WAY-100635 has high selectivity for serotonin 5-HT(1A) versus dopamine D(4) receptors

The serotonin 5-HT(1A) receptor antagonist WAY-100635 was recently reported to have potent agonist properties at dopamine D(4) receptors (Chemel et al., 2006, Psychopharmacology 188, 244-251.). Herein WAY-100635 (pK(i) at human (h) serotonin 5-HT(1A) receptors=9.51; pK(i) at dopamine hD(4.4) receptors=7.42) stimulated [(35)S]GTPgammaS incorporation in membranes of Chinese Hamster Ovary cells expressing dopamine hD(4.4) receptors with only moderate potency and modest efficacy (pEC(50)=6.63; E(max)=19% of dopamine). Moreover, in antagonism experiments, WAY-100635 had a much lower potency at dopamine hD(4.4) receptors (pK(B)=7.09), than at serotonin h5-HT(1A) receptors (pK(B)=9.47). These data demonstrate that WAY-100635 has high selectivity for serotonin h5-HT(1A)versus dopamine hD(4.4) receptors.     

5-HT_(1A)受体拮抗剂WAY-100635的合成

2-氨基吡啶和2-氯乙酰氯经酰胺化、与N'-(邻甲氧基苯基)哌嗪缩合、氢化铝锂还原得4-(邻甲氧基苯基)-1-[2-[(2-吡啶基)氨基]乙基]哌嗪,再与环己甲酰氯缩合得到5-HT1A受体拮抗剂WAY-100635,总收率35%。     

Effects of 8-OH-DPAT and WAY-100635 on performance on a time-constrained progressive-ratio schedule

Rationale. Performance on progressive-ratio schedules has been proposed as a means of assessing the effects of drugs on motivation. We have adopted a mathematical model proposed by Killeen to analyse the effects of drugs acting at 5-HT_1A receptors on progressive-ratio performance. According to this model, the relationship between response rate and ratio size is described by a bitonic (inverted-U) function. One parameter of the function, a, expresses the motivational or "activating" effect of the reinforcer (duration of activation of responding produced by the reinforcer), whereas another parameter, δ, expresses the minimum time needed to execute a response and is regarded as an index of "motor capacity". Objective. To examine the effect of the selective 5-HT_1A receptor agonist 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] and the antagonist WAY-100635 [N-[2-(4-[2-methoxyphenyl]-1-piperazinyl)ethyl]-N-2-pyridinylcyclo-hexanecarboxamide] on progressive-ratio schedule performance. Methods. Sixteen rats responded for a food-pellet reinforcer on a time-constrained progressive-ratio schedule (55-min sessions). In phase 1, they received single doses (s.c.) of 8-OH-DPAT (25, 50, 100, 200 μg kg⁻¹, four treatments at each dose) or the vehicle (0.9% saline solution). In phase 2, they received WAY-100635 (30, 100, 300 μg kg⁻¹) according to the same regimen. In phase 3, they received 8-OH-DPAT (100 μg kg⁻¹) alone or in combination with WAY-100635 (30 μg kg⁻¹). 8-OH-DPAT dose dependently increased the value of a, significant increases being seen with the 50, 100 and 200 μg kg⁻¹ doses. The highest dose also increased δ. WAY-100635 did not significantly alter either a or δ. WAY-100635 significantly attenuated the effect of 8-OH-DPAT on both a and δ. Conclusions. The results suggest that 8-OH-DPAT enhanced the activating effect of the reinforcer (the highest dose may also have induced motor debilitation). The finding that the effect of 8-OH-DPAT on a was attenuated by WAY-100635 implicates 5-HT_1A receptors in this effect. The results are consistent with previous reports that 8-OH-DPAT facilitates feeding and food-reinforced operant responding in rats and suggest that these effects may be brought about by an increase in food motivation. Electronic Publication     

BIMT 17, a 5-HT2A receptor antagonist and 5-HT1A receptor full agonist in rat cerebral cortex

