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Conservation of behavioural topography to dopamine D1-like receptor agonists in mutant mice lacking the D1A receptor implicates a D1-like receptor not coupled to adenylyl cyclase
D1-like receptors in mutant mice
Pergamon PII: S0306-4522(99)00297-3
Neuroscience Vol. 93, No. 4, pp. 1483–1489,1483 1999 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
CONSERVATION OF BEHAVIOURAL TOPOGRAPHY TO DOPAMINE D1-LIKE RECEPTOR AGONISTS IN MUTANT MICE LACKING THE D1A RECEPTOR IMPLICATES A D1-LIKE RECEPTOR NOT COUPLED TO ADENYLYL CYCLASE J. J. CLIFFORD,* O. TIGHE,† D. T. CROKE,† A. KINSELLA,‡ D. R. SIBLEY,§ J. DRAGOk and J. L. WADDINGTON*¶ Departments of *Clinical Pharmacology and †Biochemistry, Royal College of Surgeons in Ireland, St Stephen’s Green, Dublin 2, Ireland ‡Department of Mathematics, Dublin Institute of Technology, Dublin 8, Ireland §Experimental Therapeutics Branch, National Institute for Neurological Disorders and Stroke, Bethesda, MD 20892, U.S.A. k Department of Anatomy, Monash University, Clayton, Victoria 3168, Australia
Abstract—Though D1-like dopamine receptors [D1A/B] are defined in terms of linkage to the stimulation of adenylyl cyclase, with D1A assumed to be the functionally prepotent subtype, evidence suggests the existence of another, novel D1-like receptor without such coupling. To investigate these issues we challenged mutant mice having targeted gene deletion of the D1A receptor with selective agonists and used an ethologically-based assessment technique to resolve resultant behavioural topography. D1-likedependent behaviour was substantially conserved in D1A-null mice relative to wild-types following challenge with each of two selective D1-like agents: A 68930 (0.068–2.0 mg/kg s.c.) which exhibits full efficacy to stimulate adenylyl cyclase, and SKF 83959 (0.016–2.0 mg/kg s.c.) which fails to stimulate adenylyl cyclase, and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine. Furthermore, responsivity to the selective D2-like agonist RU 24213 (0.1–12.5 mg/kg s.c.) was conserved in D1A-null mice, indicating the integrity of D1-like:D2-like interactions at the level of behaviour. These data are consistent with behavioural primacy of a D1-like receptor other than D1A [or D1B] that is coupled to a transduction system other than/additional to adenylyl cyclase. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: D1-like dopamine receptors, adenylyl cyclase, mutant mice, D1A knockout, behaviour.
mice having “knock-out” of the D1A receptor. 12,29,39,40 Using a topographical, ethologically-based approach, we have reported recently 3 that grooming, the most widely accepted behavioural index of D1-like receptor function in rats and mice which has been presumed to be mediated via the D1A receptor, 36 is not lost in D1A-null mice, together with topographical shifts among several other elements of behaviour. 3,5 This profile is inconsistent with D1-like-dependent grooming having a sole basis in D1A (or D1B) receptor function and focuses attention on another putative D1-like receptor. Furthermore, in D1A-null mice the stimulation of adenylyl cyclase by dopamine is lost while stimulation of phosphoinositide hydrolysis is conserved. 14 Described here are experiments designed to address specifically whether typical behavioural effects thought to be mediated via cyclase-coupled D1-like receptors as a class, and via cyclase-coupled D1A receptors in particular, might have a basis in another D1-like receptor not coupled to adenylyl cyclase. We posed the following questions: (i) do D1A-null mice show altered grooming and other responses to the highly selective, full efficacy D1-like agonist A 68930, 6,8 and to the anomalous D1-like agent SKF 83959 1,9 which fails to stimulate adenylyl cyclase and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine, yet induces behaviour that is qualitatively indistinguishable from that induced by A 68930 and (ii) do D1A-null mice show attenuation in typical responses to the selective D2-like agonist RU 24213, 10,13 given the well-established “enabling” or “permissive” role of D1-like-mediated function in general in the expression of typical D2-like-dependent effects via cooperative/synergistic D1-like:D2-like interactions? 34,35,38
Recognition of the functional role of dopamine in mediating multiple aspects of mammalian psychomotor behaviour, and in the actions of drugs used to treat disorders such as Parkinson’s disease and schizophrenia, has not been matched by understanding of the receptor sites through which these effects occur. 17,36 Two major families of D1-like [D1A/D1, D1B/D5] and D2-like [D2L/S, D3, D4] receptors have been identified in molecular biological terms. 16,28 However, given the general paucity of drugs able to discriminate materially within each family, considerably less is known of functional parcellation among individual family members. Furthermore, within the D1-like family all members show coupling to the stimulation of adenylyl cyclase, in accordance with the original defining characteristic of D1 as opposed to D2 receptors, 21 yet there is classical pharmacological evidence for the existence of another D1-like dopamine receptor that is coupled to phosphoinositide hydrolysis rather than/additional to adenylyl cyclase; 22,31,32 however, any functional role for this receptor is uncertain, 36 and no such receptor has yet been cloned. An important new approach to the parcellation of function between receptor subtypes in the absence of selective pharmacological agents is the generation of mutant mice in which a given subtype is absent through targeted gene deletion by homologous recombination. 18,37 In relation to the D1-like family, this technique has been applied to generate mutant ¶To whom correspondence should be addressed. Abbreviations: A 68930, [1R,3S]-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman; RU 24213, (N-n-propyl-N-phenylethyl-p-3-hydroxyphenylethylamine; SKF 83959, 3-methyl-6-chloro-7,8-dihydroxy-1[3methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine. 1483
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Transgenic animals The generation of D1A-null mice was as reported previously. 12 In outline, the targeted gene deletion was constructed in 129/Sv embryonic stem cells and male chimeras mated with C57BL/6 females to produce heterozygous mutants. Homozygous mutants (D1A 2 / 2 ) and wild-type (D1A 1 / 1 ) littermates were bred and genotyped in Dublin among the progeny of heterozygous intermatings, using a polymerase chain reaction technique. 23 Because of some failure to thrive among D1A-null mice over the weaning period, 12 resulting in a slight decrease in body weight, 3,12 standard dry mouse chow was supplemented routinely with moist diet on the floor of the cage. They were housed in groups of four to five with food and water available ad libitum, and were maintained at 21 ^ 18C on a 12 h/12 h (09.00 on/21.00 off) light/dark schedule. Young black female mice from litters of the same generational age were used in behavioural assessments. All experiments on mice were performed in compliance with relevant laws and institutional guidelines; these studies were approved by the Research Committee of the Royal College of Surgeons in Ireland. Behavioural assessment On experimental days mice were removed from their home cage, placed individually in clear glass observation cages (36 × 20 × 20 cm 3) and left undisturbed for a habituation period of 3 h. Behavioural assessments were carried out in a manner similar to that which we have described previously. 