Mapping the effects of the selective dopamine D2/D3 receptor agonist quinelorane using pharmacological magnetic resonance imaging.

Dopamine agonists with a high affinity for D2 and D3 receptors have a biphasic effect on rodent locomotion, inducing hypolocomotion at low doses and hyperlocomotion at higher doses. Controversy surrounds the role of the D3 receptor in mediating the hypolocomotor response to low agonist doses. This study examines patterns of neuronal activation induced by varying doses of the D2/D3 receptor agonist quinelorane using blood oxygen level dependent (BOLD) pharmacological magnetic resonance imaging (phMRI), and compares them with corresponding behavioural responses. Quinelorane (3 microg/kg) induced hypolocomotion in rats naive to the testing environment, and in phMRI experiments increased neuronal activity within the anterior olfactory nuclei, nucleus accumbens and islets of Calleja, regions containing a high density of D3 receptors. A 30 microg/kg dose of quinelorane resulted in biphasic locomotor effects, with initial hypolocomotion followed by sustained hyperlocomotion. phMRI indicated that this higher dose increased cerebral activity within limbic and olfactory regions, as did the lower drug dose, but induced additional activation in the caudate-putamen and globus pallidus, areas dense in D2 receptors but containing few D3 receptors. The more restricted pattern of activation at low agonist doses and close temporal relationship between behavioural and BOLD signal responses to quinelorane suggest that those nuclei most dense in D3 receptors play a key role in mediating the hypolocomotor effects of quinelorane. However, the presence of D3 receptors in activated brain regions may be coincidental, and further studies are required to show definitively which class of receptors mediates agonist-induced hypolocomotion. In contrast, the activation of D2 receptors within the striatum appears necessary for quinelorane-induced hyperlocomotion.

Long-term evaluation of isolation-rearing induced prepulse inhibition deficits in rats: an update.

RATIONALE: Rats reared in social isolation from weaning show prepulse inhibition (PPI) deficits which are thought to model the sensorimotor gating deficits seen in schizophrenia and other psychiatric disorders. We have previously reported that ten cohorts of Lister Hooded rats reared in isolation showed robust and reliable PPI deficits. OBJECTIVE: Our methodology differed from those used by others (Weiss and Feldon in Psychopharmacology 156(2-3):305-326, 2001), most notably in the weaning of pups at postnatal day (PND) 28 compared with PND20-22. Since our initial report, we have studied 18 more cohorts weaned at PND28 and one cohort weaned at PND21. METHOD: At weaning, male Lister Hooded pups were singly (isolates) or group (n=5) housed (grouped). Eight weeks later, startle and PPI responses of isolates and grouped rats were investigated using conditions of fixed inter-stimulus interval (ISI) (pulse=110 dB/50 ms; prepulse (PP)=75-80 dB/30 ms; ISI=100 ms). RESULTS: Isolates from 14 of the subsequent 18 cohorts demonstrated PPI deficits, giving an overall success rate of 86% for all 28 cohorts. %PPI ranged from 12 to 26% in the isolates and from 26 to 47% in the grouped for the successful cohorts, compared to 16-30% (isolates) and 19-35% (grouped) for those that failed. Only five out of the 19 subsequent cohorts demonstrated startle hyperreactivity, which was unrelated to PPI response. The isolates from the cohort weaned at PND21 did not show a significant deficit in PPI, suggesting, in our hands at least, a requirement for weaning at PND28. CONCLUSION: The data presented here reinforce our original findings that isolation-rearing of Lister Hooded rats provides a viable, non-pharmacological model of impaired PPI.

LPA1 receptor-deficient mice have phenotypic changes observed in psychiatric disease.

