ERPKB

BrtwMocRgAChs

Kc

S(aSnpaas(tbbaist

haC

F

A

R

0d

ARTICLE IN PRESS

levated Levels of Endocannabinoids and CB1

eceptor-Mediated G-Protein Signaling in therefrontal Cortex of Alcoholic Suicide Victims

. Yaragudri Vinod, Victoria Arango, Shan Xie, Suham A. Kassir, J. John Mann, Thomas B. Cooper, andasalingappa L. Hungund

ackground: Alcoholism is often comorbid with mood disorders and suicide. We recently reported an upregulation of CB1

eceptor-mediated signaling in the dorsolateral prefrontal cortex (DLPFC) of subjects with major depression who died by suicide. Inhe present study, we sought to determine whether the changes in depressed suicides would also be present in alcoholic suicides andhether the endocannabinoid (EC) system plays a role in suicide in alcoholism.ethods: The density of CB1 receptor and its mediated [35S]GTP�S signaling were measured in membranes obtained from the DLPFC

f alcoholic suicides (AS) (n � 11) and chronic alcoholics (CA) (n � 11). The levels of ECs were measured by a liquidhromatograph/mass spectrometry (LC-MS) method in the DLPFC of these subjects.esults: The CB1 receptor density was higher in AS compared with the CA group in the DLPFC. Western blot analysis confirmed areater immunoreactivity of the CB1 receptor in AS. The CB1 receptor-mediated [35S]GTP�S binding indicated a greater signaling inS. Higher levels of N-arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG) were observed in the DLPFC of AS.onclusions: The elevated levels of ECs, CB1 receptors, and CB1 receptor-mediated [35S]GTP�S binding strongly suggest ayperactivity of endocannabinoidergic signaling in AS. The EC system may be a novel therapeutic target for the treatment of

uicidal behavior.

ey Words: Alcoholism, suicide, human prefrontal cortex, endo-annabinoids, CB1 receptor, G-protein

everal natural lipids have emerged as candidate modulatorsof nervous system functions. For example, two putativeendocannabinoids (ECs), N-arachidonyl ethanolamide

AEA) and 2-arachidonylglycerol (2-AG), were shown to act asgonists for cannabinoid (CB) receptors (Devane et al 1992;ugiura et al 1995), giving rise to the concept of an endocan-abinoid system. The CB receptor family consists of two G-rotein coupled receptors (GPCRs), CB1 and CB2. CB1 receptorsre localized primarily in neural tissue, whereas CB2 receptorsre located peripherally and are associated with the immuneystem. CB1 receptors are known to inhibit adenylyl cyclase (AC)Howlett 1995; Childers and Breivogel 1998a) via Gi-protein andhey are the most abundant neuromodulatory receptors in therain, expressed in the cortex, hippocampus, cerebellum, andasal ganglia (Herkenham et al 1991; Glass et al 1997; Childersnd Breivogel 1998b). High levels of ECs have also been foundn the cortex, basal ganglia, and limbic structures, and this mayuggest involvement of the endocannabinoid system in cogni-ive, emotional, and motivational functions (Bisogno et al 1999).

Alterations in the monoaminergic neurotransmitter systemsave long been implicated in the pathophysiology of depressionnd more recently in suicide (Arango et al 1990, 1995, 2002;allado et al 1998; Gonzalez-Maeso et al 2002). Lower seroto-

rom the Department of Analytical Psychopharmacology (KYV, SX, TBC,BLH), Nathan Kline Institute for Psychiatric Research, Orangeburg, NewYork; and Departments of Analytical Psychopharmacology (KYV, TBC,BLH) and Neuroscience (VA, SAK, JJM), New York State Psychiatric Insti-tute, and Department of Psychiatry (VA, JJM, BLH), College of Physiciansand Surgeons, Columbia University, New York, New York.

ddress reprint requests to Basalingappa L. Hungund, Ph.D., Nathan Kline Insti-tute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY10962; E-mail: Hungund@nki.rfmh.org.

eceived July 8, 2004; revised November 17, 2004; accepted November 19,

2004.

006-3223/05/$30.00oi:10.1016/j.biopsych.2004.11.033

nergic activity may, to some degree, mediate genetic and devel-opmental changes in suicide, aggression, mood, and alcoholism(for reviews, see Mann 1998, 2003). In recent years, the endo-cannabinoid system has been slowly emerging as a target ofpsychiatric research, and alterations in CB1 receptor levels inneurodegenerative diseases (Glass et al 1993; Westlake et al1994) and schizophrenia (Dean et al 2001) have been reported.Recently, we reported elevated CB1 receptor-mediated signalingin the dorsolateral prefrontal cortex (DLPFC) of depressed sui-cide victims (Hungund et al 2004). Our earlier animal studieshave also suggested the participation of the EC system inalcoholism (for a review, see Hungund et al 2002).

