The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells.

Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson's disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson's disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson's disease, providing novel opportunities for therapeutic intervention.

Proposed clinical scale for the diagnosis of acute coronary syndrome in patients with an inconclusive electrocardiogram and myocardial injury biomarkers. / Propuesta de una escala clínica para el diagnóstico de síndrome coronario agudo en pacientes con electrocardiograma y biomarcadores de lesión miocárdica no concluyentes.

RATIONALE: Acute coronary syndrome (ACS) requires improved diagnostic accuracy through useful, safe and easy-to-apply tools. OBJECTIVES: To obtain an assessment scale for the diagnosis of ACS in patients with chest pain and nondiagnostic electrocardiogram and troponin concentrations. METHODS: A prospective cohort study included 286 patients treated in the emergency department for chest pain, with normal electrocardiogram and troponin levels. Using multiple logistic regression, we obtained the independent predictors for the diagnosis of ACS. The assessment scale's discriminative power was assessed with the area under the ROC curve. RESULTS: The diagnosis of ACS was confirmed in 103 patients (36%). The final predictive model included 3 endpoints: a history of coronary artery disease, hyperlipidaemia and a score≥6 points on the Geleijnse scale. The area under the ROC curve for the final model was 0.90 (95% confidence interval [95% CI] 0.85-0.93). A threshold of 5 points achieved a sensitivity of 76.7% (95% CI 68-84), a specificity of 91.8% (95% CI 87-95), a positive likelihood ratio of 9.36 (95% CI 5.70-15.40), a negative likelihood ratio of 0.25 (95% CI 18.00-36.00) and an overall diagnostic accuracy of 86.4% (95% CI 82-90). The predictive model was superior to the Geleijnse scale alone. CONCLUSIONS: The final scale showed good discriminative capacity for diagnosing ACS and could therefore be of interest for identifying ACS in emergency departments. Nevertheless, the scale needs to be validated in larger multicentre studies.

Ketamine enhances structural plasticity in mouse mesencephalic and human iPSC-derived dopaminergic neurons via AMPAR-driven BDNF and mTOR signaling.

Among neurobiological mechanisms underlying antidepressant properties of ketamine, structural remodeling of prefrontal and hippocampal neurons has been proposed as critical. The suggested mechanism involves downstream activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which trigger mammalian target of rapamycin (mTOR)-dependent structural plasticity via brain-derived neurotrophic factor (BDNF) and protein neo-synthesis. We evaluated whether ketamine elicits similar molecular events in dopaminergic (DA) neurons, known to be affected in mood disorders, using a novel, translational strategy that involved mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. Sixty minutes exposure to ketamine elicited concentration-dependent increases of dendritic arborization and soma size in both mouse and human cultures as measured 72 hours after application. These structural effects were blocked by mTOR complex/signaling inhibitors like rapamycin. Direct evidence of mTOR activation by ketamine was revealed by its induction of p70S6 kinase. All effects of ketamine were abolished by AMPA receptor antagonists and mimicked by the AMPA-positive allosteric modulator CX614. Inhibition of BDNF signaling prevented induction of structural plasticity by ketamine or CX614. Furthermore, the actions of ketamine required functionally intact dopamine D3 receptors (D3R), as its effects were abolished by selective D3R antagonists and absent in D3R knockout preparations. Finally, the ketamine metabolite (2R,6R)-hydroxynorketamine mimicked ketamine effects at sub-micromolar concentrations. These data indicate that ketamine elicits structural plasticity by recruitment of AMPAR, mTOR and BDNF signaling in both mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. These observations are of likely relevance to the influence of ketamine upon mood and its other functional actions in vivo.

Diagnostic validity of hand gestures in chest pain of coronary origin. / Validez diagnóstica del lenguaje de las manos en el dolor torácico de origen coronario.