Several psychiatric diseases, including schizophrenia, are thought to have a developmental aetiology, but to date no clear link has been made between psychiatric disease and a specific developmental process. LPA(1) is a G(i)-coupled seven transmembrane receptor with high affinity for lysophosphatidic acid. Although LPA(1) is expressed in several peripheral tissues, in the nervous system it shows relatively restricted temporal expression to neuroepithelia during CNS development and to myelinating glia in the adult. We report the detailed neurological and behavioural analysis of mice homozygous for a targeted deletion at the lpa(1) locus. Our observations reveal a marked deficit in prepulse inhibition, widespread changes in the levels and turnover of the neurotransmitter 5-HT, a brain region-specific alteration in levels of amino acids, and a craniofacial dysmorphism in these mice. We suggest that the loss of LPA(1) receptor generates defects resembling those found in psychiatric disease.

Up-regulation of secretoneurin immunoreactivity and secretogranin II mRNA in rat striatum following 6-hydroxydopamine lesioning and chronic L-DOPA treatment.

Destruction of the nigro-striatal pathway in Parkinson's disease and treatment with L-DOPA lead to persistent alterations in basal ganglia output pathways that are poorly characterised. Differential display mRNA analysis was used to study the effects of 6-hydroxydopamine-induced lesions of the medial forebrain bundle on gene expression in the rat striatum. One up-regulated cDNA identified in two independent groups of 6-hydroxydopamine-lesioned animals was cloned and sequence analysis showed 97% homology to secretogranin II. Differential up-regulation of secretogranin II following 6-hydroxydopamine lesioning was confirmed in a further group of 6-hydroxydopamine-lesioned rats using TaqMan real time quantitative reverse transcription-polymerase chain reaction. Following chronic L-DOPA treatment of 6-hydroxydopamine-lesioned rats, secretogranin II mRNA was further up-regulated to a similar degree to that observed for preproenkephalin A mRNA expression. Immunohistochemical analysis confirmed the increase in secretogranin II peptide levels in striatal neurones in 6-hydroxydopamine-lesioned rats following chronic L-DOPA treatment. The increase in secretogranin II mRNA occurring following destruction of the nigro-striatal pathway and chronic L-DOPA treatment may result in an increase in secretoneurin levels, which could be important for the regulation of striatal output pathways.

Long-term evaluation of isolation-rearing induced prepulse inhibition deficits in rats.

RATIONALE: Rats reared in social isolation from weaning show prepulse inhibition (PPI) deficits which are thought to model the sensorimotor gating deficits seen in schizophrenia and other psychiatric disorders. However, recent studies have questioned the robustness of this paradigm. OBJECTIVE: The existence of a substantial dataset generated over 4 years in our laboratory has allowed the investigation of the robustness and reliability of the procedure under a variety of environmental conditions. The effects of atypical antipsychotics (clozapine, olanzapine and risperidone) under different experimental conditions are also reported. METHOD: At weaning, Hooded Lister pups were singly (isolates) or group (n=5) housed (grouped). Eight weeks later, the startle and PPI response of isolates and grouped rats was investigated using conditions of fixed inter-stimulus interval (ISI) (pulse=110 dB/50 ms; prepulse=80 dB/30 ms; ISI=100 ms) or variable

SB-243213; a selective 5-HT2C receptor inverse agonist with improved anxiolytic profile: lack of tolerance and withdrawal anxiety.

SB-243213 (5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-trifluoromethylindoline hydrochloride) is a new, selective 5-hydroxytryptamine (5-HT)2C receptor inverse agonist. SB-243213 has high affinity for the human 5-HT2C receptor (pK(i) 9.37) and greater than a 100-fold selectivity over a wide range of neurotransmitter receptors, enzymes and ion channels. In in vitro functional studies, SB-243213 acted as an inverse agonist at the human 5-HT2C receptor with a pK(b) of 9.8. In in vivo studies, SB-243213 was a potent inhibitor of central 5-HT2C receptor-mediated function in rats, blocking meta-chlorophenylpiperazine-induced hypolocomotion with an ID50 of 1.1 mg/kg p.o. and a long duration of action (>8 h). In rats, SB-243213 exhibited anxiolytic-like activity in both the social interaction and Geller-Seifter conflict tests. Importantly, unlike diazepam, chronic administration of SB-243213 did not result in the development of either tolerance to the anxiolytic-like effects or withdrawal anxiogenesis. Furthermore, in rodents, SB-243213 did not affect seizure threshold, did not increase body weight or induce catalepsy, but attenuated the haloperidol-induced catalepsy. SB-243213 did not affect amphetamine-, MK-801- or phencyclidine-induced hyperactivity. In conclusion, SB-243213 may possess an improved anxiolytic profile compared to benzodiazepines. SB-243213 also modulates dopaminergic transmission, lacks pro-psychotic properties and may have utility in the treatment of schizophrenia and motor disorders.

