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Am J Psychiatry 159:3, March 2002 419
Article
Higher Expression of Serotonin 5-HT
2A
Receptors
in the Postmortem Brains of Teenage Suicide Victims
Ghanshyam N. Pandey, Ph.D.
Yogesh Dwivedi, Ph.D.
Hooriyah S. Rizavi, M.S.
Xinguo Ren, M.D.
Subhash C. Pandey, Ph.D.
Christine Pesold, Ph.D.
Rosalinda C. Roberts, M.D.
Robert R. Conley, M.D.
Carol A. Tamminga, M.D.
Objective: Abnormalities of serotonin (5-
HT) receptor subtypes have been ob-
served in the postmortem brains of adult
suicide victims; however, their role in
teenage suicide is unexplored. The au-
thors examined whether 5-HT
2A
receptor
subtypes are altered in the postmortem
brains of teenage suicide victims.
Method: Levels of 5-HT
2A
receptors were
determined through examination of [
125
I]
LSD binding, protein expression (by use of
Western blotting with a specific 5-HT
2A
re-
ceptor antibody), and mRNA (by means of
quantitative reverse transcription poly-
merase chain reaction) in the prefrontal
cortex, hippocampus, and nucleus accum-
bens of 15 teenage suicide victims and 15
normal matched teenage subjects. The
cellular localization of the 5-HT
2A
recep-
tors was determined by means of gold
immunolabeling.
Results: The authors observed signifi-
cantly higher [
125
I]LSD binding in the pre-
frontal cortex and greater protein expres-
sion and mRNA levels in the prefrontal
cortex and hippocampus but not in the
nucleus accumbens of suicide victims,
compared with normal subjects. Greater
protein expression was localized on pyra-
midal cells in cortical layer V but not in
other cortical layers or in the surrounding
neuropil of the prefrontal cortex of teen-
age suicide victims.
Conclusions: The evidence indicates
higher levels of 5-HT
2A
receptor, protein,
and mRNA expression in the prefrontal
cortex and hippocampus, which have
been implicated in emotion, stress, and
cognition. There was no higher level in
the nucleus accumbens, which has been
implicated in drug dependence and crav-
ing. Our findings suggest that a higher
level of 5-HT
2A
receptors may be one of
the neurobiological abnormalities associ-
ated with teenage suicide.
(Am J Psychiatry 2002; 159:419–429)
Suicide is a major public health concern (1) and the
ninth leading cause of death; about 30,000 individuals die
by suicide each year in the United States alone (2). In the
young, it is the second most frequent cause of death. Sui-
cidal behavior is often associated with mental disorders,
such as depression, bipolar illness, and personality disor-
ders. Besides psychiatric illnesses, other risk factors in-
clude a family history of suicide, a family history of psychi-
atric disorders, psychosocial stressors, impulsivity, and
aggression (3). Abnormalities in neurobiological mecha-
nisms may be another risk factor for suicide. Several stud-
ies (4, 5) have suggested that the major driving factor lead-
ing to suicide in teenagers is aggressive and impulsive
behavior. Since abnormalities in serotonin (5-HT) function
have been implicated in impulsive/aggressive behaviors
(6), it is possible that the pathophysiology of teenage sui-
cide may be associated with abnormal serotonin function.
Studies of postmortem brain samples from adult suicide
victims have suggested that abnormalities of 5-HT recep-
tor subtypes are associated with suicide (for a review, see
Gross-Isseroff et al. [7]). In particular, it has been reported
by several investigators, although not confirmed by all,
that one of the serotonin receptor subtypes, the 5-HT
2A
re-
ceptor, is found in higher than normal levels in the post-
mortem brains of adult suicide victims (7–10). Further
support for this concept comes from studies that indicate
a greater number of 5-HT
2A
receptors in the platelets of
suicidal patients with different mental disorders; this
higher level appears to be independent of diagnosis (11,
12). On the basis of these findings, we previously proposed
that platelet 5-HT
2A
receptors might serve as a biological
marker of suicidal behavior (11).
Although serotonergic abnormalities and 5-HT
2A
recep-
tors have been studied in the brains of adult suicide vic-
tims, to our knowledge, serotonin receptor subtypes, spe-
cifically 5-HT
2A
receptors, have not been studied in the
postmortem brains of teenage suicide victims. In addition,
earlier studies of 5-HT
2A
receptors in the postmortem
brains of adult suicide victims showed higher 5-HT
2A
receptor numbers on the basis of the greater binding of
5-HT
2A
receptor ligands to their binding sites. Thus,
whether higher 5-HT
2A
receptor numbers in suicide vic-
tims are the result of more active binding sites or are asso-
ciated with abnormalities at the level of transcription and/
or translation of 5-HT
2A
receptors is not yet known.
Therefore, in the present investigation, we examined the
association of 5-HT
2A
receptors and teenage suicide, not
only by examining the binding characteristics of 5-HT
2A
420 Am J Psychiatry 159:3, March 2002
HIGH SEROTONIN IN TEENAGE SUICIDES
receptors (to replicate the findings in adult suicide vic-
tims), but also by studying their gene transcription and
translation in the prefrontal cortex, hippocampus, and
nucleus accumbens in teenage suicide victims and non-
suicide subjects. Furthermore, we localized the changes in
5-HT
2A
receptors at the cellular level in various cortical
layers by using gold immunolabeling.
Method
5-HT
2A
receptor levels were determined in the prefrontal cor-
tex (Brodmann’s areas 8/9), hippocampus, and nucleus accum-
bens (all from the right hemisphere) in brains obtained from 15
teenage suicide victims and 15 psychiatrically normal teenage
subjects who died of other causes, herein referred to as normal
subjects. Postmortem brain tissue was obtained from the Brain
Collection Program at the Maryland Psychiatric Research Center
in Baltimore, Md., in collaboration with the Medical Examiner’s
Office of the State of Maryland. Brain samples were free of neuro-
pathological abnormalities and HIV antibodies. Toxicological
data were obtained by analysis of urine and blood samples.
