Serotonin Research Marches On or Why the Michaelis-Menten Equation is Important to Psychiatrists

 

As the old saying goes – the demise of serotonin (5-HT) in the psychiatric literature has been greatly exaggerated. Another worthwhile proposition would be to get to know the detractors and their work as well as the basic scientists doing the research. The criticism is predictable after a while. I made a comment about this back in 2015 and charted the Medline references for major depression and serotonin. The update of that chart is below and there has not been a steep decline in references to serotonin in depression. There are roughly three times as many serotonin references per year for psychiatric disorders. When you read the introductions to these papers – there is generally a restatement of the importance of serotonin in psychiatric disorders up to stating that serotonergic signaling is indispensable in considering antidepressant drug development. The bulk of this research is not done by psychiatrists – but by basic scientists interested in the study of mental disorders. That is the focus of this post.

 

The paper today (1) was of great interest to me for several reasons.  First, it was focused on antidepressant mechanism of action and area that needs more work.  Second, it employed physical chemistry techniques (voltammetry and reaction kinetics) that have been of great interest to me since I was exposed to them as a Physical Chemistry (PChem) undergraduate.  Third, it discusses a system of 5-HT reuptake that is relatively unknown to most psychiatrists – but clearly important.  I hope to explain it all and provide the necessary references for further study.  And finally, it is the product of a lab and collaborators with a high level of expertise in physiological chemistry including the technology necessary for accurate measurement. I am referring to Hashemi Lab.  That contrasts significantly with many critics of serotonin work who have no similar expertise and typically do not do original research in the field.

Starting with the serotonergic systems – the paper is focused on extracellular 5-HT signaling as a common feature of antidepressant medications.  Models of this process have been around for a long time.  A basic assumption of the model is that presynaptic serotonin transporter (SERT) terminates serotonergic neurotransmission by reuptake 5-HT from the synaptic cleft to the intracellular space of the presynaptic neuron. One action of antidepressants studied over the past three decades has been to block that process.  When fluoxetine was initially marketed, there was an emphasis on this process and the term selective serotonin reuptake inhibitors (SSRIs) was born. As assays become more sensitive, it was shown that medications from some other classes of antidepressants also blocked 5-HT reuptake. 

The authors describe two uptake systems.  Uptake 1 consists of SERT and is characterized as a high affinity low-capacity system. Uptake 2 consists of norepinephrine transporter (NET), dopamine transporter (DAT), organic cation transporter (OCT), and plasma membrane monoamine transporter (PMAT) as a low affinity high-capacity system.  There has been a common view that transporters are restricted to blocking reuptake of the named substance (ie. DAT will only transport dopamine).  More recently it was discovered that these proteins are not specific and will transport other monoamines.  5-HT is taken up in both streams Uptake 1 via SERT and Uptake 2 by DAT, NET, OCT, and PMAT.  Characteristics of both systems are listed in the tables below.

Transporter
Uptake 1
SERT
Uptake 2
NET
DAT
OCT
PMAT

 

Despite observed clinical differences in antidepressants, physicians are generally taught to think of them by general class based on binding studies.  There is a tendency to view all antidepressants within a class as having the same mechanism of action.  That illusion of equivalency can give the impression that within a class – they are interchangeable apart from pharmacokinetic parameters (half-life, time to max concentration, etc) and side effects commonly attributed to effects at other receptors.

The more specific mechanism of action of antidepressants at the binding site is often not mentioned.  Reuptake proteins can be bound allosterically and orthosterically (3).  Orthosteric ligands bind to the protein at the site of the natural endogenous ligand of interest - in this case serotonin. Allosteric ligands or modulators bind to the protein at sites that are peripheral to the site of interest and can be positive, negative, or silent modulators based on their effect on their effect on the orthosteric ligand. In some cases, a molecule can be both an allosteric and orthosteric modulator. In the case of antidepressant medications that is true for escitalopram.  Studying the complexity of 5-HT reuptake systems has the potential to clarify mechanisms and potentially look at mechanisms that remain unclear such as antidepressant withdrawal symptoms.

The main technology used in this study was fast scan cyclic voltammetry (FSCV) to estimate the extracellular 5-HT in the hippocampus of mice where 5-HT release has been stimulated via the median forebrain bundle (MFB).  FSCV is a technique where an electrical current is applied and it oxidizes the compound in solution at the electrode surface.  Major neurotransmitters like 5-HT, dopamine (DA), and norepinephrine (NE) oxidize under these circumstances and the resulting current flow can be used to estimate concentration.  The lab involved in the study and principal investigator developed the measurement technology and calibrated it against standardized solutions of 5-HT (4) so that the control detection was a clean square wave signal in a flow cell.

The authors performed FSCV analysis – pre and post drug in the same mice and applied Michaelis-Menten (M-M) analysis of the resulting curves from these experiments.  The M-M equation was originally derived to study enzyme kinetics.  It shows the relationship between initial reaction velocity to maximal velocity for a certain enzyme and substrate.  The M-M equation is given below:

In enzyme work, M-M equations are typically analyzed graphically (y = mx + b) plotting v_ο vs [S ]  or the inverse of 1/v_ο vs 1/[S] to determine V_max and K_M.

In previous work the authors developed an expression for M-M kinetics for two reuptake mechanisms (7):

 

Where:

R(t) = rate of release

A(t) = fraction of stimulated autoreceptors

V_max1, V_max2 = M-M Vmax for each of the reuptake mechanisms (1-slow, 2-fast)

K_m1, K_m2 = Michaelis constant for each reuptake mechanism (1-slow, 2-fast)

α and β are constants to differentially weight the reuptake mechanisms individually and synergistically

 

In their paper the authors found that escitalopram, fluoxetine, reboxetine, and ketamine all decreased 5-HT reuptake and increased extracellular 5-HT.  They have an excellent graphic of their results as Figure S2 in the Supporting information (10).  Using the M-M analysis, fluoxetine followed an orthosteric Uptake 1 mechanism.  Reboxetine followed an Uptake 2 mechanism. Escitalopram did not fit the standard M-M analysis suggesting a more complex mechanism due to the combination of allosteric and orthosteric effects as well as SERT trafficking (overexpression and internalization). Ketamine indirectly increased extracellular 5-HT by effects of histamine.  In view of their results, the authors conclude that the direct measurement of serotonin may be an indicator of antidepressant potential.

