Mechanism of Acute Tryptophan Depletion

Molecular Psychiatry (2011) 16, 695713
& 2011 Macmillan Publishers Limited All rights reserved 1359-4184/11

www.nature.com/mp
FEATURE REVIEW
Mechanism of acute tryptophan depletion:

is it only serotonin?
EL van Donkelaar1, A Blokland2, L Ferrington3, PAT Kelly3, HWM Steinbusch1 and J Prickaerts1
1
Department of Psychiatry and Neuropsychology, Faculty of Health, Medicine and Life Sciences, School for Mental
Health and Neuroscience, Maastricht University, Maastricht, The Netherlands; 2Department of Neuropsychology and
Psychopharmacology, Faculty of Psychology and Neuroscience, Maastricht University, Maastricht, The Netherlands and
3
Cerebrovascular Research Laboratory, Centre for Cognitive and Neural Systems, University of Edinburgh, Edinburgh, UK
The method of acute tryptophan depletion (ATD), which reduces the availability of the essential
amino acid tryptophan (TRP), the dietary serotonin (5-hydroxytryptamine (5-HT)) precursor,
has been applied in many experimental studies. ATD application leads to decreased
availability of TRP in the brain and its synthesis into 5-HT. It is therefore assumed that a
decrease in 5-HT release and subsequent blunted neurotransmission is the underlying
mechanism for the behavioural effects of ATD. However, direct evidence that ATD decreases
extracellular 5-HT concentrations is lacking. Furthermore, several studies provide support for
alternative underlying mechanisms of ATD. This may question the utility of the method as a
selective serotonergic challenge tool. As ATD is extensively used for investigating the role of
5-HT in cognitive functions and psychiatric disorders, the potential of alternative mechanisms
and possible confounding factors should be taken into account. It is suggested that caution is
required when interpreting ATD effects in terms of a selective serotonergic effect.
Molecular Psychiatry (2011) 16, 695713; doi:10.1038/mp.2011.9; published online 22 February 2011
Keywords: acute stress; cerebral blood flow; cognitive dysfunction; depression; serotonin;
tryptophan
Introduction
Acute tryptophan depletion (ATD) currently represents the most established human challenge test
to investigate the involvement of the serotonin
(5-hydroxytryptamine; 5HT) system in the pathogenesis and pathophysiology of affective disorders.
The method is nontoxic and nonintrusive, thereby
providing the option to repeatedly manipulate the
central 5-HT system in vivo and assess the behavioural
effects of reduced 5-HT metabolism in the brain.1 The
reduction of brain 5-HT in a reversible manner reflects
the main methodological advantage of the tool,
permitting application of the same basic method in
both human subjects and rodents. This is considered
valuable for comparing neurophysiological changes
linked to behavioural effects across species.2
As intact 5-HT neurotransmission is necessary for a
wide range of physiological and functional processes,
a disruption in this system can easily provoke diverse
pathophysiological abnormalities, most of which are
Correspondence: Dr EL van Donkelaar, Department of Psychiatry
and Neuropsychology, Division of Neuroscience, Faculty of
Health, Medicine and Life Sciences, School for Mental Health
and Neuroscience, Maastricht University, P.O. Box 616, 6200 MD
Maastricht, The Netherlands.
E-mail: eva.vandonkelaar@maastrichtuniversity.nl
Received 12 March 2010; revised 4 January 2011; accepted 19
January 2011; published online 22 February 2011
reflected in dysfunctional behavioural output. ATDinduced behavioural changes in human subjects

and laboratory animals are normally attributed to
decreased 5-HT release, reflecting altered 5-HT
neuronal activity. However, it is not fully clear what
mechanisms underlie the neurophysiological effects
of ATD and to what extent changes in 5-HT neuronal
activity contribute to the ATD-induced functional and
behavioural alterations. Also, no convincing evidence
exists for affected central 5-HT release following ATD
in animals.3
The ATD method seems important in the investigation of 5-HT-related vulnerability factors implicated
in the onset of depression,4 and previously the
monoamine systems were considered to be primarily
responsible for the onset of depressive disorders.5
However, the lack of mood-lowering effects after ATD
in healthy subjects may not support a direct causal
relationship between acute decreased 5-HT metabolism and major depressive disorder.6 Moreover, as
will be discussed in this review, evidence exists
that ATD possibly exerts its neurochemical and
behavioural effects through other mechanisms that
might go beyond a straightforward decrease in 5-HT
metabolism. This review covers an extensive evaluation of both the methodology and the diverse neurochemical and behavioural effects of ATD, including
a critical assessment of the common parameters
used for indicating presumed ATD-induced changes
Underlying mechanisms of acute tryptophan depletion

EL van Donkelaar et al
696
in 5-HT functionality. Furthermore, this review

aims to outline alternatives for potential underlying
mechanisms of the method that might go beyond a
disturbed 5-HT system and thus draw into question
the utility of ATD as a serotonergic challenge tool in
experimental research in general and depression
research in particular.
Methodological aspects of ATD

5-HT is synthesized in a two-step reaction (Figure 1)
from the initial substrate L-tryptophan (TRP), and the
bioavailability of this essential amino acid is the
principal rate-limiting factor. Thus, variations in
dietary intake of TRP can have profound effects upon
the synthesis of this very important neurotransmitter
substance and may impact upon those aspects of
brain function that are influenced by serotonergic
neurons. It is this fact that underpins the use of ATD
as both an experimental tool and a clinical probe for
depressive illness.
From plasma to brain tryptophan
Amino acids can only be transported from the blood
through the capillary endothelial cells of the bloodbrain barrier (BBB) into the brain by carrier-mediated
transporter systems in the capillary cell plasma
membranes.7 Given that the surface area of the BBB
is much smaller compared with the surface area of
brain cell membranes, it is this initial transport
through the BBB that limits the uptake of plasma
TRP into the brain.8 The branched-chain amino acids
(leucine, isoleucine and valine) together with the
aromatic amino acids (phenylalanine, tyrosine and
TRP) are subclassified as large neutral amino acids
(LNAAs). Of the nine different amino acid transport
systems identified at the BBB, the so-called Transport
System L is only half saturated under normal
physiological conditions and mediates high-affinity,
sodium-independent uptake of all LNAAs.8,9 Consequently, in order to bind to the L-amino-acid transport
carrier and subsequent transport into the brain, TRP
has to compete heavily with the other LNAAs.1012
The availability of TRP in the brain thus depends
upon the ratio of TRP to the sum of the other LNAAs
(TRP/SLNAA), and a decrease in this ratio in plasma
is normally used as the best predictor of reduced
availability of TRP in the brain and subsequent
synthesis into 5-HT.13,14
From bound to free plasma tryptophan: the brain influx

parameter
Approximately 90% of all TRP molecules circulating
in the blood are bound to serum albumin. Although
positive correlations between serum free-TRP and
whole brain TRP levels have been reported in rats,15,16
the dissociation of TRP from albumin by endogenous
and exogenous ligands has been shown to increase
the entry of TRP into the brain, thereby enhancing
central 5-HT synthesis.17,18 This observation suggests
that only free TRP is available for transport into the
brain.15 As the changes in TRP-free levels can take
place independently of changes in total TRP levels,19
this would make a distinction between free and
bound TRP necessary for estimating its availability
in the brain. However, accumulating evidence indicates that total peripheral TRP concentrations (free
plus bound) more accurately reflect the rate of influx
of TRP into the brain. It has been shown that TRP is
only loosely bound to albumin and although albumin
itself cannot cross the BBB, it appears to be a highly
flexible protein undergoing reversible conformational
changes.20 These conformational changes, which
occur during transport of TRP from the circulating
albumin-bound pool, enhance the dissociation of TRP
from the albumin-binding sites within the cerebral
microvasculature and appear to be highly dependent
upon cerebral haemodynamics.21 Low cerebral blood
flow (CBF) is likely to increase the interaction
between the albumin-bound TRP complex and the
glycocalyx of the BBB, thereby causing more TRP
to dissociate from albumin.21,22 This implies that
temporally dynamic or spatial differences in local
CBF may influence the rate of central TRP uptake in
general and even within specific brain areas.23 Thus,
although only free TRP can eventually cross the BBB,
the amount of albumin-bound TRP in plasma must
also be taken into account to calculate the availability
of TRP in the brain, as TRP can easily dissociate from
albumin near the BBB, thereby increasing the TRPfree pool and subsequent uptake into the brain (see
also Figure 2).
Solugel
In most studies, pure amino-acid mixtures without
TRP have been used to reduce plasma TRP levels.
However, one disadvantage of the amino-acid mixture
is that differences in the distinct amino acids were
found between the control condition (TRP ) and
Figure 1 Central serotonin synthesis and metabolism. In the brain, tryptophan (TRP) is first hydroxylated into
5-hydroxytryptophan (5-HTP) by the enzyme tryptophan hydroxylase (TPH). Aromatic L-amino-acid decarboxylase (AAAD)
subsequently catalyzes the decarboxylation of 5-HTP into 5-hydroxytryptamine (5-HT). The enzymes monoamine oxidase
(MAO) and aldehyde dehydrogenase (ADH) eventually break serotonin (5-HT) down into the inactive metabolite
5-hydroxyindoleacetic acid (5-HIAA).
Molecular Psychiatry

Figure 2 Overview of tryptophan metabolism from food

intake to brain uptake and the differential effects of
carbohydrates and protein upon the availability of tryptophan (TRP) in plasma for uptake into the brain. In order to
obtain the amino-acid TRP, its inclusion in the diet is
essential. The majority of TRP is bound to plasma albumin
and only free TRP will eventually cross the blood-brain
barrier (BBB). Albumin-bound TRP, however, easily dissociates from albumin near the cerebrovasculature under
the influence of haemodynamic changes, thereby increasing
the TRP-free fraction available for uptake into the brain. The
amount of TRP in plasma eventually crossing the BBB also
depends upon the presence of other large neutral amino
acids (LNAAs) that all compete for the same amino acid
transport system L at the BBB. Because of this competition
of TRP with leucine (LEU), isoleucine (ILE), valine (VAL),
phenylalaline (PHE) and tyrosine (TYR), the ratio of TRP to
the sum of the other LNAAs (TRP/SLNAA) in plasma better
reflects the amount of central TRP available for synthesis
into 5-hydroxytryptamine (5-HT). Dietary carbohydrates
increase the uptake of the LNAAs into peripheral tissue,
thereby decreasing their levels in plasma. Together with an
increase in total TRP levels, the TRP/SLNAA ratio changes
in favour of TRP and increases its availability for transport
across the BBB. As little as 2.5% of additional proteins
counteracts the effects of carbohydrates, as the protein
ingestion-induced increase in the levels of all amino acids is
much higher than the decrease by carbohydrates. When a
TRP-free diet is administrated (acute tryptophan depletion),
all amino acids are elevated except for TRP, thereby
decreasing the TRP/SLNAA ratio and thus TRP uptake into
the brain.
a condition in which animals were treated with

saline.2,24 In that case, the control condition is not
an optimal and representative control condition. An
alternative manner to reduce central 5-HT concentrations by lowering the levels of its dietary precursor
TRP in plasma can be achieved by administration of
specific TRP-free nutritional mixtures.
Besides the TRP-free or TRP-low balanced diets
and pure amino-acid mixtures without TRP, a more
advanced technique is the oral administration of
a gelatin-based proteincarbohydrate mixture.2 By

adding a specific amount of TRP to the control
mixture, the effects of peripheral TRP suppletion, as
often observed with traditional amino-acid mixtures
in humans,25,26 are avoided and thus do not cause
misinterpretation of the ATD effects.2,27,28 Gelatin is
derived from the selective hydrolysis of collagen
protein, which is easily digestible and naturally lacks
the essential amino-acid TRP.29 The gelatin hydrolysate used for the nutritional mixture is gelatin in an
enzymatic hydrolyzed form, commercially available
as Solugel (PB Gelatins, Tessenderlo, Belgium).
Solugel no longer consists of a combination of a few
selective amino acids, but comprises a broad range of
amino acids in the form of peptides, which makes it
comparable with standard diets. Moreover, it is water
dispensable and unique for its gel-forming ability.24 In
addition, a specific amount of carbohydrate is mixed
with the Solugel, which adds an essential caloric
value, thereby making the nutritional mixture even
more similar to conventional food intake. Moreover,
mixing proteins with carbohydrates avoids any
unwanted effects upon amino-acid availability in
the blood (see below and Figure 2) as is normally
found with unbalanced diets containing high
amounts of carbohydrate or protein only.3032
697
Carbohydrate and dietary protein

