BR A IN RE S EA RCH 1 1 17 ( 20 0 6 ) 5 4 –60

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Short Communication

Semax, an analog of ACTH(4–10) with cognitive effects,

regulates BDNF and trkB expression in the rat hippocampus

Oleg V. Dolotova,⁎, Ekaterina A. Karpenkoa , Lyudmila S. Inozemtsevaa ,

Tamara S. Seredeninaa , Natalia G. Levitskayaa , Joanna Rozyczkac , Elena V. Dubyninaa ,
Ekaterina V. Novosadovaa , Lyudmila A. Andreevaa , Lyudmila Yu. Alfeevaa ,
Andrey A. Kamenskyb , Igor A. Grivennikov a , Nikolay F. Myasoedov a , Jürgen Engelec
a
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia
b
Chair of Animal and Human Physiology, M.V. Lomonosov Moscow State University, Moscow, Russia
c
Institute of Anatomy, University of Leipzig, Medical Faculty, Leipzig, Germany

A R T I C LE I N FO AB S T R A C T

Article history: The heptapeptide Semax (Met–Glu–His–Phe–Pro–Gly–Pro) is an analog of the

Accepted 29 July 2006 adrenocorticotropin fragment (4–10) which after intranasal application has profound
Available online 22 September 2006 effects on learning and exerts marked neuroprotective activities. Here, we found that a
single application of Semax (50 μg/kg body weight) results in a maximal 1.4-fold increase of
Keywords: BDNF protein levels accompanying with 1.6-fold increase of trkB tyrosine phosporylation
ACTH(4–10) levels, and a 3-fold and a 2-fold increase of exon III BDNF and trkB mRNA levels, respectively,
Hippocampus in the rat hippocampus. Semax-treated animals showed a distinct increase in the number of
BDNF conditioned avoidance reactions. We suggest that Semax affects cognitive brain functions
trkB by modulating the expression and the activation of the hippocampal BDNF/trkB system.
Cognitive effect © 2006 Elsevier B.V. All rights reserved.

Abbreviations:
ACTH, adrenocorticotropic hormone
BDNF, brain derived neurotrophic
factor
CAR, conditioned avoidance
response
CREB, cAMP/calcium-response
element binding protein
GAPDH, D-glyceraldehyde-
3-phosphate dehydrogenase
ITR, inter-trial reaction
LTP, long-term potentiation
NGF, nerve growth factor
SDS PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis

⁎ Corresponding author.
E-mail address: olegd@img.ras.ru (O.V. Dolotov).

0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2006.07.108
 BR A IN RE S E A RCH 1 1 17 ( 20 0 6 ) 5 4 –6 0 55

