Molecular Psychiatry SYSTEMATIC REVIEW OPEN The serotonin theory of depression: a systematic umbrella review of the evidence Joanna Moncrieff!?™ Ruth E. Cooper, Tom Stockmann’, Simone Amendola, Michael P, Hengartner® and Mark A. Horowitz’ (© The Author(s) 2022 ‘The serotonin hypothesis of depression is still influential, We aimed to synthesise and evaluate evidence on whether depression is associated with lowered serotonin concentration or activity in a systematic umbrella review of the principal relevant areas of research. PubMed, EMBASE and PsycINFO were searched using terms appropriate to each area of research, from their inception ‘until December 2020, Systematic reviews, meta-analyses and large data-set analyses in the following areas were identified serotonin and serotonin metabolite, 5-HIAA, concentrations in body fluids; serotonin S-HT 4 receptor binding; serotonin transporter {SERT levels measured by imaging or at post mortem; tryptophan depletion studies; SERT gene associations and SERT gene- ‘environment interactions. Studies of depression associated with physical conditions and specific subtypes of depression (eg, bipolar depression) were excluded. Two independent reviewers extracted the data and assessed the quality of included studies using the AMSTAR-2, an adapted AMSTAR-2, or the STREGA for a large genetic study. The certainty of study results was assessed Using a modified version of the GRADE, We did not synthesise results of individual meta-analyses because they included ‘overlapping studies. The review was registered with PROSPERO (CRD42020207203) 17 studies were included: 12 systematic reviews and meta-analyses, 1 collaborative meta-analysis, 1 meta-analysis of large cohort studies, 1 systematic review and narrative synthesis, 1 genetic association study and 1 umbrella review. Quality of reviews was variable with some genetic studies of high quality. Two meta-analyses of overlapping studies examining the serotonin metabolite, 5-HIAA, showed no association with depression (largest n = 1002). One meta-analysis of cohort studies of plasma serotonin showed no relationship with depression, and evidence that lowered serotonin concentration was associated with antidepressant use (n ~ 1869). Two meta-analyses of ‘overlapping studies examining the 5-HT;q receptor (largest n= 561), and three meta-analyses of overlapping studies examining ‘SERT binding (largest n = 1845) showed weak and inconsistent evidence of reduced binding in some areas, which would be Consistent with increased synaptic availablity of serotonin in people with depression, if this was the original, causal abnormaly. However, effects of prior antidepressant use were not reliably excluded. One meta-analysis of tryptophan depletion studies found ‘no effect in most healthy volunteers (7 = 566), but weak evidence of an effect in those with a family history of depression (n =75). ‘Another systematic review (n = 342) and a sample of ten subsequent studies (n = 407) found no effect in volunteers. No systematic review of typtophan depletion studies has been performed since 2007. The two largest and highest quality studies of the SERT gene, one genetic association study (n= 115,257) and one collaborative meta-analysis n= 43,165), revealed no evidence of an association with depression, or of an interaction between genotype, stress and depression. The main areas of serotonin research provide no consistent evidence of there being an association between serotonin and depression, and no support for the hypothesis that depression is caused by lowered serotonin activity or concentrations. Some evidence was consistent with the possiblity that long-term antidepressant use reduces serotonin concentration. ‘Molecular Psychiatry; https//doi.org/10.1038/s41380-022-01661-0 INTRODUCTION The idea that depression is the resuit of abnormalities in brain chemicals, particularly serotonin (5-hydroxytryptamine or S-HT), has been influential for decades, and provides an important justification for the use of antidepressants. A link between lowered serotonin and depression was fist suggested in the 1960s ['], and widely publicised from the 1980s withthe advent of the Selective Serotonin, Reuptake Inhibitor (SSR) antidepressants [2~2]. Although it has been questioned more recently (5, 5], the serotonin theory of depression remains influential, with principal English language textbooks still giving it qualified support (7, 8, leading researchers endorsing it '5-11], and much empirical research based on it (11-14). Surveys suggest that 80% or