Epileptogenesis and enhanced prepulse inhibition in GABA(B1)-deficient mice.

The recent cloning of two GABA(B) receptor subunits, GABA(B1) and GABA(B2), has raised the possibility that differences in GABA(B) receptor subunit composition may give rise to pharmacologically or functionally distinct receptors. If present, such molecular diversity could permit the selective targeting of GABA(B) receptor subtypes specifically involved in pathologies such as drug addiction, spasticity, pain, and epilepsy. To address these issues we have developed a GABA(B1) subunit knockout mouse using gene targeting techniques. In the brains of GABA(B1) null mice, all pre- and postsynaptic GABA(B) receptor function was absent demonstrating that the GABA(B1) subunit is essential for all GABA(B) receptor-mediated mechanisms. Despite this, GABA(B1) null mice appeared normal at birth, although by postnatal week four their growth was retarded and they developed a generalized epilepsy that resulted in premature death. In addition, GABA(B1) heterozygote animals showed enhanced prepulse inhibition responses compared to littermate controls, suggesting that GABA(B1) deficient mice exhibit increased sensorimotor gating mechanisms. These data suggest that GABA(B) receptor antagonists may be of benefit in the treatment of psychiatric and neurological disorders in which attentional processing is impaired.

Comparative pharmacological study of ropinirole (SKF-101468) and its metabolites in rats.

The dopamine receptor agonist ropinirole (SKF-101468) is used to treat Parkinson's disease. Ropinirole is metabolized by two routes to a series of different metabolites although the predominant pathway is species-dependent. It is unknown whether any of the metabolites contribute to its antiparkinsonian activity and whether D3 or D2 receptor agonist activity plays a preferential role. Therefore ropinirole and its primary metabolites, SKF-104557, SKF-97930 and SKF-96990, and the rat metabolite, SKF-89124 were tested in the 6-hydroxydopamine lesion model of Parkinson's disease. SKF-89124 and SKF-96990 were also assayed in radioligand binding and microphysiometer functional assays at cloned human dopamine D2 and D3. Ropinirole and SKF-89124 were equipotent in-vivo, and produced dose-related increases in circling at 0.05-0.8 mg kg(-1), s.c. (ropinirole) and 0.05-0.75 mg kg(-1), s.c. (SKF-89124). Neither SKF-96990 or SKF-97930, at doses up to 15 mg kg(-1), increased the circling rate. Some circling was observed with 15 mg kg(-1) SKF-104557 but the response was less than half that produced by ropinirole (0.8 mgkg(-1)). SKF-104557 was 150-fold less potent than ropinirole. SKF-89124 possessed-30-fold higher affinity for D3 over D2 receptors in radioligand binding studies, but was not selective in the functional microphysiometer assay. SKF-96990 was 10-fold selective for D3 over D2 receptors in the radioligand binding assay. Ropinirole and SKF-104557 are 20-fold selective for D3 over D2 receptors in radioligand binding assays whereas in microphysiometry, selectivity is 10-fold. SKF-97930 is inactive in radioligand binding and microphysiometer assays. Primary metabolites of ropinirole did not contribute significantly to its activity in this model of Parkinson's disease. The lack of dopamine D3/D2 receptor selectivity for ropinirole rules out the possibility of attributing the degree of either D2 or D3 receptor activity to the behavioural efficacy of ropinirole.

