Endogenous and exogenous cannabinoids (CBs) acting through the CB1 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 CB1 receptors and mediated signaling in the dorsolateral prefrontal cortex (DLPFC) of subjects with major depression who had died by suicides (depressed suicides, DS). [3H]CP-55,940 and CB1 receptor-stimulated [35S]GTPγS binding sites were analyzed in membranes obtained from DLPFC of DS (10) and matched normal controls (10). Upregulation (24%, P<0.0001) of CB1 receptor density (Bmax) 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 CB1 receptors in DS (38%, P<0.001) was also demonstrated by Western blot analysis. The CB1 receptor-stimulated [35S]GTPγS binding was significantly greater (45%, P<0.001) in the DLPFC of DS compared with matched controls. The observed upregulation of CB1 receptors with concomitant increase in the CB1 receptor-mediated [35S]GTPγS 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.
Cannabinoid (CB) receptors received their name as those receptors that bind cannabinoidergic drugs, such as 9-tetrahydrocannabinol (Δ9-THC), derived from Cannabis sativa and its biologically active synthetic analogs. Δ9-THC is the major psychoactive component in marijuana extracts and can produce a multiplicity of effects in humans, including alterations in mood, perception, cognition and memory.1,2,3,4 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 characterizing 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, CB1 and CB2, have been identified7,8 and have been shown to belong to G-protein coupled receptor (GPCR) family. Two endogenous cannabimimetic substances, characterized to be N-arachidonyl ethanolamide (AEA/anandamide) and 2-arachidonylglycerol (2-AG), were discovered and shown to act as agonists for CB receptors.9,10 The CB1 receptor is distributed primarily in neural tissue, whereas CB2 receptor is expressed mainly in the peripheral immune system.11,12 These receptors exhibit seven transmembrane domains, linked to Gi/o protein to inhibit adenylyl cyclase.13 Interestingly, the CB1 receptor is one of the most abundant neuromodulatory receptors in the brain and is expressed predominantly in the cerebral cortex, hippocampus, cerebellum and basal ganglia.14,15,16
Numerous studies have implicated alterations in several receptors and G-protein function in the pathophysiology of various neurological and psychiatric disorders. Decreased CB1 receptor binding in neurodegenerative diseases related to extrapyramidal function has been reported.17 Alterations in the serotonergic (for a review see, Arango et al18) and adrenergic19 receptors in the pathophysiology of depression and suicidal behavior are well documented. Recently, Dean et al,20 reported increased CB1 receptor density in dorsolateral prefrontal cortex (DLPFC) in schizophrenia.
The increased levels of endocannabinoids and downregulation of CB1 receptor in response to chronic ethanol intake in mouse brain suggest modulation of the endocannabinoidergic system by alcohol21,22 (for a review, see Hungund et al23). The mood and cognition altering ability of exogenous cannabinoids and alcohol, and the association between depression, suicide and alcohol abuse raise the question whether endogenous cannabinoidergic system plays any role in the etiology of depression and suicidal behavior. Therefore, to address this question, we studied the density of CB1 receptors and CB1 receptor-mediated [35S]GTPγS 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 (−20°C) and stored at −80°C in tightly sealed, thick plastic bags until sectioning. After a control and suicide were matched, coronal sections (20 μm) from the hemicerebrum were taken from a level just anterior to the genu of the corpus callosum with a large format Leica Cryopolycut cryostat. Interleaved sections every 200 μm were sectioned at 50 μm 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 described.24 Tissue from Brodmann area 9 (∼1.5 g) was dissected frozen, the white matter was removed as much as possible, and tissue returned immediately to –80°C until membrane preparation.
The study was conducted with approval from the NYSPI Institutional Review Board. Dorsolateral prefrontal 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 comprised 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=1) and ethanol (n=3) were detected. Two individuals received lidocaine as part of resuscitation efforts at the emergency room. All cases were free of neuropathology. Both suicides and controls were examined psychiatrically by structured interviews with family members and/or close friends. The psychiatric diagnoses 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.
Brain tissue (∼1 g) was homogenized in 20 volumes of ice-cold TME buffer (50 mM Tris-HCl, 3 mM MgCl2 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 4°C. 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 –80°C until the assay.
Determination of protein content
The protein content of the membrane fraction was determined by Lowry's method26 using bovine serum albumin (BSA) as the standard. Protein content of the membrane, also normalized by silver staining, was used for Western blot analysis.