Clinical evidence suggests that alcoholism and affective dis-orders are independently associated with suicide behavior (Roy2000; Preuss et al 2002). Alcoholism and other substance usedisorders are related to more aggression and impulsivity, alsorisk factors for suicide (Rossow and Amundson 1995; Rich et al1998; Koller et al 2002). It has been suggested that chronicalcoholics are at 60 to 120 times higher risk for suicide than thegeneral population, and alcoholism may contribute to about 25%of all suicides (Murphy and Wetzel 1990). Alcoholism andattempted suicide are familial, suggesting a role for genes andrearing (Potash et al 2000), although the responsible genesremain to be identified. An association between the CB1 receptorgene and attention-deficit/hyperactivity disorder (ADHD) in al-coholics has also been recently reported (Ponce et al 2003).However, cellular mechanisms by which alcohol and the endo-cannabinoid system produce behavioral changes in humansremain to be elucidated.

The modulation of the EC system by alcohol and thefrequency of suicidal behavior in alcoholism raises the ques-tion whether the EC system plays a role in alcoholism orsuicidal behavior per se. In this study, we have analyzed thedensity of CB1 receptor, CB1 receptor-mediated G-proteinsignaling and EC levels in the DLPFC, and CB1 receptor-mediated G-protein signaling in the occipital cortex (OC) ofalcoholic suicide victims and chronic alcoholic control sub-

jects dying from other causes.

BIOL PSYCHIATRY 2005;xx:xxx© 2005 Society of Biological Psychiatry

M

S

oDIuCtYtgwipwwwscpwidd

o[acw

T

G

12345678911M12345678911M

a); CA,

c e.

2 BIOL PSYCHIATRY 2005;xx:xxx K.Y. Vinod et al

w

ARTICLE IN PRESS

ethods and Materials

ubjects, Tissue Collection, and DissectionBrain samples of prefrontal cortex and occipital cortex were

btained from autopsy material in the Brain Collection of theepartment of Neuroscience at the New York State Psychiatric

nstitute (NYSPI) and Columbia University, New York. All tissuesed in this study was provided by the Allegheny Countyoroner in accordance with protocols approved by the Institu-

ional Review Board of the University of Pittsburgh, the Nework State Psychiatric Institute, and Columbia University. Cortical

issues from Brodmann area 9 and visual association cortex (1.5each) were dissected frozen. The meninges and white matterere removed as much as possible, and tissues were returned

mmediately to �80°C until membrane preparation. Brain sam-les from 11 chronic alcoholics (CA) (age range: 16–70 years),ho died from causes other than suicide, were studied togetherith a matched group of 11 subjects of alcoholic suicides (AS)ho had a lifetime diagnosis of alcohol dependence and died by

uicide (age range: 20–61 years). The coroner determined theause of death and made the verdict of suicide. There were nineairs of Caucasians and two pairs of African Americans. Thereere no significant differences in age, sex, and postmortem

nterval (PMI) distribution between AS and CA subjects. Theemographic variables, such as sex, age, PMI, and cause ofeath, as well as toxicology, are summarized in Table 1.

Toxicological analyses were performed on all cases, rulingut recent consumption of substances of abuse (except alcohol

EtOH]) or psychoactive drugs (an antiepileptic and a benzodi-zapine were detected in two cases [CA � 1 and AS � 1]). Allases were free of neuropathology. Both AS and CA subjects

able 1. Characteristics of Alcoholic Suicide and Chronic Alcoholic Subject

roup Sex (M/F) Age (Years)

. CA M 16

. CA M 17

. CA M 18

. CA M 24

. CA F 30

. CA F 35

. CA F 38

. CA F 58

. CA M 640. CA M 671. CA M 70ean � SD 39.7 � 21.2

. AS M 20

. AS M 20

. AS M 29

. AS M 31

. AS F 28

. AS F 39

. AS F 32

. AS F 41

. AS M 420. AS M 581. AS M 61ean � SD 36.5 � 13.5

The data are presented as mean and SD. No statistical significance was ocross the groups.

M, male; F, female; PMI, postmortem interval (rounded to nearest hoursarbon monoxide; AE, antiepileptic; BZ, benzodiazapine; AS, alcoholic suicid

ere characterized psychiatrically by interviews with family

ww.elsevier.com/locate/biopsych

members and/or close friends using our previously validatedpsychological autopsy method and a structured clinical interviewfor Axis I and Axis II diagnoses according to DSM-III-R criteria(Kelly and Mann 1996). Psychological autopsies confirmed thatall the subjects had a diagnosis of alcohol dependence or abuse.One of the suicides had Axis I diagnoses of major depression andschizoaffective disorder but was currently in remission. None of thesubjects had comorbid nonalcohol substance use disorders. Thesamples were coded to mask investigators to group assignment.

Membrane PreparationBrain tissue (�1 g) was homogenized in 20 volumes of ice-cold

TME buffer (50 mM Tris-HCl, 3 mM magnesium chloride [MgCl2],and 1 mM of ethylenediaminetetraacetic acid [EDTA], pH 7.4)containing .32 M sucrose and a freshly added protease inhibitorcocktail. The homogenate was centrifuged at 1000g for 10 minutesat 4oC. The resulting supernatant was then centrifuged at 22,000g for20 minutes. The pellet was dissolved in TME buffer and recentri-fuged at 22,000g for 20 minutes. The final pellet was dissolved inTME buffer. Aliquots of membrane fractions were made and storedat �80oC until assay. The protein content of the membrane fractionwas determined by Lowry et al (1951) method using bovine serumalbumin (BSA) as the standard.

