Agonistic properties of a series of psychotropic drugs at 5-HT1A receptors in rat and human brain membranes determined by [35S]GTPγS binding assay.

BACKGROUND: Many psychoactive compounds have been developed to have more beneficial clinical efficacy than conventional drugs by adding agonistic action at 5-HT1A receptors. The aim of the present study was to evaluate several psychotropic drugs that had been reported to behave as an agonist at 5-HT1A receptor (aripiprazole, brexpiprazole, asenapine, lurasidone, and vortioxetine) in both rat and postmortem human brain membranes. METHODS: The [35S]GTPγS binding assay for Gi/o proteins coupled with 5-HT1A receptors was performed in rat brain membranes and postmortem human brain membranes. RESULTS: The specific binding was stimulated by brexpiprazole in rat hippocampus, human hippocampus, and human prefrontal cortex. Aripiprazole also behaved as an agonist in the same brain regions. Interestingly, its potency was much higher in rat hippocampal membranes than in human brain membranes, indicating the possibility of species differences. Although vortioxetine was an efficacious stimulator at high concentrations, its potency was undeterminable because of a lack of saturability. In addition to 5-HT1A receptor agonism, involvement of other components, e.g., 5-HT1B receptor agonism, was speculated by the biphasic inhibitory effects of the selective 5-HT1A receptor neutral antagonist. Negligible stimulatory effects were obtained as to lurasidone and asenapine. CONCLUSIONS: Our previous studies have raised the concept of a psychoactive drug group with a common pharmacological mechanism of action, i.e., 5-HT1A receptor agonism, consisting of perospirone, aripiprazole, ziprasidone, clozapine, quetiapine, nemonapride, and trazodone. The present study demonstrates the data indicating that brexpiprazole and probably vortioxetine are included in this drug group. Lurasidone and asenapine are excluded from this group.

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.

5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated activation of Gαq/11 in postmortem dorsolateral prefrontal cortex of opiate addicts.

BACKGROUND: Chronic exposure to opiates causes the development of tolerance and physical dependence as well as persistent brain neuroplasticity. Despite a wealth of postmortem human studies for opiate addicts, little direct information regarding the functional status of serotonergic and cholinergic receptor-mediated signaling pathways in the human brain of opiate addicts is yet available. METHODS: Functional activation of Gαq/11 proteins coupled to 5-HT2A and M1 type muscarinic acetylcholine receptor (mAChR) was assessed by using the method named [35S]GTPγS binding/immunoprecipitation in frontal cortical membrane preparations from postmortem human brains obtained from opiate addicts and matched controls. RESULTS: Concentration-response curves for 5-HT and carbachol in individual subjects were analyzed according to a nonlinear regression model, which generated the values of maximum percent increase (%Emax), negative logarithm of the half-maximal effect (pEC50) and slope factor. As for 5-HT2A receptor-mediated Gαq/11 activation, the %Emax values were reduced significantly and the pEC50 values were decreased significantly in opiate addicts as compared to the control group. Regarding carbachol-induced Gαq/11 activation, no significant difference in %Emax or pEC50 values was detected between the both groups, whereas the slope factor was increased significantly in opiate addicts as compared to the control group. CONCLUSION: Our data demonstrate that the signaling pathways mediated by Gαq/11 proteins coupled with 5-HT2A receptors and M1 mAChRs in prefrontal cortex are functionally altered in opiate addicts in comparison with control subjects. These alterations may underpin some aspects of addictive behavior to opiate as well as neuropsychological consequences or comorbid mental disorders associated with opioid use.

5-HT2A receptor-mediated Gαq/11 activation in psychiatric disorders: A postmortem study.

