Advances in Bioscience and Biotechnology, 2013, 4, 36-44 ABB
http://dx.doi.org/10.4236/abb.2013.410A3005 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Reduction of serotonergic gene expression in the raphe
nuclei of the midbrain under positive fighting experience in
male mice
Dmitry A. Smagin1*, Ul’yana A. Boyarskikh2*, Natalya P. Bondar1, Maxim L. Filipenko2,
Natalia N. Kudryavtseva1#
1Institute of Cytology and Genetics, Siberian Department of Russian Academy of Sciences, Novosibirsk, Russia
2Institute of Chemical Biology and Basic Medicine, Siberian Department of Russian Academy of Sciences, Novosibirsk, Russia
Received 15 July 2013; revised 15 August 2013; accepted 20 September 2013
Copyright © 2013 Dmitry A. Smagin et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The concept of a major inhibitory role of serotonin in
aggressive behavior is widely accepted by investiga-
tors. There was ample evidence that a pharmacologi-
cally-induced increase in the serotonin activity at-
tenuates agonistic behavior in animals, and that the
manipulations inhibiting the brain serotonergic sys-
tem can elicit aggressiveness in male mice and rats.
Repeated experience of aggression in daily agonistic
interactions has been shown to reduce serotonin ac-
tivity in brain of victorious male mice. The study
aimed to analyze expression of the serotonergic genes
-Tph2, Sert, Maoa and Htr1a, as well as the Bdnf and
Creb genes in the midbrain raphe nuclei of male mice
with positive fighting experience in daily encounters
and male mice with the same track record of aggres-
sion followed by two-week no-fight period. It has been
shown that mRNA levels of the serotonergic and Creb
genes are reduced in the winners in comparison with
male mice without consecutive positive fighting ex-
perience of aggression. After the fighting deprivation
the Tph2, Sert, Bdnf and Creb genes recover their ex-
pression while mRNA levels of the Maoa and Htr1a
genes proceed at a significantly higher level as com-
pared with the respective controls. Downregulation of
serotonergic genes is indicative of the inhibition of
serotonergic activity under repeated experience of
aggression. Nevertheless recovering of Tph2 and Sert
gene expression and overexpression of the Maoa and
Htr1a genes after no-fight period suggest that changes
in brain serotonergic activity are not main cause of
the behavioral pathology developing in male mice in
this experimental context.
Keywords: Tph2; Sert; MaoA; Bdnf; Creb; mRNA;
Repeated Aggression; Fighting Deprivation
1. INTRODUCTION
The concept of a major inhibitory role of serotonin (5-
HT) in aggressive behavior is widely accepted by inves-
tigators. There was ample evidence that a pharmacologi-
cally-induced increase in 5-HT activity attenuates ago-
nistic behavior in animals and that the manipulations in-
hibiting the brain serotonrgic system can elicit aggres-
siveness in male mice and rats [1-7]. It has been shown
that after fights 5-HT level decreases in the prefrontal
cortex in aggressive rats [8]. Reduced cerebrospinal fluid
(CSF) level of the 5-HT major metabolite 5-hydroxyin-
doleacetic acid (5-HIAA), as well as decreased sensitiv-
ity of 5-HT1A receptors shown in prisoners or psychi-
atric patients with repeated violent or aggressive acts [9-
13], also suggests a reduced overall 5-HT function in ag-
gressive individuals.
Experimental studies in male mice with repeated ex-
perience of aggression accompanied by social victories
in daily agonistic interactions have demonstrated reduced
5-HIAA level or/and 5-HIAA/5-HT ratio [14,15] and a
decrease in the activity of rate-limiting enzyme in the 5-
HT biosynthesis, tryptophan hydroxylase, in brain areas
such as the midbrain, hippocampus and striatum [16,17].
