Ligand bias and inverse agonism on 5-HT2A receptor-mediated modulation of G protein activity in post-mortem human brain.

BACKGROUND AND PURPOSE: Whereas biased agonism on the 5-HT2A receptor has been ascribed to hallucinogenic properties of psychedelics, no information about biased inverse agonism on this receptor is available. In schizophrenia, increased 5-HT2A receptor constitutive activity has been suggested, highlighting the therapeutic relevance of inverse agonism. This study characterized the modulation of G protein activity promoted by different drugs, commonly considered as 5-HT2A receptor antagonists, in post-mortem human brain cortex. EXPERIMENTAL APPROACH: Modulation of [35S]GTPγS binding to different subtypes of Gα proteins exerted by different 5-HT2A receptor drugs was determined by scintillation proximity assays in brain from human, WT and 5-HT2A receptor KO mice. KEY RESULTS: MDL-11,939 was the only drug having no effect on the basal activity of 5-HT2A receptor. Altanserin and pimavanserin decreased basal activation of Gi1, but not Gq/11 proteins. This effect was blocked by MDL-11,939 and absent in 5-HT2A receptor KO mice. Volinanserin showed 5-HT2A receptor-mediated inverse agonism both on Gi1 and Gq/11 proteins. Ketanserin exhibited 5-HT2A receptor partial agonism exclusively on Gq/11 proteins. On the other hand, eplivanserin and nelotanserin displayed inverse agonism on Gq/11 and/or Gi1 proteins, which was insensitive to MDL-11,939 and was present in KO mice suggesting a role for another receptor. CONCLUSION AND IMPLICATIONS: The results reveal the existence of constitutively active 5-HT2A receptors in human pre-frontal cortex and demonstrate different pharmacological profiles of various 5-HT2A receptor drugs previously considered antagonists. These findings indicate that altanserin and pimavanserin possess biased inverse agonist profile towards 5-HT2A receptor activation of Gi1 proteins.

Effect of antipsychotic drugs on group II metabotropic glutamate receptor expression and epigenetic control in postmortem brains of schizophrenia subjects.

Antipsychotic-induced low availability of group II metabotropic glutamate receptors (including mGlu2R and mGlu3R) in brains of schizophrenia patients may explain the limited efficacy of mGlu2/3R ligands in clinical trials. Studies evaluating mGlu2/3R levels in well-designed, large postmortem brain cohorts are needed to address this issue. Postmortem samples from the dorsolateral prefrontal cortex of 96 schizophrenia subjects and matched controls were collected. Toxicological analyses identified cases who were (AP+) or were not (AP-) receiving antipsychotic treatment near the time of death. Protein and mRNA levels of mGlu2R and mGlu3R, as well as GRM2 and GRM3 promoter-attached histone posttranslational modifications, were quantified. Experimental animal models were used to compare with data obtained in human tissues. Compared to matched controls, schizophrenia cortical samples had lower mGlu2R protein amounts, regardless of antipsychotic medication. Downregulation of mGlu3R was observed in AP- schizophrenia subjects only. Greater predicted occupancy values of dopamine D2 and serotonin 5HT2A receptors correlated with higher density of mGlu3R, but not mGlu2R. Clozapine treatment and maternal immune activation in rodents mimicked the mGlu2R, but not mGlu3R regulation observed in schizophrenia brains. mGlu2R and mGlu3R mRNA levels, and the epigenetic control mechanisms did not parallel the alterations at the protein level, and in some groups correlated inversely. Insufficient cortical availability of mGlu2R and mGlu3R may be associated with schizophrenia. Antipsychotic treatment may normalize mGlu3R, but not mGlu2R protein levels. A model in which epigenetic feedback mechanisms controlling mGlu3R expression are activated to counterbalance mGluR loss of function is described.

The α-synuclein PET tracer [18F] ACI-12589 distinguishes multiple system atrophy from other neurodegenerative diseases.