OBJECTIVES: Serotonin-2A (5-HT2A) receptors play an important role in the regulation of many brain functions that are disturbed in patients with such psychiatric diseases as mood disorders and schizophrenia. The objective of this study was to evaluate 5-HT2A receptor-mediated signalling pathway through Gαq/11 activation in psychiatric patients by using post-mortem brain samples. METHODS: Functional activation of Gαq/11 proteins coupled to 5-HT2A receptors was determined by means of [35S]GTPγS binding/immunoprecipitation assay in post-mortem prefrontal cortex of psychiatric patients diagnosed as bipolar disorder (BP), major depressive disorder (MDD), and schizophrenia, and individually matched controls. The effects of antipsychotic treatment as well as suicide were also analysed. RESULTS: There was no significant difference in maximum percent increase (%Emax) or slope factor among the four groups. The negative logarithm of concentration eliciting the half-maximal effect (pEC50) was significantly reduced in BP and schizophrenia patients as compared to controls. These alterations were attributable to antipsychotic medication. The pEC50 values in 'non-suicide' group of schizophrenia, but not in 'suicide' group, were significantly reduced as compared with controls. CONCLUSIONS: Altered 5-HT2A receptor-mediated signalling pathway through Gαq/11 proteins in prefrontal cortex might be apparently involved in pathophysiology and pharmacotherapy of BP and schizophrenia. In schizophrenic patients, these alterations as a result of successful treatment with antipsychotic agents may help in prevention of suicidal behaviour.

Fundamental features of receptor-mediated Gαi/o activation in human prefrontal cortical membranes: A postmortem study.

To elucidate possible abnormalities in transmembrane signal transduction in psychiatric diseases, use of autopsy brain is a feasible approach. However, postmortem studies should be interpreted with caution concerning such factors as age, gender, psychotropic drug history, agonal state, postmortem delay (PMD), and storage period. In this study, agonist-induced [35S]GTPγS binding was performed in postmortem dorsolateral prefrontal cortical membranes of 40 control subjects. In addition to the previously reported G protein-coupled receptor (GPCR)-mediated Gi/o activation, κ-opioid receptor-mediated [35S]GTPγS binding was detected by using U-50,448. The responses elicited by 16 different agonists were determined, and the effects of several factors were investigated. Gender difference was negligible. Concentration-response curve of histamine H3 receptor-mediated [35S]GTPγS binding was shifted rightward in the subjects with some drugs detected at toxicological screening. Age-related alterations were minimal, except for the age-dependent supersensitivity of µ-opioid receptor-mediated Gαi/o activation, revealed by endomorphin-1- and DAMGO-stimulated [35S]GTPγS binding. Age-related increase in %Emax values was also detected as to DPDPE-induced [35S]GTPγS binding through δ-opioid receptors. With an exception of NOP receptor/G-protein coupling, GPCR-mediated [35S]GTPγS binding is relatively stable irrespective of PMD or storage period. There were many positive correlations among the %Emax values for different receptor subtypes, which might reflect formation of heterodimer complex of such GPCRs coupled to the same Gi/o proteins. These results provide us with important fundamental data in the future project using human postmortem brains from patients with psychiatric disorders.

Functional coupling of M1 muscarinic acetylcholine receptor to Gαq/11 in dorsolateral prefrontal cortex from patients with psychiatric disorders: a postmortem study.

Accumulating studies have implicated intracellular signaling through muscarinic acetylcholine receptors (mAChRs) in psychiatric illness. In the present study, carbamylcholine chloride (carbachol)-induced Gαi/o and Gαq/11 activation was identified in postmortem human prefrontal cortical membranes. The following two sample cohorts were used: subjects [1], consisting of 40 controls without neuropsychiatric disorders, and subjects [2], consisting of 20 with bipolar disorder (BP), 20 major depressive disorder (MDD), 20 schizophrenia, and 20 controls, strictly sex- and age-matched. Carbachol-stimulated [35S]GTPγS binding to human brain membranes was assessed by the two methods, i.e., conventional method using filtration techniques (Gαi/o activation coupled to M2/M4 mAChRs) applied to subjects [1], and [35S]GTPγS binding/immuno precipitation assay (Gαq/11 activation coupled to M1 mAChR) applied to subjects [1] and [2]. The concentration eliciting the half-maximal effect (EC50), maximum percent increase (%Emax), and slope factor were obtained from concentration-response curve of carbachol-induced Gαi/o and Gαq/11 activation. The pEC50 values of both carbachol-induced Gαi/o and Gαq/11 activations in subjects [1] were significantly correlated, though its implications or underlying molecular processes are unclear. The results of M1 mAChR-mediated Gαq/11 activation in subjects [2] indicated no significant disorder-specific alterations. However, the distribution patterns of the pEC50 values showed unequal variances among the groups. There was a significant inverse correlation between the %Emax values and the pEC50 values in subjects with schizophrenia, but not in those with BP or MDD, or controls. These data support the notion that schizophrenia patients consist of biologically heterogeneous subgroups with respect to M1 mAChR-mediated signaling pathways.