2,3,9 Following injection of drug or vehicle animals were assessed using a rapid time-sampling behavioural checklist technique. For this procedure, each of 10 randomly allocated mice was observed individually for 5 s periods at 1 min intervals over 15 consecutive minutes, using an extended, ethologically-based behavioural checklist. This allowed the presence or absence of the following individual behaviours (occurring alone or in any combination) to be determined in each 5 s period: sniffing; locomotion (co-ordinated movement of all four limbs producing a change in location); ponderous locomotion (a “plodding” variant induced in mice by D2-like agonists, and different from normal, fluid ambulation); total rearing (of any form); rearing from a sitting position (front paws reaching upwards with hind limbs on floor in sitting position); rearing free (front paws reaching upwards away from any cage wall while standing on hind limbs); rearing towards a cage wall (front paws reaching upwards on a cage wall while standing on hind limbs); sifting (sifting movements of the front paws through cage bedding material); grooming (of any form); intense grooming (grooming of the snout and then the face with the forepaws followed by vigorous grooming of the hind flank or anogenital region with the snout); vacuous chewing (chewing movements not directed onto any physical material); chewing (chewing movements directed onto cage bedding and/or faecal pellets without consumption); eating (chewing with consumption); climbing (jumping onto cage top with climbing along grill in inverted or hanging position); stillness (motionless, with no behaviour evident). After this 15 min assessment using the behavioural checklist, animals were evaluated using a conventional 0-to-6-point stereotypy scale: 2,10 0 asleep or inactive; 1 episodes of normal activities; 2 discontinuous activity with bursts of prominent sniffing or rearing; 3 continuous stereotyped activity such as sniffing or rearing along a fixed path; 4 stereotyped sniffing or rearing fixated in one location; 5 stereotyped behaviour with bursts of licking or gnawing; 6 continuous licking or gnawing. This cycle of assessment by behavioural checklist followed by stereotypy scale was repeated on two further occasions over a total observation period of 1 h. Mice were used on two occasions only, separated by a drug-free interval of at least one week; on each occasion mice were allocated randomly to one of the various treatment groups. All assessments were made by an observer unaware of the treatment and genotype of each animal. Materials A 68930 ([1R,3S]-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman; Abbott, U.S.A.) was dissolved in dilute acetic acid and made up to volume with distilled water; SKF 83959 (3-methyl-6-chloro7,8-dihydroxy-1[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine; RBI/NIMH, U.S.A.) was dissolved in distilled water; RU 24213 (N-n-propyl-N-phenylethyl-p-3-hydroxyphenylethylamine; HoechstMarion-Roussel, France) was dissolved in distilled water. All agents
or their respective vehicles were injected subcutaneously into the flank in a volume of 2 ml/kg. Data analysis From application of the behavioural checklist, the total counts for each individual behaviour were determined as the number of 5 s observation “windows” in which a given behaviour was evident, summed over the 1 h period, and expressed as means ^S.E.M.; stereotypy scores were averaged over the 1 h period and expressed similarly. These data were then analysed using analysis of variance, subsequent to square-root transformation for behavioural counts, with Helmert contrasts 27 to identify those particular drug doses at which responsivity differed by genotype, relative to lower doses, for a given topography of behaviour. RESULTS
Responsivity to A 68930 The characteristic response to A 68930 was a prominent, dose-dependent induction of grooming, particularly intense grooming which constitutes ethologically complete grooming syntax (0.068–2.0 mg/kg; P , 0.001 for each topography of grooming). Induction of overall grooming by A 68930 did not differ between the genotypes (no dose × genotype interaction, P . 0.05). There was also no dose × genotype interaction (P . 0.05) for intense grooming, though responsivity to the highest dose of A 68930 (2.0 mg/kg) only was somewhat reduced in D1A-null mice relative to wild-types (P , 0.01); however, in D1A-null mice intense grooming to this dose of A 68930 still occurred to significant excess (P , 0.05) relative to its own vehicle control. A 68930 failed to influence locomotion in wild-types but induced prominent locomotion in D1A-null mice (dose × genotype interaction, P , 0.01), primarily at 2.0 mg/kg (P , 0.001). There was no significant reduction in sniffing or sifting responses to A 68930, though total rearing and each topography of rearing response (free, to a wall, and from a seated position) was reduced (P , 0.01) in D1A-null mice relative to wild-types at 2.0 mg/kg (Fig. 1). No stereotyped behaviour was evident, and there was no material stimulation of any other topography of behaviour for either genotype. Responsivity to SKF 83959 Similarly, the characteristic response to SKF 83959 was induction of grooming, including intense grooming (0.016– 2.0 mg/kg; P , 0.001 for grooming, P , 0.05 for intense grooming). Induction of overall grooming by SKF 83959 was somewhat reduced in D1A-null mice relative to wildtypes (dose × genotype interaction, P , 0.01), particularly at 0.08 mg/kg (P , 0.001); however, at an intermediate dose (0.4 mg/kg), induction of overall grooming in D1A-null mice occurred to significant excess (P , 0.05) relative to its own vehicle control, and to an extent that did not differ significantly from wild-types given the same dose. However, induction of intense grooming by SKF 83959 did not differ between genotypes (no dose × genotype interaction, P . 0.05). Induction of locomotion by SKF 83959 was somewhat reduced in D1A-null mice relative to wild-types (dose × genotype interaction, P , 0.05), particularly at 0.08 and 2.0 mg/kg (P , 0.05). There was no significant reduction in sniffing or chewing responses to SKF 83959, while sifting was reduced (P , 0.05) but not abolished in D1A-null relative to wildtypes at 0.08 mg/kg. Among topographies of rearing responsivity to SKF 83959, total rearing and both rearing free and rearing
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Fig. 1. Behavioural counts for grooming, intense grooming, locomotion, total rearing and sifting responses to A 68930 (0.068–2.0 mg/kg) vs vehicle (V) in “knockout” [D1A 2 / 2 ] vs wild-type [D1A 1 / 1 ] mice. Data are means ^S.E.M. for n 8 per group; ***P , 0.001, **P , 0.01 vs wild-types.