Specific changes in pharmacological sensitivity of 5-
HT1A receptors to agonist of 5-HT1A receptor buspirone
[18] and in 5-HT1A receptor binding in the frontal cortex,
hippocampus and hypothalamus have been demonstrated
in male mice with repeated aggression [19]. All these fin-
*These authors contributed equally to this work.
#Corresponding author.

D. A. Smagin et al. / Advances in Bioscience and Biotechnology 4 (2013) 36-44 37
dings are consistent with the 5-HT deficiency hypothesis
of aggression [15].
The study aimed to elicit a possible role of the sero-
tonergic Tph2, Sert, MaoA, 5-HT1a genes, encoding the
tryptophan hydroxylase, 5-HT transporter, monoamine
oxidase A and 5-HT1A receptors, respectively, which may
be involved into regulations of positive fighting experi-
ence. Expression of the Bdnf (encoding brain-derived
neutrophic factor) and Creb (encoding cyclic AMP re-
sponse element binding protein) genes was also studied.
The focus was on the area of the midbrain raphe nuclei
containing the cell bodies of serotonergic neurons. The
mRNA levels were analyzed in male mice that had a long
positive fighting history (21 wins in daily agonistic in-
teractions). Since the behavioral data indicated that the
implications of chronic aggression persist at least two
weeks after the cessation of agonistic interactions [15,20,
21], the expression of serotonergic genes was also ana-
lyzed in a group of 21-time winners who were kept away
from agonistic interactions referred to as “no-fight pe-
riod” or “fighting deprivation” throughout. Such animals
are special as they appear to be more aggressive after no-
fight period than before. The comparison of the expres-
sion of genes in these groups of the winners helps answer
the question on whether or not the altered gene expres-
sion in the midbrain raphe nuclei of the fighting deprived
winners recovers after no-fight period to the control lev-
els.
2. MATERIALS AND METHODS
2.1. Animals
Adult male mice of the C57BL/6J strain from a stock
maintained in the Animal Facility of the Institute of Cy-
tology and Genetics, SD RAS, (Novosibirsk, Russia)
were used. The animals were housed under standard con-
ditions [12:12-h light/dark regime, switch on at 8.00 a.m.;
food (pellets) and water available ad libitum]. Mice were
weaned at one month of age and housed in groups of
eight to ten in plastic cages (36 × 23 × 12 cm). Expe-
riments were performed on mice 10 - 12 weeks of age.
All procedures were in compliance with the European
Communities Council Directive of November 24, 1986
(86/609/EEC). The study protocol was approved by the
Scientific Committee N 9 on the Ethics of Animal Ex-
periments in the Institute of Cytology and Genetics SD
RAS (Permit Number: N 613).
2.2. Sensory Contact Model for the Study of
Agonistic Behavior in Male Mice
Repeated experience of aggression accompanied by so-
cial victories in male mice was induced using the sensory
contact model [22-24]. Pairs of weight-matched animals
were each placed in a steel cage bisected by a perforated
transparent partition allowing the animals to see, hear
and smell each other, but preventing physical contact.
The animals were left undisturbed for three days to adapt
to new housing conditions and sensory contact before
they were exposed to encounters. Every afternoon (1400
- 1700 hours, local time), the cage lid was replaced by a
transparent one, and 5 min later (the period necessary for
individuals’ activation), the partition was removed for 10
minutes to encourage agonistic interactions. The superi-
ority of one of the mice was firmly established within
two or three encounters with the same opponent. The su-
perior mouse (winners) would be attacking, biting and
chasing another, who would be displaying only defensive
behavior (sideways postures, upright postures, withdra-
wal, lying on the back or freezing). As a rule, agonistic
interactions between males are discontinued by lowering
the partition if the strong aggression has lasted 3 min, in
some cases, less. Each defeated mouse (defeater, loser)
was exposed to the same winner for three days, while
afterwards each loser was placed, once a day after the
fight, in an unfamiliar cage with an unfamiliar winner
behind the partition. Each victorious mouse remained in
its original cage. This procedure was performed once a
day for 21 days and yielded an equal number of the win-
ners and losers.
