More than twelve different human behavioural traits
and whole-body medical disorders are reported to
be associated with serotonin transporter (SERT)
gene (SLC6A4) variation. Reduced transporter
expression and function resulting from variation
in the gene’s major transcriptional control region
(the serotonin-transporter-gene-linked polymorphic
region (5-HTTLPR)) is associated with anxiety- and
depression-related personality traits1. Similarly,
the 5-HTTLPR and other regulatory and structural
variations (in the non-coding and coding regions,
respectively) seem to have a role in neuropsychiatric
conditions such as bipolar disorder, depression, anxiety
disorders (especially obsessive–compulsive disorder),
suicide, eating disorders, substance-abuse disorders,
autism, attention-deficit/hyperactivity disorder and
neurodegenerative disorders2–5. In addition, therapeu-
tic responses and side effects following treatment with
selective serotonin-reuptake inhibitors (SSRIs) have
been found to be associated with SLC6A4 variants6.
Other disorders with SLC6A4 associations include
myocardial infarction, pulmonary hypertension, irri-
table bowel syndrome and sudden infant death syn-
drome (SIDS)4. Although each of these findings has
been replicated at least once or supported by recent
meta-analyses4,7–9, uncertainties remain about the
biological bases for the associations3,4.
Human SLC6A4 maps to chromosome 17q11.2 and
is composed of 14 exons that span ∼40 kb (FIG. 1a). The
sequence of the transcript predicts a protein made up
of 630 amino acids with 12 transmembrane domains.
Alternative promoters, differential splicing involving
exons 1A, 1B and 1C, and 3′-untranslated-region vari-
ability resulting in multiple mRNA species are likely
to regulate expression of the gene in humans (FIG. 1a).
The transcriptional activity of SLC6A4 is modu-
lated by a variation in the length of the 5-HTTLPR
together with two single nucleotide polymorphisms
(SNPs) in this region, rs25531 and rs25532, all of
which are located upstream of the transcription start
site1,2 (FIG. 1a). Additional variants at the SLC6A4 locus
include a variable number of tandem repeats (VNTR)
polymorphism in functional intron 2 and several
other SNPs that change the structure or function of
the transporter protein1,10–12. Most of these SNPs are
rare, but the rs25531 polymorphism has a minor-allele
frequency of 9–15% in Caucasians and 24% in African
Americans and interacts with the 5-HTTLPR to affect
SLC6A4 transcription (FIG. 1c)2,13. Several of the less-
common SNPs and their haplotypes are associated
with behavioural phenotypes or disorders, including
obsessive–compulsive disorder and autism2,10–12,14–16.
To explore the question of what genetic dis-
orders might be attributable to life-long SLC6A4
*Laboratory of Clinical
Science, Intramural Research
Program, National Institute of
Mental Health, National
Institutes of Health,
Bethesda, Maryland 20892,
USA. ‡Molecular and Clinical
of Psychiatry and
Psychotherapy, University of
Würzburg, 97080 Würzburg,
A type of genetic variation
within a DNA sequence. It
occurs when a single
nucleotide (for example,
thymine) replaces one of the
other three nucleotides (for
Targeting the murine serotonin
transporter: insights into human
Dennis L. Murphy* and Klaus-Peter Lesch‡
Abstract | Mutations resulting in reduced or completely abrogated serotonin-transporter
(SERT) function in mice have led to the identification of more than 50 different phenotypic
changes, ranging from increased anxiety and stress-related behaviours to gut dysfunction,
bone weakness and late-onset obesity with metabolic syndrome. These multiple effects,
which can be amplified by gene–environment and gene–gene interactions, are primarily
attributable to altered intracellular and extracellular serotonin concentrations during
development and adulthood. Much of the human data relating to altered expression of the
gene that encodes SERT are based on genetic-association findings or correlations and are
therefore not as robust as the experimental mouse results. Nevertheless, SERT-function-
modifying gene variants in humans apparently produce many phenotypes that are similar to
those that manifest themselves in mice.
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c SERT protein
a SLC6A4 gene
(LA, LG, SA, SG)
345 6 7
(STin2: 9, 10 or 12 repeats)
b SLC6A4/SERT variants
Structural modifications of
chromosomal regions that alter
gene activity without changing
the nucleotide sequence.
A unique learning and recall
process, mostly studied with
fear conditioning, that requires
the alteration of stimulus–
response associations such
that the organism ceases to
respond to a previously
dysfunction, mice with reduced or completely abro-
gated SERT protein following targeted disruption of
Slc6a4 were generated17, revealing a remarkable pheno-
typic pleiotropy (BOX 1; FIG. 2). Additional genetic
engineering tools were used in subsequent investiga-
tions of Slc6a4, and produced consequences that were
largely consistent with those of the original studies
of the constitutive-knockout mice18-21 (FIG. 2). Many of
these effects can now be understood on the basis
of specific developmental, neurochemical, receptor-
signalling and other molecular consequences of Slc6a4
inactivation. In this Review we describe the remark-
able extent of pleiotropy in these mice and discuss
the underlying mechanisms. we also provide some
thoughts on the relevance of these observations to
human and non-human primate neurobiology, evolu-
tion, behaviour, gene–environment interactions and
Phenotypes of Slc6a4-mutant mice
Anxiety-like behaviours. Various approaches have been
used to experimentally alter Slc6a4 expression and SERT
function in mice, including the constitutive Slc6a4 knock-
out reviewed here (FIG. 2). The 5-HTTLPR short variant,
which results in lower expression of SLC6A4, is strongly
associated with anxiety-related, harm-avoidant and
negative personality traits in humans1,3,7,8,22. Slc6a4+/–
and Slc6a4–/– mice were therefore predicted to exhibit
increased anxiety-like behaviours. This was indeed
found to be the case, according to multiple measures,
in both male and female Slc6a4–/– mice with different
genetic backgrounds23–25. Gender differences and an
intermediate phenotype in heterozygote mice have also
been observed18–20,24–26. After mild postnatal foot-shock
stress experiences27 or exposure to predator odours28,
anxiety-like behaviours are intensified in Slc6a4–/– mice
but not in wild-type mice. Latent anxiety-like behaviours
Figure 1 |?organization?of?the?human?serotonin?transporter?(serT)?gene?(SLC6A4).?a | The structure of the SLC6A4
gene, including the sites of the major functional variants: the serotonin-transporter-gene-linked polymorphic region
(5-HTTLPR), the variable number of tandem repeats (VNTR) (between 9 and 12 repeats can be found in intron 2 (STin2)) and
the single nucleotide polymorphisms (SNPs) . b | Relative potential additive SERT expression and function for the major
SLC6A4 polymorphisms. There is a theoretical 4.65-fold difference in function between individuals with a combination of
the less active allele of each variant and individuals with the most active variants1,2,4,10,11. The left-most two bars bars depict the
actual differences in SERT expression and function that were measured in human lymphoblasts from large numbers of
individuals, with the light-blue bar representing the 5-HTTLPR short/short (SS) + rs25531 genotype and the yellow bar
representing the 5-HTTLPR long/long (LL) + rs25531 genotype; the six bars on the right combine this information with the
functional consequences of the additional variants (G56A, I425V and STin2) that are predicted from in vitro measurements
(dark-blue segments). c | The SERT protein, with its 12 transmembrane (TM) segments, its extracellular loops and its
intracellular amino- and carboxy-terminal tails. SNPs that change amino acids are denoted by red circles (except the
functionally validated G56A and I425V SNPs, which are coloured yellow); those in blue are synonymous. Panels a and c
modified, with permission, from REF. 4 (2004) American Society for Pharmacology and Experimental Therapeutics. Data in
part b from REFS 1,2,4,10,11.