[3H]CP-55,940 Binding AssayAn aliquot of membrane (100 �g protein) was incubated with

TME buffer, [3H]CP-55,940 (.05–5.0 nm), and .1% fatty acid freeBSA in silicone-treated test tubes for 1 hour at 37oC. Thenonspecific binding of radioligand was defined by CP-55,940 (10�m). The reaction was terminated by the addition of 2 mLice-cold termination buffer (.1% BSA in 50 mM Tris-HCl, pH 7.4).

I (hours) Cause of Death Toxicology

09 MVA, passenger None12 MVA, passenger None13 Homicide, stabbing None09 Accident, drowning CO13 Natural, cardiovascular None13 Natural, cancer None20 Homicide, GSW None09 MVA, passenger None12 Natural, cardiovascular None09 Natural, hemorrhage None08 Accident, fall from height AE, BZ

.6 � 3.412 Suicide, GSW None24 Suicide, GSW None22 Suicide, GSW None09 Suicide, GSW CO04 Suicide, hanging AE, BZ13 Suicide, hanging None11 Suicide, poisoning None13 Suicide, hanging CO12 Suicide, GSW None12 Suicide, hanging None17 Suicide, GSW None

.4 � 5.6

ed between age (t � .43, df � 20, p � .67) and PMI (t � .95, df � 20, p � .35)

chronic alcoholic; MVA, motor vehicle accident; GSW, gunshot wound; CO,

s

PM

11

13

bserv

The reaction mixture was rapidly filtered through polyethylenei-

mpwf(imeaff

C

haal(ctrCwgatTp

A

pwpfi(humc

F(at

K.Y. Vinod et al BIOL PSYCHIATRY 2005;xx:xxx 3

ARTICLE IN PRESS

ine (.1%) pretreated glass fiber filters using a Brandel 24-osition cell harvester (Brandel, Gaithersburg, Maryland). Filtersere washed three times with the termination buffer and trans-

erred to scintillation vials containing 5 mL of scintillation cocktailICN Biochemicals, Costa Mesa, California). The vials werencubated overnight at room temperature. The radioactivity waseasured by liquid scintillation spectroscopy (Beckman) at an

fficiency of 47% for tritium. The Bmax (maximal binding sites)nd Kd (apparent dissociation constant) values were determinedrom saturation isotherms using nonlinear regression analysis toit the data to the single binding equation.

B1 Receptor Immunoblot AnalysisImmunoblot analysis was used to confirm the presence of

igher CB1 receptor protein in the DLPFC of AS victims. Anliquot of membrane protein (30 �g) separated by 12% poly-crylamide gel was electrophoretically transferred to nitrocellu-ose membrane. The membrane was treated with blocking bufferTTBS [10 mM Tris, .9% sodium chloride [NaCl]; 1% Tween 20ontaining 3% milk powder] of pH 7.4) for 1 hour at roomemperature. The membrane was incubated with human anti-CB1

eceptor antibody (1:500, Biosource International, Camarillo,alifornia) overnight at 4oC. The blot was washed three timesith TTBS and then incubated with alkaline phosphatase conju-ated anti-immunoglobulin G (IgG) for 1 hour at room temper-ture. After washing the blot for three to four times with TTBS,he immunoreactive band was visualized by CDP-Star reagent.he blot was reprobed with �-tubulin antibody to ensure equalrotein loading.

gonist-Stimulated [35S]GTP�S Binding AssayThe CB1 receptor-mediated [35S]GTP�S binding assay was

erformed as described previously (Gonzalez-Maeso et al 2000)ith minor modification. Briefly, an aliquot of membrane (50 �grotein) was preincubated in assay buffer (TME buffer and .1%atty acid free BSA and 100 mM NaCl) containing GDP (40 �m)n silicone-treated test tubes for 15 minutes. Later, [35S]GTP�S.05 nM) was added to the reaction mixture and incubated for 1our at 37oC. The CB1 receptor agonist, CP-55,940 (1 �m), wassed to study CB1 receptor-mediated [35S]GTP�S binding. Aaximum stimulation of [35S]GTP�S binding was achieved when

igure 1. Scatchard transformation of saturation binding of [3H]CP-55,940.5–5.0 nm) to the prefrontal cortical membranes of a AS (alcoholic suicide)nd CA (chronic alcoholic) subjects. The inset represents the Scatchardransformation of the same binding data.

ortical membranes were incubated with 1 �m CP-55,940 and 40

�m of GDP. The basal activity was estimated in absence ofCP-55,940. To determine the specificity of CB1 receptor-stimu-lated [35S]GTP�S binding, a concentration-response effect in thepresence of the CB1 receptor antagonist SR141716A was alsostudied. The nonspecific binding of radioligand was determinedin the presence of 10 �m GTP�S. The termination and filtration(without presoaking the filters in polyethyleneimine) of reactionmixture was performed as described for [3H]CP-55,940 bindingassay. The radioactivity was measured by liquid scintillationspectroscopy at an efficiency of 95% for 35S.

