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Upregulation of CB1 receptors and agonist-stimulated [35S]GTPS binding in the prefrontal cortex of depressed suicide victims

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Endogenous and exogenous cannabinoids (CBs) acting through the CB(1) receptors have been implicated in the regulation of several behavioral and neuroendocrine functions. Modulation of endocannabinoidergic system by ethanol in mouse brain, and the association of suicide and mood disorders with alcoholism suggest possible involvement of the cannabinoidergic system in the pathophysiology of depression and suicide. Therefore, the present study was undertaken to examine the levels of CB(1) receptors and mediated signaling in the dorsolateral prefrontal cortex (DLPFC) of subjects with major depression who had died by suicides (depressed suicides, DS). [(3)H]CP-55,940 and CB(1) receptor-stimulated [(35)S]GTPgammaS binding sites were analyzed in membranes obtained from DLPFC of DS (10) and matched normal controls (10). Upregulation (24%, P<0.0001) of CB(1) receptor density (B(max)) was observed in DS (644.6+/-48.8 fmol/mg protein) compared with matched controls (493.3+/-52.7 fmol/mg protein). However, there was no significant alteration in the affinity of receptor (DS; 1.14+/-0.08 vs control; 1.12+/-0.10 nM). Higher density of CB(1) receptors in DS (38%, P<0.001) was also demonstrated by Western blot analysis. The CB(1) receptor-stimulated [(35)S]GTPgammaS binding was significantly greater (45%, P<0.001) in the DLPFC of DS compared with matched controls. The observed upregulation of CB(1) receptors with concomitant increase in the CB(1) receptor-mediated [(35)S]GTPgammaS binding suggests a role for enhanced cannabinoidergic signaling in the prefrontal cortex of DS. The cannabinoidergic system may be a novel therapeutic target in the treatment of depression and/or suicidal behavior.
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ORIGINAL RESEARCH ARTICLE
Upregulation of CB
1
receptors and agonist-stimulated
[
35
S]GTPcS binding in the prefrontal cortex of depressed
suicide victims
BL Hungund
1,2,3,5
, KY Vinod
2,5
, SA Kassir
1
, BS Basavarajappa
1,2
, R Yalamanchili
2
, TB Cooper
1,2,3
,
JJ Mann
1,3
and V Arango
1,3,4
1
New York State Psychiatric Institute, New York, NY, USA;
2
Nathan Kline Institute for Psychiatric Research, Orangeburg, NY,
USA;
3
Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, NY, USA;
4
Department
of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY, USA
Endogenous and exogenous cannabinoids (CBs) acting through the CB
1
receptors have been
implicated in the regulation of several behavioral and neuroendocrine functions. Modulation of
endocannabinoidergic system by ethanol in mouse brain, and the association of suicide and
mood disorders with alcoholism suggest possible involvement of the cannabinoidergic
system in the pathophysiology of depression and suicide. Therefore, the present study was
undertaken to examine the levels of CB
1
receptors and mediated signaling in the dorsolateral
prefrontal cortex (DLPFC) of subjects with major depression who had died by suicides
(depressed suicides, DS). [
3
H]CP-55,940 and CB
1
receptor-stimulated [
35
S]GTPcS binding sites
were analyzed in membranes obtained from DLPFC of DS (10) and matched normal controls
(10). Upregulation (24%, Po0.0001) of CB
1
receptor density (B
max
) was observed in DS
(644.6748.8 fmol/mg protein) compared with matched controls (493.3752.7 fmol/mg protein).
However, there was no significant alteration in the affinity of receptor (DS; 1.1470.08 vs
control; 1.1270.10 nM). Higher density of CB
1
receptors in DS (38%, Po0.001) was also
demonstrated by Western blot analysis. The CB
1
receptor-stimulated [
35
S]GTPcS binding was
significantly greater (45%, Po0.001) in the DLPFC of DS compared with matched controls. The
observed upregulation of CB
1
receptors with concomitant increase in the CB
1
receptor-
mediated [
35
S]GTPcS binding suggests a role for enhanced cannabinoidergic signaling in the
prefrontal cortex of DS. The cannabinoidergic system may be a novel therapeutic target in the
treatment of depression and/or suicidal behavior.