In the search for antidepressant agents with a rapid onset of action, we have found that compound BIMT 17 (1-[2-[4-(3-trifluoromethylphenyl)piperazin1-yl]ethyl]benzimidazol-[1H]-2-one) shows a good affinity for cerebral cortical 5-HT_1A (pK _i = 7.72) and 5-HT_2A (pK _i = 6.90) receptors, with no appreciable affinity for the other 5-HT receptor subtypes, including 5-HT_2C. BIMT 17 reduced forskolin-stimulated cAMP accumulation in the cerebral cortex (pEC₅₀ = 6.09) and in the hippocampus (pEC₅₀ = 6.50), and antagonized 5-HT-induced phosphatidylinositol turnover (pK _i = 6.96) in the cerebral cortex. The effect on cAMP accumulation was blocked by the 5-HT_1A receptor antagonist tertatolol. Buspirone, 8-OH-DPAT and S 14671 {1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphtyl)]piperazine, claimed to be 5-HT_1A receptor agonists, did not reduce forskolin-stimulated cAMP formation in the cerebral cortex.On the basis of these data, it was concluded that BIMT 17 was the only compound that behaved as a full agonist with respect to the CAMP response in the cortex, while exerting concurrent agonism at 5-HT_1A receptors and antagonism at 5-HT_2A receptors. These characteristics might explain the peculiar behaviour of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).     

双重作用的多巴胺D2/5-HT2A受体拮抗剂比较药效团分析

双重作用的多巴胺D₂/5-HT_2A受体拮抗剂是开发非典型抗精神病药物的有效途径,但最新研究显示, 非典型抗精神病药物将显著增加患者因心律失常及其他心脏疾病而猝死的风险,本文对D₂/5-HT_2A受体拮抗剂的药效团模型以及可能引起心血管风险的α_1A肾上腺素受体拮抗剂和hERG K⁺通道阻断剂的药效团模型进行比较分析,从药效团模型的角度分析多靶点药物的设计。     

PSAB-OFP,a selective alpha 7 nicotinic receptor agonist,is also a potent agonist of the 5-HT3 receptor

5-Hydroxytryptamine 3 (5-HT(3)) and alpha 7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of alpha 7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective alpha 7 receptor agonists. We used a recently reported selective alpha 7 receptor agonist, (R)-(-)-5'Phenylspiro[1-azabicyclo[2.2.2] octane-3,2'-(3'H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant alpha 7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT(3) receptors. To assess the functional activity of PSAB-OFP on 5-HT(3) receptors we studied recombinant human 5-HT(3) receptors expressed in Xenopus oocytes, as well as native mouse 5-HT(3) receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca(2+) imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both alpha 7 and 5-HT(3) receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective alpha 7 receptor ligands.     

Chronic fluoxetine-induced desensitization of 5-HT1A and 5-HT1B autoreceptors: regional differences and effects of WAY-100635

Desensitization of 5-HT(1A) and 5-HT(1B) autoreceptors is thought to be the mechanism underlying the therapeutic effects of fluoxetine and other selective serotonin reuptake inhibitors when these are administered chronically. The blockade of 5-HT(1A) autoreceptors occurring on administration of a selective serotonin reuptake inhibitor together with a 5-HT(1A) autoreceptor antagonist is responsible for the acute increase in 5-hydroxytryptamine (serotonin, 5-HT) levels observed under these circumstances. The effects of repeated administration of selective serotonin reuptake inhibitors together with 5-HT(1A) receptor antagonists have not been widely studied. In this work, we found that the effects of fluoxetine (5 mg/kg, i.p., daily for 12 days) to desensitize 5-HT(1B) autoreceptors in the frontal cortex, as measured by the effect of the locally administered 5-HT(1B) receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129), and to desensitize 5-HT(1A) autoreceptors as measured by the action of the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 50 microg/kg, s.c.) to reduce 5-HT levels in cortex, were prevented by concomitant administration of the 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635; 0.3 mg/kg, s.c.). 5-HT(1B) receptor activity in the hypothalamus, as measured by the effects of locally administered CP 93129, and 5-HT(1A) autoreceptor activity, as determined by the effects of subcutaneous 8-OH-DPAT to reduce 5-HT levels in hypothalamus, were not altered either by fluoxetine alone or by fluoxetine in the presence of WAY-100635. The data suggest that the regulation of extracellular levels of 5-HT in the cortex and hypothalamus is subject to different autoregulatory mechanisms.     