[3H]CP-55,940 binding assay
An aliquot of membrane (100 μg protein) was incubated with TME buffer, [3H]CP-55,940 (0.05–5.0 nM) and 0.1% fatty acid-free BSA in silicone-treated test tubes for 1 h at 37°C. The nonspecific binding of radioligand was defined by CP-55,940 (10 μM). 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.
CB1 receptor-stimulated [35S]GTPgS binding assay
The functional coupling between CB1 receptor and G-protein was assessed by [35S]GTPγS binding assay as described previously27 with minor modification. Briefly, an aliquot of membrane (50 μg protein) was incubated in assay buffer (TME buffer and 0.1% fatty acid-free BSA and 100 mM NaCl) containing GDP (40 μM), and [35S]GTPγS (0.05 nM) in silicone-treated test tubes for 1 h at 37°C. The CB1 receptor agonist, CP-55,940 (1 μM), was used to study CB1 receptor-stimulated [35S]GTPγS binding. The nonspecific binding of radioligand was determined in the presence of 10 μM GTPγS. The termination and filtration (without presoaking the filters in polyethyleneimine) of reaction mixture was performed as described for [3H]CP-55,940 binding assay. The radioactivity was measured by liquid scintillation spectroscopy at an efficiency of 95% for 35S.
Western blot analysis
Briefly, aliquots of membrane protein (30 μg), separated by 10% polyacrylamide gel, were electrophoretically 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-CB1 receptor antibody (1 : 500) overnight at 4°C. The blot was washed three times with TTBS and then incubated with alkaline phosphatase-conjugated anti-IgG for 1h 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 α-tubulin antibody to ensure equal protein loading.
Data and statistical analysis
The Bmax (maximal binding sites) and Kd (apparent dissociation constant) values were determined from saturation isotherms using nonlinear regression analysis to fit the data to the single-site binding equation (Prism; GraphPad software). The density and affinity of CB1 receptor was expressed as fmol/mg protein and nM, respectively. The CB1 receptor-stimulated [35S]GTPγS 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 parametric (paired Student ‘t’-test). Differences were considered to be significant at P<0.05. Immunoblots were analyzed using the NIH image software program. Data are expressed as mean±SEM from two to three experiments, each run in at least duplicate unless otherwise indicated.
[35S]GTPγS and [3H]CP-55,940 were purchased from DuPont NEN (Boston, MA, USA). Fatty acid-free BSA, protease inhibitor cocktail, GDP and GTPγS 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-CB1 receptor was obtained from Biosource Internationals (California, CA, USA). Anti-α-tubulin monoclonal antibody was from Amersham Bioscience (Piscataway, NJ, USA). Alkaline phosphatase-conjugated 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.
The density of CB1 receptor
A saturation analysis suggests that [3H]CP-55,940 binding is saturable below 5.0 nM concentration. The nonspecific binding was about 15% of total [3H]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 representative saturation isotherm and Scatchard plot is shown in Figure 1.
The average density (Bmax) of CB1 receptor in DLPFC of normal control subjects was 493.3±52.7 fmol/mg protein. The apparent dissociation constant (Kd) was 1.12±0.10 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 CB1 receptor was observed in the DLPFC of DS (644.6±48.8 fmol/mg protein; 24%, P<0.0001) compared with matched controls (Figure 2). However, there was no difference in the affinity of receptor for radioligand (DS; 1.14±0.08 vs control; 1.12±0.10 nM), suggesting an upregulation of the density of receptor in the absence of altered affinity of the receptor. A significant increase (38%, P<0.001) in CB1 receptor immunoreactivity was also demonstrated by Western blot analysis. A representative CB1 receptor immunoblot of a DS and matched control (3A) and levels of CB1 receptor immunoreactivity of all the subjects (3B) are shown in Figure 3.
CB1 receptor-stimulated [35S]GTPgS binding
The CB1 receptor-stimulated [35S]GTPγS binding was used to assess the coupling efficiency between a receptor and its G-protein. Using the CB1 receptor agonist, CP-55,940 stimulated-[35S]GTPγS binding as the outcome measure, maximum stimulation of [35S]GTPγS binding was observed when cortical membranes were incubated with 1 μM CP-55,940 and 40 μM of GDP (data not shown). The increase in CB1 receptor-stimulated [35S]GTPγS binding was 45% greater in cortical membranes of DS (31.1±4.7 fmol/mg protein; P<0.001) compared with matched controls (16.9±2.5 fmol/mg protein) (Figure 4). However, no significant group difference in basal [35S]GTPγS binding was observed.
