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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 depletion: is it only ? 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 , 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 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 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 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 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 . 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 undergoing reversible conformational From plasma to brain tryptophan changes.20 These conformational changes, which Amino 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 (, and ) together with the temporally dynamic or spatial differences in local aromatic amino acids (, 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 and metabolism. In the brain, tryptophan (TRP) is first hydroxylated into 5-hydroxytryptophan (5-HTP) by the enzyme (TPH). Aromatic L-amino-acid decarboxylase (AAAD) subsequently catalyzes the of 5-HTP into 5-hydroxytryptamine (5-HT). The enzymes (MAO) and aldehyde dehydrogenase (ADH) eventually break serotonin (5-HT) down into the inactive metabolite 5-hydroxyindoleacetic acid (5-HIAA).

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 697 a gelatin-based protein– 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 of protein, which is easily digestible and naturally lacks the essential amino-acid TRP.29 The gelatin hydro- lysate 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 , 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 Figure 2 Overview of tryptophan metabolism from food more similar to conventional food intake. Moreover, intake to brain uptake and the differential effects of mixing with avoids any carbohydrates and protein upon the availability of trypto- phan (TRP) in plasma for uptake into the brain. In order to unwanted effects upon amino-acid availability in obtain the amino-acid TRP, its inclusion in the is the blood (see below and Figure 2) as is normally essential. The majority of TRP is bound to plasma albumin found with unbalanced diets containing high and only free TRP will eventually cross the blood-brain amounts of carbohydrate or protein only.30–32 barrier (BBB). Albumin-bound TRP, however, easily dis- sociates from albumin near the cerebrovasculature under Carbohydrate and dietary protein the influence of haemodynamic changes, thereby increasing Various metabolic processes are triggered when a the TRP-free fraction available for uptake into the brain. The protein/carbohydrate meal, as is the case with ATD amount of TRP in plasma eventually crossing the BBB also methods, is ingested. These processes can have effects depends upon the presence of other large neutral amino on plasma TRP levels too. After the intake of carbo- acids (LNAAs) that all compete for the same amino acid transport system L at the BBB. Because of this competition hydrates, blood glucose levels raise, thereby stimulat- of TRP with leucine (LEU), isoleucine (ILE), valine (VAL), ing the pancreas to release the anabolic phenylalaline (PHE) and tyrosine (TYR), the ratio of TRP to . The secretion of insulin stimulates glucose to the sum of the other LNAAs (TRP/SLNAA) in plasma better be taken up by the cells for subsequent normalization reflects the amount of central TRP available for synthesis of glucose levels in the blood. Concomitantly with the into 5-hydroxytryptamine (5-HT). Dietary carbohydrates insulin-induced drop in blood glucose levels, plasma increase the uptake of the LNAAs into peripheral tissue, TRP levels increase whereas the concentration of thereby decreasing their levels in plasma. Together with an most other amino acids in plasma decreases.33 When increase in total TRP levels, the TRP/SLNAA ratio changes a carbohydrate-rich meal contains no additional in favour of TRP and increases its availability for transport protein, activation of the insulin response increases across the BBB. As little as 2.5% of additional proteins counteracts the effects of carbohydrates, as the protein protein synthesis, thereby stimulating the uptake of ingestion-induced increase in the levels of all amino acids is almost all amino acids (mainly the branched-chain much higher than the decrease by carbohydrates. When a amino acids) into muscle tissue. Most of the TRP in TRP-free diet is administrated (acute tryptophan depletion), plasma is bound to serum albumin and thus is not all amino acids are elevated except for TRP, thereby available for uptake into peripheral tissues, and hence decreasing the TRP/SLNAA ratio and thus TRP uptake into the effect of insulin upon TRP is much less compared the brain. 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 a condition in which animals were treated with albumin is available because of the insulin-induced saline.2,24 In that case, the control condition is not uptake of fatty acids, which were bound to albumin. an optimal and representative control condition. An Thus, because TRP is the only amino acid that binds alternative manner to reduce central 5-HT concentra- to albumin, it is the only one that is very well tions by lowering the levels of its dietary precursor prevented from being taken out of the bloodstream TRP in plasma can be achieved by administration of and up into peripheral tissue. This is even more the specific TRP-free nutritional mixtures. case when the binding to albumin increases because Besides the TRP-free or TRP-low balanced diets of insulin secretion. Taken together, after the inges- and pure amino-acid mixtures without TRP, a more tion of carbohydrate the plasma ratio of LNAAs advanced technique is the oral administration of changes in favour of total TRP, which eventually

