European Journal of Pharmacology 708 (2013) 95–104

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Behavioural pharmacology

Strain differences in profiles of dopaminergic neurotransmission

in the prefrontal cortex of the BALB/C vs. C57Bl/6 mice:
Consequences of stress and afobazole
Elmira A. Anderzhanova a,n, Heidi Bächli c, Olga A. Buneeva d, Victor B. Narkevich b,
Alexei E. Medvedev d, Christoph K. Thoeringer e, Carsten T. Wotjak a, Vladimir S. Kudrin b
a
Max Planck Institute Psychiatry, Max-Planck-Gesellschaft, Kraepelinstrasse 2-10, 80804-D Munich, Germany
b
Institute of Pharmacology, Russian Academy of Medical Sciences, Baltiyskaya str. 8, 125315 Moscow, Russia
c
Heidelberg University Hospital, Im Neuenheimer Feld 400, 69120-D Heidelberg, Germany
d
Institute of Biomedical Chemistry, Russian Academy of Medical sciences, Pogodinskaya str. 10, 119992 Moscow, Russia
e
Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany

art ic l e i nf o a b s t r a c t

Article history: We found that in mice the basal activity of monoamine oxidase B (MAO-B) in the medial prefrontal cortex
Received 21 December 2012 (mPFC) is lower in BALB/C than in C57Bl/6J mice, whereas activity of MAO-A is similar between strains. BALB/
Received in revised form C mice, in comparison to C57Bl/6N mice, have higher basal content of dopamine in the mPFC, in both
11 March 2013
microdialysates and tissue content. Novelty stress (open field test) elicits a further increase in the
Accepted 13 March 2013
Available online 21 March 2013
microdialysate levels of dopamine in BALB/C, but not in C57Bl/6N mice; a subsequent accumulation of
extracellular 3,4-dioxyphenylacetic acid (DOPAC) reaffirms the difference in catabolic capacity of mono-
Keywords: aminergic systems between the strains. We demonstrated that in stress-susceptible BALB/C mice the novel
Stress anxiolytic afobazole, 5 mg/kg, selectively mitigates trait anxiety; however it does not change the behavioral
Dopamine
response in stress-resilient C57Bl/6N mice. Afobazole inhibits MAO-A in in vitro; it also lowers the
MAO
microdialysate DOPAC levels in both strains (which testifies to its MAO-A inhibiting activity in vivo) and
BALB/C mice
C57Bl/6 mice slightly suppresses dopamine release when elevated. Therefore, it is likely that the drug may mediate its
Anxiety anxiolytic activity via modulation of volume dopaminergic transmission at level of the mPFC.
Afobazole & 2013 Elsevier B.V. All rights reserved.
Microdialysis

1. Introduction pharmacological challenges (Griebel et al., 2000; Fowler et al.,

Afobazole, a novel drug with selective anxiolytic activity, has
been used in clinical practice in Russia since 2005 (Seredenin et al.,
2009). This drug interacts with melatonin and sigma1 receptors
(Seredenin and Voronin, 2009). The regulation of calcium home-
ostasis via sigma1 receptors is thought to be a primary mechanism
of its action (Cuevas et al., 2011). The mood stabilizing action of
afobazole can be related to its ability to tailor the monoamine
neurotransmission since the monoamine oxidase (MAO) inhibiting
activity of afobazole has been shown in vitro and ex vivo (Kudrin,
2006; Voronin et al., 2009; Davydova et al., 2010).
In behavioral tests afobazole powerfully discriminates between
the genetic backgrounds of C57Bl/6 and BALB/C mice, two strains
which differ in their stress-coping strategies (Broadhurst, 1976).
These strains are distinguished by their performance in a variety of
tasks (Crawley et al., 1997; Carola et al., 2004) and response to
n
Corresponding author. Tel.: þ49 89 30622 604; fax: þ 49 89 30622 610.
E-mail address: anderzhanova@mpipsykl.mpg.de (E.A. Anderzhanova).
2001). These differences can be determined by the peculiarities of
the biogenic monoamine systems. For instance, the expression of
MAO-A and MAO-B is lower in the BALB/C mice than C57Bl/6 mice
(Siesser et al., 2010). In turn, MAO-A and MAO-B insufficiency is
characterized with increased stress reactivity in mice (Grimsby
et al., 1997; Popova et al., 2006).
In mice with a passive stress-coping strategy (i.e. BALB/C),
afobazole administration results in active, exploration-oriented
response to novelty. At the same time, afobazole does not suppress
the characteristic exploratory response in mice with active stress-
coping strategy (C57Bl/6) which points to an absence of any
maladaptive sedative influence. This feature of afobazole contrasts
with the action of benzodiazepines, which tranquilize animals
irrespectively of the existing behavioral phenotype (Seredenin and
Blednov, 1994; Uyanaev et al., 2003; Volkova et al., 2010).
To date there is no evidence of afobazole's MAO-A and MAO-B
inhibitory action in vivo, e.g., by observing the microdialysis
levels of monoamines (which reflect a volume transmission in
targeting areas, Rice and Cragg, 2008) and their metabolites in
brain structures.