from a seated position did not differ between genotypes while rearing to a wall was reduced in D1A-null mice at 0.08 mg/kg (P , 0.05) and 2.0 mg/kg (P , 0.01) relative to wild-types (Fig. 2). No stereotyped behaviour was evident, and there was no material stimulation of any other topography of behaviour for either genotype.
lenge stereotypy ratings were elevated (P , 0.01) in D1A-null mice relative to wildtypes (Fig. 3). RU 24213 did not induce any form of grooming behaviour; indeed, it effected a dosedependent reduction in baseline levels of overall grooming which did not differ between the genotypes. There was no material stimulation of any other topography of behaviour, and no atypical behaviours were released in D1A-null mice.
Responsivity to RU 24213 The characteristic response to RU 24213 was the prominent, dose-dependent induction of stereotyped sniffing and ponderous locomotion (0.1–12.5 mg/kg; P , 0.001 for each topography of behaviour). Neither the induction of sniffing and ponderous locomotion nor increases in stereotypy score by RU 24213 differed between the genotypes (no dose × genotype interactions, P . 0.05), though following vehicle chal-
DISCUSSION
Stimulation of adenylyl cyclase was the first transduction mechanism identified for dopamine receptor activation 15,20 and remains central to dopamine receptor typology and function. Subsequent identification of a pharmacologically distinct subtype (D2, either coupled to inhibition of adenylyl cyclase or without cyclase coupling) underpinned the original
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Fig. 2. Behavioural counts for grooming, intense grooming, locomotion, total rearing and sifting responses to SKF 83959 (0.016–2.0 mg/kg) vs vehicle (V) in “knockout” [D1A 2 / 2 ] vs wild-type [D1A 1 / 1 ] mice. Data are means ^S.E.M. for n 8 per group; ***P , 0.001, **P , 0.01, *P , 0.05 vs wild-types.
proposal for dopamine receptor nosology whereby those coupled to stimulation of adenylyl cyclase were designated D1. Thereafter, following a period over which greater functional relevance was ascribed to the D2 receptor, the introduction of selective D1 agonists and antagonists readily indicated a profound role for D1 receptors in mammalian dopaminemediated function which encompasses multiple aspects of psychomotor behaviour. 17,36 In particular, the induction of grooming, including the topography of intense grooming which constitutes ethologically complete, programmed grooming syntax, 4 endures as the most widely accepted behavioural index of D1 receptor activation; such grooming topography is readily induced in a pharmacologically-specific manner by all D1 agonists identified to date. 24,33,36 Among the family of D1-like subtypes identified on a molecular biological basis (D1A and D1B in rodents; D1 and D5 as
their primate homologues), each evidences “defining” linkage to the stimulation of adenylyl cyclase, 16,28 as do D1C 30 and D1D 7 subtypes identified to date only in lower species. Within the rodent D1-like family, it is the D1A receptor that has been ascribed functional prepotence, and this includes mediation of grooming behaviour; the D1B receptor has a low density corticolimbic localization, while it is D1A receptors that are localized densely to those major, dopamine-innervated striatal/limbic striatal regions that are known to subserve typical dopaminergic behaviours and in which D1B receptors are essentially absent. 16,17,28,36 In the present context, grooming behaviour, particularly ethologically complete, intense grooming syntax, has its genesis in the D1A-rich and D1Bdeficient anterior dorsolateral striatum, 4 at which selective inactivation of D1-like receptors with 1-ethoxycarbonyl-2ethoxy-1,2-dihydroquinoline abolishes D1-like agonist-induced
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Fig. 3. Behavioural counts for sniffing, ponderous locomotion, total rearing and sifting, and stereotypy scores, in response to RU 24213 (0.1–12.5 mg/kg) vs vehicle (V) in “knockout” [D1A 2 / 2 ] vs wild-type [D1A 1 / 1 ] mice. Data are means ^S.E.M. for n 8 per group.