more of the general public now believe itis established that depression is caused by a ‘chemical imbalance 15, 15] Many general practtioners also subscribe to ths view [171 and popular websites commonly cite the theory [18), "aon of Pychinty,Uivey Coleg London, Landon, UK Reseach and Development Departmen Goodrayes Resa Nonh Ek UK acy of Education, Hea and Haman Sencar, iver of Greemith Lndan, UK “yc UK Cora UK Depart ef panic ad Cnc Pychelgy end ety Stes, Feat af Mean ne syeholgy,Splencs Unies of me, are Ray, “Oxparment of Apple Psychlogy, Zieh Unverty of Raped Scene, uy Ssuzaand emat jmoncrtiuclas Received: 21 June 2021 Revised: 31 May 2022 Accepted: 7 June 2022 Published oan: 20 July 20 SPRINGER NATUREJ Monetieff Ik is often assumed that the effects of antidepressants demonstrate that depression must be at least partially caused by a brain-based chemical abnormality, and that the apparent efficacy of SSRIs shows that serotonin is implicated. Other ‘explanations for the effects of antidepressants have been put forward, however, including the idea that they work via an amplified placebo effect or through their ability to restrict or blunt femations in general (19, 20. Despite the fact that the serotonin theory of depression has been so influential, no comprehensive review has yet synthesised the relevant evidence, We conducted an ‘umbrella’ review of the principal areas of relevant research, following the model of a similar review examining prospective biomarkers. of major depressive disorder [21]. We sought to establish whether the current evidence supports a role for serotonin in the aetiology of depression, and specially whether depression is associated with indications of lowered serotonin concentrations or activity, METHODS Search strategy and selection criteria The present umbrella review was reported in accordance with the 2009 PRISMA statement [22]. The protocol was registered with PROSPERO in December 2020. (registration number {€RD42020207203)_{hetps//wmw-crd yorkacuk/prospero/cisplay_ record php?RecordlD 207203). This was subsequently updated to reflect our decision to modify the quality rating system for some studies to more appropriately apprase thelr cual, and to inchide a modified GRADE to assess the overall certainty of the findings in each category of the umbrella review. In order to cover the different areas and to manage the large volume of research that has been conducted on the serotonin system, we conducted an ‘umbrella review, Umbrella reviews survey ‘existing systematic reviews and meta-analyses relevant to areseatch question and represent one of the highest levels of evidence synthess avallable [23]. Athough they are traditionally resticted to systematic reviews and meta-analyses, we aimed to identiy the best evidence availabe. Therefore, we also includes some large studies that combined data from individual studies but did not employ conventional systematic review methods, and one large genetic study. The latter used nationwide databases to capture more individuals than entire meta-analyses, so is lke to provide even more reliable evidence than syntheses of individual studies We first conducted a scoping review to Identify areas of research consistently held to provide support for the serotonin hypothesis of depression. Si areas were identified, addressing the following questions: (1) Serotonin and the serotonin metabolite 5- HIAA~whether there are lower levels of serotonin and 5-HIAA in body fluids in depression; (2) Receptors - whether serotonin, receptor levels are altered in people with depression; (3) The serotonin transporter (SERT) - whether there are higher levels of the serotonin transporter in people with depression (which would lower synaptic levels of serotonin); () Depletion studies - whether tryptophan depletion (which lowers available serotonin) can induce depression; (5) SERT gene - whether there are higher levels of the serotonin transporter gene in people with depression; (6) Whether there ls an interaction between the SERT gene and stress in depression, We searched for systematic reviews, mata-analyses, and large database studies in these six areas in PubMed, EMBASE and PsycINFO using the Healthcare Databases Advanced Search tool provided by Health Education England and NICE (National Institute for Health and Care Excelence). Searches were con- ducted until December 2020 ‘We used the following terms in all searches: (depress* OR affective OR mood) AND (systematic OR meta-analysis), and limited searches to tle and abstract, since not doing so