Molecular Psychiatry (2004) 9, 184–190. doi:10.1038/sj.mp.4001376
Keywords: CB
1
receptor; [
35
S]GTPgS binding; depression; suicide; prefrontal cortex
Introduction
Cannabinoid (CB) receptors received their name
as those receptors that bind cannabinoidergic
drugs, such as 9-tetrahydrocannabinol (D
9
-THC),
derived from Cannabis sativa and its biologically
active synthetic analogs. D
9
-THC is the major
psychoactive component in marijuana extracts
and can produce a multiplicity of effects in
humans, including alterations in mood, percep-
tion, cognition and memory.
1–4
Marijuana
is currently the most widely abused drug second
to alcohol. However, the functional significance
of the cannabinoidergic system in health and
disease is just beginning to emerge.
5,6
Significant progress has been made in characteriz-
ing CB receptors both centrally and peripherally, and
in studying the role of second messenger systems at
the cellular level. To date, two types of CB receptors,
CB
1
and CB
2
, have been identified
7,8
and have been
shown to belong to G-protein coupled receptor
(GPCR) family. Two endogenous cannabimimetic
substances, characterized to be N-arachidonyl etha-
nolamide (AEA/anandamide) and 2-arachidonylgly-
cerol (2-AG), were discovered and shown to act as
agonists for CB receptors.
9,10
The CB
1
receptor is
distributed primarily in neural tissue, whereas CB
2
receptor is expressed mainly in the peripheral
immune system.
11,12
These receptors exhibit seven
transmembrane domains, linked to G
i/o
protein to
inhibit adenylyl cyclase.
13
Interestingly, the CB
1
receptor is one of the most abundant neuromodula-
tory receptors in the brain and is expressed predomi-
nantly in the cerebral cortex, hippocampus,
cerebellum and basal ganglia.
14–16
Received 20 January 2003; revised 7 March 2003; accepted 10
March 2003
Correspondence: Dr BL Hungund, Nathan Kline Institute for
Psychiatric Research, Bldg 39, 140 Old Orangeburg Road,
Orangeburg, NY 10962, USA;
E-mail: hungund@nki.rfmh.org
5
BLH and KYV contributed equally to this work.
Molecular Psychiatry (2004) 9, 184 190
&
2004 Nature Publishing Group All rights reserved 1359-4184/04
$
25.00
www.nature.com/mp
Numerous studies have implicated alterations in
several receptors and G-protein function in the
pathophysiology of various neurological and psychia-
tric disorders. Decreased CB
1
receptor binding in
neurodegenerative diseases related to extrapyramidal
function has been reported.
17
Alterations in the
serotonergic (for a review see, Arango et al
18
) and
adrenergic
19
receptors in the pathophysiology of
depression and suicidal behavior are well documen-
ted. Recently, Dean et al,
20
reported increased CB
1
receptor density in dorsolateral prefrontal cortex
(DLPFC) in schizophrenia.
The increased levels of endocannabinoids and
downregulation of CB
1
receptor in response to
chronic ethanol intake in mouse brain suggest
modulation of the endocannabinoidergic system by
alcohol
21,22
(for a review, see Hungund et al
23
). The
mood and cognition altering ability of exogenous
cannabinoids and alcohol, and the association be-
tween depression, suicide and alcohol abuse raise the
question whether endogenous cannabinoidergic sys-
tem plays any role in the etiology of depression and
suicidal behavior. Therefore, to address this question,
we studied the density of CB
1
receptors and CB
1
receptor-mediated [
35
S]GTPgS binding in prefrontal
cortex of subjects with major depression who had
died by suicides. Further studies of the role of the
cannabinoidergic system in various neuropsychiatric
disorders would be of great interest.
Materials and methods
Human brain tissue
Brain samples of prefrontal cortex were obtained from
autopsy material derived from the brain tissue
collection of the Department of Neuroscience at the
New York State Psychiatric Institute (NYSPI) and
Columbia University, NY, USA. All tissue used in this
study was provided by the Allegheny County Coroner
in accordance with protocols approved by the
Institutional Review Board of the University of
Pittsburgh. Brains were collected and bisected at
autopsy. The right hemispheres were cut coronally
into 1.5-cm thick sections. Blocks were placed on a
glass slide, immersed in freon (201C) and stored at
801C in tightly sealed, thick plastic bags until
sectioning. After a control and suicide were matched,
coronal sections (20 mm) from the hemicerebrum were
taken from a level just anterior to the genu of the
corpus callosum with a large format Leica Cryopoly-
cut cryostat. Interleaved sections every 200 mm were
sectioned at 50 mm and stained with cresylecht violet
for cytoarchitectonics. Once the sectioning was
completed, Brodmann area 9 was identified using
gyral and sulcal landmarks, cytoarchitecture and a
standardized coronal atlas (Robert Perry and Edward
Bird, personal communication), as previously de-
scribed.