grooming; 26 on this basis, mice with targeted gene deletion of the D1A receptor would be expected not to evidence any typical grooming response to D1-like agonists as conceptualized currently. A 68930 is a reference D1-like agonist having high affinity and selectively for D1-like over D2-like and non-dopaminergic receptors and demonstrating full efficacy, indistinguishable from that of dopamine itself, in terms of stimulation of adenylyl cyclase. 6,8 Yet, strikingly, the prominent induction of overall grooming by A 68930 in wild-types was entirely conserved in D1A-null mice. In particular, induction of intense grooming by A 68930 was substantially unaltered; some
attenuation in (but not abolition of) intense grooming only to the highest dose of A 68930 appeared to reflect disruption of grooming syntax as response incompatibility with prominent stimulation of locomotion, which this same dose of A 68930 induced only in the D1A-null genotype. Sniffing and sifting responses to A 68930 were also little altered, though these behaviours, like locomotion, are not specific to D1-like receptor stimulation; only rearing responsivity to A 68930 was blocked in D1A-null mice. Reduced responsivity to the D1-like agonist SKF 81297 has been reported in another line of mice having deletion of the D1A receptor, 39 though this was attained using a different genetic construct. It should be noted
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that responsivity to SKF 81297 was quantified 39 in terms of photobeam interruptions, which are insensitive to many individual elements of behaviour and composite numerous others so as to give limited resolution of the actual topography of behaviour. 3 On the basis of the present studies, substantial conservation in D1A-null mice of several topographies of behavioural responsivity to A 68930, particularly of grooming and intense grooming syntax, would indicate the involvement of a D1-like receptor other than D1A (or D1B). To illuminate further the processes involved in these effects, we utilized the novel, anomalous agent SKF 83959. This agent has high affinity and selectivity for D1-like over D2-like and non-dopaminergic receptors, but fails to stimulate adenylyl cyclase, and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine; 1,10 yet, despite evidencing all the “defining” characteristics of an antagonist at cyclase-coupled D1-like receptors, it induces in rats grooming and intense grooming that is qualitatively indistinguishable from that induced by A 68930. 6,10,11 The induction of overall grooming by SKF 83959 in D1A-null mice was somewhat attenuated but not abolished, while ethologically complete, intense grooming syntax was essentially conserved in D1A-null mice relative to wild-types; though attenuation of locomotion to SKF 83959 in D1A-null mice contrasts with heightening of locomotion to A 68930, the basis of this difference is unclear. Sniffing and chewing responses to SKF 83959 were also little altered, while locomotion, sifting and rearing to a wall were attenuated in D1A-null mice. SCH 23390, an agent that shares with SKF 83959 an antagonist profile at cyclase-coupled D1-like receptors, has been reported to be less active in reducing photobeam interruptions and in inducing catalepsy in alternatively constructed D1A-null mice. 39 Thus, the distinct profiles of these drugs in mice with targeted gene deletion of the D1A receptor appear unrelated to their common profile as antagonists at cyclase-coupled D1-like receptors. On the basis of the present studies, substantial conservation in D1A-null mice of core topographies of behavioural responsivity to SKF 83959, particularly grooming and intense grooming, would indicate the involvement of a D1like receptor that is not coupled to adenylyl cyclase. In essence we report that D1A-null mice, in which D1A receptor binding and dopamine-sensitive adenylyl cyclase are essentially absent, 12,14 evidence substantial conservation of the characteristic topographical responsivity of wild-types to A 68930 and to SKF 83959, agents that act as a full agonist and antagonist, respectively, at cyclase-coupled D1-like receptors. Furthermore, well-recognized cooperative/synergistic and oppositional D1-like:D2-like interactions, through which D1A receptors have been presumed to critically “enable” or inhibit the expression of, respectively, typical
and atypical responses to the D2-like agonist RU 24213, 34– 36,38 appear also to be conserved in D1A-null mice at the level of behaviour; yet electrophysiological effects of selective dopaminergic agonists on nucleus accumbens neurons appear to be reduced therein. 40 Raised stereotypy scores in D1A-null mice given vehicle for RU 24213 were within the 0– 1 range indicative of normal, non-stereotyped behaviour, and may reflect the heightening of certain spontaneous behaviours in the absence of any treatment. 3 CONCLUSION
The present findings therefore suggest that these topographic behavioural effects do not involve the D1A or indeed any other molecular biologically characterized D1-like receptor having the “defining” characteristic of linkage to the stimulation of adenylyl cyclase, such as D1B; rather, they appear to have their basis in another, functionally prepotent D1-like receptor that is not so linked. Indeed, there are previous data that among a range of partial D1-like agonists, their efficacies both to induce grooming behaviour 25 and to inhibit cell firing in the nucleus accumbens 19 appear unrelated to their efficacies to stimulate adenylyl cyclase. Over recent years, classical pharmacological evidence has emerged to suggest the existence of an additional D1-like receptor linked to activation of phosphoinositide hydrolysis; 9,22,31,32 however, any functional role for this putative receptor at the level of behavioural regulation has remained unclear. We cannot exclude incontrovertibly some involvement of residual D1A receptors or of compensatory mechanisms consequent to the developmental absence of D1A receptors, or that behaviours at issue might involve more than one D1-like receptor or a nonD1-like receptor. However, while the present findings do not in themselves indicate directly the involvement in behavioural regulation of a D1-like receptor linked specifically to phosphoinositide hydrolysis, the action of D1-like agonists to stimulate adenylyl cyclase is lost in D1A-null mice while their action to stimulate phosphoinositide hydrolysis is conserved therein; 14 this parallelism with the present behavioural profile would be in accordance with such a mechanism and with a prominent behavioural role for this novel D1like receptor. Acknowledgements—These studies were supported by the Research Committee of the Royal College of Surgeons in Ireland and the Higher Education Authority. We thank Abbott for A 68930 and Hoechst-Marion-Roussel for RU 24213. SKF 83959 was kindly provided by Research Biochemicals International as part of the Chemical Synthesis Program of the National Institute of Mental Health, contract N01MH30003. John Drago is a Logan Research Fellow of Monash University.
