Neuropharmacology 60 (2011) 1347e1354
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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
Increased serotonin axons (immunoreactive to 5-HT transporter)
in postmortem brains from young autism donors
Efrain C. Azmitia a, b, *, Jorawer S. Singh a, b, Patricia M. Whitaker-Azmitia c
a
Dept. of Biology, New York University, 100 Washington Sq East, New York, NY 11791, United States
Dept. of Psychiatry, New York University, 100 Washington Sq East, New York, NY 11791, United States
c
Dept. of Psychology, Stony Brook University, Stony Brook, NY 11794, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 August 2010
Received in revised form
4 November 2010
Accepted 1 February 2011
Imaging studies of serotonin transporter binding or tryptophan retention in autistic patients suggest that
the brain serotonin system is decreased. However, treatment with drugs which increase serotonin (5-HT)
levels, specific serotonin reuptake inhibitors (SSRIs), commonly produce a worsening of the symptoms. In
this study we examined 5-HT axons that were immunoreactive to a serotonin transporter (5-HTT)
antibody in a number of postmortem brains from autistic patients and controls with no known diagnosis
who ranged in age from 2 to 29 years. Fine, highly branched, and thick straight fibers were found in
forebrain pathways (e.g. medial forebrain bundle, stria terminalis and ansa lenticularis). Many immunoreactive varicose fine fibers were seen in target areas (e.g. globus pallidus, amygdala and temporal
cortex). Morphometric analysis of the stained axons at all ages studied indicated that the number of
serotonin axons was increased in both pathways and terminal regions in cortex from autism donors. Our
findings provide morphological evidence to warrant caution when using serotonin enhancing drugs
(e.g. SSRIs and receptor agonist) to treat autistic children.
This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.
! 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cerebral cortex
Stress
Morphometrics
Medial forebrain bundle
Ansa lenticularis
1. Introduction
Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders defined by social and communication
deficits as well as restricted behaviors. The Centers for Disease
Control states that the average prevalence of ASDs identified among
children aged 8 years increased 57% in 10 sites in USA from 2002 to
2006. Incidence of ASD worldwide is not uniform, with regional
variations from 1.1/1000 in China (Zhang and Ji, 2005) to 9.1/1000 in
the most recent findings in the US. This is an incidence greater than
both childhood cancers and diabetes combined, yet little is known
about autism’s cause, prevention or treatment. Originally described
as one of the most heritable mental illnesses (Bailey et al., 1995),
recent re-evaluation of this description has shown methodological
biases, misinterpretations, and erroneous assumptions in much of
the original data (Chamak, 2010). The more likely explanation is that
autism occurs in response to an environmental factor.
Elevated prenatal cortisol due to stress is associated with several
negative conditions including aborted fetuses, excessive fetal
activity, delayed fetal growth and development, premature
* Corresponding author. Tel.: þ1 212 998 8235.
E-mail address: [email protected] (E.C. Azmitia).
0028-3908/$ e see front matter ! 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2011.02.002
delivery, low birth-weight, attention and temperament problems in
infancy, externalizing problems in childhood, psychopathology,
and chronic illness in adulthood (Field and Diego, 2008) and many
studies indicate that increased stress (either psychological or
physical) during pregnancy presents a significant risk factor for the
occurrence of autism. These stressful events; such as infection,
bereavement, depression, socioeconomic stress, vaginal bleeding,
and threatened miscarriage may seem diverse, but all present one
common endophenotype e activation of the homeostatic mechanisms of the hypothalamicepituitaryeadrenal (HPA) axis, elevating
blood cortisol levels. In turn, the increase in cortisol may act as an
epigenetic factor e changing the developmental trajectory of
neuronal maturation necessary for normal function.