24
Tissue from Brodmann area 9 (B1.5 g) was
dissected frozen, the white matter was removed as
much as possible, and tissue returned immediately to
–801C until membrane preparation.
The study was conducted with approval from the
NYSPI Institutional Review Board. Dorsolateral pre-
frontal cortex postmortem samples (Brodmann area 9)
from 10 normal controls (age range: 15–79 years) were
studied with a matched group of 10 subjects who had
a lifetime diagnosis of major depression and died by
suicide (age range: 13–77 years). The groups com-
prised pairs of depressed suicides (DS) and control
cases matched for age, sex, postmortem interval (PMI)
and ethnic group. There were nine pairs of caucasians
and one pair of African-Americans (2nd pair in Table
1). This distribution reflects the ethnic make-up of
Allegheny County, where the samples were collected.
There were no significant differences in age, sex and
PMI distribution between DS and control subjects.
The demographic variables, such as sex, age, PMI and
cause of death, as well as toxicology results, are
summarized in Table 1. The Coroner determined the
cause of death and reached the verdict of suicide.
Toxicological analyses were performed on all the
cases, ruling out recent consumption of substance of
abuse or psychoactive medication except in three
samples where an anxiolytic drug (n ¼ 1) and ethanol
(n ¼ 3) were detected. Two individuals received
lidocaine as part of resuscitation efforts at the
emergency room. All cases were free of neuropathol-
ogy. Both suicides and controls were examined
psychiatrically by structured interviews with family
members and/or close friends. The psychiatric diag-
noses were made according to DSM-III-R criteria.
25
The psychological autopsies revealed that all suicide
victims had a lifetime diagnosis of major depression
and all controls were free of psychopathology. The
samples were coded to mask investigators to the
diagnostic group of all subjects. All the assays were
carried out in a paired design under the same
experimental conditions.
Membrane preparation
Brain tissue (B1 g) was homogenized in 20 volumes
of ice-cold TME buffer (50 mM Tris-HCl, 3 mM MgCl
2
and 1 mM of EDTA, pH 7.4) containing 0.32 M sucrose
and freshly added protease inhibitor cocktail. The
homogenate was centrifuged at 1000 g for 10 min at
41C. The resulting supernatant was then centrifuged
at 22 000 g for 20 min. The pellet was dissolved in
TME buffer and recentrifuged at 22 000 g for 20 min.
The final pellet, dissolved in TME buffer, was made
aliquots and stored at –801C until the assay.
Determination of protein content
The protein content of the membrane fraction was
determined by Lowry’s method
26
using bovine serum
albumin (BSA) as the standard. Protein content of the
membrane, also normalized by silver staining, was
used for Western blot analysis.
[
3
H]CP-55,940 binding assay
An aliquot of membrane (100 mg protein) was incu-
bated with TME buffer, [
3
H]CP-55,940 (0.05–5.0 nM)
and 0.1% fatty acid-free BSA in silicone-treated test
CB
1
receptors-mediated signaling in brain of DS
BL Hungund et al
185
Molecular Psychiatry
tubes for 1 h at 371C. The nonspecific binding of
radioligand was defined by CP-55,940 (10 mM). The
reaction was terminated by the addition of 2 ml ice-
cold termination buffer (0.1% BSA in 50 mM Tris-
HCl, pH 7.4). The reaction mixture was rapidly
filtered through polyethyleneimine (0.1%) pretreated
glass fiber filters using a Brandel 24-position cell
harvester (Brandel, Gaithersburg, MD, USA). Filters,
washed three times with the termination buffer, were
transferred to scintillation vials containing 5 ml of
scintillation cocktail (ICN biochemicals, USA), and
were incubated overnight at room temperature. The
radioactivity was measured by liquid scintillation
spectroscopy (Beckman) at an efficiency of 47% for
tritium.
CB
1
receptor-stimulated [
35
S]GTPgS binding assay
The functional coupling between CB
1
receptor and
G-protein was assessed by [
35
S]GTPgS binding assay
as described previously
27
with minor modification.