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2013.03.015
96 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104
In the medial prefrontal cortex (mPFC) dopamine optimizes the
task-dependent balance between cognitive stability and flexibility
(Fallon et al., 2012). Its modulation affects the expression of anxiety
or, more specifically, the cognitive control of fear/anxiety expression
(Boulougouris and Tsaltas, 2008; Cools and D'Esposito, 2011).
The aim of the present study was to characterize the para-
meters of the monoaminegric system in C57Bl/6 and BALB/C mice
and to determine the effect of afobazole on the dopamine, 3,
4-dioxyphenylacetic acid (DOPAC), homovanilic acid (HVA) and
5-hydroxyphenylacetic acid (5-HIAA) accumulation in the extra-
cellular space of the mPFC in freely moving animals during mild
stress (induced by testing in the open field). We assumed that
short-term exposure to the open field is accompanied by a tonic
release of dopamine in a strain-dependent manner. Taking into
account the strain selective action of afobazole we hypothesize
that the drug can specifically modulate the dynamics of mono-
aminergic neurotransmission in C57Bl/6 and BALB/C mice.
2. Materials and methods
2.1. Subjects
Male C57Bl/6N and BABL/C mice, Wistar rats (“Stolbovaya”
breeding center of RAMS, Russia), and male C57Bl/6J, C57Bl/6N
and BALB/C mice (Charles River, Germany), 8–10 weeks old, were
individually housed under a reversed 12:12 h light–dark cycle
(lights off 09:00 h) with food and water ad libitum for at least 14
days before starting the experiments. Experimental groups con-
sisted of 6–9 mice. The specific source of animals is indicated in
each experiment. All experiments were performed in strict com-
pliance with the European community recommendations for the
care and use of laboratory animals. The authors further attest that
all efforts were made to minimize the number of animals used and
their suffering.
2.2. Microdialysis experiment schedule
One week before the microdialysis experiment, mice under-
went a stereotaxic implantation of guide cannulas. After the
surgery and during the experimental runs animals were kept in
square Plexiglas chambers (16 cm  16 cm) on sawdust bedding
under reversed light–dark cycle. One day before the experiment,
microdialysis probes were inserted into the mPFC under slight
isoflurane anesthesia. We have employed a cross-over drug
administration protocol in order to minimize number of animal
required. The animals were injected with afobazole and vehicle in
random order on day 1 and day 2 and were pooled into two groups
on the basis of the treatment they received. This approach not only
decreases possible confounding effect of injection stress, but also
the effect of continuous microdialysis procedure, where possible
changes in probe recovery are almost impossible to track and
correlate with the results. Sixty minutes after the injection, each
animal was carefully moved into the open field (a Plexiglas
chamber, square in plan, 25 cm  25 cm, with non-transparent
walls of 32 cm), which was illuminated with white light (60 lx at
floor level) and left for 10 min. Afterwards, mice were transferred
back to their home cages for an additional hour of microdialysis
fraction collection.
2.3. Drugs
Afobazole, 5-ethoxy-2-[2-(morpholino)-ethylthio]benzimida-
zole dihydrochloride (Institute of Pharmacology RAMS, Moscow,
Russia) was dissolved in saline (0.5 mg/ml) and injected i.p. at a
dose of 5 mg/kg (the mean total injection volume was about
300 μl). The dose was chosen based on a previous comprehensive
analysis of behavioral effects of afobazole (Seredenin et al., 2009).
Control animals received saline injection (300 μl).
2.4. Elevated plus maze test
Animals were obtained from the “Stolbovaya” breeding center
of RAMS, Russia. Anxiety levels were assessed by the latency to
first enter the open arm, number of entries and absolute time
spent on the open and closed arms of the elevated plus maze
(EPM) 60 min after saline or afobazole administration. The cross-
shaped platform had a center area of 5  5 cm and the length of
closed and open arms was 20 cm. The walls and floor were made
of non-transparent Plexiglas. The EPM was elevated 30 cm above
the ground and brightly illuminated (100 lx at floor level). The
walls and floor surface were cleaned between testing with 10%
ethanol. Time of behavior monitoring was 5 min.
2.5. Surgery, probe implantation, and microdialysis
Animals were obtained from Charles River, Germany. C57Bl/6N
and BABL/C mice were anesthetized with isoflurane (Abbott,
India), 2% v/v in O2 with co-administration of the analgetic drug
Metacam® (Boeringer Ingelheim, Germany) (0.5 mg/kg). A mouse
was placed on a feed-back controlled heating pad (37 1C) (Harvard
Apparatus, USA) and fixed in a stereotaxic apparatus (TSE Systems
Inc., Germany). A microdialysis probe guide cannula (MAB 4.15.IC,
Microbiotech/se AB, Sweden), which serves as a track for the probe
and allows it to avoid excessive disturbance of tissue during the
probe insertion, was implanted into the right mPFC so that the tip
was 2 mm above the targeted area. The stereotaxic coordinates for
the mPFC were AP 2.20 mm, ML 0.35 mm, and DV −1.50 mm
(Paxinos and Franklin, 2001). The cannula was fixed with fast
drying acrylic Pattern Resin LC (GC America, USA) and anchored by
two small (2 mm) custom-made stainless steel screws. A bigger
aluminum block was mounted next to the implanted guide
cannula to secure the microdialysis probe with the wire tether
(Harvard Apparatus, USA). Animals were allowed to recover in the
experimental square Plexiglas home cages for 1 week. Three
consecutive days after surgery Metacam® was added to the
drinking water for a final concentration of 0.25 mg/100 ml. The
dual channel liquid swivel TCS2-23 (Eicom, Japan) was used to
provide a free movement of animals during the experiment. One
day before the experiment mice were shortly anesthetized with
isoflurane, aluminum block and swivel were fasten with steel
wires, microdialysis probes (o.d. 0.2 mm, cuprophane membrane
2 mm of length, MAB 4.15.2.Cu, Microbiotech/se AB, Sweden) were
inserted and connected to the perfusion lines which consist of FEP
tubing (i.d. 0.157 0.05 mm). The shape of the perfusion lines was
tailored to minimize both inlet and outlet volume (this reduces the
back pressure and dead volume of tubing). From the moment of
insertion probes were kept continuously perfused with sterile
artificial cerebrospinal fluid (145 mM NaCl, 2.7 mM KCl, 1.2 mM
CaCl2, 1.0 mM MgCl2, 2.0 mM Na2HPO4, pH ¼7.4) at a flow rate of
0.5 μl/min.
Two hours before the beginning of each experimental proce-
dure the perfusion flow rate was increased to 1.0 μl/min, and
probes were left for a 2 h equilibration period. Ten minute
microdialysis fractions (6 basal, 6 post-treatment and 7 post-
“stressed”) were collected into 300 μl microtubes (Microbiotech/se
AB. Sweden) which were kept in a refrigerated Univentor 820
Microsampler (Univentor, Malta). The dead volume of the outlet
line was compensated with a delay in fraction harvesting (10 min).
Therefore, all microdialysis fractions carefully corresponded to the
actual time of the experimental schedule. At the end of each
 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104 97

experimental day, the perfusion flow rate was reduced to 0.5 μl/ limits of detection for DOPAC, HVA, and 5-HIAA were not exam-
min for ongoing overnight perfusion. ined since the microdialysate concentrations for these metabolites
are usually in the hundred nM range. Retention times for DOPAC,
2.6. Monoamine assays dopamine, 5-HIAA, HVA were 3.2, 5.5, 6.5, and 8.5 min, respec-
tively. The experimental sample monoamine levels were quanti-
Once collected, all microdialysis samples were stored at −80 1C fied by external standard curve calibration using peak area for
and analyzed within 2–3 weeks following sampling. Dopamine, quantification.
DOPAC, HVA, 5-HIAA contents were determined by reverse-phase
HPLC with an electrochemical detection system which consisted of
2.7. Histology/probe placement verification
the SunFlow100 isocratic pump (SunChrom, Germany) coupled
with the Decade amperometric detector (ANTEC Leyden, the
After completing the microdialysis procedure, mice were
Netherlands). The mobile phase contained 0.09 M Na2HPO4,
anaesthetized with isoflurane, and brains were removed. Forty
0.05 M Na citrate, 1.7 mM OSA, 0.05 mM Na2-EDTA and 10%
micrometer brain sections were stained with cresyl violet (Carl
acetonitrile (v/v) and pH was adjusted to 3.0 with 10 M NaOH.
Roth GmbH, Germany) and verified under microscope for probe
All reagents used for the mobile phase were of analytical grade
placement using Paxinos and Franklin (2001) mouse atlas (Fig. 1).
and obtained from Carl Roth, GmbH or MERCK KGaA, Germany.
When probe placement was found to be out of the targeting area,
The mobile phase was filtered through a 0.22 μm nylon filter
the respective samples sets were discarded.
(Merck Millipore, Merck KGaA, Germany) and pumped through
the system at a flow rate of 0.6 ml/min. Monoamines were
separated on an analytical column (C18, 150 mm  3.2 mm, 3 μm, 2.8. Determination of monoamine oxidase (MAO) enzymatic activity
YMC-PackProC18, YMC Europe GMBH, Germany). Detection was
performed at glassy carbon electrode at oxidation potential set at Animals were obtained from Charles River, Germany. C57Bl/6J
þ650 mV against an Ag/AgCl electrode. Injection volume was 10 μl mice and BALB/C mice were sacrificed by decapitation and the
and the limit of detection for dopamine was 1.8 fM/10 μl. The median prefrontal cortex was rapidly dissected, frozen and stored

