroxylase; GABA; Development; Pa x6; NADPH-diaphorase; Nitric Oxide; Hydrogen Sulfide; Proliferation.
1. Introduction
The fishes brain have unique peculiarity among the vertebrates.
It grows with organism during all their life. Thereby, the
fishes are an attractive animal model for investigation of the
embryonal and postembryonal central nervous system (CNS)
development and different impacts on these processes. It has
been shown, that in the adult vertebrate brain the system of
cambial el ements is pres erved. Their activity allo ws to increase
the neurons and glia population during all postnatal period [1].
Currently, the mechanisms of pre- and postnatal neurogenesis
in fishes, which have long standing fetal state, are unknown. In
recent years, considerable attention of different neuroscientists
was attracted to the gaseous mediators (NO and Н2S) participa-
tion in the brain work. Their presence was revealed in the brain
of the different vertebrates groups - from cyclostomes to
mammals. Th ese r ese arch es acqu ir e speci al meanin g in co n nec-
tion with a new data about morphogenetic role of classical and
gaseous mediato r s in the vertebrates CNS develo pment [2].
Pacific salmons were the main objects of our investigation.
They present an ancient group of vertebrates and the oldest
branch of actinopterygian fishes. Today, the availab le literature
data, concerning information about salmon brain development,
interrelations between embryonic and definitive parts in their
structure, pre- and postembryonic neurogenesis, organization
and establishment of neurotransmitter and modulated brain
system are very limited .
2. Participation of Classical Neurotransmiters in
Postembrionic Neurogenesis in the Salmon
Br ai n
The present study allows to suggest, that in the pacific salmon
brain exist two forms of intercellular communication through-
out different age periods of postembryonic development. The
first form occurs on the early stages of postembryonic devel-
opment and present intercellular interaction, which is realized
through paracrine mechanism, during which the cells do not
have full-fledged outgrowths (dendrites and axon) and synaptic
structure yet. However, such a low differentiated cells by this
time are capable t o express a sp ecific synthesises: so me neuro-
transmitters and enzymes, synthesizing them, gasotransmitters,
transcriptional factors etc (Figure 1А, C-D). We believe, that
most of signals, which are synthesized during this period, par-
ticipate in regulation of neuron-targets differentiation and spe-
cific phenotype expression, as morphogenetic factors, what
corresponds to Ugrumovs conception [2], concerning the
mammalian brain development during embryonic ontogenesis.
It is known, that a neuron begins to release typical signal mo-
lecules shortly after their formation from cellsprogenitors and
long before the formation of interneuronal connections occurs.
A large proportion of all signaling molecules are involved in
autocrine and paracrine regulation of differentiation of neu-
rons–targets and they function as morphogenetic and transcrip-
tion factors. In mammals the duration of the signal molecules
action is limited to certain periods of ontogeny, when processes
of differentiation of neuronstargets and the expression of spe-
cific phenotype are modulated by a long-term morphogenetic
Thise work was supported by the Grant Far Eastern Branch Russian
Academy of Sciences № 12-III-A-06-095.
Copyright © 2012 SciRes. E NG
influence. In adult fishes a postnatal neuro- and gliogenesis still
occurs in a periventricular area. Already on early postembryo-
nic morphogenetic stages, two systems of neurochemical sig-
naling (dopaminergic and GABA-ergic) exist simultaneously in
the Oncorhynchus masou brain. These systems exert paracrine
and possibly autocrine impacts towards the cell-targets before
synaptic contacts shaping occurs and the neurotransmission of
specific interneuronal connections begins. The maximal con-
centration of D1 dopamine receptors in the eel brain [3] was
revealed in the periventricular brain areas (morphogenetic
fields), where neurogenesis is preserved during the entire life of
the an imal. Therefore, t he cells, which are l ocated in p roliferat-
ing brain regions, constitute the targets to dopamine regulation.