To me this is a landmark work.  Just the brief list of references below indicates very active research in this area.  It shows the amount of complexity involved in signaling at the neuronal level. The authors speculate that in one case the suggested superior efficacy of escitalopram may reflect the unique mechanism of action that they suggest. It also suggests that a much more sophisticated approach is necessary when reading the antidepressant literature. Are the suggested mechanisms of action for ketamine through NMDA really the primary mechanism of action or is it the effects of inhibitory H3 receptors on 5-HT neurons?  And what about the issue of antidepressant withdrawal phenomenon?  Do the new pharmacodynamic insights provided by the research have implications for withdrawal?  That area has been primarily addressed by pharmacokinetics in the past.  Finally – even though we have been getting glimpses of the importance of 5-HT over the past 70 years, the complexity has not been sorted out. Contrary to some opinions – the is an exciting area of research and the people involved are doing brilliant work. It raises the bar for those engaged in clinical work interested in the associated pathophysiology and pharmacology.

 

George Dawson, MD, DFAPA

 

References:

1:   Witt CE, Mena S, Holmes J, Hersey M, Buchanan AM, Parke B, Saylor R, Honan LE, Berger SN, Lumbreras S, Nijhout FH, Reed MC, Best J, Fadel J, Schloss P, Lau T, Hashemi P. Serotonin is a common thread linking different classes of antidepressants. Cell Chem Biol. 2023 Dec 21;30(12):1557-1570.e6. doi: 10.1016/j.chembiol.2023.10.009. Epub 2023 Nov 21. PMID: 37992715.

2:  Hexter M, van Batenburg-Sherwood J, Hashemi P. Novel Experimental and Analysis Strategies for Fast Voltammetry: 2. A Troubleshoot-Free Flow Cell for FSCV Calibrations. ACS Meas Sci Au. 2023 Jan 11;3(2):120-126. doi: 10.1021/acsmeasuresciau.2c00059. PMID: 37090258; PMCID: PMC10120031.

3:  John CE, Jones SR. Fast Scan Cyclic Voltammetry of Dopamine and Serotonin in Mouse Brain Slices. In: Michael AC, Borland LM, editors. Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis; 2007. Chapter 4. Available from: https://www.ncbi.nlm.nih.gov/books/NBK2579/

4:  Stucky C, Johnson MA. Improved Serotonin Measurement with Fast-Scan Cyclic Voltammetry: Mitigating Fouling by SSRIs. J Electrochem Soc. 2022 Apr;169(4):045501. doi: 10.1149/1945-7111/ac5ec3. Epub 2022 Apr 11. PMID: 36157165; PMCID: PMC9491377.

5:  Jiang C, He X, Wang Y, Chen CJ, Othman Y, Hao Y, Yuan J, Xie XQ, Feng Z. Molecular Modeling Study of a Receptor–Orthosteric Ligand–Allosteric Modulator Signaling Complex. ACS Chemical Neuroscience. 2023 Jan 24;14(3):418-34.

6:  Weber BL, Beaver JN, Gilman TL. Summarizing studies using constitutive genetic deficiency to investigate behavioural influences of uptake 2 monoamine transporters. Basic Clin Pharmacol Toxicol. 2023 Nov;133(5):439-458. doi: 10.1111/bcpt.13810. Epub 2022 Nov 20. PMID: 36316031; PMCID: PMC10657738.

7:  Wood KM, Zeqja A, Nijhout HF, Reed MC, Best J, Hashemi P. Voltammetric and mathematical evidence for dual transport mediation of serotonin clearance in vivo. J Neurochem. 2014 Aug;130(3):351-9. doi: 10.1111/jnc.12733. Epub 2014 Apr 26. PMID: 24702305; PMCID: PMC4107184.

8:  Bunin MA, Wightman RM. Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission. J Neurosci. 1998 Jul 1;18(13):4854-60. doi: 10.1523/JNEUROSCI.18-13-04854.1998. PMID: 9634551; PMCID: PMC6792557.

9.  Matthäus F, Haddjeri N, Sánchez C, Martí Y, Bahri S, Rovera R, Schloss P, Lau T. The allosteric citalopram binding site differentially interferes with neuronal firing rate and SERT trafficking in serotonergic neurons. European Neuropsychopharmacology. 2016 Nov 1;26(11):1806-17.

10:  Supporting Information for Serotonin is the Common Thread Linking Different Classes of Antidepressants  (see Figures S1 and S2).

 

Read This Critique!

 

Today was a good day for psychiatric criticism. An “Umbrella Review” that essentially declared that serotonin was dead in psychiatric research (1) has essentially been refuted (2). I do not want to mischaracterize the authors conclusion so here it the direct quote from the original paper.

“This review suggests that the huge research effort based on the serotonin hypothesis has not produced convincing evidence of a biochemical basis to depression. …  We suggest it is time to acknowledge that the serotonin theory of depression is not empirically substantiated.” (1)

Ron Pies, MD and I (3) noted several problems with the paper on a historical and rhetorical basis and penned a response based on those elements of the paper.  The authors used the terms “serotonin theory” and then “chemical imbalance theory” interchangeably in the paper.  We knew that the former was a hypothesis at best and the latter really did not exist as either a hypothesis or theory in the psychiatric literature. We referenced 4 reviews of the serotonin hypothesis from 1954 to 2017 and the results that the total evidence was inconclusive or inconsistent.  We included historical quotes to illustrate that researchers investigating neurochemistry were fully aware of the complexity of psychiatric disorders and that even clear-cut evidence of a finding implicating a neurotransmitter would not rule out environmental or psychological factors in the etiology of depression.

We also discussed the complexity of serotonergic systems in the brain and the fact that it is an ongoing focus of extensive research and ongoing publications. The only possible conclusion is that the science around serotonin is not settled and that needs to be recognized.  I put a post on my blog and hoped to move to a methodological focus on the paper but never got that far.