Serotonin 1A receptor availability in patients with schizophrenia and schizo-affective disorder: a positron emission tomography imaging study with [11C]WAY 100635

Background Postmortem and positron emission tomography (PET) studies have reported several alterations in serotonin 1A receptor (5-HT_1A) binding parameters in patients with schizophrenia. This study examines 5-HT_1A availability in vivo in individuals with schizophrenia and schizo-affective disorder.Materials and methods Twenty-two medication-free individuals with schizophrenia or schizo-affective disorder and 18 healthy subjects underwent PET scans with [¹¹C]WAY 100635. Regional distribution volumes (V _T, in milliliters per gram) were derived using a two-tissue compartment kinetic model. Outcome measures for 5-HT_1A availability included binding potential (BP) and the specific to nonspecific equilibrium partition coefficient (V ₃″). Eleven brain regions with high density of 5-HT_1A were included in the analysis.Results No significant differences were observed in regional BP or V ₃″ between patients and controls. No significant relationships were observed between regional 5-HT_1A availability and symptom severity.Conclusion The postmortem literature reports increased 5-HT_1A binding in the prefrontal cortex in schizophrenia. This study did not detect differences in 5-HT_1A binding. Whereas in two recently published PET studies, one reports increased binding in the temporal lobe while the other reports decreased binding in the amygdala. These inconsistencies suggest that the alterations demonstrated in postmortem studies cannot be reliably detected at the resolution achieved with PET. This raises the question as to whether major changes in the level of expression of the 5-HT_1A receptor play a role in the pathophysiology of schizophrenia.Dr. Lombardo participated in this work while at Columbia University but subsequently has moved to Pfizer Inc., 235 east 42nd Street MS 10/33, New York, NY 10017, USA.     

BIMT 17, a 5-HT1A receptor agonist/5-HT2A receptor antagonist,directly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex

BIMT 17 (1-[2-[4-(3-trifluoromethyl phenyl) piperazin-1-yl] ethyl] benzimidazol- [1H]-2-one), a 5-HT_1A receptor agonist/5-HT_2A receptor antagonist (see Borsini et al., accompanying paper), in a dose range of 1–10 mg/kg i.v., dose-dependently inhibited the electrical activity of rat medial prefronto-cortical neurons, whereas buspirone, in a dose range of 0.1–1000 g/kg, increased it. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and 1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphthyl)] piperazine (S 14671) presented biphasic patterns of response; they increased electrical activity at doses in the range of 0.1–10 g/kg and 0.1–3 g/kg i.v. respectively, and reduced it at higher doses, 30–300 g/kg and 10–30 g/kg i.v., respectively.The inhibitory effect of BIMT 17 on the firing rate of neurons in the frontal cortex was antagonized by the 5-HT_1A antagonists tertatolol and WAY 100135, and was still present after destruction of serotonin (5-HT) containing neuronal endings by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 150 g/rat, given intraventricularly), which reduced the cortical 5-HT content by 85%. This destruction of 5-HT neurons, while suppressing the ability of 8-OH-DPAT to inhibit the firing rate at high doses, did not change the excitatory action of this compound at low doses. The addition of ritanserin, a 5-HT2A receptor antagonist, potentiated both the excitatory and inhibitory effects of 8-OHDPAT on neuronal electrical activity. Direct microiontophoretic application (100 nA/20 s) of 5-HT and BIMT 17, but not that of 8-OH-DPAT, onto medial prefronto-cortical neurons, decreased the firing rate of these neurons.These findings suggest that BIMT 17 directly inhibits the electrical activity of medial prefronto-cortical neurons through its dual mode of receptor interaction.     