produced numerous irrelevant hits. In addition, we used terms specific to SPRINGERNATURE each area of research (full detals ate provided in Table 51, Supplement). We also searched citations and consulted with exper. Inclusion criteria were designed to identify the best available evidence in each research atea and consisted of 1. Research synthesis including. systematic reviews, meta-analysis tumbrela reviews, individual patient meta-analysis and large dataset analysis 2 ‘Studies that involve people wth depressive disorders or, for fexperimentalstucies (ryptoghan depletion those in which rood Symptoms are measured as an outcome 4 Studies of expermental procedures (eyptophan depletion) inv ving a sham or contol condition 4 Studies publshed in fun peer reviewed inerature Where more than five systematic reviews o age analyses exist, the most recent five ae inched Exclusion crterla consisted of: 1 Animal studies, 2. ‘Studies exlisively concerned with depression in physical condtons (eg. pos svoke or Parkinson's dlsease) oF exclusively focusing en Specie subtypes of depression such as postpartum depression, depression in chien or depresion in bipolar esorder No language or date restrictions were applied. In areas in which no systematic review or meta-analysis had been done within the last 10 years, we also selected the ten most recent studies at the time of searching (December 2020) fo istration of more recent findings. We performed this search using the same search sting for this domain, without restricting it to systematic reviews and meta-analyses. Data analysis ach member of the team was allocated one to three domains of serotonin research to search and screen for eligible studies using abstract and fll text review. In case of uncertainty, the entre team discussed eligibility to reach consensus. For included studies, data were extracted by two reviewers working independently, and disagreement was resolved by consensus. Authors of papers were contacted for clarification when data was missing oF unclear. We extracted summary effects, confidence intervals and mes: sures of statistical significance where these were reported, and, Where relevant, we extracted data on heterogeneity, For summary effects in the non-genetic studies, preference was given to the extraction and reporting of effect sizes. Mean differences were converted to effect sizes where appropriate data were available We did not perform a meta-analysis of the individual meta- analyses in each area because they included overlapping studies (22) all extracted datais presented in Table 1. Sensitivity analyses Were reported where they had substantial bearing on interpreta- tion of findings. The quality rating of systematic reviews and meta-analyses was assessed using AMSTAR-2 (A MeaSurement Tool to Assess systematic Reviews) [25]. For two studies that did not employ conventional systematic review methods (26, 27) we used a ‘modified version of the AMSTAR-? (see Table 53) For the genetic association study based on a large database analysis we used the STREGA assessment (STrengthening the REporting of Genetic Assocation Studies) (Table $4) [23]. Each study was rated independently by at least two authors. We report ratings of individual items on the relevant measure, and the percentage of items that were adequately addressed by each study (Table 1, with further detail in Tables $3 and 54). ‘Alongside quality ratings, ‘wo team members UM, MAK) rated the certainty of the results of each study using 2 modified ‘version of the GRADE guidelines [25]. Following the approach of Molecular Peyehiaty2 Monet ta wer eae ayynSup sovawue axa pue xaue> Saue> [eedtuovoiou Bupa ers pEDNBON omauio mye so0 Suuedio> 50 unenous uquoyees syonveo se wojeasdop nod sien 01 op Sams € ‘ ioe es Sues oven aloe Te» wag saypnis woyo> azoxy ee Buen ones Kayeoy SA (Uossaidop) 9569 “YVINS Pe UIUeees (ouenays soy usa toms ue sasuaneueyp Ams “Lame SPRINGER NATURE Molecular PeyehiatyJ Monetielf a E16 LED ~~ Aas wer orb Beg siseue2y 2900 ‘sq uone>yand 2) wo >roue> fav) 09 “sree Sneed 55401) ON "WER 0 Wo) HLI= J -AyeveBoIND ‘evo oto") 680 =6 mens ‘2 0) pouodes sou ~fyau601024 Bue DN vest pogo =< Sel snes UI LN35 PeDnPe (aoa angeoudite po hopessnes woauise IeAviswd) (orate 01 wou sinyp 4) stsheue 10549 Super Kiwend — %S6) x @suobosaeh dD se) Daye Aoewng fereon 2 uepaus a fp sito ned $90/691 puedes ou sin vone>pats “uopespou odor ou pip sams 2 paves Umi paveypaw suaned Soipms 2 snes wonerpen Supug us nseou usres e620 2002 1098 94m stoe samo onunwo> “Lane Molecular Peyehiaty