A group of scientists and psychiatrists was able to do that in a publication today (2).  This paper is available online and I am not going to repeat their evidence or conclusions when you can read it yourself at the link below. It is a very brief paper and I highly recommend reading it. This group found substantial methodological problems with the paper and concluded that there were substantial errors and misinterpretation of the data in the original paper.  Their conclusion was the errors prevent readers from drawing any “reliable or valid conclusions" and:

“A more accurate, constructive conclusion would be that acute tryptophan depletion and decreased plasma tryptophan in depression indicate a role for 5-HT in those vulnerable to or suffering from depression, and that molecular imaging suggests the system is perturbed. The proven efficacy of SSRIs in a proportion of people with depression lends credibility to this position.”

The most striking aspect of this critique is that it is authored by 35 scientists – many of whom are also psychiatrists. I have read papers written by many of them on aspects of the neurobiology of the human brain in various experimental settings.  There are experts in neuropharmacology and neurobiology.  The word brain trust comes to mind when I think about a group who could have written a response to the umbrella review or even the original review itself.  In addition to the neuroscience expertise – one of the authors wrote the reference on rules for conducting an umbrella review (4). There appears to be no equivalent expertise in the original paper, and in fact very few papers have that level of expertise.  Let me conclude with some observations based on the current critique:

1: Rhetoric is an important part of both general press and scientific literature.  The authors of the original Moncrieff review are all on record as supporting positions well outside of mainstream psychiatric education and practice.  To cite an example, I critiqued a paper by Middleton and Moncrieff on this blog where I also outlined various examples of philosophical, statistical, medical, and neuroscience rhetoric that essentially could have predicted the original umbrella review and both the response by Pies and I as well as the response by Jauhar, et al.   It is probably a good general policy to avoid entrenched positions when doing systematic reviews and if that is not possible to stick to clear guidelines for objectivity.

2:  The paper today was a welcome return to what psychiatrists everywhere know to be accurate and that is serotonergic systems and the brain in general are complex and the story is incomplete at this point. For the public – proclamations about causes and mechanisms are speculative apart from the evidence reviewed in today’s paper.  When you read speculative news stories about psychiatry (they generally all are) maintain a high degree of skepticism – especially if you have found something that is working for you – in this case for depression.  Always discuss what you read in the papers with your physician before making any changes. 

And for professionals, expertise still means something. With a proliferation of meta-analyses and systematic reviews being published it is evident that many authors have never done research in the field they are attempting to analyze. There is no substitute for experience doing the research and being very familiar with the literature and experimental methods in that field.  It is much easier to criticize a clinical trial than to actually do one. That is not just my experience and opinion.  Ioannidis has concluded (5): "The production of systematic reviews and meta-analyses has reached epidemic proportions. Possibly, the large majority of produced systematic reviews and meta-analyses are unnecessary, misleading, and/or conflicted." (see the graph below for an update)

3:  Several people today suggested the “damage has been done” by the original paper and there is certainly some evidence for that.  There were some suggestions that the original paper will be retracted, but I do not see that happening. Critics of psychiatry always get much more leeway than the comparatively fewer critics of other specialties.  There are many glaring examples, most notably the Rosenhan paper about psychiatric imposters - even though it was decisively critiqued at the time of its publication and subsequently shown to have been based on highly problematic and in some cases false research.  That original paper remains in a scientific journal.

4:  The profession and this journal are fortunate for the coordinated efforts by this group of authors.  It will hopefully serve as a template for responding to similar pieces in the future. I read a lot of papers in psychiatric journals and the quality of what I read is generally not very good.  Even flagship journals are publishing articles that are basically opinion pieces that call for significant modification of the entire profession. These are all typically arguments that involve author(s) attempting to control the premise of an argument.  I have read premises that are either blatantly false or unprovable and somehow these pieces are published in journals for psychiatrists. I also read medical literature and apart from the usual pieces claiming proclaiming the greatness of managed care and administrators in the American healthcare system – there are no calls for broadly reforming any other specialty. Like every other psychiatrist out there, I went to work for 35 years and was able to make a difference by helping people, doing research, and teaching in very taxing environments. Editing and peer review both need to improve - but in an environment that encourages excessive publishing it is doubtful that either will occur. 

5:  This is also a teaching and learning moment. Resident and faculty research seminars will benefit from reading both papers and reviewing the implications.  Some of those implications include questions about why it is so easy for people both inside and outside of the professional to suggest major departures in the intellectual trajectory and practice of the field and why that does not happen in any other medical specialty. 

If someone makes a claim that the field needs an immediate change in its intellectual focus or practice – there needs to be a compelling reason.  To paraphrase Carl Sagan – extraordinary demands require extraordinary proof.  We are still waiting for the extraordinary proof for serotonin, but there is some.  Proclaiming serotonin as a dead end was as big a mistake last year as it was 8 years ago.

 

George Dawson, MD, DFAPA

 

 

 References:

1: Moncrieff J, Cooper RE, Stockmann T, Amendola S, Hengartner MP, Horowitz MA. The serotonin theory of depression: a systematic umbrella review of the evidence. Mol Psychiatry. 2022 Jul 20:1-4.

2:  Jauhar S, Arnone D, Baldwin DS, Bloomfield M, Browning M, Cleare AJ, Corlett P, Deakin JFW, Erritzoe D, Fu C, Fusar-Poli P, Goodwin GM, Hayes J, Howard R, Howes OD, Juruena MF, Lam RW, Lawrie SM, McAllister-Williams H, Marwaha S, Matuskey D, McCutcheon RA, Nutt DJ, Pariante C, Pillinger T, Radhakrishnan R, Rucker J, Selvaraj S, Stokes P, Upthegrove R, Yalin N, Yatham L, Young AH, Zahn R, Cowen PJ. A leaky umbrella has little value: evidence clearly indicates the serotonin system is implicated in depression. Mol Psychiatry. 2023 Jun 16. doi: 10.1038/s41380-023-02095-y. Epub ahead of print. PMID: 37322065.

3:  Pies R, Dawson G.  The Serotonin Fixation: Much Ado About Nothing New. Psychiatric Times. 2022 Aug 22.

4: Fusar-Poli P, Radua J. Ten simple rules for conducting umbrella reviews. Evid Based Ment Health. 2018;21:95–100.