Fig. 1. Microdialysis probe placement. Probe and cannula placements are shown in subsequent coronal sections. The computer-based atlas by Paxinos and Franklin (2001)
was used to mark probe locations.
98 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104

at −80 1C until assay. The enzymatic activity of MAO-A and MAO-B
was measured according to a previous protocol (Zhou and Panchuk-
Voloshina, 1997). Briefly, PFC regions were homogenized in 20
volumes of ice-cold 20 mM Na-phosphate buffer containing 0.2%
Triton-X 100 (w/w). For the assay of MAO-A and MAO-B the
homogenates were further diluted to 1/12.5 and 1/25, respectively,
with 20 mM phosphate buffer 0.2% Triton-X 100. Since the mouse
brain contains both MAO isoforms, homogenate aliquots destined to
MAO-A assay were pre-incubated with the selective MAO-B inhibitor
L-deprenyl (10 μM), whereas those destined to the MAO-B assay were
pre-incubated with the selective MAO-A inhibitor clorgyline (1 μM).
Serotonin (100 μM) was used as a substrate for MAO-A assay, whereas
phenylethylamine (20 μM) was used for MAO-B assay. Blanks were
prepared by adding both clorgyline and L-deprenyl to the homo-
genates. The progress of the enzymatic reaction was monitored
fluorometrically (excitation at 544 nm and emission at 590 nm) at
room temperature in a PolarStar Galaxy microplate reader (BMG
Labtech, Germany). Data were expressed in nM of substrate metabo-
lized/hour/mg protein. Protein content was measured with the Pierce
BCA protein assay reagent (Perbio Science, Switzerland).
2.9. Determination of tissue levels of monoamines
2.10. Evaluation of MAO inhibitory activity by afobazole in vitro
The mitochondria of dissected mPFC of Wistar rats (the
“Stolbovaya” breeding center of RAMS, Russia) were separated by
differential centrifugation as described in a previous publication
(Medvedev et al., 1994). Briefly, the activity of MAO was assayed
radiometrically using the following substrates: 100 μM [14C] serotonin
(sp. act. 4 Ci/mol), 5 μM [14C]PEA (sp. act. 5 Ci/mol) or 100 μM [14C]
tyramine (sp. act. 4 Ci/mol). These concentrations of serotonin and
phenylethylamine are selectively deaminated by MAO-A and MAO-B,
respectively, whereas tyramine is a common substrate for both MAO-A
and MAO-B. Protein concentration was determined by the Lowry et al.
(1951) method using bovine serum albumin as a standard. Coefficients
of variance for day-to-day and for intra-series variations were o12%.
2.11. Data analysis and statistics
All data are expressed as mean 7SEM Statistical analyses were
computed with Statistica, version 5.0 (StatSoft, Inc., Tusla, OK,
USA). All differences were considered significant at po 0.05.
The post-hoc analysis (Newman–Keuls test) followed the analysis
of variance when appropriate.

Animals were obtained from the “Stolbovaya” breeding center 3. Results

of RAMS, Russia. Naïve C57Bl/6N and BALB/C mice were injected
with saline or afobazole, decapitated 60 min later, and the brains
were removed and cortici extracted on ice. Tissue samples were
homogenized in 0.2 N HClO4 and centrifuged (20 000  g, 30 min,
41 C). A monoamine assay was performed with the supernatant as
described in the respective section.
3.1. Strain-dependent amelioration of anxiety after acute treatment
with afobazole
BALB/C and C57Bl/6N mice significantly differed in levels
of anxiety-like behavior on the elevated plus-maze. The latency

Fig. 2. BALB/C and C57Bl/6N mice show different behavior and strict selectivity of acute afobazole (5 mg/kg, i.p.) treatment in the EPM test (saline treated BALB/C, n¼ 9
afobazole treated BALB/C, n¼ 7, saline treated C57Bl/6N, n¼9, afobazole treated C57Bl/6N, n ¼9). (A) Interstrain difference in the latency to a first entry to an open arm.
(B) Number of entries in open arms. (C) Total time spent in open arms. (D) Number of entries in closed arms. *Po 0.05, ***P o 0.001, n.s. – nonsignificant.
 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104 99

to the first open arm entry (Fig. 2A) was the most sensitive
measure to distinguish the two strains and to demonstrate the
anxiolytic action of afobazole. A two-way ANOVA (strain, treat-
ment) revealed a significant main effect and factorial interaction in
changes in latency (strain, F(1,35)¼7.569, P ¼0.009, treatment, F
(1,35) ¼8.828, P ¼0.005, strain  treatment, F(1,35)¼ 22.392,
P o0.001, Fig. 2A). The same analysis for closed arm entries
showed a significance of strain (F(1,34)¼ 48.099, Po 0.001) and a
factional interaction (F(1,35)¼ 6.978, P ¼0.012) (Fig. 2D). In our
experiment, three of four parameters of mouse behavior in the
EPM point on the strain-specific afobazole action in BALB/C mice.
The decrease in the latency to a first open arm entry (Fig.2A) and
closed arm entries (Fig. 2D), as well as increase in the number of
open arm entries (Fig. 2B) (two-way ANOVA, strain, F(1,31) ¼5.792,
P ¼0.022, treatment, F(1,31) ¼ 29.194, Po0.001, strain  treatment,
F(1,35) ¼4.773, Po 0.037) confirmed the selectivity of anxiolytic
activity of the drug in BALB/C mice. However, the increase in
the open arm time in C57Bl/6N mice did not support this notion
(two-way ANOVA, strain, F(1,35) ¼1.269, P ¼0.268, treatment,
F(1,35) ¼6.706, P ¼0.014, strain  treatment, F(1,35)¼ 1.097,
P ¼0.302).
3.2. Characterization of basal monoaminergic transmission in the
mPFC of BALB/C and C57Bl/6 mice
3.2.1. MAO-A and MAO-B activity
The two mice strains differed in the activity of the two isoforms
of MAO (Fig.3A). Catalytic activity of MAO-A, which, together with
enzymatic conversion of dopamine, readily metabolizes serotonin
into 5-HIAA, was relatively low, 5.1–5.4 mM/h/mg protein.
The activity of MAO-B was higher than that of MAO-A, however
it showed a strong strain difference. Accordingly, a two-way
ANOVA (strain, enzyme isoform) revealed a difference in the levels
of MAO-A and MAO-B activity (F(1,22)¼ 216.1, P o0.0001),
whereby the value of MAO-A activity was lower than that of
MAO-B (F(1,22) ¼584.4, P o0.0001). In addition, there was a
significant factorial interaction (F(1,22)¼182.7, P o0.0001). Post-
hoc analyses confirmed that the activity of MAO-B was 2-times
higher in the C57Bl/6N mice, compare to BALB/C mice (22.5 7
2.3 mM/h/mg protein and 9.9 70.5 mM/h/mg protein (Fig. 3A).
3.2.2. Tissue level of dopamine
Total content of dopamine in the PFC was higher in the BALB/C
mice (t-test, F(1,18) ¼5.323, P¼ 0.033, Fig. 3B).
3.2.3. Basal extracellular monoamine and monoamine metabolite
levels
Analysis of the microdialysis data (fractions 1–6) revealed clear
strain difference for basal dopamine release in the PFC (two-way
ANOVA (strain, time), strain: F(1,144) ¼ 15.702, P ¼0.0013, time:
F(5,144)¼ 2.877, P¼0.0197, strain  time: F(5,144)¼1.388, P¼ 0.238,
Fig. 4). When basal samples were collapsed together BALB/C mice
showed 50% higher dopamine levels, to C57Bl/6N mice, increase in
the extracellular content of dopamine (t-test, F(1,144) ¼15.657,
Po 0.0001, Fig. 3C). Strain effects were not observed when basal
DOPAC and 5-HIAA levels in the microdialysates were considered
(data are not shown).