Various metabolic processes are triggered when a
protein/carbohydrate meal, as is the case with ATD
methods, is ingested. These processes can have effects
on plasma TRP levels too. After the intake of carbohydrates, blood glucose levels raise, thereby stimulating the pancreas to release the anabolic hormone
insulin. The secretion of insulin stimulates glucose to
be taken up by the cells for subsequent normalization
of glucose levels in the blood. Concomitantly with the
insulin-induced drop in blood glucose levels, plasma
TRP levels increase whereas the concentration of
most other amino acids in plasma decreases.33 When
a carbohydrate-rich meal contains no additional
protein, activation of the insulin response increases
protein synthesis, thereby stimulating the uptake of
almost all amino acids (mainly the branched-chain
amino acids) into muscle tissue. Most of the TRP in
plasma is bound to serum albumin and thus is not
available for uptake into peripheral tissues, and hence
the effect of insulin upon TRP is much less compared
with the other LNAAs. Moreover, insulin increases
the affinity of serum albumin for TRP and increases
the ratio of bound to free plasma TRP, as more
albumin is available because of the insulin-induced
uptake of fatty acids, which were bound to albumin.
Thus, because TRP is the only amino acid that binds
to albumin, it is the only one that is very well
prevented from being taken out of the bloodstream
and up into peripheral tissue. This is even more the
case when the binding to albumin increases because
of insulin secretion. Taken together, after the ingestion of carbohydrate the plasma ratio of LNAAs
changes in favour of total TRP, which eventually

698
leads to an increased availability of TRP in the brain

to be synthesized into 5-HT.33,34 Yet, as little as 2.5%
of additional protein is sufficient for the substantial
increase of plasma LNAAs to counteract the effect of
carbohydrate and the subsequent insulin-induced fall
in LNAAs.32,35 This is the case with the TRP-free
proteincarbohydrate nutritional mixture (see also
Figure 2).
Protein synthesis
The administration of a diet devoid of TRP depletes
plasma TRP acutely by inducing hepatic protein
synthesis.36 This results in an extracellular TRP
removal that is because of an increased incorporation
of TRP into proteins in the liver and other tissue.12,15
The ATD-induced depletion of plasma TRP can be
dose dependently blocked by administration of the
protein synthesis inhibitor cycloheximide together
with a TRP-free diet.37,38 Thus, protein synthesis, and
not the inhibition of TRP transportation into the
brain, seems to be the important initial mechanism
underlying ATD-induced decreased 5-HT in the brain.
Central versus peripheral effects of ATD

The effects of ATD on affective behaviour (for
example, depression) and various cognitive functions
(for example, memory, attention and impulsivity)
have been studied in human subjects and laboratory
animals, and several theories regarding its underlying
serotonergic mechanism and its implication for
psychiatry in general have been widely explored
and reviewed over the past 20 years.3943 In this
section we will give an overview of the peripheral and
central effects after ATD in human subjects and
rodents.
ATD: revealing vulnerability to depression
Many neurophysiological processes are known to be
regulated by the 5-HT system, including mood and
cognition, which are most prominently impaired in
clinical depression.4446 5-HT cell bodies are clustered
in the brainstem raphe nuclei sending out projections
throughout the entire central nervous system with
ascending pathways innervating anatomically and
functionally diverse regions of the cerebral cortex,
including the limbic system, the basal ganglia and
structures within the diencephalon.47 Because of this
anatomy, the neurotransmitter influences all regions
of the neuraxis, thereby modulating an extensive
range of physiological and behavioural functions.
Besides mood and cognition, appetite, emesis, endocrine function, gastrointestinal function, motor function, neurotrophism, perception, sensory function,
pain sensitivity, sex, sleep and even vascular function
are all under the control of the 5-HT system.
Consequently, disrupted 5-HT synthesis and subsequent abnormal 5-HT function can lead to a diverse
range of behavioural disturbances also implicated
in clinical depression. Consistent findings in this
respect specifically include decreased peripheral TRP
levels48 and lower levels of the inactive 5-HT

metabolite 5-hydroxyindoleacetic acid (5-HIAA) in
cerebral spinal fluid (CSF), which all reflect diminished 5-HT metabolism.49,50 The effectiveness of
serotonergic drugs used in the treatment of depression is also suggestive of an important role of
disrupted function of specific pre- and post-synaptic
receptors underlying impaired 5-HT neurotransmission and linked to specific depressive symptoms.51,52
Human subjects with genetic, pre-existing 5-HT
dysfunction may lack endogenous compensatory
capacity to deal with an acute decrease in 5-HT
metabolism, thereby exhibiting higher behavioural
sensitivity to ATD.4,26 This implies that a predisposition of so-called serotonergic vulnerability only
results in direct overt psychiatric symptoms when
these are triggered, as with ATD, by challenging the
already vulnerable 5-HT system4 up to a certain
threshold.26 In line with this hypothesis, ATDinduced transient mild mood-lowering effects, as
reflected by lower mood ratings, have been reported
in carriers of the short allelic polymorphism in the
promoter of the 5-HT transporter gene 5-HTTLPR
(serotonin-transporter-linked promoter region)53 and
in healthy subjects with a family history of depression.54 Similarly, a higher behavioural response to
ATD has been observed in women,55,56 who are
presumably predisposed to a lower 5-HT synthesis
rate compared with men.57 Moreover, ATD provokes a
relapse of depressive symptoms in healthy subjects
with a history of depression.58,59 However, this effect
is only in those subjects who were previously treated
successfully with selective serotonin re-uptake inhibitors (SSRIs) or monoamine oxidase inhibitors (MAOIs).
Remitted, medication-free depressed patients with a
positive treatment response history to antidepressants
that primarily interact with systems other than 5-HT
(for example, tricyclic antidepressants or selective
norepinephrine reuptake inhibitors) appear not to be
affected by ATD.6062
ATD in healthy human subjects: inducing cognitive
deficits
As mentioned above, it is generally believed and
accepted that ATD does not induce considerable
mood-lowering effects in healthy human subjects.59,63,64 Nevertheless, acute decreased peripheral
TRP levels and diminished 5-HIAA concentrations in
CSF are consistently reported after ATD and appear to
be similar in all subpopulations, that is, in both
healthy and the so-called serotonergic vulnerable
subjects.54,6569 Interestingly, both healthy and vulnerable subjects display cognitive dysfunctional behaviour after ATD as reported consistently between
studies.27,28,7075 It might therefore be suggested that
an acute decrease in peripheral TRP levels directly
interferes with mechanisms implicated in cognitive processing that depend less upon 5-HT functioning. Altered cognitive processing has been reported
with impairments in long-term memory formation

(in particular, consolidation processes74), decision

making, reversal learning and working memory.
Effects of ATD in rodents: affect and/or cognition?
Animal models enable direct investigation of the
relationships between brain and behaviour with the
aim of gaining insight into human behaviour and
its underlying neuronal and neuroendocrinological
processes.76 Therefore, the direct consequences of
ATD upon brain parameters like TRP and 5-HT in the
rat are generally used for interpretation of the alterations in behavioural output in accordance with the
underlying neurochemical mechanism of the method.
A large body of preclinical literature provides
evidence that ATD in rats significantly depletes the
levels of TRP in plasma, thereby reducing 5-HT
metabolism, as is suggested by the lower TRP and
5-HT levels in the rat brain tissue.24,7780 In rats,
however, the levels of peripheral and central TRP
reductions, as well as brain 5-HT concentrations,
have not been consistently reported, and other ATDinduced neurophysiological effects, as discussed
below, have been observed in the absence of central
TRP or 5-HT reductions.81,82 Similarly and in line
with the ATD effects in healthy human subjects,
ATD-induced alterations in affective behavioural
parameters in the rat appear controversial between
studies.77,8385 Object memory performance is the only
parameter consistently reported as impaired after
ATD79,83,8588 and seems even more pronounced in
rats with pre-existing abnormal 5-HT function.80
Table 1 provides an overview of the peripheral and
central neurochemical effects and other neurophysiological changes, as well as cognitive and affective
behavioural alterations after ATD induced in rodents,
mainly rats, through administration of nutritional
mixtures completely devoid of TRP.
Methodological considerations: do we actually

measure central effects?
It is generally assumed that the mood-lowering
and cognitive dysfunctional effects of ATD are mediated by decreases in 5-HT neuronal activity. Yet, as
described below, most parameters used to indicate
reduced 5-HT synthesis or release appear to merely
estimate decreases in 5-HT metabolism and neuronal
activity, respectively, thereby remaining rather
speculative.
The TRP/SLNAA ratio in plasma
The effects of ATD treatment upon 5-HT levels in the
brain cannot be directly investigated in humans. In
general therefore, the ratio TRP/SLNAA in plasma is
used to estimate the amount of TRP available in the
brain for synthesis into 5-HT. In most cases, total
peripheral TRP levels (free plus albumin bound) are
used for calculating the ratio to the sum of the other
LNAAs. Although only the relatively small fraction of
free TRP eventually crosses the BBB, TRP can easily
dissociate from albumin near the BBB, thereby
increasing the TRP-free pool and subsequent uptake

into the brain. Thus, the direct effect of physiological
factors such as hormones, exercise or mild stressors
upon the rate of dissociation of TRP from albumin can
only be taken into account if both total and free TRP
levels are actually measured,89 which is usually not
done. Therefore, based upon total peripheral TRP
levels alone, the TRP/SLNAA ratio in plasma is
probably a distorted estimation of the rate of influx of
TRP into the brain.
A decrease in the TRP/SLNAA ratio suggests that
less TRP will be available to the brain for synthesis
into 5-HT. This has been confirmed by the ATDinduced decrease in the uptake of central a-methyl-Ltryptophan (a-M-TRP) as measured by positron emission tomography in humans.57 Although this finding
supports the concept that ATD exerts central effects,
a-M-TRP uptake does not reveal anything specific
about 5-HT release,39,67 despite the fact that it is
considered a reliable indicator of 5-HT synthesis.
Finally, other LNAAs such as methionine and
threonine are generally not included in the ratio as
active competitors of plasma TRP. However, evidence
exists that together with the branched-chain amino
acids and the aromatic amino acids, methionine
and threonine share the same L-amino-acid transport
carrier at the BBB.8,9,90 Thus, calculating the TRP/
SLNAA ratio without taking into account all competitive amino acids is most likely to result in an
overestimation of brain TRP influx and subsequent
dissociation from brain TRP levels and 5-HT synthesis.
699
The 5-HIAA/5-HT ratio in the brain

In humans subjected to ATD, a decrease in the
concentration of 5-HIAA in CSF suggests that less
5-HT has been catabolyzed, which normally takes
place after release, that is, after neuronal firing.
Therefore, a decrease in the amount of 5-HIAA is
thought to reflect decreased 5-HT metabolism, as
lower intracellular 5-HT availability is presumed to
result in reduced 5-HT release. However, numerous
other peripheral factors seem to influence both the
amount of 5-HIAA produced and its transport into
and out of the CSF (see ref. 52). Moreover, 5-HT
release and neuronal firing seem not to correlate
necessarily with 5-HT metabolism,91,92 and thus the
amount of 5-HIAA in CSF seems not to be a valid
index of changes in 5-HT release.92,93
In contrast to human subjects, animal models offer
the possibility to directly measure changes in TRP,
5-HT and 5-HIAA concentration in distinct brain
areas. Significantly lower tissue levels of TRP and
5-HT have been reported after ATD compared with
TRP control conditions in animals.24,79,80,85 The
5-HIAA/5-HT ratio is normally used to calculate the
5-HT turnover rate and estimate changes in 5-HT
release, reflecting neuronal activity. However, this
seems to apply only under normal physiological
conditions when the rate of 5-HT synthesis remains
constant.94 An increase in 5-HIAA levels would then
increase the 5-HIAA/5-HT ratio and allow accurate
700
Age/sex/strain/
nutritional mixture/
dosing regime
Adult male Sprague

Dawley rats
AA mixture
1 10 ml kg1
4 months old male

Wistar rats
AA mixture
1 10 ml kg1
(5 g /kg1)
TRP : 0.153 g
absolute amount
4 months old male