SPRINGERNATURE2 Monet ta (cum angeondide sajaues uesy w pape fe saps nos 2500) sasAeuenaW / syed Banos Lopsexdop ‘aye uonemoste usnyouiied nantes onus yp Jo woniod y aw (224 ordues yr oy ao. 4) shu 29) 56) "#omeuebomoy dp m6) Daye Keung snes ueneypen ——_saneeou uucyoues stenpiaput sov‘ey SBERP LE npn ses " “Suey ip aazus8 omwoy eg Uosodop pue 406 1435 ubamog uote sdhsopms cz soya een stor" 89) stor "te w 00 fons voneposse Sno Saws wee toms panuue> “Lame SPRINGER NATURE Molecular PeychiatyJ Monetielf a sanouanny 295 (or00e) e102 sre 9 prog 385 (aoa angeoudite po hopeses woauise ravisw super Gin c pevnge ou neni ‘sajpmsssone sanjerd pauquos i (oz endures yr wou we. 1) shu 29) S60 "dysuaboinin dp 556) Bape Aoewung vn wn vm sms wonerpen, Lopsexiop uy wsyeseurfos wanLiies pu ssane ‘SSnise vonDe mu, siytoulod BL Les unseous useses ioe 7 69 wr ss=N stores Soop 18 faiaeas (orogey aioe ee aioe ee sxnouionn 903 amoung as asROGIAIAD va 102 2 sya (eroge) siz (oa) tor size ers sepiog 295 "e18 19p109 305 “0g ensesouyssans-2uan, ioe nseunson peony, e600 fons onunuo> ae Molecular Peyehiaty SPRINGERNATURE2 Monet ta nase serene 86) ee (Cees) (tet) seerine )(ententon fig. 1 Prefered Reporting items for Systematic reviews and Meta-Analyses (PRISMA) flow diagramme. Kennis etal [21], we devised six criteria relevant to the included studles: whether a unified analysis was conducted on original data; whether confounding by antidepressant use was adequately addressed; whether outcomes were pre-specified; whether results were consistent or heterogeneity was adequately addressed if present; whether there was a likelihood of publication bias; and sample size. The importance of confounding by effects of current Cr past antidepressant use has been highlighted in several studies [B0, 31] The results of each study were scored 1 or 0 according to whether they fuliled each criteria, and based on these ratings an overall judgement was made about the certainty of evidence ‘across studies in each of the six areas of research examined. The Certainty of each study was based on an algorithm that prioritised sample size and uniform analysis using original data (explained more fully in the supplementary materia, following suggestions, that these are the key aspects of reliability (27, 32]. An assessment (of the overall certainty of each domain of research examining the role of serotonin was determined by consensus of at least two authors and a direction of effect indicated, RESULTS Search results and quality rating Searching identified 361 publications across the 6 diferent areas of research, among which seventeen studies fulfilled inclusion criteria (see Fig. 1 and Table St for details ofthe selection proces). Included studies, their characteristics and results are shown in Table 1. AS no systematic review or meta-analysis had been performed within the last 10 years on serotonin depletion, we also Identified the 10 latest studies for illustration of more recent research findings (Table 2), ‘Quality ratings are suramarised in Table 1 and reported in detail in Tables 52-53. The majority (11/17) of systematic reviews and Molecular Peyehiaty meta-analyses satisfied less than 50% of criteria, Only 31% adequately assessed risk of bias in individual studies (a further 44% partially assessed this), and only 50% adequately accounted for risk of bias wher interpreting the results of the review. One collaborative meta-analysis of genetic studies was considered to be of high quality due to the inclusion of several measures to ensure consistency and relabiity (27), The large genetic analysis Of the effect of SERT polymorphisms on depression, satisfied 88% of the STREGA quality criteria [32 Serotonin and 5-HIAA Serotonin can be measured in blood, plasma, urine and CSF, butt is rapidly metabolised to S-hydroxyindoleacetic acid (5-HIAA). CSF is thought to be the ideal resource forthe study of biomarkers of DBtative brain diseases, since itis in contact with brain interstitial fui [33]. However, collecting CSF samples is invasive and carries some risk, hence large-scale studies are scarce. Three studies fulfilled inclusion criteria (Table 1). One meta- analysis of three large observational cohort. studies of post- menopausal women, revealed lower levels of plasma 5-HT in women with depression, which did not, however, reach statistical significance of p<0.05 after adjusting for multiple Comparisons. Sensitivity analyses revealed that antidepressants Were strongly associated with lower serotonin levels indepen dently of depression Two meta-analyses