Vol.2, No.3, 225-233 (2010)
doi:10.4236/health.2010.23032
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Shifts in the balance of brain tryptophan metabolism due
to age and systemic administration of lipopolysaccharide
Hideki Miura1, Tetsuya Shirokawa2, Norio Ozaki1, Kenichi Isobe3
1Department of Psychiatry, Nagoya University Graduate School of Medicine, Nagoya, Japan;
2Department of Information Technology for Human Welfare, Nihon Fukushi University, Mihama, Japan
3Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Received 9 November 2009; revised 22 December 2009; accepted 24 December 2009.
ABSTRACT
The kynurenine (KYN) pathway, which is initi-
ated by indoleamine 2, 3-dioxygenase (IDO), is a
key tryptophan (TRP) metabolic pathway. It
shares TRP with the serotonin (5-HT) pathway.
Because activation of the KYN pathway by pro-
inflammatory cytokines induces depressive
symptoms, shifts in the balance of TRP metabo-
lism to the KYN pathway are closely related to
the etiology of depression. In the present study,
the influence of age on the effect of the inflam-
mation response system (IRS) on brain TRP
metabolism was investigated. Male ICR mice
(PND21) were reared for 4 weeks (younger
group) or until they reached 1 year of age (older
group), and given an intraperitoneal (i.p.) injec-
tion of lipopolysaccharide (LPS). The TRP, KYN,
and 5-HT levels were measured in the prefrontal
cortex, hippocampus, amygdala, and dorsal
raphe nuclei. An increase in TRP and 5-HT levels
was observed with age in all brain regions,
whereas age was associated with decreases in
KYN levels in the dorsal raphe nuclei. In all brain
regions, LPS increased TRP levels, while it in-
creased KYN levels in the prefrontal cortex and
amygdala. Reduced KYN/5-HT ratios in all re-
gions were observed with age, whereas increas-
ed KYN/5-HT ratios were observed with LPS in
all regions except the dorsal raphe nuclei. Thus,
age shifted the balance between the KYN and
5-HT pathways toward the 5-HT pathway, and
countered the effects of LPS, which shifted the
balance to the KYN pathway. These effects are
relevant to the etiology of psychiatric disorders
in elderly people.
Keywords: Tryptophan; Kynurenine; Serotonin;
Age; Lipopolysaccharide
1. INTRODUCTION
Changes in tryptophan (TRP) metabolism play an im-
portant role in the brain-endocrine-immune system in-
teraction that is hypothesized to be involved in the eti-
ology and/or pathophysiology of major depression. Two
main pathways metabolize TRP. One is the kynurenine
(KYN) pathway, which is initiated by indoleamine 2,
3-dioxygenase (IDO). The other is the serotonin (5-HT)
pathway, which is initiated by the enzyme tryptophan
hydroxylase (TPH).
An induction of IDO by inflammation may occur in
depression [1]. Studies of depression induced by cytokine
therapy have indicated that the severity of depressive
symptoms is correlated with decreases in serum TRP
and/or increases in KYN [2-4]. Immunological chal-
lenges such as exposure to proinflammatory cytokines
(IL-1, IFN-, IFN-, and TNF-induce IDO activity
[5-10], which metabolizes TRP to KYN, deprives TPH of
its substrate, and may result in 5-HT depletion [11]. Be-
cause IDO can directly metabolize 5-HT [12], activated
IDO may also decrease 5-HT levels [13]. Such 5-HT
depletion is included in the monoamine hypothesis, a
major etiological hypothesis for depression that proposes
the existence of a relationship between decreases in
monoamines in the brain and the onset and symptoms of
depression [14-15].
According to the monocyte-T-lymphocyte hypothesis
[16-17], IL-1 released from activated macrophages di-
rectly stimulates corticotropin-releasing hormone (CRH)
release from the paraventricular nucleus (PVN) in the
hypothalamus. Thus, the HPA-axis hyperactivity hy-
pothesis, which proposes a relationship between HPA-
axis hyperactivity and depression [18], closely relates to
changes elicited by macrophage hyperactivity.
