Mechanism of Acute Tryptophan Depletion

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

1Department of Psychiatry and Neuropsychology, Faculty of Health, Medicine and Life Sciences, School for MentalHealth and Neuroscience, Maastricht University, Maastricht, The Netherlands; 2Department of Neuropsychology andPsychopharmacology, Faculty of Psychology and Neuroscience, Maastricht University, Maastricht, The Netherlands and3Cerebrovascular 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 essentialamino acid tryptophan (TRP), the dietary serotonin (5-hydroxytryptamine (5-HT)) precursor,has been applied in many experimental studies. ATD application leads to decreasedavailability of TRP in the brain and its synthesis into 5-HT. It is therefore assumed that adecrease in 5-HT release and subsequent blunted neurotransmission is the underlyingmechanism for the behavioural effects of ATD. However, direct evidence that ATD decreasesextracellular 5-HT concentrations is lacking. Furthermore, several studies provide support foralternative underlying mechanisms of ATD. This may question the utility of the method as aselective serotonergic challenge tool. As ATD is extensively used for investigating the role of5-HT in cognitive functions and psychiatric disorders, the potential of alternative mechanismsand possible confounding factors should be taken into account. It is suggested that caution isrequired 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 repre-sents the most established human challenge testto investigate the involvement of the serotonin(5-hydroxytryptamine; 5HT) system in the patho-genesis and pathophysiology of affective disorders.The method is nontoxic and nonintrusive, therebyproviding the option to repeatedly manipulate thecentral 5-HT system in vivo and assess the behaviouraleffects of reduced 5-HT metabolism in the brain.1 Thereduction of brain 5-HT in a reversible manner reflectsthe main methodological advantage of the tool,permitting application of the same basic method inboth human subjects and rodents. This is consideredvaluable for comparing neurophysiological changeslinked to behavioural effects across species.2

As intact 5-HT neurotransmission is necessary for awide range of physiological and functional processes,a disruption in this system can easily provoke diversepathophysiological abnormalities, most of which are

reflected in dysfunctional behavioural output. ATD-induced behavioural changes in human subjectsand laboratory animals are normally attributed todecreased 5-HT release, reflecting altered 5-HTneuronal activity. However, it is not fully clear whatmechanisms underlie the neurophysiological effectsof ATD and to what extent changes in 5-HT neuronalactivity contribute to the ATD-induced functional andbehavioural alterations. Also, no convincing evidenceexists for affected central 5-HT release following ATDin animals.3

The ATD method seems important in the investiga-tion of 5-HT-related vulnerability factors implicatedin the onset of depression,4 and previously themonoamine systems were considered to be primarilyresponsible for the onset of depressive disorders.5

However, the lack of mood-lowering effects after ATDin healthy subjects may not support a direct causalrelationship between acute decreased 5-HT metabo-lism and major depressive disorder.6 Moreover, aswill be discussed in this review, evidence existsthat ATD possibly exerts its neurochemical andbehavioural effects through other mechanisms thatmight go beyond a straightforward decrease in 5-HTmetabolism. This review covers an extensive evalua-tion of both the methodology and the diverse neuro-chemical and behavioural effects of ATD, includinga critical assessment of the common parametersused for indicating presumed ATD-induced changes

Received 12 March 2010; revised 4 January 2011; accepted 19January 2011; published online 22 February 2011

Correspondence: Dr EL van Donkelaar, Department of Psychiatryand Neuropsychology, Division of Neuroscience, Faculty ofHealth, Medicine and Life Sciences, School for Mental Healthand Neuroscience, Maastricht University, P.O. Box 616, 6200 MDMaastricht, The Netherlands.E-mail: [email protected]

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

www.nature.com/mp

in 5-HT functionality. Furthermore, this reviewaims to outline alternatives for potential underlyingmechanisms of the method that might go beyond adisturbed 5-HT system and thus draw into questionthe utility of ATD as a serotonergic challenge tool inexperimental research in general and depressionresearch 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 thebioavailability of this essential amino acid is theprincipal rate-limiting factor. Thus, variations indietary intake of TRP can have profound effects uponthe synthesis of this very important neurotransmittersubstance and may impact upon those aspects ofbrain function that are influenced by serotonergicneurons. It is this fact that underpins the use of ATDas both an experimental tool and a clinical probe fordepressive illness.

From plasma to brain tryptophanAmino acids can only be transported from the bloodthrough the capillary endothelial cells of the blood-brain barrier (BBB) into the brain by carrier-mediatedtransporter systems in the capillary cell plasmamembranes.7 Given that the surface area of the BBBis much smaller compared with the surface area ofbrain cell membranes, it is this initial transportthrough the BBB that limits the uptake of plasmaTRP into the brain.8 The branched-chain amino acids(leucine, isoleucine and valine) together with thearomatic amino acids (phenylalanine, tyrosine andTRP) are subclassified as large neutral amino acids(LNAAs). Of the nine different amino acid transportsystems identified at the BBB, the so-called TransportSystem L is only half saturated under normalphysiological conditions and mediates high-affinity,sodium-independent uptake of all LNAAs.8,9 Conse-quently, in order to bind to the L-amino-acid transportcarrier and subsequent transport into the brain, TRPhas to compete heavily with the other LNAAs.1012

The availability of TRP in the brain thus dependsupon the ratio of TRP to the sum of the other LNAAs(TRP/SLNAA), and a decrease in this ratio in plasmais normally used as the best predictor of reducedavailability of TRP in the brain and subsequentsynthesis into 5-HT.13,14

From bound to free plasma tryptophan: the brain influxparameterApproximately 90% of all TRP molecules circulatingin the blood are bound to serum albumin. Althoughpositive correlations between serum free-TRP andwhole brain TRP levels have been reported in rats,15,16

the dissociation of TRP from albumin by endogenousand exogenous ligands has been shown to increasethe entry of TRP into the brain, thereby enhancingcentral 5-HT synthesis.17,18 This observation suggeststhat only free TRP is available for transport into thebrain.15 As the changes in TRP-free levels can takeplace independently of changes in total TRP levels,19

this would make a distinction between free andbound TRP necessary for estimating its availabilityin the brain. However, accumulating evidence indi-cates that total peripheral TRP concentrations (freeplus bound) more accurately reflect the rate of influxof TRP into the brain. It has been shown that TRP isonly loosely bound to albumin and although albuminitself cannot cross the BBB, it appears to be a highlyflexible protein undergoing reversible conformationalchanges.20 These conformational changes, whichoccur during transport of TRP from the circulatingalbumin-bound pool, enhance the dissociation of TRPfrom the albumin-binding sites within the cerebralmicrovasculature and appear to be highly dependentupon cerebral haemodynamics.21 Low cerebral bloodflow (CBF) is likely to increase the interactionbetween the albumin-bound TRP complex and theglycocalyx of the BBB, thereby causing more TRPto dissociate from albumin.21,22 This implies thattemporally dynamic or spatial differences in localCBF may influence the rate of central TRP uptake ingeneral 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 mustalso be taken into account to calculate the availabilityof TRP in the brain, as TRP can easily dissociate fromalbumin near the BBB, thereby increasing the TRP-free pool and subsequent uptake into the brain (seealso Figure 2).

SolugelIn most studies, pure amino-acid mixtures withoutTRP have been used to reduce plasma TRP levels.However, one disadvantage of the amino-acid mixtureis that differences in the distinct amino acids werefound between the control condition (TRP ) and

Figure 1 Central serotonin synthesis and metabolism. In the brain, tryptophan (TRP) is first hydroxylated into5-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 metabolite5-hydroxyindoleacetic acid (5-HIAA).