A positron emission tomography (PET) tracer detecting α-synuclein pathology will improve the diagnosis, and ultimately the treatment of α-synuclein-related diseases. Here we show that the PET ligand, [18F]ACI-12589, displays good in vitro affinity and specificity for pathological α-synuclein in tissues from patients with different α-synuclein-related disorders including Parkinson's disease (PD) and Multiple-System Atrophy (MSA) using autoradiography and radiobinding techniques. In the initial clinical evaluation we include 23 participants with α-synuclein related disorders, 11 with other neurodegenerative disorders and eight controls. In vivo [18F]ACI-12589 demonstrates clear binding in the cerebellar white matter and middle cerebellar peduncles of MSA patients, regions known to be highly affected by α-synuclein pathology, but shows limited binding in PD. The binding statistically separates MSA patients from healthy controls and subjects with other neurodegenerative disorders, including other synucleinopathies. Our results indicate that α-synuclein pathology in MSA can be identified using [18F]ACI-12589 PET imaging, potentially improving the diagnostic work-up of MSA and allowing for detection of drug target engagement in vivo of novel α-synuclein targeting therapies.

Electroactive Materials Surface Charge Impacts Neuron Viability and Maturation in 2D Cultures.

Since neurons were first cultured outside a living organism more than a century ago, a number of experimental techniques for their in vitro maintenance have been developed. These methods have been further adapted and refined to study specific neurobiological processes under controlled experimental conditions. Despite their limitations, the simplicity and visual accessibility of 2D cultures have enabled the study of the effects of trophic factors, adhesion molecules, and biophysical stimuli on neuron function and morphology. Nevertheless, the impact of fundamental properties of the surfaces to which neurons adhere when cultured in vitro has not been sufficiently considered. Here, we used an electroactive polymer with different electric poling states leading to different surface charges to evaluate the impact of the net electric surface charge on the behavior of primary neurons. Average negative and positive surface charges promote increased metabolic activity and enhance the maturation of primary neurons, demonstrating the relevance of considering the composition and electric charge of the culture surfaces. These findings further pave the way for the development of novel therapeutic strategies for the regeneration of neural tissues, particularly based on dynamic surface charge variation that can be induced in the electroactive films through mechanical solicitation.

Synthesis and Preclinical Evaluation of [11C]AZ11895530 for PET Imaging of the Serotonin 1A Receptor.

The serotonin 1A (5-HT1A) receptor is a G-protein-coupled receptor implicated in the pathophysiology of several neuropsychiatric and neurodegenerative disorders. We here report the preparation of two candidate 5-HT1A radioligands, [11C]AZ11132132 ([11C]3) and [11C]AZ11895530 ([11C]4), and their subsequent evaluation in vitro using autoradiography and in vivo using positron emission tomography (PET). Compounds 3 and 4 were radiolabeled at high radiochemical purity (>99%) and high molar activity (>38 GBq/µmol) by heteroatom methylation with [11C]methyl iodide. Autoradiography on whole hemispheres from post-mortem human brain revealed substantial nonspecific binding of [11C]3, while the binding of [11C]4 to brain tissue was consistent with the distribution of 5-HT1A receptors and sensitive to co-incubation with the reference 5-HT1A antagonist WAY-100635 (10 µM). Following intravenous injection of [11C]4 into a cynomolgus monkey, brain radioactivity concentration (Cmax ∼ 2.2 SUV) was high whereafter it decreased rapidly. The regional binding potential (BPND) values were calculated using the simplified reference tissue model with cerebellum as reference region. The values varied between 0.2 and 1.0 for temporal cortex, raphe nuclei, frontal cortex, and hippocampus which is consistent with the known 5-HT1A expression pattern. After pretreatment with WAY100635 (0.5 mg/kg), a homogeneous distribution of radioactivity was observed in non-human primate (NHP) brain. Although [11C]4 fulfilled important criteria for successful in vivo neuroimaging, including good blood-brain-barrier permeability and high specific binding in vitro to human brain tissue, the regional BPND values for [11C]4 in NHP brain were low when compared to those obtained with existing radioligands and thus do not merit further investigation of [11C]4. Evaluation of structurally related analogues is underway in our laboratory to identify improved candidates for clinical imaging.