Diagnostic Methods
All study victims were diagnosed by means of the Structured
Clinical Interview for DSM-III-R (13) and the Diagnostic Evalua-
tion After Death (14); diagnoses were based on the information
obtained from interviewing at least one family member and from
all available medical records. Discrepancies were resolved
through a consensus conference between two senior psychia-
trists. Subjects were considered to be suicides only if the manner
of death was determined to be suicide by the medical examiner.
The comparison groups consisted of normal subjects who were
verified as free from mental illnesses by use of these same diag-
nostic procedures. This study was approved by the institutional
review board at our facility.
[
125
I]LSD Binding to 5-HT
2A
Receptors
Tissues were homogenized in 10 vol of hypotonic medium (5
mM of Tris and 0.1% of EDTA; pH=7.5), by using a polytron setting
of 9 for 30 seconds, and centrifuged at 1,000 g for 10 minutes at
4
°C. The resulting supernatant was centrifuged at 30,000 g for 15
minutes at 4
°C. The pellet thus obtained was washed twice with
10 vol of hypotonic buffer and centrifuged. The final pellet was
suspended in 2–5 ml of incubation buffer (50 mM of Tris, 120 mM
of sodium chloride, 5 mM of potassium chloride, 1 mM of magne-
sium chloride, and 0.05% of ascorbic acid; pH=7.4), depending on
the size of the membrane pellet. This suspension was used for the
determination of the 5-HT
2A
receptor level.
The receptor binding assay was carried out in triplicate in plas-
tic tubes containing incubation buffer, [
125
I]LSD, ranging from
0.25–3.00 nM (five to six different concentrations), and a 40-
µl
cortical membrane suspension with or without 1
µM of ket-
anserin in a total incubation volume of 100
µl and incubated at
37
°C for 1.5 hours. The incubation was terminated by rapid filtra-
tion over glass microfiber filters and washed three times with 5 ml
of ice-cold 50-mM Tris buffer (pH=7.7) containing 0.01% bovine
serum albumin. The filters were dried and then counted by a
gamma counter. Specific binding was defined as the difference
between the binding observed in the presence or absence of 1
µM
of ketanserin and ranged from 75% to 80%, depending on the
concentration of the ligand.
Polymerase Chain Reaction Analysis
The mRNA content of the 5-HT
2A
receptors was measured by
use of quantitative reverse transcription polymerase chain reac-
tion, as described earlier (15), with internal standards and these
amplification primers: forward, 772–779 bp (5
′ATCAAGTCACTC-
CAGAAAGAAGCT); reverse, 1153–1176 bp (5
′TGAAAAGGCTGAC-
CTATAGGTCTT).
Internal standard templates were generated by site-directed
mutagenesis to introduce a BglII restriction site midway between
the amplification primers so that the digestion of internal stan-
dard amplicon would generate two fragments of approximately
equal size (208 and 197 bp). The internal primer sequence was as
follows: bp=958–981, 5
′AAGGCATGCAAGAT CTGGGCATC. The
italicized bases indicate the BglII restriction site, while bold and
italicized bases indicate the mutation site. To quantitate 5-HT
2A
receptor mRNA, various amounts of cRNA were added to a l-µg
mixture of total RNA and RNA–cRNA and reverse transcribed by
using 200 U of Moloney’s murine leukemia virus reverse tran-
scriptase. The transcription was carried out in the presence of
random hexamer, 50 mM of Tris hydrochloride (pH=8.3), 74 mM
of potassium chloride, and 3 mM of magnesium chloride in a vol-
ume of 20
µl. The incubation was carried out at room temperature
for 10 minutes then at 37
°C for 1 hour; the contents were heat de-
natured at 95
°C for 5 minutes. The aliquots were amplified with 1
µM of primers, 200 mM of deoxynucleotide triphosphate, 1.5 mM
of magnesium chloride, 50 mM of Tris hydrochloride (pH=9.0), 20
mM of ammonium sulfate, 1
µCi of [
32
P]cytidine 5′-triphosphate,
and 2.5 U of Hot Tub DNA polymerase (Amersham Pharmacea
Biotech, Piscataway, N.J.) in a total volume of 100
µl. The mixture
was amplified for 30 cycles: 94
°C for 45 seconds, 60°C for 1
minute; 72
°C for 1 minute, and 72°C for 15 minutes. Fifty-one mi-
croliters of aliquots were digested with 30 U of BglII overnight at
37
°C in 50 mM of Tris hydrochloride (pH=8.0), 10 mM of magne-
sium chloride, and 100 mM of sodium chloride in a volume of 60
µl. The competitive polymerase chain reaction was analyzed by
agarose gel electrophoresis in triplicate (1.5% weight/volume) in
0.5
× Tris-borate-EDTA buffer.
To quantitate the amount of product, bands stained with ethid-
ium bromide were removed and counted. Data are presented as
the counts incorporated into the amplified cRNA standard, di-
vided by the counts incorporated into the corresponding subunit
mRNA amplification product, versus the counts from a known
amount of internal standard (cRNA) added to the test sample.
Determination of Protein Levels
Tissues were homogenized in homogenizing buffer containing
5 mM of Tris hydrochloride (pH=7.4), 0.1% of EDTA, 2
µM of leu-
peptin, 0.5 mM of phenylmethylsulfonyl fluoride, 1.45
µM of pep-
statin, 0.2 U/ml of aprotinin, and 2 mM of dithiothreitol. The ho-
mogenate was centrifuged at 3,000 rpm for 10 minutes at 4
°C. The
supernatant was recentrifuged at 32,000 rpm for 15 minutes at
4
°C. The resulting pellet was resuspended in homogenizing
buffer.