Underlying mechanisms of acute tryptophan depletionEL van Donkelaar et al

696

Molecular Psychiatry

a condition in which animals were treated withsaline.2,24 In that case, the control condition is notan optimal and representative control condition. Analternative manner to reduce central 5-HT concentra-tions by lowering the levels of its dietary precursorTRP in plasma can be achieved by administration ofspecific TRP-free nutritional mixtures.Besides the TRP-free or TRP-low balanced diets

and pure amino-acid mixtures without TRP, a moreadvanced technique is the oral administration of

a gelatin-based proteincarbohydrate mixture.2 Byadding a specific amount of TRP to the controlmixture, the effects of peripheral TRP suppletion, asoften observed with traditional amino-acid mixturesin humans,25,26 are avoided and thus do not causemisinterpretation of the ATD effects.2,27,28 Gelatin isderived from the selective hydrolysis of collagenprotein, which is easily digestible and naturally lacksthe essential amino-acid TRP.29 The gelatin hydro-lysate used for the nutritional mixture is gelatin in anenzymatic hydrolyzed form, commercially availableas Solugel (PB Gelatins, Tessenderlo, Belgium).Solugel no longer consists of a combination of a fewselective amino acids, but comprises a broad range ofamino acids in the form of peptides, which makes itcomparable with standard diets. Moreover, it is waterdispensable and unique for its gel-forming ability.24 Inaddition, a specific amount of carbohydrate is mixedwith the Solugel, which adds an essential caloricvalue, thereby making the nutritional mixture evenmore similar to conventional food intake. Moreover,mixing proteins with carbohydrates avoids anyunwanted effects upon amino-acid availability inthe blood (see below and Figure 2) as is normallyfound with unbalanced diets containing highamounts of carbohydrate or protein only.3032

Carbohydrate and dietary proteinVarious metabolic processes are triggered when aprotein/carbohydrate meal, as is the case with ATDmethods, is ingested. These processes can have effectson plasma TRP levels too. After the intake of carbo-hydrates, blood glucose levels raise, thereby stimulat-ing the pancreas to release the anabolic hormoneinsulin. The secretion of insulin stimulates glucose tobe taken up by the cells for subsequent normalizationof glucose levels in the blood. Concomitantly with theinsulin-induced drop in blood glucose levels, plasmaTRP levels increase whereas the concentration ofmost other amino acids in plasma decreases.33 Whena carbohydrate-rich meal contains no additionalprotein, activation of the insulin response increasesprotein synthesis, thereby stimulating the uptake ofalmost all amino acids (mainly the branched-chainamino acids) into muscle tissue. Most of the TRP inplasma is bound to serum albumin and thus is notavailable for uptake into peripheral tissues, and hencethe effect of insulin upon TRP is much less comparedwith the other LNAAs. Moreover, insulin increasesthe affinity of serum albumin for TRP and increasesthe ratio of bound to free plasma TRP, as morealbumin is available because of the insulin-induceduptake of fatty acids, which were bound to albumin.Thus, because TRP is the only amino acid that bindsto albumin, it is the only one that is very wellprevented from being taken out of the bloodstreamand up into peripheral tissue. This is even more thecase when the binding to albumin increases becauseof insulin secretion. Taken together, after the inges-tion of carbohydrate the plasma ratio of LNAAschanges in favour of total TRP, which eventually

Figure 2 Overview of tryptophan metabolism from foodintake to brain uptake and the differential effects ofcarbohydrates and protein upon the availability of trypto-phan (TRP) in plasma for uptake into the brain. In order toobtain the amino-acid TRP, its inclusion in the diet isessential. The majority of TRP is bound to plasma albuminand only free TRP will eventually cross the blood-brainbarrier (BBB). Albumin-bound TRP, however, easily dis-sociates from albumin near the cerebrovasculature underthe influence of haemodynamic changes, thereby increasingthe TRP-free fraction available for uptake into the brain. Theamount of TRP in plasma eventually crossing the BBB alsodepends upon the presence of other large neutral aminoacids (LNAAs) that all compete for the same amino acidtransport system L at the BBB. Because of this competitionof TRP with leucine (LEU), isoleucine (ILE), valine (VAL),phenylalaline (PHE) and tyrosine (TYR), the ratio of TRP tothe sum of the other LNAAs (TRP/SLNAA) in plasma betterreflects the amount of central TRP available for synthesisinto 5-hydroxytryptamine (5-HT). Dietary carbohydratesincrease the uptake of the LNAAs into peripheral tissue,thereby decreasing their levels in plasma. Together with anincrease in total TRP levels, the TRP/SLNAA ratio changesin favour of TRP and increases its availability for transportacross the BBB. As little as 2.5% of additional proteinscounteracts the effects of carbohydrates, as the proteiningestion-induced increase in the levels of all amino acids ismuch higher than the decrease by carbohydrates. When aTRP-free diet is administrated (acute tryptophan depletion),all amino acids are elevated except for TRP, therebydecreasing the TRP/SLNAA ratio and thus TRP uptake intothe brain.


697


leads to an increased availability of TRP in the brainto be synthesized into 5-HT.33,34 Yet, as little as 2.5%of additional protein is sufficient for the substantialincrease of plasma LNAAs to counteract the effect ofcarbohydrate and the subsequent insulin-induced fallin LNAAs.32,35 This is the case with the TRP-freeproteincarbohydrate nutritional mixture (see alsoFigure 2).

Protein synthesisThe administration of a diet devoid of TRP depletesplasma TRP acutely by inducing hepatic proteinsynthesis.36 This results in an extracellular TRPremoval that is because of an increased incorporationof TRP into proteins in the liver and other tissue.12,15

The ATD-induced depletion of plasma TRP can bedose dependently blocked by administration of theprotein synthesis inhibitor cycloheximide togetherwith a TRP-free diet.37,38 Thus, protein synthesis, andnot the inhibition of TRP transportation into thebrain, seems to be the important initial mechanismunderlying ATD-induced decreased 5-HT in the brain.

Central versus peripheral effects of ATD

The effects of ATD on affective behaviour (forexample, depression) and various cognitive functions(for example, memory, attention and impulsivity)have been studied in human subjects and laboratoryanimals, and several theories regarding its underlyingserotonergic mechanism and its implication forpsychiatry in general have been widely exploredand reviewed over the past 20 years.3943 In thissection we will give an overview of the peripheral andcentral effects after ATD in human subjects androdents.

ATD: revealing vulnerability to depressionMany neurophysiological processes are known to beregulated by the 5-HT system, including mood andcognition, which are most prominently impaired inclinical depression.4446 5-HT cell bodies are clusteredin the brainstem raphe nuclei sending out projectionsthroughout the entire central nervous system withascending pathways innervating anatomically andfunctionally diverse regions of the cerebral cortex,including the limbic system, the basal ganglia andstructures within the diencephalon.47 Because of thisanatomy, the neurotransmitter influences all regionsof the neuraxis, thereby modulating an extensiverange of physiological and behavioural functions.Besides mood and cognition, appetite, emesis, endo-crine function, gastrointestinal function, motor func-tion, neurotrophism, perception, sensory function,pain sensitivity, sex, sleep and even vascular functionare all under the control of the 5-HT system.Consequently, disrupted 5-HT synthesis and subse-quent abnormal 5-HT function can lead to a diverserange of behavioural disturbances also implicatedin clinical depression. Consistent findings in thisrespect specifically include decreased peripheral TRP

levels48 and lower levels of the inactive 5-HTmetabolite 5-hydroxyindoleacetic acid (5-HIAA) incerebral spinal fluid (CSF), which all reflect dimin-ished 5-HT metabolism.49,50 The effectiveness ofserotonergic drugs used in the treatment of depres-sion is also suggestive of an important role ofdisrupted function of specific pre- and post-synapticreceptors underlying impaired 5-HT neurotransmis-sion and linked to specific depressive symptoms.51,52