A Novel Dynamic Human In Vitro Model for Studying the Blood-Brain Barrier.

Constructing a reliable in vitro blood-brain barrier (BBB) model using human primary cells has been considered a major challenge during the past decades. These systems could provide valuable information regarding the effect of therapeutic compounds on different BBB cell types (endothelial cells, astrocytes, pericytes) and their ability to cross the barrier in order to reach the brain. Several attempts have been made to develop in vitro BBB models, but these studies mainly used rat, bovine, and porcine cells rather than human primary cells. Genetically modified cell lines have also been used, but they do not appear to maintain physiological properties of the BBB. Here, we describe a detailed protocol for co-culturing and maintaining human brain primary endothelial cells, pericytes, and astrocytes under flow to create an in vitro human BBB model, which can be used for toxicity testing and for studying cross-interaction among different cell types involved in the BBB formation.

Development of 11C-Labeled ASEM Analogues for the Detection of Neuronal Nicotinic Acetylcholine Receptors (α7-nAChR).

The homo-pentameric alpha 7 receptor is one of the major types of neuronal nicotinic acetylcholine receptors (α7-nAChRs) related to cognition, memory formation, and attention processing. The mapping of α7-nAChRs by PET pulls a lot of attention to realize the mechanism and development of CNS diseases such as AD, PD, and schizophrenia. Several PET radioligands have been explored for the detection of the α7-nAChR. 18F-ASEM is the most functional for in vivo quantification of α7-nAChRs in the human brain. The first aim of this study was to initially use results from in silico and machine learning techniques to prescreen and predict the binding energy and other properties of ASEM analogues and to interpret these properties in terms of atomic structures using 18F-ASEM as a lead structure, and second, to label some selected candidates with carbon-11/hydrogen-3 (11C/3H) and to evaluate the binding properties in vitro and in vivo using the labeled candidates. In silico predictions are obtained from perturbation free-energy calculations preceded by molecular docking, molecular dynamics, and metadynamics simulations. Machine learning techniques have been applied for the BBB and P-gp-binding properties. Six analogues of ASEM were labeled with 11C, and three of them were additionally labeled with 3H. Binding properties were further evaluated using autoradiography (ARG) and PET measurements in non-human primates (NHPs). Radiometabolites were measured in NHP plasma. All six compounds were successfully synthesized. Evaluation with ARG showed that 11C-Kln83 was preferably binding to the α7-nAChR. Competition studies showed that 80% of the total binding was displaced. Further ARG studies using 3H-KIn-83 replicated the preliminary results. In the NHP PET study, the distribution pattern of 11C-KIn-83 was similar to other α7 nAChR PET tracers. The brain uptake was relatively low and increased by the administration of tariquidar, indicating a substrate of P-gp. The ASEM blocking study showed that 11C-KIn-83 specifically binds to α7 nAChRs. Preliminary in vitro evaluation of KIn-83 by ARG with both 11C and 3H and in vivo evaluation in NHP showed favorable properties for selectively imaging α7-nAChRs, despite a relatively low brain uptake.

Differential brain ADRA2A and ADRA2C gene expression and epigenetic regulation in schizophrenia. Effect of antipsychotic drug treatment.