The zones, which synthesize dopamine and GABA in these
brain regions, are localized in a territory of major vascular
plexuses (in the forebrain and medulla oblongata). The neuro-
transmitters (dopamine and GABA) may be released into the
portal system blood flow and further into the general circulation,
impacting endocrine influence on peripheral organs [4]. Our
most recent findings suggest, that dopamine and GABA, in
undifferentiated cells of periventricular and subventricular hy-
pophysotropic areas of different age groups of O. masou, con-
stitute morphogenetic factors (inductors of the development)
Along with the paracrine signaling form mentioned, in the
salmons during ontogenesis the specific activation systems of
forebrain and system of distant (synaptic) intercellular signaling
are developed. The nuclei of preglomerular complex constitute
the source of these directed connections. The preglomerular
complex in fishes is considered a polymodal sensory center of
diencephalon, realizing transmission of visual, mechanosensory,
octavo-lateral, and acoustic information to the dorsal and ven-
tral regions of the telencephalon [5]. Information on origin,
pathways of migration, and phenotype of cells, their lifespan,
and functional integration in the course of postembryonic neu-
rogenesis remains at present rather li mited. In the br ain of a few
nonmammalian vertebrates the volume of the sensory projec-
tive zones is assumed to increase during the entire life of the
animal. This is provided at the expense of proliferation of neur-
al stem cells, lo cated in specific regions, n eurogenic nich es [6].
This is related with necessity of adaptation of the CNS of such
animals to increase in the body dimensions and, respectively,
increase in the volume of primary sensory signaling. In agree-
ment with this assumption, we suggest that dopamine, GABA-
and NO-ergic systems participate in regulation of basic histo-
genetic processes: cells migration and differentiation of neuro-
and gliospecific lines, because preglomerular nuclei contain
morphologically and neurochemically heterogeneous cell pop-
ulations [7], which represent different ontogenetic stages of
main cellular types. The cells formed in proliferative (PCNA-
contained) diencephalic zones migrate to preglomerular area,
where their further differentiation and growth take place. The
presen ce of D1 and D 2 dopamine receptors [6, 8] and benzodia-
zepine receptors B type [9] in these nuclei in the teleost fishes
brain confirms these idea. The period of the blood-brain barri er
shaping, during the first year of life [10] in the salmon brain
may be considered as a critical stage of the paracrine interrela-
tions predominance in the salmon brain. The specific connec-
tions shaping, the neuronal processes development and synap-
togenesis are occurring in the next ontogenesis period.
We consider, that cells maturing in different parts of the
salmon brain occur heterochronycally in many respects. In the
caudal brain parts t he reticulospinal cell s , raphe nucleus, V, VII,
IX and X nuclei of craniocerebral nerves cells acquire features
of phenotypical specialization earlier than in the forebrain
structures. In the medullar and spinal cord neurons of one-
and two-year-old young cherry salmon O. masou full-fledged
dendrites and axons are revealed, but their processes have
«growth cones», what present the sign of the continued growth
and development of these structures in postembryonic period
and of their further differentiation. In a three-year-old salmon O.
masou large differentiated cells, which have expressed TH,
GABA and parvalbumin in a spinal cord column motoneurons,
nuclei of craniocerebellar nerves, reticulospinal cells and dien-
cephal ic nucl ei were revealed [ 11, 12].
Recently the participation of radial glia in postembryonic
neurogenesis in a kind of asymmetric mitosis has been shown.
One cell r emain in periven tri cul ar area and have roun d ed shape,
another have a long process, which later is pulled in using som-
al translocation [13]. The presence of TH- and GABA-ir cells
in the territory of the PCNA-ir proliferative zones in one- and
two -ye a r-old young cherry salmon O. masou and neuromeric
structure of diencephalic and medullar brain parts marking,
undoubtedly show, that dopamine- and GABA-ergic signalling
participate in postembryonic neurogenesis of the O. masou
Figur e 1. A - immunolocalisation of tyrosine hydroxilase (TH) in
parvocellular preoptic nucleus (Pop), B - prol iferative nuclear an-
tigen (PCNA) in dorsal thalamus (DTh), C - neuronal nitric oxide
synthase (NOS) in pretectal (Ptn), dorsal (DTN), ventro-medial
(VMTN) thalamic nuclei, D - transcriptional factor Pax6 in peri-
ventricular diencephalon 6-mo nt h-old Onco r hy n chus masou. Im-
munonegative border of dorsal neuromers on A, delineated by a
triangle, the cluster of immunopositive cells on D, delineated by
rectangle. Inf infundibulum, FR fasciculus retroflexus, Pt
pretectum. Scale: А, C 100 μm, B, D 50 μm.