Measurement of EC Levels by Gas Chromatography/MassSpectrometry

Endocannaboid levels were measured by a liquid chromato-graph/mass spectrometry (LC-MS) method using the isotopicdilution procedure as described (Di Marzo et al 2000) with minormodifications. Briefly, brain tissue was homogenized in 4 mL ofchloroform-methanol-Tris buffer (2:1:1, pH 7.4) containing .25mM phenylmethylsulfonyl fluoride (PMSF), 20 �L of 1% buty-lated hydroxytoluene (BHT), .05 mL of AEA-d8 (50 ng), and .050mL of 2-AG-d8 (500 ng). The homogenate was centrifuged andthe organic layer removed and taken to dryness under nitrogen.The residue was dissolved in .3 mL of ethyl acetate and recen-trifuged. The supernatant was transferred to another tube anddried down under nitrogen. The residue was redissolved in ethylalcohol (40 �L) and transferred to a vial for the measurement ofAEA and 2-AG by LC-MS (Agilent 1100 series mass LC-MSD). Theseparation was achieved on a Supelcosil LC-8 column (25 cm x4.6 mm, 5 mm) using methanol-ammonium acetate-acetic acid(85:15:.05) as a mobile phase. The standard curves were fittedwith a quadratic equation with the curve encompassing a rangeof .5–50 and 50–1500 ng for AEA and 2-AG, respectively, andwere processed similarly with quality controls with each batch ofsamples.

Statistical AnalysesStatistical analysis was performed by using GraphPad Prism

(GraphPad software, San Diego, California). Experiment in each

Figure 2. The density of CB1 receptor was estimated using [3H]CP-55,940 inthe prefrontal cortical membranes of AS (alcoholic suicide, n � 11) and CA

www.elsevier.com/locate/biopsych

(chronic alcoholic, n� 11) subjects. **Significantly higher than that of CAbj t (t 3 59 df 18 � 001)

sAbiPdluv

R

TI

bnAtaaofps(daC(taACac

C

iac

Ft (chro*

4 BIOL PSYCHIATRY 2005;xx:xxx K.Y. Vinod et al

w

ARTICLE IN PRESS

ample was run in at least duplicate unless otherwise indicated.ll values presented are mean and standard deviation. Immuno-lots were analyzed using the National Institutes of Health (NIH)mage software program. The demographic variables (age andMI) were compared between CA and AS subjects with indepen-ent t tests. Differences in binding parameters, protein, and ECevels between CA and AS groups as a whole were analyzedsing independent-sample unpaired t test with two-tailed palues considered significant at p � .05.

esults

he Density of CB1 Receptor: Radioligand Binding andmmunoblot Analyses

The saturation binding analysis suggests that [3H]CP-55,940inding sites were saturable at 5.0 nm concentration (Figure 1). Theonspecific binding was 10% to 18% of total [3H]CP-55,940 binding.Scatchard analysis demonstrates high affinity (apparent dissocia-

ion constant [Kd] was 1.3 � .3 nm), saturable, monophasic binding,nd a Hill coefficient of near unity indicates a single class of receptort the concentrations used. Significantly higher (39%) density (Bmax)f the CB1 receptor was observed in the DLPFC of AS (820.1 � 139.2mol/mg protein) compared with CA (590.2 � 91.7 fmol/mgrotein) (t � 3.59, df � 18, p � .0021) (Figure 2). There was noignificant difference in the affinity of receptor between the groupst� 1.59, df� 18, p� .12), indicating the upregulation of receptorensity may not be due to its altered affinity. Western blot analysislso revealed a significantly higher (67%, t� 2.66, df� 20, p� .014)B1 receptor immunoreactivity in the DLPFC of AS than CA subjectsFigure 3B). Furthermore, significant positive correlations betweenhe Bmax (radioligand binding) and protein expression (immunore-ctivity) of the CB1 receptor were observed in both CA (r� .94) andS (r� .85) groups. A representative CB1 receptor immunoblot of aA and matched AS is provided in Figure 3A. The Western blotnalysis clearly demonstrated single immunoreactivity band, whichorresponds to the relative molecular weight of the CB1 receptor.

B1 Receptor-Mediated [35S]GTP�S BindingCB1 receptor agonist, CP-55,940-stimulated [35S]GTP�S bind-

ng assay was performed in membranes isolated from the DLPFCnd the OC of CA and AS subjects to asses the functional

igure 3. The Western blot analysis for CB1 receptor. A representative immuhe prefrontal cortical membranes of AS (alcoholic suicide, n � 11) and CASignificantly higher than that of CA subjects (t � 2.66, df � 20, p � .01).

oupling between CB1 receptor and G-protein. Greater stimula-

ww.elsevier.com/locate/biopsych

tion (34%; t � 5.38, df � 20, p � .0001) of CB1 receptor-mediated[35S]GTP�S binding was observed in cortical membranes of AS(102.1 � 10.5 fmol/mg protein) compared with CA (76.4 � 11.8fmol/mg protein) subjects (Figure 4). However, this parameterwas found to be unaltered (t� 1.30, df� 20, p� .20) in the OC ofAS victims (58.9 � 8.8) when compared with CA group (54.2 �8.1) (Figure 5). The pharmacological specificity of the CB1

receptor-mediated [35S]GTP�S binding was tested by CB1 recep-tor antagonist, SR SR141716A. One-site competition analysisindicated a concentration-dependent inhibition of CP-55,940stimulated [35S]GTP�S binding by SR141716A with a medianeffective concentration (EC50) of 11.9 nm (Figure 6).

t of the CB1 receptor (A) and levels of CB1 receptor immunoreactivity (B) innic alcoholic; n � 11) subjects expressed in arbitrary densitometric units.