N-terminal fragments of the adrenocorticotropic hormone (p < 0.001, one-way ANOVA) diurnal changes of hippocampal
(ACTH) – a member of the melanocortin family of peptides – BDNF protein levels during the light phase, probably asso-
are well known for their potent neuroregenerative and ciated with cognitive activity and circadian changes in
cognitive activities (de Wied, 1999; Strand et al., 1993). The
heptapeptide Semax (Met–Glu–His–Phe–Pro–Gly–Pro) is an
analog of the ACTH (4–10) fragment (Met–Glu–His–Phe–Arg–
Trp–Gly), completely devoid of any hormonal activity asso-
ciated with the full-length ACTH molecule, which stimulates
learning and memory formation in rodents and humans
(Kaplan et al., 1996; Ashmarin et al., 1995). In addition,
Semax profoundly interferes with several forebrain and
hippocampal functions: it increases the selective attention at
the moment of information reception, improves memory
consolidation and promotes learning abilities (Ashmarin et
al., 1995). Further major advantages of this peptide in
comparison with the ACTH (4–10) fragment, is its higher
resistance against enzymatic cleavage and a subsequent
prolonged (20 times) action in vivo (Potaman et al., 1991).
Semax is the active component of the novel drugs, “SEMAX-
0.1% Solution” and “SEMAX-1.0% Solution” which are applied
intranasally. In Russia these drugs are used for treatment of
brain hypoxia and ischemia, brain strokes, cranial and brain
traumas, and to facilitate adaptive processes to extreme (e.g.,
emergency) situations (“SEMAX-0.1% Solution” has been
included in rehabilitation sets of the rescue and search groups
of the Ministry of Civil Defence, Emergencies and Disaster
Relief of Russia), and to improve learning abilities and memory
formation.
Despite these clinical benefits, the cellular and molecular
mechanisms underlying the cognitive action of Semax, as well
as of short melanocortins (Adan et al., 1994), are widely
unknown. At the cellular level, Semax was shown to prevent
the death of cultured rat basal forebrain cholinergic neurons,
and to stimulate the activity of choline acetyltransferase
(Grivennikov et al., 1999). In addition, recently we obtained the
evidence that Semax induced a rapid 8-fold and 5-fold
increase in BDNF and NGF mRNA levels, respectively when
applied to primary glial cultures of rat basal forebrain
(Shadrina et al., 2001), thus implying that Semax might
modulate brain functions by affecting neurotrophin synthesis.
In the present study, we sought to investigate whether
intranasally applied Semax affects the expression and the
activation of the BDNF/trkB system in the rat hippocampus. In
addition, we sought to determine whether such modulatory
effects on hippocampal BDNF/trkB signaling would interfere
with the acquisition of active avoidance responses. Fig. 1 – Characterization of the effects of Semax on
We first tested whether intranasal application of Semax hippocampal BDNF protein levels. (A) Semax was applied
would affect BDNF protein levels in the rat hippocampus. To intranasally at the indicated concentrations, and
this end, animals were treated with Semax at concentrations hippocampal BDNF levels were determined after 24 h using
ranging from 0.5 to 500 μg/kg body weight, and hippocampal an immunoenzymatic assay. Average control BDNF level
BDNF protein levels were measured after 24 h. In these corresponds to 100% and equals 0.85 ng BDNF/mg total
experiments, water solutions Semax at doses of 50 and protein. Controls received equivalent volumes of distilled
500 μg/kg body weight resulted in a statistically significant, water. Data represent means ± SEM (n = 10 rats) from two
1.4-fold increase in BDNF protein levels, whereas similar independent experiments. ***p < 0.001 (one-way ANOVA with
increases were undetectable in animals which received post hoc Mann–Whitney U tests). (B) Hippocampal BDNF
lower doses of Semax (Fig. 1A). To characterize the time levels 3, 6, 24 and 48 h after intranasal application 50 μg of
course of the effects of Semax on hippocampal BDNF levels, Semax per kg body weight. Data represent mean ± SEM
Semax was applied intranasally at 50 μg/kg body weight and (n = 8 rats) from two independent experiments. *p < 0.05,
hippocampal BDNF levels were assessed after 3, 6, 24 and 48 h. **p < 0.01, ***p < 0.001 (one-way ANOVA with post hoc
As shown in Fig. 1B, water-treated controls showed significant Mann–Whitney U tests).
56 BR A IN RE S EA RCH 1 1 17 ( 20 0 6 ) 5 4 –60
hormone levels (Pollock et al., 2001), and possibly interfered
with effects of intranasal administration of water, with
highest levels in the middle and lowest levels in the beginning
of the light phase. Semax resulted in a 1.2–1.4-fold increase of
BDNF levels after 3, 24 and 48 h as compared to the respective
controls. Such an increase remained undetectable 6 h after
Semax application. As a control, BDNF levels were additionally
determined in the cerebellum 3 and 24 h after Semax