Three groups of animals were used: (1) Winners—a
group of mice with repeated experience of aggression ac-
companied by victories during 21 days of agonistic inter-
actions; (2) Fighting deprived winners-a group of chroni-
cally victorious 21-time mice who lived for 14 days after
the last encounter without agonistic interactions (period
of fighting deprivation). During this period, each of them
shared a cage with a losers, the partition between their
compartments being down at all times, to prevent physi-
cal encounters; (3) Controls—the mice without consecu-
tive experience of agonistic interactions. To the end of
experiment, animals of all experimental groups were 15 -
17 weeks of age.
To measure mRNA levels in the raphe nuclei area of
midbrain, all the mice were decapitated simultaneously:
21-time winners, 24 hours after the last agonistic interac-
tion; fighting deprived winners, immediately after 14-day
period of deprivation; and the controls. The raphe nuclei
areas were dissected in animals according to the Mouse
Brain Atlas [25]. The mouse brains were removed and
chilled rapidly on ice. Dissection of the brain regions
was made by one experimenter during two consecutive
days. Animals of all groups were represented in each day.
All biological samples were encrypted, rapidly frozen in
liquid nitrogen, and stored at −70˚C until use.
2.3. Behavioral Study
Behavior of each winner in agonistic interaction test was
videorecorded for 10 min during its last encounter, and

D. A. Smagin et al. / Advances in Bioscience and Biotechnology 4 (2013) 36-44 38
the data were documented. Furthermore, we needed to
know whether both groups of the winners could be con-
sidered identical at the time each mouse was won its last
encounter. To find out, the groups were compared in
terms of behavior. During a 10-min test, the following
were the behavioral domains analyzed in the winners: (1)
Attacks: attacking, biting and chasing; (2) Aggressive
grooming—the winner mounts onto the loser’s back, holds
it down and spends much time licking and nibbling at the
loser’s scruff of the neck. The loser is wholly immobi-
lized or sometimes stretches out the neck and again
freezes under the winner; (3) Digging—digging up and
scattering the sawdust on the loser’s territory (kick-digs:
pulling the sawdust forwards with the forepaws; push-
digs: pushing the sawdust backwards with the hind paws);
(4) Hostile behavior—the total time spent attacking, ag-
gressively grooming and digging; (5) Self-grooming-bo-
dy care activities (fur licking, head washing, nose wash-
ing). The total time and/or number of behavioral events
were measured.
2.4. Total RNA Extraction and Reverse
Transcription
Total RNA was isolated from the tissues using TRIzol
(Invitrogen) according to the manufacturer’s instructions.
The concentration of total RNA was quantified by meas-
uring the absorbance at 260 nm. The integrity of total
RNA was assessed using agarose gel electrophoresis.
cDNAs were synthesized using total RNA (1 μg), ran-
dom N9 primer (100 ng), and MoMLV reverse transcript-
tase (200 U, Biosan). Each RT reaction was run in dupli-
cate. RNA aliquots were used to confirm the absence of
any genomic DNA in each sample.
2.5. Real-Time Quantitative PCR
The Tph2, Sert, Maoa, Htr1a, Bdnf, Creb, Cph n, Gapdh,
and Hprt cDNA levels were quantified by SybrGreen-
based real-time PCR in a total volume of 25 l contain-
ing an aliquot of the RT mixture, dNTPs (200 nM), F and
R primers (300 nM), SybrGreen I (1:20,000, Invitrogen),
standard PCR buffer, and hot-start TaqDNA polymerase
(0.5 U, Biosan). Amplification was run for 3 min at 95˚C
followed by 40 cycles of 6 s at 96˚C, 6 s at 60˚C, 12 s at
72˚C. Fluorescence was monitored for 5 s after each cy-
cle at the appropriate melting temperature. To check for
the presence of nonspecific PCR products or primer
dimers, a melting curve analysis was performed after the
final PCR cycle. Cphn, Gapdh, and Hprt were considered
initially as possible housekeeping (normalizers) genes in
this study.