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For most autosomal genes
expression occurs from either
allele, whereas a small
proportion (<1%) of genes are
imprinted, meaning that
expression occurs from only
one allele. Which of the two
alleles is expressed is
dependent on the parental
have also been reported in Slc6a4+/– mice that experience
poor maternal care; brain-derived neurotrophic factor
(BDNf) has been identified as the molecular substrate
of the epigenetic programming that causes these effects29.
Repeated stressful experiences in adult Slc6a4–/– mice
lead to deficits in extinction recall following fear condi-
tioning and to depression-like behaviour30. These mice
also develop changes in the dendritic morphology and
spine density of pyramidal neurons in their infralimbic
cortex and basolateral amygdala30. finally, mice treated
with the SSRI fluoxetine during early development also
exhibit increased anxiety-like behaviour as well as other
related behaviours that include avoidance of open-field
when SERT-deficient mice are interbred with mice
that lack one copy of the BDNf gene, the anxiety-like
behaviours and tissue serotonin depletion intensify, in
keeping with data that show that BDNf is required for
the development and maintenance of the brain’s serotonin
system32. Interestingly, a marked reduction in anxiety-like
behaviours was found in transgenic mice generated using
a human yeast artificial chromosome (yAC) construct
that causes a two- to threefold overexpression of SERT
and reduced extracellular serotonin concentrations.
This appears to confirm a direct relationship between
serotonin availability and anxiety-related behaviours21.
The anxiety-like behaviour in the Slc6a4+/– and
Slc6a4–/– mice can be normalized by the serotonin 1A
receptor (5-HT1A receptor) antagonist wAy 100635,
suggesting that the postsynaptic 5-HT1A receptor is a
participant in these anxiety-like behaviours24.
Somatosensory cortex and the whisker barrel pathway.
Cytoarchitectonic changes are found in the barrelfield
layer IV cortex of Slc6a4–/– and Slc6a4+/– mice, and
gene dose-dependent reductions in the density of the
barrel images has also been observed33,34. Similar changes
are also found in mice that lack the gene that codes for
monoamine oxidase A (MAOA), an enzyme that is
involved in the metabolism of serotonin34. To evaluate
whether these morphological changes were of functional
importance, responses to whisker stimulation in corti-
cal barrelfields were investigated using local cerebral
glucose-utilization measurements35. In wild-type mice,
unilateral stimulation of whiskers leads to a significant
glucose-utilization response in the contralateral somato-
sensory cortex. The magnitude of this response is sig-
nificantly reduced in Slc6a4–/– mice, and this reduction
is also observed in other components of the trigeminal
pathway and other somatosensory pathways that connect
to the barrelfields35.
Rescue of cortical barrel architecture abnormalities in
Slc6a4–/– and Slc6a4+/– mice can be accomplished by treat-
ment with the serotonin-synthesis inhibitor parachloro-
phenylalanine (PCPA) in a narrow developmental time
window between postnatal day 1 and postnatal day 2
(REF. 33). Partial genetic rescue also occurs following inac-
tivation, in this cortical area, of Htr1b, which encodes the
serotonin 1B receptor34 (5-HT1B receptor).
Neuroendocrine and sympathoadrenal responses to
stress. Slc6a4–/– and Slc6a4+/– mice have reduced basal
plasma corticosterone levels but respond to acute stress
with greater than normal levels of adrenocorticotropic
hormone (ACTH) and oxytocin release36–39. These
exaggerated ACTH responses are further increased
in Slc6a4–/– mice that have been interbred with Bdnf+/–
mice40. ACTH elevations in response to the placement
of mice on the elevated plus maze, which increases stress,
are also exaggerated in Slc6a4–/– mice41. As well, elevated
plasma corticosterone levels are found in Slc6a4–/– mice
in response to chronic mild stress42.
Hypothalamo–pituitary and adrenomedullary
responses to restraint stress in conscious mice have
also been examined43. At baseline, adrenal and pituitary
serotonin concentrations in Slc6a4–/– mice are mark-
edly lower than in littermate controls. Restraint stress
increases plasma levels of catecholamines, ACTH and
corticosterone in all genotypes, but these responses are
exaggerated in Slc6a4–/– mice. The responses are associ-
ated with significant reductions in levels of adrenaline,
Box 1 | Pleiotropy and evolution
Difficulties in unravelling genetically complex disorders, especially those that involve
the brain, have been attributed to clinical and genetic heterogeneity, variable
expressivity (penetrance), epistasis, imprinting, epigenetics and pleiotropy. Pleiotropy
refers to the situation in which multiple traits arise from mutations in a single gene.