The samples (30
µg of protein in each lane) were resolved on
7.5% polyacrylamide gel and then blotted for 2 hours on en-
hanced chemiluminescence membranes. The membranes were
then incubated overnight with 5-HT
2A
receptor antibody (1:5000
dilution; Pharmingen, San Diego) and thereafter with a horserad-
ish peroxidase-linked secondary antibody (antimouse immuno-
globulin G [IgG], 1:3000 dilution) for 5 hours. The signal was de-
tected by treating the blots with Western blot detection reagent
(Amersham Pharmacia Biotech, Piscataway, N.J.) and exposing
them to enhanced chemiluminescent membrane. To determine
the immunolabeling of
β-actin, the membranes were stripped
and incubated with
β-actin (monoclonal β-actin antibody) at a
dilution of 1:3000 for 5 hours and with secondary antibody (anti-
mouse IgG) at a dilution of 1:5000 for 2 hours. The optical densi-
ties of the bands were quantified by using a densitometer (Loats
Image Analysis System, Westminster, Md.), and values were cor-
rected with the optical density of
β-actin protein in each sample.
Am J Psychiatry 159:3, March 2002 421
PANDEY, DWIVEDI, RIZAVI, ET AL.
Gold Immunolabeling
Brain tissue samples that were flash frozen after autopsy and
kept at –80
°C were dropped into ice-cold fixative (4% paraformal-
dehyde in 0.1 M of phosphate-buffered saline; pH=7.4) and ro-
tated for 2 hours at 4
°C. Tissues were kept in fixative at 4°C for 24
hours, embedded in 30% sucrose in phosphate-buffered saline,
and refrozen on dry ice for sectioning.
Twenty-micron sections were rinsed in phosphate-buffered sa-
line for 2 days at 4
°C and blocked with RPMI medium 1640
(Gibco-BRL, Grand Island, N.Y.) for 30 minutes. This was followed
by rinsing in 2% normal serum for 30 minutes and 1% bovine se-
rum albumin in phosphate-buffered saline for 30 minutes before
incubation in 5-HT
2A
receptor antibody (2 µg/ml) diluted in 1%
bovine serum albumin in phosphate-buffered saline for 18 hours
at 4
°C, followed by 2 hours at room temperature, while rotating.
Sections were rinsed twice in 1% bovine serum albumin in phos-
phate-buffered saline for 30 minutes, then incubated for 60 min-
utes (at room temperature) with the corresponding secondary
antibody conjugated to l nm of colloidal gold particles and di-
luted in 1% bovine serum albumin in phosphate-buffered saline
(concentration of 1:200). Sections were then rinsed several times
for 30 minutes with 1% bovine serum albumin in phosphate-buff-
ered saline, followed by distilled water. For gold particles to be vis-
ible with light microscopy, they were silver-enhanced for 12 min-
utes and rinsed repeatedly with distilled water. For comparison
sections, from which nonspecific labeling was determined, an
identical protocol was followed, except that 1% bovine serum al-
bumin in phosphate-buffered saline was substituted for the pri-
mary antibody. Sections were then mounted onto microscope
slides, air dried, and counterstained with toluidine blue.
Quantification of Gold Immunolabeling
For quantification purposes, an oil immersion objective of
100
× was coupled with a 1.6× magnification tube; therefore, the
number of immunogold particles was clearly discernible. When
there is dense immunolabeling, immunogold particles some-
times overlap. Particles are counted by using a Samba-2000 image
analysis system (Imaging Products International, R&M BioMet-
rics, Inc., Chantilly, Va.). The grain-counting program first calcu-
lates the average size of a silver-enhanced gold particle, then
quantifies the number of gold particles on the basis of their aver-
age size. For each area, gold particles were also quantified manu-
ally by an experimenter who was blind to the diagnosis to ensure
the reliability of the computerized counts. An interclass correla-
tion coefficient (ICC) with 95% confidence intervals (CIs) for the
reliability of computer-generated counts, as compared to manual
counts, for gold particle quantification were as follows: pyramidal
cells: ICC=0.9177 (95% CI=0.8194–0.9625) for normal subjects
and ICC=0.9364 (95% CI=0.8531–0.9725) for suicide victims; neu-
ropil: ICC=0.9015 (95% CI=0.7839–0.9551) for normal subjects
and ICC=0.9054 (95% CI=0.7814–0.9591) for suicide victims.
With the Samba image analysis, 120
×120-µm sampling frames
were randomly placed in each cortical layer. For pyramidal cells,
only cells that met the following criteria were measured: 1) pres-
ence of a nucleolus, 2) having a pyramidal shape, and 3) presence
of an apical dendrite. The average number of gold particles per
100
µm
2
was calculated in the cell soma of at least 10 randomly
selected cells in each cortical layer for each of three to five 20-
µm
sections of the prefrontal cortex. The mean coefficients of error
for average number of gold particles per cell was 2.8% (SD=0.5%)
for the normal subjects and 3.2% (SD=0.6%) for the suicide vic-
tims. To estimate gold immunodensity in the neuropil, we aver-
aged at least 10 measurements taken in randomly selected 100-
µm
2
cell-free regions of each cortical layer of three to five 20-µm
sections. The mean coefficients of error for average number of
gold particles per 100
µm
2
of cell-free neuropil region were 1.3%
(SD=0.2%) for the normal subjects and 1.1% (SD=0.3%) for the
suicide victims. Nonspecific labeling was determined by counting
the number of gold particles per 100
µm
2
in sections in which the
primary antibody was omitted. Background labeling was cor-
rected by subtracting nonspecific labeling on pyramidal cells
from the total labeling on pyramidal cells and likewise by sub-
tracting nonspecific labeling in the neuropil from total labeling in
the neuropil.