Amplification efficiencies were calculated using a
relative standard curve derived from threefold serial di-
lutions of pooled cDNA. In all cases, the amplification
efficiency was higher than 90%. Each sample was PCR-
amplified twice. qRT-PCR results were quantified using
the relative standard curve method. The PCR primer se-
quences are shown in Ta bl e 1 . Samples from animals of
different experimental groups were mixed before mo-
lecular study. Extraction of total RNA, reverse transcrip-
tion, and qRT-PCR were made during 2 weeks by one
experimenter in blind regimen with use of one buffer and
primers set.
2.6. Statistical Analysis
Normal distribution and homogeneity of variances were
tested by the Shapiro-Wilk’s and Levene’s tests, respec-
tively. Statistical analysis of behavioral data was per-
formed pairwise comparison of the groups with t-Student
test or the Mann-Whitney U test. Statistical analysis of
mRNA levels was performed using one-way ANOVA
with factor “groups” under the consideration—the con-
trols, winners, and fighting deprived winners—followed
by the comparison of the groups using the Bonferroni
post hoc testing or Fisher LSD test. The data are reported
as mean ± SEM. The statistical significance was set at P
≤ 0.05.
For each experimental sample, the relative amount of
mRNA is determined from the appropriate standard curve.
Since the all reference genes, Cphn, Gapdh, and Hprt,
changed their expression under repeated experience of
aggression in the midbrain raphe nuclei in the winners as
compared with the controls, we can’t use them to obtain
a normalized level of the Bdnf, Creb, Tph2, Sert, Maoa,
and Htr1a genes. The other statistical approach [26] was
used to reveal differences in serotonergic and other genes
expression between the experimental groups: the mean of
relative amount of mRNA in the control group for each
gene was taken as 100%, and changes in the winners,
fighting deprived winners, and controls were calculated
as percentage of the mean in the controls. In the control
group, the number of animals used in experiment was 16
- 17. Groups of 21-time winners and fighting deprived
winners contained 11 - 13 mice.
3. RESULTS
3.1. Analysis of the Winners’ Behavior in
Agonistic Interactions
We needed at first to know whether both groups of the
aggressive mice (winners) could be considered identical-
lat the time each mouse was won its last encounter. If
they were, all the differences in genes’ expression be-
tween group of 21-time winners and group of 21-time
winners before fighting deprivation, or lack thereof,
could solely be accounted for by deprivation. To find out,
the animals were compared in terms of aggressive be

D. A. Smagin et al. / Advances in Bioscience and Biotechnology 4 (2013) 36-44 39
Table 1. Primer sequences.
