Luis F. Callado

Identification of a serotonin/glutamate receptor complex implicated in psychosis

The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception. Some antipsychotic drugs were identified by their high affinity for serotonin 5-HT2A receptors (2AR). Drugs that interact with metabotropic glutamate receptors (mGluR) also have potential for the treatment of schizophrenia. The effects of hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide, require the 2AR and resemble some of the core symptoms of schizophrenia. Here we show that the mGluR2 interacts through specific transmembrane helix domains with the 2AR, a member of an unrelated G-protein-coupled receptor family, to form functional complexes in brain cortex. The 2AR–mGluR2 complex triggers unique cellular responses when targeted by hallucinogenic drugs, and activation of mGluR2 abolishes hallucinogen-specific signalling and behavioural responses. In post-mortem human brain from untreated schizophrenic subjects, the 2AR is upregulated and the mGluR2 is downregulated, a pattern that could predispose to psychosis. These regulatory changes indicate that the 2AR–mGluR2 complex may be involved in the altered cortical processes of schizophrenia, and this complex is therefore a promising new target for the treatment of psychosis.

HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.

Histone deacetylases (HDACs) compact chromatin structure and repress gene transcription. In schizophrenia, clinical studies demonstrate that HDAC inhibitors are efficacious when given in combination with atypical antipsychotics. However, the molecular mechanism that integrates a better response to antipsychotics with changes in chromatin structure remains unknown. Here we found that chronic atypical antipsychotics downregulated the transcription of metabotropic glutamate 2 receptor (mGlu2, also known as Grm2), an effect that was associated with decreased histone acetylation at its promoter in mouse and human frontal cortex. This epigenetic change occurred in concert with a serotonin 5-HT2A receptor–dependent upregulation and increased binding of HDAC2 to the mGlu2 promoter. Virally mediated overexpression of HDAC2 in frontal cortex decreased mGlu2 transcription and its electrophysiological properties, thereby increasing psychosis-like behavior. Conversely, HDAC inhibitors prevented the repressive histone modifications induced at the mGlu2 promoter by atypical antipsychotics, and augmented their therapeutic-like effects. These observations support the view of HDAC2 as a promising new target for schizophrenia treatment.

Effectiveness of pindolol plus serotonin uptake inhibitors in depression: a meta-analysis of early and late outcomes from randomised controlled trials

BACKGROUND Contradictory results on the efficacy of pindolol associated with selective serotonin reuptake inhibitors (SSRIs) in depressive illness have been published and no former review has produced an overall figure of its efficacy. This study aims to review the efficacy and tolerability of pindolol plus SSRIs in depressive illness. METHODS A meta-analysis of randomised controlled trials (RCTs) comparing pindolol plus SSRIs with placebo plus SSRIs. RESULTS Nine RCTs met inclusion criteria. Outcome favoured pindolol at 2 weeks time (N=5; OR=2.8; 95% CI 1.4-5.7), but not at four to 6 weeks (N=7; OR=1.4; 95% CI 0.8-2.7). Results for early outcome studies were robust to sensitivity analysis. Nineteen more studies, averaging null results, would be needed to change the overall probability (P=0.0001) to a non-significant figure. CONCLUSIONS Pindolol seems to hasten the response to SSRIs in depression with a timing window circumscribed to the first weeks of treatment.

Selective Increase of α2A‐Adrenoceptor Agonist Binding Sites in Brains of Depressed Suicide Victims

Abstract: The α2A‐ and α2C‐adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [3H]RX821002 (2‐[3H]methoxyidazoxan) suggested the presence of α2A‐adrenoceptors in the frontal cortex and both α2C‐adrenoceptors and α2A‐adrenoceptors in the caudate. The proportions in caudate were similar in controls (α2A, 86%; α2C, 14%), depressed suicides (α2A, 91%; α2C, 9%), and suicides with other diagnoses (α2A, 88%; α2C, 12%). Autoradiography of [3H]RX821002 binding under α2B/C‐adrenoceptor‐masking conditions confirmed the similar densities of α2A‐adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [3H]RX821002 binding by (−)‐adrenaline revealed a greater proportion (61 ± 9%) of α2A‐adrenoceptors in the high‐affinity conformation for agonists than in controls (39 ± 5%). Simultaneous analysis with the agonists [3H]clonidine and [3H]UK14304 and the antagonist [3H]RX821002 in the same depressed suicides confirmed the enhanced α2A‐adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high‐affinity conformation of the brain α2A‐adrenoceptors.