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(1995) Anatomical localisation of SK & F 38393-induced behaviours in rats using the irreversible monoamine receptor antagonist EEDQ. Synapse 19, 134–143. Seber G. A. F. Linear regression analysis. Wiley, Chichester. Sibley D. R. and Monsma F. J. (1992) Molecular biology of dopamine receptors. Trends pharmac. Sci. 13, 61–69. Smith D. R., Striplin C. D., Geller A. M., Mailman R. B., Drago J., Lawler C. P. and Gallagher M. (1998) Behavioural assessment of mice lacking D1A dopamine receptors. Neuroscience 86, 135–146. Sugamori K. S., Demchyshyn L. L. and Niznik H. B. (1994) D1a, D1b, and D1c dopamine receptors from Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 91, 10536–10540. Undie A. S. and Friedman E. (1990) Stimulation of a dopamine D-1 receptor enhances inositol phosphate formation in rat brain. J. Pharmac. exp. Ther. 253, 987–992. Undie A. S., Weinstock J., Sarau H. M. and Friedman E. (1994) Evidence for a distinct D1-like dopamine receptor that couples to activation of phosphoinositide metabolism in brain. J. Neurochem. 62, 2045–2048. Wachtel S. R., Brooderson R. J. and White F. J. (1992) Parametric and pharmacological analyses of the enhanced grooming response elicited by the D1 dopamine receptor agonist SKF 38393 in the rat. Psychopharmacology 109, 41–48. Waddington J. L. and Daly S. A. (1993) Regulation of unconditioned motor behaviour by D1:D2 interactions. In D1:D2 Dopamine Receptor Interactions: Neuroscience and Psychopharmacology (ed. Waddington J. L.), pp. 71–78. Academic, London. Waddington J. L., Daly S. A., McCauley P. G. and O’Boyle K. M. (1994) Levels of functional interaction between D1-like and D2-like dopamine receptor systems. In Dopamine Receptors and Transporters: Pharmacology, Structure and Function, (ed. Niznik H. B.), pp. 511–537. Marcel Dekker, New York. Waddington J. L., Daly S. A., Downes R. P., Deveney A. M., McCauley P. G. and O’Boyle K. M. (1995) Behavioural pharmacology of ‘D-1-like’ dopamine receptors: further subtyping, new pharmacological probes and interactions with ‘D-2-like’ receptors. Prog. Neuropsychopharmac. biol. Psychiat. 19, 811–831. Wei L.-N. (1997) Transgenic animals as new approaches in pharmacological studies. A. Rev. Pharmac. Toxicol. 37, 119–141. White F. J. and Hu X.-T. (1993) Electrophysiological correlates of D1:D2 interactions. In D1:D2 Dopamine Receptor Interactions: Neuroscience and Psychopharmacology (ed. Waddington J. L.), pp. 79–114. Academic, London. Xu M., Moratalla R., Gold L. H., Hiroi N., Koob G. F., Graybiel A. M., White F. J. and Tonegawa S. (1994) Dopamine D1 receptor mutant mice are deficient in striatal expression of dynorphin and in dopamine-mediated behavioural responses. Cell 79, 729–742. Xu M., Xiu-Ti H., Cooper D. C., Moratalla R., Graybiel A. M., White F. J. and Tonegawa S. (1994) Elimination of cocaine-induced hyperactivity and dopamine-mediated neurophysiological effects in dopamine D1 receptor mutant mice. Cell 79, 945–955. (Accepted 18 May 1999)
Pergamon PII: S0306-4522(99)00297-3
Neuroscience Vol. 93, No. 4, pp. 1483–1489,1483 1999 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
CONSERVATION OF BEHAVIOURAL TOPOGRAPHY TO DOPAMINE D1-LIKE RECEPTOR AGONISTS IN MUTANT MICE LACKING THE D1A RECEPTOR IMPLICATES A D1-LIKE RECEPTOR NOT COUPLED TO ADENYLYL CYCLASE J. J. CLIFFORD,* O. TIGHE,† D. T. CROKE,† A. KINSELLA,‡ D. R. SIBLEY,§ J. DRAGOk and J. L. WADDINGTON*¶ Departments of *Clinical Pharmacology and †Biochemistry, Royal College of Surgeons in Ireland, St Stephen’s Green, Dublin 2, Ireland ‡Department of Mathematics, Dublin Institute of Technology, Dublin 8, Ireland §Experimental Therapeutics Branch, National Institute for Neurological Disorders and Stroke, Bethesda, MD 20892, U.S.A. k Department of Anatomy, Monash University, Clayton, Victoria 3168, Australia
Abstract—Though D1-like dopamine receptors [D1A/B] are defined in terms of linkage to the stimulation of adenylyl cyclase, with D1A assumed to be the functionally prepotent subtype, evidence suggests the existence of another, novel D1-like receptor without such coupling. To investigate these issues we challenged mutant mice having targeted gene deletion of the D1A receptor with selective agonists and used an ethologically-based assessment technique to resolve resultant behavioural topography. D1-likedependent behaviour was substantially conserved in D1A-null mice relative to wild-types following challenge with each of two selective D1-like agents: A 68930 (0.068–2.0 mg/kg s.c.) which exhibits full efficacy to stimulate adenylyl cyclase, and SKF 83959 (0.016–2.0 mg/kg s.c.) which fails to stimulate adenylyl cyclase, and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine. Furthermore, responsivity to the selective D2-like agonist RU 24213 (0.1–12.5 mg/kg s.c.) was conserved in D1A-null mice, indicating the integrity of D1-like:D2-like interactions at the level of behaviour. These data are consistent with behavioural primacy of a D1-like receptor other than D1A [or D1B] that is coupled to a transduction system other than/additional to adenylyl cyclase. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: D1-like dopamine receptors, adenylyl cyclase, mutant mice, D1A knockout, behaviour.