Wistar rats
Solugel
2 10 ml kg1, 90 min
interval
4 months old male

Wistar rats
Solugel2 10 ml kg1,
90 min interval
Adult male Sprague

Dawley rats
Solid TRP-free diet
TRP : 0.7%
Reference
Brown et al.84
Blokland
et al.77
Lieben et al.24
Lieben et al.83
Cahir et al.78
Free plasma TRP
At T3
89%
70% at T2
65% at T4
49% at T6
TRP
fcx: 70%
hpc: 66%
remaining cortex: 63%
5-HT
fcx: 39%
hpc: 55%
remaining cortex: 41%
Not reported
At T4:
TRP hpc: 43%
5-HT hpc: 67%
5-HT striatum: 40%
5-HIAA hpc: 40%
At T5:
TRP hpc: 33%
TRP fcx (no pfc): 14%
38% at T5
71% at T2
78% at T4
At T3:
5-HT hpc: 23%
5-HIAA hpc: 39%
% Change brain
parameters compared
with control
Not reported
% Change plasma
TRP/SLNAA ratio
compared with
baseline
No differences between 1 and 3

weeks of TRP diet upon peripheral
and central parameters
Decreased 5-HT1A receptor binding
in dorsal raphe only after ATD at T3.
No effects upon 5-HT1A or
5-HT2A binding in fcx, hpc or
remaining cortex. After 3 weeks
of diet, 46% increase in 5-HT2A
binding in cortex and not, however,
in fcx or hpc
NA
Object recognition impairment at T4

No anxiety or depressive-like effects
and no effects upon spatial learning
in water maze
NA
No cognitive impairment or
depressive-like effects. Increased
anxiety at T5
TYR ctx: 44%
Lowering of 5-HT in both hpc and

striatum. Both TRP and 5-HIAA
levels only decreased compared
with control in hpc. Increase (43%)
TYR in hpc and decrease in striatum
after ATD. No effects upon TRP,
5-HT, 5-HIAA in ctx. No effect
upon DA.
ATD-induced decrease of CIT in
striatum, hpc and ctx
No cognitive impairment or changes

in locomotor activity
or analgesia
Behavioural effects
TRP suppletion effect in fcx: 5-HT

45% increase compared with water.
Decreased 5-HT after ATD in fcx
compared with TRP , but not
different from water. 5-HIAA
striatum 35% lower compared
with water, not different from TRP
Additional neurobiochemical
observations
Table 1 Overview of neurobiochemical and behavioural effects of acute tryptophan depletion in rodents induced by a pure amino-acid mixture without TRP, a TRP-free
proteincarbohydrate nutritional mixture (Solugel) or a solid TRP-free diet

Age/sex/strain/
dosing regime
Adult male Wistar

rats
Solugel
2 10 ml kg1, 90 min
interval
Adult male Sprague

Dawley rats
AA mixture
2 10 ml kg1, 90 min
interval
TRP : 2.3 g
4 months old male

Wistar rats
Solugel
1 10 ml kg1
4 months old male

and female Wistar
rats
Solugel
2 10 ml kg1, 90 min
interval
3 months old male

Brown-Norway and
Sprague Dawley rats
Solugel
2 10 ml kg1, 90 min
interval
2 months old male

Wistar rats
Solugel
2 10 ml kg1, 90 min
interval
Van der
Plasse99
Cahir et al.97
Rutten et
al.87,97,114
Jans et al.4
Jans et al.85
Jans and
Blokland86
Continued
Reference
Table 1
At T4
CMS: 60%
Control: 64%
At T4
Brown Norway:
58%
Sprague Dawley:
48%
At T4
Males:
66%
Females:
53% (pro/es)
55% (met/di)
At T1
48%
At T3
23%
Free plasma TRP

At T3.5
79%
Free plasma TRP

60% at T3
70% at T4
35% at T5
50% at T6
% Change plasma
TRP/SLNAA ratio
compared with
baseline
Not reported
- BN
fcx: 5-HT 5-HIAAk
hpc: 5-HT 5-HIAAk
- SD:
fcx: 5-HT 5-HIAAk
hpc: 5-HIAA k
5-HIAA/5-HT k
Males:
TRP fcx: 56%
Females (pro/es)
TRP fcx: 49%
TRP hpc: 53%
Not reported
Hpc
5-HT: 37%
5-HIAA: 34%
% Change brain
parameters compared
with control
Decreased ratio 5-HIAA/5-HT in hpc

and fcx only in SD rats
In general, higher peripheral TRP

levels were observed in females. No
central ATD effects in females met/
di. Higher TYR levels in female pro/
es compared with female met/di and
males. ATD-induced increased TYR
in hpc and fcx in female pro/es.
Increased TYR in hpc only in female
met/di
No effects upon 5-HT turnover in

hpc
No effects upon plasma or central
BDNF protein levels
Extracellular 5-HT and DA

transmitter concentration
(microdialysis): No differences
between TRP- and TRP treatment
at any time point
observations
Lower sucrose intake and blunted

weight gain after CMS. No anxiety
or depressive-like behaviour after
ATD or CMS. Less passive social
interaction after ATD, independent
of CMS. Object recognition impairment after ATD, independent of
CMS
Stronger ATD-induced behavioural

responses in SD rats. Object
recognition impairment only in SD
rats. Controversial anxiety effects and
trend toward increased behavioural
despair in SD rats. No anxiety/
depressive-like behaviour in BN rats
Object recognition impairment only

in female pro/es, not in males.
Generally less passive body contact
after ATD. Females generally more
active and decreased anxiety
Object recognition impairment at T1

and T3, reversed by PDE4 inhibition
NA
No differences in general activity

and no effect of novelty stimulation
upon general activity
Behavioural effects

701
At T4
TRP
SERT /
65%
SERT /
61%
SERT /
55%
ratio TRP/SLNAA
mean 50%k
SERT/ rat
(Slc6a41Hubr)
Solugel
2 10 ml kg1, 90 min
interval
6 months old male

Wistar rats
Solugel
1 10 ml kg1
4 months old male

and female Wistar
rats
Solugel
10 ml kg1, several
applications
Adult male Sprague

Dawley rats
AA mixture
2 10 ml kg1, 90 min
interval
TRP : 2.3 g
Olivier et al.80
van Donkelaar
et al.88
Jans et al.,
2009169
Ardis et al.96
Free plasma TRP
At T3.579%
Males
1 10: 48% at T2
2 10, 60 min
interval: 65% at T2
3 10, 60 min
interval:
73% at T4
54% at T6
Females
2 10, 90 min interval:
73% at T2
60% at T4
2% at T6
80% at T2 (after 4
days of daily
treatment)
81% at T4 (after 4
days of daily
treatment)
5-HT
fcx: 33%
hpc: 34%
remaining ctx: 12%
5-HIAA
fcx: 37%
hpc: 37%
remaining ctx: 43%
NA
Object recognition impairment in

males at T2, T4 and T6 after
2 10 ml kg1, 90 min interval
Not reported
Object recognition impairment

independent of genotype. No effect
after low dose ATD in SERT /
wild types. Generally strongest
effect on SERT / even after TRP
treatment. No effects on untreated
rats
Behavioural effects
Object recognition impairment at

T2, reversed by PDE2 and PDE5
inhibition
No reduction of 5-HT or 5-HIAA in

striatum. Decreased 5-HIAA/5-HT
ratio in both cortex (41%) and
striatum (34%), not in hpc
No effects upon DA, NE or the
metabolites, DOPAC and HVA
Decreased TRP/SLNAA compared

with TRP control and untreated
rats, independent of genotype.
Decreased 5-HT in both fcx and hpc,
strongest in SERT/ genotype.
No genotype effect upon peripheral
TRP, ratio or central 5-HT levels.
Increased plasma ratio after TRP
treatment in all genotypes similarly
observations
Not reported
5-HT fcx:
SERT / : 19%
SERT /: 19%
SERT /: 63%
5-HT hpc:
SERT / 13%
SERT /18%
SERT /70%
% Change brain
parameters compared
with control
702
At T2
73%
% Change plasma
TRP/SLNAA ratio
compared with
baseline
Age/sex/strain/
dosing regime
Reference
Table 1 Continued

3 months old male

Wistar rats
Solugel
2 10 ml kg1, 90 min
interval
3 months old male

Wistar rats
Solugel
2 10 ml kg1, 90 min
interval
3 months old male

Wistar rats
Solugel
2 10 ml kg1, 90 min
interval
3 months old male

Swiss and C57BL/6J
mice
Solugel
1 10, 2 10, 1 20
and 2 20 ml kg1, 30
or 60 min interval
van Donkelaar
et al.81
van Donkelaar
et al.170
van Donkelaar
et al., 200982
van Donkelaar
et al.165
No effect of ATD upon
TRP, 5-HT or 5-HIAA
concentration at any of
the time points
measured and
independent of the
specific dosing regime

TRP, 5-HT or 5-HIAA
concentration
56%
Max depletion at T1
(30 min interval)
Swiss: 2 10 ml kg1:
74%
2 20 ml kg1: 77%
C57BL/6J:
2 10 ml kg1: 40%
1 15 ml kg1:70%

TRP, 5-HT or 5-HIAA
concentration

TRP, 5-HT or 5-HIAA
concentration
40%
MDMA pretreated:
37%
control: 40%
% Change brain
parameters compared
with control
% Change plasma
TRP/SLNAA ratio
compared with
baseline
ATD application stress-mediated

changes in TRP/SLNAA ratio
No effects upon affective or

cognitive behaviour
NA
NA
Significant MDMA pretreatment

effect
ATD potentiated hyperaemia only
after MDMA pretreatment
No effect of ATD upon serum and
brain BDNF levels
Positive correlations between TRP
and BDNF in hpc and pfc
ATD application stress-mediated
decreased BDNF levels in pfc
NA
Behavioural effects
ATD-induced decrease of CIT in hpc

ATD-induced tissue oligaemia
observations
Abbreviations: ATD, acute tryptophan depletion (TRP treatment); BDNF, brain-derived neurotrophic factor; BN, Brown-Norway rat; CIT, citrulline; CMS, chronic mild
stress; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; fcx, frontal cortex; hpc, hippocampus; HVA, homovanillic acid; met-di, female rats in metestrus or
diestrus stage of the reproductive cycle; MDMA, 3,4-methylenedioxymethamphetamine, ecstasy; NA, not applicable; NE, norepinephrine; PDE 2, 4, 5,
phosphodiesterase enzyme type 2, 4, 5; pfc, prefrontal cortex; pro-es, female rats in proestrus or estrus stage of the reproductive cycle; SD, Sprague Dawley rat;
SERT/, homozygous serotonin transporter knockout rat; SERT /, heterozygous serotonin transporter knockout rat; SERT / , wild-type control of serotonin
transporter knockout rat; T, time in hours after (first) administration; TRP, tryptophan; TRP , control nutritional mixture with additional tryptophan; TRP/SLNAA,
ratio of TRP to the sum of other large neutral amino acids; TYR, tyrosine; 5-HT; 5-hydroxytryptamine (serotonin); 5-HIAA, 5-hydroxyindoleacetic acid; k, significant
decrease.
Age/sex/strain/
dosing regime
Continued
Reference
Table 1