A variety of obstetrical complications have been associated with
an increased risk of autism, including bleeding during pregnancy
(Brimacombe et al., 2007; Juul-Dam et al., 2001), low Apgar scores
(Larsson et al., 2005; Glasson et al., 2004), long labors or precipitous
labor (Glasson et al., 2004), threatened abortion (Glasson et al.,
2004), induced labor (Brimacombe et al., 2007; Glasson et al.,
2004), and breech presentation. Psychologically, pregnant women
who report sadness (Zhang et al., 2010), anxiety or adverse life
events (Beversdorf et al., 2005), physical stress (including exposure
to storms) (Kinney et al., 2008), were all at greater risk of giving
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E.C. Azmitia et al. / Neuropharmacology 60 (2011) 1347e1354
birth to a child with autism. Stress in pregnancy causes dysregulation of fetal HPA reactivity, which endures into postnatal life, even
adulthood (Egliston et al., 2007). A further indication that the HPA
axis has been perturbed in autism during development, is in the
number of studies which report that children with autism have
altered HPA Reactivity, including lower levels of cortisol (Curin
et al., 2003), altered rhythms (Corbett et al., 2008), and responses
to stress (Corbett et al., 2008).
The relationship between serotonin and stress is long standing.
One study conducted in the laboratory of Dr. Erminio Costa showed
that stress related hormones from the adrenal gland were able to
increase serotonin turnover in the brain (Azmitia et al., 1970). Using
a method involving injections of radioactive tryptophan, coupled
with HPLC measures of plasma and brain tryptophan and serotonin,
adrenalectomy (removal of endogenous glucocorticoids) resulted in
a 50% decrease in turnover that was completely reversed by
injections of corticosterone. This finding has been replicated in
a variety of forms and species over the last 40 years (Azmitia et al.,
1993; Meijer and de Kloet, 1998; Leonard, 2005). Stress acting
during early development in rodents can reduce the postsynaptic
receptors of the serotonin system and glucocorticoid receptors
throughout life (Veenema, 2009; Mitchell et al., 1990). Bioinformatic analysis identified nine binding sites in various serotonin receptors (HTR1D, HTR1F, HTR2A, HTR3A, and HTR6) for
transcription factors in the glucocorticoid receptor family indicating a new avenue to explain stress induced serotonin activation
(Falkenberg and Rajeevan, 2010). Thus, it would be expected that
the reported increase in prenatal stress in autism would lead to
a compromised function of the serotonin system.
A number of imaging studies indicate that the brain serotonin
system in autistic patients is reduced (Chugani et al., 1997, 1999;
Makkonen et al., 2008). This would support the use of serotonin
enhancing drugs such as SSRIs to treat this disorder (Kolevzon et al.,
2006; Fatemi et al., 1998; Mehlinger et al., 1990). However, large
clinical studies show that SSRIs can make autistic children worse,
not better (Brodkin et al., 1997; King et al., 2009). A large metaanalysis reached a similar conclusion; there is no evidence of effect
of SSRIs in children and emerging evidence of harm (Williams et al.,
2010). In order to help resolve this puzzle, and facilitate the
development of an effective therapy, immunocytochemical studies
of serotonin neurons were performed in pathological specimens.
Our results indicate a substantial increase in brain serotonin axons
in young autistic donors and suggest that treatment with serotonin
enhancing drugs is not indicated.
2. Materials and methods
2.1. Brain banks
The postmortem brains were obtained from the Brain Bank for Disabilities and
Aging in Staten Island, the Autism Tissue Program in Princeton New Jersey, and the
NICHD Brain and Tissue Bank for Developmental Disorders at University of Maryland, Baltimore. Results of the autopsies and the Autism Diagnostic Interviewrevised (ADIr) summaries are shown in Table 1 for the autism and “no known
psychiatric diagnosis” (NKD) control donors. The average age for the autism group
(n ¼ 10; 14.33 yrs, range 2.9e29 yr) is similar to the control group (n ¼ 9; 14.4 yr,
2.1e25.6 yr). The autism group was all male while six out of the nine control group
members were male. The postmortem interval was 15.9 h for autism and 15.3 h for
the control group. The autism group died of drowning (n ¼ 3), seizures (n ¼ 4),
respiratory distress (n ¼ 2), or cardiac failure (n ¼ 1). One autistic patient (UMB4305) died from serotonin syndrome, which includes hyperthermia, respiratory
distress and seizures. Most of the controls died of trauma (4/6) when recorded.