Figure 4. The CB1 receptor-mediated [35S]GTP�S binding was greater in theprefrontal cortical membranes of AS (alcoholic suicide, n� 11) comparedwith CA (chronic alcoholic, n� 11) subjects. Data are presented as percent-

noblo

age of stimulation over the basal. ***Significantly higher than that of CA

L

Dpn(apc

D

mv(sabsiaiepDsma

F1(s

K.Y. Vinod et al BIOL PSYCHIATRY 2005;xx:xxx 5

ARTICLE IN PRESS

evels of EndocannabinoidsLevels of both ECs, AEA and 2-AG, were measured in the

LPFC of AS and CA subjects. The levels of AEA (98.4 � 26.4mol/gm tissue) were significantly lower than 2-AG (2.20 � .80mol/gm tissue) in the DLPFC of CA subjects. Levels of both AEA79%, 176.8 � 42.5 pmol/gm tissue, t� 5.18, df� 20, p� .0001)nd 2-AG (47%, 3.24 � .94 nmol/gm tissue, t� 2.75, df� 20,�.012) were significantly higher in the DLPFC of AS whenompared with CA subjects (Figure 7).

iscussion

We previously reported higher levels of CB1 receptors and itsediated G-protein signaling in the DLPFC of depressed suicideictims compared with nondepressed nonsuicide normal subjectsHungund et al 2004). Postmortem studies of brains of depresseduicide victims, however, cannot address whether the observedlterations are due to the presence of major depression or suicidalehavior. Therefore, to further address this question, the presenttudy was designed to investigate the prefrontal cortical EC systemn alcoholics, comparing alcoholics who committed suicide tolcoholics who died by other causes. The results of this studyndicating enhanced EC function in relation to suicide and consid-ring our recent finding in depressed suicides (Hungund et al 2004)rovides further evidence linking a hyperactive EC system in theLPFC to suicide in both mood disorder and in alcoholism. The

tudy reveals elevated levels of CB1 receptors in prefrontal corticalembranes of AS as compared with CA. In addition, immunoblot

igure 5. The CB1 receptor-mediated [35S]GTP�S binding was unaltered (t�.30, df� 20, p � .20) in membranes isolated from the occipital cortex of AS

alcoholic suicide, n� 11) compared with CA (chronic alcoholic, n � 11)ubjects. Data are presented as percentage of stimulation over the basal.

nalysis demonstrated a higher immunoreactivity for CB1 receptor

in the DLPFC of AS, corroborating the results of radioligand bindingassay. The observed upregulation of CB1 receptors may reflecteither increased rate of synthesis or decreased degradation ofreceptors but is not due to altered affinity of the receptors, as Kd

values are not different between groups.G-protein coupled receptors play an important role in various

cellular functions. Several studies have shown an abnormalreceptor-G-protein coupling in the brain of suicide victims(Gurguis et al 1997; Gonzalez-Maeso et al 2000; Dwivedi et al2002). In the present study, [35S]GTP�S was employed to assesthe coupling efficiency of CB1 receptors and G-proteins incortical membranes. We observed enhanced CB1 receptor-medi-ated [35S]GTP�S binding in the DLPFC of AS compared with CAsubjects. The pharmacological specificity of the CB1 receptor-mediated [35S]GTP�S binding was confirmed by the CB1 receptorantagonist, SR SR141716A. Enhanced agonist-stimulated G-pro-tein signaling does not appear to be due to altered affinitybetween the receptor and G-protein, as basal [35S]GTP�S bindingwas similar in both the groups. It could be due to the observedhigh density of CB1 receptors in the DLPFC of AS.

The consequence of elevated levels of CB1 receptors in theDLPFC of AS observed in this study is not known. Upregulation ofCB1 receptors due to the feedback or a homeostatic response to lowlevels of ECs is not the explanation, because we observed elevatedlevels of both AEA and 2-AG in the DLPFC of AS. Elevated levels ofboth ECs (Leweke et al 1999; De Marchi et al 2003) and CB1

receptors have also been found in schizophrenic patients (Dean etal 2001), indicating that this pattern of abnormality may not beunique to suicide. However, it is not clear whether the alterationsreported in these studies were associated with suicidal behavior orwith schizophrenia. Delta-9tetrahydrocannabinol, a primary constit-uent of marijuana, which is a CB1 receptor agonist and mimics theaction of ECs, appears to increase certain forms of impulsivebehaviors in humans (McDonald et al 2003). The high levels ofECs/CB1 receptor may facilitate impulsive behavior, which is char-acteristic of chronic alcoholics and depressed subjects with suicidalbehavior. Impulsivity is thought to be part of a diathesis for suicidaland aggressive behaviors, increasing the probability of acting onsuch feelings (for a review, see Mann et al 1999).