703

704
estimation of an increase in 5-HT neuronal activity, as

it is assumed that more 5-HT has been released.
Conversely, a decrease in 5-HIAA levels under a
constant rate of 5-HT synthesis would decrease the
5-HIAA/5-HT ratio. Yet, ATD is applied to induce a
decrease in 5-HT synthesis. As a decrease in intracellular 5-HT availability most likely results in a
reduction of the amount of 5-HT available for release,
less extracellular 5-HT will then be available to be
catabolyzed into 5-HIAA. Thus, ATD is likely to
decrease both 5-HT and 5-HIAA, thereby maintaining
the 5-HIAA/5-HT ratio constant. In line with this, no
changes in the 5-HIAA/5-HT ratio were found 3 h after
ATD, whereas both 5-HT (23%) and 5-HIAA (39%)
levels appeared to be significantly decreased in rat
hippocampus.84 A decrease in this ratio after ATD
would only occur if significantly less 5-HIAA is
produced compared with the already reduced amount
of 5-HT synthesized.
As described previously, the conversion of TRP into
the 5-HTP intermediate by the TPH2 (tryptophan
hydroxylase 2) isoform is the first and rate-limiting
step in the biosynthesis of 5-HT. In the brain, this
enzyme is only 50% saturated and, therefore, the rate
at which 5-HT is synthesized is limited only by
substrate (that is, TRP) availability.95 However, it
seems difficult to directly attribute an ATD-induced
decrease in the 5-HIAA/5-HT ratio to a reduction in
TRP availability. Brain measurements are generally
taken at only one specific time point that impedes a
comparison of the central parameters with baseline
values. Thus, a decrease in 5-HIAA levels and in the
5-HIAA/5-HT ratio after ATD is normally interpreted
as evidence for an ATD-induced reduction in 5-HT
metabolism.
However, one animal study showed that 5-HT
levels might not have changed compared with TRP
treatment.82 Furthermore, information upon baseline
5-HT values is generally lacking. Although reductions
of the 5-HIAA/5-HT ratio have been repeatedly
reported after ATD in rats,85,96,97 between the studies,
the concomitant changes in 5-HIAA or 5-HT in
specific brain areas do not seem to be in accordance
with each other. In general, it appears that ATDinduced absolute changes in central 5-HT or 5-HIAA
concentrations and potential underlying mechanisms
require further examination.
A decrease in the 5-HIAA/5-HT ratio after ATD in
rats seems more likely to be caused by factors
influencing the catabolism of 5-HT, such as fluctuations in the activity of the MAO enzyme.98 An acute
decrease in MAO activity would reduce the absolute
amount of 5-HIAA produced, thereby producing a
relative increase in 5-HT levels. This would eventually lower the 5-HIAA/5-HT ratio independent of
a reduction in precursor availability per se or the
subsequent decrease in 5-HT synthesis and as such
does not reflect a decrease in 5-HT release. It could be
hypothesized that an acute change in MAO activity
implies a compensatory mechanism activated upon
the acute decrease in peripheral TRP levels.
Several rat studies, all of similar experimental

design, have directly reported ATD-induced
decreased central 5-HT levels after administration of
the same TRP-free nutritional mixture.24,80,85,99 This is
generally thought to reflect an ATD-induced decrease
in 5-HT synthesis. However, as mentioned previously,
the decrease in 5-HT after TRP treatment at one
specific time point is normally only compared with
the control TRP treatment group and not with its
own baseline concentrations. Thus, without these
baseline values before treatment, the extent to which
the significant difference in 5-HT levels between the
experimental (TRP) and control (TRP ) treatment
group actually imply a reduction in 5-HT release (that
is, activity) after ATD remains unclear. In addition,
no direct evidence exists that ATD affects central
5-HT release (see next section).
5-HT neuronal release
Alterations in neuronal activity can be measured by
in vivo microdialysis. With this technique, changes
in extracellular 5-HT concentrations can be measured
that are indicative of changes in neuronal release.
Actual reductions of basal 5-HT release after ATD
have only been reported in combination with 5-HT
reuptake inhibitors.1,100,101 A blockade after 5-HT
reuptake seems necessary to raise 5-HT to optimal
levels for detection (see ref. 67). Yet, without the
initial systemic increase in extracellular 5-HT concentrations, basal 5-HT release in the prefrontal cortex
of rats appeared not to be affected by ATD.99 This
might suggest that in the absence of de novo
synthesis, 5-HT function can largely be maintained
from transmitter being recycled into the presynaptic
cell from the synaptic cleft. A possible decrease in
5-HT might then reflect a decrease in the storage pool
of 5-HT without affecting 5-HT release.102 In line with
this, decreased levels in whole-brain 5-HT levels at
2 h after the consumption of a TRP-free diet in cats
did not parallel changes in the functional activity of
5-HTcontaining dorsal raphe cells throughout the 4 h
after ingestion.102 Only recently, the lack of evidence
for ATD-induced alterations in 5-HT release and
neuronal activity was first critically outlined by
Feenstra et al.3
Taken together, not only the parameters used to
calculate reductions in brain TRP availability and
5-HT metabolism may be somewhat inaccurate, but
also no direct evidence exists that the ATD-induced
reductions in central 5-HT levels correlate with
parallel changes in 5-HT neuronal activity under
normal physiological conditions. In general, therefore, ATD-induced behavioural alterations cannot
easily be directly attributed to changes in 5-HT
neuronal activity. Nevertheless, ATD consistently
induces cognitive impairment in both animals and
humans and triggers lowering of mood especially in
healthy subjects with a vulnerable 5-HT system. ATD
does not directly or indirectly affect other monoaminergic systems in rats24,82,84,85,96 and no behavioural changes have been observed after depletion of other

plasma amino acids.72 Moreover, administration of a

TRP-free AA mixture in primates decreased TRP and
5-HIAA concentrations in CSF without affecting
catecholamine metabolites, suggesting that the catecholamine system is not influenced by ATD.103
ATD-induced functional changes thus seem specific for the peripheral depletion of the essential amino
acid TRP, but are more likely 5-HT mediated than
5-HT induced. Moreover, the effects of changes in
TRP availability upon 5-HT synthesis rate are generally not measured under normal physiological
circumstances, that is, in the absence of any other
regulatory changes. Therefore, changes other than
substrate availability that interfere with normal 5-HT
regulation or disruptions in substrate availability
itself might act as potential confounding factors for
the ATD-induced neurochemical effects as expected
under normal circumstances.104 Thus, the exact
underlying mechanism might go beyond a straightforward alteration in the central 5-HT system itself.
Potential alternative mechanisms underlying the

effects of ATD
Decreased nitric oxide synthase (NOS) activity
The enzyme NOS is suggested to play an important role in long-term potentiation (LTP) processes
and consequently in learning and memory.105,106
As described below, several findings suggest that
ATD may affect the activity of this enzyme. NOS
catalyzes the conversion of the amino acid arginine
(ARG) into citrulline (CIT) and nitric oxide (NO;107).
Thus, NOS inhibition results in less conversion of
ARG into CIT and NO. Therefore, the amount of NO
synthesis might be directly related to the levels of
ARG and CIT. Two independent studies reported
significant lower CIT levels in the rat hippocampus
after ATD.24,81 This decrease in CIT appeared to be
independent of changes in its precursor ARG. On the
basis of the suggested interdependency of CIT and
ARG, this finding suggests that ATD might directly
affect the activity of NOS, and that decreased CIT
concentrations most likely parallel decreases in NO.
Endogenous NO can modulate neuronal function
through interference with the release of several
neurotransmitters,108 yet its precise interaction with
the 5-HT system seems rather complex. Whereas
slight increases in NO concentrations most likely
enhance 5-HT release, moderate increases appear to
decrease 5-HT release.109 The modulation of 5-HT
release by NO might therefore depend on pre-existing
NO concentrations and the effects might be differently regulated in distinct brain areas.105
A decrease in the activity of NOS and subsequent
decreased synthesis of NO after ATD could underlie
the ATD-induced object memory impairments106 as
hippocampal inhibition of NOS is known to impair
object recognition performance in rats.106,110 As the
ATD-induced decrease in brain CIT levels is seemingly caused by an interruption in NOS activity, this
same decrease in NOS activity presumably also
decreases the synthesis of NO, which could explain

the ATD-induced object recognition impairments that
are consistently reported in rodents.79,80,83,85,87,88,111
The second messenger molecules cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) have an important role in intracellular signalling and are highly involved in learning
and memory processes.110,112114 Both cAMP and
cGMP are selectively hydrolyzed by phosphodiesterase (PDE) enzymes, and inhibition of PDE appears to
be a reliable method for improving memory processes
by increasing the levels of either cAMP, cGMP or
both.115 Administration of the PDE2 inhibitor, BAY
60-7550, and the PDE5 inhibitor, zaprinast, have
shown to increase NOS activity in rat hippocampus
and striatum, and improve object recognition performance.116 Thus, besides directly increasing presynaptic cGMP levels, the enhancing effects of PDE5 and
PDE2 inhibition upon memory performance might
also be mediated by activation of NOS postsynaptically.116 As NO can freely diffuse back into the
presynapse, it can increase cGMP levels by stimulating the synthesis of soluble guanylyl cyclase.117 An
increase in NOS activity increases NO concentrations,
which might underlie the improvement of the ATDinduced memory impairment by PDE inhibitors.87,88
See also Figure 3 for an overview of how decreased
NOS activity might explain ATD-induced object
memory impairment and its attenuation through
PDE inhibition.
705
Cerebrovascular abnormalities
Although not completely clear at present, ATD also
appears to affect local cerebral blood dynamics that may
explain behavioural ATD effects. Under normal physiological conditions, the cerebral metabolic rate of glucose
(CMRG) provides an index of changes in regional
neuronal activity, and changes in glucose metabolism
are found to be closely coupled to changes in
CBF.118,119 On this basis, abnormalities in either of
these closely linked neurophysiological parameters in
depressive subjects are thought to reflect changes in
serotonergic neurotransmission in brain areas that can
be functionally linked to the complexity of depressive
symptomatology displayed.120123 However, the 5-HT
neurotransmitter is a powerful vasoconstrictor124 and
serotonergic fibres innervating cerebral arteries, arterioles and veins have been identified.47 Thus, if
depression is represented by decreased central 5-HT
neurotransmitter concentrations, an increase in CBF
would seem more likely. Similarly, the ATD-induced
reduction in 5-HT synthesis would decrease vasoconstrictor tone, thereby most likely increasing CBF
due to vasodilatation. Surprisingly, a decrease in local
CBF following ATD has been reported in human
subjects125 and was also observed in rats.81 In the
latter, the acute decrease of peripheral TRP levels
resulted in a downward resetting of the cerebral flow
metabolism coupling relationship independently
of changes in central TRP or 5-HT. This parallels
preliminary findings of an uncoupling of flow from

706
Figure 3 Potential underlying mechanism of acute tryptophan (TRP) depletion-induced rat object memory impairment and its attenuation through phosphodiesterase
inhibition. A decrease in the ratio of TRP to the sum of
other large neutral amino acids (SLNAA) induced by acute
tryptophan depletion (ATD) potentially directly affects ()
the activity of nitric oxide synthase (NOS), thereby
decreasing central citrulline (CIT) levels without affecting
its precursor arginine (ARG). A decrease in CIT most likely
parallels a reduction in nitric oxide (NO) that subsequently
affects local cerebral blood flow (CBF) in brain areas highly
implicated in object memory processes. Indicated with ( )
is the possible mechanisms behind the improvement of
object memory deficits by inhibition of either the phosphodiesterase (PDE) enzyme type 5 (PDE5-I) or type 2 (PDE2-I),
thereby directly increasing the levels of cyclic adenosine
monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP), respectively, or type 4 (PDE4-I) increasing
cAMP only. Both second messenger molecules are highly
implicated in learning and memory processes. Inhibition of
PDE5 and PDE2 also directly activates NOS, thereby
increasing NO levels that stimulate the synthesis of soluble
guanylyl cyclase (sGC) and cGMP presynaptically. The
vasodilating properties of the PDE inhibitors might additionally attenuate ATD-induced decreases in CBF. ()
Inhibition or decrease; ( ) stimulation or increase.
metabolism in unipolar depressed patients compared

with bipolar patients and healthy controls.126
Although controversy exists about the exact location
and direction of the neurophysiological abnormalities
in depressive subjects,120,127 depressive illness is
generally characterized by decreases in CBF and
CMRG in prefrontal cortex structures. However, it
seems unlikely that a decrease in 5HT alone
accounts for the specific decreases in haemodynamic
regulation.
In addition, low CBF most likely increases the
interaction between the albumin-bound TRP complex
and the glycocalyx of the BBB, thereby causing more
TRP to dissociate from albumin.21,22 This might
provide an explanation for the fact that ATD-induced
peripheral depletion of total TRP levels does not
result in a significant decrease in brain TRP availability. Although it remains unclear how changes in
peripheral TRP concentrations can interfere with the
dynamic regulation of CBF, the findings support the
notion that the underlying mechanisms of ATD might
go beyond a straightforward 5-HT-mediated mechanism (see also Figure 3).
In general, decreased local CBF is best explained by