The drug histories were available for some of the autistic patients in medical
records included in the ATP portal. These are listed in Table 2. SSRIs were given to 2
children. Seroquel, olanzapine, aripiprazole and resperidone are atypical neuroleptics that have significant serotonin receptor antagonist activity and 3 children
have a history of this class of drug.
Although residual drugs in postmortem tissue may interfere with autoradiographic analysis, this is not the case with immunocytochemistry. Statistical analysis
Table 1
Summary of autopsy report of donors from ATP website (www.ATPPortal.org). The
brain cases are identifiers are used to obtain autopsy and medical records of the
donors on the ATP portal. Diagnosis of autism uses the ADIr procedure. The postmortem delay (PMD) is the time from death to autopsy. In most cases the body is
stored at 5 $ C before autopsy. The cause of death is obtained from the autopsy
record. Note that case UMB-4305 died from the Serotonin Syndrome, which is
caused by an excess of brain serotonin.
Case no.
Diagnosis
B-6399
B-5733
HSB-4640
Autism
Autism
Autism
Cal-105
UMB-4305
UMB-4315
UMB-4899
UMB-4999
IBR 93-01
B-6994
Autism
Autism
Autism
Autism
Autism
Autism
Autism
Average
Age (yr)
Sex
PMD
(hrs.)
Cause of death
2.8
5.9
8.5
M
M
M
4
15.6
13.8
11.9
12.9
14.1
14.4
20.8
23
29
M
M
M
M
M
M
M
11
13
22
9
14
14
43
Drown
Respiratory distress
Breathing difficulty,
sweaty, seizure
Drown/foam
Serotonin syndrome
Seizures
Drown
Cardiac arrhythmia
Seizure
Seizure
14.33
15.9
BTB-4235
UMB-1706
UMB-1670
NKD
NKD
NKD
2.1
8.6
13.3
M
F
M
14
20
5
UMB-1790
UMB-4722
UMB-4638
UMB-4669
UMB-4590
UMB-3960
NKD
NKD
NKD
NKD
NKD
NKD
13.7
14.6
15.1
16.4
20.5
25.6
M
M
F
M
M
F
18
16
5
16
19
25
Average
14.4
Reject of cardiac
allograft transplant
Asphyxia, suicide?
Multiple injuries
Multiple injuries
Head injury
Head injury
15.3
showed no significant correlations of serotonin axon density and age, drug-treatment,
cause of death, PMI, or storage time in our sample.
2.2. Brain processing
The brains were hemisected and the right half was fixed in 10% formalin for at
least 30 days. In most cases, large hemispheric sections were embedded with
propylene glycol and serial 50 mm-thick sections were cut on a sliding microtome at
room temperature. The tissue slabs were dehydrated in ethyl alcohol and stored in
70% ethyl alcohol; used to prevent deterioration of tissue during several years of
storage. All brains were examined for white matter and vascular disease changes and
for evidence of infarction by general pathological procedures such as brain cutting,
sampling, and staining.
The sections used included the temporal lobe hemisphere and attached
subcortical structures from telencephalon, diencephalon and midbrain. The sections
were extensively washed and treated with H2O2. Sections stained with 5-HTT
monoclonal antibodies were incubated for 3 days and stained with DAB/nickel. The
serotonin transporter (5-HTT) antibody is a mouse monoclonal product obtained
from Abcam (#44520; Cambridge, MA) used at dilutions of 1/100e1/200. Immunocytochemical studies were performed as previously described (Nixon et al., 2005).
The 5-HT immunoreactive (IR) fibers were seen in every area and at every age
examined. The general innervation pattern resembles that described in human
cortex (Austin et al., 2002; Azmitia and Nixon, 2008) and seen in many animal
studies.
Slides were viewed with a research-quality Leica Orthoplan microscope with
several objectives (1.6# Pl; 6.3# Pl-Apo 0.2 numerical aperture (N.A.), 25# Pl Fluotar
0.6 N.A. and 63# Pl-Apo 1.40 N.A. oil, periplan GW objective of 8#) and Apl 1.25 N.A.
oil condenser with swing arm to enable Kohler illumination at all magnifications.