Pharmacological analysis of the haemodynamic effects of 5-HT1B/D receptor agonists in the normotensive rat

1. The receptors involved in mediating the haemodynamic effects of three 5-HT_1B/D receptor agonists were investigated in pentobarbitone anaesthetized rats (n=6–17 per group).
2. Cumulative intravenous (i.v.) infusions of rizatriptan and sumatriptan (from 0.63 to 2500 μg kg⁻¹; each dose over 5 min) induced dose-dependent and marked hypotension (−42±6 and −34±4 mmHg at the highest dose, respectively; both P<0.05 vs vehicle: +5±3 mmHg) and bradycardia (−85±16 and −44±12 beats min⁻¹ at the highest dose, respectively; both P<0.05 vs vehicle: +16±6 beats min⁻¹). Zolmitriptan evoked only moderate hypotension at the highest dose (−19±9 mmHg; P<0.05 vs vehicle).
3. A high dose of the 5-HT_1B/D receptor antagonist, GR 127935 (0.63 mg kg⁻¹, i.v.), failed to antagonize the hypotension and bradycardia evoked by sumatriptan (−35±6 mmHg and −52±19 beats min⁻¹, respectively; both not significant vs sumatriptan in untreated rats), but moderately reduced the hypotension and bradycardia evoked by rizatriptan (−20±5 mmHg and −30±17 beats min⁻¹, respectively; both P<0.05 vs vehicle and vs rizatriptan in untreated rats).
4. The selective 5-HT_1A receptor antagonist, WAY 100635 (0.16 and 0.63 mg kg⁻¹, i.v.), dose-dependently attenuated the haemodynamic responses evoked by rizatriptan and sumatriptan, which were almost abolished by the higher dose of WAY 100635 (−4±3 mmHg and −15±8 beats min⁻¹; both not significant vs vehicle and P<0.05 vs rizatriptan in untreated rats). A slight but statistically significant reduction in mean arterial pressure (MAP) persisted at the highest dose of sumatriptan (−13±4 mmHg following the higher dose of WAY 100635; P<0.05 vs vehicle).
5. In pithed rats with MAP normalized by angiotensin II, rizatriptan failed to induce hypotension or bradycardia (+5±4 mmHg and −6±16 beats min⁻¹, respectively; both NS vs vehicle and P<0.05 vs rizatriptan in untreated rats). Similarly, sumatriptan failed to induce bradycardia in pithed rats (+5±6 beats min⁻¹; not significant vs vehicle and P<0.05 vs sumatriptan in untreated rats), whereas a slight but statistically significant reduction in MAP, compared to controls, occurred at the highest dose (−9±9 mmHg; P<0.05 vs both vehicle and sumatriptan in untreated rats).
6. In bilaterally vagotomized and atropine-treated (1 mg kg⁻¹, i.v.) rats, the reductions in MAP and heart rate evoked by rizatriptan (−31±4 mmHg and −64 ±9 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs rizatriptan in controls) and sumatriptan (−47±8 mmHg and −56±10 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs sumatriptan in controls) were not statistically significantly different from those observed in controls.
7. In conclusion, the 5-HT_1B/D receptor agonists, rizatriptan and sumatriptan, elicit hypotension and bradycardia in the normotensive anaesthetized rat predominantly via activation of central 5-HT_1A receptors, and a consequent reduction in sympathetic outflow.