5:  Ioannidis JP. The mass production of redundant, misleading, and conflicted systematic reviews and meta‐analyses. The Milbank Quarterly. 2016 Sep;94(3):485-514.  https://onlinelibrary.wiley.com/doi/abs/10.1111/1468-0009.12210

Supplementary:

To update Ioannidis observations on the systematic reviews (SR) and meta-analyses (MA) versus randomized clinical trials (RCTs) I pulled up searches for those types of studies on PubMed and graphed them below.  The 2023 numbers are incomplete and that results in the tailing off of the graph on the right. The numbers of SR + MA compared with RCTs is striking. For the last complete year of data (2022) there were 38,422 RCTs compared with 42,738 SR and 36,614 MA.  As you might be able to estimate from the graph the inflection point where the annual production of RCTs were exceeded by SR + MA is relatively recent in about 2017, but the growth of these two groups has been exponential over the past 20 years.   That suggests to me that it is easier to talk about research rather than doing it yourself.

 

Graphics Credit:

Thanks to my colleague Eduardo Colon, MD for the sunrise photo.

 

The Umbrella Review of Serotonin

Over the past week a review was published in Molecular Psychiatry that claimed to discredit nearly all of the previous work on serotonin hypotheses of depression (there are far more than one).  Ron W. Pies, MD, and I wrote a rejoinder to this review. Whenever you consider a commentary about a published paper the level needs to be considered.  For example, if the paper is a polemic – responding to the rhetoric is one approach.  For those not familiar with the rhetoric around this issue take a look at this previous post on Chemical Imbalance Theory and you will be brought up to speed.  If you need additional information here is a second, more recent post.  If the paper is primarily scientific then responding to the science and measurements in the paper is another. These days, responding to the statistics is a third option and in the case of specialized reviews like an “umbrella review” commentary on the methodology is a third.  For our initial effort we made a conscious decision not to go “to far into the weeds” of science or statistics.

On that basis, we respond to a fair amount of rhetoric and science. I refer interested readers to our paper published this morning on the Psychiatric Times.  On that page the study I am referring to is reference 1, The serotonin theory of depression: a systematic umbrella review of the evidence.  The serotonin theory of depression is just like Fight Club – there is no serotonin theory of depression and that is one of the first points we make in the paper.

As far as the science of serotonin goes – it is fairly intense. Since 1957 when there were only 2 known serotonin receptors types, we have developed a lot of knowledge about this system.  With that knowledge there has been a mind-boggling amount of system complexity that nobody has been able to explain to date. We are basically getting glimpse of how the entire system works. It is highly likely that there are behavioral, cognitive, and autonomic correlates of these systems – but we have a way to go.  Back in the day when I was a research fellow in neuroendocrinology I tried (in vain) to find out how serotonin signaling affected the HPA axis. Practically all researchers at the time considered monoaminergic hypotheses of mood disorders to have heuristic value (see the quote below). The intervening 30 years of advanced research proved them correct. The authors of the umbrella review conclude that it is time to acknowledge that the serotonin theory of depression is unsubstantiated despite a large research effort and that this should be acknowledged.  That is difficult to do when they seem to be the only people promoting this theory.

For those interested in excellent summaries of current serotonin research I suggest the following volumes written by 41 and 128 scientists respectively.

At some point, I will take a much closer look at the methodology used in this study. Just looking at the PRISMA diagram and 360 reviews being pared down to 17 with just a few in some categories – suggests that the umbrella has collapsed.

 

George Dawson, MD, DFAPA

Reference:

Ron W. Pies, George Dawson.  The Serotonin Fixation: Much Ado About Nothing New, Psychiatric Times. August 3, 2022

https://www.psychiatrictimes.com/view/the-serotonin-fixation-much-ado-about-nothing-new

Supplementary Graphic:

When I first started to respond to the chemical imbalance theory rhetoric - I took all of the psychopharmacology books off my shelves from the past 35 years to illustrate that in all of those texts on the subject there were no references to a chemical imbalance theory and that I had never been taught such a theory by my professors (many of whom were leading psychopharmacologists).  Since the original photo, my stack of psychopharmacology journals has increased about 3/4 of a foot and that would bring the stack up to about 5 feet. I am not going to pull them all down to remeasure so I just made this graphic.

Graphics Credit:

The iceberg graphic at the top of this post was done by the following authors and I added the text only.  Full credit is listed below per Wikimedia and CC licensing:

Created by Uwe Kils (iceberg) and User:Wiska Bodo (sky)., CC BY-SA 3.0 , via Wikimedia Commons 

Genomics And Metabolomics In Psychiatry As A Combined Tool - Predicting SSRI Response

From Reference 1

The last two posts here were an introduction to some recent work that combine aspects of genomics and metabolomics.  The paper that I will briefly discuss here is done by researchers from the same groups focused on studies in genomics and metabolomics.  They present work that is quite exciting because it illustrates the amount of information necessary to analyze the biological complexity present in brain science and they produce results that may prove very useful from a clinical standpoint.  The study involves serotonin metabolism in depression. I have discussed serotonin in many places on this blog as a significant neurotransmitter that refuses to go away despite the various critics.  This work will illustrate why that may be.  The first paper (1) looks at the relationship between baseline serotonin levels and levels after treatment with selective serotonin reuptake inhibitors and what those phenotypes may be associated with at a genetic level.  A second paper (2) that I will discuss in a subsequent post looks at kynurenine metabolism in depression and the association of that metabolite with symptoms severity and the genetic correlates.  Both papers offer a new look at serotonin metabolism in depression and its genetic basis.

In paper 1, the sample included 366 patients in the Mayo Clinic Pharmacogenomics Research Network Antidepressant Medication Pharmacogenomics Study (PGRN-AMPS). 918 samples from these patients after 4 and 8 weeks of SSRI therapy were used for the metabolomics part of the study.  Baseline serotonin and changes in these levels were measured.  Patients with higher baseline levels and/or greatest drops in serotonin levels were determined to have the best response to treatment with SSRI medications.