As noted above, changes in immunological activity
have been incorporated into two main hypotheses about
the etiology and pathophysiology of major depression. A
shift in the balance between the KYN and 5-HT pathways
to the KYN pathway may occur in the brain of patients
with major depression [19]. However, patients ordinarily

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226
become depressed in response to adverse life events
and/or the loss of social support (i.e., psychological
and/or environmental factors), especially when such
events or losses are encountered at an advanced age
[20-21]. In order to investigate the shift in the balance in
the TRP metabolism to the KYN pathway, the influence
of such factors on TRP metabolism was investigated [22].
Specifically, a previously designed animal model [23-28]
was applied to assess the influence of the following three
risk factors on TRP metabolism: age, social isolation, and
activation of the inflammation response system (IRS).
The results showed that older age and social isolation
shifted the balance between the KYN and 5-HT pathways
to the 5-HT pathway, whereas novelty stress shifted the
balance to the KYN pathway [22]. Further study revealed
that immunological challenges such as intraperitoneal
(i.p.) injection of lipopolysaccharide (LPS) shifted the
balance to the KYN pathway [29]. These studies may
confirm that psychological stressors, as well as immu-
nological challenges, can shift the balance between the
KYN and 5-HT pathways toward the KYN pathway, as
expected. Although interactions between social isolation
and systemic LPS injection on TRP metabolism in the
brain have been observed [29], the influence of age on
changes elicited by systemic LPS injection remains to be
examined.
The aim of the present study was to clarify the influ-
ence of age on a shift in the balance between the KYN
and 5-HT pathways elicited by i.p. injection of LPS. For
this series, four brain regions were selected, the first three
of which possess 5-HT nerve terminals: the prefrontal
cortex, because it relates to behavioral motivation; the
amygdala, because it relates to emotion; the hippocampus,
because it regulates the HPA axis, and hyperactivity of
this axis is closely related to the etiology and patho-
physiology of depression; and the dorsal raphe nuclei,
because they contain the cell bodies of 5-HT neurons and
are the center of brain 5-HT synthesis.
2. METHODOLOGY
2.1. Animals
A total of 32 male-specific, pathogen-free (SPF) ICR
mice were used in the present experiments. At 21 post-
natal days (PND), the mice were housed in groups of
4–5 per cage, and they were reared for 4 weeks (younger
group) or until they became 1 year old (older group). On
the final day (Day 28 for the younger group, and 1 year
old for the older group), the animals were further sepa-
rated into two groups and were then given a 0 mg/kg or
1 mg/kg intraperitoneal (i.p.) injection of LPS diluted
with saline. Thus, the mice were divided into 4 groups as
follows: younger, LPS 0 mg/kg (n=8); younger, LPS 1
mg/kg (n=8); older, LPS 0 mg/kg (n=8); older, LPS 1
mg/kg (n=8).
Mice in the older group with apparent wounds were
excluded from the study, because any such wound would
itself likely have influenced the immune response, and in
turn TRP metabolism. Although this selection process
excluded apparently subordinate mice from the study, it
was more important to avoid the potential influence of
the immune responses associated with wounds on TRP
metabolism.
The cages used in this series measured 21 x 31 x 13
cm. Cage exchange was performed 2 times per week.
Food and water were provided ad libitum. The animals
were kept on a 12-h light/dark cycle (lights on at 09.00 h,
off at 21.00 h), and room temperature was maintained at
21–23˚C. All efforts were made to minimize both the
number of animals used and the degree of their suffering.
All experiments were conducted in accordance with the
European Communities Council Directive of November
24, 1986 (86/609/EEC). The study was approved by the
ethical committee of the Nagoya University Graduate
School of Medicine.