The regulatory mechanisms governing the EC system remain

Figure 6. To determine the specificity of CB1 receptor-stimulated[35S]GTP�S binding, a concentration-response effect in the presence of theCB1 receptor antagonist SR141716A was studied in the cortical membranesof CA (chronic alcoholic) subjects. Inhibition of CP-55,940-stimulated[35S]GTP�S binding by SR141716A was concentration-dependent with an

EC50 of 11.9 nm. EC50, median effective concentration.

www.elsevier.com/locate/biopsych

lirabeEahtrfs

ptarcecbestbl

P(Etbta(2dwoasH

Fh � .00( dony

6 BIOL PSYCHIATRY 2005;xx:xxx K.Y. Vinod et al

w

ARTICLE IN PRESS

argely unknown. Neuroanatomical and electrophysiological stud-es of the mammalian nervous system have revealed that the CB1

eceptors are located in presynaptic terminals of neurons (Kreitzernd Regehr 2001). Fatty acid amide hydrolase (FAAH), a membraneound serine hydrolase that inactivates ECs, appears to be prefer-ntially located in the somatodendritic compartments of neurons.ndocannabinoids are released on demand by stimulated neuronsnd activate presynaptic CB1 receptors. Based on these findings, itas been suggested that ECs are synthesized by postsynaptic cellshat act as retrograde messengers to modulate neurotransmitterelease (Kreitzer and Regehr 2001; Wilson and Nicoll 2002). There-ore, there may be elevated retrograde signaling mediated by the ECystem in the DLPFC of suicide victims.

The relevance of impaired cannabinoidergic signaling to theathophysiology of suicide remains to be elucidated. The sensitiza-ion of cannabinoidergic signaling could lead to decreased cyclicdenosine monophosphate (cAMP) content of the cell, as theseeceptors are negatively coupled to AC, resulting in the reducedAMP dependent protein kinase A (PKA) and cAMP responselement binding protein (CREB) activities. A reduced activity ofAMP/PKA and CREB signaling in the cortex of suicide brain haseen reported (Dowlatshahi et al 1999; Reiach et al 1999; Dwivedit al 2002, 2003). Many biological responses are regulated by thetate of phosphorylation of specific proteins, which in turn affectshe regulation of cellular functions. Thus, a hyperactivity of canna-inoidergic signaling may result in impairments in various physio-ogical functions that manifest in suicidal behavior.

Several studies have highlighted the effect of age, sex, andMI on the levels and function of receptors and G-proteinsDowlatshahi et al 1999) and also the effect of PMI on the levelsCs (Schmid et al 1995). However, the brain samples analyzed inhis study were closely matched with regard to sex, age, ethnicackground, and PMI, and no significant group differences inhese demographic variables were observed. Suicide is associ-ted with several psychiatric disorders and also drugs of abuseSuominen et al 1996; Rich et al 1998; Kessler et al 1999; Garlow002). However, all the subjects included in this study had aiagnosis of alcoholism and no drug of abuse other than EtOHas detected postmortem, and yet the elevation in CB1 receptorsccurred only in the suicide group. Whether the observedlterations in the DLPFC of AS is due to the pathophysiology ofuicide or psychotropic medication is of particular relevance.

igure 7. Levels of AEA (A) and 2-AG (B) were estimated in the DLPFC of AS (aigher in the levels of AEA in AS than that of CA subjects (t� 5.18, df� 20, p

t� 2.75, df� 20, p � .01). AEA, N-arachidonyl ethanolamide; 2-AG, 2-arachi

owever, our toxicological analyses revealed that only two cases

ww.elsevier.com/locate/biopsych

(one from each group) had medications (antiepileptic and ben-zodiazapine) at the time of death and no other psychoactivedrugs were detected in remaining subjects. Therefore, thepresent findings in brains of AS are unlikely to be the result ofshort-term drug effects and more likely to be related to thepathophysiology of suicide. In the present study, we included CAas a psychiatric control group for AS instead of normal controlgroups. This enabled us to identify the biochemical alterations inthe brain of alcoholic suicide victims with greater specificity.Alteration of CB1 receptor-mediated G-protein signaling seems tobe region-specific because the CB1 receptor-mediated[35S]GTP�S signaling was found to be unaltered in the OC of ASvictims. This may be due to unaltered density of CB1 receptor inthe OC of AS victims. Future studies are needed to examine thecomponents of EC system in the human postmortem brains ofalcoholics, alcoholic suicides, and normal control subjects inmore brain regions of interest to further understanding the role ofEC system in the neurobiology of alcoholism and suicide.

In conclusion, convergence of three different approachespointing to the upregulation of CB1 receptors taken together withhigher levels of ECs strongly suggests that hyperactivity of the ECsignaling in the DLPFC may be a potential factor in suicide inalcoholism. The present study corroborates our previous obser-vations and further elucidates the association between hyperac-tivity of EC system and suicide. Pharmacological agents thatmodulate EC function would be of interest in the potentialdevelopment of treatment for suicidal behavior.

This study was supported by Grants AA13003 and NARSADindependent investigator award (BLH); AA09004 and MH40210(VA); MH62185 (JJM); and AFSP Young Investigator Grant (KYV).

We thank Dr. Veeranna, Center for Dementia Research NKI,for his technical advice.

Arango V, Ernsberger P, Marzuk PM, Chen JS, Tierney H, Stanley M, et al(1990): Autoradiographic demonstration of increased serotonin 5-HT2

and beta-adrenergic receptor binding sites in the brain of suicide vic-tims. Arch Gen Psychiatry 47:1038 –1047.

Arango V, Underwood MD, Gubbi AV, Mann JJ (1995): Localized alterationsin pre- and postsynaptic serotonin binding sites in the ventrolateralprefrontal cortex of suicide victims. Brain Res 688:121–133.