Images were taken with a Canon E05 (Rebel T1i) professional digital camera with an
18.0 megapixel resolution and stored on an Apple Mac Pro. Images were coded but
not modified (e.g. contrast enhancement, sharpening) before morphometric
measures using threshold analysis. All captured images were made with identical
lighting condition.
2.3. Morphometrics
The details of this method have been previously published (Azmitia and Nixon,
2008). Using the ImageJ program, the images of brains were first converted to 8-bit.
E.C. Azmitia et al. / Neuropharmacology 60 (2011) 1347e1354
1349
Table 2
Summary of autistic donor symptoms and drug history from ATP website (www.ATPPortal.org). The symptoms are obtained from both the ADIr and medical records of the
patient provided on the portal. Drug history is available from the medical records available on line. Abbreviations: Agg e aggression; resp e respiratory, ANX e anxiety; OCD e
obsessive compulsive disorder.
Cases
Symptoms
B-6399
B-5733
HSB-4640
Cal-105
UMB-4305
UMB-4899
UMB-4999
Aggressive
High fever at 7 & 14 mo, constipated
Resp arrest, sweaty, Agg, scoliosis.
Anxious (Anx), Irritable, hypotonia
Bipolar, Agg., fecal smearing
Stereotypy
Agg., masturbation, OCD, anx, restless,
biting, head-banging, frustration, sleep
disorder, gastric distress
Agg, hyperactivity, sleep disorder
Aggressive, Rigid, Anxious, Sleep disruption
IBR 93-01
B-6994
A threshold range was set to 60e120. After the threshold was set, the “Analyze
Particles” feature of ImageJ was used. The size of particles (objects) to be measured
was set from 5 mm2 to infinity, and the circularity was set 0e1.0. The “Summarize”
option was then selected in the “Analyze Particles” menu. The data was compiled
and transferred to Microsoft Excel for statistical analysis. In Excel the area covered by
the aggregates was plotted across age, and against the average sizes of neurons
corresponding to the same subjects and brain regions.
2.4. Statistics
ANOVA, descriptive and comparative statistics were performed in Excel
worksheets using the optional statistical package.
3. Results
3.1. 5-HTT immunoreactive axons in fiber tracts
In humans there are two ascending pathways to the forebrain
cortical structures from the serotonergic neurons in the midbrain
raphe nuclei. Both pathways were heavily labeled with 5-HTT-IR
axons (Fig. 1). One is the medial forebrain pathway (MFB), which
receives fibers mainly from the median raphe nucleus, and the
other is the ansa lenticularis (Ansa Lent), which receives fibers from
Drug history
Depakote and Prednizone
Zopanax, Albuterol, Pulmacort, Prednazone
Seroquel (600 mg,d), Depakote, Clonazepam, Quetiapine, Zyprexa
Resperidone, Zoloft, Prozac Naltrexone,
Seroquel, propranolol, thioridazine
SSRI (Effexor, Paxil, Celexa, Prozac, clomipramine) but becomes more aggressive,
angry and irritable. Atypical antipsychotics (olanzapine, aripiprazole, seroquil
without any improvement. Anticonvulsants/mood stabilizers as well (lithium,
neurontin,dilantin, depakote, lamictal,omega 3 fatty acids, keppra, trileptal,
topamax) all without improvement and in some cases worsening behavior.
the dorsal raphe nucleus through the zona incerta. The MFB
projects most of its fibers to the cortex through the septum and
stria terminalis. The Ansa Lent sends fibers to the Globus Pallidus
and to the amygdala through the ventro-amygdalo-fugal pathway
(VAFP).
The density of 5-HTT-IR fibers in these two pathways is much
heavier in brains from autistic donors compared to those from
control donors (Fig. 2). In brains from individuals with no known
diagnosis (NKD) (Figs. 1 and 2), the 5-HTT-IR fibers are relatively
straight and fine, with occasional thicker fibers. In captured images
from autistic donors, the serotonin pathways were denser and the
5-HTT-IR axons appear thicker than seen in comparable control
brains (Fig. 2). The apparent increase was present at all ages
examined and density of labeling showed no significant differences
across ages in either population. The amount of labeling did not
correlate with postmortem interval, time of storage, cause of death,
or drug history of the patients (data not shown).