However, there are remarkably few extended descriptions of pleiotropy available for
humans, although pleiotropy has been well documented in non-human primates, flies
and yeast. There are also few cases in which the evolutionary molecular and functional
pathways that lead to pleiotropic traits or related disease models have been successfully
disentangled. Exceptions include the effects of the dunce mutation in Drosophila1 and
the changes that are observed after targeted disruption of serotonin receptors2, factors
regulating cell growth and differentiation (such as transforming growth factor-β, p27kip1
and CRKL), signalling molecules (such as the G protein Gsα) and transcription factors
(such as Pitx2) in mice122,123. What is striking about the Slc6a4 knockout is its minimal
impact on anatomical development. By contrast, in the other examples there is a relative
lack of evident associated behavioural, neurological and other neurobiological
abnormalities. When evolutionary selection occurs on the basis of improved fitness, the
general conception has been that the naturally selected mutational variant of the gene
relates to a single phenotype that is benefitted. The presence of more than one trait that
is influenced by a gene product does not pose a problem, as long as all traits are equally
benefitted. It is not yet clear whether this is the situation for the consequences of
spontaneous human SLC6A4 variation or for engineered murine Slc6a4 variation.
Alternate possibilities are that the phenotypes do not affect fitness (termed neutral
pleiotropy), have opposing and equal contributions (antagonistic pleiotropy) or have
consequences only after sexual maturity is reached124,125.
Some population biologists favour the view that evolutionary adaptations result from
substitutions of single genes, with large effects126,127. However, more common views of
phenotypic divergence favour very small changes in many genes124,128. In the face of this
enormous pleiotropy in the Slc6a4-modified mouse, an obvious question is why there is
only one serotonin transporter. SLC6A4’s amino-acid coding structure is highly
conserved4, and the divergence of even the most closely related transporters (for
dopamine and noradrenaline) appears to pre-date vertebrate speciation, suggesting
that these transporters are at least as far from human serotonin transporter (SERT) in
evolution as Drosophila melanogaster or Caenorhabditis elegans SERT4.
What has constrained the evolution of SERT? A few theorists have discussed how trait
variation would be evolutionarily constrained by pleiotropy124,129. Thus, although any
two traits can be complimentary, some traits have been found to be or have been
considered to be antagonistic, and are therefore subject to selection pressures that act
on each one separately. In other words, the sheer number of consequences that arise
from any functional genetic variation in SERT or similar molecules would be expected
to decrease overall fitness and thus reduce the survival of the new genetic variant.
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Targeted disruption at the 5′ end
by exon 2 deletion: ‘knockout’
Insertional mutation at the 3′ end
by gene trapping, replacing exon 14
500 kb human YAC:
100% loss of function
Two- to threefold increase
in human SERT expression
Cylindrical columns of neurons
that are found in layer IV of the
rodent neocortex. Each barrel
receives sensory input from a
single whisker follicle and the
topographical organization of
the barrels corresponds
precisely to the arrangement of
whisker follicles on the face.
A test for social interaction and
offensive aggressive behaviour
in rodents. An unfamiliar
mouse (the ‘intruder’) is
introduced into the cage of a
mouse that has been kept
isolated in its ‘resident’ cage
for several months.
noradrenaline and serotonin in the adrenal glands, and
pituitary tissue ACTH is also significantly reduced43.
These differences suggest that one function of SERT is
to restrain adrenomedullary activation in response to
severe stress44. The usual increase in tyrosine-hydroxylase
transcription and adrenomedullary angiotensin-II-
receptor expression that follows adrenal adrenaline and
noradrenaline release in response to restraint and other
forms of stress does not occur in Slc6a4–/– mice45. Thus,
exaggerated adrenomedullary responses seem to be an
autonomic correlate of the anxiety-like behaviours and
exaggerated hypothalamo–pituitary responses that occur
in Slc6a4–/– mice43–45.
Aggression. Slc6a4–/– mice are less aggressive than control
mice in the isolated-resident/intruder test46. Additionally,
Slc6a4–/– and Slc6a4+/– mice fail to shorten the latency
time to first attack in a second intruder-encounter
experiment, unlike wild-type mice. This confirms a role
for altered SERT levels in aggressive behaviours and
indicates that Slc6a4–/– and Slc6a4+/– mice have altered
emotional learning in comparison with wild-type mice.
The findings might also be related to other social-
interaction deficits in the Slc6a4–/– and Slc6a4+/– mice
and suggest that these mice might be a useful model of
some aspects of social anxiety and autism46,47. Reports
of altered aggression in mice with a deletion of one
Bdnf allele suggest that studies of Slc6a4 × Bdnf double-
mutant mice might provide further evidence of specific
epistasis that is similar to that which is observed for
anxiety-like behaviours and neuroendocrine responses
in these mice40,48,49.
Sleep, brain excitability, body temperature and gut
motility. Slc6a4–/– and Slc6a4+/– mice have substan-
tially increased rapid eye movement (REM) sleep time50.
furthermore, brain excitability (as reflected in their
susceptibility to pentylenetetrazole-induced seizures)
is reduced in these mice51, whereas baseline body tem-
perature is increased52. Gut physiological function is
also abnormal in Slc6a4–/– mice, which have increased
colonic motility and other symptoms that resemble
the symptoms of human irritable bowel syndrome53.
These changes have also been found to accompany the
serotonin-precursor (5-hydroxytryptophan; 5-HTP)-
induced serotonin syndrome54.
Sensory, bladder and vascular function. Sensory function
is reduced in Slc6a4–/– mice, as reflected by their reduced
spinal reflexes and reduced responses to mildly painful
thermal or nerve-crush injuries49,55. Likewise, bladder
responses to stretching are reduced in female Slc6a4–/–
mice56. However, basic neurological and motor testing
reveals no overt changes in other reflexes, including
the eye-blink, whisker and righting reflexes46.
Both blood plasma and platelets are devoid of sero-
tonin in Slc6a4–/– mice53,57. furthermore, most peripheral
organs have markedly depleted serotonin levels, con-
firming that the normal presence of serotonin in most
peripheral tissues is dependent on SERT58. The elevated
blood pressure that normally develops in response to
reduced oxygen availability at simulated high altitudes
is reduced or absent in Slc6a4–/– mice. This might
provide an explanation for the protection against the
development of human pulmonary hypertension that
is apparently afforded by the lesser-expressing short
allele and short/short genotype of the 5-HTTLPR4,57.