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 698 leads to an increased availability of TRP in the brain levels48 and lower levels of the inactive 5-HT to be synthesized into 5-HT.33,34 Yet, as little as 2.5% metabolite 5-hydroxyindoleacetic acid (5-HIAA) in of additional protein is sufficient for the substantial cerebral spinal fluid (CSF), which all reflect dimin- increase of plasma LNAAs to counteract the effect of ished 5-HT metabolism.49,50 The effectiveness of carbohydrate and the subsequent insulin-induced fall serotonergic drugs used in the treatment of depres- in LNAAs.32,35 This is the case with the TRP-free sion is also suggestive of an important role of protein–carbohydrate nutritional mixture (see also disrupted function of specific pre- and post-synaptic Figure 2). receptors underlying impaired 5-HT neurotransmis- sion and linked to specific depressive symptoms.51,52 Protein synthesis Human subjects with genetic, pre-existing 5-HT The administration of a diet devoid of TRP depletes dysfunction may lack endogenous compensatory plasma TRP acutely by inducing hepatic protein capacity to deal with an acute decrease in 5-HT synthesis.36 This results in an extracellular TRP metabolism, thereby exhibiting higher behavioural removal that is because of an increased incorporation sensitivity to ATD.4,26 This implies that a predisposi- of TRP into proteins in the and other tissue.12,15 tion of so-called serotonergic vulnerability only The ATD-induced depletion of plasma TRP can be results in direct overt psychiatric symptoms when dose dependently blocked by administration of the these are triggered, as with ATD, by challenging the protein synthesis inhibitor cycloheximide together already vulnerable 5-HT system4 up to a certain with a TRP-free diet.37,38 Thus, protein synthesis, and threshold.26 In line with this hypothesis, ATD- not the inhibition of TRP transportation into the induced transient mild mood-lowering effects, as brain, seems to be the important initial mechanism reflected by lower mood ratings, have been reported underlying ATD-induced decreased 5-HT in the brain. 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 Central versus peripheral effects of ATD in healthy subjects with a family history of depres- The effects of ATD on affective behaviour (for sion.54 Similarly, a higher behavioural response to example, depression) and various cognitive functions ATD has been observed in women,55,56 who are (for example, memory, attention and impulsivity) presumably predisposed to a lower 5-HT synthesis have been studied in human subjects and laboratory rate compared with men.57 Moreover, ATD provokes a animals, and several theories regarding its underlying relapse of depressive symptoms in healthy subjects serotonergic mechanism and its implication for with a history of depression.58,59 However, this effect psychiatry in general have been widely explored is only in those subjects who were previously treated and reviewed over the past 20 years.39–43 In this successfully with selective serotonin re-uptake inhi- section we will give an overview of the peripheral and bitors (SSRIs) or monoamine oxidase inhibitors (MAOIs). central effects after ATD in human subjects and Remitted, medication-free depressed patients with a rodents. positive treatment response history to that primarily interact with systems other than 5-HT ATD: revealing vulnerability to depression (for example, tricyclic antidepressants or selective Many neurophysiological processes are known to be reuptake inhibitors) appear not to be regulated by the 5-HT system, including mood and affected by ATD.60–62 cognition, which are most prominently impaired in clinical depression.44–46 5-HT cell bodies are clustered in the brainstem sending out projections ATD in healthy human subjects: inducing cognitive throughout the entire central nervous system with deficits ascending pathways innervating anatomically and As mentioned above, it is generally believed and functionally diverse regions of the cerebral cortex, accepted that ATD does not induce considerable including the limbic system, the basal ganglia and mood-lowering effects in healthy human sub- structures within the diencephalon.47 Because of this jects.59,63,64 Nevertheless, acute decreased peripheral anatomy, the neurotransmitter influences all regions TRP levels and diminished 5-HIAA concentrations in of the neuraxis, thereby modulating an extensive CSF are consistently reported after ATD and appear to range of physiological and behavioural functions. be similar in all subpopulations, that is, in both Besides mood and cognition, appetite, emesis, endo- healthy and the so-called serotonergic vulnerable crine function, gastrointestinal function, motor func- subjects.54,65–69 Interestingly, both healthy and vulner- tion, neurotrophism, perception, sensory function, able subjects display cognitive dysfunctional beha- pain sensitivity, sex, sleep and even vascular function viour after ATD as reported consistently between are all under the control of the 5-HT system. studies.27,28,70–75 It might therefore be suggested that Consequently, disrupted 5-HT synthesis and subse- an acute decrease in peripheral TRP levels directly quent abnormal 5-HT function can lead to a diverse interferes with mechanisms implicated in cogni- range of behavioural disturbances also implicated tive processing that depend less upon 5-HT function- in clinical depression. Consistent findings in this ing. Altered cognitive processing has been reported respect specifically include decreased peripheral TRP with impairments in long-term memory formation

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 699 (in particular, consolidation processes74), decision increasing the TRP-free pool and subsequent uptake making, reversal learning and working memory. into the brain. Thus, the direct effect of physiological factors such as , exercise or mild stressors Effects of ATD in rodents: affect and/or cognition? upon the rate of dissociation of TRP from albumin can Animal models enable direct investigation of the only be taken into account if both total and free TRP relationships between brain and behaviour with the levels are actually measured,89 which is usually not aim of gaining insight into human behaviour and done. Therefore, based upon total peripheral TRP its underlying neuronal and neuroendocrinological levels alone, the TRP/SLNAA ratio in plasma is processes.76 Therefore, the direct consequences of probably a distorted estimation of the rate of influx of ATD upon brain parameters like TRP and 5-HT in the TRP into the brain. rat are generally used for interpretation of the altera- A decrease in the TRP/SLNAA ratio suggests that tions in behavioural output in accordance with the less TRP will be available to the brain for synthesis underlying neurochemical mechanism of the method. into 5-HT. This has been confirmed by the ATD- A large body of preclinical literature provides induced decrease in the uptake of central a-methyl-L- evidence that ATD in rats significantly depletes the tryptophan (a-M-TRP) as measured by positron emis- levels of TRP in plasma, thereby reducing 5-HT sion tomography in humans.57 Although this finding metabolism, as is suggested by the lower TRP and supports the concept that ATD exerts central effects, 5-HT levels in the rat brain tissue.24,77–80 In rats, a-M-TRP uptake does not reveal anything specific however, the levels of peripheral and central TRP about 5-HT release,39,67 despite the fact that it is reductions, as well as brain 5-HT concentrations, considered a reliable indicator of 5-HT synthesis. have not been consistently reported, and other ATD- Finally, other LNAAs such as and induced neurophysiological effects, as discussed are generally not included in the ratio as below, have been observed in the absence of central active competitors of plasma TRP. However, evidence TRP or 5-HT reductions.81,82 Similarly and in line exists that together with the branched-chain amino with the ATD effects in healthy human subjects, acids and the aromatic amino acids, methionine ATD-induced alterations in affective behavioural and threonine share the same L-amino-acid transport parameters in the rat appear controversial between carrier at the BBB.8,9,90 Thus, calculating the TRP/ studies.77,83–85 Object memory performance is the only SLNAA ratio without taking into account all compe- parameter consistently reported as impaired after titive amino acids is most likely to result in an ATD79,83,85–88 and seems even more pronounced in overestimation of brain TRP influx and subsequent rats with pre-existing abnormal 5-HT function.80 dissociation from brain TRP levels and 5-HT synthesis. Table 1 provides an overview of the peripheral and central neurochemical effects and other neurophysio- The 5-HIAA/5-HT ratio in the brain logical changes, as well as cognitive and affective In humans subjected to ATD, a decrease in the behavioural alterations after ATD induced in rodents, concentration of 5-HIAA in CSF suggests that less mainly rats, through administration of nutritional 5-HT has been catabolyzed, which normally takes mixtures completely devoid of TRP. 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 Methodological considerations: do we actually lower intracellular 5-HT availability is presumed to measure central effects? result in reduced 5-HT release. However, numerous It is generally assumed that the mood-lowering other peripheral factors seem to influence both the and cognitive dysfunctional effects of ATD are medi- amount of 5-HIAA produced and its transport into ated by decreases in 5-HT neuronal activity. Yet, as and out of the CSF (see ref. 52). Moreover, 5-HT described below, most parameters used to indicate release and neuronal firing seem not to correlate reduced 5-HT synthesis or release appear to merely necessarily with 5-HT metabolism,91,92 and thus the estimate decreases in 5-HT metabolism and neuronal amount of 5-HIAA in CSF seems not to be a valid activity, respectively, thereby remaining rather index of changes in 5-HT release.92,93 speculative. In contrast to human subjects, animal models offer the possibility to directly measure changes in TRP, The TRP/SLNAA ratio in plasma 5-HT and 5-HIAA concentration in distinct brain The effects of ATD treatment upon 5-HT levels in the areas. Significantly lower tissue levels of TRP and brain cannot be directly investigated in humans. In 5-HT have been reported after ATD compared with general therefore, the ratio TRP/SLNAA in plasma is TRP þ control conditions in animals.24,79,80,85 The used to estimate the amount of TRP available in the 5-HIAA/5-HT ratio is normally used to calculate the brain for synthesis into 5-HT. In most cases, total 5-HT turnover rate and estimate changes in 5-HT peripheral TRP levels (free plus albumin bound) are release, reflecting neuronal activity. However, this used for calculating the ratio to the sum of the other seems to apply only under normal physiological LNAAs. Although only the relatively small fraction of conditions when the rate of 5-HT synthesis remains free TRP eventually crosses the BBB, TRP can easily constant.94 An increase in 5-HIAA levels would then dissociate from albumin near the BBB, thereby increase the 5-HIAA/5-HT ratio and allow accurate