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes.

Although multiple roles of dopamine through D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via Gs/olf or Gi/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gαq activation in rat brain membranes was investigated. Agonist-induced Gαq activation was assessed by increase in guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding to Gαq determined by [35S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gαq functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D1-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D2-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D1-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT2A receptor, but not by dopamine D1-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D1-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT2A receptor has been mentioned.

Functional activation of Gαq/11 protein via α1 -adrenoceptor in rat cerebral cortical membranes.

Although it is recognized that α1 -adrenoceptors are coupled to diverse intracellular signalling pathways, its primary transduction mechanisms are evoked by activating phospholipase C in the cell membrane through Gαq/11 , resulting in production of inositol 1,4,5-trisphosphate and diacylglycerol. However, there have been few studies that indicate directly the involvement of Gαq/11 proteins in this signalling pathway in the central nervous system. In the current study, we tried to pharmacologically characterize (-)-adrenaline-stimulated [35 S]GTPγS binding to Gαq/11 in rat brain membranes. Functional activation of Gαq/11 coupled to α1 -adrenoceptor was investigated by using [35 S]GTPγS binding/immunoprecipitation assay in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. The specific [35 S]GTPγS binding to Gαq/11 was stimulated by (-)-adrenaline in a concentration-dependent and saturable manner in rat cerebral cortical membranes. In hippocampal or striatal membranes, the stimulatory effects of (-)-adrenaline were scarce. The effect of (-)-adrenaline was potently inhibited by prazosin, a potent and selective α1 -adrenoceptor antagonist, but not by yohimbine, a selective α2 -adrenoceptor antagonist. The response was mimicked by cirazoline, but not by R(-)-phenylephrine. Although oxymetazoline also stimulated the specific [35 S]GTPγS binding to Gαq/11 as an apparent "super-agonist", detailed pharmacological characterization revealed that its agonistic properties in this experimental system were derived from off-target effects on 5-HT2A receptors, but not via α1 -adrenoceptors. In conclusion, functional coupling of α1 -adrenoceptors to Gαq/11 proteins are detectable in rat brain membranes by means of [35 S]GTPγS binding/immunoprecipitation assay. It is necessary to interpret the experimental data with caution when oxymetazoline is included as an agonist at α1 -adrenoceptors.

Optimization and pharmacological characterization of receptor-mediated Gi/o activation in postmortem human prefrontal cortex.

The biochemical abnormalities in transmembrane signal transduction mediated through G protein-coupled receptors (GPCRs) have been postulated as underlying pathophysiology of psychiatric diseases such as schizophrenia and mood disorders. In the present study, the experimental conditions of agonist-induced [35 S]GTPγS binding in postmortem human brain membranes were optimized, and the responses induced by a series of agonists were pharmacologically characterized. The [35 S]GTPγS binding assay was performed in postmortem human prefrontal cortical membranes by means of filtration techniques, and standardized as to GDP concentration, membrane protein content, MgCl2 and NaCl concentrations in assay buffer, incubation period and effect of white matter contamination. Under the standard assay conditions, the specific [35 S]GTPγS binding was stimulated by the addition of 15 compounds in a concentration-dependent manner. Of these agonists, R(+)-8-OH-DPAT, UK-14,304, DAMGO and DPDPE showed apparently biphasic concentration-response curves. As for these four responses, only higher-potency site was pharmacologically characterized. The receptors involved in the responses investigated were 5-HT1A receptor (probed with R(+)-8-OH-DPAT or 5-HT), α2A -adrenoceptor (UK-14,304 or (-)-epinephrine), M2 /M4 mAChRs (carbachol), adenosine A1 receptor (adenosine), histamine H3 receptor (histamine), group II mGlu (l-glutamate), GABAB receptor (baclofen), µ-opioid receptor (DAMGO or endomophin-1), δ-opioid receptor (DPDPE or SNC-80) and NOP (nociceptin). Although dopamine also activated specific [35 S]GTPγS binding, this response was likely mediated via α2A -adrenoceptor, but not dopamine receptor subtypes. The present study provides us with fundamental aspects of the strategy for elucidation of probable abnormalities of neural signalling mediated by G proteins activated through multiple GPCRs in the brain of psychiatric patients.