2.2. LPS Injection
For the LPS injection, LPS from E. coli (Sigma, St.
Louis, MO) was diluted with saline (2 mg/10 ml) and
i.p.-injected (0 mg/kg or 1 mg/kg) into the mice (saline
only in the 0 mg/kg group). The time of LPS injection
was between 11.00 h and 14.00 h. The injection volume
was 5 ml/kg. After having received the LPS injection,
the mice were returned to their rearing cages until brain
sample preparation.
2.3. Sample Preparation
Four brain regions (prefrontal cortex, hippocampus,
amygdala, and dorsal raphe nuclei) were dissected from
the whole brain.
The mice were sacrificed by decapitation 4 h after the
i.p.-injection of LPS; mice were decapitated under brief
ether anesthesia. The brains were removed, and four
brain regions were dissected out as quickly as possible
on glass plates over ice [22]. The samples were weighed
and treated with 1000 µl of an ice-cold 0.2 M tri-
chloroacetic acid (TCA) solution containing 0.2 mM
sodium pyrosulfite, 0.01% EDTA-2Na, and 0.5 µM iso-
proterenol (ISO) and 3-nitro-L-tyrosine (3-NTYR) as an
internal standard per 100 mg of wet tissue. The solution
was sonicated and then centrifuged at 10,000g for 20
min at 4˚C. The supernatant was filtered through a Mil-
lipore HV filter (0.45 µm pore size) and then subjected
to both high-performance liquid chromatography (HPLC)
with electrochemical detection (ECD) of 5-HT, and
HPLC with fluorimetric detection (FD) of TRP as well
as ultraviolet (UV) detection of KYN.
The standard solution was prepared using the above-
mentioned ice-cold 0.2 M TCA solution containing
0.5-µM internal standards (ISO, 3-NTYR), and the solu-

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227
tion concentrations were adjusted to 0.5 µM for 5-HT
and KYN, and to 10 µM for TRP.
2.4. HPLC Determination of 5-HT Levels in
the Brain
The levels of 5-HT in the brain extracts were measured
by HPLC with ECD. The HPLC-ECD system employed
here consisted of a CMA/200 autosampler (CMA/Mic-
rodialysis AB, Stockholm, Sweden), a micro LC pump
(BAS, West Lafayette, IN), an LC-4C ECD (BAS), a
Bio-Phase ODS-4 51-6034 column (4.0x110 mm; BAS),
a CR-6A recorder (Shimadzu, Kyoto, Japan), an LC-26A
vacuum degasser (BAS), and a CTO-10A column heater
set at 35˚C (Shimadzu). The mobile-phase solution con-
sisted of 0.1 M tartaric acid-0.1 M sodium acetate buffer
(pH 3.2), containing 0.5 mM EDTA-2Na, 555 µM so-
dium 1-octane sulfonate, and 5% acetonitrile. The flow
rate was 700 µl/min. The concentration of each com-
pound was calculated by comparison with both internal
(ISO) and external standards. The sensitivity of the 5-HT
measurements was 150 f mol.
2.5. HPLC Determination of Brain Levels of
TRP and KYN
The levels of TRP and KYN were measured according to
the methods of Widner and colleagues [30-31]. The
HPLC pump was an LC-10AD (Shimadzu). For separa-
tion, reversed-phase column LiChroCART 55-4 car-
tridges filled with Purospher STAR Rp-18e (55-mm
length, 3-µm grain size) together with a reverse-phase
LiChroCART 4–4 precolumn filled with Purospher
STAR RP-18e (5-µm grain size; Merck, Rahway, NJ)
were used. In this series, TRP was detected by RF-535
FD (Shimadzu) at an excitation wavelength of 285 nm
and an emission wavelength of 365 nm. KYN and
3-NTYR were detected by a SPD-10A UV-detector
(Shimadzu) at a wavelength of 360 nm. Both detectors
were connected in a series to enable simultaneous meas-
urement. The mobile-phase solution consisted of a
15-mM L-acetic acid-sodium acetate buffer, pH 4.0,
containing 2.7% acetonitrile. The flow rate was 900
µL/min at room temperature. The sensitivities of the
TRP and KYN measurements were, respectively, 50 f
mol and 100 f mol.