Briefly, an aliquot of membrane (50 mg protein) was
incubated in assay buffer (TME buffer and 0.1% fatty
acid-free BSA and 100 mM NaCl) containing GDP
(40 mM), and [
35
S]GTPgS (0.05 nM) in silicone-treated
test tubes for 1 h at 371C. The CB
1
receptor agonist,
CP-55,940 (1 mM), was used to study CB
1
receptor-
stimulated [
35
S]GTPgS binding. The nonspecific bind-
ing of radioligand was determined in the presence of
10 mM GTPgS. The termination and filtration (without
presoaking the filters in polyethyleneimine) of
reaction mixture was performed as described for
[
3
H]CP-55,940 binding assay. The radioactivity was
measured by liquid scintillation spectroscopy at an
efficiency of 95% for
35
S.
Western blot analysis
Briefly, aliquots of membrane protein (30 mg), sepa-
rated by 10% polyacrylamide gel, were electrophor-
etically transferred to nitrocellulose membrane. The
membrane was treated with blocking buffer (TTBS
(10 mM Tris, 0.9% NaCl; 1% Tween 20 containing 3%
milk powder) of pH 7.4) for 1 h at room temperature.
The membrane was incubated with human anti-CB
1
receptor antibody (1 : 500) overnight at 41C. The blot
was washed three times with TTBS and then
incubated with alkaline phosphatase-conjugated
anti-IgG for 1 h at room temperature. After washing
the blot for 3–4 times with TTBS, the immunoreactive
band was visualized by CDP-star reagent. The blot
was reprobed with a-tubulin antibody to ensure equal
protein loading.
Data and statistical analysis
The B
max
(maximal binding sites) and K
d
(apparent
dissociation constant) values were determined from
saturation isotherms using nonlinear regression ana-
lysis to fit the data to the single-site binding equation
(Prism; GraphPad software). The density and affinity
of CB
1
receptor was expressed as fmol/mg protein and
nM, respectively. The CB
1
receptor-stimulated
Table 1 Demographic characteristics of DS and their matched control subjects
Diagnosis Sex (M/F) Age at death (years) PMI (h) Cause of death Toxicology
1. MDD M 13 18 Suicide, NaOH poisoning None
Control M 15 16 Homicide, GSW None
2. MDD M 14 16 Suicide, hanging None
Control M 17 14 Homicide, GSW Alcohol
3. MDD M 17 17 Suicide, GSW None
Control M 16 18 Accident, asphyxia None
4. MDD M 25 18 Suicide, GSW Alcohol
Control M 25 18 Accident, explosion Alcohol
5. MDD F 28 19 Suicide, fall from height None
Control F 27 15 MVA, pedestrian None
6. MDD F 34 04 Suicide, hanging None
Control F 38 07 Homocide, GSW None
7. MDD F 43 12 Suicide, poisoning ANX, OPIA, ACE
Control F 42 16 Homicide, GSW None
8. MDD F 61 11 Suicide, hanging None
Control F 56 18 MVA, passenger None
9. MDD M 64 19 Suicide, GSW LIDO
Control M 66 19 Natural, cardiovascular None
10. MDD M 77 18 Suicide, hanging None
Control M 79 10 Natural, cardiovascular LIDO
MDD (10) 6M/4F 37.677.2 15.971.4
Control (10) 6M/4F 38.17 7.1 15.071.2
The data are presented as mean7SEM. MDD, major depressive disorder; GSW, gun shot wound; MVA, motor vehicle
accident; PMI, postmortem interval (rounded to near hours); ANX, anxiolytic; LIDO, lidocaine; OPIA, opiates; ACE,
acetaminophen; Low level of alcohol was detected in three cases (0.02–0.03%).
CB
1
receptors-mediated signaling in brain of DS
BL Hungund et al
186
Molecular Psychiatry
[
35
S]GTPgS binding expressed as fmol/mg protein’ is
a percentage of stimulation over the basal activity.
Statistical analysis performed using nonparametric
analysis of variance (Mann–Whitney U) and para-
metric (paired Student t’-test). Differences were
considered to be significant at Po0.05. Immunoblots
were analyzed using the NIH image software program.
Data are expressed as mean7SEM from two to three
experiments, each run in at least duplicate unless
otherwise indicated.