Human subjects with genetic, pre-existing 5-HTdysfunction may lack endogenous compensatorycapacity to deal with an acute decrease in 5-HTmetabolism, thereby exhibiting higher behaviouralsensitivity to ATD.4,26 This implies that a predisposi-tion of so-called serotonergic vulnerability onlyresults in direct overt psychiatric symptoms whenthese are triggered, as with ATD, by challenging thealready vulnerable 5-HT system4 up to a certainthreshold.26 In line with this hypothesis, ATD-induced transient mild mood-lowering effects, asreflected by lower mood ratings, have been reportedin carriers of the short allelic polymorphism in thepromoter of the 5-HT transporter gene 5-HTTLPR(serotonin-transporter-linked promoter region)53 andin healthy subjects with a family history of depres-sion.54 Similarly, a higher behavioural response toATD has been observed in women,55,56 who arepresumably predisposed to a lower 5-HT synthesisrate compared with men.57 Moreover, ATD provokes arelapse of depressive symptoms in healthy subjectswith a history of depression.58,59 However, this effectis only in those subjects who were previously treatedsuccessfully with selective serotonin re-uptake inhi-bitors (SSRIs) or monoamine oxidase inhibitors (MAOIs).Remitted, medication-free depressed patients with apositive treatment response history to antidepressantsthat primarily interact with systems other than 5-HT(for example, tricyclic antidepressants or selectivenorepinephrine reuptake inhibitors) appear not to beaffected by ATD.6062

ATD in healthy human subjects: inducing cognitivedeficits

As mentioned above, it is generally believed andaccepted that ATD does not induce considerablemood-lowering effects in healthy human sub-jects.59,63,64 Nevertheless, acute decreased peripheralTRP levels and diminished 5-HIAA concentrations inCSF are consistently reported after ATD and appear tobe similar in all subpopulations, that is, in bothhealthy and the so-called serotonergic vulnerablesubjects.54,6569 Interestingly, both healthy and vulner-able subjects display cognitive dysfunctional beha-viour after ATD as reported consistently betweenstudies.27,28,7075 It might therefore be suggested thatan acute decrease in peripheral TRP levels directlyinterferes with mechanisms implicated in cogni-tive processing that depend less upon 5-HT function-ing. Altered cognitive processing has been reportedwith impairments in long-term memory formation


698


(in particular, consolidation processes74), decisionmaking, reversal learning and working memory.

Effects of ATD in rodents: affect and/or cognition?Animal models enable direct investigation of therelationships between brain and behaviour with theaim of gaining insight into human behaviour andits underlying neuronal and neuroendocrinologicalprocesses.76 Therefore, the direct consequences ofATD upon brain parameters like TRP and 5-HT in therat are generally used for interpretation of the altera-tions in behavioural output in accordance with theunderlying neurochemical mechanism of the method.A large body of preclinical literature providesevidence that ATD in rats significantly depletes thelevels of TRP in plasma, thereby reducing 5-HTmetabolism, as is suggested by the lower TRP and5-HT levels in the rat brain tissue.24,7780 In rats,however, the levels of peripheral and central TRPreductions, as well as brain 5-HT concentrations,have not been consistently reported, and other ATD-induced neurophysiological effects, as discussedbelow, have been observed in the absence of centralTRP or 5-HT reductions.81,82 Similarly and in linewith the ATD effects in healthy human subjects,ATD-induced alterations in affective behaviouralparameters in the rat appear controversial betweenstudies.77,8385 Object memory performance is the onlyparameter consistently reported as impaired afterATD79,83,8588 and seems even more pronounced inrats with pre-existing abnormal 5-HT function.80

Table 1 provides an overview of the peripheral andcentral neurochemical effects and other neurophysio-logical changes, as well as cognitive and affectivebehavioural alterations after ATD induced in rodents,mainly rats, through administration of nutritionalmixtures completely devoid of TRP.

Methodological considerations: do we actuallymeasure central effects?

It is generally assumed that the mood-loweringand cognitive dysfunctional effects of ATD are medi-ated by decreases in 5-HT neuronal activity. Yet, asdescribed below, most parameters used to indicatereduced 5-HT synthesis or release appear to merelyestimate decreases in 5-HT metabolism and neuronalactivity, respectively, thereby remaining ratherspeculative.

The TRP/SLNAA ratio in plasmaThe effects of ATD treatment upon 5-HT levels in thebrain cannot be directly investigated in humans. Ingeneral therefore, the ratio TRP/SLNAA in plasma isused to estimate the amount of TRP available in thebrain for synthesis into 5-HT. In most cases, totalperipheral TRP levels (free plus albumin bound) areused for calculating the ratio to the sum of the otherLNAAs. Although only the relatively small fraction offree TRP eventually crosses the BBB, TRP can easilydissociate from albumin near the BBB, thereby

increasing the TRP-free pool and subsequent uptakeinto the brain. Thus, the direct effect of physiologicalfactors such as hormones, exercise or mild stressorsupon the rate of dissociation of TRP from albumin canonly be taken into account if both total and free TRPlevels are actually measured,89 which is usually notdone. Therefore, based upon total peripheral TRPlevels alone, the TRP/SLNAA ratio in plasma isprobably a distorted estimation of the rate of influx ofTRP into the brain.A decrease in the TRP/SLNAA ratio suggests that

less TRP will be available to the brain for synthesisinto 5-HT. This has been confirmed by the ATD-induced decrease in the uptake of central a-methyl-L-tryptophan (a-M-TRP) as measured by positron emis-sion tomography in humans.57 Although this findingsupports the concept that ATD exerts central effects,a-M-TRP uptake does not reveal anything specificabout 5-HT release,39,67 despite the fact that it isconsidered a reliable indicator of 5-HT synthesis.Finally, other LNAAs such as methionine and

threonine are generally not included in the ratio asactive competitors of plasma TRP. However, evidenceexists that together with the branched-chain aminoacids and the aromatic amino acids, methionineand threonine share the same L-amino-acid transportcarrier at the BBB.8,9,90 Thus, calculating the TRP/SLNAA ratio without taking into account all compe-titive amino acids is most likely to result in anoverestimation of brain TRP influx and subsequentdissociation from brain TRP levels and 5-HTsynthesis.

The 5-HIAA/5-HT ratio in the brainIn humans subjected to ATD, a decrease in theconcentration of 5-HIAA in CSF suggests that less5-HT has been catabolyzed, which normally takesplace after release, that is, after neuronal firing.Therefore, a decrease in the amount of 5-HIAA isthought to reflect decreased 5-HT metabolism, aslower intracellular 5-HT availability is presumed toresult in reduced 5-HT release. However, numerousother peripheral factors seem to influence both theamount of 5-HIAA produced and its transport intoand out of the CSF (see ref. 52). Moreover, 5-HTrelease and neuronal firing seem not to correlatenecessarily with 5-HT metabolism,91,92 and thus theamount of 5-HIAA in CSF seems not to be a validindex of changes in 5-HT release.92,93

In contrast to human subjects, animal models offerthe possibility to directly measure changes in TRP,5-HT and 5-HIAA concentration in distinct brainareas. Significantly lower tissue levels of TRP and5-HT have been reported after ATD compared withTRP control conditions in animals.24,79,80,85 The5-HIAA/5-HT ratio is normally used to calculate the5-HT turnover rate and estimate changes in 5-HTrelease, reflecting neuronal activity. However, thisseems to apply only under normal physiologicalconditions when the rate of 5-HT synthesis remainsconstant.94 An increase in 5-HIAA levels would thenincrease the 5-HIAA/5-HT ratio and allow accurate


699


Table

1Overview

ofneurobiochemicalandbehaviouraleffectsofacute

tryptophandepletionin

rodentsinducedbyapure

amino-acid

mixture

withoutTRP,aTRP-free

proteincarbohydrate

nutritionalmixture

(Solugel)orasolidTRP-freediet

Reference

Age/sex/strain/

nutritionalmixture/

dosingregime

%Changeplasm

aTRP/SLNAAratio

comparedwith

baseline

%Changebrain

parameters

compared

withcontrol

Additionalneurobiochemical

observations

Behaviouraleffects

Brownetal.84

Adultmale

Sprague

Dawleyrats

AAmixture

110mlkg1

Notreported

AtT3:

5-HThpc:2

3%

5-HIAAhpc:3

9%

TRPsuppletioneffectin

fcx:5-HT

45%

increase

comparedwithwater.