2.6. Statistical Analyses
To examine differences in the levels of TRP, 5-HT, and
KYN, and in the ratios of KYN/TRP, 5-HT/TRP, and
KYN/5-HT, two-way MANOVA (Wilks’s lambda) for
age and LPS was conducted on dependent measures in
each brain region, followed by the Tukey-Kramer test.
Data are presented as the means ± S.E.M. All P values of
less than 0.05 were accepted as significant.
3. RESULTS
3.1. Prefrontal Cortex
The results of the two-way MANOVA for age and LPS
were as follows: age (F (6, 23) = 6.917; P = 0.0003) and
LPS (F (6, 23) = 11.020; P < 0.0001) significantly al-
tered the dependent measures. The interaction between
age and LPS (F (6, 23) = 3.810; P = 0.0088) was sig-
nificant. The results of the post-hoc test are shown in
Figure 1(a) and Table 1(a). Age significantly decrea-
sed the KYN/5-HT ratio (p < 0.01, Table 1(a)), whereas
it did not alter that of KYN/TRP (Table 1(a)). Thus, age
shifted the balance between KYN and 5-HT pathways
toward the 5-HT pathway. Whereas LPS significantly
increased the KYN/5-HT ratio (p < 0.01, Table1(a)), it
did not alter the KYN/TRP ratio (Table 1(a)). Thus, LPS
shifted the balance between the KYN and 5-HT path-
ways toward the KYN pathway.
3.2. Hippocampus
The results of the two-way MANOVA for age and LPS
were as follows: age (F (6, 23) = 10.114; P < 0.0001)
and LPS (F (6, 23) = 10.393; P < 0.0001) significantly
altered the dependent measures. The interaction between
age and LPS (F (6, 23) = 3.676; P = 0.0105) was sig-
nificant. The results of the post-hoc test are shown in
Figure 1(b) and Table 1(b). Age significantly decreased
the ratios of KYN/TRP (p < 0.01, Table 1(b)) and
KYN/5-HT (p < 0.01, Table 1(b). Thus, age shifted the
balance between the KYN and 5-HT pathways to the
5-HT pathway. Although LPS significantly decreased the
KYN/TRP ratio (p < 0.05, Table 1(b)), it increased the
KYN/5-HT ratio (p < 0.01, Table 1(b)). Thus, LPS
shifted the balance between KYN and 5-HT pathways
toward the KYN pathway.
3.3. Amygdala
The results of the two-way MANOVA for age and LPS
were as follows: age (F (6, 23) = 13.776; P < 0.0001)
and LPS (F (6, 23) = 14.523; P < 0.0001) significantly
altered the dependent measures. The interaction between
age and LPS (F (6, 23) = 5.904; P = 0.0008) was sig-
nificant. The results of the post-hoc test are shown in the
Figure 1(c) and Table 1(c). Age significantly decreas-
ed the KYN/5-HT ratio (p < 0.01, Table 1(c)), whereas it
did not alter the KYN/TRP ratio (Table 1(c)). Thus, age
shifted the balance between the KYN and 5-HT path-
ways to the 5-HT pathway. Whereas LPS significantly
increased the KYN/5-HT ratio (p < 0.01, Table 1(c)), it
did not alter the KYN/TRP ratio (Table 1(c)). Thus, LPS
shifted the balance between the KYN and 5-HT path-
ays toward the KYN pathway. w

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228
0
30
Younger Older
(n mol/g)
0
0.5
Younger Older
(n mol/g)
0
5
Younger Older
(n mol/g)
TRP KYN 5-HT
+ ++
** **
0
30
Younger Older
(n mol/g)
0
0.5
Younger Older
(n mol/g)
0
5
Younger Older
(n mol/g)
TRP KYN5-HT
++ ++
**
(a) (b)
0
30
Younger Older
(n mol/g)
0
0.5
Younger Older
(n mol/g)
0
5
Younger Older
(n mol/g)
TRP KYN 5-HT
++ ++
**
**
0
30
Younger Older
(n mol/g)
0
0.5
Younger Older
(n mol/g)
0
5
Younger Older
(n mol/g)
TRPKYN5-HT
++ ++
**
(c) (d)
Figure 1. Changes in tryptophan (TRP), kynurenine (KYN), and serotonin (5-HT) levels elicited by age and LPS. Each bar indicates
a group defined by age and LPS (n=8). Younger, younger group; Older, older group. White bar, LPS 0 mg/kg; black bar, LPS 1 mg/kg.