     

WAY100635 prevents the changes induced by fluoxetine upon the 5-HT1A receptor functionality

5-HT1A receptor-mediated signalling in rat brain was evaluated after chronic administration (14 days; s.c.) of the selective serotonin reuptake inhibitor (SRRI) fluoxetine (10 mg/kg/day) alone, or in combination with the 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg/day). The density of 5-HT1A binding sites was unchanged following fluoxetine, WAY100635, or the combination of fluoxetine and WAY100635. However, the net stimulation of [35S]GTPgammaS binding induced by the 5-HT1A agonist 8-OH-DPAT was significantly attenuated in dorsal raphe nucleus (DRN), but not in hippocampus, after chronic fluoxetine. Moreover, depending of the area analysed, the basal binding of [35S]GTPgammaS was differentially affected by this treatment: increased in DRN and decreased in hippocampal dentate gyrus. Interestingly, the changes in [35S]GTPgammaS basal binding and on 5-HT1A receptors functionality were prevented by the concomitant administration of WAY100635. The inhibition of dorsal raphe firing by 8-OH-DPAT was also attenuated in fluoxetine-treated rats (ED50 = 2.12 +/- 0.32 microg/kg and 4.34 +/- 0.09 microg/kg, for vehicle and fluoxetine respectively), an effect which was also prevented by the concomitant administration of WAY100635 (ED50 = 2.10 +/- 0.58 microg/kg). Chronic administration of WAY100635 alone did not affect the 5-HT1A receptor-induced stimulation of [35S]GTPgammaS binding, nor the 8-OH-DPAT-induced inhibition of 5-HT neuron firing. These results demonstrate that the concomitant blockade of 5-HT1A receptors when administering fluoxetine prevents those adaptive changes of 5-HT1A receptor function associated with the chronic administration of this antidepressant. These findings could be relevant from the therapeutic point of view, and further support the potential benefit of treatments with a SSRI/5-HT1A receptor antagonist combination.     

Putative antipsychotics with pronounced agonism at serotonin 5-HT1A and partial agonist activity at dopamine D2 receptors disrupt basal PPI of the startle reflex in rats

INTRODUCTION: Prepulse inhibition (PPI) of the startle reflex has been extensively studied because it is disrupted in several psychiatric diseases, most notably schizophrenia. In rats, and to a lesser extent, in humans, PPI can be diminished by dopamine (DA) D(2)/D(3) and serotonin 5-HT(1A) receptor agonists. A novel class of potential antipsychotics (SSR181507, bifeprunox, and SLV313) possess partial agonist/antagonist properties at D(2) receptors and various levels of 5-HT(1A) activation. MATERIALS AND METHODS: It thus appeared warranted to assess, in Sprague-Dawley rats, the effects of these antipsychotics on basal PPI. RESULTS: SSR181507, sarizotan, and bifeprunox decreased PPI, with a near-complete abolition at 2.5-10 mg/kg; SLV313 had a significant effect at 0.16 mg/kg only. Co-treatment with the 5-HT(1A) receptor antagonist WAY100,635 (0.63 mg/kg) showed that the 5-HT(1A) agonist activity of SSR181507 was responsible for its effect. By contrast, antipsychotics with low affinity and/or efficacy at 5-HT(1A) receptors, such as aripiprazole (another DA D(2)/D(3) and 5-HT(1A) ligand), and established typical and atypical antipsychotics (haloperidol, clozapine, risperidone, olanzapine, quetiapine, and ziprasidone) had no effect on basal PPI (0.01-2.5 to 2.5-40 mg/kg). DISCUSSION: The present data demonstrate that some putative antipsychotics with pronounced 5-HT(1A) agonist activity, coupled with partial agonist activity at DA D(2) receptors, markedly diminish PPI of the startle reflex in rats. CONCLUSIONS: These data raise the issue of the influence of such compounds on sensorimotor gating in humans.     

The behavioural responses to 8-OH-DPAT,ipsapirone and the novel 5-HT1A receptor agonist Bay Vq 7813 in the pig