Genes Primer sequences Functions
Bdnf F 5’-CAAACAAGACACATTACCTTCCT-3’ Brain-derived neurotrophic factor
R 5’-ATGGTCATCACTCTTCTCACCT-3’
Creb F 5’-CAGCCACAGATTGCCACATTAG-3’ Cyclic AMP response element binding protein
R 5’-CTTATGGAGACTGGATAACTGATG-3’
Tph2 F 5’-CACCATTGTGACCCTGAATCC-3’ Tryptophan hydroxylase 2-rate-limiting enzyme of 5-HT synthesis
R 5’-AAGCTCGGTGCCGTACATGAG-3’
Sert F 5’-GCTGAGATGAGGAACGAAGAC-3’ Serotonin transporter protein
R 5’-AGGAAGAAGATGATGGCAAAG-3’
Maoa F 5’-GAATGTCAATGAGCGTCTAGTTC-3’ Monoamine oxidase A, enzyme of 5-HT catabolism
R 5’-ATGGTGCATCAACAGGGATCTC-3’
5ht1a F 5’-TTGGAACTACTTTGGGTTATGG-3’ Serotonin 5-HT1A receptors
R 5’-ATTGTCAATTTCTTTGGTGAGTG-3’
Cphn F 5’-GTTTTTTATCTGCACTGCCAAG-3’ PPIA—peptidylprolyl isomerase A (cyclophilin A);
enzyme accelerates folding of proteins
R 5’-TTCTTGCTGGTCTTGCCATTC-3’
Gapdh F 5’-TGTTCCAGTATGACTCCACTCA-3’ Glyceraldehyde-3-phosphate dehydrogenase; glycolysis enzyme
R 5’-GACACCAGTAGACTCCACGACA-3’
Hprt F 5’-TGAAAAGGACCTCTCGAAGTGT-3’
Hypoxanthine-guanine phosphoribosyltransferase; synthesis of purine
nucleotides through the purine salvage pathway
R 5’-CACTAATGACACAAACGTGATTC-3’
grooming, digging etc. No differences were found be-
tween these groups of the winners in any of the individ-
ual or social behaviors measured after the respective 21-
day periods of agonistic interactions (P > 0.05, Table 2).
3.2. Cphn, Gapdh, and Hprt Genes
Data obtained revealed a change in expression of the
so-called housekeeping genes which we were going to
use for normalization for the qRT PCR data: the expres-
sion of the Cphn, Gapdh, and Hprt genes used as puta-
tive reference genes decreased significantly in the mid-
brain raphe nuclei in the 21-time winners as compared
with the controls. Similarly to previous study [26] as it
turns out, the use of these genes as reference genes in the
analysis of expression of other genes of interest was
hardly possible. The method for calculating changes in
the expression of each gene in the winners before and
after deprivation versus its expression in the respective
controls was used to reveal differences in all genes ex-
pression between the experimental groups [26].
One-way ANOVA revealed a significant influence of
the factor groups (the control, winners and fighting
deprived winners) on mRNA level of the Cphn (F2,38 =
9.58; P < 0.001), Gapdh (F2,37 = 4.77; P < 0.014), and
Hprt (F2,39 = 10.50; P < 0.001) genes (Figure 1). Based
on the post hoc Bonferroni test as compared to the re-
spective levels in the controls, mRNA levels of the Cphn,
Gapdh, and Hprt genes were decreased in the 21-time
winners (P < 0.001, P < 0.013, P < 0.007, respectively).
In fighting deprived winners the Cphn mRNA level was
significantly less (P < 0.035) and expression of the Ga-
pdh and Hprt genes did not differ significantly in com-
parison with the controls. However expression of Hprt
gene in the fighting deprived winners was significantly
higher than in the winners before deprivation (P <
0.001).
3.3. Bdnf and Creb Genes
One way ANOVA revealed a significant influence of the
factor groups on the mRNA levels of the Bdnf (F2,39 =
4.76; P < 0.014) and Creb (F2,38 = 8.88; P < 0.001) genes.
Based on the Bonferroni post hoc test (Figure 2) as
compared to the respective levels in the controls, mRNA
levels of the Creb gene were decreased (P < 0.008) in the
21-time winners. In the fighting deprived winners, the
Bdnf and Creb mRNA level was higher than in the
inners before deprivation (P < 0.012 and P < 0.001, w

D. A. Smagin et al. / Advances in Bioscience and Biotechnology 4 (2013) 36-44 40
Tab l e 2 . Behavioral data from mice to become no-deprived winners and in mice to become post-deprived winners during their re-
spective last encounter.