Values are shown as means ± S.E.M. The results of the Tukey-Kramer test for age and LPS are shown. Effects of age are shown: +, p
< 0.05; ++, p < 0.01. Effects of LPS are shown: *, p < 0.05; **, p < 0.01. (a) Prefrontal cortex; (b) hippocampus; (c) amygdala; (d)
dorsal raphe nuclei.
Table 1. Changes in the KYN/TRP, 5-HT/TRP, and KYN/5-HT ratios elicited by age and LPS. Eight animals were used in each group
(as defined by age and LPS). Values are shown as means ± S.E.M. The results of the Tukey-Kramer test for age and LPS are shown.
Effects of age are shown: +, p < 0.05; ++, p < 0.01. Effects of LPS are shown: *, p < 0.05; **, p < 0.01. (a) prefrontal cortex; (b)
hippocampus; (c) amygdala; (d) dorsal raphe nuclei.
(a) Prefrontal cortex
Age LPS KYN/TRP 5-HT/TRP KYN/5-HT
Younger 0 mg/kg 0.016 ± 0.002 0.180 ± 0.014 0.093 ± 0.011
Younger 1 mg/kg 0.012 ± 0.001 0.068 ± 0.010 0.204 ± 0.028
Older 0 mg/kg 0.009 ± 0.001 0.211 ± 0.046 0.054 ± 0.009
Older 1 mg/kg 0.015 ± 0.002 0.150 ± 0.015+, ** 0.106 ± 0.016 ++, **
(b) Hippocampus
Age LPS KYN/TRP 5-HT/TRP KYN/5-HT
Younger 0 mg/kg 0.018 ± 0.002 0.089 ± 0.008 0.213 ± 0.035
Younger 1 mg/kg 0.014 ± 0.001 0.033 ± 0.006 0.500 ± 0.079
Older 0 mg/kg 0.013 ± 0.002 0.144 ± 0.023 0.107 ± 0.018
Older 1 mg/kg 0.009 ± 0.001 ++, * 0.142 ± 0.025 ++ 0.079 ± 0.019 ++, **
(c) Amygdala
Age LPS KYN/TRP 5-HT/TRP KYN/5-HT
Younger 0 mg/kg 0.013 ± 0.001 0.235 ± 0.024 0.060 ± 0.006
Younger 1 mg/kg 0.011 ± 0.001 0.070 ± 0.007 0.175 ± 0.022
Older 0 mg/kg 0.010 ± 0.002 0.195 ± 0.017 0.056 ± 0.012
Older 1 mg/kg 0.010 ± 0.001 0.163 ± 0.011 ** 0.062 ± 0.005 ++, **
(d) Dorsal raphe nuclei
Age LPS KYN/TRP 5-HT/TRP KYN/5-HT
Younger 0 mg/kg 0.021 ± 0.004 0.119 ± 0.023 0.193 ± 0.029
Younger 1 mg/kg 0.016 ± 0.002 0.073 ± 0.007 0.223 ± 0.028
Older 0 mg/kg 0.013 ± 0.002 0.152 ± 0.012 0.133 ± 0.052
Older 1 mg/kg 0.009 ± 0.001 ++ 0.148 ± 0.021 ++ 0.071 ± 0.017 ++

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3.4. Dorsal Raphe Nuclei
The results of the two-way MANOVA for age and LPS
were as follows: age (F (6, 23) = 10.978; P < 0.0001) and
LPS (F (6, 23) = 5.751; P = 0.0009) significantly altered
the dependent measures. The interaction between age and
LPS (F (6, 23) = 1.757; P = 0.1529) was not significant.