Arango V, Underwood MD, Mann JJ (2002): Serotonin brain circuits involved

lic suicide, n � 11) and CA (chronic alcoholic, n� 11) subjects. aSignificantly01). bSignificantly higher in the levels of 2-AG in AS than that of CA subjectslglycerol; DLPFC, dorsolateral prefrontal cortex.

lcoho

in major depression and suicide. Prog Brain Res 136:443– 453.

B

C

C

C

D

D

D

D

D

D

D

D

G

G

G

G

G

G

H

H

H

K.Y. Vinod et al BIOL PSYCHIATRY 2005;xx:xxx 7

ARTICLE IN PRESS

isogno T, Berrendero F, Ambrosino G, Cebeira M, Ramos JA, Fernandez-Ruiz JJ, et al (1999): Brain regional distribution of endocannabinoids:Implications for their biosynthesis and biological function. Biochem Bio-phys Res Commun 256:377–380.

allado LF, Meana JJ, Grijalba B, Pazos A, Sastre M, Garcia-Sevilla JA (1998):Selective increase of alpha2A-adrenoceptor agonist binding sites inbrains of depressed suicide victims. J Neurochem 70:1114 –1123.

hilders SR, Breivogel CS (1998a): Cannabis and endogenous cannabinoidsystems. Drug Alcohol Depend 51:173–187.

hilders SR, Breivogel CS (1998b): The functional neuroanatomy of braincannabinoid receptors. Neurobiol Dis 5:417– 431.

e Marchi N, De Petrocellis L, Orlando P, Daniele F, Fezza F, Di Marzo V(2003): Endocannabinoid signalling in the blood of patients with schizo-phrenia. Lipids Health Dis 19:2–5.

ean B, Sundram S, Bradbury R, Scarr E, Copolov D (2001): Studies on[3H]CP-55940 binding in the human central nervous system: Regionalspecific changes in density of cannabinoid-1 receptors associated withschizophrenia and cannabis use. Neuroscience 103:9 –15.

evane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, et al(1992): Isolation and structure of a brain constituent that binds to thecannabinoid receptor. Science 258:1946 –1949.

i Marzo V, Breivogel CS, Tao Q, Bridgen DT, Razdan RK, Zimmer AM, et al(2000): Levels, metabolism, and pharmacological activity of anandamidein CB1 cannabinoid receptor knockout mice: Evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain. J Neuro-chem 75:2434 –2444.

owlatshahi D, MacQueen GM, Wang JF, Reiach JS, Young LT (1999): GProtein-coupled cyclic AMP signaling in postmortem brain of subjectswith mood disorders: Effects of diagnosis, suicide, and treatment at thetime of death. J Neurochem 73:1121–1126.

wivedi Y, Conley RR, Roberts RC, Tamminga CA, Pandey GN (2002):[3H]cAMP binding sites and protein kinase A activity in the prefrontalcortex of suicide victims. Am J Psychiatry 159:66 –73.

wivedi Y, Rao JS, Rizavi HS, Kotowski J, Conley RR, Roberts RC, et al (2003):Abnormal expression and functional characteristics of cyclic adenosinemonophosphate response element binding protein in postmortembrain of suicide subject. Arch Gen Psychiatry 60:273–282.

wivedi Y, Rizavi HS, Conley RR., Roberts RC, Tamminga CA, Pandey GN(2002): mRNA and protein expression of selective alpha subunits of Gproteins are abnormal in prefrontal cortex of suicide victims. Neuropsy-chopharmacology 27:499 –517.

arlow SJ (2002): Age, gender, and ethnicity differences in patterns of co-caine and ethanol use preceding suicide. Am J Psychiatry 159:615– 619.

lass M, Dragunow M, Faull RL (1997): Cannabinoid receptors in the humanbrain: A detailed anatomical and quantitative autoradiographic study inthe fetal, neonatal and adult human brain. Neuroscience 77:299 –318.

lass M, Faull RL, Dragunow M (1993): A significant loss of cannabinoidreceptors in the substantia nigra in Huntington’s disease. Neuroscience56:523–527.

onzalez-Maeso J, Rodriguez-Puertas R, Gabilondo J, Meana JJ (2000): Char-acterization of receptor mediated [35S]GTP�S binding to cortical mem-brane from post-mortem human brain. Eur J Pharmacol 390:25–36.

onzalez-Maeso J, Rodriguez-Puertas R, Meana JJ, Garcia-Sevilla JA, GuimonJ (2002): Neurotransmitter receptor-mediated activation of G-proteins inthe brains of suicide victims with mood disorders: Selective supersensi-tivity of �2A-adrenoceptors. Mol Psychiatry 7:755–767.

urguis GN, Turkka J, George DT, Linnoila M (1997): Beta-adrenoreceptorcoupling to GS protein in alcohol dependence, panic disorder, and pa-tients with both conditions. Neuropsychopharmacology 16:69 –76.

erkenham M, Lynn AB, Johnson MR, Melvin LS, deCost BR, Rice KC (1991):Characterization and localization of cannabinoid receptors in rat brain; aquantitative in vitro autoradiographic study. J Neurosci 16:8057– 8066.

owlett AC (1995): Pharmacology of cannabinoid receptors. Annu Rev Phar-macol Toxicol 35:607– 634.

ungund BL, Basavarajappa BS, Vadasz C, Kunos G, Rodriguez de Fonseca F,

Colombo G, et al (2002): Ethanol, endocannabinoids, and the cannabi-noidergic signaling system. Alcohol Clin Exp Res 26:565–574.