Postsynaptic α2A-adrenoceptor density is enhanced in the dorsolateral prefrontal cortex (DLPFC) of antipsychotic-treated schizophrenia subjects. This alteration might be due to transcriptional activation, and could be regulated by epigenetic mechanisms such as histone posttranslational modifications (PTMs). The aim of this study was to evaluate ADRA2A and ADRA2C gene expression (codifying for α2-adrenoceptor subtypes), and permissive and repressive histone PTMs at gene promoter regions in the DLPFC of subjects with schizophrenia and matched controls (n = 24 pairs). We studied the effect of antipsychotic (AP) treatment in AP-free (n = 12) and AP-treated (n = 12) subgroups of schizophrenia subjects and in rats acutely and chronically treated with typical and atypical antipsychotics. ADRA2A mRNA expression was selectively upregulated in AP-treated schizophrenia subjects (+93%) whereas ADRA2C mRNA expression was upregulated in all schizophrenia subjects (+53%) regardless of antipsychotic treatment. Acute and chronic clozapine treatment in rats did not alter brain cortex Adra2a mRNA expression but increased Adra2c mRNA expression. Both ADRA2A and ADRA2C promoter regions showed epigenetic modification by histone methylation and acetylation in human DLPFC. The upregulation of ADRA2A expression in AP-treated schizophrenia subjects might be related to observed bivalent chromatin at ADRA2A promoter region in schizophrenia (depicted by increased permissive H3K4me3 and repressive H3K27me3) and could be triggered by the enhanced H4K16ac at ADRA2A promoter. In conclusion, epigenetic predisposition differentially modulated ADRA2A and ADRA2C mRNA expression in DLPFC of schizophrenia subjects.

Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

Di-aryl guanidinium derivatives: Towards improved α2-Adrenergic affinity and antagonist activity.

Compounds with excellent receptor engagement displaying α2-AR antagonist activity are useful not only for therapeutic purposes (e.g. antidepressants), but also to help in the crystallization of this particular GPCR. Therefore, based on our broad experience in the topic, we have prepared eighteen di-aryl (phenyl and/or pyridin-2-yl) mono- or di-substituted guanidines and 2-aminoimidazolines. The in vitro α2-AR binding affinity experiments in human brain tissue showed the advantage of a 2-aminoimidazolinium cation, a di-arylmethylene core, a conformationally locked pyridin-2-yl-guanidine and a di-substituted guanidinium to achieve good α2-AR engagement. After different in vitro [35S]GTPγS binding experiments in human prefrontal cortex tissue, it was possible to identify that compounds 7a, 7b and 7c were α2-AR partial agonist, whereas 8h was a potent α2-AR antagonist. Docking and MD studies with a model of α2A-AR and two crystal structures suggest that antagonism is achieved by compounds carrying a di-substituted guanidine which substituent occupy a pocket adjacent to TM5 without engaging S2005.42 or S2045.46, and a mono-substituted cationic group, which favorably interacts with E942.65.

Identification and in vitro characterization of C05-01, a PBB3 derivative with improved affinity for alpha-synuclein.

The neuropathological hallmark of Parkinsons disease, multiple system atrophy and dementia with Lewy bodies is the accumulation of α-synuclein. The development of an imaging biomarker for α-synuclein is an unmet need. To date, no selective α-synuclein imaging agent has been identified, though initial studies suggest that the tau tracer [11C]PBB3 displays some degree of binding to α-synuclein. In this study, a series of compounds derived from the PBB3 scaffold were examined using fluorescence imaging and tissue microarrays (TMAs) derived from brain samples with different proteinopathies. One compound, C05-01, was selected based on its higher fluorescence signal associated with Lewy body aggregates compared with other PBB3 analogues. In vitro binding assays using human brain homogenates and recombinant fibrils indicated that C05-01 had higher affinity for α-synuclein (KD/Ki 25 nM for fibrils, Ki 3.5 nM for brain homogenates) as compared with PBB3 (KD 58 nM). In autoradiography (ARG) studies using fresh frozen human tissue and TMAs, [3H]C05-01 displayed specific binding in cases with α-synuclein pathology. C05-01 is the first PBB3 analogue developed as a potential compound targeting α-synuclein. Despite improved affinity for α-synuclein, C05-01 showed specific binding in AD tissue with Amyloid ß and tau pathology, as well as relatively high non-specific and off-target binding. Additional efforts are needed to optimize the pharmacological and physicochemical properties of this series of compounds as ligands for α-synuclein. This study also showed that the construction of TMAs from different proteinopathies provides a tool for evaluation of fluorescent or radiolabelled compounds binding to misfolded proteins.