Fig. 3. Characterization of dopaminergic system in the BALB/C and C57Bl/6 mice. (A) MAO-A and MAO-B activities were measured in the homogenates of mPCF of BALB/C
(n¼ 7) and C57Bl/6J (n¼ 6) mice. The metabolizing activity was estimated by utilization of benzylamine. (B) Tissue content of dopamine in the BALB/C (n ¼7) and C57Bl/6N
(n¼ 7) mice. (C) Extracellular levels of dopamine in the BALB/C (n¼ 13) and C57Bl/6N (n¼ 13) mice. *P o0.05, ***Po 0.001.
100 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104
Fig. 4. Changes in the dopamine content in microdialysates of PFC of BALB/C and
C57Bl/6N mice was monitored during baseline, after saline (circles) or afobazole,
5 mg/kg (squares), administration (arrow), and subsequent open field test (filled
bar). Saline treated BALB/C, n¼ 6, afobazole treated BALB/C, n¼ 7, saline treated
C57Bl/6N, n¼ 7, afobazole treated C57Bl/6N, n¼ 6. * depicts a significant difference
(Po 0.05) between open field-corresponding value and each basal value of
dopamine in the saline treated BALB/C group.
3.3. Afobazole treatment selectively affects dopamine, but not
serotonin metabolizing profile in the mPFC of the BALB/C
3.3.1. The effect of afobazole on extracellular accumulation
of dopamine before and during open field performance
Microdialysis data showed a higher reactivity of dopamine release
in the mPFC of BALB/C mice. After saline or drug administration
(Fig. 4A, fractions 6–12) dopamine tended to increase in BALB/C mice,
however not significantly. Two way ANOVA showed neither signifi-
cant time (F(6,30)¼1.826, P¼0.127) nor treatment (F(1,30)¼0.005,
P¼0.9428) effects nor an interaction of these factors (F(6,30)¼0.009,
P¼0.849). Afobazole treatment (5 mg/kg) normalized the significant
increase in dopamine content following exposure to the open field
(Fig. 4A, fractions 12–19). Statistical analysis showed the effects of
time (F(7,49¼4.306, Po0.001) and treatment (F(1,49)¼ 5.821, P¼
0.046), but not a significant interaction (F(7,49)¼ 1.384, P¼0.232).
The stress-resilient C57Bl/6N mice failed to show any changes in
the absolute content of dopamine after saline/drug administration
(time F(6,24)¼2.035, P¼0.099, treatment F(1,24)¼0.300, P¼0.612),
treatment  time F(6,24)¼ 0.932, P¼0.490) or after the open novelty
stress (time F(7,14)¼1.446, P¼0.263), treatment F(1,14)¼ 0.022,
P¼0.894), treatment  time (F(7,14)¼0.503, P¼0.817) (Fig. 4B).
3.3.2. Effect of afobazole on DOPAC, HVA, dopamine turnover and
5-HIAA extracellular accumulation before and during open field
performance
A three-way ANOVA showed a strong interaction of strain, treat-
ment, and time factors (F(72,421)¼2.198, P¼0.008) in determining
extracellular DOPAC values in the mPFC of mice. To acquire more
specific information concerning effects of genotype and treatment
we performed a cross-over paired analysis of changes in the DOPAC
content. First, we revealed that injection stress did not evoke any
changes in DOPAC levels (Fig. 5A, fractions 6–12) both in BALB/C and
C57Bl/6N saline treated animals (time F(6,18)¼1.249 P¼ 0.328, strain
F(1,18)¼0.062, P¼0.818, time  strain F(6,18)¼1.461, P¼0.246). Sec-
ond, the magnitude of increase in the extracellular content of DOPAC
(Fig. 5A, fractions 12–19) in saline treated mice induced by open field
novelty is determined by mouse strain and time interaction (time F
(7,21)¼ 5.335, P¼0.001, strain F(1,21)¼0.062, P¼0.818, time  strain
F(7,21)¼ 3.684, P¼ 0.009). Third, the decrease in extracellular DOPAC
levels after afobazole treatment was not influenced by genotype
(Fig. 5B, fractions 6–12: time F(6,36¼4.505, P¼ 0.001), strain
F(1,36)¼ 0.910, P¼0.498, time  strain F(6,36)¼0.259, P¼ 0.628; frac-
tions 12–19: time F(7,49)¼0.842, P¼0.558, strain F(1,49)¼2.099,
P¼ 0.190, time  strain F(7,36)¼0.480, P¼ 0.844; fractions 1–19: time
F(18,54)¼7.966, Po0.001, strain F(1,54)¼0.001, P¼0.976, time  -
strain F(18,54)¼1.061, P¼0.413).
Fourth, in BALB/c mice the difference in the DOPAC time course
following drug vs. saline administration was not reliably detected
(Fig. 5C, fractions 6–12: time F(6,30) ¼0.289, P ¼0.937, treatment
F(1,30)¼1.047, P¼0.353, time  treatment F(6,30)¼4.462, P¼ 0.002).
Changes induced by open field novelty were not significant (Fig. 5C,
fractions 12–19: time F(7,49)¼1.467, P¼0.201, treatment F(1,49)¼
4.527, P¼0.071, time  treatment F(6,49)¼ 0.801, P¼0.590). Fifth, in
C57Bl/6N mice afobazole tended to decline in the extracellular
content of DOPAC (Fig. 5D, fractions 6–12: time F(6,24)¼0.977, P¼
0.461). This tendency resulted in a strict difference between afoba-
zole- and saline-treated animals after open field novelty exposure
(Fig. 5D, fractions 12–19: time F(7,21)¼6.189, Po0.001, treatment
F(1,21)¼ 30.793, P¼0.011, time  treatment F(6,21)¼4.408, P¼0.004).
There were no changes in the extracellular HVA level (Fig. 6C).
The dopamine turnover index, calculated as ([DOPAC] þ[HVA])/
[dopamine], was increased during the open field test performance
to the same degree in both strains. Afobazole effectively returned
this short term elevation to basal values irrespective of the strain
(Fig. 6D). This was proven by a two-way ANOVA (treatment, strain)
of turnover indices under separate time point (fraction 16):
(treatment: F(1,9) ¼6.684, P ¼0.029; strain: F(1,9)¼0.481,
P¼ 0.505; treatment  time: F(1,9) ¼0.128, P ¼0.728). There were
no effects of strain, open field exposure, treatment, and factorial
interactions on the serotonin metabolite, 5-HIAA (Fig. 6F).
3.3.3. Tissue level of 5-HIAA in the PFC of BALB/C and C57Bl/6N mice
treated with afobazole
5-HIAA total content in the PFC was sensitive to afobazole
treatment (treatment: F(1,22)¼4.771, P¼ 0.039. This effect can be
attributed to C57Bl/6N mice, which however was not statistically
significant (treatment  strain interaction: F(1,22)¼2.430, P¼0.133)
(Fig. 6E).
3.3.4. In vitro MAO-A inhibiting activity of afobazole
We examined the MAO-A and MAO-B inhibiting capacity of
afobazole in vitro and observed a dose-dependent reduction only
in MAO-A activity in the mitochondria of rat mPFC in the presence
of the drug with an IC50 ¼3.6 71.5 mM (Fig. 6A). The inhibiting
activity was low in comparison to μM range of IC50 in the case of
hydrazine and non-hydrazine non-selective MAO-inhibitors (data
are not shown), but may significantly contribute the overall
efficacy of dopamine and serotonin neurotransmission. Afobazole
did not show any ability to inhibit MAO-B at the same preparations
(data are not shown).
 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104 101

