[35S]GTPγS (Guanosine-5'-O-(γ-thio)triphosphate-[35S]) Binding Scintillation Proximity Assay Experiments in Postmortem Brain Tissue.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are the very first effector in signal transduction events triggered by G-protein-coupled receptors (GPCRs). One of the most widely used approaches for determining GPCR activity in native tissue is based on the binding of [35S]GTPγS. Classically, an heterogeneous procedure including a filtration step has been used, but a modification of the protocol including an immunoprecipitation step has allowed the specific discrimination of the contribution of the different Gα subunit subtypes to the effect of each ligand. Nowadays, that the concept of functional selectivity has been demonstrated for several ligands and GPCRs, information obtained from this methodological approach will be very useful for broadening the knowledge of GPCRs signaling profiles and describing the effect of different ligands over them. In this chapter we will describe the detailed protocol of antibody-capture [35S]GTPγS scintillation proximity assay (SPA) in order to provide the reader with comprehensive guidelines to study receptor-mediated functional activation of different Gα-protein subtypes in native mammalian brain membranes. In addition, advantages and limitations of this method will be described, as well as future direction in the application of this approach indicated.

FADD adaptor and PEA-15/ERK1/2 partners in major depression and schizophrenia postmortem brains: basal contents and effects of psychotropic treatments.

Enhanced brain apoptosis (neurons and glia) may be involved in major depression (MD) and schizophrenia (SZ), mainly through the activation of the intrinsic (mitochondrial) apoptotic pathway. In the extrinsic death pathway, pro-apoptotic Fas-associated death domain (FADD) adaptor and its non-apoptotic p-Ser194 FADD form have critical roles interacting with other death regulators such as phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) and extracellular signal-regulated kinase (ERK). The basal status of FADD (protein and messenger RNA (mRNA)) and the effects of psychotropic drugs (detected in blood/urine samples) were first assessed in postmortem prefrontal cortex of MD and SZ subjects (including a non-MD/SZ suicide group). In MD, p-FADD, but not total FADD (and mRNA), was increased (26%, n=24; all MD subjects) as well as p-FADD/FADD ratio (a pro-survival marker) in antidepressant-free MD subjects (50%, n=10). In contrast, cortical FADD (and mRNA), p-FADD, and p-FADD/FADD were not altered in SZ brains (n=21) regardless of antipsychotic medications (except enhanced mRNA in treated subjects). Similar negative results were quantified in the non-MD/SZ suicide group. In MD, the regulation of multifunctional PEA-15 (i.e., p-Ser116 PEA-15 blocks pro-apoptotic FADD and PEA-15 prevents pro-survival ERK action) and the modulation of p-ERK1/2 were also investigated. Cortical p-PEA-15 was not changed whereas PEA-15 was increased mainly in antidepressant-treated subjects (16-20%). Interestingly, cortical p-ERK1/2/ERK1/2 ratio was reduced (33%) in antidepressant-free when compared to antidepressant-treated MD subjects. The neurochemical adaptations of brain FADD (increased p-FADD and pro-survival p-FADD/FADD ratio), as well as its interaction with PEA-15, could play a major role to counteract the known activation of the mitochondrial apoptotic pathway in MD.

The inverse agonist effect of rimonabant on G protein activation is not mediated by the cannabinoid CB1 receptor: evidence from postmortem human brain.

Rimonabant (SR141716) was the first potent and selective cannabinoid CB1 receptor antagonist synthesized. Several data support that rimonabant behaves as an inverse agonist. Moreover, there is evidence suggesting that this inverse agonism may be CB1 receptor-independent. The aim of the present study was to elucidate whether the effect of rimonabant over G protein activation in postmortem human brain is CB1 dependent or independent. [(35)S]GTPγS binding assays and antibody-capture [(35)S]GTPγS scintillation proximity assays (SPA) were performed in human and mice brain. [(3)H]SR141716 binding characteristics were also studied. Rimonabant concentration-dependently decreased basal [(35)S]GTPγS binding to human cortical membranes. This effect did not change in the presence of either the CB1 receptor agonist WIN 55,212-2, the CB1 receptor neutral antagonist O-2050, or the CB1 allosteric modulator Org 27569. [(35)S]GTPγS binding assays performed in CB1 knockout mice brains revealed that rimonabant inhibited the [(35)S]GTPγS binding in the same manner as it did in wild-type mice. The SPA combined with the use of specific antibody-capture of G(α) specific subunits showed that rimonabant produces its inverse agonist effect through G(i3), G(o) and G(z) subtypes. This effect was not inhibited by the CB1 receptor antagonist O-2050. Finally, [(3)H]SR141716 binding assays in human cortical membranes demonstrated that rimonabant recognizes an additional binding site other than the CB1 receptor orthosteric binding site recognized by O-2050. This study provides new data demonstrating that at least the inverse agonist effect observed with >1µM concentrations of rimonabant in [(35)S]GTPγS binding assays is not mediated by the CB1 receptor in human brain.

Gene expression patterns in brain cortex of three different animal models of depression.