Receptor⁻Receptor Interactions in Multiple 5-HT1A Heteroreceptor Complexes in Raphe-Hippocampal 5-HT Transmission and Their Relevance for Depression and Its Treatment.

Due to the binding to a number of proteins to the receptor protomers in receptor heteromers in the brain, the term "heteroreceptor complexes" was introduced. A number of serotonin 5-HT1A heteroreceptor complexes were recently found to be linked to the ascending 5-HT pathways known to have a significant role in depression. The 5-HT1A⁻FGFR1 heteroreceptor complexes were involved in synergistically enhancing neuroplasticity in the hippocampus and in the dorsal raphe 5-HT nerve cells. The 5-HT1A protomer significantly increased FGFR1 protomer signaling in wild-type rats. Disturbances in the 5-HT1A⁻FGFR1 heteroreceptor complexes in the raphe-hippocampal 5-HT system were found in a genetic rat model of depression (Flinders sensitive line (FSL) rats). Deficits in FSL rats were observed in the ability of combined FGFR1 and 5-HT1A agonist cotreatment to produce antidepressant-like effects. It may in part reflect a failure of FGFR1 treatment to uncouple the 5-HT1A postjunctional receptors and autoreceptors from the hippocampal and dorsal raphe GIRK channels, respectively. This may result in maintained inhibition of hippocampal pyramidal nerve cell and dorsal raphe 5-HT nerve cell firing. Also, 5-HT1A⁻5-HT2A isoreceptor complexes were recently demonstrated to exist in the hippocampus and limbic cortex. They may play a role in depression through an ability of 5-HT2A protomer signaling to inhibit the 5-HT1A protomer recognition and signaling. Finally, galanin (1⁻15) was reported to enhance the antidepressant effects of fluoxetine through the putative formation of GalR1⁻GalR2⁻5-HT1A heteroreceptor complexes. Taken together, these novel 5-HT1A receptor complexes offer new targets for treatment of depression.

Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.

Disturbances in the FGFR1-5-HT1A Heteroreceptor Complexes in the Raphe-Hippocampal 5-HT System Develop in a Genetic Rat Model of Depression.

The FGFR1-5-HT1A heteroreceptor complexes are involved in neuroplasticity in the rat hippocampus and in the mesencephalic raphe 5-HT nerve cells. There exists a 5-HT1A protomer enhancement of FGFR1 protomer signaling. Acute and 10 day treatment with intracerebroventricular (i.c.v.) FGF-2 and the 5-HT1A agonist 8-OH-DPAT produced enhanced antidepressant effects in the forced swim test (FST). We studied in the current work the disturbances in the FGFR1-5-HT1A heterocomplexes in a genetic rat model of depression, the Flinders sensitive line (FSL) rats of Sprague-Dawley (SD) origin, by means of neurochemical, neurophysiological and behavioral techniques. In control SD rats, the FGFR1 agonist SUN11602 and FGF2 produced a significant reduction of G protein-coupled inwardly rectifying K+ channel (GIRK) currents induced by 8-OH-DPAT in the CA1 area of the hippocampus. In FSL rats, only i.c.v. 8-OH-DPAT alone treatment produced a significant reduction in the immobility time. The combined i.c.v. treatment (FGF2 + 8-OH-DPAT) in FSL rats did not cause a significant decrease in immobility time in the FST. However, in the SD rats this combined treatment produced a significant reduction. Furthermore, in the FSL rat a significant increase in the density of FGFR1-5-HT1A proximity ligation assay (PLA) positive clusters was only found after i.c.v. 8-OH-DPAT treatment alone in the CA2 and CA3 areas. In the SD rat a significant increase in the density of specific PLA clusters was only observed in the CA2 area of the i.c.v. combined treatment (FGF2 + 8-OH-DPAT) group. No treatment led to significant changes in the PLA clusters of the dorsal raphe in the FSL rat. However, significant changes in the density of specific PLA clusters were only found in the dorsal raphe of SD rats after combined treatment and treatment with 8-OH-DPAT alone. The results indicate that in FSL rats compared with SD rats alterations may develop in the ability of 8-OH-DPAT and combined FGFR1 and 5-HT1A agonist treatment to increase the density of FGFR1-5-HT1A heteroreceptor complexes of the dorsal raphe. It is proposed that such deficits in FSL rats may possibly reflect a failure of the combined agonist treatment to uncouple the 5-HT1A autoreceptors from the GIRK channels. This may contribute to the failure of producing antidepressant-like effects in the FSL rat by combined agonist treatment as seen in the SD rat. The antidepressant-like effects seen with the 5-HT1A agonist alone treatment in FSL but not in SD rats may instead involve significant increases in the FGFR1-5-HT1A complexes of the CA2 and CA3 areas of the hippocampus.

