Marble Burying in NMRI Male Mice Is Preferentially Sensitive to Pre- Versus Postsynaptic 5-HT1A Receptor Biased Agonists.

Obsessive compulsive disorder (OCD) is a psychiatric disorder characterized by excessive and repetitive thoughts and gestures, mainly treated pharmacologically with selective serotonin reuptake inhibitors (SSRIs). The marble burying test in mice is commonly used to model OCD and has been shown to be sensitive to SSRIs, which decrease burying behavior. The activity of SSRIs in this model is mediated through activation of 5-hydroxytryptamine (5-HT) 1A receptors, but the respective implication of pre- versus postsynaptic 5-HT1A receptors has not been elucidated. Here, we investigated marble burying behavior by male NMRI mice following acute administration of 3 biased agonists, which preferentially activate presynaptic 5-HT1A receptors (F13714) or postsynaptic receptors (NLX-101) or which exhibit balanced activation of both pre- and postsynaptic 5-HT1A receptors (NLX-112). When administered at the dose of 2.5 mg/kg i.p., all 3 biased agonists completely or nearly completely abolished marble burying behavior. However, they varied in their potency with minimal effective doses of 0.16, 0.63, and 2.5 mg/kg i.p., for F13714, NLX-112, and NLX-101, respectively. The selective 5-HT1A receptor antagonist, WAY100,635 was inactive up to 2.5 mg/kg. These results suggest that marble burying behavior in male NMRI mice is preferentially sensitive to activation of pre- versus postsynaptic 5-HT1A receptors. Moreover, they suggest that targeting 5-HT1A receptors with biased agonists could provide an innovative therapeutic approach to combat OCD.

The clozapine metabolite N-desmethylclozapine displays variable activity in diverse functional assays at human dopamine D2 and serotonin 5-HT1A receptors.

N-desmethylclozapine (NDMC or norclozapine) is the major active metabolite of the antipsychotic clozapine in humans. The activity of NDMC differs from clozapine at a number of neurotransmitter receptors, probably influencing the pharmacological effects of clozapine treatment. Here, we tested the properties of NDMC in comparison with clozapine at recombinant human dopamine D(2) and serotonin 5-HT(1A) receptors, using a panel of functional assays implicating diverse signalling pathways. At dopamine D(2) receptors, NDMC as well as clozapine did not display agonist activity in measures of G protein activation by [(35)S]GTPγS binding and in the sensitive Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation assay. In contrast, there were weak partial agonist actions of NDMC (but not of clozapine) for dopamine D(2)-dependent activation of Ca(2+) liberation via coexpressed chimeric Gα(q/o) proteins and for G protein-coupled inward rectifier potassium channel (GIRK) current induction in Xenopus oocytes. Intriguingly, GIRK currents induced by NDMC via dopamine D(2) receptors showed a rapid and transient time course, strikingly different from currents recorded with other receptor agonists. At serotonin 5-HT(1A) receptors, NDMC was a more efficacious partial agonist than clozapine for [(35)S]GTPγS binding, ERK1/2 phosphorylation and GIRK activation. Respective low and moderate partial agonist properties of NDMC at dopamine D(2) and serotonin 5-HT(1A) receptors thus differentiate the metabolite from its parent drug and may contribute to the overall effects of clozapine pharmacotherapy.

F15063, a potential antipsychotic with dopamine D(2)/D(3) receptor antagonist and 5-HT(1A) receptor agonist properties: influence on immediate-early gene expression in rat prefrontal cortex and striatum.