Chemicals
[
35
S]GTPgS and [
3
H]CP-55,940 were purchased from
DuPont NEN (Boston, MA, USA). Fatty acid-free BSA,
protease inhibitor cocktail, GDP and GTPgS were
procured from Sigma Co (St Louis, MO, USA). Glass
fiber filters (GF/B) were purchased from Brandel Inc.
(Gaithersburg, MD, USA). CP-55,940 was a gift from
Pfizer Pharmaceutical (Groton, CT, USA). Human
anti-CB
1
receptor was obtained from Biosource Inter-
nationals (California, CA, USA). Anti-a-tubulin
monoclonal antibody was from Amersham Bioscience
(Piscataway, NJ, USA). Alkaline phosphatase-conju-
gated anti-IgG was obtained from Promega (Madison,
WI, USA). CDP-star chemiluminescence kit was
purchased from Tropix (Bedford, MA, USA). Other
chemicals, of analytical grade, were purchased from
standard commercial sources.
Results
The density of CB
1
receptor
A saturation analysis suggests that [
3
H]CP-55,940
binding is saturable below 5.0 nM concentration.
The nonspecific binding was about 15% of total
[
3
H]CP-55,940 binding. A Scatchard analysis of the
binding data indicates a monophasic binding of
radioligand, and Hill’s coefficient of near unity
suggests the binding of radioligand to a single class
of receptor at the concentration used. A representa-
tive saturation isotherm and Scatchard plot is shown
in Figure 1.
The average density (B
max
)ofCB
1
receptor in DLPFC
of normal control subjects was 493.3752.7 fmol/mg
protein. The apparent dissociation constant (K
d
) was
1.1270.10 nM, suggesting a high affinity of the
receptor for the radioligand. All the comparisons
shown below utilized Mann–Whitney U and paired
t’-tests. Greater density of CB
1
receptor was observed
in the DLPFC of DS (644.6748.8 fmol/mg protein;
24%, Po0.0001) compared with matched controls
(Figure 2). However, there was no difference in the
affinity of receptor for radioligand (DS; 1.1470.08 vs
control; 1.1270.10 nM), suggesting an upregulation of
the density of receptor in the absence of altered
affinity of the receptor. A significant increase (38%,
Po0.001) in CB
1
receptor immunoreactivity was also
demonstrated by Western blot analysis. A representa-
tive CB
1
receptor immunoblot of a DS and matched
control (3A) and levels of CB
1
receptor immunoreac-
tivity of all the subjects (3B) are shown in Figure 3.
CB
1
receptor-stimulated [
35
S]GTPgS binding
The CB
1
receptor-stimulated [
35
S]GTPgS binding was
used to assess the coupling efficiency between a
receptor and its G-protein. Using the CB
1
receptor
agonist, CP-55,940 stimulated-[
35
S]GTPgS binding as
the outcome measure, maximum stimulation of
[
35
S]GTPgS binding was observed when cortical
membranes were incubated with 1 mM CP-55,940
and 40 mM of GDP (data not shown). The increase in
CB
1
receptor-stimulated [
35
S]GTPgS binding was 45%
greater in cortical membranes of DS (31.174.7 fmol/
mg protein; Po0.001) compared with matched con-
trols (16.972.5 fmol/mg protein) (Figure 4). However,
no significant group difference in basal [
35
S]GTPgS
binding was observed.
Discussion
Although our understanding of clinical aspects of
depression has advanced, the precise underlying
neurobiological basis of this disorder remains to be
elucidated. Disturbances in pre- and postsynaptic
proteins in DS have been reported.
28
A number of
0 100 200 300 400 500 60
0
0
200
400
600
Bound
Bound / Free
0 1 2 3 4 5
0
200
400
600
DS
Control
[
3
H
]
CP-55,940
(
nM
)
Specific binding (fmol/mg protein)
Figure 1 The saturation binding of [
3
H]CP-55,940 (0.5–
5.0 nM) to prefrontal cortical membrane of DS and matched
control. The inset represents the Scatchard transformation
of the same binding data.
0
250
500
750
1000
Control
DS
***
Density CB
1
receptor
(fmol/mg protein)
Figure 2 The density of CB
1
receptor was estimated in
prefrontal cortical membranes of DS (10) and matched
controls (10). Data are mean7SEM of two to three experi-
ments, each assayed in duplicate. ***Po0.0001.
CB
1
receptors-mediated signaling in brain of DS
BL Hungund et al
187
Molecular Psychiatry
studies have found differences in serotonergic and
adrenergic receptors in the prefrontal cortex of DS
victims.