mice having “knock-out” of the D1A receptor. 12,29,39,40 Using a topographical, ethologically-based approach, we have reported recently 3 that grooming, the most widely accepted behavioural index of D1-like receptor function in rats and mice which has been presumed to be mediated via the D1A receptor, 36 is not lost in D1A-null mice, together with topographical shifts among several other elements of behaviour. 3,5 This profile is inconsistent with D1-like-dependent grooming having a sole basis in D1A (or D1B) receptor function and focuses attention on another putative D1-like receptor. Furthermore, in D1A-null mice the stimulation of adenylyl cyclase by dopamine is lost while stimulation of phosphoinositide hydrolysis is conserved. 14 Described here are experiments designed to address specifically whether typical behavioural effects thought to be mediated via cyclase-coupled D1-like receptors as a class, and via cyclase-coupled D1A receptors in particular, might have a basis in another D1-like receptor not coupled to adenylyl cyclase. We posed the following questions: (i) do D1A-null mice show altered grooming and other responses to the highly selective, full efficacy D1-like agonist A 68930, 6,8 and to the anomalous D1-like agent SKF 83959 1,9 which fails to stimulate adenylyl cyclase and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine, yet induces behaviour that is qualitatively indistinguishable from that induced by A 68930 and (ii) do D1A-null mice show attenuation in typical responses to the selective D2-like agonist RU 24213, 10,13 given the well-established “enabling” or “permissive” role of D1-like-mediated function in general in the expression of typical D2-like-dependent effects via cooperative/synergistic D1-like:D2-like interactions? 34,35,38
Recognition of the functional role of dopamine in mediating multiple aspects of mammalian psychomotor behaviour, and in the actions of drugs used to treat disorders such as Parkinson’s disease and schizophrenia, has not been matched by understanding of the receptor sites through which these effects occur. 17,36 Two major families of D1-like [D1A/D1, D1B/D5] and D2-like [D2L/S, D3, D4] receptors have been identified in molecular biological terms. 16,28 However, given the general paucity of drugs able to discriminate materially within each family, considerably less is known of functional parcellation among individual family members. Furthermore, within the D1-like family all members show coupling to the stimulation of adenylyl cyclase, in accordance with the original defining characteristic of D1 as opposed to D2 receptors, 21 yet there is classical pharmacological evidence for the existence of another D1-like dopamine receptor that is coupled to phosphoinositide hydrolysis rather than/additional to adenylyl cyclase; 22,31,32 however, any functional role for this receptor is uncertain, 36 and no such receptor has yet been cloned. An important new approach to the parcellation of function between receptor subtypes in the absence of selective pharmacological agents is the generation of mutant mice in which a given subtype is absent through targeted gene deletion by homologous recombination. 18,37 In relation to the D1-like family, this technique has been applied to generate mutant ¶To whom correspondence should be addressed. Abbreviations: A 68930, [1R,3S]-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman; RU 24213, (N-n-propyl-N-phenylethyl-p-3-hydroxyphenylethylamine; SKF 83959, 3-methyl-6-chloro-7,8-dihydroxy-1[3methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine. 1483
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Transgenic animals The generation of D1A-null mice was as reported previously. 12 In outline, the targeted gene deletion was constructed in 129/Sv embryonic stem cells and male chimeras mated with C57BL/6 females to produce heterozygous mutants. Homozygous mutants (D1A 2 / 2 ) and wild-type (D1A 1 / 1 ) littermates were bred and genotyped in Dublin among the progeny of heterozygous intermatings, using a polymerase chain reaction technique. 23 Because of some failure to thrive among D1A-null mice over the weaning period, 12 resulting in a slight decrease in body weight, 3,12 standard dry mouse chow was supplemented routinely with moist diet on the floor of the cage. They were housed in groups of four to five with food and water available ad libitum, and were maintained at 21 ^ 18C on a 12 h/12 h (09.00 on/21.00 off) light/dark schedule. Young black female mice from litters of the same generational age were used in behavioural assessments. All experiments on mice were performed in compliance with relevant laws and institutional guidelines; these studies were approved by the Research Committee of the Royal College of Surgeons in Ireland. Behavioural assessment On experimental days mice were removed from their home cage, placed individually in clear glass observation cages (36 × 20 × 20 cm 3) and left undisturbed for a habituation period of 3 h. Behavioural assessments were carried out in a manner similar to that which we have described previously. 2,3,9 Following injection of drug or vehicle animals were assessed using a rapid time-sampling behavioural checklist technique. For this procedure, each of 10 randomly allocated mice was observed individually for 5 s periods at 1 min intervals over 15 consecutive minutes, using an extended, ethologically-based behavioural checklist. This allowed the presence or absence of the following individual behaviours (occurring alone or in any combination) to be determined in each 5 s period: sniffing; locomotion (co-ordinated movement of all four limbs producing a change in location); ponderous locomotion (a “plodding” variant induced in mice by D2-like agonists, and different from normal, fluid ambulation); total rearing (of any form); rearing from a sitting position (front paws reaching upwards with hind limbs on floor in sitting position); rearing free (front paws reaching upwards away from any cage wall while standing on hind limbs); rearing towards a cage wall (front paws reaching upwards on a cage wall while standing on hind limbs); sifting (sifting movements of the front paws through cage bedding material); grooming (of any form); intense grooming (grooming of the snout and then the face with the forepaws followed by vigorous grooming of the hind flank or anogenital region with the snout); vacuous chewing (chewing movements not directed onto any physical material); chewing (chewing movements directed onto cage bedding and/or faecal pellets without consumption); eating (chewing with consumption); climbing (jumping onto cage top with climbing along grill in inverted or hanging position); stillness (motionless, with no behaviour evident). After this 15 min assessment using the behavioural checklist, animals were evaluated using a conventional 