Copyright © 2012 SciRes. ENG
Along with classical neuromediators systems, immunoloca-
lisation of transcriptional factor Pax6 were studied by us. Pax6
is a marker for pro gen ito r cell s; the l abel in g of P ax6 ad equately
reflects the neuromeric structure of the salmon brain in the
different age groups. The results of Pax6 labeling of the salmon
brain have shown that this marker is expressed in early
youngsters, in the age of 3 and 6 months, as well as in one-year
old and adult animals. In one-year-old salmon, we have ob-
served a specific labeling of periventricularly localized cells,
creating clusters and domens (Fig. 1D). The investigation of the
late-age stages of the salmon has revealed specific accumula-
tions of cells, owing long radially oriented outgrowths, the cells
bodies, localized near the brain ventricle lumen or along im-
munopositive fibres [12].
The PCNA labeling in 1-year-old and salmon adults have
shown the presence of a vast population of proliferating cells in
periventricul ar areas of the dienceph alon and central grey layer
of the medulla (Figure 1B). Moreover, on the level of the fore-
brain distribution of ТН-, GABA-, and Pax6-ir cells have la-
beled a neuromeric construction of the brain; this is confirmed
by PCNA labeling of proliferative zones. On the border be-
tween the dorsal prosomers Р2-Р3, we have not detected the
immunopositive labeling with ТН, GABA, Pax6 and PCNA
(Figure 1A) In the diencephalon localization of proliferating
PCNA-immunogenic zones have соrresponded to the proso-
meric construction of the forebrain. Thus, we have revealed
several active PCNA-ir zones of proliferation, including ТН,
GAB A a nd NADPH-d (Figure 2A, B) expressi ng cells.
The Pax6 expression was revealed in the glomerular and
preglomerular nuclei, what indicate the morphogenetic
processes course on a territory of this major sensory centre
during the salmon postembryonal development. In glomerular
nucleus the Pax6 immunolocalization was revealed in the de-
fined cells populations, which, by our opinion; corresponds to
neuroanatomical zones, where differentiation of neurons, pass-
ing different types of sensory signaling, take place. We suggest
that Pax6 participate in the brain structure regionalization in
postembryonic period too. The Pax6 expression in the brain of
different salmon age groups indicate, that neurodetermination
and migration of cells, occurred in proliferative zones in these
ages periods, are regulated by transcriptional factor itself [12].
We hypot hesi ze, th at t he an ter io r and medi al p reglo merul ar an d
glomerular nuclei of the salmon constitute the zones of post-
embryonic morphogenesis, in which postmitotic neuroblasts are
participating in formation of a definitive structure of this main
sensor y center an d migrate fro m cerebral zo nes, owing p rimary
3. Participation of Gasotransmitters in
Postembrionic Neurogenesis in the Fishes
Br ai n
In cont rast to mammals, the fishes brain has high neuronal plas-
ticity and is capable to produce new cells during the entire life
of the animal [1]. The results of our investigation indicate the
presence of nNOS and NADPH-d activity both in neurons and
glial cells in the O. masou brain. It is probable, that NO in these
cells participate in the paracrine control of postembryonic neu-
rogenesis and is functioning as morphogenetic factors (induc-
tors of the development); the similar situation was demonstrat-
ed in the mammalian brain [14]. The involvement of NO in
postnatal neurogenesis was found in different vertebrates [15].
Two main neurogenesis sites have been identified in the adult
mammalian brain. They inclu de the subvent ricular zon e (SVZ),
and the subgranular zone (SGZ) of dentate gyrus (DG). The
process of neurogenesis is composed of three main steps, which
include precursor proliferation, migration; differentiation, inte-
gration and survival. It has been demonstrated, that SVZ of
mammals is surrounded by nNOS positive neurons [16], and
cells expressing nNOS also have been identified in neuronal
precursors in DG [17]. These findings suggest nNOS might
take part in neurogenesis regulation.
The results of our study allows to suggest, that NO in the O.
masou salmon medulla periventricular area, which contains
PCNA-ir proliferating cells too, can act in different age periods
as a regulator of adult neurogenesis, what confirms data on
Endogenously H2S is synthesized from L-cysteine by pyri-
doxal-5'-phosphate-dependent enzymes, cystathionine
β-synthase (CBS), and cystathionine γ-lyase (CSE), which are
expressed in many tissues. The analysis of localization of CBS,
which is an immunohistochemical marker of H2S in the brain of
bony fishes, has been never made before. We found that, in the
sa l mon O. masou, CBS labels neurons of the reticular forma-
tion, vessels, neurons of the ventral spinal column, and climb-
ing fibers in the cerebellum [18].