Also, Slc6a4–/– mice display lower left cardiac ventricu-
lar weight relative to body weight and develop cardiac
fibrosis as well as valvulopathy57,59. Thus, serotonin and
SERT have a role not only in visceral function but also
in cardiovascular and respiratory function.
Bone and muscle strength, and agility. The ability to
cling to a wire-mesh screen (a test of strength) and
to maintain a grasp on a rotating metal rod is reduced
in Slc6a4–/– mice46. Likewise, bone weight, thickness
and structural resistance to fracture in vitro are also
significantly reduced60,61. One plausible basis for these
impairments is the lifetime reduction in physical activity
and exercise that results from reduced horizontal, verti-
cal and risk-assessment behaviours (including standing
and surveying and sniffing the environment) in SERT-
deficient mice46,62. There do not appear to have been any
Figure 2 |?Molecular?manipulations?that?alter?the?expression?and?function?of?
serotonin?transporter?(serT)?in?mice.?a–d | The central part of the schematic is a linear
depiction of the 14 exons and the interconnecting introns of Slc6a4. The sites and
strategies of the four major genetic modifications that have been performed, along with
the resulting effects on SERT expression and function, are illustrated and summarized
in the text boxes. a | For the constitutive knockout, two similar targeting constructs
directed towards exon 2 of Slc6a4 (the deleted region was replaced with a neo cassette)
have been used to disrupt the gene, both resulting in a complete abrogation of brain
SERT protein and labelled-ligand binding to SERT (that is, an abrogation of SERT binding
sites)17,18. b | In addition, a reduction or complete abrogation of SERT has been achieved
using a gene-trap approach that replaced exon 14 with a non-functional sequence20.
c | Short-interfering RNA (siRNA) has also been used to partially reduce (knockdown)
SERT levels19. d | Finally, a human yeast artificial chromosome (YAC) construct that
resulted in a two- to threefold overexpression of SERT and a reduction in extracellular
serotonin concentrations has also been created21. e | Also shown is one theoretical
model of how SERT components might insert into the lipid bilayer membrane to yield
cross-membrane facilitated transport.
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Body weight (g)
Plasma leptin (ng per ml)
6 8 10 12 14 16
+/+ +/– –/–
Plasma insulin (ng per ml)
A characteristic of the
interactions between two or
more genetic loci. Negative
epistasis occurs when the
combined phenotypic effect of
two or more loci is less than
the sum of the effects at
individual loci, whereas positive
epistasis occurs when the
combined effect of the two or
more loci is greater than the
sum at individual loci.
Rapid eye movement (REM)
The period of sleep during
which dreaming is thought to
occur. REM sleep is
characterized by increased
brain-wave activity, bursts of
rapid eye movement,
accelerated respiration and
heart rate, and muscle
relaxation to the point of
comparable evaluations of the effect of human SLC6A4-
variant associations on physical fitness or strength.
However, similar results would be predicted from the
observations in Slc6a4–/– and Slc6a4+/– mice, particularly
because reductions in bone mineral density are found in
humans receiving SSRI treatment63,64.
Body weight, locomotor activity and obesity. An obes-
ity phenotype emerges in Slc6a4–/– mice at approxi-
mately 3 months of age and becomes more exaggerated
throughout life62,65 (FIG. 3a,b). Obesity is associated with
increased plasma levels of insulin and leptin62 (FIG. 3c,d).
Obese adult Slc6a4–/– mice also exhibit hyperglycaemia
(despite normal glucose clearance) and reduced basal
corticosterone levels. Plasma triglycerides and choles-
terol are also elevated in Slc6a4–/– mice, and prelimi-
nary data indicate the presence of insulin resistance, as
reflected by the smaller-than-normal glucose reductions
that occur following insulin administration (Holmes, A.
and D.L.M., unpublished observations). Daily food con-
sumption is normal in Slc6a4–/– mice, indicating that the
obesity is not simply dietary in origin46. Indeed, daily
home-cage locomotor activity (measured concomitantly
with food consumption) is lower in both pre-obese and
obese Slc6a4–/– mice26,46. These observations contrast
with the normal motor activity that is observed in
other laboratory assessments, including habituation to
This obesity phenotype resembles that of type 2 dia-
betes with obesity — ‘metabolic syndrome’ — in humans.
The lack of hyperphagia and lack of differential response
to a high-fat diet, together with normal metabolic func-
tion, suggest that reduced serotonin signalling affects the
development of obesity through reduced motor activity
— a ‘couch potato’ syndrome. However, a confirmed
basis for the locomotor-activity deficit is unknown
at present, as are the potential contributions of some
endocrine and other metabolic factors67,68.
Responses to drugs. Many pharmacologic agents that
act through SERT (including 3,4-methylenedioxymeth-
amphetamine (MDMA), 2′-NH2-1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (2′-NH2-MPTP) and SSRIs)
or through serotonin receptors (including 8-hydroxy-
dipropylaminotetralin (8-OH-DPAT), Ru24969 and 2,5-
dimethoxy-4-iodoamphetamine (DOI)) have reduced
efficacy in Slc6a4–/– and Slc6a4+/– mice, reflecting the
reduced availability or downregulated function of these
drug targets17,37,46,51,69,70. MDMA self-administration
is absent in Slc6a4–/– mice, and alcohol preference and
ingestion are reduced (although alcohol’s sedative effects
are increased)71–74. By contrast, cocaine preference is
enhanced in Slc6a4–/– and Slc6a4+/– mice75. However, in
Slc6a4-mutant mice that have been interbred with mice
that lack the dopamine transporter, cocaine preference
is abolished66,75. Behavioral changes and large changes in
body temperature that result from treatment with 5-HT
alone or 5-HT and monoamine oxidase inhibitors occur
as components of exaggerated serotonin syndrome in
Slc6a4–/– and Slc6a4+/– mice. These behavioural changes
encompass a wide range of cognitive, autonomic and
somatic features76,77. They suggest that humans carrying
the lesser-expressing 5-HTTLPR short variant could
be at a higher risk for developing the human serotonin
The basis of SERT pleiotropy. An overview of the
multiple phenotypes that are observed in Slc6a4–/– and
Slc6a4+/– mice is provided in FIG. 4, together with a
comprehensive summary of the directions of the experi-
mental differences that are seen in these mice compared
with wild-type mice. One evident basis for these mul-
tiple changes is the anatomy of the serotonin system,
which is best considered as four interacting subsystems
(FIG. 5b). Of these, the serotonin subsystem in the CNS
has been the most comprehensively studied (FIG. 5a).