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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 protein–carbohydrate nutritional mixture (Solugel) or a solid TRP-free diet

Reference Age/sex/strain/ % Change plasma % Change brain Additional neurobiochemical Behavioural effects nutritional mixture/ TRP/SLNAA ratio parameters compared observations dosing regime compared with with control baseline

Brown et al.84 Adult male Sprague Not reported At T3: TRP suppletion effect in fcx: 5-HT No cognitive impairment or changes Dawley rats 5-HT hpc: À23% 45% increase compared with water. in locomotor activity depletion tryptophan acute of mechanisms Underlying AA mixture 5-HIAA hpc: À39% Decreased 5-HT after ATD in fcx or analgesia 1 Â 10 ml kgÀ1 compared with TRP þ, but not different from water. 5-HIAA striatum À35% lower compared with water, not different from TRP þ Blokland 4 months old male À38% at T5 At T5: TYR ctx: þ 44% No cognitive impairment or et al.77 Wistar rats TRP hpc: À33% depressive-like effects. Increased AA mixture TRP fcx (no pfc): À14% anxiety at T5 1 Â 10 ml kgÀ1 (5 g /kgÀ1 ) Donkelaar van EL TRP þ : 0.153 g absolute amount Lieben et al.24 4 months old male À71% at T2 At T4: Lowering of 5-HT in both hpc and NA Wistar rats À78% at T4 TRP hpc: À43% striatum. Both TRP and 5-HIAA

Solugel 5-HT hpc: À67% levels only decreased compared al et 2 Â 10 ml kgÀ1 ,90min 5-HT striatum: À40% with control in hpc. Increase (43%) interval 5-HIAA hpc: À40% 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 Lieben et al.83 4 months old male À70% at T2 Not reported Object recognition impairment at T4 Wistar rats À65% at T4 No anxiety or depressive-like effects Solugel2 Â 10 ml kgÀ1 , À49% at T6 and no effects upon spatial learning 90 min interval in water maze Cahir et al.78 Adult male Sprague Free plasma TRP TRP No differences between 1 and 3 NA Dawley rats At T3 fcx: À70% weeks of TRP diet upon peripheral Solid TRP-free diet À89% hpc: À66% and central parameters TRP þ : 0.7% remaining cortex: À63% Decreased 5-HT1A receptor binding 5-HT in dorsal raphe only after ATD at T3. fcx: À39% No effects upon 5-HT1A or hpc: À55% 5-HT2A binding in fcx, hpc or remaining cortex: À41% remaining cortex. After 3 weeks of diet, 46% increase in 5-HT2A binding in cortex and not, however, in fcx or hpc Table 1 Continued

Reference Age/sex/strain/ % Change plasma % Change brain Additional neurobiochemical Behavioural effects nutritional mixture/ TRP/SLNAA ratio parameters compared observations dosing regime compared with with control baseline

Van der Adult male Wistar Free plasma TRP Extracellular 5-HT and DA No differences in general activity Plasse99 rats À60% at T3 transmitter concentration and no effect of novelty stimulation Solugel À70% at T4 (microdialysis): No differences upon general activity 2 Â 10 ml kgÀ1 ,90min À35% at T5 between TRP- and TRP þ treatment interval À50% at T6 at any time point Cahir et al.97 Adult male Sprague Free plasma TRP Hpc No effects upon 5-HT turnover in NA Dawley rats At T3.5 5-HT: À37% hpc AA mixture À79% 5-HIAA: À34% No effects upon plasma or central 2 Â 10 ml kgÀ1 ,90min BDNF protein levels interval TRP þ : 2.3 g Rutten et 4 months old male At T1 Not reported Object recognition impairment at T1 al.87,97,114 Wistar rats À48% and T3, reversed by PDE4 inhibition Solugel At T3 1 Â 10 ml kgÀ1 À23% Jans et al.4 4 months old male At T4 Males: In general, higher peripheral TRP Object recognition impairment only

and female Wistar Males: TRP fcx: À56% levels were observed in females. No in female pro/es, not in males. depletion Donkelaar tryptophan van acute EL of mechanisms Underlying rats À66% Females (pro/es) central ATD effects in females met/ Generally less passive body contact Solugel Females: TRP fcx: À49% di. Higher TYR levels in female pro/ after ATD. Females generally more 2 Â 10 ml kgÀ1 ,90min À53% (pro/es) TRP hpc: À53% es compared with female met/di and active and decreased anxiety interval À55% (met/di) males. ATD-induced increased TYR in hpc and fcx in female pro/es.