In carp Cyprinus carpio, the periventricular area of the me-
dulla oblongata and ventral and lateral areas of the cerebellum
have contained strongly CBS-stained cells without any out-
growths (Figure 2C, D). These CBS-positive cells were found
in periventricular zone, corresponding to the area of primary
proliferation [18]. Hence, it is logical to hypothesize that H2S
may also work as a regulator of postnatal neurogenesis in the
carp br ain.
Figur e 2. A clusters of NADPH-d-producing cells (delineated by
rectangles) in periventricular area of medulla oblongata of Oncor-
hynchus masou; on B in a large magnification. C - cystathionine
β-synthase (CBS) producing cells (red arrows) in periventricular
area of Cyprinus carpio brain, on D in a large magnification. LX
lobus of vagal nerve, IV forth ventricle, MLF medial longitu-
dinal fasci cle . Sc a le: А, C 200 μm, B, D 50 μm.
Copyright © 2012 SciRes. E NG
In cyprinoid s a periventr icular area is free o f NADPH/ nNOS
activity. It seems, that H2S may function as a si gnal molecul e in
a periventricul ar area of carp. The resu lts of a stud y performed
allows to suggest, that NO in the salmon medulla periventricu-
lar area can act as a regulator of adult neurogenesis, while in a
periven tricular area o f medulla o blongata and ventral an d later-
al zones of cerebellum of capr we have found cells, owing
strong CBS immunolabeling (Figure 2D). It seems, that NO
and H2S may function as a signal molecules in periventricular
area and they can act as a regulators of the adult neurogen esis.
4. Conclusion
The data provided by this study add to our general understand-
ing, that peculiarities of distribution of classical neuromediators
(GABA, catecholamines) and gasotransmitters (NO and H2S)
are directly connected with ability of the fishes brain to grow
during the animal entire life. We suggest, that some classical
neuromediators (GABA, catecholamenes) and gasotransmitters
(NO and H2S) not only regulate functional activity of neurons
and modulate synaptic transmission in mature neural networks,
but also are regarded as inductors of the fishes brain develop-
ment (morphogenetic factors) in postembryonic ontogenesis.
This confirmation is proved by finding of the phenotypically
immature elements, expressing the above mentioned molecules
in proliferating brain areas, in the three-year-old salmon brain,
and of elements, which owe morphology of radial glia. The
presen ce of enzyme s, synt hesizin g gasot ransmitters in the brain
areas, which are expressin g PC NA, have proved th eir partici pa-
tion in regulation of postembryonic neurogenesis.
In the fishes, which preserve fetal state during long time
(salmon and carp), such markers as NO and H2S in periventri-
cular proliferative areas may present in different ratios. This is
consistent with the hypothesis that in functionally similar com-
plexes in animals the different signal transduction systems may
be involved. In contrast to widespread neurogenetic model D.
rerio, the development of the salmon nervous system occur
during long time. As it follows from our data, the development
of different CNS structures in the O. masou brain is characte-
rized by evident heterochrony, so the cells of caudal brain re-
gions gai n features o f ph enot ypical sp eciali zatio n earl ier than i n
the foreb rain structures. We suggest that the brain o f these ani-
mals du r ing a long t ime preserves t he signs of fetal organization
and low differentiated cells presence confirms this hypothesis.
The data p resented in this study open a new trend in investi-
gation of cellular mechanisms of shaping in structural organiza-
tion in the postembryonic fishes brain and in examination of
morpho-functional manifestations concerning histogenetic
processes in different periods of postembrionic ontogenesis.
The new prio rity data recei ved are conn ected with development
of nervous tissue in the pacific salmon brain and with dynamic
of the brain shaping and distribution of classical neurotransmit-
ters and gaseous mediators in a context of incessant postem-
bryonic neurogenesis.
[1] G.K. Zupanc, “Towards brain repair: Insights from teleost fish,”
Semin. Cell Dev. B iol., vol. 20, pp. 683-690. 200 9.
[2] M.V. Ugrumov, “Developing brain as an endocrine organ: a
paradoxical reality,” Neurochem. Res., vol. 35, pp. 837-850.