application (data not shown). At both time points, BDNF levels
were indistinguishable from those of untreated with Semax
controls.
To examine whether Semax-induced increase of hippo-
campal BDNF levels activates BDNF/trkB intracellular signal-
ing system, we tested tyrosine phosphorylation of trkB
receptors 3 h after Semax intranasal application. Western
blot analysis (Fig. 2) revealed that Semax induced a 1.5-fold
increase of hippocampal trkB phosphorylation as compared to
control.
The BDNF gene contains four 5′ non-coding exons (exons I–
IV) which all possess their own distinct promoter and hence
allow for a selective control of BDNF expression (Metsis et al.,
1993). Diurnal changes of hippocampal BDNF levels have been
previously associated with BDNF exon III (Berchtold et al.,
1999). RT-PCR using specific primers for BDNF exon III revealed
a 3-fold increase of hippocampal BDNF mRNA levels 3 h after
intranasal Semax application (Fig. 3). After 24 h, mRNA levels
were indistinguishable from those of controls (data not
shown).
To further determine whether Semax would also affect the
expression of the high affinity BDNF receptor, trkB, Semax-
treated animals were analyzed for trkB mRNA levels by RT-
PCR. This analysis was performed 24 h after Semax treatment,
when the increase in BDNF expression was highest. At this
time point, Semax-treated animals showed a 2-fold increase
in trkB mRNA levels as compared to controls (Fig. 3).
Taken together, these findings provide evidence that
Semax is capable to modulate hippocampal BDNF/trkB
signaling.
In a next set of experiments, we sought to determine
whether Semax-induced activation of BDNF signaling would
influence conditioned behavior as assessed by active avoid-
ance responses. In these experiments, Semax treatment
(50 μg/kg body weight) was started 20 h prior to the first
training and repeated every 24 h. As shown in Fig. 4, the
number of CARs (conditioned avoidance responses) increased
(F1,29 = 4,59; p < 0.05, two-way ANOVA) and number of ITRs
(inter-trial reactions) decreased (F1,29 = 4,31; p < 0.05, two-way
ANOVA) in the Semax-treated animal group as compared to
control. Post hoc analysis revealed significant Semax effects
on the number of CARs on the 2nd and 4th days of learning
and on the number of ITRs on the 3rd and 4th days of learning.
The number of CARs is a measure of acquired avoidance
behavior whereas ITR indicates the fear level in the avoidance
situation (Bohus and Lisak, 1968). Consequently, these data
suggest that Semax facilitates learning and at the same time
suppresses fear during avoidance conditioning.
Our present series of experiments reveals that intranasal
application of the ACTH(4–10) analog, Semax, stimulates
hippocampal BDNF and trkB expression in normal adult rats
and facilitates learning.
Fig. 2 – Effect of Semax on tyrosine phosphorylation of trkB
receptors in the rat hippocampus. Tyrosine phosphorylation
of trkB receptors in hippocampi excised from animals 3 h
after intranasal Semax application (50 μg per kg body weight)
was detected after immunoprecipitation of tissue lysates
(200 μg of total protein per lane) with anti-phosphotyrosine
antibody 4G10 and Western blot with anti-trkB antibody.
(A) Representative blot showing hippocampal tyrosine
phosphorylation of trkB receptors. Lane 1, controls. Lane 2,
Semax-treated rats. (B) Quantitative results of Western blot
analysis of hippocampal tyrosine phosphorylation of trkB
receptors. Open columns, controls. Filled columns,
Semax-treated animals. Data represent the mean ± SEM
(n = 6 rats) from two independent experiments. *p < 0.05
(one-way ANOVA with post hoc Mann–Whitney U tests).
Interestingly, Semax treatment did not increase hippo-
campal BDNF levels beyond maximal BDNF levels detected in
controls, suggesting that Semax modulates hippocampal
BDNF expression within its physiological (possibly, circadian)
range. Moreover, in contrast to the hippocampus, Semax
failed to affect BDNF levels in the cerebellum, hence, pointing
to a brain region-specific action of this peptide. In further
support of this notion, we observed recently that Semax only
induces a small (1.2-fold) and timely restricted (3 h) increase of
BDNF levels in the basal forebrain (Dolotov et al., 2006). Finally,
in the hippocampus, Semax resulted in the increased tran-
scription of BDNF exon III mRNA, which is known to be CREB
(cAMP-response element binding protein)-dependent (Tao et
al., 1998). Our knowledge on the Semax binding sites is
currently rather limited. So far, we obtained evidence that
Semax binding to basal forebrain cell membranes is calcium-
dependent (Dolotov et al., 2006). The pronounced effects of
Semax on BDNF exon III mRNA expression now further point
to the possibility that Semax acts through G-protein coupled
receptors. Our current findings do not allow to discern
whether the increases in trkB mRNA expression seen in
Semax-treated animals result from a direct action of this
 BR A IN RE S E A RCH 1 1 17 ( 20 0 6 ) 5 4 –6 0 57