OBJECTIVES: To determine the frequency of 3 hand gestures by patients with chest pain and determine the diagnostic validity of the gestures in acute coronary syndrome. PATIENTS AND METHODS: A prospective study was conducted on 383 adult patients treated for chest pain in an emergency department. We observed certain hand gestures, previously referred to in the medical literature as characteristic of coronary pain (gesture 1: a clenched fist held over the sternal area or Levine's sign; gesture 2: open hand located over the same area; gesture 3: both hands placed in the centre of the chest), as well as other gestures. We analysed their association with the coronary origin of the pain. RESULTS: We confirmed the coronary origin of the pain in 164 (43%) patients (ACS group). The other 219 patients (57%) did not have a coronary origin for the pain (nonACS group). Eighty-nine percent of the patients identified their pain with one of the 3 classical gestures. The most frequent gesture was number 2, both overall (59%) and by group (60% ACS group; 57.5% nonACS group). There was no significant association between the type of gesture and the final diagnosis (P=.172). The greater specificity corresponded to Levine's sign (90%), followed by other gestures (86%) and gesture 3 (81%). CONCLUSIONS: Although 89% of the patients expressed their chest pain with one of the 3 manual gestures classically associated with coronary pain, none achieved sufficient diagnostic accuracy to be used as indicative of this type of pain.

Deconstructing vulnerability for psychosis: Meta-analysis of environmental risk factors for psychosis in subjects at ultra high-risk.

BACKGROUND: Subjects at ultra high-risk (UHR) for psychosis have an enhanced vulnerability to develop the disorder but the risk factors accounting for this accrued risk are undetermined. METHOD: Systematic review of associations between genetic or environmental risk factors for psychosis that are widely established in the literature and UHR state, based on comparisons to controls. RESULTS: Forty-four studies encompassing 170 independent datasets and 54 risk factors were included. There were no studies on association between genetic or epigenetic risk factors and the UHR state that met the inclusion criteria. UHR subjects were more likely to show obstetric complications, tobacco use, physical inactivity, childhood trauma/emotional abuse/physical neglect, high perceived stress, childhood and adolescent low functioning, affective comorbidities, male gender, single status, unemployment and low educational level as compared to controls. CONCLUSIONS: The increased vulnerability of UHR subjects can be related to environmental risk factors like childhood trauma, adverse life events and affective dysfunction. The role of genetic and epigenetic risk factors awaits clarification.

Limited beneficial effects of piceatannol supplementation on obesity complications in the obese Zucker rat: gut microbiota, metabolic, endocrine, and cardiac aspects.

Resveratrol is beneficial in obese and diabetic rodents. However, its low bioavailability raises questions about its therapeutic relevance for treating or preventing obesity complications. In this context, many related natural polyphenols are being tested for their putative antidiabetic and anti-obesity effects. This prompted us to study the influence of piceatannol, a polyhydroxylated stilbene, on the prevention of obesity complications in Zucker obese rats. A 6-week supplementation was followed by the determination of various markers in plasma, liver, adipose tissue and heart, together with a large-scale analysis of gut microbiota composition. When given in doses of 15 or 45 mg/kg body weight/day, piceatannol did not reduce either hyperphagia or fat accumulation. It did not modify the profusion of the most abundant phyla in gut, though slight changes were observed in the abundance of several Lactobacillus, Clostridium, and Bacteroides species belonging to Firmicutes and Bacteroidetes. This was accompanied by a tendency to reduce plasma lipopolysaccharides by 30 %, and by a decrease of circulating non-esterified fatty acids, LDL-cholesterol, and lactate. While piceatannol tended to improve lipid handling, it did not mitigate hyperinsulinemia and cardiac hypertrophy. However, it increased cardiac expression of ephrin-B1, a membrane protein that contributes to maintaining cardiomyocyte architecture. Lastly, ascorbyl radical plasma levels and hydrogen peroxide release by adipose tissue were similar in control and treated groups. Thus, piceatannol did not exhibit strong slimming capacities but did limit several obesity complications.

Gut microbiota: a key player in health and disease. A review focused on obesity.