From Reference 1

When a GWAS study was performed looking at the baseline serotonin concentrations as the phenotype and at 4 and 8 weeks of treatment a significant single nucleotide polymorphism (SNP) cluster was noted at the Tetraspanin 5 (TSPAN5) gene on chromosome 4 and a number of SNPs were noted at the Glutamate-rich 3 (ERICH3) gene on chromosome 1.  Both are highlighted in the Manhattan plot at the top of this post. Both of these genes were noted to be novel genes involved in serotonin metabolism and plasma serotonin concentrations.

The SNPs 5' of the TSPAN5 gene were cis regulatory elements for that gene (referred to in the paper as cis-expression quantitative trait loci or eQTLs).  In the ERICH3 cluster SNPs, two were variants that were associated with proteosome mediated degradation of ERICH3.  Changes in the expression of this gene were correlated with plasma serotonin concentrations but the serotonin pathway expression was unaltered.  One of the SNPs was also associated with clinical response in the STAR*D study.

The study is interesting because of the plasma serotonin concentration phenotypes, positive treatment response, and identification of associated genes and SNPs. What has not been determined is the specific mechanism of the drop in serotonin levels and the specific genetic mechanisms.  In the experimental section of their paper they show how the serotonin concentrations in the periphery could also affect levels in the CNS by looking at TSPAN5 and ERICH3 expression in neuroblastoma cells.  The authors were able to demonstrate that both mRNA and protein fold changes of enzymes involved in the synthesis, degradation, and transport of serotonin were affected by knockdown (KD) - (a technique of gene silencing by the introduction of doublestranded interfering RNAs (siRNAs)) or overexpression (OE) (a technique leading to enhanced gene transcription by the introduction of regulatory elements for transcription.) genes.     

The paper is an excellent example of molecular psychiatry and a possible application in the field of precision medicine.  The ultimate goal is to determine the treatment in psychiatry that has a high probability of working as soon as possible and eliminate the long trials that many people have to endure before they find a medication that is effect for (in this case) depression.  Along the way it will be evident that just as clinical psychiatrists have know for some time -  the general categories of psychiatric disorders - just like all polygenic illnesses are really a collection of diverse disorders at the omic levels.

The rapid identification of these many subtypes will not only lead to more rapid and efficient treatment - but also prevent unnecessary exposure to medications that can be both intolerable and ineffective.

In closing, some will question the utility of reading papers that contain a lot of terminology from what we used to call molecular biology.  I like to read these papers because it continues to consolidate what I learned in medical school and add to that knowledge. In my biochemistry seminars back then - there was still a lot of emphasis on enzymatic pathways and protein function. We had to know the basics of nucleic acid structure, function, and analysis - but nothing like the details presented in this paper.  As an example, we knew the synthetic pathways and enzymes for serotonin biosynthesis discussed in this paper - but the idea of analyzing human DNA with a chip encompassing 7.5 million SNPs across hundreds of research subjects would have been mind blowing and in many ways it still is.  Reading papers like this one also assures that you are not stuck in serotonin metabolism and receptor theory from the 1980s.  If that is all you know these days - it is not enough!

I realize this is not for everybody - but for some of us it is very exciting stuff.

 
George Dawson, MD, DFAPA

Graphics Credit:

All of the above figures are from reference 1, per a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License.  The graphics are unaltered and are taken from the paper as they are printed in reference 1 below.  This is a non-commercial and not-for-profit blog.




References:

1:  Gupta M, Neavin D, Liu D, Biernacka J, Hall-Flavin D, Bobo WV, Frye MA, Skime M, Jenkins GD, Batzler A, Kalari K, Matson W, Bhasin SS, Zhu H, Mushiroda T, Nakamura Y, Kubo M, Wang L, Kaddurah-Daouk R, Weinshilboum RM. TSPAN5, ERICH3 and selective serotonin reuptake inhibitors in major depressive disorder: pharmacometabolomics-informed pharmacogenomics. Mol Psychiatry. 2016 Dec;21(12):1717-1725. doi: 10.1038/mp.2016.6. Epub 2016 Feb 23. PubMed PMID: 26903268

2: Liu D, Ray B, Neavin DR, Zhang J, Athreya AP, Biernacka JM, Bobo WV,Hall-Flavin DK, Skime MK, Zhu H, Jenkins GD, Batzler A, Kalari KR, Boakye-Agyeman F, Matson WR, Bhasin SS, Mushiroda T, Nakamura Y, Kubo M, Iyer RK, Wang L, Frye MA, Kaddurah-Daouk R, Weinshilboum RM. Beta-defensin 1, aryl hydrocarbon receptor and plasma kynurenine in major depressive disorder: metabolomics-informed genomics. Transl Psychiatry. 2018 Jan 10;8(1):10. doi: 10.1038/s41398-017-0056-8. PubMed PMID: 29317604.


3: Ji Y, Biernacka JM, Hebbring S, Chai Y, Jenkins GD, Batzler A, Snyder KA, Drews MS, Desta Z, Flockhart D, Mushiroda T, Kubo M, Nakamura Y, Kamatani N, Schaid D, Weinshilboum RM, Mrazek DA. Pharmacogenomics of selective serotonin reuptake inhibitor treatment for major depressive disorder: genome-wide associations and functional genomics. Pharmacogenomics J. 2013 Oct;13(5):456-63. doi: 10.1038/tpj.2012.32. Epub 2012 Aug 21. PubMed PMID: 22907730.

4: Mrazek DA, Biernacka JM, McAlpine DE, Benitez J, Karpyak VM, Williams MD, Hall-Flavin DK, Netzel PJ, Passov V, Rohland BM, Shinozaki G, Hoberg AA, Snyder KA, Drews MS, Skime MK, Sagen JA, Schaid DJ, Weinshilboum R, Katzelnick DJ. Treatment outcomes of depression: the pharmacogenomic research network antidepressant medication pharmacogenomic study. J Clin Psychopharmacol. 2014 Jun;34(3):313-7. doi: 10.1097/JCP.0000000000000099. Erratum in: J Clin Psychopharmacol. 2014 Oct;34(5):558. PubMed PMID: 24743713.