The results of the post-hoc test are shown in Figure 1(d)
and Table 1(d). Age significantly decreased the KYN/
TRP (p < 0.01, Table 1(d)) and the KYN/5-HT (p < 0.01,
Table 1(d)) ratios. Thus, age shifted the balance between
the KYN and 5-HT pathways to the 5-HT pathway. More-
over, LPS did not alter the KYN/TRP, 5-HT/TRP, or
KYN/5-HT ratios (Table 1(d)). Thus, LPS did not shift
the balance between the KYN and 5-HT pathways.
4. DISCUSSION
In the present study, the influences of age on the balance
between the KYN and 5-HT pathways of brain TRP me-
tabolism were investigated. This balance has been known
to shift in favor of the KYN pathway in response to a
systemic injection of LPS.
4.1. Effects of Age on Brain TRP Metabolism
Age shifted the balance between the KYN and 5-HT
pathways to the 5-HT pathway, as was expected based on
the results of our previous study [22]. Animal experi-
ments have yielded controversial findings on the effects
of age on KYN pathway activity. One previous study
suggested that age activates the KYN pathway, espe-
cially as regards the synthesis of kynurenic acid (KYNA),
a neuroprotective product of the KYN pathway [32].
However, another study suggested that age reduces IDO
activity in the liver, kidneys, and small intestines of rats
[33]. In a human study, Alzheimer’s disease patients and
age-matched controls exhibited a decrease in plasma
TRP levels and an increase in the KYN/TRP ratio, as
compared to those of a younger control group [34]. Fur-
thermore, the serum KYN/TRP ratio in healthy individu-
als was found to increase with older age [35], and the
serum KYN/TRP ratio in nonagenarians was signifi-
cantly higher than that of controls [36]. As the serum
KYN/TRP ratio has been found to consistently rise with
increasing age, it serves as a marker of IDO activity in
humans. In the present study, no evidence of activation
of the KYN pathway elicited by age was found. Instead,
the KYN/TRP ratio decreased in the hippocampus and
dorsal raphe nuclei, as age was associated with increased
TRP levels, while KYN levels remained unaltered or to
some extent decreased. Apparently, increases in age are
associated with increases in brain 5-HT levels. The pre-
sent findings regarding the abovementioned shifts are
based primarily on an increase in 5-HT levels. However,
previous studies have reported increases [37-38], no
change [39-40], or even decreases [41-42] in 5-HT levels
elicited by age. Thus, the influence of age on brain 5-HT
levels remains controversial. A previous rat-model study
revealed a decrease in the 5-HT turnover ratio elicited by
age [24], and therefore decreased 5-HT turnover is sus-
pected to have been the cause of observed increases in
5-HT levels. Although the effects of age on TRP metabo-
lism were discussed in a previous study, the age of the
mice used (6 months) in that study was not sufficient to
investigate the effects of age [22]. In the present study,
older (1-year-old) mice were used in order to clarify this
point. The results of our previous study regarding the
effects of age on TRP metabolism were confirmed here.