Statistical Analyses
Data analyses were performed by using the SPSS 8.0 statistical
software package (SPSS, Inc., Chicago). [
125
I]LSD binding, pro-
tein, and mRNA 5-HT
2A
receptor levels were compared between
suicide victims and normal subjects by using analysis of covari-
ance, in which race was used as covariate. Group differences be-
tween normal subjects and suicide victims with or without a his-
tory of mental disorders were tested in one-way analysis of
variance, followed by Bonferroni’s multiple comparison proce-
dure where significant main effects were present. The relation-
ships among postmortem interval, age, and 5-HT
2A
receptor
measures were determined by Pearson product-moment correla-
tion analyses. Comparisons of 5-HT
2A
receptor measures with
gender or race were performed with independent t tests. Age and
postmortem interval were compared between suicide victims
and normal subjects with independent t tests. The level of signifi-
cance was alpha
≤0.05.
Results
Effects of Characteristics on 5-HT
2A
Receptors
The demographic characteristics, postmortem intervals,
and toxicology of the suicide victims and normal subjects
are shown in Table 1. There were no significant differences
in age or postmortem interval between the suicide victims
and the normal subjects. We did not observe any signifi-
cant effects of age on [
125
I]LSD binding (r=0.24, df=28, p=
0.49), protein expression of 5-HT
2A
receptor immunolabel-
ing (r=0.26, df=28, p=0.16), or mRNA expression levels (r=
0.11, df=28, p=0.56) in the prefrontal cortex. Similarly, there
were no significant effects of postmortem interval on
[
125
I]LSD binding (r=0.11, df=28, p=0.55), mRNA expres-
sion levels (r=0.17, df=28, p=0.37), or protein expression
levels (r=0.19, df=28, p=0.31) in the prefrontal cortex. There
were no significant correlations between gender and
[
125
I]LSD binding (r=0.07, df=28, p=0.71), mRNA levels (r=
0.03, df=28, p=0.88), or protein expression (r=0.03, df=28,
p=0.88) in the prefrontal cortex. As in the prefrontal cortex,
we did not find any significant effects of age, postmortem
interval, or gender on [
125
I]LSD binding, mRNA expression,
or protein levels of 5-HT
2A
receptors in the hippocampus
or nucleus accumbens. Although we did not observe any
significant correlation between age and 5-HT
2A
receptors,
such a relationship could not be ruled out, since the age
range of our subjects was so narrow (i.e., 13 to 19 years).
The group of suicide victims was composed of one His-
panic, two blacks, and 12 whites; the group of normal sub-
jects was composed of 10 blacks and five whites. No signif-
icant effect of race was observed on any of the measures of
5-HT
2A
receptors in either the prefrontal cortex, hippo-
campus, or nucleus accumbens.
422 Am J Psychiatry 159:3, March 2002
HIGH SEROTONIN IN TEENAGE SUICIDES
[
125
I]LSD Binding Sites
Earlier studies have examined 5-HT
2A
receptors by
means of radioligand binding techniques with different
radioligands. Since a higher level of 5-HT
2A
receptor bind-
ing sites has been reported in the brains of adult suicide
victims, it was of interest to examine whether similar
changes were present in the prefrontal cortex of teenage
suicide victims. We determined 5-HT
2A
binding sites by
Scatchard analysis, using [
125
I]LSD as the ligand and a sat-
uration isotherm and Scatchard plot for a normal subject
and a suicide victim, as shown in Figure 1. After analyzing
the binding data, we observed that the mean B
max
value of
[
125
I]LSD binding was significantly greater in this brain
area of teenage suicide victims than in this area of normal
subjects, without any significant difference in K
D
values
(Table 2).
Levels of mRNA Expression
We studied earlier the distribution of 5-HT
2A
receptor
mRNA in various areas of the human brain and observed
that 5-HT
2A
receptor mRNA levels are highly expressed in
various cortical areas, the hippocampus, amygdala, and
nucleus accumbens (14); therefore, we next quantitated
5-HT
2A
receptor mRNA in the prefrontal cortex, hippo-
campus, and nucleus accumbens obtained from teenage
suicide victims and normal subjects, using the quantita-
tive reverse transcription polymerase chain reaction
technique. A representative autoradiogram of the gel
electrophoresis of 5-HT
2A
receptors in the prefrontal cor-
TABLE 1. Demographic and Clinical Characteristics of 15 Teenage Suicide Victims and 15 Psychiatrically Normal Teenagers
Who Died of Other Causes
Group and
Subject Psychiatric Diagnosis
Age
(years) Race Gender
Postmortem
Interval
(hours) Cause of Death Drug Toxicology Findings
Suicide
victims
1 Alcohol abuse 15 White Female 7 Gunshot wound Ethanol
2 Major depression 12 Black Male 10 Hanging None
3 None 15 White Female 11 Drug overdose Phenylpropanolamine,
chlorpheniramine, codeine,
salicylate, acetaminophen
4 Personality disorder 13 White Male — Hanging None
5 Hyperactivity, attention deficit
disorder
13 White Male 18 Hanging Methylphenidate
6 None 13 Black Male 11 Hanging None
7 Major depression 15 White Male 11 Asphyxia None
8 Major depression, hyperactivity,
attention deficit disorder
15 White Female 17 Drug overdose Imipramine, desipramine
9 Alcohol abuse 20 White Male 32 Hanging Ethanol
10 None 16 Hispanic Male 20 Hanging None
11 Adjustment disorder 17 White Female 25 Drug overdose Verapamil
12 Adjustment disorder with
depressed mood
15 White Male 27 Gunshot wound Pseudoephedrine,
phenylpropanolamine
13 None 16 White Female 18 Gunshot wound Amitriptyline
14 Adjustment disorder 16 White Female 33 Gunshot wound None
15 Adjustment disorder, conduct
disorder
16 White Male 24 Hanging None
Mean 15.13 18.86
SD 1.96 8.38
Normal
subjects
16 19 Black Male 6 Gunshot wound None
17 16 Black Male 6 Gunshot wound None
18 19 Black Male 11 Gunshot wound None
19 17 Black Male 11 Gunshot wound None
20 16 White Male 10 Stab wounds None
21 18 Black Male 11 Gunshot wound None
22 17 Black Male 10 Gunshot wound None
23 13 Black Male 22 Gunshot wound None
24 14 Black Male 18 Gunshot wound None
25 18 Black Male 27 Drowning None
26 16 White Male 21 Hanging None
27 18 White Female 35 Accident (multiple
injuries)
None
28 17 Black Female 26 Accident (multiple
injuries)
None
29 19 White Female 30 Cardiac
arrhythmia
None
30 18 White Male 19 Drowning None
Mean 17.00 17.53
SD 1.77 9.12
Am J Psychiatry 159:3, March 2002 423
PANDEY, DWIVEDI, RIZAVI, ET AL.