of a total of 19 studies of S-HIAA in CSF (seven studies were included in both) found no evidence of an association between 5-HIAA concentrations and depression. Receptors Fourteen different serotonin receptors have been identified, with most research on depression focusing on the S-HTyq receptor (11, 34), Since the functions of other S-HT receptors and their SPRINGER NATUREJ Monetielf aye oN pay oN, ay on aye on ay ON aye ON 40 96) voye Kewuns suns pe mat toa yo Avs Ane ue leuosied yim aydoad 99, 102 "Ie proqway Sto2 “Ie 3 ou.0W osssaidap uma aydoad st aw 30 A stor "ree usuEM, Arey © no 28 I A OF 9102 aspu260H nor 902 “fey sano nts 90 “te 1 mst doe “ew usen Hse 8102“ ofmewyer0q payaeds 20N AHoE 0702 "ew sywuow ¢ 3589) 38 Jo) uonerjpau >1d0.204>45d ON 9p sdnoiB pesed 0702 =ae39H fous 200) u59p feu, sms wone1poW dphied —_pejlene> pesiuopuey ‘foms ey>- (UY WweYS) YUUP a>URIeG PDE OUNE yA HYP UoREIdaP URYdordAn aYM>e BuLEEWOD F>xPMIE LOND IUO>>Y— aEL Molecular Peyehiaty SPRINGERNATURE2 Monet ta "8 1 ‘Soup mercer) 290 1435 10) boy ou jo suapina "AueU=> YO ret oy Aion, Augace uoyies poses. jo ssugpna ‘aupeoes mor hie, ‘payp ou jo suapa “Rove sespOn uewop Jo ‘aun emi ' t ° ‘ vn © Sloe" sara ' L vm L vn 208 ws 4) opp ue yo buapin Aeuns wel An ° ‘ ° ‘ wn 2026 as Jo) aya ue jo aovapine hed wor Koh ° ° ° ° vn 2436 1435 op aye ue jo spuapie Aayjeus> mo] Aan ° ‘ ' ° wn ‘ ‘ ‘ ‘ vn ‘ L wn . vn Uossaudp pue 406 1435 uBawuag uoneDesse 206 135 002 1 ° ° ° ‘ wn ° nessa ° nase uuoroies parea.u yo souapie Aujeuso mo] in ° Boye ou yo anuapia Kueus> Moy ' ‘pays ou yo souppi Kuss aaeopor 1 srcapaue a pawns ysoioi par) Ga ‘payp ou jo uepne Kuleus> ae=PoW ' ‘ ° t t L ‘en asain jo a sawioning hq Supuneue Pou fons ‘puapino yo upBuoas Jo Bune: jsano aip pue Ams Yea 20) BuReL IAA POYIPOW “E amEL ouepine yo Ares SPRINGER NATURE Molecular PeyehiatyJ Monetieff relationship to depression have not been well characterised, we restricted our analysis to data on S-HTsq receptors (11, 24) 5-lTaq : recep, in ts autorecepts HIE the tees ot i, ferotoninpressynaptically (31, therefore, if depression is the a result of reduced Serotonin actvy caused by abnormaltis inthe as S:HTyq receptor, people with depression would be expected to & show increased activity of S-HTyq receptors compared to those swthou 36) Two meta-analyses satisfied incision criteria, involving ve of alle the same studies (27,38) (see Table 1) The majority of resus Ba & across the two analyses suggested either no difference in 5-HTiq alae receptors between people with depression and contol, of'@ ele Tower level of these inhibitory receptors, which would imp fe: higher concentrations or activity of serotonin in people with Hee depression Both metaranalyes were based on studies that ae predominantly involved patents who were taking or had recent £3 $ ‘taken (within 1-3 weeks of scanning) antidepressants or other | § ge types or psychiatric rnedieation and both sets of authors 3 ag ts us medication on findings. in adcition, one analysis was of very : Peeuey Tow qualty (57, including not reporting on the numbers invalved > babas in'each analysis and using one-sided pales, and one was pogo strongly influenced by three studies and publication bias was 5 eisisl present 2 ‘The serotonin transporter (SERT) The serotonin transporter protein (SERT) transports serotonin out of the synapse, thereby lowering the availabilty of serotonin in the synapse (39, 40), Animals with an inactivated gene for SERT have higher levels of extra-cellular serotonin in the brain than normal [#1~43] and SSRIs are thought to work by inhibiting the action of SERT, and thus increasing levels of serotonin in the synaptic cleft [44], Although changes in SERT may be a marker for ther abnormalities, I depression is caused by low serotonin Availabilty or activity, and if SERT is the origin of that deficit then the amount or activity of SERT would be expected to be higher in ppeople with depression compared to those without (40). SERT binding potential isan index of the concentration of the serotonin transporter protein and SERT concentrations can also. be ‘measured post-mortem, ‘Three overlapping meta-analyses based on a total of 40 Individual “studies fulfilled inclusion criteria (See Table 1) i 5 i ale 57 39,4], Overall the data indicated possi reductions in SET Binding in some brain ares, though areas in which effects were detected were not consistent across the reviews In action elects of amidepressats and other medication cannot be ruled ext, since most inlused studies mainly or excisely involved