Molecular Psychiatry (2011) 16, 695–713 & 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 1Department 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 3Cerebrovascular 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, 695–713; doi:10.1038/mp.2011.9; published online 22 February 2011 Keywords: acute stress; cerebral blood flow; cognitive dysfunction; depression; serotonin; tryptophan Introduction reflected in dysfunctional behavioural output. ATD- induced behavioural changes in human subjects Acute tryptophan depletion (ATD) currently repre- and laboratory animals are normally attributed to sents the most established human challenge test decreased 5-HT release, reflecting altered 5-HT to investigate the involvement of the serotonin neuronal activity. However, it is not fully clear what (5-hydroxytryptamine; 5–HT) system in the patho- mechanisms underlie the neurophysiological effects genesis and pathophysiology of affective disorders. of ATD and to what extent changes in 5-HT neuronal The method is nontoxic and nonintrusive, thereby activity contribute to the ATD-induced functional and providing the option to repeatedly manipulate the behavioural alterations. Also, no convincing evidence central 5-HT system in vivo and assess the behavioural exists for affected central 5-HT release following ATD effects of reduced 5-HT metabolism in the brain.1 The in animals.3 reduction of brain 5-HT in a reversible manner reflects The ATD method seems important in the investiga- the main methodological advantage of the tool, tion of 5-HT-related vulnerability factors implicated permitting application of the same basic method in in the onset of depression,4 and previously the both human subjects and rodents. This is considered monoamine systems were considered to be primarily valuable for comparing neurophysiological changes responsible for the onset of depressive disorders.5 linked to behavioural effects across species.2 However, the lack of mood-lowering effects after ATD As intact 5-HT neurotransmission is necessary for a in healthy subjects may not support a direct causal wide range of physiological and functional processes, relationship between acute decreased 5-HT metabo- a disruption in this system can easily provoke diverse lism and major depressive disorder.6 Moreover, as pathophysiological abnormalities, most of which are will be discussed in this review, evidence exists that ATD possibly exerts its neurochemical and Correspondence: Dr EL van Donkelaar, Department of Psychiatry behavioural effects through other mechanisms that and Neuropsychology, Division of Neuroscience, Faculty of might go beyond a straightforward decrease in 5-HT Health, Medicine and Life Sciences, School for Mental Health metabolism. This review covers an extensive evalua- and Neuroscience, Maastricht University, P.O. Box 616, 6200 MD tion of both the methodology and the diverse neuro- Maastricht, The Netherlands. E-mail: [email protected] chemical and behavioural effects of ATD, including Received 12 March 2010; revised 4 January 2011; accepted 19 a critical assessment of the common parameters January 2011; published online 22 February 2011 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 From bound to free plasma tryptophan: the brain influx aims to outline alternatives for potential underlying parameter mechanisms of the method that might go beyond a Approximately 90% of all TRP molecules circulating disturbed 5-HT system and thus draw into question in the blood are bound to serum albumin. Although the utility of ATD as a serotonergic challenge tool in positive correlations between serum free-TRP and experimental research in general and depression whole brain TRP levels have been reported in rats,15,16 research in particular. 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 Methodological aspects of ATD central 5-HT synthesis.17,18 This observation suggests 5-HT is synthesized in a two-step reaction (Figure 1) that only free TRP is available for transport into the 15 from the initial substrate L-tryptophan (TRP), and the brain. As the changes in TRP-free levels can take bioavailability of this essential amino acid is the place independently of changes in total TRP levels,19 principal rate-limiting factor. Thus, variations in this would make a distinction between free and dietary intake of TRP can have profound effects upon bound TRP necessary for estimating its availability the synthesis of this very important neurotransmitter in the brain. However, accumulating evidence indi- substance and may impact upon those aspects of cates that total peripheral TRP concentrations (free brain function that are influenced by serotonergic plus bound) more accurately reflect the rate of influx neurons. It is this fact that underpins the use of ATD of TRP into the brain. It has been shown that TRP is as both an experimental tool and a clinical probe for only loosely bound to albumin and although albumin depressive illness. itself cannot cross the BBB, it appears to be a highly flexible protein undergoing reversible conformational From plasma to brain tryptophan changes.20 These conformational changes, which Amino acids can only be transported from the blood occur during transport of TRP from the circulating through the capillary endothelial cells of the blood- albumin-bound pool, enhance the dissociation of TRP brain barrier (BBB) into the brain by carrier-mediated from the albumin-binding sites within the cerebral transporter systems in the capillary cell plasma microvasculature and appear to be highly dependent membranes.7 Given that the surface area of the BBB upon cerebral haemodynamics.21 Low cerebral blood is much smaller compared with the surface area of flow (CBF) is likely to increase the interaction brain cell membranes, it is this initial transport between the albumin-bound TRP complex and the through the BBB that limits the uptake of plasma glycocalyx of the BBB, thereby causing more TRP TRP into the brain.8 The branched-chain amino acids to dissociate from albumin.21,22 This implies that (leucine, isoleucine and valine) together with the temporally dynamic or spatial differences in local aromatic amino acids (phenylalanine, tyrosine and CBF may influence the rate of central TRP uptake in TRP) are subclassified as large neutral amino acids general and even within specific brain areas.23 Thus, (LNAAs). Of the nine different amino acid transport although only free TRP can eventually cross the BBB, systems identified at the BBB, the so-called Transport the amount of albumin-bound TRP in plasma must System L is only half saturated under normal also be taken into account to calculate the availability physiological conditions and mediates high-affinity, of TRP in the brain, as TRP can easily dissociate from sodium-independent uptake of all LNAAs.8,9 Conse- albumin near the BBB, thereby increasing the TRP- quently, in order to bind to the L-amino-acid transport free pool and subsequent uptake into the brain (see carrier and subsequent transport into the brain, TRP also Figure 2). has to compete heavily with the other LNAAs.10–12 The availability of TRP in the brain thus depends Solugel upon the ratio of TRP to the sum of the other LNAAs In most studies, pure amino-acid mixtures without (TRP/SLNAA), and a decrease in this ratio in plasma TRP have been used to reduce plasma TRP levels. is normally used as the best predictor of reduced However, one disadvantage of the amino-acid mixture availability of TRP in the brain and subsequent is that differences in the distinct amino acids were synthesis into 5-HT.13,14 found between the control condition (TRP þ ) and Figure 1 Central serotonin synthesis