Functional activation of Gαq coupled to 5-HT2A receptor and M1 muscarinic acetylcholine receptor in postmortem human cortical membranes.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) play a pivotal role in a wide range of signal transduction pathways, and receptor/G-protein coupling has been implicated in the pathophysiology of mental disorders. In this study, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation assay for Gαq was applied to postmortem human brains. After its optimization for human prefrontal cortical membranes, we selected 5-hydroxytryptamine (5-HT) and carbachol as efficient agonists for subsequent experiments. The concentration-response curve of 5-HT shifted towards the right by the addition of increasing concentrations of ketanserin (with a pA 2 value of 9.18), indicating the involvement of the 5-HT2A receptor. Besides, the carbachol-stimulated [35S]GTPγS binding to Gαq was competitively antagonized by telenzepine (with a pA 2 value of 8.81), indicating the involvement of the M1 muscarinic acetylcholine receptor (mAChR). Concentration-response curves of 5-HT2A receptor- and M1 mAChR-mediated Gαq activation were determined in 40 subjects. The mean maximum percentage increase (%E max) was 155 and 470%, respectively, and the mean half-maximal effect concentration (EC50) was 131 nM and 15.2 µM, respectively. When the pharmacological parameters were correlated with age, postmortem delay, freezing storage period, and tissue pH, no statistically significant correlation was observed except for the negative correlation between age and %E max value of carbachol-stimulated [35S]GTPγS binding to Gαq. The %E max values for 5-HT2A receptor- and M1 mAChR-mediated Gαq activation also tended to correlate with each other. These results provide fundamental information of Gαq-coupled 5-HT2A receptor and M1 mAChR in native human brains, and lay the foundation for future studies in mental disorder patients.

A Case of Persistent Generalized Retrograde Autobiographical Amnesia Subsequent to the Great East Japan Earthquake in 2011.

Functional retrograde autobiographical amnesia is often associated with physical and/or psychological trauma. On 11 March 2011, the largest earthquake on record in Japan took place, and subsequent huge tsunami devastated the Pacific coast of northern Japan. This case report describes a patient suffering from retrograde episodic-autobiographical amnesia for whole life, persisting for even more than five years after the disaster. A Japanese man, presumably in his 40s, got police protection in April 2016 but was unable to respond to question about his own name. He lost all information about his personal identity, and his memory was wholly lost until the disaster on 11 March 2011. He was able to recall his life after the disaster, and semantic memories and social abilities were largely preserved. A medical examination performed on 1 November 2016 verified that he was awake, alert, and oriented to time, place, and person (except for himself). General physical and neurological examinations revealed no pathological findings. He also experienced some symptoms associated with posttraumatic stress disorder (PTSD), such as intrusive thoughts, flashbacks, and nightmares. No abnormalities were detected by biochemical test and brain magnetic resonance imaging (MRI). Physicians and other professionals who take care of victims of disaster should be aware of dissociative spectrum disorders, such as psychogenic amnesia.

Comparative analysis of pharmacological properties of xanomeline and N-desmethylclozapine in rat brain membranes.