Pharmacological actions of a novel, high-affinity, and selective human dopamine D(3) receptor antagonist, SB-277011-A.

SB-277011-A (trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolininecarboxamide), is a brain-penetrant, high-affinity, and selective dopamine D(3) receptor antagonist. Radioligand-binding experiments in Chinese hamster ovary (CHO) cells transfected with human dopamine D(3) or D(2 long) (hD(3), hD(2)) receptors showed SB-277011-A to have high affinity for the hD(3) receptor (pK(i) = 7.95) with 100-fold selectivity over the hD(2) receptor and over 66 other receptors, enzymes, and ion channels. Similar radioligand-binding data for SB-277011-A were obtained from CHO cells transfected with rat dopamine D(3) or D(2). In the microphysiometer functional assay, SB-277011-A antagonized quinpirole-induced increases in acidification in CHO cells overexpressing the hD(3) receptor (pK(b) = 8.3) and was 80-fold selective over hD(2) receptors. Central nervous system penetration studies showed that SB-277011-A readily entered the brain. In in vivo microdialysis studies, SB-277011-A (2. 8 mg/kg p.o.) reversed the quinelorane-induced reduction of dopamine efflux in the nucleus accumbens but not striatum, a regional selectivity consistent with the distribution of the dopamine D(3) receptor in rat brain. SB-277011-A (2-42.3 mg/kg p.o.) did not affect spontaneous locomotion, or stimulant-induced hyperlocomotion. SB-277011-A (4.1-42.2 mg/kg p.o.) did not reverse prepulse inhibition deficits in apomorphine- or quinpirole-treated rats, but did significantly reverse the prepulse inhibition deficit in isolation-reared rats at a dose of 3 mg/kg p.o. SB-277011-A (2.5-78. 8 mg/kg p.o.) was noncataleptogenic and did not raise plasma prolactin levels. Thus, dopamine D(3) receptor blockade produces few of the behavioral effects characteristic of nonselective dopamine receptor antagonists. The effect of SB-277011-A on isolation-induced prepulse inhibition deficit suggests that blockade of dopamine D(3) receptors may benefit the treatment of schizophrenia.

Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource.

Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol-SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations-with consequent benefits for the analysis of gene function and the identification of novel pathways.

Design and synthesis of trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide (SB-277011): A potent and selective dopamine D(3) receptor antagonist with high oral bioavailability and CNS penetration in the rat.

A selective dopamine D(3) receptor antagonist offers the potential for an effective antipsychotic therapy, free of the serious side effects of currently available drugs. Using clearance and brain penetration studies as a screen, a series of 1,2,3, 4-tetrahydroisoquinolines, exemplified by 13, was identified with high D(3) affinity and selectivity against the D(2) receptor. Following examination of molecular models, the flexible butyl linker present in 13 was replaced by a more conformationally constrained cyclohexylethyl linker, leading to compounds with improved oral bioavailability and selectivity over other receptors. Subsequent optimization of this new series to improve the cytochrome P450 inhibitory profile and CNS penetration gave trans-N-[4-[2-(6-cyano-1, 2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarbo xamide (24, SB-277011). This compound is a potent and selective dopamine D(3) receptor antagonist with high oral bioavailability and brain penetration in the rat and represents an excellent new chemical tool for the investigation of the role of the dopamine D(3) receptor in the CNS.

Evaluation of the spontaneously hypertensive rat as a model of attention deficit hyperactivity disorder: acquisition and performance of the DRL-60s test.

CD. However, the pattern of responding at DRL-60s suggested poor schedule control for the WKY rats. Therefore, the performance of SHR in the DRL test does not appear to represent a valid model of ADHD. Further, our findings with the WKY rat suggest that this strain is a poor behavioural control for the SHR.

Attenuation of haloperidol-induced catalepsy by a 5-HT2C receptor antagonist.