mentally inactivating endogenous BDNF (Mu et al., 1999;

Mizuno et al., 2000) or upon stress-induced impairment of
hippocampal BDNF levels (Smith et al., 1995). Consequently,
it is tempting to speculate that Semax exerts its cognitive
effects by modulating BDNF/trkB signaling in the hippocam-
pus. A direct link between both events remains, however, to
be established. The effect of Semax in the intact brain might
well differ from that in the injured brain, and a significance
of the effects presented here for the neuroprotective
activities of Semax is unclear. It should be noted that brain
injury increases the expression of BDNF and trkB in the
hippocampus, and it is suggested that endogenous BDNF can
have a neuroprotective effect after brain insults (Hicks et al.,
1999; Kokaia et al., 1996). Semax-induced increase of
hippocampal BDNF protein levels after 3 h and 24 h was
comparable to the previously reported increase of this
neurotrophin levels at 6 h after the insult in the rat dentate
gyrus, the hippocampal region of the highest ischemia-
induced BDNF protein expression and of the highest
resistance to ischemic damage (Kokaia et al., 1996). Thus
the possible neuroprotective interference of Semax on
ischemia-induced hippocampal BDNF expression needs to
be investigated.

Fig. 3 – Effects of Semax on trkB and BDNF exon III containing

mRNA levels in the rat hippocampus. BDNF exon III
containing mRNA and trkB mRNA levels were determined by
RT-PCR in hippocampi excised from animals 3 h and 24 h,
respectively, after intranasal Semax application (50 μg per kg
body weight). Exon III BDNF and trkB mRNA levels were
determined by RT-PCR using GAPDH and β-actin,
respectively, as an internal standard. (A) Representative
agarose gels showing hippocampal exon III BDNF, trkB,
GAPDH and β-actin cDNA bands. Lanes 1, 1a, 4 and 4a,
controls. Lanes 2, 2a, 5 and 5a, Semax-treated rats. Lanes 3
and 3a, pUC19 DNA/MspI ladder. Lanes 6 and 6a, 100 bp
ladder. (B) Quantitative results of RT-PCR for exon III BDNF
(GAPDH normalization) and trkB (β-actin normalization) in
the rat hippocampus. Open columns, controls. Filled
columns, Semax-treated animals. Data represent the mean ±
SEM (n = 8 rats) from two independent experiments. *p < 0.05
(one-way ANOVA with post hoc Mann–Whitney U tests).