Brain region-specific modulation of immediate-early gene (IEG) may constitute a marker of antipsychotic drug-like activity. We investigated the effects of the putative antipsychotic drug N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine (F15063), a compound that targets both dopamine D(2) and serotonin 5-HT(1A) receptors, in comparison with haloperidol and clozapine on rat mRNA expression of IEG i.e. the zinc-fingered transcription factors c-fos, fosB, zif268, c-jun and junB, two transcription factors of the nuclear receptor family nur77 and nor1, and the effector IEG arc. F15063 (10 mg/kg) and clozapine (10 mg/kg), but not haloperidol (0.63 mg/kg), induced c-fos and fosB mRNA expression in prefrontal cortex, a region associated with control of cognition and negative symptoms of schizophrenia. In striatum, only c-fos, fosB, junB and nur77 were induced by clozapine whereas all IEG mRNAs were increased by haloperidol and F15063 (from 2.5 mg/kg) with similar high efficacy despite a total absence of F15063-induced catalepsy. However, at 0.63 mg/kg, F15063 induced a lower degree of striatal IEG mRNA expression than haloperidol and pretreatment with the serotonin 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride (WAY100635) (0.63 mg/kg) increased the level of IEG mRNA induction by F15063. Furthermore, (+)-8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] at 0.16 mg/kg decreased haloperidol-induced striatal IEG mRNA expression although it exerted no effects on its own. These results are consistent with an activation of serotonin 5-HT(1A) receptors by F15063, thus reducing D(2) blockade-induced striatal IEG mRNA. Furthermore, the substantial F15063-induced expression of IEGs such as c-fos in striatum is not related to cataleptogenic activity and may act more as a marker of efficacious dopamine D(2) receptor blockade.

Activation of G proteins and extracellular signal-regulated kinase 1/2 phosphorylation via human dopamine D4.4 receptors: differential pathway-dependent potencies of receptor agonists.

Agonist activity at recombinant human dopamine D4.4 receptors was compared in stably transfected CHO cells using two functional readouts: G protein activation by [35S]GTPgammaS binding and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2). Results with a large series of agonists reveal markedly higher relative agonist efficacy in the pERK1/2 assay compared with [35S]GTPgammaS binding, while potencies were generally higher in the latter readout. Whereas efficacies were highly correlated when comparing both tests, potencies determined using the pERK1/2 assay were neither correlated with those for G protein activation nor with binding affinities. In order to examine if these differences may be attributable to distinct assay conditions (5 min incubation for pERK1/2 compared with binding equilibrium conditions for [35S]GTPgammaS), selected compounds were tested in a modified short-duration [35S]GTPgammaS binding assay. In these experiments, potencies were generally reduced; however, compounds exhibiting comparably high potency in the pERK1/2 assay were not affected by this duration-dependent potency shift. We conclude that assay parameters such as signal amplification and incubation time have to be considered with respect to the appropriate choice of experimental approaches that best reflect agonist activity at dopamine D4 receptors in vivo.

Effects of antipsychotics and reference monoaminergic ligands on marble burying behavior in mice.

Antipsychotics constitute efficacious augmenting agents in the treatment of anxiety disorders, including refractory obsessive-compulsive disorder. We examined the effects of 36 compounds, including typical, atypical and novel antipsychotics with dual dopamine D2/5-hydroxytryptamine 1A (D2/5-HT1A) actions on marble burying behavior in mice, a putative preclinical test for anxiety disorders. One hour after drug administration, male NMRI mice were placed individually in cages containing 20 marbles, and the total number of marbles buried after 30 min was counted. The selective serotonin reuptake inhibitors, citalopram (2.5-40 mg/kg), fluoxetine (2.5-10 mg/kg) and the benzodiazepine diazepam (2.5-10 mg/kg), reduced the number of buried marbles. The atypical antipsychotic, clozapine (0.16-10 mg/kg), but not its congener olanzapine, was effective in this test. Haloperidol, a typical antipsychotic, also reduced the number of buried marbles, albeit not in a dose-dependent manner. The atypical risperidone was partially active (0.16-0.63 mg/kg), as was the benzamide derivative, amisulpride, albeit at high (10-40 mg/kg) doses. Among the 'third-generation' antipsychotics possessing combined D2/5-HT1A properties, bifeprunox was active at 0.0025 mg/kg, whereas SLV313 and aripiprazole were active only at the highest doses (2.5 and 10 mg/kg, respectively). SSR181507, F15063 and the antidyskinetic agent, sarizotan, were without any effect. Among a series of receptor subtype-selective ligands, only the 5-HT1A agonist, (+)-8-OH-DPAT (0.63-2.5 mg/kg) and the 5-HT2A/2B/2C antagonist, ritanserin (0.63-2.5 mg/kg) were active. Among novel antipsychotics with dual D2/5-HT1A properties, only bifeprunox was able to potently reduce the number of buried marbles. Inhibition of marble burying behavior may result from the interplay of several receptor systems, including 5-HT2 receptor blockade, dopamine D2 partial agonism and serotonin 5-HT1A agonism.