5: Ji Y, Schaid DJ, Desta Z, Kubo M, Batzler AJ, Snyder K, Mushiroda T, Kamatani N, Ogburn E, Hall-Flavin D, Flockhart D, Nakamura Y, Mrazek DA, Weinshilboum RM. Citalopram and escitalopram plasma drug and metabolite concentrations: genome-wide associations. Br J Clin Pharmacol. 2014 Aug;78(2):373-83. doi: 10.1111/bcp.12348. PubMed PMID: 24528284; PubMed Central PMCID: PMC4137829.

6: Athreya A, Iyer R, Neavin D, Wang L, Weinshilboum R, Kaddurah-Daouk R, Rush J, Frye M, Bobo W. Augmentation of Physician Assessments with Multi-Omics Enhances Predictability of Drug Response: A Case Study of Major Depressive Disorder. IEEE Comput Intell Mag. 2018 Aug;13(3):20-31. doi: 10.1109/MCI.2018.2840660. Epub 2018 Jul 20. PubMed PMID: 30467458; PubMed Central PMCID: PMC6241311.

The Heuristic is Dead - Long Live The Heuristic

One of the latest non-scandals to rock monolithic psychiatry and psychiatrists everywhere is David Healy's commentary on serotonin and depression (1).  Certainly nothing new there from the Healian perspective - SSRIs don't work, SSRIs don't work for melancholic depression, no evidence that SSRIs raise or lower serotonin levels, SSRIs pushed tricyclic antidepressants out of the market, and SSRIs forced the abandonment of research on biological disturbances in depression like hypercortisolemia.  The only thing I did not see was the idea that SSRIs are somehow addicting because they cause discontinuation symptoms in a subgroup of people who take them.  Feel free to call that a withdrawal syndrome, but withdrawal does not constitute an addiction.  All of these premises allow him to reject serotonin and it's role in depression on the vaguest possible grounds while stating that it is not irrelevant:

"Serotonin is not irrelevant.  Just as with noradrenaline, dopamine, and other neurotransmitters, we can expect some correlation with personality and temperament."

Indeed - but I expect those to be a very weak correlations probably not on par with the mood disorder work.  I was glad to see his reference to the late Marrku Linnoila (2) or more specifically Linnoila and Virkkunen.  Linnoila had done some of the outstanding work in this area and work on serotonin metabolites (CSF HIAA) in alcohol use disorders remains a classic.  He talks about the serotonin based research running into the sand.  I did a Medline search of serotonin and major depression and plotted the papers per year and there does not seem to be any precipitous fall off (the 2015 references are through March).  If anything I would expect the research to increase with the availability of ligands for additional receptors and the further characterization of serotonin transporter (SERT) variants.

But the main problem with Healy's argument is that he is talking about serotonin as if there is a chemical floating around in the brain and that is it.  He is basically describing his own brand of chemical imbalance theory.  So if he is saying there is no serotonin chemical imbalance theory I would of course have to wholeheartedly agree with him.  And in fact, I would tell him the same thing I told the Prozac rep back in the 1980's:  "The brain is far more than a bag of chemicals.  Unless you can say something about brain structure and physiology naming a neurotransmitter is meaningless."  There is no such thing as a chemical imbalance theory and reading through the index of any psychopharmacology text printed in the past 30 years will confirm that.

Healy can't stop himself at that level.  He goes on to describe the impact of his chemical imbalance theory on clinicians:

"This history raises a questions about the weight doctors and others put on biological and epidemiological plausibility.  Does a plausible (but mythical) account of biology and treatment let everyone put aside clinical trial data that show no evidence of lives saved or restored function?"

Things get very tenuous at that point.  As a clinician who has made a career out of treating patients with the most severe problems  I don't care at all about the "biological and epidemiological plausibility" of a theory of a medication.  I got past that in the 1990s when the elegant Nobel laureate mechanism of action of aminophylline was debunked and the former first line drug for asthma and COPD exacerbations was suddenly relegated to tertiary status.  And even then, with the people I was running up on gurneys from the emergency department while calculating the parameters of their aminophylline drip - not a patient of mine was lost.  I suppose you could say it was all an elaborate placebo response, treating all of those people with acute shortness of breath bad enough to be brought in by paramedics.  I was after all using a tertiary treatment with a disproven molecular mechanism.  But really?  And my living, breathing pulmonary patients did not seem to mind.

The only thing that matters to me is if the medication works in my hands, has few side effects, and I am using it exclusively to save lives and restore functioning.  And without keeping anyone in suspense, I do prescribe SSRIs and they do work, and I do fully acknowledge to anyone willing to listen that the mechanism of action is unknown. Not surprisingly many patients want more than that and I can discuss speculative mechanisms as well as the next psychiatrist.  I don't think that makes me a drug company stooge or an idiot, because I am the only person with any accountability in the entire scheme of things.  It really doesn't matter to me what David Healy or anybody else thinks.  I am seeing very ill people and it is my job to get them better and not cause them side effects with the medication and not have the drug interact with one of their underlying medical conditions.  When you get right down to it, I don't need a mechanism of action to use a medication - only the approval of a regulatory body like the FDA.  At some point somebody may say that  SSRIs are no better for depression than aminophylline was for asthma, but so far (apart from Healy and various sympathizers) that has not happened.  I just attended a CME conference last weekend and they were still recommended.  As far as I know the FDA has not sent out any letter to doctors telling them not to prescribe them any more.

Healy also asks the irrelevant question:  "Do clinical trial data marketed as evidence of effectiveness make it easier to adopt a mythical account of biology ?"  Or I guess the real question is do these data make it easier to accept this strawman?  I strongly encourage any psychiatrist to read and reread the FDA approved package insert on any drug they prescribe and any drug their patient happens to be taking.  I encourage reading and rereading those package inserts especially if they are updated and sent out in a mass mailing by the FDA.  These package inserts need to be studied because they are not the final word in safe prescribing.  If you are an experienced psychiatrist you will routinely be called on to decide if a medication (based on all of the available evidence) is safe to prescribe to a patient with cardiac, liver and renal disease.  You will have minimal data to go by in most cases, because people with significant medical illness are typically eliminated from clinical trials.   Any psychiatrist reading those package inserts will recognize that for some time now, the package inserts have contained clinical trials data and that data is very useful.  It's utility has nothing to do with putative biological mechanisms, it has to do with safely monitoring and prescribing the medication.  The other consideration implicit in this question is that clinical psychiatrists are unaware that clinical trials are really a primitive technology.  Given that awareness exists, why would anyone accept clinical trials data as proof of much of anything.  How in the world does a double-blind placebo controlled study with an intent to treat analysis have anything at all to do with my practice of psychiatry?  It is totally irrelevant clinically and even less relevant theoretically.  Its only use is to get a new drug in my hands that my patients and I will accept as useful or reject as too toxic or worthless.