Summary In pigs, behavioural responses were examined after administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a full agonist at 5-hydroxytryptamine (5-HT) receptors of the 5-HT_1A subtype, and the pyrimidinylpiperazine derivatives ipsapirone and Bay Vq 7813 (2-[4-(2-pyrimidinyl)-1-piperazinylpropyl]-1,2-benzisothiazol-3(2H)one-1,1-dioxide), which act as partial agonists at 5-HT_1A receptors. The most prominent behavioural response examined after 8-OH-DPAT, 0.5 mg/kg i. m., ipsapirone, 2–5 mg/kg i.m., and Bay Vq 7813, 0.5–2 mg/kg i.m. or i.v., were head shakes. The potency of the three drugs to induce this behaviour correlated with their activity at 5-HT_1A receptors as determined by inhibition of forskolin-stimulated adenylate cyclase, substantiating that the head shake response has potential as a quantitative probe of in vivo receptor function. The 5-HT₂/5-HT_1C receptor antagonist ritanserin did not counteract the head shakes induced by ipsapirone, suggesting that neither 5-HT₂ nor 5-HT_1C receptors are involved in mediation of this response to this 5-HT_1A receptor agonist in pigs. Once daily administration of Bay Vq 7813 or ipsapirone for 3–5 days led to a reduction in the head shake response. 1-Pyrimidinylpiperazine (1-PP), a pharmacologically active metabolite shared by ipsapirone, Bay Vq 7813, and related pyrimidinylpiperazine derivatives, did not induce behavioural alterations in pigs. The data provide further evidence that marked species differences exist in functional responses to 5-HT receptor ligands. Send offprint requests to W. Löscher at the above address     

Antagonistic cannabinoid CB1/dopamine D2 receptor interactions in striatal CB1/D2 heteromers. A combined neurochemical and behavioral analysis

In vitro results show the ability of the CB(1) receptor agonist CP 55,940 to reduce the affinity of D(2) receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D(2) receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB(1)-D(2) receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB(1)/D(2) receptor-receptor interactions may occur in CB(1)/D(2) formed heteromers. Antagonistic CB(1)/D(2) interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB(1) receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB(1) receptor antagonist rimonobant but also the specific A(2A) receptor antagonist MSX-3 blocked the inhibitory effect of CB(1) receptor agonist on D(2)-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB(1)/D(2) receptor-receptor interactions within CB(1)/D(2) heteromers in which A(2A) receptors may also participate.     

Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices

1. The actions of N-(2-(-4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5-hydroxytryptamine_1A (5-HT_1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices.
2. WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (R_in), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum.
3. Bath application of either 5-hydroxytryptamine (5-HT, 10–30 μM) or 5-carboxamidotryptamine (5-CT, 300 nM) hyperpolarized the membrane potential (ΔV_m=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced R_in (−25±8% and −18±1%, respectively). 5-HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials.
4. 5-HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABA_A receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5-CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05).
5. WAY 100635 fully antagonized the hyperpolarization, the reduction of R_in, and the decrease in amplitude of e.p.s.ps elicited by 5-HT, while it did not affect the action of 5-HT on the action potential frequency adaptation. In the presence of WAY 100635, 5-HT elicited a depolarization which was blocked by 10–30 μM RS 23597-190, a selective 5-HT₄ receptor antagonist.
6. Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5-HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5-HT₄ receptor-mediated response. Since WAY 100635 selectively antagonizes 5-HT_1A receptor-mediated actions of 5-HT, our data also demonstrate that the inhibitory action of 5-HT on excitatory synaptic transmission in CA1 is mediated by 5-HT_1A receptors.

     

mCPP-induced hyperactivity in 5-HT2C receptor mutant mice is mediated by activation of multiple 5-HT receptor subtypes