tex is given in Figure 2A. As expected, we observed the
amplification product arising from the mRNA template at
405 bp and the corresponding digestion products arising
from the cRNA at 208+197 bp. As shown in Table 2, we
found that the mean 5-HT
2A
mRNA levels were signifi-
cantly greater in the prefrontal cortex of the 15 teenage
suicide victims than in the 15 normal subjects. We also
found that the mean mRNA levels in the hippocampus of
the suicide victims were significantly higher than in the
normal subjects, as shown in Table 2.
To determine if the higher levels of 5-HT
2A
mRNA ob-
served in the prefrontal cortex and hippocampus were
generalized abnormalities or were specific to certain rele-
vant areas of the brain, we determined 5-HT
2A
receptor
mRNA levels in the nucleus accumbens, which also is rich
in 5-HT
2A
receptor mRNA (14). However, the mean 5-HT
2A
receptor mRNA levels in the nucleus accumbens of suicide
victims were not significantly different from those of the
normal subjects, as shown in Table 2, indicating that the
higher 5-HT
2A
receptor mRNA in suicide victims is specific
to certain brain areas, such as the prefrontal cortex and
hippocampus.
Immunolabeling of 5-HT
2A
Receptors
To further examine if the changes in the gene transcrip-
tion (mRNA levels) of 5-HT
2A
receptors also occur at the
translational level, we sought to determine the immunola-
beling of 5-HT
2A
receptor protein, using a specific anti-
body. A representative autoradiogram showing a Western
blot of 5-HT
2A
receptors is depicted in Figure 2B, which
shows that the levels of 5-HT
2A
receptor protein are much
higher in the prefrontal cortex from three teenage suicide
victims than in the brains of three normal subjects. Com-
parison of the mean 5-HT
2A
receptor protein levels in the
prefrontal cortex of 15 suicide victims and 15 normal sub-
jects revealed that the protein expression levels of 5-HT
2A
receptors were significantly higher in these suicide victims
than in the normal subjects (Table 2). The mean 5-HT
2A
protein levels were also significantly higher in the hippo-
campus, but not in the nucleus accumbens, of the suicide
victims compared with the normal subjects (Table 2), sug-
gesting that abnormal 5-HT
2A
protein levels are specific to
the prefrontal cortex and hippocampus and that this
larger amount is not a generalized abnormality, since
FIGURE 1. Representative Saturation Isotherms of [
125
I]LSD Binding to 5-HT
2A
Receptors in the Brains of a Teenage Suicide
Victim and a Psychiatrically Normal Teenager Who Died of Another Cause
a
a
Each point is the mean of triplicate determinations. For this particular experiment, the binding indices for the normal subject were as follows:
K
D
=0.32 nM, B
max
=298 fmol/mg protein, r=0.97. For the suicide victim, they were as follows: K
D
=0.51 nM, B
max
=425 fmol/mg protein, r=
0.99.
TABLE 2. Measures of Serotonin 5-HT
2A
Receptors in the Brains of 15 Teenage Suicide Victims and 15 Psychiatrically Nor-
mal Teenagers Who Died of Other Causes
Measure
Prefrontal Cortex Hippocampus Nucleus Accumbens
Normal Subjects Suicide Victims Normal Subjects Suicide Victims Normal Subjects Suicide Victims
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
[
125
I]LSD binding
B
max
(fmol/mg protein) 292.80 105.07 410.00
a
129.96 — — — — — — — —
K
D
(nM) 0.37 0.08 0.48 0.23 — ———— — ——
Protein (percent of normal
subjects’ level) 100.00 17.49 167.47
b
38.97 100.00 14.66 159.27
c
48.36 100.00 24.03 108.80 23.54
mRNA (attomol/
µg of total RNA) 545.20 334.23 1036.27
d
506.45 192.87 72.21 268.33
e
92.71 261.74 223.13 293.43 275.64
a
Significant difference between groups (F=5.85, df=1, 27, p<0.03).
b
Significant difference between groups (F=30.05, df=1, 27, p<0.0001).
c
Significant difference between groups (F=13.12, df=1, 27, p<0.001).
d
Significant difference between groups (F=6.67, df=1, 27, p<0.02).
e
Significant difference between groups (F=5.97, df=1, 27, p<0.03).
Concentration of [
125
I]LSD (nM)
Bound [
125
I]LSD (fmol/mg protein)
Bound [
125
I]LSD (fmol/mg protein)
Bound/Free [
125
I]LSD
0
100
200
300
400
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0
400
800
200
600
1000
0 100 200 300 400 500
Normal subject
Suicide victim
424 Am J Psychiatry 159:3, March 2002
HIGH SEROTONIN IN TEENAGE SUICIDES
these levels appeared to be normal in the nucleus accum-
bens of the suicide victims.