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: Selective biased agonism via Gαi1-proteins.

Serotonin 5-HT2A receptors (5-HT2ARs) have been implicated in schizophrenia. However, postmortem studies on 5-HT2ARs expression and functionality in schizophrenia are scarce. The 5-HT2AR mRNA and immunoreactive protein expression were evaluated in postmortem tissue from dorsolateral prefrontal cortex (DLPFC) of antipsychotic-free (n = 18) and antipsychotic-treated (n = 9) subjects with schizophrenia, and matched controls (n = 27). Functional coupling of 5-HT2AR to G-proteins was tested by measuring the activation induced by the agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride ((±)DOI) in antibody-capture [35S]GTPγS scintillation proximity assays (SPA). In antipsychotic-free schizophrenia subjects, 5-HT2AR mRNA expression and protein immunoreactivity in total homogenates was similar to controls. In contrast, in antipsychotic-treated schizophrenia subjects, lower mRNA expression (60±9% vs controls) and a trend to reduced protein immunoreactivity (86±5% vs antipsychotic-free subjects) just in membrane-enriched fractions was observed. [35S]GTPγS SPA revealed a significant ~6% higher stimulation of Gαi1-protein by (±)DOI in schizophrenia, whereas activation of the canonical Gαq/11-protein pathway by (±)DOI remained unchanged. Expression of Gαi1- and Gαq/11-proteins did not differ between groups. Accordingly, in rats chronically treated with clozapine, but not with haloperidol, a 30-40% reduction was observed in 5-HT2AR mRNA expression, 5-HT2AR protein immunoreactivity and [3H]ketanserin binding in brain cortical membranes. Overall, the data suggest a supersensitive 5-HT2AR signaling through inhibitory Gαi1-proteins in schizophrenia. Together with previous results, a dysfunctional pro-hallucinogenic agonist-sensitive 5-HT2AR conformation in postmortem DLPFC of subjects with schizophrenia is proposed. Atypical antipsychotic treatment would contribute to counterbalance this 5-HT2AR supersensitivity by reducing receptor expression.

Synthesis, 3 H-labelling and in vitro evaluation of a substituted dipiperidine alcohol as a potential ligand for chemokine receptor 2.

The immune system is implicated in the pathology of neurodegenerative disorders. The C-C chemokine receptor 2 (CCR2) is one of the key targets involved in the activation of the immune system. A suitable ligand for CCR2 could be a useful tool to study immune activation in central nervous system (CNS) disorders. Herein, we describe the synthesis, tritium radiolabelling, and preliminary in vitro evaluation in post-mortem human brain tissue of a known potent small molecule antagonist for CCR2. The preparation of a tritium-labelled analogue for the autoradiography (ARG) study gave rise to an intriguing and unexpected side reaction profile through a novel amination of ethanol and methanol in the presence of tritium. After successful preparation of the tritiated radioligand, in vitro ARG measurements on human brain sections revealed nonspecific binding properties of the selected antagonist in the CNS.

A novel dynamic multicellular co-culture system for studying individual blood-brain barrier cell types in brain diseases and cytotoxicity testing.