Fig. 5. Changes in the DOPAC extracellular content in the mPFC of BALB/C and C57Bl/6N mice were monitored during baseline, after saline (circles) or afobazole, 5 mg/kg
(squares), administration (arrow), and subsequent open field test (filled bar). Saline treated BALB/C, n¼ 6, afobazole treated BALB/C, n¼ 7, saline treated C57Bl/6N, n¼ 7,
afobazole treated C57Bl/6N, n¼ 6. (A) Between strain comparisons in the saline treated mice. (B) Between strain comparisons in the afobazole treated mice. (C) Between
treatment group comparisons in BALB/C mice. (D) Between treatment group comparisons in C57Bl/6N mice. *P o0.05, ***Po 0.001.

4. Discussion
The PFC a brain area which is primarily employed in maintain-
ing behavioral flexibility and learning (Williams and Goldman-
Rakic, 1995; Floresco et al., 2009; Goto et al., 2010), can in part
determine adaptive response to environmental changes (Cerqueira
et al., 2007). In addition, the PFC seems to be involved in the
tailoring of fear and trait anxiety expression (Holmes and
Wellman, 2009). A proposed dual-mode of dopamine function in
the PFC would be regarded as a powerful mechanism for proces-
sing of information which is necessary for selecting an appropriate
behavior in response to actual demand (Durstewitz and Seamans,
2008). The main task of our study was to characterize the
parameters of the dopaminergic system in the PFC of mice
showing difference in stress-coping strategies (i.e. C57Bl/6 and
BALB/C mice) and explore possible monoaminergic mechanisms of
afobazole action.
We confirmed that C57Bl/6N and BALB/C mice show different
levels of trait anxiety (e.g., Kastenberger et al., 2012), with BALB/C
mice displaying higher anxiety levels. Concerning the role of dopa-
mine in the regulation of anxiety and fear expression (Grimsby et al.,
1997; Stein et al., 2002; Seamans and Yang, 2004; Popova et al.,
2006) we analyzed tissue and extracellular dopamine contents in
respect to MAO-A and MAO-B activity in the PFC of the two strains.
First, we found a strict difference in the activity of MAO-B between
C57Bl/6J and BALB/C mice that concurs with previous observations of
MAO-B expression in the C57Bl/6J and BALB/C mice (Siesser et al.,
2010). Second, we observed that both the total content of dopamine
and it's extracellular concentration inversely related to the total
MAO-B activity. Moreover, the lower MAO-B activity and higher
dopamine contents were observed in the stress-susceptible BALB/C
mice (Fig. 3). The implied correlation between MAO-B activity and
the behavioral phenotype is in contrast to a report from Bortolato
et al. (2009), where MAO-B knockout mice showed reduced anxiety
levels. However, in this study the behavior assessments were done
under different conditions and one must also consider that behavior
in knockout animals may be highly confounded by compensatory
processes.
The extracellular content of dopamine is generally maintained
by the dopamine transporter. Because of the limited expression of
the dopamine transporter in the PFC (Garris and Wightman, 1994),
the extracellular level of dopamine is also under control of an MAO
and norepinephrine transporter (Gesi et al., 2001).
MAO-B is mainly localized in non-dopaminergic cells, specifi-
cally, in glia and serotoninergic neurons (Levitt et al., 1982). MAO-
B would be expected to minimally influence extracellular level of
dopamine. However, dopamine, which readily spills over the
synapse, can be effectively metabolized by MAO-B. Indeed, the
neurotransmitter has a similar affinity to MAO-A and MAO-B
(Chen and Shih, 1998; Shih et al., 1999). From our data we can
conclude that MAO-B contributes to the regulation of basal
dopamine content in the mPFC.
Following i.p. injections and during open field stress dopamine
tended to increase in the stress susceptible BALB/C mice (Fig. 4A),
but not in the stress-resilient C57Bl/6N (Fig. 4B). In saline treated
BALB/C mice elevation in the extracellular dopamine content
during the open field novelty stress achieved statistical signifi-
cance when compared to basal values. When changes in the
extracellular levels of DOPAC were assessed, we found that both
C57Bl/6N and BALB/C mice showed an increase in the dopamine
metabolite in response to open field exposure (Fig. 5A). In C57Bl/
6N mice this increase was more prominent and lasting.
A rise in DOPAC levels usually accompanies dopamine release
evoked by an increase in neuronal firing and therefore reflects
neuronal activity (Roth et al., 1976). Once released dopamine is
taken up in the dopaminergic neuron, where it can be metabolized
102 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104

Fig. 6. Changes in monoamine metabolite levels after afobazole treatment. Dynamics of accumulation of monoamine metabolites in microdialysates of the PFC of BALB/C and
C57Bl/6N mice was monitored during baseline, after saline (circles) or afobazole, 5 mg/kg (squares), administration (arrow), and subsequent open field test (filled bar). Saline
treated BALB/C, n ¼6, afobazole treated BALB/C, n¼ 7, saline treated C57Bl/6N, n¼7, afobazole treated C57Bl/6N, n¼ 6. Tissue content was obtained in BALB/C (n ¼7/7) and
C57Bl/6N (n ¼7/7) mice. (A) Afobazole inhibits brain MAO-A with IC50 ¼3.6 71.5 mM (n¼ 4). The metabolizing activity was estimated by utilization of [14C]-serotonin
creatinine sulfate. (B) Changes in the tissue content of DOPAC. (C) Changes in the extracellular content of HVA. (D) Dopamine turnover index changes over course of the
experiment. (E) Total concentration of 5-HIAA. (F) Changes in the extracellular content of 5-HIAA.