people wino ale Fada history of toking antidepressants or othe. psychatic medications. Only one meta-analyss tested effects of antsepres Peek Sts, and akhough rests were not infiuenced by the percentage Pills of drugmnaive patents in each sty, numbers were small s0 5 Tiel Unlikely thot mediation telted effects would have been reliably Hi detected (45), All three reviews cited evidence from animal studies Halsss that antidepressant treatment reduces SERT 46-3. None ofthe S5sis 2 3 = analyses corrected for multiple testing, and one review was of very low quality [37] If the results do represent a postive finding that is ‘Where no'efect found or where prior use of medication i nt relevant as Pe independent of medication, they would suggest that depression is 38 associated with higher concentrations or activity of seretonin, HE Depletion studies mp Je fo Jo Tryptophan depletion using etary means or chemicals, such as g 4 parachiorophenyialanine (PCPA), is thought to reduce serotonin g 3 Fevels. Since PCPA is potentially toxic. reversible tryptophan 3 fe 2 depletion using an amino acid dink that lacks Wyptophan the a2 i ‘most commonly used method and i thought to affect serotonin te = 3 within 5-7h of ingestion, Questions remain, however, about ai se § Whether elther method reliably reduces brain serotonin, and about other effects including changes in brain nitrous oxide, SPRINGERNATURE Molecular Peyehiatycerebrovascular changes, reduced BONF and amino acid imbal- lances that may be produced by the manipulations and might explain observed effects independent of possible changes in serotonin activity (49) ‘One meta-analysis and one systematic review fuliled inclusion criteria (see Table 1). Data from studies involving volunteers mostly showed no effect, including a meta-analysis of parallel ‘group studies [50]. In 2. small meta-analysis of within-subject Studies involving 75 people with a positive family history, a minor effect was found, with people given the active depletion showing ‘larger decrease in mood than those wno had a sham procedure [50]. Across both reviews, studies involving people diagnosed with depression showed slightly greater mood reduction following tryptophan depletion than sham treatment overall, but most paricipants had taken or were taking antidepressants and participant numbers were small (50, 51] Since these research syntheses were conducted more than 10 yeats ago, we searched for a systematic sample of ten recently published studies (Table 2). Eight studies conducted with healthy Volunteers showed no effects of tryptophan depletion on mood, including the only two parallel group studies. One study presented effects in people with and without a family history of depression, and no differences were apparent in ether group [52] Two cross-over studies involving people with depression and current oF recent use of antidepressants showed no convin- cing effects of a depletion drink (53, 54], although one study is reported a5 positive mainly due to finding an improvement in mood in the group given the sham drink [54 SERT gene and gene-stress interactions ‘A possible link between depression and the repeat length polymorphism in the promoter region ofthe SERT gene (5-HTTLPR, Specifically the presence ofthe short repeats version, which causes lower SERT mRNA expression, has been proposed [551 Interestingly, lower levels of SERT would produce higher levels of synaptic serotonin. However, more recently, this hypothesis has been Superseded by focus on the interaction affect between this polymorphism, depression andl stress, with the idea that the short ‘version of the polymorphism may only give rise to depression in the presence of stressful life events [55, 56]. Unlke other areas of Serotonin research, numerous systematic reviews and meta-analyses fof genetic studies have been conducted, and most recently a Very large analysis based on a sample ftom two genetic databanks Details of the five mast recent studies that have addressed the association between the SERT gene and depression, and the interaction effect are detales in Table 1 ‘Although some earlier meta-analyses of case-control studies showed 2 statistical significant association between the S-HTTLPR and depression in some ethnic groups (57, 58), two recent large, high quality studies did not find an association between the SERT gene polymorphism and depression (27, 32] These two studies consist of by far the largest and most comprehensive study to date [32] and a high-quality meta-analysis that involved a consistent reanalysis