Cell bodies in the raphe nuclei provide modulatory
serotonergic input to multiple neuronal circuits in the
brain through the midbrain and upper pons cell groups,
plus input to the spinal cord from its brainstem caudal
cell groups (FIG. 5a). In the periphery, a partially separate
serotonergic enteric neural system (ENS) exists within
the gut78,79 and interacts with other CNS and peripheral
serotonergic mechanisms (FIG. 5b). furthermore, sev-
eral other tissues without any prominent serotonergic
innervations (such as lung, heart, blood-vessel and
pancreatic tissue, as well as platelets) contain functional
SERT. These tissues normally take up and store sero-
tonin in vesicles, and can release it in response to local
stimuli. Thus, they can be considered to be a peripheral
serotonergic system. A neuroendocrine serotonergic
Figure 3 |?Association?of?age-dependent?obesity?with?increased?plasma?insulin?and?
leptin?in?Slc6a4-mutant?mice.?a | Body weight as a function of age in Slc6a4–/–, Slc6a4+/–
and control mice. Across all ages the Slc6a4–/– and Slc6a4+/– mice had greater body weight
than the control mice. b | An obese Slc6a4–/– mouse (top) and a control mouse (bottom).
c,d | Plasma leptin and insulin concentrations are higher in Slc6a4–/– and Slc6a4+/– mice
than in control mice. *, significant change; **, highly significant change. Data from REF. 62.
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Slc6a4–/– and Slc6a4+/– mice: overview of major phenotypes
resistant, platelet dysfunction,
cardiac fibrosis, valvulopathy
Late-onset obesity (+20%),
type 2 diabetes, dyslipidaemia
irritable bowel syndrome
(thermal, nerve injury)
Stress responses (ACTH,
temperature and motor responses)
Gut motility (diarrhoea, constipation)
Brain glucose utilization
Bone mass and strength
Nociception (nerve injury and thermal)
Raphe serotonin neuron firing rate
EEG power spectra, ‘bursting’
Learned helplessness (forced-swim
and tail-suspension tests)
Acoustic startle response
Somatosensory and visual cortex
Infralimbic cortex (dendrite
Apoptosis in neonatal brain
Cell density in neonatal cortex
Pyramidal neuron spine density in
Effect of SSRIs (inescapable stress
and 5-HT clearance)
Effect of ipsapirone (serotonin synthesis ↓
and DRN firing rate)
Effect of RU24969 (motor function)
Effect of CP 93129 (serotonin clearance)
Effect of DOI (head twitch)
Effect of pentylenetetrazole (seizure)
Effect of MDMA (motor function and
Effect of 2′-NH2-MPTP (motor
function and temperature)
Effect of alcohol (10-day intake)
Effect of 8-OH-DPAT (temperature
and neuronal activity)
Effect of cocaine (preference)
Effect of alcohol (motor function)
SERT binding sites
Serotonin content in brain
Extracellular fluid serotonin
5-HT1A receptor sites and mRNA
5-HT2A and 5-HT2C receptors
system responds to serotonin release with diverse sig-
nalling through hormones such as prolactin, ACTH,
corticosterone and oxytocin.
The primary functional basis for the multiple pheno-
typic changes that occur in Slc6a4+/– and Slc6a4–/– mice is
a profound alteration in serotonin neurochemistry and
regulatory responses17,58. These abnormalities act in some-
what different ways during development (when serotonin
has neurotrophic and morphogenic effects80) and in
adulthood (when the continuing excess of extracellular
serotonin seems most important). Thus, this pleiotropy
(BOX 1) can be considered to result from several distinct
or overlapping pathways. furthermore, there are possible
differences in pathway formation that lead to different
pleiotropic consequences based on developmental time-
lines: for example, the same reductions in SERT function
might lead to independent and different consequences
in the brain (for example, anxiety-like behaviours) and in
the gut (for example, hypermotility) — this would be
‘true’ pleiotropy. Alternatively, the consequences of SERT
dysfunction in the brain might cause or contribute to gut
hypermotility directly, possibly through increased anxi-
ety. Less likely, but conceivably, gut dysfunction might
cause or contribute to brain and behaviour dysfunction,
Figure 4 |?overview?of?the?major?pleiotropic?central?and?peripheral?phenotypes?discovered?in?Slc6a4–/– and?
Slc6a4+/–?mice.?Explicit, specific experimental results are summarized using symbols to indicate increases (↑),
decreases (↓) or other types of significant qualitative changes (∆) in Slc6a4–/– and Slc6a4+/– mice compared with control
mice. For a more comprehensive evaluation and review of pharmacological features, see REF. 51. 2′-NH2-MPTP, 2′-NH2-
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 5-HT, 5-hydroxytryptamine; 8-OH-DPAT, 8-hydroxy-dipropylamino-
tetralin; ACTH, adrenocorticotropic hormone; DOI, 2,5-dimethoxy-4-iodoamphetamine; DRN, dorsal raphe nucleus;
EEG, electroencephalogram; MDMA, 3,4-methylenedioxymethamphetamine; NS, changes were not significant; NT, not
tested; OCT3, organic cation transporter 3; REM, rapid eye movement; SERT, serotonin transporter; SSRI, selective
90 | fEBRuARy 2008 | VOLuME 9
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Nature Reviews | Neuroscience
(B6, B7) Hippocampus
a Central serotonergic system
b Peripheral serotonergic system
An uncommon but potentially
life-threatening adverse drug
reaction that is caused by
excess serotonergic activity at
CNS and peripheral serotonin
receptors. It can result from
therapeutic drug use or
between drugs such as SSRIs
and monoamine oxidase
inhibitors. It can also occur
spontaneously in a mild form in
Slc6a4–/– mice, and Slc6a4–/–
and Slc6a4+/– mice are more
susceptible to developing the
syndrome following the
administration of relatively low
doses of many drugs.
including anxiety. Of course, different mechanisms
might also coexist and interact. Likewise, such interactive
mechanisms, possibly involving allelic variation of SERT
function, have also been postulated in multiple studies
of humans, as illustrated by one recent twin study that
directly examined the basis for the well-documented co-
occurrence of SERT-variation-related mood disorders
and coronary artery disease81,82.