in a multistage process, including degradation with MAO-A.
In contrast to dopamine, which is released in the zone of synaptic
contact, a product of dopamine degradation, DOPAC, diffuses
outside of the synapse. It can be presumed that observed differ-
ence in the extracellular DOPAC accumulation is determined by a
higher degree of phasic release of dopamine in the C57Bl/6N mice
(Winstanley et al., 2006). Together with this tempting hypothesis a
more simple explanation of the difference in the profile of
extracellular DOPAC accumulation between the strains may be
based on the difference in the MAO-B activity. Third, as known, in
the mPFC conversion to DOPAC also follow a primarily degradation
of dopamine to HVA. We did not observe any changes in the
extracellular HVA level; therefore, the possible influence of in
catechol-o-methyltransferase activity on the difference in DOPAC
level was ruled out.
It is difficult to define the key factor linking an increase in
dopamine neurotransmission and activation of anxiety mechanisms.
The correlation between the increase in the extracellular levels of
dopamine and stress could equally mean that either dopamine
contributes to stress mechanisms or that it reflects the compensatory
response to stress.
In the present study, we aimed not only to examine interac-
tions between the expression of anxiety and changes in dopamine
neurotransmission, but also to figure out possible monoaminergic
mechanisms of the strain-selective anxiolytic action of afobazole.
Afobazole decreased anxiety-related behavior in BALB/C mice as
can be seen in changes in the latency to a first open arm entry and
number of open arm entries (Fig. 2A and B). The indirect measure
of locomotor activity in the EPM test, the closed-arm entry
numbers (Fig.2D), suggests that afobazole does not affect locomo-
tion in C57Bl/6N mice and may suppress locomotion in BALB/C
mice. However, this measure may have a limited value (File, 2001).
Analysis for the open arm time (Fig.2C) did not reveal either the
basal difference between strains or selectivity of afobazole action.
 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104
The analysis for individual data brings to a certain skepticism
concerning the increase in the open arm time in C57Bl/6N mice. It
can be expected that an increase in animal number in experi-
mental group would clarify the situation. Despite the lack in “four
out of four” proof, our data do not contradict with the previous
reports and do not reject selectivity of abobazole anxiolytic action.
The interpretation of mouse behavior in the EPM was done under
the assumption that afobazole does not show sedative action
(Seredenin and Blednov, 1994). Thus, in mice with a passive
stress-coping strategy (i.e. BALB/C), afobazole administration
results in active, exploration-oriented response to novelty. In mice
with active stress-coping strategy (C57Bl/6N) afobazole does not
suppress the characteristic exploratory response which points to
the absence of any maladaptive sedative influence. Depending on
the strain, afobazole also differentially affects the heart rate
variability in the open field test (Kaverina et al., 2009), BDNF
expression (Seredin et al., 2006), and monoamine metabolism
(Kudrin, 2006). We used an open field test paradigm as a novelty
stress model, therefore, we did not perform any additional
behavioral analysis. It is worth mentioning that evaluations of
locomotor activity would help characterize basic mouse behavior,
however even a detailed analysis could not fully confirm the
difference in anxiety levels between the lines or changes in
anxiety levels following drug treatment (Ferguson et al., 2004;
Kuzmin et al., 2006; Lalonde and Strazielle, 2008).
The primary goal of the present study was an evaluation of
differences in the monoamine system as a basis of the selective
anxiolytic action of afobazole administered at dose which was
validated as anxiolytic. Afobazole binds to sigma1 receptors in vivo
(Seredenin and Voronin, 2009). As previously reported, sigma1
ligands show antidepressant properties that corresponded to
changes in monoaminergic neurotransmission. While the MAO-A
and MAO-B inhibitory activity of igmesine and OPC-14523 was
very low, the drugs blocked the norepinephrine transporter
(Akunne et al., 2001; Tottori et al., 2001). Interestingly, two
substances of plant origin, hypericin and berberin, which exert
antidepressant activity, show MAO inhibitory activity (Kulkarni
and Dhir, 2008; Raffa, 1998). Sigma-ligand antidepressant proper-
ties seem to depend on the function of N-methyl-D-aspartate
receptor (Bergeron et al., 1993) which may confirm an importance
of Caþþ-mediated mechanisms in the regulation of MAO and
monoamine transporter activity (Monnet, 2005).
In our in vitro experiment we showed that afobazole selectively
and reversible inhibits MAO-A in PFC preparations, however with
low efficacy (Fig. 6A). The inhibiting activity was confirmed in vivo
when afobazole counteracted an increase in the extracellular
DOPAC during the open field exposure in C57Bl/6N mice
(Fig. 5D). The dopamine turnover index, ([DOPAC]þ [HVA])/[dopa-
mine] was elevated during open field exposure and decreased
under afobazole treatment similarly in both strains (Fig. 6D).
Increase in the dopamine turnover index coincides with stress.
This measure is particularly sensitive, when examined in the PFC.
We did not observe the previously reported difference between
BALB/C and C57Bl/6 mice in dopamine turnover which can be seen
after open field stress (Le Moal and Simon, 1991). However,
anxiolytic effect of afobazole is confirmed by drop in the dopamine
turnover. Since basal activities of MAO-A were comparable in two
strains it can be suggested that the decrease in the DOPAC level is
due to the selective inhibition of MAO-A. The significance of MAO-
A inhibition by afobazole in the regulation of extracellular dopa-
mine levels during its facilitated release was demonstrated in
BALB/C mice where afobazole treated animals did not show a
significant elevation in extracellular dopamine during the open
field novelty stress. A possible impact of afobazole on serotonin
turnover (Davydova et al., 2010) was not confirmed. Monitoring of
5-HIAA failed to reveal significant strain or general effect.
103
Accordingly, our data suggest that afobazole modulates elevated
dopamine levels in the mPFC in the anxiety susceptibility
phenotype.
We can speculate that the difference in basal levels of dopa-
mine and MAO-B activity between the two strains significantly