Blood brain barrier (BBB) cells play key roles in the physiology and pathology of the central nervous system (CNS). BBB dysfunction is implicated in many neurodegenerative diseases, including Alzheimer's disease (AD). The BBB consists of capillary endothelial cells, pericytes encircling the endothelium and surrounding astrocytes extending their processes towards it. Although there have been many attempts to develop in vitro BBB models, the complex interaction between these cell types makes it extremely difficult to determine their individual contribution to neurotoxicity in vivo. Thus, we developed and optimised an in vitro multicellular co-culture model within the Kirkstall Quasi Vivo System. The main aim was to determine the optimal environment to culture human brain primary endothelial cells, pericytes and astrocytes whilst maintaining cellular communication without formation of a barrier in order to assess the contribution of each cell type to the overall response. As a proof of concept for the present system, the effects of amyloid-beta 25-35 peptide (Aß25-35), a hallmark of AD, were explored. This multicellular system will be a valuable tool for future studies on the specific roles of individual BBB cell type (while making connection with each other through medium) in CNS disorders as well as in cytotoxicity tests.

Substituted conformationally restricted guanidine derivatives: Probing the α2-adrenoceptors' binding pocket.

In this paper we report the design, synthesis and pharmacological evaluation of new N-substituted 2-amino-1,4-dihydroquinazolines, 2-amino-1,4-dihydropyridopyrimidines and 2-amino-4,5-dihydro-1,3-benzodiazepines as α2-adrenoceptors ligands. Computational studies show that the proposed substitutions and guanidine-containing ring size will probe an extensive area of the active site. Preparation of these molecules involved novel routes than those previously utilised in our laboratory for the preparation of the acyclic aryl-guanidine counterparts. Compounds 8b and 18c showed the highest affinity and antagonistic activity, within their series, towards the α2-adrenoceptor in human brain tissue in vitro experiments. Structure-activity relationships have been established for the design and biological evaluation of novel α2-adrenoceptor ligands.

Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the Gq/11-coupled serotonin 5-HT2A receptor and the Gi/o-coupled metabotropic glutamate 2 (mGlu2) receptor-GPCRs that are involved in signaling alterations associated with psychosis-assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2-5-HT2A heteromers with an mGlu2 agonist led to activation of Gq/11 proteins by the 5-HT2A receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to Gi/o proteins, and we determined the relative location of the Gi/o-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of Gq/11, but not Gi/o, was reduced in the frontal cortex of 5-HT2A knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry.

Evaluation of 5-HT2A and mGlu2/3 receptors in postmortem prefrontal cortex of subjects with major depressive disorder: effect of antidepressant treatment.

Several studies have demonstrated alterations in serotonin 5-HT2A (5-HT2AR) and glutamate metabotropic mGlu2 (mGlu2R) receptors in depression, but never in the same sample population. Recently it has been shown that both receptors form a functional receptor heterocomplex that is altered in schizophrenia. The present study evaluates the gene expression and protein density of 5-HT2AR and mGlu2/3R in the postmortem prefrontal cortex of subjects with major depressive disorder (n = 14) compared with control subjects (n = 14) in a paired design. No significant differences between subjects with depression and controls in the relative mRNA levels of the genes HTR2A, GRM2 and GRM3 were observed. The 5-HT2AR density evaluated by [(3)H]ketanserin binding was significantly lower in antidepressant-treated subjects (Bmax = 313 ± 17 fmol/mg protein; p < 0.05) compared to controls (Bmax = 360 ± 12 fmol/mg protein) but not in antidepressant-free subjects (Bmax = 394 ± 16 fmol/mg protein; p > 0.05). In rats, chronic treatment with citalopram (10 mg/kg/day) and mirtazapine (5 mg/kg/day) decreased mRNA expression and 5-HT2AR density whereas reboxetine (20 mg/kg/day) modified only mRNA expression. The mGlu2/3R density evaluated by [(3)H]LY341495 binding was not significantly different between depression and control subjects. The present results demonstrate no changes in expression and density of both 5-HT2AR and mGlu2/3R in the postmortem prefrontal cortex of subjects with major depressive disorder under basal conditions. However, antidepressant treatment induces a decrease in 5-HT2AR density. This finding suggests that 5-HT2AR down-regulation may be a mechanism for antidepressant effect.