Gut microbiota, its evolutive dynamics and influence on host through its protective, trophic and metabolic actions, has a key role in health and opens unique opportunities for the identification of new markers of the physiopathological state of each individual. Alterations in gut microbiota composition have been associated with plenty disorders. Of interest, the vast number of studies demonstrates the role of microbiota in obesity, a serious public health problem that has reached epidemic proportions in many developed and middle-income countries. The economic and health costs of this condition and its comorbidities such as fatty liver, insulin resistance/diabetes, or cardiovascular events are considerable. Therefore, every strategy designed to reduce obesity would imply important savings. Targeting microbiota, in order to restore/modulate the microbiota composition with antibiotics, probiotics, prebiotics, or even fecal transplants, is considered as a promising strategy for the development of new solutions for the treatment of obesity. However, there is still lot to do in this field in order to identify the exact composition of microbiota in "health" and the specific mechanisms that regulate the host-microbiotal crosstalk. In addition, it is important to note that changes not only in the gut microbiota profile (abundance) but also in its metabolism and functions need to be taken into account in the context of contribution in the physiopathology of obesity and related disorders.

Local inactivation of Gpr88 in the nucleus accumbens attenuates behavioral deficits elicited by the neonatal administration of phencyclidine in rats.

Gpr88, an orphan G-protein-coupled receptor, is highly and almost exclusively expressed in the medium spiny projection neurons of the striatum, and may thus participate in the control of motor functions and cognitive processing that are impaired in neuropsychiatric disorders such as Parkinson's disease or schizophrenia (SZ). This study investigated the relevance of Gpr88 to SZ-associated behavior by knocking down Gpr88 gene expression in the ventral striatum (nucleus accumbens) in a neurodevelopmental rat model of SZ, generated by neonatal treatment with phencyclidine (PCP). In this model, we compared the effects of the local inactivation in the adult animal of the expression of Gpr88 and of Drd2, a gene strongly implicated in the etiology of SZ and coding for the dopamine receptor type 2 (D2). To inactivate specifically Gpr88 and D2 expression, we used the lentiviral vector-mediated microRNA silencing strategy. The neonatal PCP treatment induced in the adult rat hyperlocomotion in response to amphetamine (Amph) and social novelty discrimination (SND) deficits. The inactivation of D2 did not modify the locomotor response to Amph or the cognitive deficits induced by PCP, whereas the silencing of Gpr88 inhibited the Amph-induced hyperlocomotion and reduced the impairment of SND elicited by neonatal exposure to PCP. These observations suggest a role for Gpr88 in the regulation of cognitive and motor functions, and support its relevance to the pathophysiology and treatment of SZ and other disorders involving dysfunction of the accumbens-striatal complex.

Agomelatine: mechanism of action and pharmacological profile in relation to antidepressant properties.

Agomelatine behaves both as a potent agonist at melatonin MT1 and MT2 receptors and as a neutral antagonist at 5-HT2C receptors. Accumulating evidence in a broad range of experimental procedures supports the notion that the psychotropic effects of agomelatine are due to the synergy between its melatonergic and 5-hydroxytryptaminergic effects. The recent demonstration of the existence of heteromeric complexes of MT1 and MT2 with 5-HT2C receptors at the cellular level may explain how these two properties of agomelatine translate into a synergistic action that, for example, leads to increases in hippocampal proliferation, maturation and survival through modulation of multiple cellular pathways (increase in trophic factors, synaptic remodelling, glutamate signalling) and key targets (early genes, kinases). The present review focuses on the pharmacological properties of this novel antidepressant. Its mechanism of action, strikingly different from that of conventional classes of antidepressants, opens perspectives towards a better understanding of the physiopathological bases underlying depression.

Valproate improves prepulse inhibition deficits induced by corticotropin-releasing factor independent of GABA(A) and GABA(B) receptor activation.