Increased TYR in hpc only in female al et met/di Jans et al.85 3 months old male At T4 -BN Decreased ratio 5-HIAA/5-HT in hpc Stronger ATD-induced behavioural Brown-Norway and Brown Norway: fcx: 5-HT þ 5-HIAAk and fcx only in SD rats responses in SD rats. Object Sprague Dawley rats À58% hpc: 5-HT þ 5-HIAAk recognition impairment only in SD Solugel Sprague Dawley: - SD: rats. Controversial anxiety effects and 2 Â 10 ml kgÀ1 ,90min À48% fcx: 5-HT þ 5-HIAAk trend toward increased behavioural interval hpc: 5-HIAA k despair in SD rats. No anxiety/ 5-HIAA/5-HT k depressive-like behaviour in BN rats Jans and 2 months old male At T4 Not reported Lower intake and blunted Blokland86 Wistar rats CMS: À60% weight gain after CMS. No anxiety Solugel Control: À64% or depressive-like behaviour after 2 Â 10 ml kgÀ1 ,90min ATD or CMS. Less passive social interval interaction after ATD, independent oeua Psychiatry Molecular of CMS. Object recognition impair- ment after ATD, independent of CMS 701 702 oeua Psychiatry Molecular

Table 1 Continued

Reference Age/sex/strain/ % Change plasma % Change brain Additional neurobiochemical Behavioural effects nutritional mixture/ TRP/SLNAA ratio parameters compared observations dosing regime compared with with control baseline

Olivier et al.80 SERTÀ/À rat At T4 5-HT fcx: Decreased TRP/SLNAA compared Object recognition impairment (Slc6a41Hubr ) TRP SERT þ / þ : À19% with TRP þ control and untreated independent of genotype. No effect Solugel SERT þ / þ SERT þ /À : À19% rats, independent of genotype. after low dose ATD in SERT þ / þ depletion tryptophan acute of mechanisms Underlying 2 Â 10 ml kgÀ1 ,90min À65% SERT À/À : À63% Decreased 5-HT in both fcx and hpc, wild types. Generally strongest interval SERT þ /À 5-HT hpc: strongest in SERTÀ/À genotype. effect on SERT À/À even after TRP þ À61% SERT þ / þ À13% No genotype effect upon peripheral treatment. No effects on untreated SERT À/À SERT þ /À À18% TRP, ratio or central 5-HT levels. rats À55% SERT À/À À70% Increased plasma ratio after TRP þ ratio TRP/SLNAA treatment in all genotypes similarly mean 50%k van Donkelaar 6 months old male At T2 Not reported Object recognition impairment at et al.88 Wistar rats À73% T2, reversed by PDE2 and PDE5 Solugel inhibition Donkelaar van EL 1 Â 10 ml kgÀ1 Jans et al., 4 months old male Males Not reported Object recognition impairment in 2009169 and female Wistar 1 Â 10: À48% at T2 males at T2, T4 and T6 after rats 2 Â 10, 60 min 2 Â 10 ml kgÀ1 , 90 min interval

Solugel interval: À65% at T2 al et 10 ml kgÀ1 , several 3 Â 10, 60 min applications interval: À73% at T4 À54% at T6 Females 2 Â 10, 90 min interval: À73% at T2 À60% at T4 þ 2% at T6 À80% at T2 (after 4 days of daily treatment) À81% at T4 (after 4 days of daily treatment) Ardis et al.96 Adult male Sprague Free plasma TRP 5-HT No reduction of 5-HT or 5-HIAA in NA Dawley rats At T3.5À79% fcx: À33% striatum. Decreased 5-HIAA/5-HT AA mixture hpc: À34% ratio in both cortex (À41%) and 2 Â 10 ml kgÀ1 ,90min remaining ctx: À12% striatum (À34%), not in hpc interval 5-HIAA No effects upon DA, NE or the TRP þ : 2.3 g fcx: À37% metabolites, DOPAC and HVA hpc: À37% remaining ctx: À43% Table 1 Continued

Reference Age/sex/strain/ % Change plasma % Change brain Additional neurobiochemical Behavioural effects nutritional mixture/ TRP/SLNAA ratio parameters compared observations dosing regime compared with with control baseline

van Donkelaar 3 months old male À40% No effect of ATD upon ATD-induced decrease of CIT in hpc NA et al.81 Wistar rats TRP, 5-HT or 5-HIAA ATD-induced tissue oligaemia Solugel concentration 2 Â 10 ml kgÀ1 ,90min interval van Donkelaar 3 months old male MDMA pretreated: No effect of ATD upon Significant MDMA pretreatment NA et al.170 Wistar rats À37% TRP, 5-HT or 5-HIAA effect Solugel control: À40% concentration ATD potentiated hyperaemia only 2 Â 10 ml kgÀ1 ,90min after MDMA pretreatment interval van Donkelaar 3 months old male À56% No effect of ATD upon No effect of ATD upon serum and NA et al., 200982 Wistar rats TRP, 5-HT or 5-HIAA brain BDNF levels Solugel concentration Positive correlations between TRP depletion Donkelaar tryptophan van acute EL of mechanisms Underlying 2 Â 10 ml kgÀ1 ,90min and BDNF in hpc and pfc interval ATD application stress-mediated decreased BDNF levels in pfc van Donkelaar 3 months old male Max depletion at T1 No effect of ATD upon ATD application stress-mediated No effects upon affective or 165 et al. Swiss and C57BL/6J (30 min interval) TRP, 5-HT or 5-HIAA changes in TRP/SLNAA ratio cognitive behaviour al et mice Swiss: 2 Â 10 ml kgÀ1 : concentration at any of Solugel À74% the time points 1 Â 10, 2 Â 10, 1 Â 20 2 Â 20 ml kgÀ1 : À77% measured and and 2 Â 20 ml kgÀ1 ,30 C57BL/6J: independent of the or 60 min interval 2 Â 10 ml kgÀ1 : À40% specific dosing regime 1 Â 15 ml kgÀ1 :À70%