Action of novel antipsychotics at human dopamine D3 receptors coupled to G protein and ERK1/2 activation.

The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.

Differential profile of antipsychotics at serotonin 5-HT1A and dopamine D2S receptors coupled to extracellular signal-regulated kinase.

The effects of antipsychotics targeting dopamine D2 and serotonin 5-HT1A receptors were compared with conventional antipsychotics on phosphorylation of Extracellular signal-Regulated Kinase 1/2 (ERK 1/2) in CHO cell lines stably expressing either the human serotonin 5-HT1A or human dopamine D2S receptor. All antipsychotics except haloperidol and olanzapine exhibited agonist properties at serotonin 5-HT1A receptors. Emax values (% effect of 10 microM 5-HT) were: bifeprunox (74), SSR181507 (73), SLV313 (72), aripiprazole (60), ziprasidone (56), clozapine (33). At dopamine D2S receptors, partial agonist activity (% effect of 10 microM dopamine) was observed for bifeprunox (76), SSR181507 (66) and aripiprazole (59). Other antipsychotics attenuated dopamine-induced ERK phosphorylation, with pK(B) values of : SLV313 (8.5), haloperidol (8.1), olanzapine (7.8), ziprasidone (7.7), and clozapine (6.4). Amongst the dopamine D2/serotonin 5-HT1A receptor compounds, aripiprazole acts as a partial dopamine D2S and serotonin 5-HT1A receptor agonist. SSR181507 and bifeprunox possess a profile of action similar to each other, efficaciously stimulating both serotonin 5-HT1A and dopamine D2S receptors. In contrast, SLV313, also an efficacious serotonin 5-HT1A receptor agonist, acted as a high potency dopamine D2 receptor antagonist. Thus, antipsychotics display varying efficacies at serotonin 5-HT1A and dopamine D2S receptors which may play a major role in their differential functional profiles in blocking the diverse symptoms of schizophrenia.

Novel antipsychotic agents with 5-HT(1A) agonist properties: role of 5-HT(1A) receptor activation in attenuation of catalepsy induction in rats.

Compounds possessing 5-HT(1A) agonist properties attenuate catalepsy induced by D(2) receptor blockade. Here we examined the role of 5-HT(1A) receptor agonism in the reduced cataleptogenic potential of several novel antipsychotic agents in the crossed leg position (CLP) and the bar catalepsy tests in rats. When administered alone, ziprasidone produced marked catalepsy, whereas aripiprazole, bifeprunox, SLV313, SSR181507 and sarizotan did not. However, when 5-HT(1A) receptors were blocked with the selective antagonist, WAY100635 (0.63 mg/kg, SC), robust cataleptogenic properties of SLV313, bifeprunox and sarizotan were unmasked and the catalepsy induced by ziprasidone was accentuated. In contrast, only modest catalepsy was induced by aripiprazole and SSR181507, even following a higher dose of WAY100635 (2.5 mg/kg). This suggests that these compounds possess other anti-cataleptic properties, such as partial agonism at dopamine D(2) receptors. The capacity to reverse neuroleptic-induced catalepsy was investigated in interaction studies with haloperidol (2.5 mg/kg, SC). Whereas ziprasidone and aripiprazole did not markedly reduce the effects of haloperidol, SLV313 and sarizotan attenuated CLP catalepsy. In contrast, SSR181507 and bifeprunox potently inhibited both CLP and bar catalepsy. Taken together, these data show that 5-HT(1A) receptor activation reduces the cataleptogenic potential of novel antipsychotic agents but indicate marked diversity in the contribution of 5-HT(1A) and/or other mechanisms to the profiles of the drugs.

Effects of novel antipsychotics with mixed D(2) antagonist/5-HT(1A) agonist properties on PCP-induced social interaction deficits in the rat.