Neurochemical characteristics. Basal extracellular fluid
serotonin concentrations are markedly increased in the
striatum and cortex of Slc6a4+/– and Slc6a4–/– mice83.
Serotonin clearance in the CA3 region of the hippo-
campus is prolonged in Slc6a4–/– mice, to the point
where its removal cannot be distinguished from diffu-
sion alone, and is prolonged to an intermediate extent in
Slc6a4+/– mice73,83. These markedly increased extracellu-
lar serotonin concentrations thus amplify and extend the
duration of serotonin signalling at serotonin receptors,
significantly altering the functional state of the entire
In contrast to the extracellular fluid levels, serotonin
brain-tissue concentrations are decreased by 40–60% in
Slc6a4–/– mice; furthermore, serotonin concentrations are
reduced to <10% of normal in most peripheral tissues
where serotonin is not synthesized17,38,43,58. In all of these
tissues, and in others that express SERT, the reductions
in tissue serotonin content seem to be a direct result of
a failure of this specific SERT-mediated uptake system,
with a lack of or inadequate compensation by other
As a consequence of the deficient recycling of sero-
tonin by its transporter, serotonin synthesis and turnover
are increased across brain regions in Slc6a4–/– mice, with
the greatest increase in female mice58. Dopaminergic neu-
rons in the substantia nigra accumulate excess serotonin
in Slc6a4–/– mice through the dopamine transporter84.
furthermore, expression of the organic cation transport-
ers OCT1 and OCT3, which are also low-affinity trans-
porters of monoamines, is increased53,84–86, indicative of
some partial but inefficient attempt at compensation
through heterologous transporters.
Slc6a4+/– mice have fewer specific SERT binding
sites (sites to which a labelled ligand for SERT binds),
decreased serotonin clearance and elevated extracel-
lular serotonin levels; however, they have unchanged
tissue serotonin concentrations in the brain and in the
periphery and have unchanged brain serotonin synthesis
and turnover17,58,73,83. Thus the loss of one Slc6a4 allele
leads to a decrease in transporter function, but a single
copy of Slc6a4 is adequate to maintain overall tissue
The spontaneous firing rate of serotonergic neurons
in the dorsal raphe of anesthesized Slc6a4–/– mice is ~35%
of that of wild-type control mice, with intermediate
values (~60%) in Slc6a4+/– mice69. In addition, the time
required for CA3 hippocampal neurons to return to nor-
mal firing rates following microiontophoretic applica-
tion of serotonin is prolonged. This probably represents
the failure of the rapid clearance of serotonin, as has
been documented in the CA3 hippocampal region69,73.
As expected, the SSRI paroxetine further increased the
serotonin-induced prolongation of hippocampal neuron
recovery time in Slc6a4+/– mice69.
5-HT1A receptors in the brainstem raphe area are
substantially (>60%) decreased in Slc6a4–/– female mice,
with somewhat smaller reductions in males and inter-
mediate values in Slc6a4+/– mice. female but not male
Slc6a4–/– mice also show modest reductions in 5-HT1A
receptors in the hypothalamus and in some areas of the
amygdala and septum, but show no changes in the cortex
or hippocampus36,37,87,88. Thus, Slc6a4+/– and Slc6a4–/– mice
display a downregulation of 5-HT1A receptors at presyn-
aptic somatodendritic sites, accompanied by insensitivity
of these receptors and of 5-HT1A-mediated neuroendo-
crine responses. This is similar to the changes that are
observed in rodents chronically treated with SSRIs.
Figure 5 |?central?and?peripheral?serotonergic?systems.?a | CNS serotonin neuron
cell-body groups in the nine raphe nuclei, B1–B9. The more caudal nuclei (B1–B3) in the
medulla project axons to the spinal cord and the periphery, whereas the more rostral
raphe nuclei contain the principal dorsal raphe groups (B6 and B7; depicted in yellow) and
the median raphe groups (B5 and B8; depicted in green), which project to different but
overlapping brain areas. b | Serotonin also functions in the enteric nervous system (ENS),
the hypothalamo–pituitary–adrenocortical (HPA) system, the adrenomedullary
neuroendocrine serotonin system (NSS) and the peripheral serotonin system (PSS), which
includes the lungs, the heart, the blood vessels, the pancreas and platelets36,37,43,78,130,131.
DRN, dorsal raphe nucleus; MFB, medial frontal bundle; MRN, median raphe nucleus.
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* * *
* * *
LL LS SS
* * *
(nCi per mg tissue)
(fmol per mg protein)
SLC6A4 5-HTTLPR genotypes
SERT binding sitesSERT binding sites
Serotonin uptake by SERT
Locomotor activity following MDMA
(photobeam breaks per 60 min)
Reporter-gene activity of L and S variant
Serotonin uptake by SERT
(pmol per min
per mg protein)
Cumulative locomotor activity
(pmol per 107 cells)
(cpm per µg protein)
Reflex actions that occur
relatively quickly because the
sensory neurons do not pass
directly into the brain; rather,
they synapse in the spinal
cord. This characteristic
bypasses the delay of routing
signals through the brain,
although the brain does receive
sensory input while the reflex
5-HT2A receptors are decreased in the striatum,
claustrum and cortex, but increased in the septum
and hypothalamus of Slc6a4–/– mice38,89. By contrast,
5-HT2C receptors are increased in the amygdala and
choroid plexus of Slc6a4–/– mice, but unchanged in
other regions38. Chronic SSRI treatment also increases
5-HT2C-receptor expression, but does not usually
affect 5-HT2A-receptor levels90.