Abbreviations: ATD, acute tryptophan depletion (TRP treatment); BDNF, brain-derived neurotrophic factor; BN, Brown-Norway rat; CIT, ; CMS, chronic mild stress; DA, ; DOPAC, 3,4-dihydroxyphenylacetic acid; fcx, frontal cortex; hpc, hippocampus; HVA, ; 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; SERTÀ/À , homozygous serotonin transporter knockout rat; SERT þ /À , heterozygous serotonin transporter knockout rat; SERT þ / þ , wild-type control of serotonin transporter knockout rat; T, time in hours after (first) administration; TRP, tryptophan; TRP þ, 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 oeua Psychiatry Molecular decrease. 703 Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 704 estimation of an increase in 5-HT neuronal activity, as Several rat studies, all of similar experimental it is assumed that more 5-HT has been released. design, have directly reported ATD-induced Conversely, a decrease in 5-HIAA levels under a decreased central 5-HT levels after administration of constant rate of 5-HT synthesis would decrease the the same TRP-free nutritional mixture.24,80,85,99 This is 5-HIAA/5-HT ratio. Yet, ATD is applied to induce a generally thought to reflect an ATD-induced decrease decrease in 5-HT synthesis. As a decrease in intra- in 5-HT synthesis. However, as mentioned previously, cellular 5-HT availability most likely results in a the decrease in 5-HT after TRP treatment at one reduction of the amount of 5-HT available for release, specific time point is normally only compared with less extracellular 5-HT will then be available to be the control TRP þ treatment group and not with its catabolyzed into 5-HIAA. Thus, ATD is likely to own baseline concentrations. Thus, without these decrease both 5-HT and 5-HIAA, thereby maintaining baseline values before treatment, the extent to which the 5-HIAA/5-HT ratio constant. In line with this, no the significant difference in 5-HT levels between the changes in the 5-HIAA/5-HT ratio were found 3 h after experimental (TRP–) and control (TRP þ ) treatment ATD, whereas both 5-HT (À23%) and 5-HIAA (À39%) group actually imply a reduction in 5-HT release (that levels appeared to be significantly decreased in rat is, activity) after ATD remains unclear. In addition, hippocampus.84 A decrease in this ratio after ATD no direct evidence exists that ATD affects central would only occur if significantly less 5-HIAA is 5-HT release (see next section). produced compared with the already reduced amount of 5-HT synthesized. 5-HT neuronal release As described previously, the conversion of TRP into Alterations in neuronal activity can be measured by the 5-HTP intermediate by the TPH2 (tryptophan in vivo microdialysis. With this technique, changes hydroxylase 2) isoform is the first and rate-limiting in extracellular 5-HT concentrations can be measured step in the of 5-HT. In the brain, this that are indicative of changes in neuronal release. enzyme is only 50% saturated and, therefore, the rate Actual reductions of basal 5-HT release after ATD at which 5-HT is synthesized is limited only by have only been reported in combination with 5-HT substrate (that is, TRP) availability.95 However, it reuptake inhibitors.1,100,101 A blockade after 5-HT seems difficult to directly attribute an ATD-induced reuptake seems necessary to raise 5-HT to optimal decrease in the 5-HIAA/5-HT ratio to a reduction in levels for detection (see ref. 67). Yet, without the TRP availability. Brain measurements are generally initial systemic increase in extracellular 5-HT con- taken at only one specific time point that impedes a centrations, basal 5-HT release in the prefrontal cortex comparison of the central parameters with baseline of rats appeared not to be affected by ATD.99 This values. Thus, a decrease in 5-HIAA levels and in the might suggest that in the absence of de novo 5-HIAA/5-HT ratio after ATD is normally interpreted synthesis, 5-HT function can largely be maintained as evidence for an ATD-induced reduction in 5-HT from transmitter being recycled into the presynaptic metabolism. cell from the synaptic cleft. A possible decrease in However, one animal study showed that 5-HT 5-HT might then reflect a decrease in the storage pool levels might not have changed compared with TRP þ of 5-HT without affecting 5-HT release.102 In line with treatment.82 Furthermore, information upon baseline this, decreased levels in whole-brain 5-HT levels at 5-HT values is generally lacking. Although reductions 2 h after the consumption of a TRP-free diet in cats of the 5-HIAA/5-HT ratio have been repeatedly did not parallel changes in the functional activity of reported after ATD in rats,85,96,97 between the studies, 5-HTcontaining dorsal raphe cells throughout the 4 h the concomitant changes in 5-HIAA or 5-HT in after ingestion.102 Only recently, the lack of evidence specific brain areas do not seem to be in accordance for ATD-induced alterations in 5-HT release and with each other. In general, it appears that ATD- neuronal activity was first critically outlined by induced absolute changes in central 5-HT or 5-HIAA Feenstra et al.3 concentrations and potential underlying mechanisms Taken together, not only the parameters used to require further examination. calculate reductions in brain TRP availability and A decrease in the 5-HIAA/5-HT ratio after ATD in 5-HT metabolism may be somewhat inaccurate, but rats seems more likely to be caused by factors also no direct evidence exists that the ATD-induced influencing the catabolism of 5-HT, such as fluctua- reductions in central 5-HT levels correlate with tions in the activity of the MAO enzyme.98 An acute parallel changes in 5-HT neuronal activity under decrease in MAO activity would reduce the absolute normal physiological conditions. In general, there- amount of 5-HIAA produced, thereby producing a fore, ATD-induced behavioural alterations cannot relative increase in 5-HT levels. This would even- easily be directly attributed to changes in 5-HT tually lower the 5-HIAA/5-HT ratio independent of neuronal activity. Nevertheless, ATD consistently a reduction in precursor availability per se or the induces cognitive impairment in both animals and subsequent decrease in 5-HT synthesis and as such humans and triggers lowering of mood especially in does not reflect a decrease in 5-HT release. It could be healthy subjects with a vulnerable 5-HT system. ATD hypothesized that an acute change in MAO activity does not directly or indirectly affect other mono- implies a compensatory mechanism activated upon aminergic systems in rats24,82,84,85,96 and no behaviour- the acute decrease in peripheral TRP levels. al changes have been observed after depletion of other