Considerable interest has arisen in identifying antipsychotic agents with improved efficacy against negative symptoms, such as social withdrawal. In rats, a social interaction deficit can be induced by the NMDA antagonist phencyclidine (PCP). Here, we examined the effects of antipsychotics, reported to exert dual 5-HT(1A)/D(2) actions, on PCP-induced social interaction deficits. Drugs were administered daily for 3 days in combination with either vehicle or PCP (2.5mg/kg, SC) and social interaction was measured on the last day of drug treatment. Pairs of unfamiliar rats receiving the same treatment were placed in a large open field for 10 min and the number of social behaviors were scored. The results indicate that: (1) PCP significantly reduced social interaction by over 50% compared with vehicle-treated controls; (2) haloperidol (0.0025-0.16 mg/kg, SC) and clozapine (0.04-10mg/kg, IP) did not reverse PCP-induced social interaction deficits; (3) the substituted benzamide remoxipride reversed PCP-induced deficits at 0.63 and 2.5mg/kg (4) the 5-HT(1A) agonist 8-OH-DPAT was inactive (at 0.01-0.63 mg/kg, SC); (5) among compounds reported to exert dual 5-HT(1A)/D(2) actions, SSR181507 (at 0.16 mg/kg, SC) and aripiprazole (at 0.04 and 0.16 mg/kg, IP), but not ziprasidone (0.04-2.5mg/kg, IP), SLV313 (0.0025-0.16 mg/kg, SC) or bifeprunox (0.01-0.63 mg/kg, IP), significantly reversed PCP-induced social interaction deficits; and (6) the 5-HT(1A) receptor antagonist WAY100635 blocked the effects of SSR181507 and aripiprazole. These findings indicate that the balance of activity at 5-HT(1A) and D(2) receptors profoundly influences the activity of antipsychotics in this model of social withdrawal, and their potential benefit on at least some of the negative symptoms of schizophrenia.

Tolerance and inverse tolerance to the hyperalgesic and analgesic actions, respectively, of the novel analgesic, F 13640.

5-HT(1A) receptor activation by the very-high-efficacy, selective 5-HT(1A) receptor agonist F 13640 [(3-Chloro-4-fluoro-phenyl)-[4-fluoro-4-([(5-methyl-pyridin-2-ylmethyl)-amino]-methyl)piperidin-1-yl]-methanone] was recently discovered to constitute a novel central mechanism of broad-spectrum analgesia that, remarkably, grows rather than decays with chronicity. However, in rodents not exposed to nociception, F 13640 induces its analgesic effect only after having initially induced hyperalgesia. Numerical simulations implementing a signal transduction theory here show that the progressive increase in the intensity of nociceptive stimulation which F 13640 presumably mimics should eventually produce a large analgesic effect without initially causing marked pain. In vivo studies examined the effects of progressively increasing doses of F 13640 on the threshold of mechanically induced vocalization and, also, on the 5-HT syndrome in rats. The infusion of increasing (0.04-0.63 mg/rat/day) doses of F 13640 over a 5-week period induced a large analgesia preceded by a hyperalgesic effect that was small and comparable to that induced by initial exposure to a low, 0.04 mg/rat/day dose. Furthermore, increasing the dose of F 13640 induced tachyphylaxis to the 5-HT syndrome. Producing the mirror opposite of morphine's neuroadaptive actions, F 13640 causes an analgesia that becomes more powerful with chronic administration, and this at the expense of the initial hyperalgesia which it may also produce.

Tacrine-scopolamine interactions on state-dependent retrieval.

OBJECTIVES: This study examined the effects of tacrine on scopolamine-induced state-dependence. METHODS: Rats were trained to complete an FR10 schedule of lever presses for milk reward within 120 s after the onset of an operant session and were subsequently tested for the retrieval of the response in either the same or a different, pharmacologically defined, state. RESULTS: In rats trained with 2.5 mg/kg scopolamine, the pre-test administration of 10 mg/kg tacrine prevented scopolamine from enabling the retrieval that otherwise occurred when animals were both trained and tested with scopolamine. However, retrieval of the response was also hampered in animals that were trained with tacrine-scopolamine co-administration and tested with saline, and vice versa, indicating that the co-administration of tacrine and scopolamine did not induce the saline-associated, presumably normal state. At >/=2.5 mg/kg doses, tacrine itself induced state-dependence with both tacrine-to-saline and saline-to-tacrine state changes. CONCLUSION: The findings indicate that tacrine is unable to normalize the particular mnesic state induced by scopolamine. The data may elucidate tacrine's limited therapeutic efficacy insofar as scopolamine's mnesic actions both model human pathology and are due to scopolamine producing state-dependence.