a loss of dilator tone. Besides the effect of 5-HT on
vasodilation, decreased local CBF has also been
reported after direct inhibition of endothelial or
neuronal NOS.128,129 As mentioned previously, direct
inhibition of NOS in the hippocampus is known to
impair object memory performance.106,110 Together,
these findings support the suggestion that the ATDinduced impairments in object memory performance
of rats could be related to one common mechanism,
that is, a decrease in NO. The involvement of NO can
be explained by the ATD-induced changes in brain
levels of CIT as described earlier. All the findings
together suggest that ATD-induced object memory
impairments are most likely caused by a decrease in
NO that reduces local CBF in hippocampal areas
highly implicated in memory processing. Additionally, this is in line with the fact that PDE2 and PDE5
inhibition have been found to increase NOS activity
in the hippocampus, which explains their potential to
attenuate the ATD-induced object memory impairments. In addition, PDE inhibitors increase central
cAMP and cGMP concentrations, which are both
well-known vasodilators.130 Their strong vasodilating
properties possibly contribute to the improvement of
object memory performance through attenuation of
the ATD-induced cerebrovascular effects (see also
Figure 3). Interestingly, low-dose PDE4 or PDE5
inhibition did not directly affect cerebrovascular
dynamics 30 min after administration.131 However,
this parallels the failure of the specific low-dosing
regimes to reverse an object memory impairment in
rodents.87,88
Decreased brain-derived neurotrophic factor (BDNF)
BDNF could also be considered as a possible factor
that is influenced by ATD. There are indications that
an ATD effect on BDNF is indirect as ATD itself did
not have a direct effect on peripheral and central
BDNF levels.82,97 Disruption of BDNF regulation has
been implicated in both depressive symptomatology
and cognitive dysfunction. Also, there are coregulating mechanisms between BDNF and the 5-HT system
in general.132 The highest levels of BDNF mRNA have
been reported in the dentate gyrus and hippocampal
CA3 and CA2 layers.133,134 Interestingly, another
study81 showed an ATD-induced decrease in local
CBF in similar areas (dentate PO, CA3 and CA2 region
of the hippocampus). Regional changes in BDNF
protein levels have been found after brief cerebrovascular events,135 consistent with the fact that BDNF has
an important neuroprotective role.136,137 Therefore, it
can be suggested that ATD, cerebrovascular effects
and BDNF could be interlinked.
In addition, BDNF has been implicated as having an
important role in neuronal plasticity, including
LTP.138 As LTP is assumed to be the underlying
substrate of learning and memory processes,139 BDNF
is thought to be a potential mediator of memory
formation in general, and is known to be required
specifically for memory consolidation.140 ATD in

human subjects selectively impairs memory consolidation,74 which has been suggested to be caused by
lower 5-HT levels in hippocampal areas.73 However,
in rats, the ATD-induced cerebrovascular changes
were independent of changes in central 5-HT levels.81
This suggests that decreased CBF in brain regions
normally high in BDNF levels could also be considered underlying BDNF-mediated alterations in
learning and memory processing after ATD.
Kynurenine (KYN) metabolites
Under normal physiological conditions, only 1 to 2%
of the amount of ingested TRP is used by the body
for the synthesis of 5-HT.19 The majority of total
ingested TRP is catabolyzed into KYN by induction
of tryptophan pyrrolase in the liver.141 Induction of
pyrrolase by the enzymes IDO (indolamine 2,3dioxygenase) and TDO (tryptophan 2,3-dioxygenase)
in the liver reduces TRP availability142 and therefore
5-HT synthesis is also influenced by IDO and TDO
activity.143,144 Stimulation of these enzymes by proinflammatory cytokines, in particular interferon-g,145
enhances the catabolism of TRP,142 thereby decreasing
the amount of TRP eventually available for 5-HT
synthesis in the brain. Moreover, TDO activity can
also be induced by corticoids.146
Tryptophan pyrrolase is the first rate-limiting
enzyme of the KYN pathway and KYN is the major
degradation product of TRP.144 KYN is further
converted into potentially neuroactive metabolites
such as kynurenine acid and quinolinic acid.147
Independently of each other, both metabolites exert
specific effects upon N-methyl-D-aspartate (NMDA)
receptors,148 which have an important role in LTP and
memory formation.149,150 NMDA receptor antagonists
have been shown to inhibit LTP and selectively
impair learning and memory,151 but antagonists can
also have a neuroprotective effect.152 Kynurenine acid
has an antagonistic effect on the NMDA receptor and
has been shown to have neuroprotective effects.153
Conversely, quinolinic acid depolarizes neurons by
activating NMDA receptors.153 As a result, quinolinic
acid can lead to neurotoxicity, similar to that found in
hypoxia and ischaemia.154156 Thus, an ATD-induced
change in the amount of KYN metabolites could
provide an explanation for the observed cerebral
oligaemia paralleling decreased peripheral TRP
levels,81 and as such KYN metabolites have already
been suggested to additionally account for the consistently reported memory impairments after ATD.2
Confounding stress effects
ATD application-related procedures might produce
stress, which might interfere with TRP metabolism,
and subsequent 5-HT synthesis as brain 5-HT,
together with other monoamines, is critically involved in the mediation of the central response to
stressors and subsequent behavioural adaptation.157
Acute stressors stimulate hypothalamicpituitary
adrenal axis activity, thereby increasing central 5-HT
necessary for stress coping.158 In rodents, overnight
food deprivation, repeated oral administrations by

gavage, blood sampling and the immobilization
necessary for applying the former are well-known
stressors.159162 Yet, these experimental procedures
are inevitably implicated in the application of the
ATD method in rats and mice. In rats, both acute and
repeated exposure to stressful stimuli have been
shown to increase glucocorticoid levels and alter
5-HT turnover and release in both the hippocampus
and frontal cortex.163 Thus, ATD application-related
procedures in animals might interfere with normal
brain TRP metabolism and subsequent 5-HT synthesis, thereby acting as confounding stress factors for
the pharmacokinetic and behavioural effects of ATD.
Stress-induced changes in the breakdown of fat
stored in fat cells (lipolysis) may also alter brain
TRP concentrations. In the same way that insulin
stimulates the uptake of albumin-bound fatty acids
by fat cells, thereby decreasing the fraction of free
TRP in plasma, stress can result in the reverse; stress
increases lipolysis and thus the amount of plasma
free-fatty acids.164 This increases the affinity of
albumin for fatty acids, thereby displacing TRP from
its albumin-binding sites. The resultant increase in
the fraction of free TRP in plasma might increase the
availability of TRP for uptake into the brain (see
Figure 4). In a recent study with mice it was found
that oral treatment by gavage combined with blood
sampling and food deprivation, which are the
standard experimental procedures, increased the
TRP/SLNAA ratio in plasma within 20 min.165 Thus,
acute stress effects might explain the moderate
depletion effects after ATD as a failure to considerably
reduce central 5-HT levels eventually. Furthermore, it
could be suggested that comparing TRP (ATD) and
TRP (control) conditions may not compare normal
versus low levels of TRP, but instead compare
moderate versus elevated TRP levels.
The effects of acute stress, including the ATDrelated application procedures, upon 5-HT autoreceptor binding might additionally provide an explanation
for the consistently reported ATD-induced memory
dysfunction as indicated by object recognition impairment in rats.79,83,8588 Several 5-HT receptors,
including 5-HT1A, are known to be highly involved
in memory function166 and ATD has been shown to
decrease 5-HT1A receptor binding.78 Also, acute
corticosterone administration directly decreases
5-HT1A autoreceptor functioning.167 Thus, a more
generalized stress effect of ATD application-related
procedures, possibly linked to a decrease in 5-HT1A
receptor function as described above, might underlie
the ATD-induced impaired object recognition performance for which intact 5-HT1A receptor function
seems to be crucial (see also Figure 4).
As a final note, it should be mentioned that these
stress effects may be especially related to the experimental procedures used in animal research. In human
research the effects of stress may be less as the
procedure of the treatment is less stressful than
the procedures used in animal research. Nevertheless,
707

708
Figure 4 Potential confounding stress effects of acute

tryptophan (TRP) depletion application-related procedures.
The method of acute TRP depletion (ATD) requires several
highly stressful procedures, such as overnight food deprivation, repeated blood sampling, oral administrations by
gavage and immobilization. Acute stress stimulates lipolysis, thereby increasing the amount of free-fatty acids in
plasma that displace TRP from its albumin-binding sites.
The resultant rise in free plasma TRP changes the ratio of
TRP to the sum of the other large neutral amino acids (TRP/
SLNAA) in favour of TRP, thereby increasing the amount of
TRP available for uptake into the brain and its subsequent
synthesis into 5-hydroxytryptamine (5-HT). Stress stimulates the secretion of corticosterone (CORT) that decreases
brain-derived neurotrophic factor (BDNF) and 5-HT1A
receptor binding. Both BDNF and 5-HT1A receptor functioning are highly implicated in learning and memory processes. The acute stress of ATD application-related
procedures might thus explain the failure of ATD to
considerably decrease 5-HT metabolism and its negative
effect upon object memory performance through increased
CORT levels.
the intake of the amino-acid drink in humans has

frequently been reported as unpleasant and some
associated stress cannot be ruled out.31,162164
Serotonin, depression and ATD

The 5-HT system is highly implicated in a wide range
of functional processes. Whereas mood-lowering
effects after ATD are only experienced in depression-free subjects predisposed to genetic 5-HT
abnormalities, altered cognitive processing after
ATD is observed in both healthy and genetically
vulnerable
subjects.
Moreover,
ATD-induced
impaired object memory performance as measured
by the object recognition task is consistently reported
in rats79,80,83,85,86 even after only moderate peripheral
TRP depletion or a single administration.87,88 For
adequate cognitive processing, the 5-HT system
interacts highly with other neurophysiological systems that might all be interrupted after ATD.81,82 This
further supports the notion that an acute decrease
in peripheral TRP levels directly interferes with
mechanisms implicated in cognitive processing that

presumably depend less upon 5-HT functioning
per se. The potential alternative mechanisms underlying ATD effects might therefore provide an explanation for the consistently reported ATD-induced
cognitive deficits across species.
Chronic exposure to stressful situations is one of
the main contributing factors to the onset of depressive illness,158 and an intact 5-HT system is critical for
mediating an adequate neurophysiological stress
response and subsequent behavioural adaptation.157
At the level of somatodendritic 5-HT autoreceptors,
adaptations to stress might be reflected by a reduction
of these receptors, thereby reducing serotonergic
feedback in dorsal raphe projection areas in an
attempt to counteract an ATD-induced decrease in
central 5-HT.78 Such an adaptive mechanism to an
acute stressor might be less efficient in subjects
predisposed to 5-HT dysfunction, which might
account for the mood-lowering effects after ATD in
these subjects.
As corticosteroid modulation of 5-HT function has
a central role in mood disorders and cognitive
functioning is highly impaired in clinical depression,
the ATD findings reviewed in this article might have
important implications regarding the mechanisms of
adaptation to stress, and the implication of the 5-HT
system in cognitive processing. As the 5-HT system is
primarily modulatory, it is not surprising that in the
clinical situation, most 5-HT-based treatments result
in only a partial symptomatic improvement. The
reviewed ATD findings thus support new insights for
alternative treatment strategies along other pathways
that interact highly with the 5-HT system. Upregulation of the cAMP and BDNF systems has already
resulted in a novel model for the mechanism of action
of antidepressants and new targets for the development of therapeutic targets.168
For an adequate interpretation of data resulting
from application of the method in both clinical and
preclinical settings, the described potential alternative mechanisms should be taken into account.
Abnormal physiological conditions, including ATD
application-related confounding stressors possibly
interfere with the high number of cellular processes
that are involved in 5-HT synthesis and metabolism,
and might trigger other neurophysiological systems
that generally interact with the 5-HT system. This
possibly also reflects the heterogeneity of major
depressive disorder in general, as all these neurophysiological processes might eventually be altered in
clinical conditions and thus potentially be treated
along different pharmacological pathways. In general,
several findings support the fact that depression may
not be caused solely by an abnormality of 5-HT
function, but more likely by a dysfunction of other
systems or brain regions modulated by 5-HT or
interacting with its dietary precursor. Similarly, the
ATD method does not seem to challenge the 5-HT
system per se, but rather triggers 5-HT-mediated
adverse events.