5-HTT immunoreactive axons in cortex: Examination of
composite captured images made from two postmortem brains
reveals the detailed regional analysis of serotonin axons. The two
images are dark-field composites made of the 5-HTT-IR axons
(appearing white) extending from Layer I down to Layer VI and into
the white matter. In the cortical section from a 25-year-old control
donor (Fig. 3A), 5-HTT-IR fibers were seen in all layers with an
apparent higher density in the upper layers, especially Layer I. A
similar distribution of serotonergic fibers was reported in primates
(Austin et al., 2002; Morrison et al., 1982). In the cortical section
from the 29-year-old autism donor, dense distribution of 5-HTT-IR
axons was seen in all layers. The density of axons appears much
greater than seen in the control donor in all layers. Many fibers
appear to be entering from the ventral white matter. Several
apparent dystrophic fibers were seen in layer III in the autism brain
(arrows, Fig. 3B).
3.2. Morphometrics of immunoreactive axons in tracts and
globus pallidus
Fig. 1. The picture shows 5-HTT immunoreactive (IR) serotonin axons in two main
ascending pathways into the forebrain in a control brain of no known diagnosis donor
of 2.1 years of age. The medial forebrain bundle (MFB) lies in the ventromedial aspects
of the brainstem. The ansa lenticularis (Ansa Lent), also called the (Dorsal Raphe
Cortical Tract See Azmitia and Gannon, 1986) lies in the dorsolateral aspect of the
hypothalamus, extending from the zona incerta and lying just below the globus pallidus. Scale bar is 100 mm.
In all brains, counts of the immunoreactive axons were assessed
and the fraction of each picture that contained label was assessed.
The results for 3 pairs are shown in Fig. 4. The counts for 3 fiber
tracts: the MFB, Ansa Lenticularis, and stria terminalis were all
increased at all time points. In addition, the number of axons is
increased in the globus pallidus of autism donors. These findings
were also seen when considering area fraction. Therefore, not only
Fig. 2. The series of pictures are grouped by matching age of control:autism matches: 2.1 vs. 2.8 years; 8.6 vs. 8.5 years; and 25.6 vs. 29 years, respectively. AT all ages, the number of
5-HTT immunoreactive fibers was greater in the brain from an autism donor than in brains from the control NKD donors.
E.C. Azmitia et al. / Neuropharmacology 60 (2011) 1347e1354
Fig. 3. The two composites of the fusiform cortices were made from dark-field
photographs. The pictures show the 5-HTT immunoreactive fibers as white against
a black background. In the brain from the control donor (A), aged 25.6 years, the
serotonergic fibers are very dense in layer I and gradually decrease in number as they
move deeper into the tissue. In the cortex from the autism donor (B), aged 29 years, the
serotonergic fibers show a much denser and broader innervations pattern. Occasional
patches of dystrophic fibers can be seen in layer III of the autism tissue (arrows). Scale
bar is equal to 100 mm.
are there more axons, but these axons take up more of the neuropil
in the images of the brain sections.
3.3. Morphometric analysis of the superior temporal cortex
In the final analysis, photographs were taken of the Upper
Layer (I and II) of the superior temporal cortex from autism and
NKD control donors. Staining was seen in all sections analyzed.
The counts and area fraction were calculated and expressed as age
of donor. There was no change across time in either the donor or
NKD control brains (Fig. 5). However, there was a consistent and
dramatic increase in autism compared to control cortex in both
the number of axons per unit section and in the area fraction
(Fig. 5).
4. Discussion
The increase of serotonin axons in the brains at all ages from
autistic donors compared to NKD control donors was dramatic not
only in the captured images, but also in the morphometric analysis.
The increased presence of serotonin axons was seen in three of the
major serotonin pathways: MFB, Ansa Lent, and Stria Terminalis.