Decreased5-HTafterATDin

fcx

comparedwithTRP,

butnot

differentfrom

water.5-HIAA

striatum3

5%

lower

compared

withwater,notdifferentfrom

TRP

Nocognitiveim

pairmentorchanges

inlocomotoractivity

oranalgesia

Blokland

etal.77

4monthsold

male

Wistarrats

AAmixture

110mlkg1

(5g/kg1)

TRP:0.153g

absolute

amount

38%

atT5

AtT5:

TRPhpc:3

3%

TRPfcx(nopfc):1

4%

TYRctx:44%

Nocognitiveim

pairmentor

depressive-likeeffects.Increased

anxiety

atT5

Liebenetal.24

4monthsold

male

Wistarrats

Solugel

210mlkg1,90min

interval

71%

atT2

78%

atT4

AtT4:

TRPhpc:4

3%

5-HThpc:6

7%

5-HTstriatum:4

0%

5-HIAAhpc:4

0%

Loweringof5-HTin

both

hpcand

striatum.Both

TRPand5-HIAA

levelsonly

decreasedcompared

withcontrolin

hpc.Increase(43%)

TYRin

hpcanddecrease

instriatum

afterATD.NoeffectsuponTRP,

5-HT,5-HIAAin

ctx.Noeffect

uponDA.

ATD-induceddecrease

ofCIT

instriatum,hpcandctx

NA

Liebenetal.83

4monthsold

male

Wistarrats

Solugel210mlkg

1,

90min

interval

70%

atT2

65%

atT4

49%

atT6

Notreported

Objectrecognitionim

pairmentatT4

Noanxiety

ordepressive-likeeffects

andnoeffectsuponspatiallearning

inwatermaze

Cahiretal.78

Adultmale

Sprague

Dawleyrats

SolidTRP-freediet

TRP:0.7%

Freeplasm

aTRP

AtT3

89%

TRP

fcx:7

0%

hpc:6

6%

remainingcortex:6

3%

5-HT

fcx:3

9%

hpc:5

5%

remainingcortex:4

1%

Nodifferences

betw

een1and3

weeksofTRPdietuponperipheral

andcentral

parameters

Decreased5-HT1Areceptorbinding

indorsalrapheonly

afterATDatT3.

Noeffectsupon5-HT1Aor

5-HT2Abindingin

fcx,hpcor

remainingcortex.After3weeks

ofdiet,46%

increasein

5-HT2A

bindingin

cortexandnot,however,

infcxorhpc

NA


700


Table

1Continued

Reference

Age/sex/strain/

nutritionalmixture/

dosingregim

e

%Changeplasm

aTRP/SLNAAratio

comparedwith

baseline

%Changebrain

parameters

compared

withcontrol

Additionalneurobiochem

ical

observations

Behaviouraleffects

Vander

Plasse99

Adultmale

Wistar

rats

Solugel

210mlkg1,90min

interval

Freeplasm

aTRP

60%

atT3

70%

atT4

35%

atT5

50%

atT6

Extracellular5-HTandDA

transm

itterconcentration

(microdialysis):Nodifferences

betw

eenTRP-andTRP

treatm

ent

atanytimepoint

Nodifferencesin

generalactivity

andnoeffectofnoveltystim

ulation

upongeneralactivity

Cahiretal.97

Adultmale

Sprague

Dawleyrats

AAmixture

210mlkg1,90min

interval

TRP:2.3g

Freeplasm

aTRP

AtT3.5

79%

Hpc

5-HT:3

7%

5-HIAA:3

4%

Noeffectsupon5-HTturnoverin

hpc

Noeffectsuponplasm

aorcentral

BDNFprotein

levels

NA

Ruttenet

al.87,97,114

4monthsold

male

Wistarrats

Solugel

110mlkg1

AtT1

48%

AtT3

23%

Notreported

Objectrecognitionim

pairmentatT1

andT3,reversedbyPDE4inhibition

Jansetal.4

4monthsold

male

andfemaleWistar

rats

Solugel

210mlkg1,90min

interval

AtT4

Males:

66%

Females:

53%

(pro/es)

55%

(met/di)

Males:

TRPfcx:5

6%

Females(pro/es)

TRPfcx:4

9%

TRPhpc:5

3%

Ingeneral,higherperipheralTRP

levelswere

observedin

females.No

centralATDeffectsin

femalesmet/

di.HigherTYRlevelsin

femalepro/

escomparedwithfemalemet/diand

males.ATD-inducedincreasedTYR

inhpcandfcxin

female

pro/es.

IncreasedTYRin

hpconly

infemale

met/di

Objectrecognitionim

pairmentonly

infemalepro/es,notin

males.

Generallyless

passivebodycontact

afterATD.Femalesgenerallymore

activeanddecreasedanxiety

Jansetal.85

3monthsold

male

Brown-Norw

ayand

SpragueDawleyrats

Solugel

210mlkg1,90min

interval

AtT4

BrownNorw

ay:

58%

SpragueDawley:

48%

-BN

fcx:5-HT

5-HIAAk

hpc:5-HT

5-HIAAk

-SD:

fcx:5-HT

5-HIAAk

hpc:5-HIAAk

5-HIAA/5-HTk

Decreasedratio5-HIAA/5-HTin

hpc

andfcxonly

inSDrats

StrongerATD-inducedbehavioural

responsesin

SDrats.Object

recognitionim

pairm

entonly

inSD

rats.Controversialanxietyeffectsand

trendtowardincreasedbehavioural

despairin

SDrats.Noanxiety/

depressive-likebehaviourin

BNrats

Jansand

Blokland86

2monthsold

male

Wistarrats

Solugel

210mlkg1,90min

interval

AtT4

CMS:6

0%

Control:6

4%

Notreported

Lowersucrose

intakeandblunted

weightgainafterCMS.Noanxiety

ordepressive-likebehaviourafter

ATDorCMS.Less

passivesocial

interactionafterATD,independent

ofCMS.Objectrecognitionim

pair-

mentafterATD,independentof

CMS


701


Table

1Continued

Reference

Age/sex/strain/

nutritionalmixture/

dosingregime

%Changeplasm

aTRP/SLNAAratio

comparedwith

baseline

%Changebrain

parameters

compared

withcontrol


observations

Behaviouraleffects

Olivieretal.80

SERT/rat

(Slc6a41Hubr )

Solugel

210mlkg1,90min

interval

AtT4

TRP

SERT

/

65%

SERT

/

61%

SERT/

55%

ratioTRP/SLNAA

mean50%k

5-HTfcx:

SERT

/:1

9%

SERT

/:1

9%

SERT/:6

3%

5-HThpc:

SERT

/1

3%

SERT

/1

8%

SERT/7

0%

DecreasedTRP/SLNAAcompared

withTRP

controlanduntreated

rats,independentofgenotype.

Decreased5-HTin

both

fcxandhpc,

strongestin

SERT/genotype.

Nogenotypeeffectuponperipheral

TRP,ratioorcentral

5-HTlevels.