Although the age of the mice in the present study may
still have been insufficient for studying the effects of age,
the older (1-year-old) mice did exhibit more of a shift in
the balance between the KYN and 5-HT pathways to the
5-HT pathway, as well as more of a decrease in the
KYN/TRP ratio in the hippocampus and dorsal raphe
nuclei, and more of an increase in the 5-HT/TRP ratio in
all regions (except for the amygdala), than the shift ob-
served in young adult (6-month-old) mice (i.e., increased
5-HT/TRP ratio in the prefrontal cortex and hippocam-
pus) [22].
4.2. Effects of Systemic LPS Administration
on Brain TRP Metabolism
A shift in the balance between the KYN and 5-HT path-
ways to the KYN pathway after LPS exposure was ob-
served, as LPS increased brain TRP and KYN levels.
These results confirmed previously reported data regard-
ing the influence of LPS on TRP metabolism [29]. The
putative mechanisms of these changes elicited by LPS
are as follows.
The elevation in TRP is associated with the following
mechanisms. One mechanism regulating brain TRP lev-
els is the free fatty acid (FFA) levels induced by the
sympathetic nerve system. The activated sympathetic
nerve increases plasma FFA, which binds to albumin.
The albumin bound to FFA reduces its affinity for TRP
[43], resulting in an increase in free plasma TRP levels
[44-45]. The other mechanism regulating brain TRP lev-
els is the competition between TRP and the so-called
“large neutral” amino acids (LNAA), which include
aromatic amino acids and branched-chain amino acids
(BCAA), at uptake sites at the blood-brain barrier (BBB).
Elevation of the plasma TRP/LNAA ratio regulates TRP
uptake activity at the BBB [45-47]. Thus, LPS may in-
crease plasma FFA, which would in turn bind to albumin
and reduce its affinity for TRP, resulting in an increase in
free plasma TRP. Due to the increase in the TRP/LNAA
ratio at BBB amino acid-uptake sites, LPS may lead to
increase brain TRP levels [19].
In the present study, systemic LPS administration ac-

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230
tually increased brain KYN levels. Systemic LPS ad-
ministration is known to increase both brain [48] and
peripheral [49] IDO activity. Because KYN is thought to
enter the brain by the same system that transports TRP
and other large amino acids [50], blood KYN may enter
the brain. In fact, 60% of the total KYN pool in the nor-
mal brain is derived from blood [51]. In gerbils, 78% of
the brain KYN is derived from blood, whereas brain
KYN is derived exclusively from the blood after sys-
temic LPS injection [52]. Thus, most of the increase in
brain KYN elicited by systemic LPS injection in these
studies may have originated from outside of the brain.
However, in the present study, immune-activated brain
microglia and/or astrocytes may have synthesized KYN,
thus accounting for a small portion of the increase in
brain KYN. Here, the systemic administration of LPS did
not directly increase the brain KYN/TRP ratio. It is likely
that the increase in free TRP in the plasma preceded the
newly synthesized KYN by peripherally activated IDO.
A recent study has indicated that the effects of i.p. injec-
tion of LPS on the mRNA expression of IDO in the hip-
pocampus and hypothalamus reached a peak at 6 h after
injection [53]. Another study has suggested that i.p.-LPS
injection actually increased the KYN/TRP ratio in the
whole brain at 28 h following injection [54]. Thus, the
brain KYN/TRP ratio at 4 h after LPS injection may not
have reflected the maximum elevation of IDO activity
elicited by LPS.
In the present study, LPS did not significantly reduce
the level of 5-HT, although a previous study revealed a
reduction in 5-HT levels with the administration of LPS
[29]. Because LPS clearly decreased 5-HT levels in all
brain regions except the dorsal raphe nuclei of the
younger group (i.e., the Tukey-Kramer test showed sig-
nificant decreases, data not shown), the unchanged 5-HT
levels may be primarily attributable to a lack of LPS-
alteration of 5-HT levels in the older group of mice. The
mechanisms by which different 5-HT levels are produced
at different ages post-LPS administration remain to be
elucidated in the future studies.