Corticotropin-releasing factor (CRF) is implicated in the pathogenesis of bipolar disorder, an illness associated with deficits in prepulse inhibition (PPI) of the acoustic startle response. Valproate is used in the treatment of bipolar disorder and may alter CRF activity via a GABA(A)-ergic mechanism. This study determined the effect of valproate on CRF-disrupted PPI and examined the role of the hypothalamic-pituitary-adrenal axis and GABA-ergic signaling in the effect of valproate. Valproate (60-240 mg/kg) dose-dependently reversed PPI deficits displayed by transgenic mice overexpressing CRF (CRFtg), and normalized PPI deficits induced by CRF i.c.v. infusion in 129Sv mice. Valproate enhanced corticosterone secretion more effectively in CRFtg than in wild-type mice. The effect of valproate on PPI was not blocked by the GABA(A) receptor antagonist bicuculline, the GABA(B) receptor antagonists phaclofen and SCH 50911 or combined administration of a GABA(A) and GABA(B) receptor antagonist. The beneficial effect of valproate on PPI was not mimicked by the GABA(A) receptor agonist muscimol, the GABA transaminase inhibitor vigabatrin, the histone deacetylase (HDAC) inhibitor sodium butyrate or by the mood stabilizers lithium, carbamazepine, lamotrigine or topiramate. Thus, we showed that valproate improves CRF-induced PPI deficits, albeit via a so far unknown mechanism. These marked beneficial effects of valproate on CRF-induced sensorimotor gating deficits suggest that valproate may be of particular value in specific subgroups of bipolar patients that are characterized by alterations in the CRF system.

CRF1 receptor antagonists do not reverse pharmacological disruption of prepulse inhibition in rodents.

RATIONALE: As enhanced corticotropin-releasing factor (CRF) transmission is associated with induction of sensorimotor gating deficits, CRF1 receptor antagonists may reverse disrupted prepulse inhibition (PPI), an operational measure of sensorimotor gating. OBJECTIVES: To determine the effects of CRF1 receptor antagonists in pharmacological models of disrupted PPI and to determine if long-term elevated central CRF levels alter sensitivity towards PPI disrupting drugs. METHODS: CP154,526 (10-40 mg/kg), SSR125543 (3-30 mg/kg) and DMP695 (40 mg/kg) were tested on PPI disruption provoked by D-amphetamine (2.5, 3 mg/kg), ketamine (5, 30 mg/kg) and MK801 (0.2, 0.5 mg/kg) in Wistar rats, C57Bl/6J and CD1 mice, and on spontaneously low PPI in Iffa Credo rats and DBA/2J mice. PPI-disrupting effects of D-amphetamine (2.5-5 mg/kg) and MK801 (0.3-1 mg/kg) were examined in CRF-overexpressing (CRFtg) mice, which display PPI deficits. Finally, we determined the influence of CP154,526 on D-amphetamine-induced dopamine outflow in nucleus accumbens and prefrontal cortex of CRFtg mice using in vivo microdialysis. RESULTS: No CRF1-antagonists improved PPI deficits in any test. CRFtg mice showed blunted PPI disruption in response to MK801, but not D-amphetamine. Further, D-amphetamine-induced dopamine release was less pronounced in CRFtg versus wild-type mice, a response normalized by pretreatment with CP154,526. CONCLUSION: The inability of CRF1 receptor antagonists to block pharmacological disruption of sensorimotor gating suggests that the involvement of CRF1 receptors in the modulation of dopaminergic and glutamatergic neurotransmission relevant for sensory gating is limited. Furthermore, the alterations observed in CRFtg mice support the notion that long-term elevated central CRF levels induce changes in these neurotransmitter systems.

Alpha1-adrenoceptors mediate dihydroxyphenylalanine-induced activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaques.