Atypical neuroleptics produce fewer extrapyramidal side-effects (EPS) than typical neuroleptics. The pharmacological profile of atypical neuroleptics is that they have equivalent or higher antagonist affinity for 5-HT2 than for dopamine D2 receptors. Our aim was to identify which 5-HT2 receptor contributed to the atypical profile. Catalepsy was defined as rats remaining immobile over a horizontal metal bar for at least 30 s, 90 min after dosing. Radioligand binding assays were carried out with homogenates of human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors expressed in Human Embryo Kidney (HEK293) cells. Haloperidol (1.13 mg kg(-1) i.p.) induced catalepsy in all experiments. The selective 5-HT2C/2B receptor antagonist, SB-228357 (0.32-10 mg kg(-1) p.o.) significantly reversed haloperidol-induced catalepsy whereas the 5-HT2A and 5-HT2B receptor antagonists, MDL-100907 (0.003-0.1 mg kg(-1) p.o.) and SB-215505 (0.1-3.2 mg kg(-1) p.o.) respectively did not reverse haloperidol-induced catalepsy. The data suggest a role for 5-HT2C receptors in the anticataleptic action of SB-228357.

5-HT4 receptor antagonism does not affect motor and reward mechanisms in the rat.

5-HT4 receptors are concentrated in areas of the brain which are rich in dopamine neuronal markers, which may suggest that they influence motor and reward processes. We tested this hypothesis by examining the effects of a 5-HT4 receptor antagonist, 8-amino-7-chloro-(N-butyl-4-piperidyl)methylbenzo-1,4-dioxan-5-car boxylate hydrochloride (SB-204070-A) on amphetamine- and nicotine-induced locomotor stimulation in intact rats. In rats with unilateral 6-hydroxydopamine-induced lesions of the ascending nigrostriatal dopaminergic projection, SB-204070-A was tested for its effects on amphetamine-induced rotation. SB-204070-A was also tested for its effects on rewarded behaviour maintained by intracranial self-stimulation. SB-204070-A did not alter behaviour under any of these conditions, suggesting a lack of involvement of the 5-HT4 receptor in motor and reward processes.

Evidence to suggest that agonist modulation of hyperlocomotion is via post-synaptic dopamine D2 or D3 receptors.

It has been suggested that a sub-population of dopamine D3 receptors is located pre-synaptically and these serve as autoreceptors in dopamine projection areas such as the nucleus accumbens/ventral striatum. To study further the physiological role and synaptic location of the dopamine D3 receptor, we have investigated the in vivo effect of the D3/D2 receptor agonist quinelorane on amphetamine-induced hyperactivity and extracellular dopamine release from the nucleus accumbens of the conscious rat. Amphetamine increased dopamine release to 202 +/- 34% of pre-injection control values, but quinelorane at 2.5 micrograms/kg, a dose which effectively blocked amphetamine-induced hyperlocomotion, had no significant effect on amphetamine-induced dopamine release. These data suggest that hyperlocomotion is mediated via post-synaptic rather than pre-synaptic dopamine receptors. Since quinelorane has significant affinity for the dopamine D3 receptor, these effects may be via post-synaptic D3 receptors; however, D2 receptor effects cannot be disregarded. In summary, these data indicate that the quinelorane effect on amphetamine-stimulated hyperlocomotion is not mediated via D3 or D2 autoreceptors, but rather a population of receptors located post-synaptically, which appear to mediate the inhibition of rat locomotor activity.

Localization of serotonin-4 receptors in the striatonigral pathway in rat brain.