The serotonin receptor agonist mCPP induces hyperlocomotion in 5-HT2C receptor knockout (KO) mice or in the presence of a 5-HT2C receptor antagonist. In the present group of experiments, we evaluate the role of 5-HT1A, 5-HT1B and 5-HT2A receptors in mCPP-induced hyperactivity in 5-HT2C KO mice. We also assess the ability of agonists at these receptors to induce hyperactivity in wildtype (WT) mice pre-treated with a selective 5-HT2C receptor antagonist. As previously reported, mCPP (3 mg/kg) induced hyperactivity in 5-HT2C KO mice. A combination of the 5-HT1B receptor agonist CP-94,253 (20 mg/kg) and the 5-HT1A receptor agonist 8-OH-DPAT (0.5 mg/kg) induced marked hyperactivity in WT but not in 5-HT2C KO mice, nor in mice treated with the selective 5-HT2C receptor antagonist, SB 242084 (1.5 mg/kg). Neither CP-94,253 nor 8-OH-DPAT had any intrinsic effect on locomotion in WTs. mCPP-induced hyperactivity was attenuated in 5-HT2C KO mice by the 5-HT1B receptor antagonist SB 224289 (2.5 mg/kg), and the 5-HT2A receptor antagonists ketanserin (0.3 mg/kg) and M100907 (0.01 mg/kg) but not by the 5-HT1A receptor antagonist WAY 100635 (1 mg/kg). The 5-HT(2A/2B/2C) receptor agonist, Ro 60-0175 (3 mg/kg), induced a modest increase in locomotor activity in WT mice pre-treated with SB 242084. However, the combination of Ro 60-0175 with CP-94,253 induced a substantial increase in activity in 5-HT2C KO mice, an effect comparable to mCPP-induced hyperactivity. Thus, joint activation of 5-HT1A and 5-HT1B receptors stimulates locomotion in WT mice but this response is dependent on a functional 5-HT2C receptor population and hence is absent in 5-HT2C KO mice. By contrast, mCPP-induced hyperactivity depends on the inactivation of a separate 5-HT2C receptor population and is mediated by 5-HT2A and 5-HT1B receptor activation.     

Cloning,pharmacological characterisation and tissue distribution of a novel 5-HT<Subscript>4</Subscript> receptor splice variant, 5-HT<Subscript>4(i)</Subscript>

5-HT₄ receptor pre-mRNA is alternatively spliced in human (h) tissue to produce several splice variants, called 5-HT_4(a) to 5-HT_4(h) and 5-HT_4(n). Polymerase chain reaction (PCR) with primers designed to amplify both 5-HT_4(a) and 5-HT_4(b) amplified three additional bands in different tissues, two representing different mRNA species both encoding 5-HT_4(g) and one representing mRNA for a novel splice variant named 5-HT_4(i), cloned from testis and pancreas respectively. Primary and nested PCR detected both 5-HT_4(g) and 5-HT_4(i) in multiple tissues. Whereas 5-HT_4(i), was found in all cardiovascular tissues analysed, 5-HT_4(g) was mainly present in atria. However, quantitative RT-PCR indicated 5-HT_4(g) expression also in cardiac ventricle. The pharmacological profiles and ability to activate adenylyl cyclase (AC) were compared between four recombinant h5-HT₄ splice variants (a, b, g and i) expressed transiently and stably in HEK293 cells. Displacement of [³H]GR113808 with ten ligands revealed identical pharmacological profiles (affinity rank order: GR125487, SB207710, GR113808>SB203186>serotonin, cisapride, tropisetron>renzapride, 5-MeOT>5-CT). In transiently transfected HEK293 cells cisapride was a partial agonist compared to serotonin at 5-HT_4(b), 5-HT_4(g) and 5-HT_4(i) receptors. In membranes from HEK293 cells stably expressing 5-HT_4(g) (3,000 fmol/mg protein) or 5-HT_4(i) (500 fmol/mg protein), serotonin and 5-MeOT were full agonists while cisapride was full agonist at 5-HT_4(g) and partial agonist at 5-HT_4(i), probably due to different receptor expression levels. At both 5-HT_4(g) and 5-HT_4(i), the behaviour of 5-HT₄ receptor antagonists was dependent on receptor level. At high receptor levels, tropisetron and SB207710 and to a variable extent SB203186 and GR113808 displayed some partial agonist activity, whereas GR125487 and SB207266 reduced the AC activity below basal, indicating both receptors to be constitutively active. We conclude that the novel 5-HT_4(i) receptor splice variant is pharmacologically indistinguishable from other 5-HT₄ splice variants and that the 5-HT_4(i) C-terminal tail does not influence coupling to AC.     