Why are reasonable people interested in serotonin despite Healy's commentary?

The complexity of serotonin or more appropriately serotonergic signaling for one.  Absolute levels of neurotransmitters are rarely the issue in complex disorders.  In the case of serotonin any bare bones discussion needs to consider the fact that there are 14 serotonin (5-HT) receptor subtypes coded by 18 genes.  Those receptors are organized into 7 families, 6 of which are G-protein receptor linked superfamily.  The remaining family is part of the ligand-gated ion channel superfamily.  The combinatorics of these receptor types, their specific locations, and their genetic variants can lead to a dizzying number of signaling variations that can easily be found in psychiatric research (4).  As an example, this is from a 2013 review of serotonin signaling in depression and suicide by Mann (3):

".......Part of the neurobiology observed in recurrent major depression, that is present between episodes as well as during episodes, is a series of abnormalities in the serotonergic system [19]. Cerebrospinal fluid (CSF) levels of the main serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), are low in more lethal suicide attempters, and predict the risk for future suicide with an odds ratio of 4.6 [20].  Convergent evidence comes from some reports of low CSF 5-HIAA in suicide attempters with other diagnoses such as schizophrenia [21,22], bipolar disorder [23] and personality disorders, although there are fewer studies and less consensus in these other disorders [24,25]. Nevertheless, if correct, such findings suggest that less serotonin transmission, as implied by less 5-HIAA, appears to be associated with suicidal behaviour across psychiatric diagnostic boundaries, as would be expected if it were associated with the diathesis for suicidal behaviour instead a specific psychiatric disorder. A review of post-mortem brain studies of suicides has found much the same thing: most studies find suicides have lower levels of serotonin and/or 5-HIAA in the brainstem serotonin neurons compared with psychiatric-matched groups [26–28]."
   
Apart from all of the disputed technical details, what is the importance of the Healy commentary?  It follows the political tradition of other commentaries mentioned on this blog by prominent psychiatrists.  People have sent me e-mails and asked me: "What do you mean by political?"  In the simplest analysis it is taking one end of a polarized position and running with it and bringing everything that you can to support that position.  That is the classic way that politicians work.  They are fueled by ideology.  On the surface, it seems that science operates like that as well, but ongoing scientific arguments don't include comments about marketing or words like "neurobabble".  They don't make assumptions about how clinicians think or whether they are confusing science and hypothesis testing with clinical work.  The best science encompasses complexity and contradictory data and we are past the point where neuroscience can be watered down for the masses in a 2 minute sound bite.

Neurobabble happens when a neuroscience discussion is reduced to a sound bite and debated the way that Democrats and Republicans discuss just about anything.  That is what happens when we are all polled "Serotonin - yes or no?"

If you really want to get your opinion out in the press that way that is one thing.  Let's not pretend that psychiatrists really think this way or that psychiatrists are just flunkies for Big Pharma talking about some crazy chemical imbalance theory or that we based our clinical practice on heuristics or for that matter marketing.

Psychiatrists are really bright people, time to stop pretending that we are not.


 George Dawson, MD, DFAPA



References:

1: Healy D. Serotonin and depression. BMJ. 2015 Apr 21;350:h1771. doi: 10.1136/bmj.h1771. PubMed PMID: 25900074.

2:  View my collection, "Marku Linnoila" from NCBI

3: Mann JJ. The serotonergic system in mood disorders and suicidal behaviour. Philos Trans R Soc Lond B Biol Sci. 2013 Feb 25;368(1615):20120537. doi: 10.1098/rstb.2012.0537. Print 2013. Review. PubMed PMID: 23440471

4:  KEGG.  The Serotonergic synapse.

5: Albert PR, Benkelfat C. The neurobiology of depression--revisiting the serotonin hypothesis. II. Genetic, epigenetic and clinical studies. Philos Trans R Soc Lond B Biol Sci. 2013 Feb 25;368(1615):20120535. doi: 10.1098/rstb.2012.0535. Print 2013. PubMed PMID: 23440469

6: Albert PR, Benkelfat C, Descarries L. The neurobiology of depression--revisiting the serotonin hypothesis. I. Cellular and molecular mechanisms. Philos Trans R Soc Lond B Biol Sci. 2012 Sep 5;367(1601):2378-81. doi: 10.1098/rstb.2012.0190. PubMed PMID: 22826338

Serotonin Syndrome at The Tipping Point

I spend a lot of my time trying to prevent Serotonin Syndrome (SSyn) and in recognizing it early enough prevent major complications.  I think that I have a fairly good record of doing this, but the statement is qualified by the fact that the likely incidence of SSyn is very low.  What is even more amazing is that if you look at the graphic, a large number of very common medications are implicated in serotonin (5-HT) turnover, signaling, and metabolism.  It is very common for me to see combinations of an SSRI or SNRI antidepressant and trazodone.  Some patients are also taking serotonergic migraine medications like sumatriptan, rizatriptan, zolmitriptan, frovatriptan, naratriptan, and almotriptan.  These migraine drugs are all 5-HT1 receptor agonists with varying affinities for subtypes of this receptor.  They are commonly encountered in clinical psychiatry in patients with chronic depression, chronic headaches and poorly controlled migraines.  In some cases these patients are also on valproate for migraine prophylaxis.  Any computerized drug interaction search will frequently flag 3 or 4 medication combinations that increase the risk for SSyn.  Reading the literature and practicing psychiatry results in a broad appreciation of SSyn including the broad range of precipitants and presentations.  The syndrome can be triggered by as little as a single dose of an SSRI type antidepressant.  On the other hand millions if not tens of millions of people tolerate combinations of serotonergic medications as suggested in the above diagram and in fact some of these combinations are recommended by experts for treating depression.