of primary data across all conducted studles, including previously unpublished data, and ‘other comprehensive quality checks (27, 59] (see Table 1). Similarly, early studies based on tens of thousands of participants suggested a statistically significant interaction Between the SERT gene, forms of stress or maltreatment and depression (60-52), with a small odds ratio in the only study that reported this (1-18, 95% CI 1.09 to 1.28) [62]. However, the two recent large, high-quality stucies did not find an interaction between the SERT gene and stress in depression (Border et al [22] and Culverhouse et al) [27] (see Table *) Overall results Table 3 presents the modified GRADE ratings for each study and the overall rating ofthe strength of evidence in each area, Areas of Molecular Peyehiaty 2 Monet ta research that provided moderate or high certainty of evidence such as the studies of plasma serotonin and metabolites and the genetic and gene-stess_ interaction studies all showed no association between markers of serotonin activity and depression. Some other areas suggested findings consistent with increased serotonin activity, but evidence was of very law certainty, mainly due to small sample sizes and posse residual confounding by current or past antidepressant use. One area - the tryptophan depletion studies - showed very Iow certainty evidence of lowered serotonin activity or availabilty ina subgroup of volunteers with a family history of depression, This evidence was considered very low certainty as it derived from a subgroup of within-subject studies, numbers were small, and there was no information on medication use, which may have influenced results. Subsequent research has not confirmed an effect with numerous negative studies in volunteers DISCUSSION (Our comprehensive review of the major strands of research on serotonin shows there is no convincing evidence that depression | associated with, oF caused by, lower Serotonin concentrations or activity. Most studies found no evidence of reduced serotonin activity in people with depression compared to people without, land methods to reduce serotonin availabilty using tryptophan depletion do not consistently lower mood in volunteers. High quality, well-powered genetic studies effectively exclude an association between genotypes related to the serotonin system and depression, incliding a proposed interaction with stress Weak evidence from some studles of serotonin S-HT,, receptors and levels oF SERT points towards a possible association between Increased serotonin activity and depression. However, these results are likely 1 be influenced by prior use of antidepressants and its effects on the serotonin system [30, 31]. The effects of tryptophan depletion in some cross-over studies involving people with depression may also be mediated by antidepressants, although these are not consistently found [63], The chemical imbalance theory of depression is still put forward by professionals [17], and the serotonin theory, in particular, has formed the basis of a considerable research effort over the last few decades [14]. The general public widely believes that depression has been convincingly demonstrated to be the result of serotonin fr other chemical abnormalities (15, 16], and this belief shapes hhow people understand theit moods, leading to a pessimistic ‘outlook on the outcome of depression and negative expectancies about the possibilty of selfregulation of mood [64~55]. The idea that depression is the result of a chemical imbalance also influences decisions about whether to take or continue anti= depressant medication and may discourage people from dis- continuing treatment, potentially leading to lifelong dependence fon these drugs [67,58] [As with all research synthesis, the findings of this umbrella review are dependent on the quality ofthe included studies, and susceptible to ther Iitations. Most of the included studies were rated as low quality on the AMSTAR-2, but the GRADE approach suggested some findings were reasonably robust. Most of the rnon-genetic studies did not reliably exclude the potential effects of previous antidepressant use and were based on relatively small numbers of participants. The genetic studles, in particular ilustrate the importance of methodological rigour and sample size. Whereas some earlier, lower quality, mostly smaller studies produced marginally postive findings, these were not confirmed In better-conducted, larger and more recent studies (27, 32). The identification of depression and assessment of