Immunocytochemical Localization
In accordance with previously published reports (16–
18) making use of immunogold labeling, we found the
expression of 5-HT
2A
receptors to be most dense in pyra-
midal neurons in layers III, V, and VI and their apical
dendrites (Figure 3). Immunogold labeling for 5-HT
2A
re-
ceptors was also observed in a small subgroup of γ-ami-
nobutyric acid (GABA)-ergic neurons, and a moderate
expression was also found in glial cells. To localize the
changes in the expression density of 5-HT
2A
receptors at
the cellular level, we quantified the number of immu-
nogold particles on pyramidal cell somata and in soma-
free regions of the neuropil. Because of its localization in
only a subset of GABA-ergic interneurons, the expression
density of 5-HT
2A
receptors was not quantified on this cell
type. Figure 3 shows low-magnification photomicro-
graphs of 20-µm sections of Brodmann’s area 9 from a nor-
mal teenage subject and a teenage suicide victim that were
immunogold-labeled for 5-HT
2A
receptors. Higher magni-
fication of layer V pyramidal neurons shows a higher
expression density of 5-HT
2A
receptors in the teenage
suicide victim than in the normal subject. The mean ex-
pression levels of 5-HT
2A
receptors were significantly
higher in the pyramidal cells of layer V of the teenage sui-
cide victims (N=9) than in the normal subjects (N=9),
whereas no changes in expression levels of 5-HT
2A
recep-
tors were found in the pyramidal cells of other cortical lay-
ers (layers III and VI) or in the surrounding neuropil (i.e.,
layers IV, V, and VI) (Figure 4). The mean values of positive
neurons of 5-HT
2A
receptor (number of gold particles/100
µm
2
) in cortical layers in the normal subjects and the sui-
cide victims were as follows: pyramidal cells (layer III—
normal subjects: mean=537.23 gold particles/100 µm
2
,
SD=143.51, suicide victims: mean=502.92, SD=85.91; layer
V—normal subjects: mean=341.75, SD=54.06, suicide vic-
tims: mean=542.73, SD=82.34; layer VI—normal subjects:
mean=442.87, SD=74.75, suicide victims: mean=445.62,
SD=78.69); neuropil (layer IV—normal subjects: mean=
476.90, SD=107.19, suicide victims: mean=437.85, SD=
78.69; layer V—normal subjects: mean=436.62, SD=91.75,
suicide victims: mean=381.27, SD=58.70; layer VI—nor-
mal subjects: mean=413.82, SD=68.65, suicide victims:
mean=396.56, SD=70.80). Thus, it appears that the higher
expression of 5-HT
2A
receptors in the postmortem brains
of teenage suicide victims is restricted to the pyramidal
cells of the somata of cortical layer V.
Several studies (7) have suggested an association be-
tween mental disorders and serotonin receptors in post-
mortem brains of adult suicide victims. Therefore, we ex-
amined the association between 5-HT
2A
receptors and
mental illness in teenage suicide victims. Five suicide vic-
tims had no history of mental illness, eight had a history of
mental illness (depression, adjustment disorder), and two
had a history of alcohol abuse. Although there were no sig-
nificant differences in any of the 5-HT
2A
receptor mea-
sures, as shown in Figure 5 and Figure 6, in teenage suicide
victims with or without mental illness in any of the brain
areas studied, the mean mRNA (attomoles/µg of total
RNA) in the prefrontal cortex tended to be higher in the
suicide victims with mental illness (mean=1197.00
attomol/µg, SD=604.11) than in the suicide victims with
no mental illness (mean=883.80 attomol/µg, SD=372.74).
Because of the small group size, we could not examine the
relationship between specific mental illness, such as de-
pression, and 5-HT
2A
indices. Our results, however, do in-
dicate that 5-HT
2A
receptor expression is higher in the pre-
frontal cortex of suicide victims, independent of a history
of mental disorders.
FIGURE 2. Representative Gel Electrophoresis Depicting
5-HT
2A
Receptor mRNA in the Prefrontal Cortex of a Psy-
chiatrically Normal Teenager Who Died From a Nonsuicide
Cause (A)
a
and Representative Western Blots From the Pre-
frontal Cortex of Three Teenage Suicide Victims and Three
Normal Subjects (B)
b
a
Total RNA (1 µg) and decreasing concentrations of cRNA were re-
verse transcribed and amplified with polymerase chain reaction by
using primers specific for the 5-HT
2A
receptor. The samples were
then digested with BgIII restriction enzyme and electrophoresed on
1.5% agarose gel. The upper band depicts 5-HT
2A
receptor RNA (405
bp). The lower bands depict BgIII-digested internal standard cRNA
(208+197).
b
Protein samples (30 µg) were resolved with 7.5% acrylamide gel
electrophoresis and transferred to nitrocellulose membrane, which
was then incubated with 5-HT
2A
receptor antibody. The mem-
branes were stripped and reprobed with
β-actin antibody. The ratio
of optical densities of the bands of 5-HT
2A
receptors to β-actin were
calculated.
Molecular
Weight
(kDa)
506
396
344
298 208+197
405
46 kDa
56 kDa
Normal
Subjects
Suicide
Victims
β-Actin
5-HT
2A
Receptors
220
A
B
Am J Psychiatry 159:3, March 2002 425
PANDEY, DWIVEDI, RIZAVI, ET AL.
Discussion
We have examined the role of 5-HT
2A
receptors in teenage
suicide, using postmortem brain samples. Although it is not
clear if the etiology or the factors underlying teenage suicide
are similar to those found in adult suicide, several factors
lend credence to a relationship between serotonin and teen-
age suicide. Most teenage suicide is driven by impulsive or
aggressive behavior, stress, or anxiety, which have been
shown to be related to abnormalities in serotonergic mech-
anisms (6); hence, it is quite likely that teenage suicide may
be related to abnormalities in serotonergic mechanisms.
We found a striking difference in 5-HT
2A
receptors be-
tween teenage suicide victims and normal subjects. First,
FIGURE 3. Low-Magnification Photomicrographs of 20-µm Brain Sections Showing the Six Cortical Layers of the Prefrontal
Cortex, Immunogold-Labeled for Serotonin 5-HT
2A
Receptors, and High-Magnification Photomicrographs of Pyramidal
Cells in Brain Layer V From a Teenage Suicide Victim and a Psychiatrically Normal Teenager Who Died of Another Cause
a
a
Note the higher level of 5-HT
2A
immunogold labeling on the somata, proximal, and apical dendrites of pyramidal cells in layer V of the pre-
frontal cortex of the suicide victim.