Conclusions
ATD currently represents the most established human
challenge test to investigate the involvement of the
5-HT system in the pathogenesis and pathophysiology
of affective disorders, including cognitive dysfunctional behaviour. However, the exact mechanism by
which ATD exerts its neurophysiological effects, and
to what extent changes in 5-HT neuronal activity
contribute to the ATD-induced functional and behavioural alterations, remain unresolved issues. This
pivotal lack of information impedes an adequate
interpretation of the results arising from application
of the method in both clinical and preclinical studies.
As most biochemical brain values are merely indicative and thus speculative in human studies, animal
models provide a better means for the exploration of
ATD-induced neurobiochemical alterations. However,
even in rats, challenging the 5-HT system by ATD
introduces speculation because of the highly controversial results between studies on both the peripheral, central and behavioural level. Moreover, no
convincing evidence exists that ATD induces alterations in central 5-HT release and subsequent neuronal
activity. Several findings support the contribution of
alternative mechanisms that go beyond a decreased
5-HT release, such as reduced NOS activity and
cerebrovascular abnormalities. In addition, experimental procedures related to the application of the
ATD method seem highly stressful and potentially
interfere with TRP metabolism, thereby confounding
ATD neurochemical and behavioural results, especially in rodents. As a decrease in CBF and confounding stress effects provide an explanation for
both the consistently reported behavioural effects and
the absent effects, it seems most likely that the
underlying mechanism of the method goes beyond a
disturbed 5-HT system. It is thus suggested that
caution is required when interpreting ATD effects in
terms of a selective serotonergic effect.
Conflict of interest
The authors declare no conflict of interest.
References
1 Fadda F, Cocco S, Stancampiano R. A physiological method to
selectively decrease brain serotonin release. Brain Res Brain Res
Protoc 2000; 5: 219222.
2 Blokland A, Lieben C, Deutz NEP, Schmitt J. Acute Tryptophan
depletion: comparing the effects of an amino acid mixture with
a gelatin-based protein in man and rats. Curr Top Nutraceut R
2004; 2: 18.
3 Feenstra MG, van der Plasse G. Tryptophan depletion and
serotonin release - a critical reappraisal. In: Muller CP, Jacobs B
(eds). Handbook of the Behavioral Neurobiology of Serotonin,
vol. 21. Academic Press: London, 2010, pp 249258.
4 Jans LA, Riedel WJ, Markus CR, Blokland A. Serotonergic
vulnerability and depression: assumptions, experimental evidence and implications. Mol Psychiatry 2007; 12: 522543.
5 Booij L, Van der Does AJ, Riedel WJ. Monoamine depletion in
psychiatric and healthy populations: review. Mol Psychiatry
2003; 8: 951973.
6 Ruhe HG, Mason NS, Schene AH. Mood is indirectly related

to serotonin, norepinephrine and dopamine levels in humans:
a meta-analysis of monoamine depletion studies. Mol Psychiatry
2007; 12: 331359.
7 Oldendorf WH. Brain uptake of radiolabeled amino acids,
amines, and hexoses after arterial injection. Am J Physiol 1971;
221: 16291639.
8 Pardridge WM. Blood-brain barrier carrier-mediated transport
and brain metabolism of amino acids. Neurochem Res 1998; 23:
635644.
9 Smith QR. Transport of glutamate and other amino acids at the
blood-brain barrier. J Nutr 2000; 130(4S Suppl): 1016S1122S.
10 Fernstrom JD, Wurtman RJ. Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 1971;
173: 149152.
11 Fernstrom JD, Wurtman RJ. Brain serotonin content: physiological regulation by plasma neutral amino acids. Science 1972; 178:
414416.
12 Gessa GL, Biggio G, Fadda F, Corsini GU, Tagliamonte A. Effect of
the oral administration of tryptophan-free amino acid mixtures
on serum tryptophan, brain tryptophan and serotonin metabolism. J Neurochem 1974; 22: 869870.
13 Fernstrom JD. Role of precursor availability in control of
monoamine biosynthesis in brain. Physiol Rev 1983; 63: 484546.
14 Fernstrom JD. Diet-induced changes in plasma amino acid
pattern: effects on the brain uptake of large neutral amino acids,
and on brain serotonin synthesis. J Neural Transm Suppl 1979;
15: 5567.
15 Biggio G, Fadda F, Fanni P, Tagliamonte A, Gessa GL. Rapid
depletion of serum tryptophan, brain tryptophan, serotonin and
5-hydroxyindoleacetic acid by a tryptophan-free diet. Life Sci
1974; 14: 13211329.
16 Oldendorf WH, Szabo J. Amino acid assignment to one of three
blood-brain barrier amino acid carriers. Am J Physiol 1976; 230:
9498.
17 Tagliamonte A, Biggio G, Vargiu L, Gessa GL. Free tryptophan in
serum controls brain tryptophan level and serotonin synthesis.
Life Sci 2 1973; 12: 277287.
18 Gessa GL, Tagliamonte A. Possible role of free serum tryptophan
in the control of brain tryptophan level and serotonin synthesis.
Adv Biochem Psychopharmacol 1974; 11: 119131.
19 Bender DA. Biochemistry of tryptophan in health and disease.
Mol Aspects Med 1983; 6: 101197.
20 Reed RG, Burrington CM. The albumin receptor effect may be due
to a surface-induced conformational change in albumin. J Biol
Chem 1989; 264: 98679872.
21 Pardridge WM, Fierer G. Transport of tryptophan into brain from
the circulating, albumin-bound pool in rats and in rabbits.
J Neurochem 1990; 54: 971976.
22 Smith QR, Momma S, Aoyagi M, Rapoport SI. Kinetics of neutral
amino acid transport across the blood-brain barrier. J Neurochem
1987; 49: 16511658.
23 Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Lowry CA.
Tryptophan metabolism in the central nervous system: medical
implications. Expert Rev Mol Med 2006; 8: 127.
24 Lieben CK, Blokland A, Westerink B, Deutz NE. Acute tryptophan
and serotonin depletion using an optimized tryptophan-free
protein-carbohydrate mixture in the adult rat. Neurochem Int
2004; 44: 916.
25 Fusar-Poli P, Allen P, McGuire P, Placentino A, Cortesi M, Perez J.
Neuroimaging and electrophysiological studies of the effects of
acute tryptophan depletion: a systematic review of the literature.
Psychopharmacology (Berl) 2006; 188: 131143.
26 Van der Does AJ. The mood-lowering effect of tryptophan
depletion: possible explanation for discrepant findings. Arch
Gen Psychiatry 2001; 58: 200202.
27 Evers EA, Tillie DE, van der Veen FM, Lieben CK, Jolles J,
Deutz NE et al. Effects of a novel method of acute tryptophan
depletion on plasma tryptophan and cognitive performance in
healthy volunteers. Psychopharmacology 2005; 178: 9299.
28 Sambeth A, Riedel W, Tillie D, Blokland A, Postma A, Schmitt J.
Memory impairments in humans after acute tryptophan
depletion using a novel gelatin-based protein drink. J Psychopharmacol 2009; 23: 5664.
709

710
29 Djagny VB, Wang Z, Xu S. Gelatin: a valuable protein for food and

pharmaceutical industries: review. Crit Rev Food Sci Nutr 2001;
41: 481492.
30 Markus CR, Panhuysen G, Tuiten A, Koppeschaar H, Fekkes D,
Peters ML. Does carbohydrate-rich, protein-poor food prevent a
deterioration of mood and cognitive performance of stress-prone
subjects when subjected to a stressful task? Appetite 1998; 31:
4965.
31 Markus CR. Effects of carbohydrates on brain tryptophan
availability and stress performance. Biol Psychol 2007; 76: 8390.
32 Benton D. Carbohydrate ingestion, blood glucose and mood.
Neurosci Biobehav Rev 2002; 26: 293308.
33 Fernstrom JD, Wurtman RJ. Elevation of plasma tryptophan by
insulin in rat. Metabolism 1972; 21: 337342.
34 Fernstrom JD, Fernstrom MH, Grubb PE. Twenty-four-hour
variations in rat blood and brain levels of the aromatic and
branched-chain amino acids: chronic effects of dietary protein
content. Metabolism 1987; 36: 643650.
35 Teff KL, Young SN, Blundell JE. The effect of protein or
carbohydrate breakfasts on subsequent plasma amino acid levels,
satiety and nutrient selection in normal males. Pharmacol,
Biochem Behav 1989; 34: 829837.
36 Harper AE, Benevenga NJ, Wohlhueter RM. Effects of ingestion of
disproportionate amounts of amino acids. Physiol Rev 1970; 50:
428558.
37 Gessa GL, Biggio G, Fadda F, Corsini GU, Tagliamonte A.
Tryptophan-free diet: a new means for rapidly decreasing brain
tryptophan content and serotonin synthesis. Acta Vitaminol
Enzymol 1975; 29: 7278.
38 Moja EA, Restani P, Corsini E, Stacchezzini MC, Assereto R,
Galli CL. Cycloheximide blocks the fall of plasma and tissue
tryptophan levels after tryptophan-free amino acid mixtures.
Life Sci 1991; 49: 11211128.
39 Neumeister A. Tryptophan depletion, serotonin, and depression: where do we stand? Psychopharmacol Bull 2003; 37:
99115.
40 Bell CJ, Hood SD, Nutt DJ. Acute tryptophan depletion. Part II:
clinical effects and implications. Aust N Z J Psychiatry 2005; 39:
565574.
41 Hood SD, Bell CJ, Nutt DJ. Acute tryptophan depletion. Part I:
rationale and methodology. Aust N Z J Psychiatry 2005; 39:
558564.
42 Young SN. The use of diet and dietary components in the study of
factors controlling affect in humans: a review. J Psychiatry
Neurosci 1993; 18: 235244.
43 Reilly JG, McTavish SF, Young AH. Rapid depletion of plasma
tryptophan: a review of studies and experimental methodology.
J Psychopharmacol 1997; 11: 381392.
44 Leonard BE. Fundamentals of Psychopharmacology, 2nd edn.
Wiley and Sons: New York, 1997.
45 Maes M, Meltzer HY. The Serotonin Hypothesis of Major
Depression. Raven Press, Ltd: New York, 1995, pp 933944.
46 Meltzer HY. Role of serotonin in depression. Ann NY Acad Sci
1990; 600: 486499; discussion 499500.
47 Steinbusch HW. Distribution of serotonin-immunoreactivity in
the central nervous system of the rat-cell bodies and terminals.
Neuroscience 1981; 6: 557618.
48 Cowen PJ, Parry-Billings M, Newsholme EA. Decreased plasma
tryptophan levels in major depression. J Affect Disord 1989; 16:
2731.
49 Asberg M, Traskman L, Thoren P. 5-HIAA in the cerebrospinal
fluid. A biochemical suicide predictor? Arch Gen Psychiatry
1976; 33: 11931197.
50 van Praag HM, de Haan S. Central serotonin metabolism and
frequency of depression. Psychiatry Res 1979; 1: 219224.
51 Naughton M, Mulrooney JB, Leonard BE. A review of the role
of serotonin receptors in psychiatric disorders. Hum Psychopharmacol 2000; 15: 397415.
52 Cryan JF, Leonard BE. 5-HT1A and beyond: the role of serotonin
and its receptors in depression and the antidepressant response.
Hum Psychopharmacol 2000; 15: 113135.
53 Neumeister A, Konstantinidis A, Stastny J, Schwarz MJ,
Vitouch O, Willeit M et al. Association between serotonin transporter gene promoter polymorphism (5HTTLPR) and behavioral
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
responses to tryptophan depletion in healthy women with and