The mechanisms underlying actions of dihydroxyphenylalanine (L-DOPA) in Parkinson's disease remain to be fully elucidated. Noradrenaline formed from L-DOPA may stimulate alpha(1)-adrenoceptors. We assessed the involvement of alpha(1)-adrenoceptors in actions of L-DOPA in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaques. In each animal, the minimal dose of L-DOPA required to alleviate parkinsonian symptoms was defined (12.5-25 mg/kg p.o.). The effects of coadministration of the alpha(1)-adrenoceptor antagonist prazosin ([4-(4-amino-6,7-dimethoxy-quinazolin-2-yl) piperazin-1-yl]-(2-furyl)methanone) on motor activity, parkinsonism, and dyskinesia were assessed. Antiparkinsonian benefit was accompanied by mild dyskinesia. L-DOPA also elicited hyperactivity, i.e., activity greater than that seen in normal animals. Coadministration of prazosin (0.16-0.63 mg/kg p.o.) with L-DOPA did not significantly affect either its antiparkinsonian actions or dyskinesia. However, prazosin significantly and dose-dependently attenuated L-DOPA-induced activity, reducing it to a level equivalent to that of normal animals. More specifically, during periods of pronounced L-DOPA-induced activity, prazosin attenuated the total and duration of activity by 80 and 76%, respectively. These actions of prazosin were expressed in the absence of sedation. Although activation of alpha(1)-adrenoceptors plays no major role in the antiparkinsonian and dyskinetic effects of L-DOPA per se, it does contribute to the induction of hyperactivity. alpha(1)-Adrenoceptors may be involved in pathological responses to L-DOPA treatment, including the dopamine dysregulation syndrome.

An immunocapture/scintillation proximity analysis of G alpha q/11 activation by native serotonin (5-HT)2A receptors in rat cortex: blockade by clozapine and mirtazapine.

Though transduction mechanisms recruited by heterologously expressed 5-HT(2A) receptors have been extensively studied, their interaction with specific subtypes of G-protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5-HT, the prototypical 5-HT(2A) agonist, DOI, and Ro60,0175 all enhanced [(35)S]GTPgammaS binding to G alpha q/11 in rat cortex with pEC(50) values of 6.22, 7.24 and 6.35, respectively. No activation of G o or G s/olf was seen at equivalent concentrations of DOI. Stimulation of G alpha q/11 by 5-HT (30 microM) and DOI (30 microM) was abolished by the selective 5-HT(2A) vs. 5-HT(2C)/5-HT(2B) antagonists, ketanserin (pK(B) values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5-HT-induced [(35)S]GTPgammaS binding to G alpha q/11 was only weakly inhibited by the preferential 5-HT(2C) receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5-HT(2B) receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5-HT(2A) receptors, blocked the recruitment of G alpha q/11 by 5-HT and DOI with pK(B) values of 8.54 and 8.14, respectively. Its actions were mimicked by the "atypical" antidepressant and 5-HT(2A) receptor antagonist, mirtazapine, which likewise blocked 5-HT and DOI-induced G alpha q/11 protein activation with pK(B) values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5-HT(2A) receptors in rat frontal cortex specifically recruit G alpha q/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5-HT(2A) receptor-mediated G alpha q/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent.

Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity.

The spatiotemporal regulation of neurotransmitter transporters involves proteins that interact with their intracellular domains. Using a proteomic approach, we identified several proteins that interact with the C terminus of the serotonin transporter (SERT). These included neuronal nitric oxide synthase (nNOS), a PSD-95/Disc large/ZO-1 (PDZ) domain-containing protein recruited by the atypical PDZ binding motif of SERT. Coexpression of nNOS with SERT in HEK293 cells decreased SERT cell surface localization and 5-hydroxytryptamine (5-HT) uptake. These effects were absent in cells transfected with SERT mutated in its PDZ motif to prevent physical association with nNOS, and 5-HT uptake was unaffected by activation or inhibition of nNOS enzymatic activity. 5-HT uptake into brain synaptosomes was increased in both nNOS-deficient and wild-type mice i.v. injected with a membrane-permeant peptidyl mimetic of SERT C terminus, which disrupted interaction between SERT and nNOS, suggesting that nNOS reduces SERT activity in vivo. Furthermore, treating cultured mesencephalic neurons with the mimetic peptide similarly increased 5-HT uptake. Reciprocally, indicating that 5-HT uptake stimulates nNOS activity, NO production was enhanced on exposure of cells cotransfected with nNOS and SERT to 5-HT. This effect was abolished by 5-HT uptake inhibitors and absent in cells expressing SERT mutated in its PDZ motif. In conclusion, physical association between nNOS and SERT provides a molecular substrate for their reciprocal functional modulation. In addition to showing that nNOS controls cell surface localization of SERT, these findings provide evidence for regulation of cellular signaling (NO production) by a substrate-carrying transporter.