[3] M. Kapsimali, B. Vidal, A. Gonzalez, S. Dufour, P. Vernier,
“Distribution of the mRNA encoding the four dopamine D, re-
ceptor subtypes in the brain of the european eel (Anguilla an-
guitta): comparative approach to the function of D, receptors in
vertebra tes,” J . Comp. Neu r ol., vol. 419, pp. 320-343. 2000.
[4] V.L. Trudeau, “Neuroendocrine regulation of gonadotrophin II
release and gonadal growth in the goldfish, Carassius auratus,”
Rev. of Reprod., vol. 2, pp. 5568. 1997.
[5] R.G. Northcutt, “Forebrai n evolution in bony fishes,” Br. Res.
Bull. , vol. 75, pp. 191-205. 2008.
[6] J. Kaslin, J. Ganz, M. Brand, “Proliferation, neurogenesis and
regeneration in the non-mammalian vertebrate brain,” Philos.
Trans. R. Soc. Lond. Biol. Sci., vol. 363, pp. 101-122. 2008.
[7] E.V. Puschina, “Neurochemical organization and connections
of the cerebral preglomerular complex of the masu salmon,”
Neurophysiology, vol. 43, №. 6. pp. 437-451 . 2011.
[8] P. Vernier, M.F. Wullimann, “Evolution of the posterior tuber-
culum and preglomerular nuclear complex,” in Encyclopedia of
Neurosciences, Part 5, M.D. Binder, N. Hirokawa, U.
Windhorst. Eds. Berlin: Springer-Verlag. 2009, pp. 1404-1413.
[9] T. Mueller, S. Guo, “The distribution of GAD67-mRNA in the
adult zebrafish (teleost) forebrain reveals a prosomeric pattern
and suggest s previou sly unid entifi ed homologies to t etrapod s,” J.
Comp. Neurol. , vol. 516. pp . 553-568. 2009.
[10] T. E. Horsberg, “Avermectin use in aquaculture,” Curr. Pharm.
Biotechnol., May vol. 13, pp . 1095-1102. 2012.
[11] E.V. Puschina, A.A. Varaksin, “Hydrogen sulfide-, parvalbumin-,
and GABA-producing systems in the masu salmon brain,” Neu-
rophysiology, vol. 43, № 2. pp. 90-102. 2011.
[12] Ye. V. Pushchina, Obukhov D. K., A. A. Varaksin, “Neuro-
chemical markers of cells of the periventricular brain area in the
Masu Salmon Oncorhynchus masou (Salmonidae),” Rus. J. of
Devel. Biol., vol. 43, №. 1, pp. 35–48. 2012.
[13] S.C. Noctor, V. Martinez-Cerdeno, L. Ivic, A.R. Kriegstein,
“Cortical neurons arise in symmetric and asymmetric division
zones and migrate through specific phases,” Nat. Neurosci., vol.
7, pp. 1 36144. 2004.
[14] J.C. Platel, S. Stamboulian, I. Nguyen, A. Bordey, “Neurotrans-
mitter signaling in postnatal neurogenesis: the first leg,” Brain
Res. Rev., vol. 63, pp. 60-71. 2010.
[15] G. Bicker, “Stop and go with NO: nitric oxide as regulator of cell
motility in simple brains,” BioEssays, vol. 27, pp. 495-505.
[16] C. Romero-Grimaldi, B. Moreno-Lуpez, C. Estrada,
“Age -dependent effect of nitric oxide on subventricular zone and
olfactory bulb neural precursor proliferation,” J. Comp. Neurol.,
vol. 506, pp. 339346. 2008.
[17] A.T. Islam, A. Kuraoka, M. Kawabuchi, “Morphological basis of
nitri c oxide production and its correlation with the polysialylated
precursor cells in the dentate gyrus of the adult guinea pig hip-
pocampus,” Anat. Sci. Int., vol. 78, pp. 98-103. 2003.
[18] E.V. Pushchina, A.A. Varaksin, D.K. Obukhov, “Cystathionine
β-synthase in the CNS of Masu salmon Oncorhynchus masou
(Salmonidae) and Carp Cyprinus carpio (Cyprinidae),” Neuro-
chem. J., vol. 5, № 1. pp. 24-34.2011.