Immunodensity and mRNA expression of A2A adenosine, D2 dopamine, and CB1 cannabinoid receptors in postmortem frontal cortex of subjects with schizophrenia: effect of antipsychotic treatment

RationaleDopamine D2 receptors are the main target of antipsychotic drugs. In the brain, D2 receptors coexpress with adenosine A2A and CB1 cannabinoid receptors, leading to functional interactions.ObjectivesThe protein and messenger RNA (mRNA) contents of A2A, D2, and CB1 receptors were quantified in postmortem prefrontal cortex of subjects with schizophrenia.Materials and methodsThe study was performed in subjects suffering schizophrenia (n = 31) who mainly died by suicide, matched with non-schizophrenia suicide victims (n = 13) and non-suicide controls (n = 33). The density of receptor proteins was evaluated by immunodetection techniques, and their relative mRNA expression was quantified by quantitative real-time polymerase chain reaction.ResultsIn schizophrenia, the densities of A2A (90 ± 6%, n = 24) and D2-like receptors (95 ± 5%, n = 22) did not differ from those in controls (100%). Antipsychotic treatment did not induce changes in the protein expression. In contrast, the immunodensity of CB1 receptors was significantly decreased (71 ± 7%, n = 11; p < 0.05) in antipsychotic-treated subjects with schizophrenia but not in drug-free subjects (104 ± 13%, n = 11). The relative mRNA amounts encoding for A2A, D2, and CB1 receptors were similar in brains of drug-free, antipsychotic-treated subjects with schizophrenia and controls.ConclusionsThe findings suggest that antipsychotics induce down-regulation of CB1 receptors in brain. Since A2A, D2, and CB1 receptors coexpress on brain GABAergic neurons and reductions in markers of GABA neurotransmission have been identified in schizophrenia, a lower density of CB1 receptor induced by antipsychotics could represent an adaptative mechanism that reduces the endocannabinoid-mediated suppression of GABA release, contributing to the normalization of cognitive functions in the disorder.

Identification of Three Residues Essential for 5-Hydroxytryptamine 2A-Metabotropic Glutamate 2 (5-HT2A·mGlu2) Receptor Heteromerization and Its Psychoactive Behavioral Function

Background: The 5-HT2A·mGlu2 receptor heterocomplex is involved in psychosis. Results: Substitution of Ala-6774.40, Ala-6814.44, and Ala-6854.48 in mGlu2 abolishes the behavioral effects of hallucinogenic 5-HT2A agonists. Conclusion: Three residues at transmembrane domain 4 of mGlu2 are necessary to form the 5-HT2A·mGlu2 receptor heterocomplex. Significance: These results provide insight into the structure and behavioral function of the 5-HT2A·mGlu2 receptor heterocomplex. Serotonin and glutamate G protein-coupled receptor (GPCR) neurotransmission affects cognition and perception in humans and rodents. GPCRs are capable of forming heteromeric complexes that differentially alter cell signaling, but the role of this structural arrangement in modulating behavior remains unknown. Here, we identified three residues located at the intracellular end of transmembrane domain four that are necessary for the metabotropic glutamate 2 (mGlu2) receptor to be assembled as a GPCR heteromer with the serotonin 5-hydroxytryptamine 2A (5-HT2A) receptor in the mouse frontal cortex. Substitution of these residues (Ala-6774.40, Ala-6814.44, and Ala-6854.48) leads to absence of 5-HT2A·mGlu2 receptor complex formation, an effect that is associated with a decrease in their heteromeric ligand binding interaction. Disruption of heteromeric expression with mGlu2 attenuates the psychosis-like effects induced in mice by hallucinogenic 5-HT2A agonists. Furthermore, the ligand binding interaction between the components of the 5-HT2A·mGlu2 receptor heterocomplex is up-regulated in the frontal cortex of schizophrenic subjects as compared with controls. Together, these findings provide structural evidence for the unique behavioral function of a GPCR heteromer.

Comparison of the effects of alpha-methyl-p-tyrosine and a tyrosine-free amino acid load on extracellular noradrenaline in the rat hippocampus in vivo.

