Okajimas Folia Anat. Jpn.

, 83(1): 1–6, May, 2006

Effects of Chronic Ethanol Administration on the Expression

Levels of Neurotrophic Factors in the Rat Hippocampus
By

Hanayo OKAMOTO1, Takanori MIKI2, Kyoung-Youl LEE2, Toshifumi

YOKOYAMA2, Hiromi KUMA1, He GU2, Hong-Peng LI2, Yoshiki
MATSUMOTO2, Ippei YAMAOKA2, Kazutoshi FUSUMADA2, Tomohiro
IMAGAWA3, Zhi-Yu WANG2, Yu NAKAMURA1 and Yoshiki TAKEUCHI2

1Department of Neuropsychiatry, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan

2Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan
3Department of Veterinary Anatomy, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan

– Received for Publication, December 21, 2005 –

Key Words: Ethanol, Hippocampus, BDNF, GDNF, Real-time PCR

Summary: Chronic ethanol consumption has adverse effects on the central nervous system. Hippocampus is one of the
target sites of ethanol neurotoxicity. Hippocampal damage is known to result in impairment of learning and memory.
This study was aimed to determine whether chronic ethanol consumption could alter the expression levels of brain-
derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) mRNAs in the hippocampus. Male
Wistar rats were given unrestricted access to a liquid diet containing 5% (v/v) ethanol as the sole fluid source for 19
weeks beginning at 10 weeks of age. The expression levels of BDNF and GDNF mRNAs in the hippocampus were
analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. The present study revealed
that chronic ethanol consumption did not result in significant changes in the expression levels of BDNF and GDNF
mRNAs. Our present results showed no significant alteration in the expression of these neurotrophic factors; these re-
sults will lead to further studies to examine the possible alterations in the gene expression of various neurotrophins that
are related to hippocampal functions including learning and memory.

Chronic ethanol ingestion has adverse effects on
the central nervous system (CNS). In fact, numer-
ous studies on laboratory animals and humans have
demonstrated that long-term ethanol consumption
can result in impairment of cognition, learning, and
memory1–3). It is now well known that even un-
complicated alcoholics without specific neurological
or hepatic problems exhibit signs of regional brain
damage and cognitive dysfunction. The hippo-
campus, which is known as one of the target sites
for neurotoxic effects, is more sensitive than other
brain regions and plays an important role in learn-
ing and memory processing.
We have recently reported that in mice that
showed intoxication signs following ethanol treat-
ment for several days, the neurons and astrocytes in
the hippocampus showed altered immunoreactiv-
ities to calbindin-D28k and glial fibrillary acidic
protein (GFAP), leading to altered neuron-glial in-
teractions4). This has led to the speculation that the
altered immunoreactivities in the hippocampus are
associated with various neurological symptoms seen
in alcoholic patients, such as cognitive dysfunction
and disabilities in orientation or learning. We have
now extended this study to evaluate these alter-
ations in the hippocampus with respect to changes
in the expression levels of mRNAs of various
neurotrophic factors.
Brain-derived neurotrophic factor (BDNF) and
glial-derived neurotrophic factor (GDNF) are
members of the neurotrophin family of neuro-
trophic factors that are abundantly and widely dis-

* Corresponding author: Takanori Miki, Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, 1750-1
Ikenobe, Miki-cho, Kagawa 761-0793, Japan. E-mail address: mikit@med.kagawa-u.ac.jp