Dopamine D1 receptor coupling to Gs/olf and Gq in rat striatum and cortex: a scintillation proximity assay (SPA)/antibody-capture characterization of benzazepine agonists.

Cloned, human dopamine D(1) receptors recruit multiple effectors but the G-protein subtype(s) activated by cerebral populations remain poorly defined, a question addressed using a rapid immunocapture technique. In rat striatum, dopamine (DA) and four selective, benzazepine agonists at D(1) receptors concentration-dependently enhanced [(35)S]GTPgammaS binding to Galphas/olf. For all drugs, Galphaq was also recruited with similar potencies and efficacies. Comparable observations were made in the cortex wherein profiles of Galphas/olf vs Galphaq activation were also highly correlated. In contrast to Galphas/olf and Galphaq, Galphao and Galphai were activated neither in the striatum nor in the cortex, except for SKF82958. As compared to DA, both SKF81297 and SKF82958 were full agonists at Gs/olf and Gq in cortex and striatum, whereas SKF38393 behaved as a partial agonist. Likewise, the "atypical" agonist, SKF83959 only partially activated Galphaq and also Gs/olf in these two regions. In both striatum and cortex, the selective D(1) receptor antagonist, SCH23390, abolished the recruitment of Galphaq and Galphas by DA, and the action of DA was partially attenuated by SKF83959. These findings demonstrate that, in native CNS tissue, DA and other D(1) receptor agonists activate Galphas and Galphaq with similar potencies and efficacies, suggesting their recruitment via pharmacologically-indistinguishable populations of D(1) receptors, and show that SPA technology is well-adapted to study the coupling of native DA receptors.

Serotonin1A autoreceptor activation by S 15535 enhances circadian activity rhythms in hamsters: evaluation of potential interactions with serotonin2A and serotonin2C receptors.