1351
Fig. 4. In this graft, the morphometric counts and area fractions in the MFB, Ansa Lent
and globus pallidus (G Pal) of the 5-HTT immunoreactivity is displayed by aged as
indicated in Fig. 3. The series of pictures are grouped by matching age of control:autism matches: 2.1 vs. 2.8 years (A); 8.6 vs. 8.5 years (B); and 25.6 vs. 29 years (C),
respectively. There is a greater difference at the earlier ages than seen in the last panel
(compare A and B with C). The increases noted in globus pallidus appear smaller than
those seen in the fiber pathways.
This consistent increase indicates that the results observed are not
confined to one portion of the serotonin system, but rather is
a global increase in all ascending pathways. The very dense number
of fibers at all ages would be consistent with a general activation of
the serotonin system, which would be consistent with the stated
hypothesis e that increased prenatal stress would lead to increased
activation of the serotonergic system.
The data used in this study comes from immunocytochemical
staining of postmortem tissue. This is the best material to study
cellular details of neurons and glial cells in the human brain. While
brain imaging is an excellent resource for monitoring binding of the
transporter ligands in the brains of living humans, the transporter
protein levels is not directly measured. Furthermore, the difficulties
in establishing the proper conditions for binding are not appropriate for cellular analysis (see Meyer, 2007).
Postmortem brain tissue is an excellent resource for detailed
cellular analysis. In this report, and in our previous study the
distribution, morphology and number of serotonin axons in the
diseased brain were studied (Azmitia and Nixon, 2008). However,
there are a number of problems with postmortem studies. (1) It is
difficult to obtain suitable numbers of brain material from
diseased patients from a single source. However, there are several
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E.C. Azmitia et al. / Neuropharmacology 60 (2011) 1347e1354
Fig. 5. In this figure the counts and are fraction of the 5-HTT immunoreactivity is shown across ages for superior temporal cortex, layers I and II. It can be seen that the values from
autism donors are consistently higher than those seen in the control NKD donors. This consistent increase is seen even though the brain samples studied came from three different
brain banks.
excellent brain banks available (e.g. Autism Tissue Program, IBR
Brain Bank in Staten Island, NY, and NICHD Brain and Tissue Bank
for Developmental Disorders at the University of Maryland) which
may be used. (2) There is a scarcity of good control brains from
donors with no known diagnosis (NKD). This is a major problem
for all work with postmortem tissue. (3) There can be problems
with the proper identification of regional anatomy. Although many
good atlases are available, because of the cut of the tissue expertise is needed to isolate consistent brain regions between samples.
(4) The tissue is immersion fixed in formalin and consequently,
long postmortem intervals (PMI) and storage times can adversely
affect its staining. Sometimes a brain with a relatively short PMI
will produce poor staining while tissue with very long times can
produce excellent staining. In this paper, tissues were obtained
from a variety of banks, and studied while encoded to minimize
experimenter bias. It is concluded that postmortem tissue remains
a very valuable and unique source of material to study neural
cellular interactions.
There are reports from living subjects indicating that the serotonin system may be reduced in autism (see Zafeiriou et al., 2009).
For instance, methyl tryptophan retention was reduced in left
frontal cortex and thalamus in 5 of 7 boys and in the right frontal
cortex and thalamus in the 2 remaining autistic boys. Makkonen
et al., 2008 studied 15 children (mean age 8 years 8 months) with
autism and 10 non-autistic comparison children (mean age 9 years
10 months) using single-photon emission computed tomography
(SPECT) with [123I] nor-b-CIT. These authors found a significant
decrease only in the medial frontal cortex area (p ¼ 0.002) and the
reduction was more evident in adolescence than in earlier childhood. The most recent study using PET with the 5-HTT ligand
(carbon 11 (11)C)-labeled trans-1,2,3,5,6,10-beta-hexahydro-6[4-(methylthio)phenyl]pyrrolo-[2,1-a]isoquinoline ([(11)C](þ)McN5652) studied 20 men with autism (18e26 years) (Nakamura et al.,
2010). Images of the [11C]McN-5652 ratio index, which reflect the
binding potential of [11C]McN-5652, showed a significant decrease
in every region of the brain. This general decline reflects a loss of
function of the system and not a structural decrease.