influence not only anxiety expression, but also sensitivity of BALB/
C mice to afobazole treatment. Thus, a possible facilitation of
phasic dopamine release in the mPFC would favor the specific
neuronal activity and mediate adaptive response, as had been
proposed for dopamine (Grace, 2000) and norepinephrine (Aston-
Jones and Cohen, 2005) action in the PFC. An resulting increase in
the level of vigilance and wakefulness would improve cognitive
control over the stressful situation (Benchenane et al., 2010;
Moustafa and Gluck, 2011; Fallon et al., 2012). In the BALB/C mice
this may prevent behavioral responses to stress, whereas in the
C57Bl/6N mice this would increase the level of exploratory activity
and facilitate learning.
Taken together our data provide evidence (1) that BALB/C and
C57Bl/6 mice show pronounced differences in the levels of MAO-B
activity, total tissue and extracellular dopamine content in the
mPFC, (2) that BALB/C mice which are characterized by higher
anxiety level show a slightly enhanced stimulated dopamine
release in comparison to that in C57Bl/6N mice, and (3) that
dopaminergic mechanisms may contribute to the strain selectivity
in the anxiolytic activity of afobazole. Considering its MAO-A
inhibitory activity, a specific involvement of afobazole in the
regulation of monoaminergic neurotransmission requires further
investigation.
Acknowledgments
The authors thank Mr. Edilio Borroni from Hoffmann la Roche
for his highly professional analysis of MAO-A and MAO-B activity
in the brain tissue, Professor Sergey B. Seredenin for considerable
scientific discussion and Ms. Caitlin Riebe for her invaluable help
with manuscript preparation.
References
Akunne, H.C., Zoski, K.T., Whetzel, S.Z., Cordon, J.J., Brandon, R.M., Roman, F.,
Pugsley, T.A., 2001. Neuropharmacological profile of a selective sigma ligand,
igmesine: a potential antidepressant. Neuropharmacology 41, 138–149.
Aston-Jones, G., Cohen, J.D., 2005. An integrative theory of locus coeruleus-
norepinephrine function: adaptive gain and optimal performance. Ann. Rev.
Neurosci. 28, 403–450.
Benchenane, K., Peyrache, A., Khamassi, M., Tierney, P.L., Gioanni, Y., Battaglia, F.P.,
Wiener, S.I., 2010. Coherent theta oscillations and reorganization of spike
timing in the hippocampal-prefrontal network upon learning. Neuron 24,
921–936.
Bergeron, R., Debonnel, G., De Montigny, C., 1993. Modification of the N-methyl-D-
aspartate response by antidepressant sigma receptor ligands. Eur. J. Pharmacol.
240, 319–323.
Bortolato, M., Godar, S.C., Davarian, S., Chen, K., Shih, J.C., 2009. Behavioral
disinhibition and reduced anxiety-like behaviors in monoamine oxidase B-
deficient mice. Neuropsychopharmacology 34, 2746–2757.
Boulougouris, V., Tsaltas, E., 2008. Serotonergic and dopaminergic modulation of
attentional processes. Prog. Brain Res. 172, 17–42.
Broadhurst, P.L., 1976. The Maudsley reactive and nonreactive strains of rats: a
clarification. Behav. Genet. 6, 363–365.
Carola, V., D'Olimpio, F., Brunamonti, E., Bevilacqua, A., Renzi, P., Mangia, F., 2004.
Anxiety-related behaviour in C57BL/6 o - 4 BALB/c chimeric mice. Behav.
Brain Res. 150, 25–32.
Cerqueira, J.J., Mailliet, F., Almeida, O.F.X., Jay, T.M., Sousa, N., 2007. The prefrontal
cortex as a key target of the maladaptive response to stress. J. Neurosci. 27,
2781–2787.
Chen, K., Shih, J., 1998. Monoamineoxidase A and B: structure, function, and
behavior. In: Goldstein, D.S., Eisenhofer, D., McCarty, R. (Eds.), Cathechola-
mines. Driving Basis Science with Clinical Medicine. Academic Press, San Diego,
pp. 292–296.
Cools, R., D'Esposito, M., 2011. Inverted-U-shaped dopamine actions on human
working memory and cognitive control. Biol. Psychiatry 69, 113–125.
104 E.A. Anderzhanova et al. / European Journal of Pharmacology 708 (2013) 95–104
Cuevas, J., Behensky, A., Deng, W., Katnik, C., 2011. Afobazole modulates neuronal
response to ischemia and acidosis via activation of {sigma}-1 receptors.
J. Pharmacol. Exp. Ther. 339, 152–160.
Crawley, J.N., Belknap, J.K., Collins, A., Crabbe, J.C., Frankel, W., Henderson, N.,
Hitzemann, R.J., Maxson, S.C., Miner, L.L., Silva, A.J., Wehner, J.M., Wynshaw-
Boris, A., Paylor, R., 1997. Behavioural phenotypes of inbred mouse strains:
implications and recommendations for molecular studies. Psychopharmacology
132, 107–124.
Davydova, A.I., Klodt, P.M., Kudrin, V.S., Kuznetsova, E.A., Narkevich, V.B., 2010.
Neurochemical study of effects of the new anxiolytic drugs afobazol and
ladasten on the synthesis and metabolism of monoamines and their metabo-
lites in the brain structures of Wistar rat on the model of monoamine synthesis
blockade induced by aromatic amino acid decarboxylase inhibitor NSD-1015.
Eksp. Klin. Farmakol. 73, 2–6.
Durstewitz, D., Seaman, K., 2008. The dual-state theory of prefrontal cortex
dopamine function with relevance to catechol-o-methyltransferase genotypes
and schizophrenia. Biol. Psychiatry 64, 739–749.
Fallon, S.J., Williams-Gray, C.H., Barker, R.A., Owen, A.M., Hampshire, A., 2012.
Prefrontal dopamine levels determine the balance between cognitive stability
and flexibility. Cereb. Cortex, PMID: 22351648.
Ferguson, G.D., Herschman, H.R., Storm, D.R., 2004. Reduced anxiety and
depression-like behavior in synaptotagmin IV (−/−) mice. Neuropharmacology
47, 604–611.
File, S.E., 2001. Factors controllingmeasures of anxiety and responses to novelty in
the mouse. Behav. Brain. Res. 125, 151–157.
Floresco, S.B., Zhang, Y., Enomoto, T., 2009. Neural circuits subserving behavioral
flexibility and their relevance to schizophrenia. Behav. Brain. Res. 204, 396–409.
Fowler, S.C., Zarcone, T.J., Vorontsova, E., 2001. Haloperiodol-induced microcata-
lepsy differs in CD-1, BALB/c, and C57BL/6 mice. Exp. Clin. Psychopharmacol. 9,
277–284.
Garris, P.A., Wightman, R.M., 1994. Different kinetics govern dopaminergic trans-
mission in the amygdala, prefrontal cortex and striatum: and in vivo voltam-
metric study. J. Neurosci. 14, 442–450.
Gesi, M., Santinami, A., Ruffoli, R., Conti, G., Fornai, F., 2001. Novel aspects of
dopamine oxidative metabolism (confounding outcomes take place of certain-
ties). Pharm. Toxicol. 89, 217–224.
Goto, Y., Yang, C.R., Otani, S., 2010. Functional and dysfunctional synaptic plasticity
in prefrontal cortex: roles in psychiatric disorders. Biol. Psychiatry 67, 199–207.
Grace, A.A., 2000. The tonic/phasic model of dopamine system regulation and its
implications for understanding alcohol and psychostimulant craving. Addict.