peptide or are indirectly mediated through BDNF (Ferrer et al.,

1998).
In addition to its effects on BDNF/trkB signaling, Semax
facilitated acquisition of active avoidance behavior. Likewise,
other studies revealed that Semax improves the acquisition
of the food-reinforced T-maze task (Ashmarin et al., 1995). It
is well established that BDNF is a potent modulator of short-
term synaptic transmission and synaptic plasticity, e.g.,
long-term potentiation, by activating tyrosine kinase trkB Fig. 4 – Effects of Semax on acquisition of active avoidance
receptors (Tyler et al., 2002). It is further generally accepted behavior in rats. Semax was applied at 50 μg per body weight
that these BDNF-dependent processes are essential for 20 h prior to the first testing and every 24 h thereafter. During
learning and memory formation. For example, contextual the trials, number of CARs and number of ITRs were recorded
learning in rats is associated with a rapid increase in daily. Open circles, controls. Filled circles, Semax-treated
hippocampal BDNF-levels (Hall et al., 2000), whereas learning animals. Data represent the means ± SEM (n = 15 rats).
and memory formation is severely impaired upon experi- *p < 0.05 (two-way ANOVA with Mann–Whitney U test).
58 BR A IN RE S EA RCH 1 1 17 ( 20 0 6 ) 5 4 –60
The observed modulatory effects of Semax on the hippo-
campal BDNF/trkB system additionally point to potential
benefits of Semax in the therapy of certain mental disorders.
In fact, BDNF is known to be involved in the pathology of
depression, a process further considered to be stress-depen-
dent (Duman, 2004).
The heptapeptide Semax was synthesized at the Institute
of Molecular Genetics, Russian Academy of Sciences, Moscow,
Russia. The purity of each batch was usually not less then 99%
as assessed by HPLC analysis. Working solutions were set up
at 10, 50, 500 and 5000 μg of Semax per ml double-distilled
water.
For all experiments, adult male Wistar rats (body weight
250–300 g) were used. Six animals were housed together under
standard conditions at a 12:12 dark:light cycle (light cycle
lasted from 9 am to 9 pm) with food and water available ad
libitum. BDNF protein and mRNA levels were determined after
a single intranasal application of 100 μl of the respective
Semax solution per kg of body weight. For active avoidance
task experiments, Semax was applied 20 h prior to the first
training and thereafter every 24 h. Control animals received an
equivalent volume of distilled water. Due to the known
circadian changes of brain BDNF levels, Semax application
started at 9:30 am and continued for 30 min. All experiments
were approved by the local animal ethical committee of
Biological Department of M.V. Lomonosov Moscow State
University.
At the indicated time points after intranasal application of
Semax, animals were sacrificed by decapitation. For determi-
nation of BDNF protein levels and trkB phosphorylation, brain
tissues were dissected, immediately frozen in liquid nitrogen
and stored at − 196 °C. For BDNF and trkB mRNA level
determination, removed brain tissues were transferred to
RNA stabilization solution, EverFresh SOLID (Clonogene,
Russia) and stored at − 20 °C.
Brain tissue was homogenized in 1 ml of ice-cold (4 °C)
100 mM Tris buffer (400 mM NaCl, 0.4% Triton X-100, 2% “Block
and Sample buffer” (BDNF Emax® ImmunoAssay System;
Promega), 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml
aprotinin, 10 μg/ml leupeptin, 1 mM benzamidin, pH 7.7) per
100 mg of wet tissue. Homogenates were centrifuged at
14,000 × g for 15 min at 4 °C and supernatants were stored at
−80 °C. BDNF levels contained in the supernatants were
determined using the BDNF Emax® ImmunoAssay System
(Promega) according to the manufacturer's instructions. Total
protein concentrations of samples were determined by a
modified method of Lowry (Peterson, 1977).
Table 1 – RT-PCR primers and predicted product length
Target mRNA Primer sequence
(forward/reverse)
BDNF exon III 5′-CTCCGCCATGCAATTTCCACT-3′
5′-GCCTTCATGCAACCGAAGTA-3′
trkB 5′-CACCAACCATCACATTTCTC-3′
5′-ATCTGTCTCTCGTCCTTCCC-3′
β-actin 5′-CTACAATGAGCTGCGTGTGGC-3′
5'-CAGGTCCAGACGCAGGATGGC-3′
GAPDH 5′-TCCATGACAACTTTGGCATTGTGG-3′
5′-GTTGCTGTTGAAGTCGCAGGAGAC-3′
Hippocampi were lysed at 4 °C in 0.5 ml of lysis buffer
(20 mM Tris, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1%
NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethyl-
sulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin,