Functional selectivity of dopamine D1 receptor agonists in regulating the fate of internalized receptors

Recently, we demonstrated that D(1) agonists can cause functionally selective effects when the endpoints of receptor internalization and adenylate cyclase activation are compared. The present study was designed to probe the phenomenon of functional selectivity at the D(1) receptor further by testing the hypothesis that structurally dissimilar agonists with efficacies at these endpoints that equal or exceed those of dopamine would differ in ability to influence receptor fate after internalization, a functional endpoint largely unexplored for the D(1) receptor. We selected two novel agonists of therapeutic interest that meet these criteria (the isochroman A-77636, and the isoquinoline dinapsoline), and compared the fates of the D(1) receptor after internalization in response to these two compounds with that of dopamine. We found that dopamine caused the receptor to be rapidly recycled to the cell surface within 1h of removal. Conversely, A-77636 caused the receptor to be retained intracellularly up to 48 h after agonist removal. Most surprisingly, the D(1) receptor recovered to the cell surface 48 h after removal of dinapsoline. Taken together, these data indicate that these agonists target the D(1) receptor to different intracellular trafficking pathways, demonstrating that the phenomenon of functional selectivity at the D(1) receptor is operative for cellular events that are temporally downstream of immediate receptor activation. We hypothesize that these differential effects result from interactions of the synthetic ligands with aspects of the D(1) receptor that are distal from the ligand binding domain.     

Cocaine conditioning: reversal by autoreceptor dose levels of 8-OHDPAT

In order to investigate the contribution of serotonergic effects of cocaine to Pavlovian conditioning of cocaine locomotor stimulant effects, two experiments were conducted in which groups of rats (N = 10) received cocaine treatments (10 mg/kg) paired or unpaired to placement in an open-field environment. Initially, a cocaine conditioned locomotion stimulant effect was established. Next, additional Coc-P and Coc-UP pairings were carried out in conjunction with pretreatment injections of the 5-HT_1A agonist, 8-OHDPAT (0.01, 0.025 and 0.05 mg/kg) or saline. In experiment 1, the Coc-P group which received the saline pretreatment again exhibited conditioning but in the 8-OHDPAT pretreatment Coc-P group conditioning was eliminated. In the second experiment, the protocol of the first experiment was repeated but expanded in the post-conditioning phase to include an 8-OHDPAT plus the 5-HT_1A antagonist pretreatment Coc-P group. As in the first experiment, the 8-OHDPAT pretreatment Coc-P group did not exhibit a cocaine conditioned locomotion stimulant effect; whereas, the saline pretreatment Coc-P and the 8-OHDPAT plus WAY-100635 pretreatment Coc-P groups did exhibit the cocaine conditioned locomotion stimulant effect. These findings are consistent with an important role for serotonin in the maintenance of cocaine Pavlovian conditioned effects.     

Functional crosstalk and heteromerization of serotonin 5-HT2A and dopamine D2 receptors

The serotonin 5-HT_2A receptor (5-HT_2AR) and dopamine D₂ receptor (D₂R) are high-affinity G protein-coupled receptor targets for two different classes of antipsychotic drugs used to treat schizophrenia. Interestingly, the antipsychotic effects are not based on the regulation of same signaling mediators since activation of the 5-HT_2AR and of the D₂R regulate G_q/11 protein and G_i/o protein, respectively. Here we use radioligand binding and second messenger production assays to provide evidence for a functional crosstalk between 5-HT_2AR and D₂R in brain and in HEK293 cells. D₂R activation increases the hallucinogenic agonist affinity for 5-HT_2AR and decreases the 5-HT_2AR induced inositol phosphate production. In vivo, 5-HT_2AR expression is necessary for the full effects of D₂R antagonist on MK-801-induced locomotor activity. Co-immunoprecipitation studies show that the two receptors can physically interact in HEK293 cells and raise the possibility that a receptor heterocomplex mediates the crosstalk observed. The existence of this 5-HT_2AR-D₂R heteromer and crosstalk may have implications for diseases involving alterations of serotonin and dopamine systems and for the development of new classes of therapeutic drugs.