Increasedplasm

aratioafterTRP

treatm

entin

allgenotypessimilarly

Objectrecognitionim

pairment

independentofgenotype.Noeffect

afterlow

dose

ATDin

SERT

/

wildtypes.Generallystrongest

effectonSERT/evenafterTRP

treatm

ent.Noeffectsonuntreated

rats

vanDonkelaar

etal.88

6monthsold

male

Wistarrats

Solugel

110mlkg1

AtT2

73%

Notreported

Objectrecognitionim

pairmentat

T2,reversedbyPDE2andPDE5

inhibition

Jansetal.,

2009169

4monthsold

male

andfemale

Wistar

rats

Solugel

10mlkg1,several

applications

Males

110:4

8%

atT2

210,60min

interval:6

5%

atT2

310,60min

interval:

73%

atT4

54%

atT6

Females

210,90min

interval:

73%

atT2

60%

atT4

2%

atT6

80%

atT2(after

4daysofdaily

treatm

ent)

81%

atT4(after

4daysofdaily

treatm

ent)

Notreported

Objectrecognitionim

pairmentin

malesatT2,T4andT6after

210mlkg1,90min

interval

Ardisetal.96

Adultmale

Sprague

Daw

leyrats

AAmixture

210mlkg1,90min

interval

TRP:2.3g

Freeplasm

aTRP

AtT3.57

9%

5-HT

fcx:3

3%

hpc:3

4%

remainingctx:1

2%

5-HIAA

fcx:3

7%

hpc:3

7%

remainingctx:4

3%

Noreductionof5-HTor5-HIAAin

striatum.Decreased5-HIAA/5-HT

ratioin

both

cortex(41%)and

striatum

(34%),notin

hpc

NoeffectsuponDA,NEorthe

metabolites,DOPACandHVA

NA


702


Table

1Continued

Reference

Age/sex/strain/

nutritionalmixture/

dosingregim

e

%Changeplasm

aTRP/SLNAAratio

comparedwith

baseline

%Changebrain

parameters

compared

withcontrol


observations

Behaviouraleffects

vanDonkelaar

etal.81

3monthsold

male

Wistarrats

Solugel

210mlkg

1,90min

interval

40%

NoeffectofATDupon

TRP,5-HTor5-HIAA

concentration

ATD-induceddecrease

ofCIT

inhpc

ATD-inducedtissueoligaemia

NA

vanDonkelaar

etal.170

3monthsold

male

Wistarrats

Solugel

210mlkg

1,90min

interval

MDMApretreated:

37%

control:4

0%

NoeffectofATDupon

TRP,5-HTor5-HIAA

concentration

SignificantMDMApretreatm

ent

effect

ATDpotentiatedhyperaemia

only

afterMDMApretreatm

ent

NA

vanDonkelaar

etal.,200982

3monthsold

male

Wistarrats

Solugel

210mlkg

1,90min

interval

56%

NoeffectofATDupon

TRP,5-HTor5-HIAA

concentration

NoeffectofATDuponserum

and

brain

BDNFlevels

Positivecorrelationsbetw

eenTRP

andBDNFin

hpcandpfc

ATDapplicationstress-m

ediated

decreasedBDNFlevelsin

pfc

NA

vanDonkelaar

etal.165

3monthsold

male

SwissandC57BL/6J

mice

Solugel

110,210,120

and220mlkg

1,30

or60min

interval

MaxdepletionatT1

(30min

interval)

Swiss:210mlkg1:

74%

220mlkg1:7

7%

C57BL/6J:

210mlkg1:4

0%

115mlkg1:70%

NoeffectofATDupon

TRP,5-HTor5-HIAA

concentrationatanyof

thetimepoints

measuredand

independentofthe

specific

dosingregim

e

ATDapplicationstress-m

ediated

changesin

TRP/SLNAAratio

Noeffectsuponaffectiveor

cognitivebehaviour

Abbreviations:ATD,acutetryptophandepletion(TRPtreatm

ent);BDNF,brain-derivedneurotrophicfactor;BN,Brown-Norw

ayrat;CIT,citrulline;CMS,chronicmild

stress;DA,dopamine;DOPAC,3,4-dihydroxyphenylaceticacid;fcx,frontalcortex;hpc,hippocampus;HVA,homovanillicacid;met-di,female

rats

inmetestrusor

diestrus

stage

ofthe

reproductive

cycle;MDMA,3,4-m

ethylenedioxymethamphetamine,ecstasy;

NA,notapplicable;NE,norepinephrine;PDE

2,4,5,

phosphodiesterase

enzymetype2,4,5;pfc,prefrontalcortex;pro-es,

female

rats

inproestrusorestrusstageofthereproductivecycle;SD,SpragueDawleyrat;

SERT/,homozygousserotonin

transporterknockoutrat;SERT/,heterozygousserotonin

transporterknockoutrat;SERT/,wild-typecontrolofserotonin

transporterknockoutrat;T,timein

hours

after(first)administration;TRP,tryptophan;TRP,

controlnutritionalmixture

withadditionaltryptophan;TRP/SLNAA,

ratioofTRPto

thesum

ofotherlargeneutralaminoacids;TYR,tyrosine;5-HT;5-hydroxytryptamine(serotonin);5-HIAA,5-hydroxyindoleaceticacid;k,significant

decrease.


703


estimation of an increase in 5-HT neuronal activity, asit is assumed that more 5-HT has been released.Conversely, a decrease in 5-HIAA levels under aconstant rate of 5-HT synthesis would decrease the5-HIAA/5-HT ratio. Yet, ATD is applied to induce adecrease in 5-HT synthesis. As a decrease in intra-cellular 5-HT availability most likely results in areduction of the amount of 5-HT available for release,less extracellular 5-HT will then be available to becatabolyzed into 5-HIAA. Thus, ATD is likely todecrease both 5-HT and 5-HIAA, thereby maintainingthe 5-HIAA/5-HT ratio constant. In line with this, nochanges in the 5-HIAA/5-HT ratio were found 3h afterATD, whereas both 5-HT (23%) and 5-HIAA (39%)levels appeared to be significantly decreased in rathippocampus.84 A decrease in this ratio after ATDwould only occur if significantly less 5-HIAA isproduced compared with the already reduced amountof 5-HT synthesized.As described previously, the conversion of TRP into

the 5-HTP intermediate by the TPH2 (tryptophanhydroxylase 2) isoform is the first and rate-limitingstep in the biosynthesis of 5-HT. In the brain, thisenzyme is only 50% saturated and, therefore, the rateat which 5-HT is synthesized is limited only bysubstrate (that is, TRP) availability.95 However, itseems difficult to directly attribute an ATD-induceddecrease in the 5-HIAA/5-HT ratio to a reduction inTRP availability. Brain measurements are generallytaken at only one specific time point that impedes acomparison of the central parameters with baselinevalues. Thus, a decrease in 5-HIAA levels and in the5-HIAA/5-HT ratio after ATD is normally interpretedas evidence for an ATD-induced reduction in 5-HTmetabolism.However, one animal study showed that 5-HT

levels might not have changed compared with TRPtreatment.82 Furthermore, information upon baseline5-HT values is generally lacking. Although reductionsof the 5-HIAA/5-HT ratio have been repeatedlyreported after ATD in rats,85,96,97 between the studies,the concomitant changes in 5-HIAA or 5-HT inspecific brain areas do not seem to be in accordancewith each other. In general, it appears that ATD-induced absolute changes in central 5-HT or 5-HIAAconcentrations and potential underlying mechanismsrequire further examination.A decrease in the 5-HIAA/5-HT ratio after ATD in

rats seems more likely to be caused by factorsinfluencing the catabolism of 5-HT, such as fluctua-tions in the activity of the MAO enzyme.98 An acutedecrease in MAO activity would reduce the absoluteamount of 5-HIAA produced, thereby producing arelative increase in 5-HT levels. This would even-tually lower the 5-HIAA/5-HT ratio independent ofa reduction in precursor availability per se or thesubsequent decrease in 5-HT synthesis and as suchdoes not reflect a decrease in 5-HT release. It could behypothesized that an acute change in MAO activityimplies a compensatory mechanism activated uponthe acute decrease in peripheral TRP levels.

Several rat studies, all of similar experimentaldesign, have directly reported ATD-induceddecreased central 5-HT levels after administration ofthe same TRP-free nutritional mixture.24,80,85,99 This isgenerally thought to reflect an ATD-induced decreasein 5-HT synthesis. However, as mentioned previously,the decrease in 5-HT after TRP treatment at onespecific time point is normally only compared withthe control TRP treatment group and not with itsown baseline concentrations. Thus, without thesebaseline values before treatment, the extent to whichthe significant difference in 5-HT levels between theexperimental (TRP) and control (TRP ) treatmentgroup actually imply a reduction in 5-HT release (thatis, activity) after ATD remains unclear. In addition,no direct evidence exists that ATD affects central5-HT release (see next section).