500 µm
10 µm
VI
V
IV
I
II
III
Prefrontal Cortex
Layer V Pyramidal Cells
Normal Subject Suicide Victim
426 Am J Psychiatry 159:3, March 2002
HIGH SEROTONIN IN TEENAGE SUICIDES
we observed that 5-HT
2A
receptor binding sites are more
numerous in the prefrontal cortex of teenage suicide vic-
tims than in normal subjects. The observation of a higher
level of 5-HT
2A
receptors in the prefrontal cortex of teenage
suicide victims is similar to that observed in adults in pre-
vious studies (7). However, we also observed a higher gene
expression of 5-HT
2A
receptors and of its cognate protein
in the prefrontal cortex and hippocampus, but not in the
nucleus accumbens, in the postmortem brains of teenage
suicide victims than in normal subjects. These differences
were not related to postmortem interval or age, and the ab-
sence of correlation between age and 5-HT
2A
receptors
could be due to the narrow age range of our subjects.
Since it has recently been reported that 5-HT
2A
receptors
are highly expressed in the pyramidal neurons in cortical
layers III, V, and VI of the primate and rat brain (18, 19), we
determined whether the observed higher protein levels of
5-HT
2A
receptors were restricted to these specific areas or
whether this change was localized throughout all the corti-
cal layers. Similar to findings in the primate brain, in this
study we found that 5-HT
2A
receptors were densely ex-
pressed in pyramidal neurons in layers III, V, and VI and to
a lesser extent in the surrounding neuropil. Our compari-
son of the expression of 5-HT
2A
receptors in various corti-
cal layers of the brains of teenage suicide victims and nor-
mal subjects led to an interesting observation that 5-HT
2A
receptor protein expression is higher only in the pyramidal
cells of cortical layer V but not in cortical layers III or VI or
in the surrounding neuropil. Our findings thus show that a
greater number of active binding sites of 5-HT
2A
receptors
is not merely a consequence of more binding sites but also
that gene expression contributes to this greater number.
More important, this higher level is restricted to a specific
cortical layer. This is an important observation because py-
ramidal neurons occupy a unique position as they modu-
late and integrate neuronal functions mediated by sero-
tonergic, glutamatergic, GABA-ergic, and dopaminergic
systems (20). It is also important because it has been
shown that the soma and dendrites of pyramidal neurons
of layer V receive synaptic contact from dopamine termi-
nals and GABA-containing neurons. Since it has been
shown that GABA-ergic mechanisms, including benzodi-
azepine receptors, may be altered in the postmortem
brains of suicide victims (21), it is quite possible that higher
levels of 5-HT
2A
receptors in pyramidal cells may cause an
imbalance between the 5-HT
2A
and the GABA-ergic sys-
tems and may play an important role in suicidal behavior.
Jakab and Goldman-Rakic (20) wrote that “the apical den-
dritic field proximal to the pyramidal cells soma is the hot
spot for 5-HT
2A
receptor mediated physiological actions
relevant to neuronal and psychotic functional states of the
cerebral cortex.” They also observed from in vivo record-
ings in the prefrontal cortex of monkeys that 5-HT facili-
tates excitatory neurotransmission in the pyramidal neu-
rons engaged in information processing.
Another important observation of this study pertains to
the higher 5-HT
2A
receptor expression specifically in the
prefrontal cortex and hippocampus but not in the nucleus
accumbens of suicide victims. The prefrontal cortex and
hippocampus have been implicated in emotion, stress,
and cognitive functions; the nucleus accumbens has been
associated with craving, drug dependence, and psycho-
motor-stimulated behavioral sensitization (22). Both
emotion and stress play important roles in suicidal behav-
ior, especially in youth. The higher 5-HT
2A
receptor indi-
ces specifically in the prefrontal cortex and hippocampus
thus further strengthen the association of higher levels of
5-HT
2A
receptors and suicidal behavior.
5-HT
2A
receptors have been previously studied in the
postmortem brains of adult suicide victims with depres-
sion and also in nonsuicidal schizophrenia victims.
Whereas higher levels of 5-HT
2A
receptors have been re-
ported in depressed suicide victims (9), a lower number of
5-HT
2A
receptor binding sites and lower mRNA levels have
been reported in patients with schizophrenia (23). From
these studies, it is not clear if higher 5-HT
2A
receptor levels
are associated with suicide, depression, or suicidal schizo-
phrenia, since insufficient data exist for suicidal schizo-
phrenia patients. In our study of teenage suicide victims,
we examined the relationship of suicide and/or mental
disorders with 5-HT
2A
receptors. Since the numbers of vic-
tims with specific psychiatric diagnoses was small, we
grouped all victims with a history of mental disorders to-
gether, including victims with mood and/or adjustment/
conduct disorders. A significant number of victims had no
history of mental disorders. Since we did not find any sig-
nificant differences in any of the 5-HT
2A
receptor measures
(mRNA, protein expression, or binding) between suicide
victims with or without a history of mental disorders in any
of the brain areas we studied, it appears that the higher
level of 5-HT
2A
receptors is specific to suicide and most
FIGURE 4. Mean Number of Gold Particles Labeling Seroto-
nin 5-HT
2A
Receptors in 100-µm
2
Sections From Cortical
Layers III–VI of Teenage Suicide Victims and Psychiatrically
Normal Teenagers Who Died of Other Causes
a
Significant difference between groups (F=21.82, df=1, 14, p<0.0001).
600
a
500
400
300
Number of Gold Particles/100 µm
2
Pyramidal Cells Neuropil
200
100
III V VI IV V VI
Suicide victims (N=9)
Normal subjects (N=9)
Am J Psychiatry 159:3, March 2002 427
PANDEY, DWIVEDI, RIZAVI, ET AL.
likely independent of psychiatric diagnosis. However,
these 5-HT
2A
receptor levels were still significantly higher
in both of these groups, compared with the normal sub-
jects. Although this finding is consistent with our previous
report that the higher level of platelet 5-HT
2A
receptors ob-
served in suicidal victims is independent of diagnosis (11),
confirmation in a larger number of suicide victims with
specific diagnoses of psychiatric disorders is needed. It is
also possible that in teenage suicide victims, higher levels
of 5-HT
2A
receptors may be related to impulsive-aggressive
behavior. However, this could not be ascertained in this
study group, since no data regarding aggressive/impulsive
behavior in these victims were available.