Hungund BL, Vinod KY, Kassir SA, Basavarajappa BS, Yalamanchili R, CooperTB, et al (2004): Upregulation of CB1 receptors and agonist-stimulated[35S]GTP�S binding in the prefrontal cortex of depressed suicide victims.Mol Psychiatry 9:184 –190.

Kelly TM, Mann JJ (1996): Validity of DSM-III-R diagnosis by psychologicalautopsy: A comparison with antemortem diagnosis. Acta Psychiatr Scand94:337–343.

Kessler RC, Borges G, Walters EE (1999): Prevalence of risk factors for lifetimesuicide attempts in the National Comorbidity Survey. Arch Gen Psychiatry56:617– 626.

Koller G, Preuss UW, Bottlender M, Wenzel K, Soyka M (2002): Impulsivity andaggression as predictors of suicide attempts in alcoholics. Eur Arch Psy-chiatry Clin Neurosci 252:155–160.

Kreitzer AC, Regehr WG (2001): Retrograde inhibition of presynaptic calciuminflux by endogenous cannabinoids at excitatory synapses onto Pur-kinje cells. Neuron 29:717–727.

Leweke FM, Giuffrida A, Wurster U, Emrich HM, Piomelli D (1999): Elevatedaspects of the metabolism in schizophrenia. Neuroreport 10:1665–1667.

Lowry OH, Rosebrough NJ, Farr AG, Randall RJ (1951): Protein measurementwith folin phenol reagent. J Biol Chem 193:265–275.

Mann JJ (1998): The neurobiology of suicide. Nat Med 1:25–30.Mann JJ (2003): Neurobiology of suicidal behaviour. Nat Rev Neurosci 4:819 –

828.Mann JJ, Waternaux C, Haas GL, Malone KM (1999): Toward a clinical model

of suicidal behavior in psychiatric patients. Am J Psychiatry 156:181–189.McDonald J, Schleifer L, Richards JB, de Wit H (2003): Effects of THC on

behavioral measures of impulsivity in humans. Neuropsychopharmacol-ogy 28:1356 –1365.

Murphy GE, Wetzel RD (1990): The lifetime risk of suicide in alcoholism. ArchGen Psychiatry 47:383–392.

Ponce G, Hoenicka J, Rubio G, Ampuero I, Jimenez-Arriero MA, Rodriguez-Jimenez R, et al (2003): Association between cannabinoid receptor gene(CNR1) and childhood attention deficit/hyperactivity disorder in Spanishmale alcoholic patients. Mol Psychiatry 8:466 – 467.

Potash JB, Kane HS, Chiu YF, Simpson SG, McKinnon DF, McInnis MG, et al(2000): Attempted suicide and alcoholism in bipolar disorder: Clinicaland familial relationships. Am J Psychiatry 157:2048 –2050.

Preuss UW, Schuckit MA, Smith TL, Danko GR, Dasher AC, Hesselbrock MN, et al(2002): A comparison of alcohol-induced and independent depression inalcoholics with histories of suicide attempts. J Stud Alcohol 63:498–502.

Reiach JS, Li PP, Warsh JJ, Kish SJ, Young LT (1999): Reduced adenylyl cyclaseimmunolabeling and activity in postmortem temporal cortex of de-pressed suicide victims. J Affect Disord 56:141–151.

Rich CL, Dhossche DM, Ghani S, Isacsson G (1998): Suicide methods andpresence of intoxicating abusable substances: Some clinical and publichealth implications. Ann Clin Psychiatry 10:169 –175.

Rossow I, Amundsen A (1995): Alcohol abuse and suicide: A 40-year prospec-tive study of Norwegian conscripts. Addiction 90:685– 691.

Roy A (2000): Relation of family history of suicide to suicide attempts inalcoholics. Am J Psychiatry 157:2050 –2051.

Schmid PC, Krebsbach RJ, Perry SR, Dettmer TM, Maasson JL, Schmid HH (1995):Occurrence and postmortem generation of anandamide and other long-chain N-acylethanolamines in mammalian brain. FEBS Lett 375:117–120.

Sugiura T, Konda S, Sukagawa A, Nakane S, Shinoda A, Itoh K, et al (1995):2-Arachidonoylglycerol: A possible endogenous cannabinoid receptorligand in brain. Biochem Biophys Res Commun 215:89 –97.

Suominen K, Henriksson M, Suokas J, Isometsa E, Ostamo A, Lonnqvist J(1996): Mental disorders and comorbidity in attempted suicide. ActaPsychiatr Scand 94:234 –240.

Westlake TM, Howlett AC, Bonner TI, Matsuda LA, Herkenham M (1994):Cannabinoid receptor binding and messenger RNA expression in hu-man brain: An in vitro receptor autoradiography and in situ hybridiza-tion histochemistry study of normal aged and Alzheimer’s brains. Neu-roscience 63:637– 652.

Wilson RI, Nicoll RA (2002): Endocannabinoid signaling in the brain. Science

296:678 – 682.

www.elsevier.com/locate/biopsych