5-HT neuronal releaseAlterations in neuronal activity can be measured byin vivo microdialysis. With this technique, changesin extracellular 5-HT concentrations can be measuredthat are indicative of changes in neuronal release.Actual reductions of basal 5-HT release after ATDhave only been reported in combination with 5-HTreuptake inhibitors.1,100,101 A blockade after 5-HTreuptake seems necessary to raise 5-HT to optimallevels for detection (see ref. 67). Yet, without theinitial systemic increase in extracellular 5-HT con-centrations, basal 5-HT release in the prefrontal cortexof rats appeared not to be affected by ATD.99 Thismight suggest that in the absence of de novosynthesis, 5-HT function can largely be maintainedfrom transmitter being recycled into the presynapticcell from the synaptic cleft. A possible decrease in5-HT might then reflect a decrease in the storage poolof 5-HTwithout affecting 5-HT release.102 In line withthis, decreased levels in whole-brain 5-HT levels at2 h after the consumption of a TRP-free diet in catsdid not parallel changes in the functional activity of5-HTcontaining dorsal raphe cells throughout the 4hafter ingestion.102 Only recently, the lack of evidencefor ATD-induced alterations in 5-HT release andneuronal activity was first critically outlined byFeenstra et al.3

Taken together, not only the parameters used tocalculate reductions in brain TRP availability and5-HT metabolism may be somewhat inaccurate, butalso no direct evidence exists that the ATD-inducedreductions in central 5-HT levels correlate withparallel changes in 5-HT neuronal activity undernormal physiological conditions. In general, there-fore, ATD-induced behavioural alterations cannoteasily be directly attributed to changes in 5-HTneuronal activity. Nevertheless, ATD consistentlyinduces cognitive impairment in both animals andhumans and triggers lowering of mood especially inhealthy subjects with a vulnerable 5-HT system. ATDdoes not directly or indirectly affect other mono-aminergic systems in rats24,82,84,85,96 and no behaviour-al changes have been observed after depletion of other


704


plasma amino acids.72 Moreover, administration of aTRP-free AA mixture in primates decreased TRP and5-HIAA concentrations in CSF without affectingcatecholamine metabolites, suggesting that the cate-cholamine system is not influenced by ATD.103

ATD-induced functional changes thus seem speci-fic for the peripheral depletion of the essential aminoacid TRP, but are more likely 5-HT mediated than5-HT induced. Moreover, the effects of changes inTRP availability upon 5-HT synthesis rate are gene-rally not measured under normal physiologicalcircumstances, that is, in the absence of any otherregulatory changes. Therefore, changes other thansubstrate availability that interfere with normal 5-HTregulation or disruptions in substrate availabilityitself might act as potential confounding factors forthe ATD-induced neurochemical effects as expectedunder normal circumstances.104 Thus, the exactunderlying mechanism might go beyond a straight-forward alteration in the central 5-HT system itself.

Potential alternative mechanisms underlying theeffects of ATD

Decreased nitric oxide synthase (NOS) activityThe enzyme NOS is suggested to play an impor-tant role in long-term potentiation (LTP) processesand consequently in learning and memory.105,106

As described below, several findings suggest thatATD may affect the activity of this enzyme. NOScatalyzes the conversion of the amino acid arginine(ARG) into citrulline (CIT) and nitric oxide (NO;107).Thus, NOS inhibition results in less conversion ofARG into CIT and NO. Therefore, the amount of NOsynthesis might be directly related to the levels ofARG and CIT. Two independent studies reportedsignificant lower CIT levels in the rat hippocampusafter ATD.24,81 This decrease in CIT appeared to beindependent of changes in its precursor ARG. On thebasis of the suggested interdependency of CIT andARG, this finding suggests that ATD might directlyaffect the activity of NOS, and that decreased CITconcentrations most likely parallel decreases in NO.Endogenous NO can modulate neuronal functionthrough interference with the release of severalneurotransmitters,108 yet its precise interaction withthe 5-HT system seems rather complex. Whereasslight increases in NO concentrations most likelyenhance 5-HT release, moderate increases appear todecrease 5-HT release.109 The modulation of 5-HTrelease by NO might therefore depend on pre-existingNO concentrations and the effects might be differ-ently regulated in distinct brain areas.105

A decrease in the activity of NOS and subsequentdecreased synthesis of NO after ATD could underliethe ATD-induced object memory impairments106 ashippocampal inhibition of NOS is known to impairobject recognition performance in rats.106,110 As theATD-induced decrease in brain CIT levels is see-mingly caused by an interruption in NOS activity, thissame decrease in NOS activity presumably also

decreases the synthesis of NO, which could explainthe ATD-induced object recognition impairments thatare consistently reported in rodents.79,80,83,85,87,88,111

The second messenger molecules cyclic adenosinemonophosphate (cAMP) and cyclic guanosine mono-phosphate (cGMP) have an important role in intra-cellular signalling and are highly involved in learningand memory processes.110,112114 Both cAMP andcGMP are selectively hydrolyzed by phosphodiester-ase (PDE) enzymes, and inhibition of PDE appears tobe a reliable method for improving memory processesby increasing the levels of either cAMP, cGMP orboth.115 Administration of the PDE2 inhibitor, BAY60-7550, and the PDE5 inhibitor, zaprinast, haveshown to increase NOS activity in rat hippocampusand striatum, and improve object recognition perfor-mance.116 Thus, besides directly increasing presynap-tic cGMP levels, the enhancing effects of PDE5 andPDE2 inhibition upon memory performance mightalso be mediated by activation of NOS postsynapti-cally.116 As NO can freely diffuse back into thepresynapse, it can increase cGMP levels by stimulat-ing the synthesis of soluble guanylyl cyclase.117 Anincrease in NOS activity increases NO concentrations,which might underlie the improvement of the ATD-induced memory impairment by PDE inhibitors.87,88

See also Figure 3 for an overview of how decreasedNOS activity might explain ATD-induced objectmemory impairment and its attenuation throughPDE inhibition.

Cerebrovascular abnormalitiesAlthough not completely clear at present, ATD alsoappears to affect local cerebral blood dynamics that mayexplain behavioural ATD effects. Under normal physio-logical conditions, the cerebral metabolic rate of glucose(CMRG) provides an index of changes in regionalneuronal activity, and changes in glucose metabolismare found to be closely coupled to changes inCBF.118,119 On this basis, abnormalities in either ofthese closely linked neurophysiological parameters indepressive subjects are thought to reflect changes inserotonergic neurotransmission in brain areas that canbe functionally linked to the complexity of depressivesymptomatology displayed.120123 However, the 5-HTneurotransmitter is a powerful vasoconstrictor124 andserotonergic fibres innervating cerebral arteries, arter-ioles and veins have been identified.47 Thus, ifdepression is represented by decreased central 5-HTneurotransmitter concentrations, an increase in CBFwould seem more likely. Similarly, the ATD-inducedreduction in 5-HT synthesis would decrease vaso-constrictor tone, thereby most likely increasing CBFdue to vasodilatation. Surprisingly, a decrease in localCBF following ATD has been reported in humansubjects125 and was also observed in rats.81 In thelatter, the acute decrease of peripheral TRP levelsresulted in a downward resetting of the cerebral flowmetabolism coupling relationship independentlyof changes in central TRP or 5-HT. This parallelspreliminary findings of an uncoupling of flow from


705


metabolism in unipolar depressed patients comparedwith bipolar patients and healthy controls.126

Although controversy exists about the exact locationand direction of the neurophysiological abnormalitiesin depressive subjects,120,127 depressive illness isgenerally characterized by decreases in CBF andCMRG in prefrontal cortex structures. However, itseems unlikely that a decrease in 5HT aloneaccounts for the specific decreases in haemodynamicregulation.In addition, low CBF most likely increases the

interaction between the albumin-bound TRP complexand the glycocalyx of the BBB, thereby causing moreTRP to dissociate from albumin.21,22 This mightprovide an explanation for the fact that ATD-inducedperipheral depletion of total TRP levels does notresult in a significant decrease in brain TRP avail-ability. Although it remains unclear how changes inperipheral TRP concentrations can interfere with thedynamic regulation of CBF, the findings support thenotion that the underlying mechanisms of ATD mightgo beyond a straightforward 5-HT-mediated mecha-nism (see also Figure 3).