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 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 abnormalities associated with depression is continuing.
Recent studies from our laboratory have suggested the participation of cannabinoidergic system in alcoholism and related behaviors.21,22,23 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 CB1 receptor density and coupling between these receptors and Gi-protein in DLPFC of DS subjects. CB1 receptor immunoblot analysis found more CB1 receptor protein immunoreactivity, substantiating the radioligand binding results.
Many transmembrane signaling processes of extracellular hormone and neurotransmitters are mediated by receptor interaction with heterotrimeric (α,β,γ) guanosine nucleotide binding proteins (G-protein). The receptor activation alters the conformation of G-proteins leading to the exchange of GDP by GTP on Gα-subunit. The conformational change promotes the dissociation of G-protein into active Gα-GTP and Gβγ-subunits.30 These two subunits later regulate the activity of several effector molecules within the cell. Recently, a nonhydrolyzable GTP analogue, [35S]GTPγS, 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 CB1 receptor-stimulated [35S]GTPγS binding in DLPFC of DS compared to matched controls. The observed increase in [35S]GTPγS binding may be due to more CB1 receptors. Interestingly, we observed low percentage of stimulation of CB1 receptor-mediated [35S]GTPγS binding either in DS or matched controls. Despite the high density of CB1 receptors, the reason for low agonist-stimulated [35S]GTPγS binding is not known at this time. However, lower efficiency of CB1 receptor coupling to Gi-protein, compared to other GPCRs has been suggested.31 This is borne out from direct comparison between the efficacy of cannabinoids and opiates in which opiates signaling was found to be 20-fold more efficient than cannabinoid signaling.32 Therefore, it is speculated that the CB1 receptor signaling functions as a subtle, fine-tuning mechanism for cells. The high density of receptors makes the CB1 receptor highly sensitive to agonists; however, the poor coupling efficiency ensures that overactivation of the system will not occur.31
The consequence of elevated CB1 receptor-mediated signaling in the pathophysiology of depression is not known. Abnormalities in cAMP signaling in depressive disorders have been reported. Dowlatshahi et al,33 found decreased cAMP signaling in the brain of depressed suicides. The increased CB1 receptor density and its mediation in [35S]GTPγS binding suggest the sensitization of cannabinoidergic signaling, 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 alterations in many neurotransmitter receptors. Aged rats exhibited a marked decrease in CB1 receptors and its mediated [35S]GTPγS 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 responsible 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 suicidality is often associated with multiple depressive symptoms, future studies should test for the differences between suicide victims with a history of major depression and nondepressed suicides and or depressed and normal subjects who died by similar cause of death. The next question whether the observed abnormalities in DS victims are a consequence 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 palmitoylethanolamide, were shown to be increased in the cerebrospinal fluid (CSF) of schizophrenics.38 Increased CB1 receptor density in the DLPFC of schizophrenia has also been recently reported.20 It has been suggested that the clinical signs of chronic cannabis consumption may resemble negative symptoms of schizophrenia.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 CB1 receptor and mediated signaling may be a pathological consequence of depression and/or schizophrenia. However, the reported elevation of endocannabinoids in CSF of schizophrenics38 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 endocannabinoid levels in different brain regions is essential and such studies are currently underway.
Regulation of receptor sensitization and desensitization is a complex phenomenon. The consequence of elevated CB1 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 CB1 receptor activation suppresses neurotransmitter release by inhibiting a calcium-dependent step in vesicle release,41 thus decreasing the local release of synaptic vesicles42,43,44 However, we cannot rule out increased endocannabinoid levels, which, if combined with observed hyperactivity of CB1 receptor-mediated signaling, and hence elevated retrograde cannabinoidergic neurotransmission in the pathophysiology of depression or suicidal behavior.
In summary, the upregulation of CB1 receptors with concomitant increase in the CB1 receptors-mediated [35S]GTPγS binding strongly suggests a role for the participation of abnormal endocannabinoidergic neurotransmission in the etiology of depression and suicide. The pharmacological manipulation of endocannabinoid system may serve as a new therapeutic target in the treatment of depression.
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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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Cite this article
Hungund, B., Vinod, K., Kassir, S. et al. Upregulation of CB1 receptors and agonist-stimulated [35S]GTPγS binding in the prefrontal cortex of depressed suicide victims. Mol Psychiatry 9, 184–190 (2004). https://doi.org/10.1038/sj.mp.4001376
- CB1 receptor
- [35S]GTPγS binding
- prefrontal cortex
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