without family history of depression. Arch Gen Psychiatry 2002;
59: 613620.
Klaassen T, Riedel WJ, van Someren A, Deutz NE, Honig A,
van Praag HM. Mood effects of 24-hour tryptophan depletion in
healthy first-degree relatives of patients with affective disorders.
Biol Psychiatry 1999; 46: 489497.
Smith KA, Fairburn CG, Cowen PJ. Relapse of depression after
rapid depletion of tryptophan. Lancet 1997; 349: 915919.
Ellenbogen MA, Young SN, Dean P, Palmour RM, Benkelfat C.
Mood response to acute tryptophan depletion in healthy
volunteers: sex differences and temporal stability. Neuropsychopharmacology 1996; 15: 465474.
Nishizawa S, Benkelfat C, Young SN, Leyton M, Mzengeza S,
de Montigny C et al. Differences between males and females in
rates of serotonin synthesis in human brain. Proc Natl Acad Sci
USA 1997; 94: 53085313.
Neumeister A, Nugent AC, Waldeck T, Geraci M, Schwarz M,
Bonne O et al. Neural and behavioral responses to tryptophan
depletion in unmedicated patients with remitted major depressive disorder and controls. Arch Gen Psychiatry 2004; 61:
765773.
Moreno FA, Gelenberg AJ, Heninger GR, Potter RL, McKnight KM,
Allen J et al. Tryptophan depletion and depressive vulnerability.
Delgado PL, Charney DS, Price LH, Aghajanian GK, Landis H,
Heninger GR. Serotonin function and the mechanism of antidepressant action. Reversal of antidepressant-induced remission
by rapid depletion of plasma tryptophan. Arch Gen Psychiatry
1990; 47: 411418.
Delgado PL, Price LH, Miller HL, Salomon RM, Licinio J,
Krystal JH et al. Rapid serotonin depletion as a provocative
challenge test for patients with major depression: relevance to
antidepressant action and the neurobiology of depression.
Psychopharmacol Bull 1991; 27: 321330.
Delgado PL, Miller HL, Salomon RM, Licinio J, Krystal JH,
Moreno FA et al. Tryptophan-depletion challenge in depressed
patients treated with desipramine or fluoxetine: implications for
the role of serotonin in the mechanism of antidepressant action.
Delgado PL, Charney DS, Price LH, Landis H, Heninger GR.
Neuroendocrine and behavioral effects of dietary tryptophan
restriction in healthy subjects. Life Sci 1989; 45: 23232332.
Young SN, Smith SE, Pihl RO, Ervin FR. Tryptophan depletion
causes a rapid lowering of mood in normal males. Psychopharmacology (Berl) 1985; 87: 173177.
Carpenter LL, Anderson GM, Pelton GH, Gudin JA, Kirwin PD,
Price LH et al. Tryptophan depletion during continuous CSF
sampling in healthy human subjects. Neuropsychopharmacology
1998; 19: 2635.
Evers EA, van der Veen FM, Jolles J, Deutz NE, Schmitt JA.
Acute tryptophan depletion improves performance and modulates the BOLD response during a Stroop task in healthy females.
Neuroimage 2006; 32: 248255.
Moore P, Landolt HP, Seifritz E, Clark C, Bhatti T, Kelsoe J et al.
Clinical and physiological consequences of rapid tryptophan
depletion. Neuropsychopharmacology 2000; 23: 601622.
Williams WA, Shoaf SE, Hommer D, Rawlings R, Linnoila M.
Effects of acute tryptophan depletion on plasma and cerebrospinal fluid tryptophan and 5-hydroxyindoleacetic acid in
normal volunteers. J Neurochem 1999; 72: 16411647.
Moreno FA, McGavin C, Malan TP, Gelenberg AJ, Heninger GR,
Mathe AA et al. Tryptophan depletion selectively reduces CSF
5-HT metabolites in healthy young men: results from single
lumbar puncture sampling technique. Int J Neuropsychopharmacol 2000; 3: 277283.
Booij L, Van der Does AJ, Haffmans PM, Riedel WJ, Fekkes D,
Blom MJ. The effects of high-dose and low-dose tryptophan
depletion on mood and cognitive functions of remitted depressed
patients. J Psychopharmacol 2005; 19: 267275.
Sambeth A, Blokland A, Harmer CJ, Kilkens TO, Nathan PJ,
Porter RJ et al. Sex differences in the effect of acute tryptophan
depletion on declarative episodic memory: a pooled analysis of
nine studies. Neurosci Biobehav Rev 2007; 31: 516529.

72 Klaassen T, Riedel WJ, Deutz NE, van Someren A, van Praag HM.
Specificity of the tryptophan depletion method. Psychopharmacology 1999; 141: 279286.
73 Riedel WJ. Cognitive changes after acute tryptophan depletion:
what can they tell us? Psychol Med 2004; 34: 38.
74 Riedel WJ, Klaassen T, Deutz NE, van Someren A, van Praag HM.
Tryptophan depletion in normal volunteers produces selective
impairment in memory consolidation. Psychopharmacology
(Berl) 1999; 141: 362369.
75 Mendelsohn D, Riedel WJ, Sambeth A. Effects of acute tryptophan depletion on memory, attention and executive functions:
a systematic review. Neurosci Biobehav Rev 2009; 33: 926952.
76 van der Staay FJ. Animal models of behavioral dysfunctions:
basic concepts and classifications, and an evaluation strategy.
Brain Res Rev 2006; 52: 131159.
77 Blokland A, Lieben C, Deutz NE. Anxiogenic and depressivelike effects, but no cognitive deficits, after repeated moderate
tryptophan depletion in the rat. J Psychopharmacol 2002; 16:
3949.
78 Cahir M, Ardis T, Reynolds GP, Cooper SJ. Acute and chronic
tryptophan depletion differentially regulate central 5-HT1A and
5-HT 2A receptor binding in the rat. Psychopharmacology 2007;
190: 497506.
79 Jans LA, Lieben CK, Blokland A. Influence of sex and estrous
cycle on the effects of acute tryptophan depletion induced by a
gelatin-based mixture in adult Wistar rats. Neuroscience 2007;
147: 304317.
80 Olivier JD, Jans LA, Korte-Bouws GA, Korte SM, Deen PM,
Cools AR et al. Acute tryptophan depletion dose dependently
impairs object memory in serotonin transporter knockout rats.
81 van Donkelaar EL, Ferrington L, Blokland A, Steinbusch HW,
Prickaerts J, Kelly PA. Acute tryptophan depletion in rats alters
the relationship between cerebral blood flow and glucose
metabolism independent of central serotonin. Neuroscience
2009; 163: 683694.
82 van Donkelaar EL, van den Hove DL, Blokland A, Steinbusch HW,
Prickaerts J. Stress-mediated decreases in brain-derived neurotrophic factor as potential confounding factor for acute tryptophan
depletion-induced neurochemical effects. Eur Neuropsychopharmacol 2009; 19: 812821.
83 Lieben CK, van Oorsouw K, Deutz NE, Blokland A. Acute
tryptophan depletion induced by a gelatin-based mixture impairs
object memory but not affective behavior and spatial learning in
the rat. Behav Brain Res 2004; 151: 5364.
84 Brown CM, Fletcher PJ, Coscina DV. Acute amino acid loads that
deplete brain serotonin fail to alter behavior. Pharmacol Biochem
Behav 1998; 59: 115121.
85 Jans L, Korte-Bouws G, Korte S, Blokland A. The effects of acute
tryptophan depletion on affective behaviour and cognition in
Brown Norway and Sprague Dawley rats. J Psychopharmacol
2008; 24: 605614.
86 Jans LA, Blokland A. Influence of chronic mild stress on the
behavioural effects of acute tryptophan depletion induced by
a gelatin-based mixture. Behav Pharmacol 2008; 19: 706715.
87 Rutten K, Lieben C, Smits L, Blokland A. The PDE4 inhibitor
rolipram reverses object memory impairment induced by acute
tryptophan depletion in the rat. Psychopharmacology (Berl) 2007;
192: 275282.
88 van Donkelaar EL, Rutten K, Blokland A, Akkerman S,
Steinbusch HW, Prickaerts J. Phosphodiesterase 2 and 5 inhibition attenuates the object memory deficit induced by acute
tryptophan depletion. Eur J Pharmacol 2008; 600: 98104.
89 Badawy AB. Plasma free tryptophan revisited: what you need to
know and do before measuring it. J Psychopharmacol 2010; 24:
809815.
90 Oldendorf WH, Szabo J. Amino acid assignment to one of three
blood-brain barrier amino acid carriers. Am J Physiol 1976; 230:
9498.
91 Crespi F. In vivo voltammetry with micro-biosensors for analysis
of neurotransmitter release and metabolism. J Neurosci Methods
1990; 34: 5365.
92 Crespi F, Garratt JC, Sleight AJ, Marsden CA. In vivo evidence
that 5-hydroxytryptamine (5-HT) neuronal firing and release are
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
not necessarily correlated with 5-HT metabolism. Neuroscience

1990; 35: 139144.
Westerink BH. Brain microdialysis and its application for the
study of animal behaviour. Behav Brain Res 1995; 70: 103124.
Shannon NJ, Gunnet JW, Moore KE. A comparison of biochemical
indices of 5-hydroxytryptaminergic neuronal activity following
electrical stimulation of the dorsal raphe nucleus. J Neurochem
1986; 47: 958965.
Boadle-Biber MC. Regulation of serotonin synthesis. Prog Biophys
Mol Biol 1993; 60: 115.
Ardis TC, Cahir M, Elliott JJ, Bell R, Reynolds GP, Cooper SJ.
Effect of acute tryptophan depletion on noradrenaline and
dopamine in the rat brain. J Psychopharmacol 2009; 23: 5155.
Cahir M, Ardis TC, Elliott JJ, Kelly CB, Reynolds GP, Cooper SJ.
Acute tryptophan depletion does not alter central or plasma
brain-derived neurotrophic factor in the rat. Eur Neuropsychopharmacol 2008; 18: 317322.
Fernstrom JD, Hirsch MJ. Brain serotonin synthesis: reduction in
corn-malnourished rats. J Neurochem 1977; 28: 877979.
van der Plasse G, Meerkerk DT, Lieben CK, Blokland A,
Feenstra MG. Lack of evidence for reduced prefrontal cortical
serotonin and dopamine efflux after acute tryptophan depletion.
Bel N, Artigas F. Reduction of serotonergic function in rat brain
by tryptophan depletion: effects in control and fluvoxaminetreated rats. J Neurochem 1996; 67: 669676.
Stancampiano R, Melis F, Sarais L, Cocco S, Cugusi C, Fadda F.
Acute administration of a tryptophan-free amino acid mixture
decreases 5-HT release in rat hippocampus in vivo. Am J Physiol
1997; 272(3 Part 2): R991R994.
Trulson ME. Dietary tryptophan does not alter the function of
brain serotonin neurons. Life Sci 1985; 37: 10671072.
Young SN, Ervin FR, Pihl RO, Finn P. Biochemical aspects of
tryptophan depletion in primates. Psychopharmacology (Berl)
1989; 98: 508511.
Lenard NR, Dunn AJ. Mechanisms and significance of the
increased brain uptake of tryptophan. Neurochem Res 2005; 30:
15431548.
Smith JC, Whitton PS. Nitric oxide modulates N-methyl-Daspartate-evoked serotonin release in the raphe nuclei and
frontal cortex of the freely moving rat. Neurosci Lett 2000; 291:
58.
Blokland A, Prickaerts J, Honig W, de Vente J. State-dependent
impairment in object recognition after hippocampal NOS inhibition. NeuroReport 1998; 9: 42054208.
Dawson VL, Dawson TM. Nitric oxide actions in neurochemistry.
Neurochem Int 1996; 29: 97110.
Prast H, Philippu A. Nitric oxide as modulator of neuronal
function. Prog Neurobiol 2001; 64: 5168.
Kaehler ST, Singewald N, Sinner C, Philippu A. Nitric oxide
modulates the release of serotonin in the rat hypothalamus. Brain
Res 1999; 835: 346349.
Prickaerts J, de Vente J, Honig W, Steinbusch HW, Blokland A.
cGMP, but not cAMP, in rat hippocampus is involved in early
stages of object memory consolidation. Eur J Pharmacol 2002;
436: 8387.
Lieben CK, Blokland A, Sik A, Sung E, van Nieuwenhuizen P,
Schreiber R. The selective 5-HT6 receptor antagonist Ro4368554
restores memory performance in cholinergic and serotonergic
models of memory deficiency in the rat. Neuropsychopharmacology 2005; 30: 21692179.
Bernabeu R, Schmitz P, Faillace MP, Izquierdo I, Medina JH.
Hippocampal cGMP and cAMP are differentially involved in
memory processing of inhibitory avoidance learning. NeuroReport 1996; 7: 585588.
Bernabeu R, Schroder N, Quevedo J, Cammarota M, Izquierdo I,
Medina JH. Further evidence for the involvement of a hippocampal cGMP/cGMP-dependent protein kinase cascade in
memory consolidation. NeuroReport 1997; 8: 22212224.
Rutten K, Prickaerts J, Hendrix M, van der Staay FJ, Sik A,
Blokland A. Time-dependent involvement of cAMP and cGMP in
consolidation of object memory: studies using selective phosphodiesterase type 2, 4 and 5 inhibitors. Eur J Pharmacol 2007;
558: 107112.
711