In general, decreased local CBF is best explained bya loss of dilator tone. Besides the effect of 5-HT onvasodilation, decreased local CBF has also beenreported after direct inhibition of endothelial orneuronal NOS.128,129 As mentioned previously, directinhibition of NOS in the hippocampus is known toimpair object memory performance.106,110 Together,these findings support the suggestion that the ATD-induced impairments in object memory performanceof rats could be related to one common mechanism,that is, a decrease in NO. The involvement of NO canbe explained by the ATD-induced changes in brainlevels of CIT as described earlier. All the findingstogether suggest that ATD-induced object memoryimpairments are most likely caused by a decrease inNO that reduces local CBF in hippocampal areashighly implicated in memory processing. Addition-ally, this is in line with the fact that PDE2 and PDE5inhibition have been found to increase NOS activityin the hippocampus, which explains their potential toattenuate the ATD-induced object memory impair-ments. In addition, PDE inhibitors increase centralcAMP and cGMP concentrations, which are bothwell-known vasodilators.130 Their strong vasodilatingproperties possibly contribute to the improvement ofobject memory performance through attenuation ofthe ATD-induced cerebrovascular effects (see alsoFigure 3). Interestingly, low-dose PDE4 or PDE5inhibition did not directly affect cerebrovasculardynamics 30min after administration.131 However,this parallels the failure of the specific low-dosingregimes to reverse an object memory impairment inrodents.87,88

Decreased brain-derived neurotrophic factor (BDNF)BDNF could also be considered as a possible factorthat is influenced by ATD. There are indications thatan ATD effect on BDNF is indirect as ATD itself didnot have a direct effect on peripheral and centralBDNF levels.82,97 Disruption of BDNF regulation hasbeen implicated in both depressive symptomatologyand cognitive dysfunction. Also, there are coregulat-ing mechanisms between BDNF and the 5-HT systemin general.132 The highest levels of BDNF mRNA havebeen reported in the dentate gyrus and hippocampalCA3 and CA2 layers.133,134 Interestingly, anotherstudy81 showed an ATD-induced decrease in localCBF in similar areas (dentate PO, CA3 and CA2 regionof the hippocampus). Regional changes in BDNFprotein levels have been found after brief cerebrovas-cular events,135 consistent with the fact that BDNF hasan important neuroprotective role.136,137 Therefore, itcan be suggested that ATD, cerebrovascular effectsand BDNF could be interlinked.In addition, BDNF has been implicated as having an

important role in neuronal plasticity, includingLTP.138 As LTP is assumed to be the underlyingsubstrate of learning and memory processes,139 BDNFis thought to be a potential mediator of memoryformation in general, and is known to be requiredspecifically for memory consolidation.140 ATD in

Figure 3 Potential underlying mechanism of acute trypto-phan (TRP) depletion-induced rat object memory impair-ment and its attenuation through phosphodiesteraseinhibition. A decrease in the ratio of TRP to the sum ofother large neutral amino acids (SLNAA) induced by acutetryptophan depletion (ATD) potentially directly affects ()the activity of nitric oxide synthase (NOS), therebydecreasing central citrulline (CIT) levels without affectingits precursor arginine (ARG). A decrease in CIT most likelyparallels a reduction in nitric oxide (NO) that subsequentlyaffects local cerebral blood flow (CBF) in brain areas highlyimplicated in object memory processes. Indicated with ( )is the possible mechanisms behind the improvement ofobject memory deficits by inhibition of either the phospho-diesterase (PDE) enzyme type 5 (PDE5-I) or type 2 (PDE2-I),thereby directly increasing the levels of cyclic adenosinemonophosphate (cAMP) and/or cyclic guanosine monopho-sphate (cGMP), respectively, or type 4 (PDE4-I) increasingcAMP only. Both second messenger molecules are highlyimplicated in learning and memory processes. Inhibition ofPDE5 and PDE2 also directly activates NOS, therebyincreasing NO levels that stimulate the synthesis of solubleguanylyl cyclase (sGC) and cGMP presynaptically. Thevasodilating properties of the PDE inhibitors might addi-tionally attenuate ATD-induced decreases in CBF. ()Inhibition or decrease; ( ) stimulation or increase.


706


human subjects selectively impairs memory consoli-dation,74 which has been suggested to be caused bylower 5-HT levels in hippocampal areas.73 However,in rats, the ATD-induced cerebrovascular changeswere independent of changes in central 5-HT levels.81

This suggests that decreased CBF in brain regionsnormally high in BDNF levels could also be con-sidered underlying BDNF-mediated alterations inlearning and memory processing after ATD.

Kynurenine (KYN) metabolitesUnder normal physiological conditions, only 1 to 2%of the amount of ingested TRP is used by the bodyfor the synthesis of 5-HT.19 The majority of totalingested TRP is catabolyzed into KYN by inductionof tryptophan pyrrolase in the liver.141 Induction ofpyrrolase by the enzymes IDO (indolamine 2,3-dioxygenase) and TDO (tryptophan 2,3-dioxygenase)in the liver reduces TRP availability142 and therefore5-HT synthesis is also influenced by IDO and TDOactivity.143,144 Stimulation of these enzymes by pro-inflammatory cytokines, in particular interferon-g,145

enhances the catabolism of TRP,142 thereby decreasingthe amount of TRP eventually available for 5-HTsynthesis in the brain. Moreover, TDO activity canalso be induced by corticoids.146

Tryptophan pyrrolase is the first rate-limitingenzyme of the KYN pathway and KYN is the majordegradation product of TRP.144 KYN is furtherconverted into potentially neuroactive metabolitessuch as kynurenine acid and quinolinic acid.147

Independently of each other, both metabolites exertspecific effects upon N-methyl-D-aspartate (NMDA)receptors,148 which have an important role in LTP andmemory formation.149,150 NMDA receptor antagonistshave been shown to inhibit LTP and selectivelyimpair learning and memory,151 but antagonists canalso have a neuroprotective effect.152 Kynurenine acidhas an antagonistic effect on the NMDA receptor andhas been shown to have neuroprotective effects.153

Conversely, quinolinic acid depolarizes neurons byactivating NMDA receptors.153 As a result, quinolinicacid can lead to neurotoxicity, similar to that found inhypoxia and ischaemia.154156 Thus, an ATD-inducedchange in the amount of KYN metabolites couldprovide an explanation for the observed cerebraloligaemia paralleling decreased peripheral TRPlevels,81 and as such KYN metabolites have alreadybeen suggested to additionally account for the con-sistently reported memory impairments after ATD.2

Confounding stress effectsATD application-related procedures might producestress, which might interfere with TRP metabolism,and subsequent 5-HT synthesis as brain 5-HT,together with other monoamines, is critically in-volved in the mediation of the central response tostressors and subsequent behavioural adaptation.157