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 705 plasma amino acids.72 Moreover, administration of a decreases the synthesis of NO, which could explain TRP-free AA mixture in primates decreased TRP and the ATD-induced object recognition impairments that 5-HIAA concentrations in CSF without affecting are consistently reported in rodents.79,80,83,85,87,88,111 metabolites, suggesting that the cate- The second messenger molecules cyclic adenosine cholamine system is not influenced by ATD.103 monophosphate (cAMP) and cyclic guanosine mono- ATD-induced functional changes thus seem speci- phosphate (cGMP) have an important role in intra- fic for the peripheral depletion of the essential amino cellular signalling and are highly involved in learning acid TRP, but are more likely 5-HT mediated than and memory processes.110,112–114 Both cAMP and 5-HT induced. Moreover, the effects of changes in cGMP are selectively hydrolyzed by phosphodiester- TRP availability upon 5-HT synthesis rate are gene- ase (PDE) enzymes, and inhibition of PDE appears to rally not measured under normal physiological be a reliable method for improving memory processes circumstances, that is, in the absence of any other by increasing the levels of either cAMP, cGMP or regulatory changes. Therefore, changes other than both.115 Administration of the PDE2 inhibitor, BAY substrate availability that interfere with normal 5-HT 60-7550, and the PDE5 inhibitor, zaprinast, have regulation or disruptions in substrate availability shown to increase NOS activity in rat hippocampus itself might act as potential confounding factors for and striatum, and improve object recognition perfor- the ATD-induced neurochemical effects as expected mance.116 Thus, besides directly increasing presynap- under normal circumstances.104 Thus, the exact tic cGMP levels, the enhancing effects of PDE5 and underlying mechanism might go beyond a straight- PDE2 inhibition upon memory performance might forward alteration in the central 5-HT system itself. also be mediated by activation of NOS postsynapti- cally.116 As NO can freely diffuse back into the presynapse, it can increase cGMP levels by stimulat- Potential alternative mechanisms underlying the 117 effects of ATD ing the synthesis of soluble guanylyl cyclase. An increase in NOS activity increases NO concentrations, Decreased (NOS) activity which might underlie the improvement of the ATD- The enzyme NOS is suggested to play an impor- induced memory impairment by PDE inhibitors.87,88 tant role in long-term potentiation (LTP) processes See also Figure 3 for an overview of how decreased and consequently in learning and memory.105,106 NOS activity might explain ATD-induced object As described below, several findings suggest that memory impairment and its attenuation through ATD may affect the activity of this enzyme. NOS PDE inhibition. catalyzes the conversion of the amino acid (ARG) into citrulline (CIT) and nitric oxide (NO;107). Cerebrovascular abnormalities Thus, NOS inhibition results in less conversion of Although not completely clear at present, ATD also ARG into CIT and NO. Therefore, the amount of NO appears to affect local cerebral blood dynamics that may synthesis might be directly related to the levels of explain behavioural ATD effects. Under normal physio- ARG and CIT. Two independent studies reported logical conditions, the cerebral metabolic rate of glucose significant lower CIT levels in the rat hippocampus (CMRG) provides an index of changes in regional after ATD.24,81 This decrease in CIT appeared to be neuronal activity, and changes in glucose metabolism independent of changes in its precursor ARG. On the are found to be closely coupled to changes in basis of the suggested interdependency of CIT and CBF.118,119 On this basis, abnormalities in either of ARG, this finding suggests that ATD might directly these closely linked neurophysiological parameters in affect the activity of NOS, and that decreased CIT depressive subjects are thought to reflect changes in concentrations most likely parallel decreases in NO. serotonergic neurotransmission in brain areas that can Endogenous NO can modulate neuronal function be functionally linked to the complexity of depressive through interference with the release of several symptomatology displayed.120–123 However, the 5-HT ,108 yet its precise interaction with neurotransmitter is a powerful vasoconstrictor124 and the 5-HT system seems rather complex. Whereas serotonergic fibres innervating cerebral arteries, arter- slight increases in NO concentrations most likely ioles and veins have been identified.47 Thus, if enhance 5-HT release, moderate increases appear to depression is represented by decreased central 5-HT decrease 5-HT release.109 The modulation of 5-HT neurotransmitter concentrations, an increase in CBF release by NO might therefore depend on pre-existing would seem more likely. Similarly, the ATD-induced NO concentrations and the effects might be differ- reduction in 5-HT synthesis would decrease vaso- ently regulated in distinct brain areas.105 constrictor tone, thereby most likely increasing CBF A decrease in the activity of NOS and subsequent due to vasodilatation. Surprisingly, a decrease in local decreased synthesis of NO after ATD could underlie CBF following ATD has been reported in human the ATD-induced object memory impairments106 as subjects125 and was also observed in rats.81 In the hippocampal inhibition of NOS is known to impair latter, the acute decrease of peripheral TRP levels object recognition performance in rats.106,110 As the resulted in a downward resetting of the cerebral flow– ATD-induced decrease in brain CIT levels is see- metabolism coupling relationship independently mingly caused by an interruption in NOS activity, this of changes in central TRP or 5-HT. This parallels same decrease in NOS activity presumably also preliminary findings of an uncoupling of flow from

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 706 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 ATD- induced 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 Figure 3 Potential underlying mechanism of acute trypto- levels of CIT as described earlier. All the findings phan (TRP) depletion-induced rat object memory impair- together suggest that ATD-induced object memory ment and its attenuation through phosphodiesterase impairments are most likely caused by a decrease in inhibition. A decrease in the ratio of TRP to the sum of NO that reduces local CBF in hippocampal areas other large neutral amino acids (SLNAA) induced by acute highly implicated in memory processing. Addition- tryptophan depletion (ATD) potentially directly affects (À) the activity of nitric oxide synthase (NOS), thereby ally, this is in line with the fact that PDE2 and PDE5 decreasing central citrulline (CIT) levels without affecting inhibition have been found to increase NOS activity its precursor arginine (ARG). A decrease in CIT most likely in the hippocampus, which explains their potential to parallels a reduction in nitric oxide (NO) that subsequently attenuate the ATD-induced object memory impair- affects local cerebral blood flow (CBF) in brain areas highly ments. In addition, PDE inhibitors increase central implicated in object memory processes. Indicated with ( þ ) cAMP and cGMP concentrations, which are both is the possible mechanisms behind the improvement of well-known vasodilators.130 Their strong vasodilating object memory deficits by inhibition of either the phospho- properties possibly contribute to the improvement of diesterase (PDE) enzyme type 5 (PDE5-I) or type 2 (PDE2-I), object memory performance through attenuation of thereby directly increasing the levels of cyclic adenosine the ATD-induced cerebrovascular effects (see also monophosphate (cAMP) and/or cyclic guanosine monopho- Figure 3). Interestingly, low-dose PDE4 or PDE5 sphate (cGMP), respectively, or type 4 (PDE4-I) increasing cAMP only. Both second messenger molecules are highly inhibition did not directly affect cerebrovascular 131 implicated in learning and memory processes. Inhibition of dynamics 30 min after administration. However,