Behavioral parameters Mice to become no-deprived winners Mice to become post-deprived winners Unpaired t-test M-W test
Attacks
Latency, s 54.7 ± 15.7 55.4 ± 17.8 t = 0.030; P < 0.97
Number 11.2 ± 1.9 9.0 ± 1.7 t = 0.848; P < 0.40
Total time, s 50.5 ± 12.1 43.7 ± 7.8 t = 0.848; P < 0.64
Aggressive grooming, s 27.4 ± 17.2 23.4 ± 16.9 U = 76; P < 0.98
Diggings, s 18.1 ± 3.5 18.6 ± 2.4 t = 0.126; P = 0.90
Hostile behavior, s 95.9 ± 18.8 85.7 ± 17.8 t = 0.386; P < 0.70
Self-grooming, N 19.2 ± 3.7 14.9 ± 3.6 t = 0.817; P = 0.42
Number of animals 14 11
0
20
40
60
80
100
120
140
Cphn HprtGpdh
**
*
*** *
+++
Figure 1. Cphn, Gapdh, and Hprt mRNA levels in the
raphe nuclei of the midbrain in the controls (light blue
columns), winners (blue columns), and fighting de-
prived winners (dark blue columns). Data are pre-
sented as percentage of the mean in the controls. *P <
0.05; **P < 0.01; ***P < 0.001 vs the controls; and +++P
< 0.001 vs the winners.
respectively).
3.4. Tph2, Sert, Maoa, and Htr1a Genes
One-way ANOVA revealed a significant influence of the
factor groups on the mRNA level of the Tph2 (F2,36) =
5.36; P < 0.009), Sert (F2,38 = 4.79; P < 0.014), Maoa,
(F2,39 = 15.32; P < 0.001) and Htr1a (F2,38 = 37.84; P <
0.001) genes. Based on the Bonferroni post hoc test
(Figure 3) as compared to the respective controls, the
mRNA levels of the Maoa and Htr1a genes were
decreased in the 21-time winners (P < 0.007 and P <
0.037, respectively) and was increased in fighting de-
prived winners (P < 0.033 and P < 0.001, respectively).
Significant differences were found between the winners
before and after deprivation for the Tph2 (P < 0.007),
Sert (P < 0.012), Maoa (P < 0.001), and Htr1a (P <
0.001) mRNA levels. Additionally, Fisher LSD test had
demonstrated significant differences between the con-
0
20
40
60
80
100
120
140
Bdnf Creb
**
++++
Figure 2. Bdnf and Creb mRNA levels in the raphe
nuclei of the midbrain in the controls (light blue co-
lumns), winners (blue columns), and fighting de-
prived winners (dark blue columns). Data are pre-
sented as percentage of the mean in the controls. *P
< 0.05; **P < 0.01; vs the controls; +P < 0.05; +++P
< 0.001; vs the winners.
0
50
100
150
200
250
Tph2 5ht1aSert Maoa
** *
*** ++ +
*+++
++ ++
*
Figure 3. Tph2, Sert, Maoa, and Htrt1a mRNA levels in the
raphe nuclei of the midbrain in the controls (light blue co-
lumns), winners (blue columns), and fighting deprived winners
(dark blue columns). Data are presented as % of the mean in
the controls. *P < 0.05; **P < 0.01 and ***P < 0.01 vs the con-
trols; +P < 0.05; ++P < 0.01; +++P < 0.001 vs the winners.
trols and winners for Sert mRNA levels (P < 0.043).

D. A. Smagin et al. / Advances in Bioscience and Biotechnology 4 (2013) 36-44 41
4. DISCUSSION
Variability of the reference genes expression in different
tissues and experimental situations was demonstrated in
studies of last years [27-32]. We also found in the 21
time winners decrease in functional activity of the genes
involved in the processes of intracellular protein trans-
port (Cphn) and glycolysis (Gapdh), presented in all nu-
cleated cells (Hprt), which suggests a profound influence
of positive fighting experience on different metabolic
and cellular processes in the raphe nuclei of the midbrain.
It is noteworthy that 2 week no-fight period is enough for
the Gapdh and Hprt genes but not for the Cphn gene to
recover their expression to the control levels.