0-to-6-point stereotypy scale: 2,10 0 asleep or inactive; 1 episodes of normal activities; 2 discontinuous activity with bursts of prominent sniffing or rearing; 3 continuous stereotyped activity such as sniffing or rearing along a fixed path; 4 stereotyped sniffing or rearing fixated in one location; 5 stereotyped behaviour with bursts of licking or gnawing; 6 continuous licking or gnawing. This cycle of assessment by behavioural checklist followed by stereotypy scale was repeated on two further occasions over a total observation period of 1 h. Mice were used on two occasions only, separated by a drug-free interval of at least one week; on each occasion mice were allocated randomly to one of the various treatment groups. All assessments were made by an observer unaware of the treatment and genotype of each animal. Materials A 68930 ([1R,3S]-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman; Abbott, U.S.A.) was dissolved in dilute acetic acid and made up to volume with distilled water; SKF 83959 (3-methyl-6-chloro7,8-dihydroxy-1[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine; RBI/NIMH, U.S.A.) was dissolved in distilled water; RU 24213 (N-n-propyl-N-phenylethyl-p-3-hydroxyphenylethylamine; HoechstMarion-Roussel, France) was dissolved in distilled water. All agents
or their respective vehicles were injected subcutaneously into the flank in a volume of 2 ml/kg. Data analysis From application of the behavioural checklist, the total counts for each individual behaviour were determined as the number of 5 s observation “windows” in which a given behaviour was evident, summed over the 1 h period, and expressed as means ^S.E.M.; stereotypy scores were averaged over the 1 h period and expressed similarly. These data were then analysed using analysis of variance, subsequent to square-root transformation for behavioural counts, with Helmert contrasts 27 to identify those particular drug doses at which responsivity differed by genotype, relative to lower doses, for a given topography of behaviour. RESULTS
Responsivity to A 68930 The characteristic response to A 68930 was a prominent, dose-dependent induction of grooming, particularly intense grooming which constitutes ethologically complete grooming syntax (0.068–2.0 mg/kg; P , 0.001 for each topography of grooming). Induction of overall grooming by A 68930 did not differ between the genotypes (no dose × genotype interaction, P . 0.05). There was also no dose × genotype interaction (P . 0.05) for intense grooming, though responsivity to the highest dose of A 68930 (2.0 mg/kg) only was somewhat reduced in D1A-null mice relative to wild-types (P , 0.01); however, in D1A-null mice intense grooming to this dose of A 68930 still occurred to significant excess (P , 0.05) relative to its own vehicle control. A 68930 failed to influence locomotion in wild-types but induced prominent locomotion in D1A-null mice (dose × genotype interaction, P , 0.01), primarily at 2.0 mg/kg (P , 0.001). There was no significant reduction in sniffing or sifting responses to A 68930, though total rearing and each topography of rearing response (free, to a wall, and from a seated position) was reduced (P , 0.01) in D1A-null mice relative to wild-types at 2.0 mg/kg (Fig. 1). No stereotyped behaviour was evident, and there was no material stimulation of any other topography of behaviour for either genotype. Responsivity to SKF 83959 Similarly, the characteristic response to SKF 83959 was induction of grooming, including intense grooming (0.016– 2.0 mg/kg; P , 0.001 for grooming, P , 0.05 for intense grooming). Induction of overall grooming by SKF 83959 was somewhat reduced in D1A-null mice relative to wildtypes (dose × genotype interaction, P , 0.01), particularly at 0.08 mg/kg (P , 0.001); however, at an intermediate dose (0.4 mg/kg), induction of overall grooming in D1A-null mice occurred to significant excess (P , 0.05) relative to its own vehicle control, and to an extent that did not differ significantly from wild-types given the same dose. However, induction of intense grooming by SKF 83959 did not differ between genotypes (no dose × genotype interaction, P . 0.05). Induction of locomotion by SKF 83959 was somewhat reduced in D1A-null mice relative to wild-types (dose × genotype interaction, P , 0.05), particularly at 0.08 and 2.0 mg/kg (P , 0.05). There was no significant reduction in sniffing or chewing responses to SKF 83959, while sifting was reduced (P , 0.05) but not abolished in D1A-null relative to wildtypes at 0.08 mg/kg. Among topographies of rearing responsivity to SKF 83959, total rearing and both rearing free and rearing
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Fig. 1. Behavioural counts for grooming, intense grooming, locomotion, total rearing and sifting responses to A 68930 (0.068–2.0 mg/kg) vs vehicle (V) in “knockout” [D1A 2 / 2 ] vs wild-type [D1A 1 / 1 ] mice. Data are means ^S.E.M. for n 8 per group; ***P , 0.001, **P , 0.01 vs wild-types.
from a seated position did not differ between genotypes while rearing to a wall was reduced in D1A-null mice at 0.08 mg/kg (P , 0.05) and 2.0 mg/kg (P , 0.01) relative to wild-types (Fig. 2). No stereotyped behaviour was evident, and there was no material stimulation of any other topography of behaviour for either genotype.
lenge stereotypy ratings were elevated (P , 0.01) in D1A-null mice relative to wildtypes (Fig. 3). RU 24213 did not induce any form of grooming behaviour; indeed, it effected a dosedependent reduction in baseline levels of overall grooming which did not differ between the genotypes. There was no material stimulation of any other topography of behaviour, and no atypical behaviours were released in D1A-null mice.
Responsivity to RU 24213 The characteristic response to RU 24213 was the prominent, dose-dependent induction of stereotyped sniffing and ponderous locomotion (0.1–12.5 mg/kg; P , 0.001 for each topography of behaviour). Neither the induction of sniffing and ponderous locomotion nor increases in stereotypy score by RU 24213 differed between the genotypes (no dose × genotype interactions, P . 0.05), though following vehicle chal-
DISCUSSION
Stimulation of adenylyl cyclase was the first transduction mechanism identified for dopamine receptor activation 15,20 and remains central to dopamine receptor typology and function. Subsequent identification of a pharmacologically distinct subtype (D2, either coupled to inhibition of adenylyl cyclase or without cyclase coupling) underpinned the original
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Fig. 2. Behavioural counts for grooming, intense grooming, locomotion, total rearing and sifting responses to SKF 83959 (0.016–2.0 mg/kg) vs vehicle (V) in “knockout” [D1A 2 / 2 ] vs wild-type [D1A 1 / 1 ] mice. Data are means ^S.E.M. for n 8 per group; ***P , 0.001, **P , 0.01, *P , 0.05 vs wild-types.