Rats were injected unilaterally with 6-hydroxydopamine either in the medial forebrain bundle or in the dorsolateral substantia nigra. Another group was injected unilaterally with kainate in the striatum. The loss of neurons was assessed by a reduction in tyrosine hydroxylase-like immunoreactivity for dopaminergic neurons, and choline acetyltransferase-like and glutamate decarboxylase-like immunoreactivities for cholinergic and GABAergic neurons, respectively. Brain sections also were analysed by autoradiography on 20 micron sections with the radio-iodinated serotonin-4 receptor antagonist [125I]SB 207710 [Brown A. M. et al. (1993) Br. J. Pharmac. 110, 10P]. Kainate injections in the striatum resulted in loss of choline acetyltransferase- and glutamate decarboxylase-like immunoreactive cell bodies in this area. There was also a decrease in glutamate decarboxylase-like immunoreactivity on the ipsilateral side in the substantia nigra and entopeduncular nucleus. These changes were accompanied by substantial (> 50%) decreases in [125I]SB 207710 binding in both the ipsilateral striatum (confined to the lesioned area) and substantia nigra, with no change in either the nucleus accumbens or the globus pallidus. There was also significant loss of [125I]SB 207710 binding in the ipsilateral entopeduncular nucleus. 6-Hydroxydopamine lesions placed either in the medial forebrain bundle or in the substantia nigra failed to decrease [125I]SB 207710 binding in any of these areas, although there was total loss of tyrosine hydroxylase-like immunoreactive terminals in the striatum and cell bodies in the nigra. We conclude that serotonin-4 receptors are present on projection neurons, both on their perikarya in the striatum and terminals in the nigra and entopeduncular nucleus. It is likely that these receptors are located on the GABAergic projection neurons and possibly on cholinergic and GABAergic interneurons. However, serotonin-4 receptors are not located on dopaminergic neurons, either on their cell bodies in the substantia nigra or terminals in the striatum.

Effect of haloperidol and (-)-sulpiride on dopamine agonist-induced hypoactivity.

High doses of dopamine D2 receptor agonists produce hyperactivity in rodents whereas low doses suppress activity. In this study, low doses of a range of dopamine agonists were examined for their effects on locomotor activity in rats. All agonists caused a dose-related hypolocomotor effect with a rank order of potency of quinelorane > (-)-quinpirole > 7-OHDPAT > PBTO. (-)-Sulpiride (1.6-160 mumol/kg), a neuroleptic with atypical properties caused a dose-related reversal of the hypolocomotor effect produced by all four agonists whereas the typical neuroleptic haloperidol (0.005-0.16 mumol/kg) did not reverse hypolocomotion. Neither sulpiride (5-16 mumol/kg) nor haloperidol (0.005-0.16 mumol/kg) affected locomotor activity per se, although haloperidol (1.6-5 mumol/kg) did reduce locomotor activity. The different behavioural profiles shown by (-)-sulpiride and haloperidol in these tests may reflect some of the clinical characteristics of these neuroleptics. The question of whether these effects can be ascribed to differential actions at dopamine receptor subtypes will only be answered when more selective dopamine receptor antagonists are developed.

Pharmacological characterization of the discriminative stimulus properties of the dopamine D1 agonist, SKF 81297.

A range of selective dopamine D1 and D2 receptor agonists and antagonists was used to characterize to the discriminative stimuli produced by d-amphetamine (0.5mg/kg) and the D1 agonists SKF 81297 (0.1mg/kg). In rats trained to discriminate d-amphetamine (0.5mg/kg) from saline, d-amphetamine produced a dose-related increase in per cent drug lever responding, and SKF 81297 did not show any d-amphetamine-like discriminative stimulus effects; neither did SKF 81297 potentiate nor antagonize the d-amphetamine discriminative stimulus. In rats trained to discriminate SKF 81297 (0.1mg/kg) from saline, SKF 81297 produced a dose-related increase in per cent drug lever responding, and SKF 38393 and SKF 83565 elicited full SKF 81297-like effects despite the fact that these compounds have widely differing efficacies for stimulating adenylate cyclase. SKF 81297 had a 25-fold greater potency than SKF 38393 in this assay. The D2 agonists, PHNO and ropinirole, did not display any SKF 81297-like discriminative stimulus effects. The SKF 81297 discriminative stimulus was completely blocked by the D1 antagonist SCH 23390 but was not blocked by the D2 antagonist BRL 34778.