BACKGROUND: 3(3-Hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) and N-desmethylclozapine are of special interest as promising antipsychotics with better efficacy, especially for negative symptoms and/or cognitive/affective impairment. METHODS: The guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding experiments were performed using (1) conventional filtration technique, (2) antibody-capture scintillation proximity assay, and (3) immunoprecipitation method, in brain membranes prepared from rat cerebral cortex, hippocampus, and striatum. RESULTS: Xanomeline had agonistic activity at the M1 muscarinic acetylcholine receptor (mAChR) in all brain regions, as well as at the 5-HT1A receptor in the cerebral cortex and hippocampus. On the other hand, N-desmethylclozapine exhibited slight agonistic effects on the M1 mAChR, and agonistic properties at the 5-HT1A receptor in the cerebral cortex and hippocampus. This compound also behaved as an agonist at the δ-opioid receptor in the cerebral cortex and striatum. In addition, the stimulatory effects of N-desmethylclozapine on [(35)S]GTPγS binding to Gαi/o were partially mediated through mAChRs (most likely M4 mAChR subtype), at least in striatum. CONCLUSIONS: The agonistic effects on the mAChRs (particularly M1 subtype, and also probably M4 subtype), the 5-HT1A receptor and the δ-opioid receptor expressed in native brain tissues, some of which are common to both compounds and others specific to either, likely shape the unique beneficial effectiveness of both compounds in the treatment for schizophrenic patients. These characteristics provide us with a clue to develop newer antipsychotics, beyond the framework of dopamine D2 receptor antagonism, that are effective not only on positive symptoms but also on negative symptoms and/or cognitive/affective impairment.

Adenosine A1( )receptors are selectively coupled to Gα(i-3) in postmortem human brain cortex: Guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding/immunoprecipitation study.

By means of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay combined with immunoprecipitation using anti-Gα subunit antibody, we recently reported 5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated Gαq activation in rat cerebral cortical membranes (Odagaki et al., 2014). In the present study, this method has been applied to postmortem human brains, with focusing on adenosine receptor-mediated G-protein activation. In the exploratory experiments using a series of agonists and the antibodies specific to each Gα subtypes in the presence of low (10 nM) or high (50 µM) concentration of GDP, the most prominent increases in specific [(35)S]GTPγS binding in the membranes prepared from human prefrontal cortex were obtained for the combinations of adenosine (1mM)/anti-Gαi-3 in the presence of 50 µM GDP as well as 5-HT (100 µM)/anti-Gαq and carbachol (1mM)/anti-Gαq in the presence of 10nM GDP. Adenosine-induced activation of Gαi-3 emerged only when GDP concentrations were increased higher than 10 µM, and the following experiments were performed in the presence of 300 µM GDP. Adenosine increased specific [(35)S]GTPγS binding to Gαi-3 in a concentration-dependent manner to 251.4% of the basal unstimulated binding, with an EC50 of 1.77 µM. The involvement of adenosine A1 receptor was verified by the experiments using selective agonists and antagonists at adenosine A1 or A3 receptor. Among the α subunits of Gi/o class (Gαi-1, Gαi-2, Gαi-3, and Gαo.), only Gαi-3 was activated by 1mM adenosine, indicating that human brain adenosine A1 receptor is coupled preferentially, if not exclusively, to Gαi-3.

Functional activation of G-proteins coupled with muscarinic acetylcholine receptors in rat brain membranes.

The functional activation of Gi/o proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[(35)S]thio) triphosphate ([(35)S]GTPγS) binding assay in rat brain membranes. The most efficacious stimulation elicited by acetylcholine or carbachol (CCh) was obtained in striatal membranes. The pharmacological properties of mAChR-mediated [(35)S]GTPγS binding determined with a series of muscarinic agonists and antagonists were almost identical among the three brain regions investigated, i.e., cerebral cortex, hippocampus, and striatum, except for the apparent partial agonist effects of (αR)-α-cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate (J 104129) observed only in the hippocampus, but not in the other two regions. Among the muscarinic toxins investigated, only MT3 attenuated CCh-stimulated [(35)S] GTPγS binding. The highly selective allosteric potentiator at the M4 mAChR subtype, 3-amino-N-[(4-chlorophenyl)methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (VU 10010), shifted the concentration-response curve for CCh leftwards as well as upwards. On the other hand, neither thiochrome nor brucine N-oxide was effective. The increases induced by CCh and 5-HT were essentially additive, though not completely, indicating that the mAChRs and 5-HT1A receptors were coupled independently to distinct pools of Gi/o proteins. Collectively, all of the data suggest that functional activation of Gi/o proteins coupled to mAChRs, especially the M4 subtype, is detectable by means of CCh-stimulated [(35)S]GTPγS binding assay in rat discrete brain regions.

Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes.

Functional coupling between serotonin2A (5-HT2A) receptors and Gαq proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-Gαq antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-Gαq antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [35S]GTPγS binding to Gαq in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of Gαq proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (±)-baclofen. When 5-HT2A receptors and M1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of Gαq proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via Gαq proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M1 receptors.