712
115 Blokland A, Schreiber R, Prickaerts J. Improving memory: a role

for phosphodiesterases. Curr Pharm Des 2006; 12: 25112523.
116 Domek-Lopacinska K, Strosznajder JB. The effect of selective
inhibition of cyclic GMP hydrolyzing phosphodiesterases 2 and 5
on learning and memory processes and nitric oxide synthase
activity in brain during aging. Brain Res 2008; 1216: 6877.
117 Murad F, Mittal CK, Arnold WP, Katsuki S, Kimura H. Guanylate
cyclase: activation by azide, nitro compounds, nitric oxide, and
hydroxyl radical and inhibition by hemoglobin and myoglobin.
Adv Cyclic Nucleotide Res 1978; 9: 145158.
118 Kuschinsky W. Coupling of function, metabolism, and blood flow
in the brain. Neurosurg Rev 1991; 14: 163168.
119 Sokoloff L. Relationships among local functional activity, energy
metabolism, and blood flow in the central nervous system. Fed
Proc 1981; 40: 23112316.
120 Drevets WC. Neuroimaging studies of mood disorders. Biol
Psychiatry 2000; 48: 813829.
121 Drevets WC, Frank E, Price JC, Kupfer DJ, Holt D, Greer PJ et al.
PET imaging of serotonin 1A receptor binding in depression.
122 Drevets WC, Ongur D, Price JL. Reduced glucose metabolism
in the subgenual prefrontal cortex in unipolar depression.
Mol Psychiatry 1998; 3: 190191.
123 Drevets WC, Ongur D, Price JL. Neuroimaging abnormalities
in the subgenual prefrontal cortex: implications for the pathophysiology of familial mood disorders. Mol Psychiatry 1998; 3:
220226, 190191.
124 Edvinsson L, MacKenzie ET. Amine mechanisms in the cerebral
circulation. Pharmacol Rev 1976; 28: 275348.
125 Talbot PS, Cooper SJ. Anterior cingulate and subgenual prefrontal
blood flow changes following tryptophan depletion in healthy
males. Neuropsychopharmacology 2006; 31: 17571767.
126 Dunn RT, Willis MW, Benson BE, Repella JD, Kimbrell TA,
Ketter TA et al. Preliminary findings of uncoupling of flow and
metabolism in unipolar compared with bipolar affective illness
and normal controls. Psychiatry Res 2005; 140: 181198.
127 Soares JC, Mann JJ. The functional neuroanatomy of mood
disorders. J Psychiatr Res 1997; 31: 393432.
128 Kelly PA, Ritchie IM, Arbuthnott GW. Inhibition of neuronal
nitric oxide synthase by 7-nitroindazole: effects upon local
cerebral blood flow and glucose use in the rat. J Cereb Blood
Flow Metab 1995; 15: 766773.
129 Kelly PA, Thomas CL, Ritchie IM, Arbuthnott GW. Cerebrovascular autoregulation in response to hypertension induced by
NG-nitro-L-arginine methyl ester. Neuroscience 1994; 59: 1320.
130 Dundore RL, Clas DM, Wheeler LT, Habeeb PG, Bode DC,
Buchholz RA et al. Zaprinast increases cyclic GMP levels in
plasma and in aortic tissue of rats. Eur J Pharmacol 1993; 249:
293297.
131 Rutten K, Van Donkelaar EL, Ferrington L, Blokland A, Bollen E,
Steinbusch HW et al. Phosphodiesterase inhibitors enhance
object memory independent of cerebral blood flow and glucose
utilization in rats. Neuropsychopharmacology 2009; 34:
19141925.
132 Mattson MP, Maudsley S, Martin B. BDNF and 5-HT: a dynamic
duo in age-related neuronal plasticity and neurodegenerative
disorders. Trends Neurosci 2004; 27: 589594.
133 Ernfors P, Wetmore C, Olson L, Persson H. Identification of cells
in rat brain and peripheral tissues expressing mRNA for members
of the nerve growth factor family. Neuron 1990; 5: 511526.
134 Smith MA, Makino S, Kvetnansky R, Post RM. Stress and
glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus.
J Neurosci 1995; 15(3 Part 1): 17681777.
135 Kokaia Z, Nawa H, Uchino H, Elmer E, Kokaia M, Carnahan J
et al. Regional brain-derived neurotrophic factor mRNA and
protein levels following transient forebrain ischemia in the rat.
Brain Res Mol Brain Res 1996; 38: 139144.
136 Larsson E, Nanobashvili A, Kokaia Z, Lindvall O. Evidence for
neuroprotective effects of endogenous brain-derived neurotrophic factor after global forebrain ischemia in rats. J Cereb
Blood Flow Metab 1999; 19: 12201228.
137 Lindvall O, Kokaia Z, Bengzon J, Elmer E, Kokaia M. Neurotrophins and brain insults. Trends Neurosci 1994; 17: 490496.
138 Lu Y, Christian K, Lu B. BDNF: a key regulator for protein

synthesis-dependent LTP and long-term memory? Neurobiol
Learn Mem 2008; 89: 312333.
139 Bliss TV, Collingridge GL. A synaptic model of memory:
long-term potentiation in the hippocampus. Nature 1993; 361:
3139.
140 Lee JL, Everitt BJ, Thomas KL. Independent cellular processes
for hippocampal memory consolidation and reconsolidation.
Science 2004; 304: 839843.
141 Stone TW, Darlington LG. Endogenous kynurenines as targets for
drug discovery and development. Nat Rev Drug Discov 2002; 1:
609620.
142 Botting NP. Chemistry and neurochemistry of the kynurenine
pathway of tryptophan metabolism. Chem Soc Rev 1995; 24:
401412.
143 Smith SA, Pogson CI. The metabolism of L-tryptophan by
isolated rat liver cells. Effect of albumin binding and amino acid
competition on oxidatin of tryptophan by tryptophan 2,3dioxygenase. Biochem J 1980; 186: 977996.
144 Moroni F. Tryptophan metabolism and brain function: focus on
kynurenine and other indole metabolites. Eur J Pharmacol 1999;
375: 87100.
145 Hu B, Hissong BD, Carlin JM. Interleukin-1 enhances indoleamine 2,3-dioxygenase activity by increasing specific mRNA
expression in human mononuclear phagocytes. J Interferon
Cytokine Res 1995; 15: 617624.
146 Turner EH, Loftis JM, Blackwell AD. Serotonin a la carte:
supplementation with the serotonin precursor 5-hydroxytryptophan. Pharmacol Ther 2006; 109: 325338.
147 Young SN, Leyton M. The role of serotonin in human mood and
social interaction. Insight from altered tryptophan levels.
Pharmacol Biochem Behav 2002; 71: 857865.
148 Stone TW. Neuropharmacology of quinolinic and kynurenic
acids. Pharmacol Rev 1993; 45: 309379.
149 Morris RG. Long-term potentiation and memory. Philos Trans R
Soc Lond B Biol Sci 2003; 358: 643647.
150 Morris RG, Davis S, Butcher SP. Hippocampal synaptic plasticity
and NMDA receptors: a role in information storage? Philos Trans
R Soc Lond B Biol Sci 1990; 329: 187204.
151 Morris RG, Anderson E, Lynch GS, Baudry M. Selective impairment of learning and blockade of long-term potentiation by
an N-methyl-D-aspartate receptor antagonist, AP5. Nature 1986;
319: 774776.
152 Lipton SA. Paradigm shift in neuroprotection by NMDA receptor
blockade: memantine and beyond. Nat Rev Drug Discov 2006; 5:
160170.
153 Stone TW, Perkins MN. Quinolinic acid: a potent endogenous
excitant at amino acid receptors in CNS. Eur J Pharmacol 1981;
72: 411412.
154 Stone TW, Forrest CM, Mackay GM, Stoy N, Darlington LG.
Tryptophan, adenosine, neurodegeneration and neuroprotection.
Metab Brain Dis 2007; 22: 337352.
155 Stone TW. Endogenous neurotoxins from tryptophan. Toxicon
2001; 39: 6173.
156 Schwarcz R, Whetsell Jr WO, Mangano RM. Quinolinic acid:
an endogenous metabolite that produces axon-sparing lesions in
rat brain. Science 1983; 219: 316318.
157 de Kloet ER, Joels M, Holsboer F. Stress and the brain: from
adaptation to disease. Nat Rev Neurosci 2005; 6: 463475.
158 van Praag HM. Can stress cause depression? Prog Neuropsychopharmacol Biol Psychiatry 2004; 28: 891907.
159 Curzon G, Joseph MH, Knott PJ. Effects of immobilization and
food deprivation on rat brain tryptophan metabolism. J Neurochem 1972; 19: 19671974.
160 Chamas FM, Underwood MD, Arango V, Serova L, Kassir SA,
Mann JJ et al. Immobilization stress elevates tryptophan hydroxylase mRNA and protein in the rat raphe nuclei. Biol Psychiatry
2004; 55: 278283.
161 Nakahara D, Nakamura M. Differential effect of immobilization
stress on in vivo synthesis rate of monoamines in medial
prefrontal cortex and nucleus accumbens of conscious rats.
Synapse 1999; 32: 238242.
162 Jahng JW, Kim JG, Kim HJ, Kim BT, Kang DW, Lee JH.
Chronic food restriction in young rats results in depression- and

163
164
165
166
anxiety-like behaviors with decreased expression of serotonin

reuptake transporter. Brain Res 2007; 1150: 100107.
Chaouloff F. Physiopharmacological interactions between stress
hormones and central serotonergic systems. Brain Res Brain Res
Rev 1993; 18: 132.
McMenamy RH. Binding of indole analogues to human serum
albumin. Effects of fatty acids. J Biol Chem 1965; 240: 42354243.
van Donkelaar EL, Blokland A, Lieben CK, Kenis G, Ferrington L,
Kelly PA et al. Acute tryptophan depletion in C57BL/6 mice does
not induce central serotonin reduction or affective behavioural
changes. Neurochem Int 2010; 56: 2134.
King MV, Marsden CA, Fone KC. A role for the 5-HT(1A),
5-HT(4) and 5-HT(6) receptors in learning and memory. Trends
Pharmacol Sci 2008; 29: 482492.
167 Laaris N, Haj-Dahmane S, Hamon M, Lanfumey L. Glucocorticoid

receptor-mediated inhibition by corticosterone of 5-HT1A autoreceptor functioning in the rat dorsal raphe nucleus. Neuropharmacology 1995; 34: 12011210.
168 Duman RS. Novel therapeutic approaches beyond the serotonin
receptor. Biol Psychiatry 1998; 44: 324335.
169 Jans LA, Lieben CK, Smits LT, Blokland A. Pharmacokinetics of
acute tryptophan depletion using a gelatin-based protein in male
and female Wistar rats. Amino Acids 2009; 37: 349357.
170 van Donkelaar EL, Kelly PA, Dawson N, Blokland A, Prickaerts J,
Steinbusch HW et al. Acute tryptophan depletion potentiates
3,4-methylenedioxymethamphetamine-induced cerebrovascular
hyperperfusion in adult male Wistar rats. J Neurosci Res 2010;
88: 15571568.
713

Mechanism of Acute Tryptophan Depletion

Загружено:

Сведения о документе

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

Mechanism of Acute Tryptophan Depletion

Загружено:

Авторское право:

Доступные форматы

Molecular Psychiatry (2011) 16, 695713

& 2011 Macmillan Publishers Limited All rights reserved 1359-4184/11

Mechanism of acute tryptophan depletion:

reflected in dysfunctional behavioural output. ATDinduced behavioural changes in human subjects

Underlying mechanisms of acute tryptophan depletion

in 5-HT functionality. Furthermore, this review

Methodological aspects of ATD

From bound to free plasma tryptophan: the brain influx

Underlying mechanisms of acute tryptophan depletion

Figure 2 Overview of tryptophan metabolism from food

a condition in which animals were treated with

a gelatin-based proteincarbohydrate mixture.2 By

Carbohydrate and dietary protein

Underlying mechanisms of acute tryptophan depletion

leads to an increased availability of TRP in the brain

Central versus peripheral effects of ATD

levels48 and lower levels of the inactive 5-HT

Underlying mechanisms of acute tryptophan depletion

(in particular, consolidation processes74), decision

Methodological considerations: do we actually

increasing the TRP-free pool and subsequent uptake

The 5-HIAA/5-HT ratio in the brain

Adult male Sprague

4 months old male

4 months old male

4 months old male

Adult male Sprague

No differences between 1 and 3

Object recognition impairment at T4

TYR ctx: 44%

Lowering of 5-HT in both hpc and

No cognitive impairment or changes

TRP suppletion effect in fcx: 5-HT

Underlying mechanisms of acute tryptophan depletion

Adult male Wistar

Adult male Sprague

4 months old male

4 months old male

3 months old male

2 months old male

Free plasma TRP

Free plasma TRP

Decreased ratio 5-HIAA/5-HT in hpc

In general, higher peripheral TRP

No effects upon 5-HT turnover in

Extracellular 5-HT and DA

Lower sucrose intake and blunted

Stronger ATD-induced behavioural

Object recognition impairment only

Object recognition impairment at T1

No differences in general activity

Underlying mechanisms of acute tryptophan depletion

6 months old male

4 months old male

Adult male Sprague

Object recognition impairment in

Object recognition impairment

Object recognition impairment at

No reduction of 5-HT or 5-HIAA in

Decreased TRP/SLNAA compared

Underlying mechanisms of acute tryptophan depletion

3 months old male

3 months old male

3 months old male