18,19
Recently, Gurevich et al,
29
suggested that
alterations in the postmodification regulation of gene
expression of serotonin might play a role in the
etiology of major depression. It is likely that the
pathobiology of depression cannot be attributed to
dysfunction in a single neurotransmitter pathway.
Therefore, the search for other neurochemical ab-
normalities associated with depression is continuing.
Recent studies from our laboratory have suggested
the participation of cannabinoidergic system in
alcoholism and related behaviors.
21–23
Existence of
comorbidity between alcoholism and depression led
us to investigate the role of cannabinoid signaling in
depression. Indeed, the present study for the first time
reveals greater CB
1
receptor density and coupling
between these receptors and G
i
-protein in DLPFC of
DS subjects. CB
1
receptor immunoblot analysis found
more CB
1
receptor protein immunoreactivity, sub-
stantiating the radioligand binding results.
Many transmembrane signaling processes of extra-
cellular hormone and neurotransmitters are mediated
by receptor interaction with heterotrimeric (a,b,g)
guanosine nucleotide binding proteins (G-protein).
The receptor activation alters the conformation of
G-proteins leading to the exchange of GDP by GTP
on G
a
-subunit. The conformational change promotes
the dissociation of G-protein into active G
a
-GTP and
G
bg
-subunits.
30
These two subunits later regulate
the activity of several effector molecules within the
cell. Recently, a nonhydrolyzable GTP analogue,
[
35
S]GTPgS, has been employed to asses the coupling
efficacy of several neurotransmitter receptors and G-
proteins in cortical membranes of human postmortem
brain.
27
The results of the present study suggest greater CB
1
receptor-stimulated [
35
S]GTPgS binding in DLPFC of
DS compared to matched controls. The observed
increase in [
35
S]GTPgS binding may be due to more
CB
1
receptors. Interestingly, we observed low percen-
tage of stimulation of CB
1
receptor-mediated
[
35
S]GTPgS binding either in DS or matched controls.
Despite the high density of CB
1
receptors, the reason
for low agonist-stimulated [
35
S]GTPgS binding is not
known at this time. However, lower efficiency of CB
1
receptor coupling to G
i
-protein, compared to other
GPCRs has been suggested.
31
This is borne out from
direct comparison between the efficacy of cannabi-
noids and opiates in which opiates signaling was
found to be 20-fold more efficient than cannabinoid
signaling.
32
Therefore, it is speculated that the CB
1
receptor signaling functions as a subtle, fine-tuning
mechanism for cells. The high density of receptors
makes the CB
1
receptor highly sensitive to agonists;
however, the poor coupling efficiency ensures that
overactivation of the system will not occur.
31
The consequence of elevated CB
1
receptor-mediated
signaling in the pathophysiology of depression is not
known. Abnormalities in cAMP signaling in depres-
sive disorders have been reported. Dowlatshahi
0
200
400
600
800
**
DS
Control
CB
1
receptor immunoreactivity
(Arbitrary number)
183
114
81
64
50
38
26
kDa
Control
CB1R
α−
α−
Tubulin
DS
a
b
Figure 3 (a) A representative immunoblot of the CB
1
receptor (top) and the same blot was reporbed with a-
tubulin (bottom) to ensure equivalent total protein loading.
(b) Levels of CB
1
receptor immunoreactivity in prefrontal
cortical membranes of DS (10) and normal controls (10)
reexpressed in mean7SEM of arbitrary densitometric units.
**Po0.001.
0
10
20
30
40
50
Control
DS
**
% Stimulation
(fmol/mg protein)
Figure 4 CB
1
receptor-stimulated [
35
S]GTPgS binding was
done in prefrontal cortical membranes of DS (10) and
matched controls (10). Data, presented as percentage of
stimulation over the basal, are mean7SEM values of two to
three experiments, each assayed in triplicate. **Po0.001.
CB
1
receptors-mediated signaling in brain of DS
BL Hungund et al
188
Molecular Psychiatry
et al,
33
found decreased cAMP signaling in the brain
of depressed suicides. The increased CB
1
receptor
density and its mediation in [
35
S]GTPgS binding
suggest the sensitization of cannabinoidergic signal-
ing, which may lead to the decreased cAMP content
of the cell as these receptors are negatively coupled
to AC.