PDE5 and PDE2 also directly activates NOS, thereby this parallels the failure of the specific low-dosing increasing NO levels that stimulate the synthesis of soluble regimes to reverse an object memory impairment in guanylyl cyclase (sGC) and cGMP presynaptically. The rodents.87,88 vasodilating properties of the PDE inhibitors might addi- tionally attenuate ATD-induced decreases in CBF. (À) Decreased brain-derived neurotrophic factor (BDNF) Inhibition or decrease; ( þ ) stimulation or increase. 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 metabolism in unipolar depressed patients compared not have a direct effect on peripheral and central with bipolar patients and healthy controls.126 BDNF levels.82,97 Disruption of BDNF regulation has Although controversy exists about the exact location been implicated in both depressive symptomatology and direction of the neurophysiological abnormalities and cognitive dysfunction. Also, there are coregulat- in depressive subjects,120,127 depressive illness is ing mechanisms between BDNF and the 5-HT system generally characterized by decreases in CBF and in general.132 The highest levels of BDNF mRNA have CMRG in prefrontal cortex structures. However, it been reported in the dentate gyrus and hippocampal seems unlikely that a decrease in 5–HT alone CA3 and CA2 layers.133,134 Interestingly, another accounts for the specific decreases in haemodynamic study81 showed an ATD-induced decrease in local regulation. CBF in similar areas (dentate PO, CA3 and CA2 region In addition, low CBF most likely increases the of the hippocampus). Regional changes in BDNF interaction between the albumin-bound TRP complex protein levels have been found after brief cerebrovas- and the glycocalyx of the BBB, thereby causing more cular events,135 consistent with the fact that BDNF has TRP to dissociate from albumin.21,22 This might an important neuroprotective role.136,137 Therefore, it provide an explanation for the fact that ATD-induced can be suggested that ATD, cerebrovascular effects peripheral depletion of total TRP levels does not and BDNF could be interlinked. result in a significant decrease in brain TRP avail- In addition, BDNF has been implicated as having an ability. Although it remains unclear how changes in important role in neuronal plasticity, including peripheral TRP concentrations can interfere with the LTP.138 As LTP is assumed to be the underlying dynamic regulation of CBF, the findings support the substrate of learning and memory processes,139 BDNF notion that the underlying mechanisms of ATD might is thought to be a potential mediator of memory go beyond a straightforward 5-HT-mediated mecha- formation in general, and is known to be required nism (see also Figure 3). specifically for memory consolidation.140 ATD in

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 707 human subjects selectively impairs memory consoli- food deprivation, repeated oral administrations by dation,74 which has been suggested to be caused by gavage, blood sampling and the immobilization lower 5-HT levels in hippocampal areas.73 However, necessary for applying the former are well-known in rats, the ATD-induced cerebrovascular changes stressors.159–162 Yet, these experimental procedures were independent of changes in central 5-HT levels.81 are inevitably implicated in the application of the This suggests that decreased CBF in brain regions ATD method in rats and mice. In rats, both acute and normally high in BDNF levels could also be con- repeated exposure to stressful stimuli have been sidered underlying BDNF-mediated alterations in shown to increase glucocorticoid levels and alter learning and memory processing after ATD. 5-HT turnover and release in both the hippocampus and frontal cortex.163 Thus, ATD application-related (KYN) metabolites procedures in animals might interfere with normal Under normal physiological conditions, only 1 to 2% brain TRP metabolism and subsequent 5-HT syn- of the amount of ingested TRP is used by the body thesis, thereby acting as confounding stress factors for for the synthesis of 5-HT.19 The majority of total the pharmacokinetic and behavioural effects of ATD. ingested TRP is catabolyzed into KYN by induction Stress-induced changes in the breakdown of of tryptophan pyrrolase in the liver.141 Induction of stored in fat cells (lipolysis) may also alter brain pyrrolase by the enzymes IDO (indolamine 2,3- TRP concentrations. In the same way that insulin ) and TDO (tryptophan 2,3-dioxygenase) stimulates the uptake of albumin-bound fatty acids in the liver reduces TRP availability142 and therefore by fat cells, thereby decreasing the fraction of free 5-HT synthesis is also influenced by IDO and TDO TRP in plasma, stress can result in the reverse; stress activity.143,144 Stimulation of these enzymes by pro- increases lipolysis and thus the amount of plasma inflammatory cytokines, in particular interferon-g,145 free-fatty acids.164 This increases the affinity of enhances the catabolism of TRP,142 thereby decreasing albumin for fatty acids, thereby displacing TRP from the amount of TRP eventually available for 5-HT its albumin-binding sites. The resultant increase in synthesis in the brain. Moreover, TDO activity can the fraction of free TRP in plasma might increase the also be induced by corticoids.146 availability of TRP for uptake into the brain (see Tryptophan pyrrolase is the first rate-limiting Figure 4). In a recent study with mice it was found enzyme of the KYN pathway and KYN is the major that oral treatment by gavage combined with blood degradation product of TRP.144 KYN is further sampling and food deprivation, which are the converted into potentially neuroactive metabolites standard experimental procedures, increased the such as kynurenine acid and .147 TRP/SLNAA ratio in plasma within 20 min.165 Thus, Independently of each other, both metabolites exert acute stress effects might explain the moderate specific effects upon N-methyl-D-aspartate (NMDA) depletion effects after ATD as a failure to considerably receptors,148 which have an important role in LTP and reduce central 5-HT levels eventually. Furthermore, it memory formation.149,150 NMDA receptor antagonists could be suggested that comparing TRP– (ATD) and have been shown to inhibit LTP and selectively TRP þ (control) conditions may not compare normal impair learning and memory,151 but antagonists can versus low levels of TRP, but instead compare also have a neuroprotective effect.152 Kynurenine acid moderate versus elevated TRP levels. has an antagonistic effect on the NMDA receptor and The effects of acute stress, including the ATD- has been shown to have neuroprotective effects.153 related application procedures, upon 5-HT autorecep- Conversely, quinolinic acid depolarizes neurons by tor binding might additionally provide an explanation activating NMDA receptors.153 As a result, quinolinic for the consistently reported ATD-induced memory acid can lead to neurotoxicity, similar to that found in dysfunction as indicated by object recognition im- hypoxia and ischaemia.154–156 Thus, an ATD-induced pairment in rats.79,83,85–88 Several 5-HT receptors, change in the amount of KYN metabolites could including 5-HT1A, are known to be highly involved provide an explanation for the observed cerebral in memory function166 and ATD has been shown to 78 oligaemia paralleling decreased peripheral TRP decrease 5-HT1A receptor binding. Also, acute levels,81 and as such KYN metabolites have already corticosterone administration directly decreases 167 been suggested to additionally account for the con- 5-HT1A autoreceptor functioning. Thus, a more sistently reported memory impairments after ATD.2 generalized stress effect of ATD application-related

procedures, possibly linked to a decrease in 5-HT1A Confounding stress effects receptor function as described above, might underlie ATD application-related procedures might produce the ATD-induced impaired object recognition perfor- stress, which might interfere with TRP metabolism, mance for which intact 5-HT1A receptor function and subsequent 5-HT synthesis as brain 5-HT, seems to be crucial (see also Figure 4). together with other monoamines, is critically in- As a final note, it should be mentioned that these volved in the mediation of the central response to stress effects may be especially related to the experi- stressors and subsequent behavioural adaptation.157 mental procedures used in animal research. In human Acute stressors stimulate hypothalamic–pituitary– research the effects of stress may be less as the adrenal axis activity, thereby increasing central 5-HT procedure of the treatment is less stressful than necessary for stress coping.158 In rodents, overnight the procedures used in animal research. Nevertheless,