Peripheral administration of an amino acid load lacking tyrosine and its precursor, phenylalanine, causes a lowering of central tyrosine levels. The aim of the present study was to examine the effects of tyrosine depletion on extracellular noradrenaline using microdialysis. Extracellular noradrenaline was measured in hippocampus of the anaesthetized rat under both baseline conditions (with reuptake inhibitor, desipramine, in the perfusion medium) and following administration of the a2-adrenoreceptor antagonist, idazoxan. The tyrosine free amino acid load did not alter either baseline noradrenaline or the twofold rise in noradrenaline evoked by idazoxan compared with saline controls. In contrast, the catecholamine synthesis inhibitor, a-methyl-p-tyrosine, caused a marked reduction in baseline extracellular noradrenaline and abolished the rise induced by idazoxan. In conclusion, the present data indicate that under the conditions used, a tyrosine-free amino acid mixture may not be an effective means to interfere with central noradrenaline function. This contrasts with recent findings demonstrating that the tyrosine-depletion approach can be used to decrease presynaptic dopamine function.

Non-adrenoceptor [3H]idazoxan binding sites (I2-imidazoline sites) are increased in postmortem brain from patients with Alzheimer's disease ☆

The I2-imidazoline site (a non-adrenergic mitochondrial site for which a glial location has been proposed and that is associated with the B-form of the enzyme monoamine oxidase) was evaluated in postmortem cortical membranes from 9 subjects with Alzheimers disease (AD) and 9 matched-controls by using [3H]idazoxan (0.6-30 nM) in the presence of 10(-6) M (-)-adrenaline to prevent binding to alpha 2-adrenoceptors. In AD the density (Bmax) of I2-imidazoline sites was significantly higher (+63%) than in controls whereas no differences were apparent in affinity values (Kd). The results support the hypothesis that the I2 imidazoline site has a major location on glial (astrocyte) cells.

Characterization of CB1 cannabinoid receptor immunoreactivity in postmortem human brain homogenates

The CB1 cannabinoid receptor (CB1) is the predominant type of cannabinoid receptor in the CNS, in which it displays a unique anatomical distribution and is present at higher densities than most other known seven transmembrane domain receptors. Nevertheless, as with almost all seven transmembrane domain receptors, the tertiary and quaternary structure of this receptor is still unknown. Studies of CB1 in rat cerebral tissue are scarce, and even less is known regarding the expression of CB1 in the human brain. Thus, the aim of the present work was to characterize CB1 expression in membranes from postmortem human brain using specific antisera raised against this protein. Western blot analysis of P1 and P2 fractions, and crude plasma membrane preparations from the prefrontal cortex showed that CB1 migrated as a 60 kDa monomer under reducing conditions. These data were confirmed by blotting experiments carried out with human U373MG astrocytoma cells as a positive control for CB1 expression and wild-type CHO cells as negative control. In addition, when proteins were solubilized in the absence of dithiothreitol, the anti-human CB1 antiserum detected a new band migrating at around 120 kDa corresponding in size to a putative CB1 dimer. This band was sensitive to reducing agents (50 mM dithiothreitol) and showed sodium dodecylsulphate stability, suggesting the existence of disulfide-linked CB1 dimers in the membrane preparations. Important differences in the anatomical distribution of CB1 were observed with regard to that described previously in monkey and rat; in the human brain, CB1 levels were higher in cortex and caudate than in the cerebellum.

α2-Adrenoceptor subtypes in the human brain: a pharmacological delineation of [3H]RX-821002 binding to membranes and tissue sections

In order to study the characterization and localization of [3H]RX-821002 (2-methoxy-idazoxan) binding to alpha 2-adrenoceptor subtypes in several regions of the human brain, we have carried out competition studies using both autoradiography and membrane binding assays. The alpha 2A-adrenoceptor subtype was found to be predominant in the different layers of the frontal cortex, cerebellum and hippocampal formation, while in the neostriatum it was the non-alpha 2A- (alpha 2B- and alpha 2C-) adrenoceptor subtype. In the frontal cortex, in addition to binding to the alpha 2A-adrenoceptor subtype, [3H]RX-821002 bound also to a small portion of alpha 2B- and alpha 2C-adrenoceptors in layer III, and to an unidentified binding site in the external layers. In the hippocampus, both alpha 2A- and non-alpha 2A- (alpha 2B- and alpha 2C-) adrenoceptors were labelled in the dentate gyrus and the CA1 field, together with 5-HT1A receptors. 5-HT1A receptors were labelled predominantly in the stratum pyramidale layer. These results, in addition to delineate the relative presence of alpha 2-adrenoceptor subtypes, indicate that caution is needed when analyzing RX 821002 binding to human brain tissue.