The reasons for the higher levels of 5-HT
2A
receptors in
suicide victims are not known, and it is unclear if higher
levels of 5-HT
2A
receptors in the brains of suicide victims
indicate a higher vulnerability for suicide. In a recent
study, Du et al. (24) examined the 5-HT
2A
receptor gene in
platelets obtained from suicidal and nonsuicidal de-
pressed patients and normal comparison subjects. They
found a significant association between the 102C allele in
the 5-HT
2A
receptor gene and major depression, particu-
larly in patients with suicidal ideation. Since the same
gene encodes both brain and platelet 5-HT
2A
receptors
(25), our observation of higher 5-HT
2A
receptor levels in
the prefrontal cortex may represent a greater genetic vul-
nerability for suicide.
However, it is possible that the higher 5-HT
2A
receptor
levels in suicide victims is secondary to changes in other
systems, such as abnormalities in the hypothalamic-pitu-
itary-adrenal (HPA) axis. Abnormal HPA axis function and
higher levels of cortisol have been consistently observed
in patients with depression (26, 27) and suicidal behavior
(28, 29). Interaction between the HPA axis and the sero-
tonergic system has also been clearly demonstrated (re-
viewed by Chauloff [30], Kuroda et al. [31], Fernandes et al.
[32], and Mikuni et al. [33]). Moreover, it has been shown
that the administration of dexamethasone or corticoster-
one causes an increase in 5-HT
2A
receptor levels in the rat
cortex (31). Recently, Farisse et al. (34) demonstrated that
transgenic mice bearing a transgene coding for glucocor-
ticoid receptor antisense mRNA, which partially blocks
glucocorticoid receptor expression, show lower glucocor-
ticoid receptor numbers and a higher level of 5-HT
2A
re-
FIGURE 5. Effects of Mental Disorders on 5-HT
2A
Receptor Measures in the Prefrontal Cortex of Teenage Suicide Victims
a
Significant difference between groups (F=3.75, df=3, 26, p<0.03).
b
Significant difference between groups (F=13.89, df=3, 26, p<0.001).
c
Significant difference between groups (F=3.45, df=3, 26, p<0.04).
1400
1000
800
600
5-HT
2A
Receptor mRNA Level
(attomol/µg total RNA)
a
400
200
0
1200
200
160
120
Immunolabeling of 5-HT
2A
Receptors
(percent of normal subjects' level)
b
80
40
0
B
max
of 5-HT
2A
Receptors
(fmol/mg protein)
c
500
0
400
300
200
100
Psychiatrically normal
subjects who died of
nonsuicide causes (N=15)
All suicide victims
(N=15)
Suicide victims with
mental disorder (N=8)
Suicide victims with
no mental disorder (N=5)
FIGURE 6. Effects of Mental Disorders on mRNA and Pro-
tein Levels of 5-HT
2A
Receptors in the Hippocampus of
Teenage Suicide Victims
a
Significant difference between groups (F=3.86, df=3, 19, p<0.03).
b
Significant difference between groups (F=4.72, df=3, 19, p<0.02).
350
250
200
150
5-HT
2A
Receptor mRNA Level
(attomol/µg total RNA)
a
100
50
0
300
200
160
120
Immunolabeling of 5-HT
2A
Receptors
(percent of normal subjects’ values)
b
80
40
0
Psychiatrically normal
subjects who died of
nonsuicide causes (N=15)
All suicide victims
(N=15)
Suicide victims with
mental disorder (N=8)
Suicide victims with
no mental disorder (N=5)
428 Am J Psychiatry 159:3, March 2002
HIGH SEROTONIN IN TEENAGE SUICIDES
ceptors. Since cortisol levels are higher in patients with de-
pression and during periods of stress or anxiety, the higher
levels of 5-HT
2A
receptors in the brain of suicide victims
may be due to an abnormal HPA axis. It is also quite possi-
ble that genetic and environmental factors (higher cortisol
level) may in combination cause up-regulation of cortical
5-HT
2A
receptors.
In summary, this is the first study to our knowledge that
demonstrates higher levels of gene expression of 5-HT
2A
receptors and of the encoded 5-HT
2A
receptor protein in
the prefrontal cortex of suicide victims (either teenage or
adult); it shows that these higher levels are restricted to the
pyramidal cells of layer V, which are considered a “hotspot”
for 5-HT
2A
receptor-mediated physiological actions. This
higher level of 5-HT
2A
receptors may play a vital role in sui-
cidal behavior and may represent a greater risk for suicide.
Received Nov. 29, 2000; revisions received April 16 and Aug. 1,
2001; accepted Aug. 27, 2001. From the Psychiatric Institute, Depart-
ment of Psychiatry, University of Illinois at Chicago; and Maryland
Psychiatric Research Center, Baltimore, Md. Address reprint requests
to Dr. G.N. Pandey, Psychiatric Institute, University of Illinois at Chi-
cago, 1601 W. Taylor St., Chicago, IL 60612; gnpandey@psych.uic.edu
(e-mail).
Supported by an NIMH grant (MH-48153) to Dr. Pandey. Brain col-
lection was supported in part by an NIMH grant (MH-40279) to Dr.
Carpenter.
The authors thank Dr. John Smialek, chief medical examiner, and
Dr. Dennis Chute, assistant medical examiner, for the collection of
brain samples; Boris Lapidus, M.D., and Christopher Grove for dissec-
tions; Terri U’Prichard for help with psychological autopsies; and Bar-
bara Brown, Janardhana R. Jonnalagadda, and Miljana Petkovic for
technical assistance.
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