While this is a surprising discrepancy between postmortem
cellular/protein levels of serotonin neurons and PET/SPECT
imaging studies in living subjects, it is also apparent in studies
of depressed patients. Depression is strongly associated with
decreased serotonin transporter binding in vivo but with
increased expression of an immunocytochemical visualization of
raphe neurons using tryptophan hydroxylase antibody in postmortem suicide studies (Boldrini et al., 2005). Interestingly, an
increased expression of the tryptophan hydroxylase immunocytochemistry in midbrain raphe neurons was also found in rats
exposed to a stress hormone (Azmitia et al., 1993), further
evidence of a specific cellular effect of stress hormones on serotonin. Further support for our findings of an increase in 5-HT
axons is the reports that BDNF, a serotonin trophic factor, is
increased in autism (Correia et al., in press; Sheikh et al., 2010).
Based on the assumption that the serotonin system is reduced,
many neuropharmacological studies have pharmacologically
increased serotonin levels to treat autistic children. There were
reports that serotonin drugs may be helpful in alleviating some of the
symptoms of autism (Kolevzon et al., 2006; Fatemi et al., 1998;
Mehlinger et al., 1990). However treatment of autistic children with
SSRIs can have severe adverse effects. Treatment of 35 young adults
with clomipramine for 12 weeks produced 13 cases of clinically
significant adverse effects (37%), including 3 cases of seizures, 3 of
agitation, constipation and 3 of weight gains (Brodkin et al., 1997). In
a larger study, one hundred forty-nine volunteers 5e17 years old
were randomized to receive citalopram (n ¼ 73) or placebo (n ¼ 76).
Citalopram use was significantly more likely to be associated with
adverse events, particularly increased energy level, impulsiveness,
decreased concentration, hyperactivity, stereotypy, diarrhea,
insomnia, and dry skin or pruritus (King et al., 2009). This suggests
that an overactive serotonin system can produce clinical manifestations similar to those seen by an underactive system, yet they would
have diametrically opposing responses to neuropharmacological
treatments that increase serotonin activity either by increasing 5-HT
levels (SSRIs) or directly stimulating the receptors (5-HT agonist). In
other words, when developing serotonin neurons decrease, the
receptors are not being stimulated and would be expected to be
upregulated because of a lack of serotonin. When serotonin neurons
are increased, the receptors are not being stimulated because they are
downregulated as a result of too much serotonin. We have shown that
E.C. Azmitia et al. / Neuropharmacology 60 (2011) 1347e1354
5-HT antagonist acting as reverse agonist may be useful in increasing
receptor expression (Abbas et al., 2007; Nishi and Azmitia, 1999).
Interestingly the two most recent drugs for treatment of altered
serotonin symptoms in autism are Aripiprazole (Abilify) and Risperidone (McCracken et al., 2002; Owen et al., 2009) and these are
antagonist to 5-HT2A receptors (Davies et al., 2004; Richelson and
Souder, 2000).
Prenatal events can produce permanent decreases in serotonin
receptor numbers and function (Whitaker-Azmitia et al., 1987). The
findings reported here indicate that the serotonin system is overstimulated during the early stages and the receptors associated
with serotonin may be reduced. The early actions of serotonin
receptors on developing brain systems are well known (see Azmitia
and Whitaker-Azmitia, 1991; Whitaker-Azmitia, 2005). Moreover,
the increase in 5-HTT immunoreactivity is not likely due to an
increased preponderance of any allelic form of the transporter gene
in autism, as the differing allelic forms of the gene do not lead to
expression changes in the postmortem human brain (Sugden et al.,
2009). The over-activity of serotonin neuronal growth poses
a problem for therapy but it does suggest that the use of serotonin
antagonists may be a more advantageous therapeutic strategy.
More work is needed to help identify the serotonin receptor
structural/functional changes associated with autism and to
develop neuroplasticity strategies for dealing with the consequence
of too much serotonin during early stages of life.
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