1
2 H. Okamoto et al.
tributed in the hippocampus5,6). BDNF is involved
in the survival of neurons and maintenance of neu-
ronal functions and participates in neuroprotection
against various insults. GDNF is a member of the
transforming growth factor superfamily and is dis-
tributed throughout multiple brain regions; it also
has neuroprotective functions7–9).
Various insults such as alcoholic, epileptic, is-
chemic, and traumatic insults can induce marked
changes in the level of gene expression of neuro-
trophins, ultimately leading to functional alter-
ations in the CNS10). It is of interest to focus upon
BDNF and GDNF separately since they have been
known to possess different signal transduction sys-
tems7). The purpose of the present study was to as-
certain whether chronic ethanol administration may
alter the level of gene expression of the two differ-
ent neurotrophic factors, i.e., BDNF and GDNF.
Materials and Methods
Male Wistar rats obtained from CLEA Japan
(Tokyo, Japan) were used in the present study.
The rats were divided into two groups, ethanol-
fed and pair-fed control. The experimental rats
(n ¼ 6) were given unrestricted access to a liquid
diet (Oriental yeast, Tokyo, Japan) containing 5%
(v/v) ethanol (99.5%; Wako, Osaka, Japan) as the
sole fluid source for 19 weeks (beginning at 10
weeks of age). The pair-fed control rats (n ¼ 6)
were fed an identical liquid diet, except that sucrose
was substituted isocalorically for ethanol. This
study was conducted in compliance with the guide-
lines for experimental use and care of laboratory
animals established by the Kagawa University Ani-
mal Ethics Committee.
At 29 weeks of age, the rats in both groups were
anesthetized with sodium pentobarbital (60 mg/kg,
i.p.) and perfused intracardially with medical-grade
physiological saline (Otsuka, Tokyo, Japan). Using
a vibratome, the brains were sectioned in the hori-
zontal plane to yield 1-mm-thick slices. The hippo-
campal region was removed in chilled physiological
saline by using a dissecting microscope. Total RNA
was extracted by homogenizing these hippocampal
slices in TRIzol reagent (Invitrogen, Carlsbad,
USA). The concentration and purity of the ex-
tracted RNA were evaluated by optical density
measurements at 260 nm and 280 nm using a spec-
trophotometer. The RNA samples were stored at

80 C until use.
Real-time reverse transcription-polymerase
chain reaction (Real-time RT-PCR) analysis was
performed as described previously11). In brief, re-
verse transcription was carried out at 37 C for
60 min, followed by 65 C for 10 min using Ready-
To-Go You-Prime First-Strand Beads (Amersham
Biosciences, Piscataway, USA). For a 33-mL reac-
tion mixture, the following reagents were used:
1 mL of sample RNA, 29 mL of RNase-free water,
Ready-To-Go You-Prime First-Strand Beads, and
3 mL of oligo dT primer (10 mM). Real-time PCR
was performed using a LightCycler rapid thermal
cycler system (Roche Diagnostics Ltd, Lewes, UK).
The reactions were performed in a 20-mL volume
with 2 mL of the cDNA diluted ten times, 0.5 mM
primers, and reagents included in the LightCycler-
FastStart DNA Master SYBR Green I mix (Roche
Diagnostics GmbH, Mannheim, Germany). The
amplification protocol consisted of one cycle at
95 C for 10 min, followed by 30 cycles at 95 C for
10 s, 65 C for 10 s, 72 C for 20 s, and 87 C for 2 s.
Detection of the fluorescent products was carried
out at the end of the 87 C extension period. Re-
garding GDNF, the temperature for annealing
was set at 58 C. To assess an appropriate inter-
nal control, coamplification of the housekeeping
gene glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was performed in each sample. The fol-
lowing forward (F) and reverse (R) primers were
used in the present study: BDNF (gene accession
number, X67108), F: GAT GAG GAC CAG AAG
GTT CG, R: GAT TGG GTA GTT CGG CAT
TG; GDNF (NM019139), F: CCC GAA GAT TAT
CCT GAC CA, R: TAG CCC AAA CCC AAG
TCA GT; GAPDH (AB017801), F: GTA TTG
GGC GCC TGG TCA CC, R: CGC TCC TGG
AAG ATG GTG ATG G. The above series of real-
time PCR procedure was performed three times.
Melting curve analysis and electrophoresis were
performed to confirm amplification specificity and
exclude genomic contamination, respectively, as
described previously11). For the melting curve
analysis, the PCR products were melted by gradu-
ally increasing the temperature beginning at 65 C
in 0.2 C steps. Electrophoresis of the PCR products
amplified from cDNA using BDNF, GDNF, and
GAPDH primers was carried out on a 2% agarose
gel, and the gel was then stained with ethidium
bromide. A similar electrophoresis of the amplifi-
cation products without RT was also performed for
each sample as a negative control.
Statistical analysis of the body and brain weights
was performed using Mann-Whitney U test or Stu-
dent’s t-test. The quantification data for mRNA
was analyzed using the LightCycler analysis soft-
ware. Data on the mRNA of BDNF and GDNF
were expressed as the ratio of the expression levels
of BDNF or GDNF mRNA to that of GAPDH
mRNA. Statistical analysis with Mann-Whitney
U test or Student’s t-test was performed by using
 Chronic Ethanol Administration and Neurotrophic Factors 3

Table 1. Data on the body weights (g) of chronic ethanol-fed SigmaStat software (Systat Software, Point Rich-
and pair-fed control rats at 10 and 29 weeks of age mond, USA). When the data was not normally dis-
tributed and the variances between groups were not
Group n 10 weeks of age 29 weeks of age
homogeneous, the square root transformation of
Chronic ethanol-fed 6 295.0 G 1.8 393.3 G 20.9* the data was performed prior to statistical analysis.
Pair-fed control 6 294.2 G 2.0 527.5 G 3.6

Each value is represented as mean G SEM.