confounders and interaction affects were limited by the data avaiable in the ‘original studies on which the included reviews and meta-analyses Were based. Common methods such as the categorisation of Continuous measures and application of linear models to non- SPRINGER NATURE ThaR J Monetieff linear data may have led to over-estimation or underestimation of effects (59, 70], including the interaction between stress and the SERT gene. The latest systematic review of tryptophan depletion studies was conducted in 2007, and there has been considerable research produced since then, Hence, we provided a snapshot of the most recent evidence at the time of writing, but this area requites an up to date, comprehensive data synthesis. However, the recent studies Were consistent with the earlier meta-analysis ith ite evidence for an effect of tryptophan depletion on mood. ‘Although umbrella reviews typically restrict themselves to systematic reviews and meta-analyses, we aimed to provide the most comprehensive possible overview. Therefore, we chose to include meta-analyses that did not involve a systematic review and a large genetic association study on the premise that these studies contribute important data on the question of whether the serotonin hypothesis of depression is supported. As a result, the AMSTAR-2 quality rating scale, designed to evaluate the quality of conventional systematic reviews, was not easily applicable to all studies and had to be modified or replaced in ‘One study in this review found that antidepressant use was associated with a reduction of plasma serotonin [26], and itis possible that the evidence for reductions in SERT density and 5slT.q receptors in some of the included imaging study reviews may reflect compensatory adaptations to serotonin-lowering effects of prior antidepressant use. Authors of one meta-analysis also highlighted evidence of S-HIAA levels being reduced after long-term antidepressant treatment [77], These findings suggest that in the long-term antidepressants might produce compensa- tory changes [72] that are opposite to thelr acute effects (73, 74 Lowered serotonin availabilty has also been demonstrated in, animal studies fellowing prolonged antidepressant administration {75}. Further research is required to claity the effects of different drugs on neurochemical systems, including the serotonin system, especially during and after long-term use, as well as the physical and psychological consequences of such effects ‘Ths review suggests that the huge research effort based on the serotonin hypothesis has not produced convincing evidence of a biochemical basis to depression, This is consistent with research fon many other biological markers [21]. We suggest itis time to acknowledge that the serotonin theory of depression is not empirically substantiated DATA AVAILABILITY [A exraced eats aalble im the paper and suplenentay materia, Further Inforavon aout the eecionralng fr eah ating fr catego of te AMSTARE REFERENCES, 1 Coppen AL The blocenisty of affective derdes 8 J. Psychiat 2 hmean, Pryce Asocation, What Is Pychiaty? 2021 3. Glaosmtkine. Pol x 208 we Paclecom Gene lenge valase Last Feb 3056 5. 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Shep T.ntercton between selective 5 ose Tanke MAC Jngsia ME, Cees TH Jagtnan E Petersen CY, dlseontnuaton in rate of setotann snes, Newachen In 2010579857 AUTHOR CONTRIBUTIONS JN conceived he ie forthe uy. M, MA, MPH, TS ane SA degen ey IN MAM, MEHL Tape SA screened als and absactd data. JM fied the vest of the manuscript IM MAK, MPH, TS, SA and REC contbuted to the the dha nthe sudy and had al responsi for he Sets to submit for pubcon, FUNDING There wat no specie funding for thi review. MAH it supported by & cial Reseach Flows rom North East London WHS Fondation rst (ELF, Tht corning of the rer. COMPETING INTERESTS ‘Aluthos have completed the Unified Competing heres frm at hp slot dcsuend! lable on request tom the coressendng author. Sk elves no confi of ners Mires being funder ofa camper Al reyes tom Palgrave Macro, London UK for hs bok plished in December, 2121, caled“EdenceSasea AniepressontPrescpon” JM rece oles fet boots abet psychi ups reports grant om the Naina state of Heath Pychaty Network (an noma group of poy) an 2 baad ener ofthe Anded eration the Counc fer Edencebuied Pychiaty Bath ae pad SPRINGER NATURE aJ Monetielf a @ ADDITIONAL INFORMATION Supplementary inormation The one veron contain spplkmentary mate saab at htpsidotorg 0 r3ss138002201661-0. 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