1 mM benzamidin, pH 7.4) per 100 mg of wet tissue. At
lysis, the lysates were spun at 14,000 × g for 30 min, and the
supernatants were collected. Total protein concentrations of
samples were determined by a modified method of Lowry
(Peterson, 1977), and samples were equalized for protein
concentration (2 mg/ml). Equal amounts of lysates (200 μg
of total protein per lane) were incubated for 2 h at 4 °C
with anti-phosphotyrosine, clone 4G10, antibodies (Upstate
Biotechnology, USA) and then incubated with pre-washed
with lysis buffer protein-G-sepharose beades (Imtek, Russia)
overnight at 4 °C. The beads were washed four times with
ice-cold lysis buffer, boiled for 5 min in Laemmli sample
buffer and then subjected to SDS-PAGE. Proteins were
separated on 8% acrylamide gels and electrotransferred to
Hybond-P PVDF membranes (Amersham). Blots were
blocked in TBS containing 3% nonfat milk and incubated
with anti-trkB (N-20) polyclonal antibodies (Santa Cruz
Biotechnology, Inc.) in the same solution overnight at
4 °C. For detection, an ECL chemiluminiscence system
(Amersham) was used with HRP-conjugated secondary
antibodies (Imtek, Russia). Optical densities of the bands
corresponding to 145 kDa proteins were determined using
BioDocAnalyze system (Biometra) and taken as a measure
of protein contents.
Total hippocampal mRNA was isolated using the PeqGold
isolation kit RNAPure (Peqlab, Germany) according to the
manufacturer's instructions. Total RNA concentration was
measured by spectrophotometric absorbance at 260 nm. A
total of 1 μg of RNA was reverse-transcribed in a 20 μl using
200 units of Moloney murine leukemia virus reverse tran-
scriptase (Invitrogen) and 0.5 μg of random hexamer primers
(ThermoHybaid, Germany). Obtained templates were ampli-
fied in a final volume of 25 μl using 1 μl of the RT-reaction
mixture, 10 pmol of each (forward and reverse) primers for
target mRNA or 5 pmol of each primers for reference genes.
Primer sequences and predicted product lengths are shown in
Table 1. In preliminary experiments, the number of PCR cycles
and annealing temperatures were tested to find out a linear
working range for all PCR products. The amplification protocol
consisted of a first round at 95 °C for 5 min and then 24 (β-
actin), 26 (GAPDH), 32 (BDNF exon III) or 26 (trkB) cycles of
denaturation for 1 min at 95 °C, annealing for 1 min at 68 °C
and extension for 1 min at 72 °C. The PCR products were
Product length, bp Reference
276 Tabuchi et al., 2002
265 Lindqvist et al., 2002
271 Figiel and Engele, 2000
376 Tabuchi et al., 2002
 BR A IN RE S E A RCH 1 1 17 ( 20 0 6 ) 5 4 –6 0
visualized by ethidium bromide staining under UV light after
electrophoresis separation on a 1.5% agarose gel. Optical
densities of the bands were determined by fluorescent image
scanning using AlphaImager2000 (Alpha Innotech Corpora-
tion) and BioDocAnalyze (Biometra). The ratios of optical
densities for BDNF exon III, trkB and β-actin or GAPDH as a
reference were calculated and taken as a measure of the
respective target mRNA.
Active avoidance conditioning was performed in a one-
compartment cage of 30 × 22 × 35 cm with a shelf fixed 20 cm
above the cage floor. The cage was covered with a transpa-
rent lid. A 3 s lasting acoustic stimulus was used as
conditioned stimulus (CS). After a pause of 2 s, the uncondi-
tioned stimulus (US) was given. This US consisted in an
electrical stimulus (0.5 mA) applied to the floor bars of the
cage. The maximum duration of the footshock was limited to
30 s to prevent injury of animal's paws during multiple
measurements. During four consecutive days, 10 conditional
trials were performed per animal and day. The inter-trial
interval varied from 15 to 30 s. During the trials, number of
conditioned avoidance responses (CARs, jumps onto the shelf
in response to CS) and number of goal-directed inter-trials
responses (ITRs, jumps onto the shelf at inter-trial interval)
were recorded daily.
Acknowledgments
This study was partially supported by the Russian Foundation
for Basic Research (Grants 02-04-08048, 03-04-48582, 05-04-
49187, 06-04-49646), INTAS Young Scientist Fellowship YSF
2002-0336, Ministry for Science and Technology of the Russian
Federation (the grant for international cooperation with
Germany) and the grant of the Program of the Russian
Academy of Sciences “Molecular and cellular biology”.
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