The presentation of SSyn will vary on who you ask.  If you are asking acute care internists or hospitalists, they are likely to say that it is an acute mental status change - usually delirium accompanying a toxic reaction to a drug.  If you ask movement disorder experts (1, 2), they may describe it as confusion, myoclonus, rigidity, and restlessness and list it in the differential diagnosis of those movement disorders and ataxia.  Psychiatrists should have the lowest threshold since we are prescribing more serotonergic medications, using augmentation strategies, and generally follow patients more closely.  I have seen it develop very gradually with onset of muscle pain secondary to hypertonia.  At that point the patient describes clear cut muscle pain and may have difficulty walking or with balance.  In consult settings, I have seen people with acute delirium completely unresponsive with ataxic breathing to the point I had to suggest an ICU setting and mechanical ventilation.

Harvey Sternbach is credited with coming up with the first diagnostic criteria for SSyn (3).  At the time his article was published in 1991, there were many descriptions of syndromes that were thought to be due to serotonergic hyperstimulation, both in humans and animals.  He analyzed 12 case reports covering 38 patients.  In the majority of these patients, the manifestations lasted for a period of 6 hours to 4 days.  There was one fatality in this case series,  where the patient developed seizures, hypotension, and disseminated intravascular coagulation.  Sternbach also discusses a second death from the literature where the patient presented with probably SSyn that was mistaken for Neuroleptic Malignant Syndrome (NMS).  As the patient was treated for NMS she developed disseminated intravascular coagulation, renal failure, hepatic failure and died.  In both of those cases, the patients were taking monoamine oxidase inhibitors with tryptophan and fluoxetine or lithium.  This was about the time that tryptophan was removed as a supplement from the American market and the author comments that this might reduce future risk.  Sternbach laid out his Suggested Diagnostic Criteria for Serotonin Syndrome based on that analysis:  

"A. Coincident with the addition of or increase in a known serotonergic agent to an established medication regimen, at least three of the following clinical features are present: 1) mental status changes (confusion, hypomania) 2) agitation 3) myoclonus 4) hyperreflexia 5) diaphoresis 6) shivering 7) tremor 8) diarrhea 9) incoordination 10) fever 
B. Other etiologies (e.g., infectious, metabolic, substance abuse or withdrawal) have been ruled out. C. A neuroleptic had not been started or increased in dosage prior to the onset of the signs and symptoms listed" (from reference 3, p. 713)

SSyn appears to be a major omission in DSM-5 (6).  The manual contains a cursory description of NMS in the section Medication-Induced Movement Disorders and Other Adverse Effects of Medication (pp. 709-714) but the only reference is in the differential diagnosis of NMS:

"Neuroleptic malignant syndrome also must be distinguished from similar syndromes resulting from the use of other substances or medications, such as serotonin syndrome; parkinsonian hyperthermia syndrome following abrupt discontinuation of dopamine agonists; alcohol or sedative withdrawal; malignant hyperthermia occurring during anesthesia; hyperthermia associated with abuse of stimulants and hallucinogens; and atropine poisoning from anticholinergics." (p. 711).

A syndrome this important needs at least equal time in the DSM-5.  In average clinical practice serotonergic medications and the flagged side effects in electronic databases probably far exceeds the concerns about medications for Parkinson's Disease and antipsychotic medications causing NMS.  The role of antidepressants in movement disorders especially myoclonus and akathisia should not be underestimated.

The most recent approach to diagnosis of SSyn has been the application of the Hunter Criteria (4).  In their original paper the authors point out that the diagnosis was typically made on the basis of recognizing 3/10 of Sternbach's criteria, but those features had low specificity and were present in other toxidromes.  The Hunter Criteria were developed by a toxicology service observing people with overdoses of serotonergic agents and were therefore more specific.  A single feature like spontaneous clonus or combinations of features like tremor and hyperreflexia or inducible clonus and diaphoresis leads to a diagnosis of serotonin toxicity (see the paper for all of the variations).

As a practicing and teaching clinical psychiatrist who has worked across a number of settings, it is critical that all psychiatrists known about SSyn and familiarize themselves with the diagnosis and acute treatment.  I have listed some of my preferred strategies in the table below on prevention and early recognition.  It is safe to say that the earlier the recognition, the better the outcome.  That is another reason why I don't suggest to anyone that they need to "get used to" a medication or even take an antidepressant if they tell me that they can't tolerate one.  It is also why the toxicity of the medications needs to be carefully explained to anyone taking them, and as a physician I have to believe that either that person or their representative will call me if there are problems.  There also needs to be a high level of vigilance for new agents that can potentially precipitate SSyn.  I recently investigated Suboxone and found a case report that when it was added to tricyclic antidepressants it precipitated the syndrome (5).
 

George Dawson, MD, DFAPA



1:  Stanley Fahn,  Joseph Jancovic.  Principles and Practice of Movement Disorders.  Churchill Livingstone Elsevier.  Philadelphia, PA, 2007.

2:  Mark Forrest Gordon,  Adena Leder.  Serotonin Syndrome.  in Movement Disorder Emergencies; Steven J Frucht, Stanley Fahn (eds).  Humana Press, Inc; Totawa, New Jersey 2005: pp 175-193.

3:  Sternbach H. The serotonin syndrome. Am J Psychiatry. 1991 Jun;148(6):705-13. Review. PubMed PMID: 2035713.

4:  Isbister GK, Buckley NA, Whyte IM. Serotonin toxicity: a practical approach to diagnosis and treatment. Med J Aust. 2007 Sep 17;187(6):361-5. Review. PubMed PMID: 17874986.

5:  Isenberg D, Wong SC, Curtis JA. Serotonin syndrome triggered by a single dose of suboxone. Am J Emerg Med. 2008 Sep;26(7):840.e3-5. doi: 10.1016/j.ajem.2008.01.039. PubMed PMID: 18774063.

6:  American Psychiatric Association.  DSM-5  Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition.  Arlington, VA, American Psychiatric Association, 2013.


Supplementary 1:

For an additional graphic of Serotonin Syndrome with a glossary of the abbreviations take a look at this link.