These relatively modest changes in receptor numbers
are accompanied by much greater changes in response
to selective receptor agonists. for example, changes
in hippocampal neuron firing rates, temperature and
other responses to 8-OH-DPAT and ipsapirone (5-HT1A
agonists), altered locomotor-behaviour responses to
Ru 24969, and responses to DOI (a 5-HT2A/2C agonist with
hallucinogenic effects) are essentially absent in Slc6a4–/–
mice, with intermediate changes in Slc6a4+/– mice on some
measures40,86,91. These diminished receptor-mediated
responses are not attributable to abnormalities in G-protein
coupling, but recent evidence indicates that signalling in
the 5-HT2A/2C–phospholipase A/arachidonic acid pathway
is markedly reduced in the cortex, striatum and substantia
nigra of Slc6a4+/– and Slc6a4–/– mice36,38,69,70,91.
Relevance for murine and human disease
As described above, serotonin functions as both a short-
range neurotransmitter and a long-range signalling
modulator, with multiple effects on whole-organism func-
tions across many species4,92,93. This extensive pleiotropy is
clearly demonstrated in this Review of the multiple mouse
phenotypes that result from a single gene alteration (of
Slc6a4). Short-range serotonin signals (within nanometer
distances inside synapses) activate pre- and postsynaptic
serotonin receptors94. Long-range signals are mediated by
hormone systems both locally, within tissues that express
SERT (such as the adrenal gland43,44), and through changes
in plasma hormone levels. Additionally, blood serotonin,
which circulates within platelets and is released into
microcirculatory beds of capillaries and arterioles, can
regulate regional blood flow95; thus, the absence of sero-
tonin in the platelets and blood of Slc6a4–/– mice might
contribute to the widespread reductions in brain glucose
utilization that are observed35.
Many of the phenotypes that have been discovered
in this SERT-targeted mouse have also been glimpsed in
studies that examined the association of human dis-
eases or traits with particular human SLC6A4 variants,
such as those involving the 5-HTTLPR (including
the SNPs rs25531 and rs25532 within it), the intron-2
VNTR, and the I425V, I425L and G56A variants in
SLC6A4 coding regions. The higher-functioning alleles
of these variants, if their potential additive influence
(FIG. 1b) were verified by analytical techniques such as
those used in studies of mice83, could confer a four- to
fivefold greater serotonin transport capacity than that
of the lower-functioning alleles. furthermore, as sub-
stitution of SERT P339L for SERT 339L yields an ~80%
reduction in serotonin uptake11, a theoretical range of
differences in serotonin transport capacities across
individuals could be as great as 15- to 20-fold if com-
binations of common and rare SLC6A4 acted together
in influencing SERT expression and function.
Humans with the 5-HTTLPR short/short genotype
closely resemble Slc6a4+/– mice with regards to levels of
SERT expression and function (FIG. 6). These similarities
allow predictive appraisals of phenotypes across spe-
cies3,4,26,39,48,51. Thus, it is no surprise to find that anxiety-
and depression-related personality traits and affective
disorders1,8,96, alcohol and other drug dependencies97,
sleep and temperature disorders50,98, irritable bowel syn-
drome (IBS)53,99, pulmonary hypertension and chronic
obstructive pulmonary disease57,100 are associated with
SLC6A4 variants in humans and other species, especially
when interactions between SLC6A4 and life stress are
taken into account9,22,101,102. Some of the human disorders
that have been found to be associated with SLC6A4 have
Figure 6 |?comparison?of?serotonin-transporter?(serT)?expression?and?function?in?
Differential reductions in SERT binding sites (sites to which a labelled ligand for SERT
binds) (a), serotonin transport (b) and function (c; as reflected in MDMA-elicited
locomotor activity (in the mice) or as measured by reporter-gene activity in lymphoblasts
(in the humans)) in Slc6a4–/–, Slc6a4+/– and control mice and in humans with different
5-HTTLPR genotypes of SLC6A4 (long/long (LL), long/short (LS) or short/short (SS)).**,
significant change; ***, highly significant change. SLC6A4 data modified, with
permission, from REF. 1 (1996) American Association for the Advancement of Science.
Slc6a4 data modified, with permission, from REF. 17 (1998) American Society for
Pharmacology and Experimental Therapeutics.
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© 2008 Nature Publishing Group
not yet been investigated in Slc6a4–/– and Slc6a4+/– mice,
for example, SIDS and myocardial infarction103,104.
Likewise, some of the Slc6a4–/– mouse phenotypes,
including the obese and type-2-diabetic phenotypes, have
yet to be studied for SLC6A4-variant frequency distor-
tions in cohorts of human patients; this would seem to
be a high priority for investigation.
A genetic contribution to temperament and behav-
ioural traits, including anxiety, dominance and alcohol
and drug preferences, has been established in humans and
several other species, probably reflecting selective
forces among our ancestors. Recent research efforts in
this area have therefore been focused on non-human
primates, especially rhesus macaques, and are currently
proceeding towards the elaboration of an interdiscipli-
nary perspective that will blend behavioural genetics
and evolutionary psychology, as well as cognitive and
social neuroscience105. In this non-human-primate
model, environmental influences might be less complex
and thus less likely to confound associations between
behaviour and genes. Maternal-separation studies in
rhesus macaques have demonstrated that genes and
environment interact to produce effects on the asso-
ciation of central serotonin turnover and behavioural
traits, including stress reactivity and alcohol preference
and dependence, with a repeat-length variation that
is orthologous to the human 5-HTTLPR106,107. This is
in keeping with the notion that the 5-HTTLPR might
influence the risk for affective and other behavioural
disorders through gene–environment interactions,
although the molecular and neural mechanisms that
underlie the interplay of genes and environmental
adversity constituting disease risk remain incompletely
finally, gene–gene interactions have been studied in
models in which Slc6a4-mutant mice have been interbred
with mice that lacked either one or both copies of the
dopamine transporter gene (Slc6a3), the noradrenaline-
transporter gene (Slc6a2), the MAOA gene (Maoa), the
5-HT1B-receptor gene (Htr1b) or the BDNf gene (Bdnf ),
with consequent amplified, reduced or qualitatively dif-
ferent new phenotypes resulting23,34,48,66,75,109. These stud-
ies of ‘experimental epistasis’ complement those in which
the Slc6a4 knockout was placed on congenic C57BL/6J
or 129S6 background strains to yield more subtle
SERT-related phenotype differences23.