Suppl. 2, S119–128.
Griebel, G., Belzung, C., Perrault, G., Sanger, D.J., 2000. Differences in anxiety-
related behaviours and sensitivity to diazepam in inbred and outbred strains of
mice. Psychopharmacology 148, 164–170.
Grimsby, J., Toth, M., Chen, K., Kumazawa, T., Klaidman, L., Adams, J.D., Karoum, F.,
Gal, J., Shih, J.C., 1997. Increased stress response and beta-phenylethylamine in
MAOB-deficient mice. Nat. Genet. 17, 206–210.
Holmes, A., Wellman, C.L., 2009. Stress-induced prefrontal reorganization and
executive dysfunction in rodents. Neurosci. Behav. Rev. 333, 773–783.
Kastenberger, I., Lutsch, C., Herzog, H., Schwarzer, C., 2012. Influence of sex and
genetic background on anxiety-related and stress-induced behaviour of
prodynorphin-deficient mice. PLoS One 7 (3), e34251, PMID: 22479578.
Kaverina, N.V., Popova, E.P., Iarkova, M.A., Seredenin, S.B., 2009. Afobazole effects on
heart rate variability in rats with different behaviors in the “open field” test.
Eksp. Klin. Farmakol. 72, 33–40.
Kudrin, V.S., 2006. A comparative study of the effect of afobazole on brain
monoamine systems in BALB/C and C57Bl/6 mice. Eksp. Klin. Farmakol. 69,
7–10.
Kulkarni, S.K., Dhir, A., 2008. On the mechanism of antidepressant-like action of
berberine chloride. Eur. J. Pharmacol. 589, 163–172.
Kuzmin, A., Madjid, N., Terenius, L., Ogren, S.O., Bakalkin, G., 2006. Big dynorphin, a
prodynorphin-derived peptide produces NMDA receptor-mediated effects on
memory, anxiolytic-like and locomotor behavior in mice. Neuropsychophar-
macology 31, 1928–1937.
Lalonde, R., Strazielle, C., 2008. Relations between open-field, elevated plus-maze,
and emergence tests as displayed by C57/BL6J and BALB/c mice. J. Neurosci.
Methods 171, 48–52.
Le Moal, M., Simon, H., 1991. Mesocorticolimbic dopaminergic network: functional
and regulatory roles. Physiol. Rev. 71, 155–234.
Levitt, P., Pinta, J.E., Breakefiels, X.O., 1982. Immunocytochemical demonstration of
monoamineoxidase B in brain astrocites and serotoninergic neurons. Proc. Natl.
Acad. Sci. USA 79, 6385–6389.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement
with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Medvedev, A.E., Kirkel, A.Z., Kamyshanskaya, N.S., Axenova, L.N., Moskvitina, T.A.,
Gorkin, V.Z., Andreeva, N.I., Golovina, S.M., Mashkovsky, M.D., 1994. Inhibition
of monoamine oxidase by novel antidepressant tetrindole. Biochem. Pharma-
col. 47, 303–308.
Monnet, F.P., 2005. Sigma-1 receptor as regulator of neuronal intracellular Ca2 þ :
clinical and therapeutic relevance. Biol. Cell 97, 873–883.
Moustafa, A.A., Gluck, M.A., 2011. A neurocomputational model of dopamine and
prefrontal-striatal interactions during multicue category learning by Parkinson
patients. Neural Networks 24, 575–591.
Paxinos, G., Franklin, K.B.J., 2001. A Stereotaxic Atlas of the Mouse Brain. Academic
Press, San Diego, CA.
Popova, N.K., Maslova, L.N., Morosova, E.A., Bulygina, V.V., Seif, I., 2006. MAO-A
knockout attenuates adrenocortical response to various kinds of stress. Psy-
choneuroendocrinology 31, 179–186.
Raffa, R.B., 1998. Screen of receptor and uptake-site activity of hypericin component
of St. John's work reveals sigma receptor binding. Life Sci. 62, PL265–270.
Rice, M.E., Cragg, S.J., 2008. Dopamine spillover after quantal release: rethinking
opamine transmission in the nigrostriatal pathway. Brain Res. Rev. 58, 303–313.
Roth, R.H., Murrin, L.C., Waiters, J., 1976. Central dopaminergic neurons: effects of
alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid.
Eur. J. Pharmacol. 36, 163–171.
Seamans, J.K., Yang, C.R., 2004. The principal features and mechanisms of dopamine
modulation in the prefrontal cortex. Prog. Neurobiol. 74, 1–58.
Seredenin, S.B., Blednov, Yu.A., 1994. Individual variability in the effects of single-
dose tranquilizers in neurotic patients: clinical, electroencephalographic and
prognostic aspects. In: Seredenin, SB, Longo, V, Gaviraghi, G (Eds.), Biological
Basis of Individual Sensitivity to Psychotropic Drugs. Grasham Press, Edinburgh,
pp. 293–298.
Seredenin, S.B., Molodavkin, G.M., Voronin, M.V., Voronina, T.A., 2009. Antidepres-
sant properties of afobazole in Porsolt and Nomura tests. Eksp. Klin. Farmakol.
72, 19–21.
Seredenin, S.B., Voronin, M.V., 2009. Neuroreceptor mechanisms of the afobazole
effect. Eksp. Klin. Farmakol. 72, 3–11.
Seredin, S.B., Melkumian, D.S., Val'dman, E.A., Iarkova, M.A., Seredina, T.C., Voronin,
M.V., Lapitskaia, A.S., 2006. Effects of afobazole on the BDNF content in brain
structures of inbred mice with different phenotypes of emotional stress
reaction. Eksp. Klin. Farmakol. 69, 3–6.
Shih, J.C., Chen, K., Ridd, M.J., 1999. Role of MAO A and B in neurotransmitter
metabolism and behavior. Pol. J. Pharmacol. 51, 25–29.
Siesser, W.B., Zhang, X., Jacobsen, J.P., Sotnikova, T.D., Gainetdinov, R.R., Caron, M.G.,
2010. Tryptophan hydroxylase 2 genotype determines brain serotonin synthesis
but not tissue content in C57Bl/6 and BALB/c congenic mice. Neurosci. Lett. 481,
6–11.
Stein, D.J., Westenberg, H.G., Liebowitz, M.R., 2002. Social anxiety disorder and
generalized anxiety disorder: serotonergic and dopaminergic neurocircuitry.
J. Clin. Psychiatry 63 (Suppl 6), 12–19.
Tottori, K., Miwa, T., Uwahodo, Y., Yamada, S., Nakai, M., Oshiro, Y., Kikuchi, T., Altar,
C.A., 2001. Antidepressant-like responses to the combined sigma and 5-HT1A
receptor agonist OPC-14523. Neuropharmacology 41, 976–988.
Uyanaev, A.A., Fisenko, V.P., Khitrov, N.K., 2003. Effect of nopept and afobazole on
the development of neurosis of learned helplessness in rats. Bull. Exp. Biol.
Med. 136, 162–164.
Volkova, A.V., Kalinina, T.C., Voronina, T.A., 2010. Comparative study of the
interoceptive effects of afobazole and diazepam. Eksp. Klin. Farmakol. 73, 2–6.
Voronin, M.V., Aksenova, L.N., Buneena, O.A., Medvedev, A.E., 2009. Effect of
afobazole on mitochondrial monoamine oxidase A activity in vitro. Bull. Exp.
Biol. Med. 148, 23–25.
Williams, G.V., Goldman-Rakic, P.S., 1995. Modulation of memory fields by
dopamine Dl receptors in prefrontal cortex. Nature 376, 572–575.
Winstanley, C.A., Theobald, D.E., Dalley, J.W., Cardinal, R.N., Robbins, T.W., 2006.
Double dissociation between serotonergic and dopaminergic modulation of
medial prefrontal and orbitofrontal cortex during a test of impulsive choice.
Cereb. Cortex 16, 106–114.
Zhou, M., Panchuk-Voloshina, N., 1997. A one-step fluorometric method for the
continuous measurement of monoamine oxidase activity. Anal. Biochem. 253,
169–174.