Recent studies have suggested age-dependent al-
terations in many neurotransmitter receptors. Aged
rats exhibited a marked decrease in CB
1
receptors and
its mediated [
35
S]GTPgS binding sites in rat brain.
34
In
this study, we also observed (data not shown) reduced
receptor density associated with increasing age in
normal control subject, suggesting that receptor losses
are related to the aging process. This observation is
consistent with previous reports.
15,16
Several studies have suggested that age, sex, PMI
and psychoactive drug medications may be respon-
sible for the alterations in neurotransmitter receptors
and G-proteins. The brain samples analyzed in this
study were well matched with regards to sex, age,
ethnic background and postmortem interval. Suicide,
however, is often associated with major depression,
35
and postmortem studies are often unable to resolve
whether the observed abnormalities are due to the
presence of major depression or whether they reflect
abnormalities that characterize suicidal behavior. In
this study, we are unable to tease out the effect of
suicide vs the effect of depression. Although suicid-
ality is often associated with multiple depressive
symptoms, future studies should test for the differ-
ences between suicide victims with a history of major
depression and nondepressed suicides and or de-
pressed and normal subjects who died by similar
cause of death. The next question whether the
observed abnormalities in DS victims are a conse-
quence of pathobiology or antidepressant medication
is of particular relevance. However, in the present
study, only three patients had medications or alcohol
at the time of death and no psychoactive drugs were
detected in the remaining patients. Therefore, the
present findings in brains of DS are related to the
illness, be it suicide or major depression, rather than
to antemortem drug treatment.
It has been suggested that cannabis use aggravates
existing psychosis.
36,37
Two endocannabinoids, which
act on CB receptor, anadamide and palmitoylethano-
lamide, were shown to be increased in the cerebrosp-
inal fluid (CSF) of schizophrenics.
38
Increased CB
1
receptor density in the DLPFC of schizophrenia has
also been recently reported.
20
It has been suggested
that the clinical signs of chronic cannabis consump-
tion may resemble negative symptoms of schizophre-
nia.
39
Several common symptomatologies do exist
between schizophrenia and mood disorders. From a
neuopharmacological standpoint, the psychoses of
schizophrenia and the mania of bipolar disorder can
both be treated with antipsychotic drugs. Some
prominent negative symptoms of schizophrenia such
as affective flattering, alogia and avolition are most
commonly observed in depression.
40
Therefore, it
may be assumed that the observed elevated CB
1
receptor and mediated signaling may be a pathologi-
cal consequence of depression and/or schizophrenia.
However, the reported elevation of endocannabinoids
in CSF of schizophrenics
38
reflects the overall
metabolism of brain, rather than region-specific
alteration. To understand the overall status of the
endocannabinoidergic system in depression and
other psychiatric illnesses, the study on endocanna-
binoid levels in different brain regions is essential
and such studies are currently underway.
Regulation of receptor sensitization and desensiti-
zation is a complex phenomenon. The consequence of
elevated CB
1
receptor-mediated response observed in
this study is not known. The hyperactivity of
cannabinoidergic signaling could be an adaptive
feedback in response to the decreased levels of
endocannabinoids. The mechanism, physiological
role and regulation of endocannabinoidergic system
are yet to be understood. Recently, it has been shown
that endocannabinoids are involved in retrograde
signaling, and CB
1
receptor activation suppresses
neurotransmitter release by inhibiting a calcium-
dependent step in vesicle release,
41
thus decreasing
the local release of synaptic vesicles
42–44
However, we
cannot rule out increased endocannabinoid levels,
which, if combined with observed hyperactivity of
CB
1
receptor-mediated signaling, and hence elevated
retrograde cannabinoidergic neurotransmission in the
pathophysiology of depression or suicidal behavior.
In summary, the upregulation of CB
1
receptors with
concomitant increase in the CB
1
receptors-mediated
[
35
S]GTPgS binding strongly suggests a role for the
participation of abnormal endocannabinoidergic neu-
rotransmission in the etiology of depression and
suicide. The pharmacological manipulation of endo-
cannabinoid system may serve as a new therapeutic
target in the treatment of depression.
Acknowledgements
This study was supported by Grants AA13003 and
NARSAD independent investigator award (BLH);
AA09004 and MH40210 (VA); MH62185 (JJM). The
preliminary findings of this study were presented at
Neuroscience meeting. We thank Dr Veeranna, Centre
for Dementia Research, NKI, for his technical advice.
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