Because novelty stress was also found to shift the bal-
ance to the KYN pathway [22], these results suggest an
overlap between the neurochemical changes elicited by
stressor and immune challenges, as has been frequently
noted elsewhere [55-59]. The activation of the IRS by
either direct immunological activation or psychological
stress activates IDO and shifts the balance to the KYN
pathway [19].
4.3. Influence of Age on Changes in Brain
TRP Metabolism Elicited by Systemic
LPS Administration
Although there were clear effects of age on TRP metabo-
lism that led to a shifting of the balance between KYN
and 5-HT pathways to the 5-HT pathway, the precise
underlying mechanisms of these effects remain unknown.
However, age was found to shift the balance of TRP me-
tabolism in a direction opposed to that elicited by LPS.
In other words, age suppressed the changes in TRP me-
tabolism elicited by LPS (i.e., inhibited a shift in the
balance between the 5-HT and KYN pathways to the
KYN pathway). Suppression of this shift is potentially
important in terms of the etiology of psychiatric disor-
ders such as delirium elicited by physical illness, as well
as depression in the elderly [60-61], because elderly
people are known to be more vulnerable than younger
people to delirium elicited by systemic inflammation
[62]. It is likely that the combined inhibition of stress and
immunological responses is closely related to such vul-
nerability, because sufficient-but not prolonged-responses
to stress and immunological challenge are needed to
quickly recover from these stimuli. Further evidence will
still be needed to clarify this point.
4.4. Limitations of the Study Design
An important limitation of the present study was that the
immune system activation elicited by a single systemic
injection of LPS was acute, not chronic. Because the type
of immunological activation in cases of depression is
thought to be chronic rather than acute, the behavioral
changes elicited by acute immune system activation has
been referred to as “sickness behavior” and is not di-
rectly comparable to depression. This limitation was
present in the protocol design of the present study.
However, a recent study using mice demonstrated that
the peripheral administration of LPS activated IDO, and
culminated in a distinct depressive-like behavioral syn-
drome, as measured by increased duration of immobility
in both a forced-swim test at 24 h and a tail-suspension
test at 28 h following the administration of LPS [54].
Because the present study was designed independently of
this new study, only the early-phase changes in TRP me-
tabolism (i.e., 4 h after LPS injection) were examined.
Thus, the time course of changes in TRP metabolism
until the appearance of a depressive-like behavioral syn-
drome was not investigated. Although acute systemic
LPS injection increased brain TRP levels, it is believed
that depression is accompanied by lowered plasma and
brain TRP levels [63-64], and depletion of TRP by the
administration of high doses of BCAAs may induce de-
pression in some vulnerable subjects and depressed sub-
jects who are in remission [65]. The discrepancy between
the TRP increase elicited by a single systemic admini-
stration of LPS and TRP reduction seen in cases of de-
pression suggest that the present results do not directly
correspond with the pathophysiology of depression.
5. CONCLUSIONS
The influences of age and the acute systemic administra-

H. Miura et al. / HEALTH 2 (2010) 225-233
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
231
tion of LPS on the balance between the KYN and 5-HT
pathways of brain TRP metabolism were investigated.
Whereas age shifted the balance of TRP metabolism to
the 5-HT pathway, the acute systemic administration of
LPS shifted the balance of these pathways in favor of the
KYN pathway. Age countered the effects of LPS. Thus,
age attenuated the normal, sufficient immunological
brain TRP-metabolism response, which is thought to be
an essential part of the normal stress response. These
effects will be important to consider as they might be
related to the etiology of psychiatric disorders in elderly
people.
6. ACKNOWLDGEMENTS
We would like to thank Mr. Ogiso, a technician in the Division of Ex-
perimental Animals, Nagoya University, who kindly advised us regard-
ing the protocol for the animal experiments. This research was sup-
ported by a grant from the Japanese Ministry of Health, Labor, and
Welfare for Comprehensive Research on Aging and Health.
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