Acute stressors stimulate hypothalamicpituitaryadrenal axis activity, thereby increasing central 5-HTnecessary for stress coping.158 In rodents, overnight

food deprivation, repeated oral administrations bygavage, blood sampling and the immobilizationnecessary for applying the former are well-knownstressors.159162 Yet, these experimental proceduresare inevitably implicated in the application of theATD method in rats and mice. In rats, both acute andrepeated exposure to stressful stimuli have beenshown to increase glucocorticoid levels and alter5-HT turnover and release in both the hippocampusand frontal cortex.163 Thus, ATD application-relatedprocedures in animals might interfere with normalbrain TRP metabolism and subsequent 5-HT syn-thesis, thereby acting as confounding stress factors forthe pharmacokinetic and behavioural effects of ATD.Stress-induced changes in the breakdown of fat

stored in fat cells (lipolysis) may also alter brainTRP concentrations. In the same way that insulinstimulates the uptake of albumin-bound fatty acidsby fat cells, thereby decreasing the fraction of freeTRP in plasma, stress can result in the reverse; stressincreases lipolysis and thus the amount of plasmafree-fatty acids.164 This increases the affinity ofalbumin for fatty acids, thereby displacing TRP fromits albumin-binding sites. The resultant increase inthe fraction of free TRP in plasma might increase theavailability of TRP for uptake into the brain (seeFigure 4). In a recent study with mice it was foundthat oral treatment by gavage combined with bloodsampling and food deprivation, which are thestandard experimental procedures, increased theTRP/SLNAA ratio in plasma within 20min.165 Thus,acute stress effects might explain the moderatedepletion effects after ATD as a failure to considerablyreduce central 5-HT levels eventually. Furthermore, itcould be suggested that comparing TRP (ATD) andTRP (control) conditions may not compare normalversus low levels of TRP, but instead comparemoderate versus elevated TRP levels.The effects of acute stress, including the ATD-

related application procedures, upon 5-HT autorecep-tor binding might additionally provide an explanationfor the consistently reported ATD-induced memorydysfunction as indicated by object recognition im-pairment in rats.79,83,8588 Several 5-HT receptors,including 5-HT1A, are known to be highly involvedin memory function166 and ATD has been shown todecrease 5-HT1A receptor binding.

78 Also, acutecorticosterone administration directly decreases5-HT1A autoreceptor functioning.

167 Thus, a moregeneralized stress effect of ATD application-relatedprocedures, possibly linked to a decrease in 5-HT1Areceptor function as described above, might underliethe ATD-induced impaired object recognition perfor-mance for which intact 5-HT1A receptor functionseems 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 experi-mental procedures used in animal research. In humanresearch the effects of stress may be less as theprocedure of the treatment is less stressful thanthe procedures used in animal research. Nevertheless,


707


the intake of the amino-acid drink in humans hasfrequently been reported as unpleasant and someassociated stress cannot be ruled out.31,162164

Serotonin, depression and ATD

The 5-HT system is highly implicated in a wide rangeof functional processes. Whereas mood-loweringeffects after ATD are only experienced in depres-sion-free subjects predisposed to genetic 5-HTabnormalities, altered cognitive processing afterATD is observed in both healthy and geneticallyvulnerable subjects. Moreover, ATD-inducedimpaired object memory performance as measuredby the object recognition task is consistently reportedin rats79,80,83,85,86 even after only moderate peripheralTRP depletion or a single administration.87,88 Foradequate cognitive processing, the 5-HT systeminteracts highly with other neurophysiological sys-tems that might all be interrupted after ATD.81,82 Thisfurther supports the notion that an acute decreasein peripheral TRP levels directly interferes with

mechanisms implicated in cognitive processing thatpresumably depend less upon 5-HT functioningper se. The potential alternative mechanisms under-lying ATD effects might therefore provide an explana-tion for the consistently reported ATD-inducedcognitive deficits across species.Chronic exposure to stressful situations is one of

the main contributing factors to the onset of depres-sive illness,158 and an intact 5-HT system is critical formediating an adequate neurophysiological stressresponse and subsequent behavioural adaptation.157

At the level of somatodendritic 5-HT autoreceptors,adaptations to stress might be reflected by a reductionof these receptors, thereby reducing serotonergicfeedback in dorsal raphe projection areas in anattempt to counteract an ATD-induced decrease incentral 5-HT.78 Such an adaptive mechanism to anacute stressor might be less efficient in subjectspredisposed to 5-HT dysfunction, which mightaccount for the mood-lowering effects after ATD inthese subjects.As corticosteroid modulation of 5-HT function has

a central role in mood disorders and cognitivefunctioning is highly impaired in clinical depression,the ATD findings reviewed in this article might haveimportant implications regarding the mechanisms ofadaptation to stress, and the implication of the 5-HTsystem in cognitive processing. As the 5-HT system isprimarily modulatory, it is not surprising that in theclinical situation, most 5-HT-based treatments resultin only a partial symptomatic improvement. Thereviewed ATD findings thus support new insights foralternative treatment strategies along other pathwaysthat interact highly with the 5-HT system. Upregula-tion of the cAMP and BDNF systems has alreadyresulted in a novel model for the mechanism of actionof antidepressants and new targets for the develop-ment of therapeutic targets.168

For an adequate interpretation of data resultingfrom application of the method in both clinical andpreclinical settings, the described potential alterna-tive mechanisms should be taken into account.Abnormal physiological conditions, including ATDapplication-related confounding stressors possiblyinterfere with the high number of cellular processesthat are involved in 5-HT synthesis and metabolism,and might trigger other neurophysiological systemsthat generally interact with the 5-HT system. Thispossibly also reflects the heterogeneity of majordepressive disorder in general, as all these neuro-physiological processes might eventually be altered inclinical conditions and thus potentially be treatedalong different pharmacological pathways. In general,several findings support the fact that depression maynot be caused solely by an abnormality of 5-HTfunction, but more likely by a dysfunction of othersystems or brain regions modulated by 5-HT orinteracting with its dietary precursor. Similarly, theATD method does not seem to challenge the 5-HTsystem per se, but rather triggers 5-HT-mediatedadverse events.

Figure 4 Potential confounding stress effects of acutetryptophan (TRP) depletion application-related procedures.The method of acute TRP depletion (ATD) requires severalhighly stressful procedures, such as overnight food depriva-tion, repeated blood sampling, oral administrations bygavage and immobilization. Acute stress stimulates lipoly-sis, thereby increasing the amount of free-fatty acids inplasma that displace TRP from its albumin-binding sites.The resultant rise in free plasma TRP changes the ratio ofTRP to the sum of the other large neutral amino acids (TRP/SLNAA) in favour of TRP, thereby increasing the amount ofTRP available for uptake into the brain and its subsequentsynthesis into 5-hydroxytryptamine (5-HT). Stress stimu-lates the secretion of corticosterone (CORT) that decreasesbrain-derived neurotrophic factor (BDNF) and 5-HT1Areceptor binding. Both BDNF and 5-HT1A receptor function-ing are highly implicated in learning and memory pro-cesses. The acute stress of ATD application-relatedprocedures might thus explain the failure of ATD toconsiderably decrease 5-HT metabolism and its negativeeffect upon object memory performance through increasedCORT levels.


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Conclusions

ATD currently represents the most established humanchallenge test to investigate the involvement of the5-HTsystem in the pathogenesis and pathophysiologyof affective disorders, including cognitive dysfunc-tional behaviour. However, the exact mechanism bywhich ATD exerts its neurophysiological effects, andto what extent changes in 5-HT neuronal activitycontribute to the ATD-induced functional and beha-vioural alterations, remain unresolved issues. Thispivotal lack of information impedes an adequateinterpretation of the results arising from applicationof the method in both clinical and preclinical studies.As most biochemical brain values are merely indica-tive and thus speculative in human studies, animalmodels provide a better means for the exploration ofATD-induced neurobiochemical alterations. However,even in rats, challenging the 5-HT system by ATDintroduces speculation because of the highly con-troversial results between studies on both the periph-eral, central and behavioural level. Moreover, noconvincing evidence exists that ATD induces altera-tions in central 5-HT release and subsequent neuronalactivity. Several findings support the contribution ofalternative mechanisms that go beyond a decreased5-HT release, such as reduced NOS activity andcerebrovascular abnormalities. In addition, experi-mental procedures related to the application of theATD method seem highly stressful and potentiallyinterfere with TRP metabolism, thereby confoundingATD neurochemical and behavioural results, espe-cially in rodents. As a decrease in CBF and con-founding stress effects provide an explanation forboth the consistently reported behavioural effects andthe absent effects, it seems most likely that theunderlying mechanism of the method goes beyond adisturbed 5-HT system. It is thus suggested thatcaution is required when interpreting ATD effects interms of a selective serotonergic effect.

Conflict of interest

The authors declare no conflict of interest.

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Mechanism of Acute Tryptophan Depletion