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 708 mechanisms implicated in cognitive processing that presumably depend less upon 5-HT functioning per se. The potential alternative mechanisms under- lying ATD effects might therefore provide an explana- tion 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 depres- sive 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 Figure 4 Potential confounding stress effects of acute central 5-HT.78 Such an adaptive mechanism to an tryptophan (TRP) depletion application-related procedures. acute stressor might be less efficient in subjects The method of acute TRP depletion (ATD) requires several predisposed to 5-HT dysfunction, which might highly stressful procedures, such as overnight food depriva- account for the mood-lowering effects after ATD in tion, repeated blood sampling, oral administrations by these subjects. gavage and immobilization. Acute stress stimulates lipoly- As corticosteroid modulation of 5-HT function has sis, thereby increasing the amount of free-fatty acids in plasma that displace TRP from its albumin-binding sites. a central role in mood disorders and cognitive The resultant rise in free plasma TRP changes the ratio of functioning is highly impaired in clinical depression, TRP to the sum of the other large neutral amino acids (TRP/ the ATD findings reviewed in this article might have SLNAA) in favour of TRP, thereby increasing the amount of important implications regarding the mechanisms of TRP available for uptake into the brain and its subsequent adaptation to stress, and the implication of the 5-HT synthesis into 5-hydroxytryptamine (5-HT). Stress stimu- system in cognitive processing. As the 5-HT system is lates the secretion of corticosterone (CORT) that decreases primarily modulatory, it is not surprising that in the brain-derived neurotrophic factor (BDNF) and 5-HT1A clinical situation, most 5-HT-based treatments result receptor binding. Both BDNF and 5-HT1A receptor function- in only a partial symptomatic improvement. The ing are highly implicated in learning and memory pro- reviewed ATD findings thus support new insights for cesses. The acute stress of ATD application-related procedures might thus explain the failure of ATD to alternative treatment strategies along other pathways considerably decrease 5-HT metabolism and its negative that interact highly with the 5-HT system. Upregula- effect upon object memory performance through increased tion of the cAMP and BDNF systems has already CORT levels. resulted in a novel model for the mechanism of action of antidepressants and new targets for the develop- ment of therapeutic targets.168 For an adequate interpretation of data resulting the intake of the amino-acid drink in humans has from application of the method in both clinical and frequently been reported as unpleasant and some preclinical settings, the described potential alterna- associated stress cannot be ruled out.31,162–164 tive mechanisms should be taken into account. Abnormal physiological conditions, including ATD application-related confounding stressors possibly Serotonin, depression and ATD interfere with the high number of cellular processes The 5-HT system is highly implicated in a wide range that are involved in 5-HT synthesis and metabolism, of functional processes. Whereas mood-lowering and might trigger other neurophysiological systems effects after ATD are only experienced in depres- that generally interact with the 5-HT system. This sion-free subjects predisposed to genetic 5-HT possibly also reflects the heterogeneity of major abnormalities, altered cognitive processing after depressive disorder in general, as all these neuro- ATD is observed in both healthy and genetically physiological processes might eventually be altered in vulnerable subjects. Moreover, ATD-induced clinical conditions and thus potentially be treated impaired object memory performance as measured along different pharmacological pathways. In general, by the object recognition task is consistently reported several findings support the fact that depression may in rats79,80,83,85,86 even after only moderate peripheral not be caused solely by an abnormality of 5-HT TRP depletion or a single administration.87,88 For function, but more likely by a dysfunction of other adequate cognitive processing, the 5-HT system systems or brain regions modulated by 5-HT or interacts highly with other neurophysiological sys- interacting with its dietary precursor. Similarly, the tems that might all be interrupted after ATD.81,82 This ATD method does not seem to challenge the 5-HT further supports the notion that an acute decrease system per se, but rather triggers 5-HT-mediated in peripheral TRP levels directly interferes with adverse events.

Molecular Psychiatry Underlying mechanisms of acute tryptophan depletion EL van Donkelaar et al 709 Conclusions 6 Ruhe HG, Mason NS, Schene AH. Mood is indirectly related ATD currently represents the most established human to serotonin, norepinephrine and dopamine levels in humans: challenge test to investigate the involvement of the a meta-analysis of monoamine depletion studies. Mol Psychiatry 2007; 12: 331–359. 5-HT system in the pathogenesis and pathophysiology 7 Oldendorf WH. Brain uptake of radiolabeled amino acids, of affective disorders, including cognitive dysfunc- , and hexoses after arterial injection. Am J Physiol 1971; tional behaviour. However, the exact mechanism by 221: 1629–1639. which ATD exerts its neurophysiological effects, and 8 Pardridge WM. Blood-brain barrier carrier-mediated transport to what extent changes in 5-HT neuronal activity and brain metabolism of amino acids. Neurochem Res 1998; 23: 635–644. contribute to the ATD-induced functional and beha- 9 Smith QR. Transport of glutamate and other amino acids at the vioural alterations, remain unresolved issues. This blood-brain barrier. JNutr2000; 130(4S Suppl): 1016S–1122S. pivotal lack of information impedes an adequate 10 Fernstrom JD, Wurtman RJ. Brain serotonin content: physio- interpretation of the results arising from application logical dependence on plasma tryptophan levels. Science 1971; 173: 149–152. of the method in both clinical and preclinical studies. 11 Fernstrom JD, Wurtman RJ. Brain serotonin content: physiologi- As most biochemical brain values are merely indica- cal regulation by plasma neutral amino acids. Science 1972; 178: tive and thus speculative in human studies, animal 414–416. models provide a better means for the exploration of 12 Gessa GL, Biggio G, Fadda F, Corsini GU, Tagliamonte A. Effect of ATD-induced neurobiochemical alterations. 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