Transcription of the Bdnf gene is dependent on cAMP
response element binding protein (CREB). In the mid-
brain raphe nuclei of the winners before and after fight-
ing deprivation the expression of the Bdnf and Creb genes,
encoding the proteins associated with neurotransmission,
neurogenesis and synaptic plasticity [33], was shown to
undergo changes similar to those in genes involved into
the metabolic and cellular processes. Reduced Creb
mRNA level was found in the raphe nuclei of the 21-time
winners as compared with the controls. It is notable that
the mRNA levels of both genes were significantly higher
after a no-fight period than before it. Dynamic changes
in the expression of these genes are likely to take place
under experience of aggression and no-fight periods.
It has been shown many times that the Bdnf and Creb
genes are involved in the brain mechanism of chronic so-
cial defeat stress [26,34-38]. At the same time, the expe-
rimental evidence of the role of these genes in the me-
chanisms of aggression is scarce and inconclusive. It has
been shown that BDNF knockout mice exhibit elevated
conspecific aggression and social dominance [39]. Schi-
zophrenic patients with first-episode psychosis have
lower serum concentrations of the BDNF and score hi-
gher on the Aggression-Hostility scale than healthy indi-
viduals [40]. Winning animals have significantly more
Bdnf mRNA in the dentate gyrus of the dorsal hippo-
campus than losing animals and home cage controls [41].
Authors support a model in which BDNF-mediated plas-
ticity within the hippocampus may instantiate aspects of
winning such as control of a territory by dominant ani-
mals. The Bdnf mRNA level increases in ventral tegmen-
tal area of 21-time winners [42]. Obviously, further stud-
ies are needed to explore the role of the Bdnf and Creb
genes in mechanisms of social aggression.
Research evidence shows that BDNF promotes the
survival and differentiation of the 5-HT neurons [43]. It
is reasonable to assume that changes in the functional
activity of the Creb and Bdnf genes in the raphe nuclei
containing the cell bodies of serotonergic neurons are at-
tributable to changes in serotonergic activity of the brain
shaped by repeated experience of aggression. The expe-
riment demonstrated a significantly reduced expression
of the Maoa, 5ht1a, Sert, and lesser reduction-Tph2 mRNA
levels in the mice winning 21 successive encounters each.
It is noteworthy that earlier a decrease in the activity of
rate-limiting enzyme in the biosynthesis of 5-HT, tryp-
tophan hydroxylase, in the midbrain was shown for the
winners [16,17]. Thus, a chronic manifestation of aggres-
sion, which, as supposed earlier, is accompanied by the
inhibition of the brain serotonergic system [15], decreas-
es the expression of the serotonergic genes, whose prod-
ucts are responsible for the inactivation and reception of
5-HT. After no-fight period expression of the Tph2 and
Sert genes increases significantly versus the period be-
fore deprivation and to a larger degree for the Maoa, and
5ht1a genes versus the respective controls. Overexpres-
sion of genes after deprivation may be considered as
common feedback mechanism initiated by cessation of
agonistic interactions. The data obtained so far strongly
support the statement that the serotonergic genes are in-
volved in the mechanisms of repeated aggression and do
not contradict the serotonin deficiency hypothesis of ag-
gression [4,6], which associates the decreased 5-HT func-
tion and increased aggression. From this statement, it
follows that serotonin inhibits aggression. Based on our
data it is plausible to assume that the repeated experience
of aggression inhibits the brain serotonergic activity and
changes the 5-HT metabolism, reception and serotoner-
gic gene expression.