Mammalian circadian activity rhythms are generated by pacemaker cells in the suprachiasmatic nucleus (SCN). As revealed by the actions of diverse agonists, serotonergic input from raphe nuclei generally inhibits photic signaling in the suprachiasmatic nucleus. In contrast, the serotonin (5HT)1A partial agonist, 4-(benzodioxan-5-yl)1-(indan2-yl)piperazine (S 15535), was found to enhance the phase-shifting influence of light on hamster circadian rhythms [Gannon, Neuroscience 119 (2003) 567]. Herein, we extend this observation in showing that S 15535 (5.0 mg/kg, i.p.) markedly (275%) enhanced the light-induced phase shift in circadian activity rhythms: further, this action was dose-dependently abolished by the highly-selective 5HT1A receptor antagonist, WAY 100,635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]N-2-pyridinyl-cyclohexane-carboxamide maleate) (0.1-0.5 mg/kg, i.p.). WAY 100,635, which was inactive alone, shares the antagonist actions of S 15535 at postsynaptic 5HT1A sites, yet blocks its effects at their presynaptic counterparts. Thus, 5HT1A autoreceptor activation must be involved in this effect of S 15535 which contrasts with the opposite, inhibitory influence upon phase shifts of the "full" agonist, 8-OH-DPAT, which acts by stimulation of postsynaptic 5HT1A receptors [Rea et al., J Neurosci 14 (1994) 3635]. Despite the occurrence of 5HT2A and 5HT2C receptors in the (rat) suprachiasmatic nucleus, their influence on circadian rhythms is unknown since actions of selective ligands have never been evaluated. This issue was investigated with the most selective agents currently available. However, the 5HT2A agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.25 and 0.5 mg/kg), and the 5HT2C agonist, alphaS-6-chloro-5-fluoro-a-methyl-1H-indole-1-ethanamine fumarate (Ro-60-0175) (1.0 and 5.0 mg/kg), failed to affect light-induced phase shifts in hamsters. Moreover, even over broad dose-ranges, the 5HT2A antagonist, (+)-(2,3-dimethoxy-phenyl)-[1-[2-(4-fluoro-phenyl)-ethyl]-piperidin-4-yl]methanol (MDL 100,907) (0.1-1.0 mg/kg), and the 5HT2C antagonist, 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl carbamoyl]indoline (SB 242,084) (1.0-10.0 mg/kg), were likewise inactive. In view of evidence that 5HT2A and 5HT2C sites functionally interact with 5HT1A receptors, we also examined the influence of these agents upon the actions of S 15535, but no significant alteration was seen in its enhancement of rhythms. In conclusion, S 15535 elicits a striking enhancement of light-induced phase shifts in circadian rhythms by specifically recruiting 5HT1A autoreceptors, which leads to suppression of serotonergic input to the suprachiasmatic nucleus. Surprisingly, no evidence for a role of 5HT2A or 5HT2C sites was found, though comparable functional studies remain to be undertaken in rats. Indeed, the present work underlines the importance of comparative studies of circadian rhythms in various species, as well as the need for further study of potential interactions among 5HT receptor subtypes in their control.

Selective blockade of D(3) dopamine receptors enhances the anti-parkinsonian properties of ropinirole and levodopa in the MPTP-lesioned primate.

To date, the lack of highly selective antagonists at the dopamine D(3) receptor has hampered clarification of their involvement in the actions of currently used therapies in Parkinson's disease. However, the novel benzopyranopyrrole, S33084, displays greater than 100-fold selectivity as an antagonist for D(3) versus D(2) receptors and all other sites tested. S33084 was administered to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmosets previously primed with levodopa to elicit dyskinesia. Administered alone, S33084 exerted a modest, but significant, anti-parkinsonian effect without provoking dyskinesia. At low D(3)-selective doses (0.16 and 0.64 mg/kg), S33084 potentiated, though to different extents and in qualitatively different ways, the anti-parkinsonian actions of both ropinirole and levodopa. At these doses, S33084 did not significantly modify levodopa-induced or ropinirole-induced dyskinesia. These data suggest that ropinirole and levodopa do not exert their anti-parkinsonian or pro-dyskinetic actions via D(3) receptor stimulation. Indeed, stimulation of D(3) receptors may be detrimental to the anti-parkinsonian properties of D(2)/D(3) agonists. Selectivity for stimulation of D(2), over D(3), receptors may therefore be a beneficial property of dopamine receptor agonists in management of motor symptoms of Parkinson's disease patients with established dyskinesia.

Discriminative stimulus properties of antidepressant agents: a review.