Results
n: number of animals examined.
The body weight data were analyzed using Mann-Whitney U test
because the variances were not homogeneous. The mean G SEM values of the body weights of
* p < 0.01 chronic ethanol-fed and pair-fed control rats are
shown in Table 1. At the beginning of ethanol
treatment (10 weeks of age), no significant differ-
ence in body weight was observed between the two
Table 2. Data on the brain weights (g) of chronic ethanol-fed groups; however, at the end of 19 weeks of ethanol
and pair-fed control rats at 29 weeks of age
treatment, a significant difference was observed.
Group n Brain weights (g) Table 2 shows the mean G SEM values of the brain
weights of chronic ethanol-fed and pair-fed control
Chronic ethanol-fed 6 2.05 G 0.01* rats at the end of ethanol treatment (29 weeks of
Pair-fed control 6 2.23 G 0.02 age); statistical analysis using Mann-Whitney U test
Each value is represented as mean G SEM.
revealed a significant difference between their brain
n: number of animals examined. weights.
* p < 0.01 Figure 1 shows a representative electrophoresis

Fig. 1. A representative agarose gel electrophoresis of the PCR products amplified from cDNA using BDNF, GDNF, and GAPDH
primers; the electrophoresis shows a single band. No band was observed when a similar amplification procedure was per-
formed without RT (data not shown). A similar electrophoresis of the PCR products was carried out for each sample in order
to exclude genomic contamination. Molecular weight markers are shown in the left lane.
4 H. Okamoto et al.
Table 3. Data on the ratio (10 2 ) of the expression levels of
BDNF and GDNF mRNAs to that of GAPDH
mRNA in chronic ethanol-fed and age-matched pair-
fed control rats
Group n BDNF GDNF
Chronic ethanol-fed 6 33:1 G 0:7 138:6 G 4:8
Pair-fed control 6 36:5 G 0:8 212:8 G 10:2
Each value is represented as mean G SEM.
n: number of animals examined, BDNF: brain-derived neuro-
trophic factor, GDNF: glial-derived neurotrophic factor.
of the PCR products on 2% agarose gel; a single
band confirmed that specific amplification had been
accomplished. When RT was excluded, the amplifi-
cation products were not detected. The above se-
ries of procedures was performed for each sample.
Additionally, the melting curves of the PCR prod-
ucts amplified using BDNF, GDNF, and GAPDH
primer pairs showed a single and sharp transition.
This confirms that a single PCR product was pres-
ent, and primer dimer formation was a rare occur-
rence within the number of cycles required for
quantification. These results provide a strong con-
firmation that the primers used in this study amplify
the target genes BDNF, GDNF, and GAPDH and
exclude genomic DNA contamination.
Table 3 shows the mean G SEM ratio (10 2 ) of
the expression levels of BDNF and GDNF mRNAs
to that of GAPDH mRNA in chronic ethanol-fed
and pair-fed control rats. No significant differences
in both BDNF and GDNF mRNA levels were ob-
served between chronic ethanol-fed and pair-fed
control rats at the end of ethanol treatment.
Discussion
Chronic ethanol ingestion is known to cause
brain damage and cognitive impairment in humans
and laboratory animals12–17). Since various neuro-
trophic factors are implicated in normal develop-
ment, functional maintenance, plasticity, and re-
covery from neuronal damage, these functional
disorders in the hippocampus are closely related
to alterations in the expression levels of various
neurotrophins, including BDNF and GDNF. How-
ever, our results demonstrated that chronic ethanol
treatment for 19 weeks resulted in no significant
alterations in the expression levels of BDNF and
GDNF mRNAs in the rat hippocampus.
Despite numerous studies on the effects of
chronic ethanol treatment on various neurotrophic
factors in the CNS, consistent results have not been
obtained. The differences in results across labo-
ratories may arise due to variations in experimental
designs such as the length of ethanol treatment,
ethanol concentration in the diet, sampling time
points, and rat strains. For example, one study
showed that the hippocampal BDNF content ini-
tially increased after 6–12 weeks of ethanol admin-
istration and subsequently decreased after 24 weeks
of ethanol administration as a liquid diet containing
8.1%–9.4% (v/v) ethanol18,19). However, in an-
other study, 6.6% (w/v) ethanol administered as a
liquid diet for 6 weeks resulted in no alterations
in the hippocampal BDNF and Trk B mRNA
levels20). Similarly, no change in neurotrophin pro-
tein and mRNA levels in the rat hippocampus was
observed after ethanol treatment for 28 weeks21).
The precise reasons for these discrepancies are un-
clear but may arise due to significant variations in
the ethanol treatment and the sampling times used
in the various studies. One of the possible ex-
planations for this discrepancy is the differences in
the rat strains. For example, the Long-Evans strain
of rats used by the MacLennan’ group18) may be
more sensitive to ethanol in terms of hippocampal
neurotrophin responsiveness than the Sprague-
Dawley rats20).
It has been reported that GDNF protects neu-
rons against ethanol-induced neuronal damage22).
We hypothesized that chronic ethanol treatment
may increase the expression levels of glial cell-
associated proteins, including GDNF, since glial
cells are activated by various extrinsic insults. Con-
trary to our hypothesis, the present study demon-
strated that chronic ethanol administration for 19
weeks did not result in a significant change in the
expression level of GDNF mRNA when compared
with that in controls. Recently, it has been reported
that ethanol inhibits GDNF protein release mainly
from astrocytes but does not affect the mRNA
expression of this protin23). Our present data on
GDNF appear to be consistent with this report.
Since GDNF is mainly released from astrocytes
in the CNS23,24), it is likely that astrocytes are less
influenced by chronic ethanol exposure. Further
studies are required to understand how GDNF
might act against the neurotoxic effects of ethanol.
The alterations in the expression levels of BDNF
and GDNF mRNAs after ethanol administration
may be explained by several potential mechanisms.
One possible explanation is that GDNF and BDNF
act in concert through common intracellular targets
such as the Ras/Erk pathway and together activate
the survival response to the neurotoxic effects of