It is noteworthy, that reduced mRNA levels of the
genes of interest in this area have been found in the ani-
mals with repeated experience of defeats [26]. Similarity
of changes in male mice with alternative social behaviors,
manifested by chronic defeats or aggression casts doubt
on the specificity of such changes for aggression. Earlier
we assumed [44] that similar changes in the winners and
losers may be explained by influence of agonistic inter-
actions stress common for both participants of social
conflicts or by development of enhanced level of anxiety
shown both in the winners [24] and losers [45]. However
we should also take into consideration the different dy-
namics of gene expression changes during period without
agonistic interactions. In the losers period of 2 weeks of
relative rest is not enough to recover expression of all
serotonergic genes as well as the Creb gene to the control
levels [26]. In the winners during no-fight period most
genes fully restore their expression. The Maoa and Htr1a
genes are expressed at significantly higher levels. Thus,
different mechanisms of brain serotonergic hypofunction
can be assumed in the mice with alternative social beha-
viors. In the losers, strong decrease of serotonergic activ-
ity may be attributed to the development of a depression-
like condition with the symptoms, sensitivity to antide-
pressants and neurochemical changes similar to those in
depressive patients [36,45,46]. In the winners behavioral

D. A. Smagin et al. / Advances in Bioscience and Biotechnology 4 (2013) 36-44 42
psychopathology developing under positive fighting ex-
perience [15] is associated with inhibition of brain sero-
tonergic system. Recovering of the Tph2 and Sert gene
expression and overexpression of Maoa and Htr1a genes
after no-fight period in the winners suggests that changes
in brain serotonergic activity are not the main or only
cause of the behavioral pathology developing in male
mice in this experimental context. Obviously, other neu-
rotransmitter systems may also be the factors, for exam-
ple, the brain dopaminergic systems, which have been
found to alter in the 21-time winners. Activation of the
brain dopaminergic systems has been demonstrated in
male mice with prolonged positive fighting experience as
elevated DOPAC (3,4-dihydroxyphenyleacetic acid) lev-
els or/and increased DOPAC/DA (dopamine) ratios in
different brain areas [15]. The DA level was increased in
the prefrontal cortex during and after fights in aggressive
rats [8]. Upregulation of the Th, and Dat1 gene expres-
sion has also been found in the ventral tegmental area of
the winners [23,47]. In the fighting deprived winners
these genes retained increased expression as compared
with the controls. Interestingly, in the losers’ ventral teg-
mental area lack of significant changes in the Th and
Dat1 gene expression was found [23]. Activation of the
Th and Comt gene expression was also found in the ce-
rebellum, frontal cortex, hippocampus, midbrain, and
striatum [48]. Thus, all data support the idea that repeat-
ed experience of aggression results in disbalance be-
tween activities of two neurotransmitter systems—sero-
tonergic and dopaminergic [15], namely the inhibition of
the former and activation of the latter, which are part of
the catecholamine systems responsible for several func-
tions, including excitation of nervous processes. In such
circumstances a low threshold for aggressive behavior
has been established, and, therefore, aggression can be de-
monstrated even in low provocative conditions. There is a
lot of experimental evidences for the relationship be-
tween increased impulsivity and, as a consequence, in-
creased aggression, and reduced serotonergic activity in
humans and animals [9-11,49]. The results of our studies
are consistent with these data.
As mentioned above, chronic positive fighting experi-
ence is accompanied by development of behavioral pa-
thology symptoms [15,49] in male mice such as ab-
normal aggression, hostility, pronounced anxiety, distur-
bances in social recognition and motivated behavior, hy-
peractivity, stereotypic and hyperkinetic reactions etc.
After fighting deprivation the male mice have been shown
to demonstrate higher aggressiveness as compared with a
period before deprivation [20,21]. Therefore, it can be
assumed that serotonergic system is not responsible for
this phenomenon since serotonergic genes recover their
expression after cessation of agonistic interactions. The
brain dopaminergic and opioidergic systems retain their
changed functional activity [15,47] and may be suspect-
ed as being the key factors of increased aggression after
no-fight periods.
5. ACKNOWLEDGEMENTS
This work was supported partly by Research Program from the Russian
Academy of Sciences “Molecular and Cellular Biology”, grant no 6.25,
and Russian Foundation for Basic Research, grant no 13-04-00072a.
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