Though drug discrimination techniques have proven invaluable in characterizing the interoceptive properties of drugs of abuse, antipsychotics and anxiolytics, with the exception of some fragmentary data with tricyclic agents, surprisingly few studies have been undertaken with antidepressants. Nevertheless, the preferential dopamine (DA) reuptake inhibitor, bupropion, elicits a robust discriminative stimulus in rodents. Moreover, in rats trained on a two-lever FR-10 schedule for food reward, the selective serotonin (5-HT) reuptake inhibitor (SSRI), citalopram, and the noradrenaline (NA) reuptake inhibitor (NARI), reboxetine, elicit discriminative stimuli at doses that selectively elevate extracellular levels of 5-HT and NA, respectively. In generalization tests, mixed inhibitors of 5-HT and NA reuptake, such as venlafaxine, substitute for both citalopram and reboxetine, while SSRIs substitute for citalopram but not for reboxetine. Intriguingly, selective NARIs appear to substitute both for reboxetine and for citalopram though, owing to long-term instability of the citalopram cue, the latter observation will require confirmation. Bupropion and the atypical antidepressant, mirtazapine - a 5-HT2/alpha2-adrenoceptor (AR) antagonist devoid of affinity for 5-HT and NA reuptake sites - substitute for neither citalopram nor reboxetine, indicating that 'antidepressant' effects per se do not account for their interoceptive properties. Moreover, mirtazapine abolishes the citalopram cue, an action mimicked by the selective 5-HT2C antagonist, SB242,084. The discriminative stimulus elicited by reboxetine is blocked by the alpha1-AR antagonist, prazosin. In contrast, it is not significantly attenuated by the alpha2-AR antagonist, RX821,002, nor by betaxolol or ICI118,551, antagonists at alpha1- and alpha2-ARs, respectively. These observations indicate that 5-HT2C receptors and alpha1-ARs contribute to the discriminative stimulus properties of SSRIs and NARIs, respectively. The present article reviews the literature devoted to the discriminative stimulus properties of antidepressant agents as training drugs, focusing in particular upon novel data with citalopram and reboxetine. In addition, several open questions and future research directions are evoked. It would be of considerable interest to extend such drug discrimination studies to other classes of antidepressants or potential antidepressants, including venlafaxine, mirtazapine and antagonists at neuropeptide (corticotropin releasing factor1 and neurokinin1) receptors.

Comparison of hippocampal G protein activation by 5-HT(1A) receptor agonists and the atypical antipsychotics clozapine and S16924.

This study employed [(35)S]guanosine 5'- O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to compare the actions of antipsychotic agents known to stimulate cloned, human 5-HT(1A) receptors with those of reference agonists at postsynaptic 5-HT(1A) receptors. In rat hippocampal membranes, the following order of efficacy was observed (maximum efficacy, E(max), values relative to 5-HT=100): (+)8-OH-DPAT (85), flesinoxan (62), eltoprazine (60), S14506 (59), S16924 (48), buspirone (41), S15535 (22), clozapine (22), ziprasidone (21), pindolol (7), p-MPPI (0), WAY100,635 (0), spiperone (0). Despite differences in species and tissue source, the efficacy and potency (pEC(50)) of agonists (with the exception of clozapine) correlated well with those determined previously at human 5-HT(1A) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast, clozapine was more potent at hippocampal membranes. The selective antagonists p-MPPI and WAY100,635 abolished stimulation of binding by (+)8-OH-DPAT, clozapine and S16924 (p-MPPI), indicating that these actions were mediated specifically by 5-HT(1A) receptors. Clozapine and S16924 also attenuated 5-HT- and (+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding, consistent with partial agonist properties. In [(35)S]GTPgammaS autoradiographic studies, 5-HT-induced stimulation, mediated through 5-HT(1A) receptors, was more potent in the septum (pEC(50) approximately 6.5) than in the dentate gyrus of the hippocampus (pEC(50) approximately 5) suggesting potential differences in coupling efficiency or G protein expression. Though clozapine (30 and 100 microM) did not enhance [(35)S]GTPgammaS labelling in any structure, S16924 (10 micro M) modestly increased [(35)S]GTPgammaS labelling in the dentate gyrus. On the other hand, both these antipsychotic agents attenuated 5-HT (10 microM)-stimulated [(35)S]GTPgammaS binding in the dentate gyrus and septum. In conclusion, clozapine, S16924 and ziprasidone act as partial agonists for G protein activation at postsynaptic 5-HT(1A) receptors in the hippocampus. These data support a role of postsynaptic 5-HT(1A) receptors in the functional profiles of certain antipsychotic agents.