Evolutionary aspects of sERt biology
This Review of the consequences of an engineered
change in one murine gene, Slc6a4, documents over 50
phenotypic alterations. This seemingly marked interfer-
ence with nature could be considered only an artefactual
curiosity in light of its production by several types of
drastic genetic engineering17,18,20,21. However, the findings
in fact provide a partial delineation of all of the geneti-
cally influenced serotonergic traits that are regulated by
SERT in mice. Additionally they provide a glimpse and,
maybe, a preview of what is being discovered in humans
with SLC6A4 variants. Most of these murine traits are
altered by the loss of a single Slc6a4 allele — the changes
often being intermediate to those that are produced
by full knockout of the gene. furthermore, the attend-
ant changes in expression levels (~50% decrease) and
function of murine SERT are similar to those that result
from 5-HTTLPR variation and related polymorphisms
in human and rhesus SLC6A4 (FIG. 6). This indicates that
pleiotropy with some similarities to that found in mice
is likely to exist not only in humans and non-human
primates but also in other species, including other
rodents, flies and worms that have known spontaneous
or targeted genetic variation of the serotonergic path-
way106,110–112. Indeed, as discussed above, many condi-
tions that are clearly analogous to those that arise in the
Slc6a4+/– and Slc6a4–/– mice have been reported in studies
of human medical disorders.
It is evident that some of the consequences of this
engineered Slc6a4 mutation are the result of changes that
occur downstream in the serotonergic systems17,51,70,73,83.
However, other consequences, although generally relatable
to altered availability of serotonin and reduced or absent
SERT, seem to be qualitatively distinct traits. for instance,
it is hard to immediately relate bone, heart, lung, bladder
and gut structural and functional changes to alterations in
aggression or the startle reflex, or to preferences for alco-
hol, MDMA and cocaine — although a psychosomatocist
might give it a try. Thus, multiple mechanisms, some going
beyond those that have already been documented17,69,70,73,83,
are predicted by this pleiotropy.
from the most global point of view, the preponder-
ance of changes resulting from the engineered reduction
in SERT can be considered deleterious. Differences in
SERT expression and function have not yet been stud-
ied across mouse species to specifically evaluate how
they alter global fitness, and it is clear that more is now
known about SLC6A4 variants in humans and non-
human primates than about murine Slc6a4 mutational
consequences in the real world.
Although the mouse Slc6a4 and the human SLC6A4
possess a similar set of three splice variants113,114, other
rodents and many non-human primates possess a more
basic regulatory region. furthermore, humans seem to
have multiple and more complex regulatory elements
that are capable of providing both a broader range of
and a more finely tunable regulatory capacity for SERT
expression and function, as reflected in the 5-HTTLPR
variants, the rs25531 and rs25532 SNPs and the intron-2
VNTR, as well as the rarer functional elements (for
example, the I425V variant)4. One could speculate that
this more finely tuned regulatory capacity of SERT is
another example of evolution-based development, like
primate and human brain development, serving to
enhance fitness to succeed in subsequent eons; however,
more data are needed to confirm this.
The coding region of SLC6A4 has changed over evo-
lutionary time4, but the non-coding regulatory regions
of the gene have not yet been carefully evaluated for
such changes beyond the few examples mentioned in
this review16. Similarly, no attempt has yet been made
to quantitate SERT expression and function in relation to
fitness in more complex and thus relatively ‘higher’
organisms, for example, chimpanzees and rhesus
macaques. SERT is also present in ‘simple’ organisms
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that have only a neural net as a brain115, as well as in
insects, worms and many other vertebrates4, but little is
known about the relative functional capacity of SERT
across these species. The crystal structure of a bacte-
rial leucine transporter was recently elucidated116 and
has been used as a predictive model for the structure
of SERT117–120. However, although a coherent and exten-
sive tabulation of neurotransmitter sodium symporter
(NSS) families that include the SLC group has become
available121, an experimental evolutionary-biology-based
study of these bacterial amino-acid transporters has not
yet been conducted.
In conclusion, a regulatory variation in the human
gene that encodes SERT, the master controller in the
fine-tuning of serotonin signalling, is not only associated
with anxiety-related traits but also modifies the risk for
a wide range of disorders. However, much of the human
data to date is based on genetic-association studies alone.
Mutations that result in reduced or absent SERT func-
tion in mice have led to the identification of more than
50 different phenotypic changes, ranging from increased
anxiety and stress-related behaviours to gut dysfunc-
tion, bone weakness and late-onset obesity with meta-
bolic syndrome. Although the effects are not as robust
as those in the experimental mice, SERT-function-
modifying gene variants in humans influence many of
the same phenotypes. The need to blend behavioural
genetics and evolutionary biology, as well as cognitive
and social neuroscience, now defines the future chal-
lenges for the biosocial sciences in terms of the evolv-
ing genetic architecture of emotional behaviour, social
interaction and disease in humans.
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We thank all of the close associates and friends in our laborato-
ries who contributed to the original reports published during
the past 10 years, as well as our colleagues elsewhere who
contributed to studies of genetically-engineered Slc6a4-mutant
mice, and T. B. DeGuzman for assistance with the manuscript
and figures. The authors’ related research was supported by the
Intramural Research Program of the National Institute of
Mental Health (D.L.M.) and by the European Commission
(NEWMOOD LSHM-CT-2003-503474), the Bundesministerium
für Bildung und Forschung (IZKF 01 KS 9603) and the Deutsche
Forschungsgemeinschaft (LE629/4-2, SFB 581, KFO 125)
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
5-HT1A receptor | 5-HT2A receptor | 5-HT1B receptor |
5-HT2C receptor | BDNF | MAOA | SLC6A4 | Slc6a4 |
Dennis L. Murphy’s homepage: http://intramural.nimh.nih.
Klaus-Peter Lesch’s homepage: http://www.psychobiologie.
Tabulation of the neurotransmitter sodium symporter
(NSS) family: http://www.biology.ucsd.edu/~msaier/
Discussion of reference 1 by K.P.L.: http://www.in-cites.
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