ethanol25–29). Our data showing unaltered expres-
sion levels of BDNF and GDNF mRNAs appear
to be consistent with these reports. However, it is
equally likely that BDNF and GDNF act indepen-
dently30). The precise reason for this discrepancy is
unclear at present. Further studies are required to
clarify this discrepancy.
Neuropathological changes in human alcoholic
patients were reviewed in detail by Harper31). The
brain weight of alcoholics is generally lower than
that of controls, and the degree of brain atrophy
correlates with the rate and amount of alcohol
consumption over the lifetime. The reduction in
brain weight and volume is strongly correlated with
a reduction in white matter volume in the pre-
frontal cortex32). The mechanism for the ethanol-
induced loss of white matter volume remains un-
known at present; however, it is strongly speculated
that both myelination and axonal integrity are in-
volved in this phenomenon to a certain extent.
Demyelination is one of the neuropathological
changes observed in human alcoholics31). Myelin-
associated proteins such as oligodendrocyte myelin
glycoprotein (OMgp), myelin-associated glyco-
protein (MAG), and Nogo are categorized as
myelin-associated inhibitors and belong to the
Nogo receptor (NgR) ligand family. These proteins
are expressed by oligodendrocytes and ultimately
lead to degeneration following neuronal dam-
age33–37). We have recently found that ethanol ad-
ministration to rats for 19 weeks resulted in a sig-
nificant decrease in the expression levels of OMgp
mRNA in the hippocampus (unpublished data). It
is speculated that the decreased expression level of
OMgp mRNA is attributed to the damage to the
oligodendrocytes/myelin sheath induced by chronic
ethanol consumption. Our data on OMgp mRNA
appear to be consistent with the neuropathological
changes in alcoholics, which are characterized by
demyelination.
In summary, chronic ethanol administration for
19 weeks did not significantly affect the expression
levels of BDNF and GDNF mRNAs in the rat hip-
pocampus. However, the functional implications of
alterations in the expression levels of these mRNAs
are uncertain at present. It is speculated that vari-
ous neurological symptoms seen in human alco-
holics are closely related to alterations in the ex-
pression levels of these important neurotrophic
factors. Further experiments that include parame-
ters such as different periods of ethanol adminis-
tration, multiple sampling points, and various etha-
nol concentrations in the diet are required to clarify
the effects of chronic ethanol administration on al-
terations in the levels of gene expression of neuro-
trophins.
Chronic Ethanol Administration and Neurotrophic Factors 5
Acknowledgements
This work was partly supported by a Grant-in-
Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technol-
ogy of Japan (B2, 16390310).
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