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 vο vs [S ] or the inverse of 1/vο vs 1/[S] to determine Vmax and KM.
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
Vmax1, Vmax2
= M-M Vmax
for each of the reuptake mechanisms (1-slow, 2-fast)
Km1, Km2
= 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).