Animal models represent a very useful tool for the study of depressive-like behavior and for the evaluation of the therapeutic efficacy of antidepressants. Nevertheless, gene expression patterns of these different animal models and whether genes classically associated with human major depression are present in these genetic profiles remain unknown. Gene expression was evaluated in three animal models of depression: acute treatment with reserpine, olfactory bulbectomy and chronic treatment with corticosterone. Gene expression analysis was carried out using the Affymetrix GeneChip technology. The results were evaluated using the GeneChip Operating software (Gcos 1.3) and analyzed with the GeneSpring GX v7.3 bioinformatics software (Agilent) and dChip 2005 software. Expression changes were validated with quantitative real-time polymerase chain reaction (RT-PCR) assays. Many transcripts were differentially expressed in the cortex of depressed-like animals in each model. Gene ontology analysis showed that significant gene changes were clustered primarily into functional neurochemical pathways associated with apoptosis and neuronal differentiation. When expression profiles were compared among the three models, the number of transcripts differentially expressed decreased and only two transcripts (complement component 3 and fatty acid-binding protein 7) were differentially expressed in common. Both genes were validated with RT-PCR. Moreover, five (Htr2a, Ntrk3, Crhr1, Ntrk2 and Crh) of the genes classically related to human major depression were differentially expressed in at least one of these models. The different animal models of depression share relevant characteristics although gene expression patterns are different among them. Moreover, some of the classical genes related to human major depression are differentially expressed in these models.

Pharmacological characterization and autoradiographic distribution of alpha2-adrenoceptor antagonist [3H]RX 821002 binding sites in the chicken brain.

Knowledge about the noradrenergic system in birds is very scarce even though their biological diversity and complex social behavior make them an excellent model for studying neuronal functions and developmental biology. While the role of norepinephrine has been described in depth in a large number of central and peripheral functions in mammals, reports for avian species are limited. The radioligand [(3)H]RX 821002 ([(3)H]1,4-[6,7(n)3H]-benzodioxan-2-methoxy-2-yl)-2-imidazol) has been used to map and characterize alpha(2)-adrenoceptors through the chicken brain using in vitro autoradiography and membrane homogenates binding assays. [(3)H]RX 821002 showed a saturable and high affinity binding to a site compatible with alpha(2)-adrenoceptor, and to a serotonergic component. The autoradiographic assays displayed a similar alpha(2)-adrenoceptor distribution than those previously reported in birds using other radioligands such as [(3)H]UK 14304 ([(3)H]5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) or [(3)H]clonidine. [(3)H]RX 821002 binding pharmacological characterization was carried out in different chicken brain regions using membrane homogenates for competition assays with different alpha(2)-adrenoceptor agonists and antagonists drugs (oxymetazoline, BRL 44408 [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazole] ARC 239 [2-(2-4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione], prazosin, UK 14304 and RX 821002). The results showed alpha(2A) as the predominant alpha(2)-adrenoceptor subtype in the chicken brain while alpha(2B)- and/or alpha(2C)-adrenoceptor subtypes were detected only in the telencephalon. RX 821002, serotonin (5-HT) and 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] competition assays, and competition binding assays performed in the presence of serotonin demonstrated that [(3)H]RX 821002 binds with higher affinity to a serotonergic component, probably 5-HT(1A) receptors, than to the alpha(2)-adrenoceptors. Similar pharmacological properties for the alpha(2)-adrenoceptor component were observed both in rat and chicken brain. The results demonstrate that the different alpha(2)-adrenoceptor subtypes are present in chicken brain and suggest that these receptors are highly conserved through evolution.

Cannabinoid system in the budgerigar brain.

Cannabinoid receptor density and cannabinoid receptor-mediated G protein stimulation were studied by autoradiographic techniques throughout the budgerigar (Melopsittacus undulatus) brain. The maximal CB(1) receptor density value (using [(3)H]CP55,940 as radioligand) was found in the molecular layer of the cerebellum (Mol), and high binding values were observed in the nucleus taeniae amygdalae (TnA), nucleus preopticus medialis, and nucleus pretectalis. The highest net-stimulated [(35)S]GTPgammaS binding values induced by the selective CB(1) receptor agonist WIN55,212-2 were observed in the nucleus paramedianus internus thalami, and high values of [(35)S]GTPgammaS binding were observed in the TnA, Mol, arcopallium dorsale and arcopallium intermedium. The distribution data suggest that in the budgerigar, as previously indicated in mammals, cannabinoid receptors may be related to the control of several brain functions in the motor system, memory, visual system, and reproductive behavior. The discrepancies between the cannabinoid receptor densities and the cannabinoid receptor-mediated stimulation found in several budgerigar brain nuclei support the hypothesis, previously described for mammals, of the existence of different G(i/o) protein populations able to associate with the cannabinoid receptors, depending on the brain structure, and could reflect the relative importance that cannabinoid transmission could exerts in each cerebral area.

Norepinephrine, epinephrine and MHPG levels in chick brain development.

In this study we determined the norepinephrine (NE), epinephrine and methoxy-hydroxy-phenyl-glycol (MHPG) levels in dissected chick telencephalon, diencephalon/mesencephalon and cerebellum in a number of stages from the late embryonic period (E16, E17, E18 and E19) and post-hatching period (P1, P2, P3, P4, P5, P15 and P30) using HPLC coupled with a coulometric detection system. A mobile phase which permits the detection of NE, epinephrine and MHPG simultaneously is also described. During development, NE levels increase dramatically after hatching in all brain structures studied and are not correlated in the same period with an increase in the MHPG/NE ratio. The values obtained for epinephrine and MHPG were significantly lower than the NE values in all the structures and stages studied. Our results support the notion of a specific role for NE during the first days after hatching.