proposal for dopamine receptor nosology whereby those coupled to stimulation of adenylyl cyclase were designated D1. Thereafter, following a period over which greater functional relevance was ascribed to the D2 receptor, the introduction of selective D1 agonists and antagonists readily indicated a profound role for D1 receptors in mammalian dopaminemediated function which encompasses multiple aspects of psychomotor behaviour. 17,36 In particular, the induction of grooming, including the topography of intense grooming which constitutes ethologically complete, programmed grooming syntax, 4 endures as the most widely accepted behavioural index of D1 receptor activation; such grooming topography is readily induced in a pharmacologically-specific manner by all D1 agonists identified to date. 24,33,36 Among the family of D1-like subtypes identified on a molecular biological basis (D1A and D1B in rodents; D1 and D5 as
their primate homologues), each evidences “defining” linkage to the stimulation of adenylyl cyclase, 16,28 as do D1C 30 and D1D 7 subtypes identified to date only in lower species. Within the rodent D1-like family, it is the D1A receptor that has been ascribed functional prepotence, and this includes mediation of grooming behaviour; the D1B receptor has a low density corticolimbic localization, while it is D1A receptors that are localized densely to those major, dopamine-innervated striatal/limbic striatal regions that are known to subserve typical dopaminergic behaviours and in which D1B receptors are essentially absent. 16,17,28,36 In the present context, grooming behaviour, particularly ethologically complete, intense grooming syntax, has its genesis in the D1A-rich and D1Bdeficient anterior dorsolateral striatum, 4 at which selective inactivation of D1-like receptors with 1-ethoxycarbonyl-2ethoxy-1,2-dihydroquinoline abolishes D1-like agonist-induced
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Fig. 3. Behavioural counts for sniffing, ponderous locomotion, total rearing and sifting, and stereotypy scores, in response to RU 24213 (0.1–12.5 mg/kg) vs vehicle (V) in “knockout” [D1A 2 / 2 ] vs wild-type [D1A 1 / 1 ] mice. Data are means ^S.E.M. for n 8 per group.
grooming; 26 on this basis, mice with targeted gene deletion of the D1A receptor would be expected not to evidence any typical grooming response to D1-like agonists as conceptualized currently. A 68930 is a reference D1-like agonist having high affinity and selectively for D1-like over D2-like and non-dopaminergic receptors and demonstrating full efficacy, indistinguishable from that of dopamine itself, in terms of stimulation of adenylyl cyclase. 6,8 Yet, strikingly, the prominent induction of overall grooming by A 68930 in wild-types was entirely conserved in D1A-null mice. In particular, induction of intense grooming by A 68930 was substantially unaltered; some
attenuation in (but not abolition of) intense grooming only to the highest dose of A 68930 appeared to reflect disruption of grooming syntax as response incompatibility with prominent stimulation of locomotion, which this same dose of A 68930 induced only in the D1A-null genotype. Sniffing and sifting responses to A 68930 were also little altered, though these behaviours, like locomotion, are not specific to D1-like receptor stimulation; only rearing responsivity to A 68930 was blocked in D1A-null mice. Reduced responsivity to the D1-like agonist SKF 81297 has been reported in another line of mice having deletion of the D1A receptor, 39 though this was attained using a different genetic construct. It should be noted
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that responsivity to SKF 81297 was quantified 39 in terms of photobeam interruptions, which are insensitive to many individual elements of behaviour and composite numerous others so as to give limited resolution of the actual topography of behaviour. 3 On the basis of the present studies, substantial conservation in D1A-null mice of several topographies of behavioural responsivity to A 68930, particularly of grooming and intense grooming syntax, would indicate the involvement of a D1-like receptor other than D1A (or D1B). To illuminate further the processes involved in these effects, we utilized the novel, anomalous agent SKF 83959. This agent has high affinity and selectivity for D1-like over D2-like and non-dopaminergic receptors, but fails to stimulate adenylyl cyclase, and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine; 1,10 yet, despite evidencing all the “defining” characteristics of an antagonist at cyclase-coupled D1-like receptors, it induces in rats grooming and intense grooming that is qualitatively indistinguishable from that induced by A 68930. 6,10,11 The induction of overall grooming by SKF 83959 in D1A-null mice was somewhat attenuated but not abolished, while ethologically complete, intense grooming syntax was essentially conserved in D1A-null mice relative to wild-types; though attenuation of locomotion to SKF 83959 in D1A-null mice contrasts with heightening of locomotion to A 68930, the basis of this difference is unclear. Sniffing and chewing responses to SKF 83959 were also little altered, while locomotion, sifting and rearing to a wall were attenuated in D1A-null mice. SCH 23390, an agent that shares with SKF 83959 an antagonist profile at cyclase-coupled D1-like receptors, has been reported to be less active in reducing photobeam interruptions and in inducing catalepsy in alternatively constructed D1A-null mice. 39 Thus, the distinct profiles of these drugs in mice with targeted gene deletion of the D1A receptor appear unrelated to their common profile as antagonists at cyclase-coupled D1-like receptors. On the basis of the present studies, substantial conservation in D1A-null mice of core topographies of behavioural responsivity to SKF 83959, particularly grooming and intense grooming, would indicate the involvement of a D1like receptor that is not coupled to adenylyl cyclase. In essence we report that D1A-null mice, in which D1A receptor binding and dopamine-sensitive adenylyl cyclase are essentially absent, 12,14 evidence substantial conservation of the characteristic topographical responsivity of wild-types to A 68930 and to SKF 83959, agents that act as a full agonist and antagonist, respectively, at cyclase-coupled D1-like receptors. Furthermore, well-recognized cooperative/synergistic and oppositional D1-like:D2-like interactions, through which D1A receptors have been presumed to critically “enable” or inhibit the expression of, respectively, typical
and atypical responses to the D2-like agonist RU 24213, 34– 36,38 appear also to be conserved in D1A-null mice at the level of behaviour; yet electrophysiological effects of selective dopaminergic agonists on nucleus accumbens neurons appear to be reduced therein. 40 Raised stereotypy scores in D1A-null mice given vehicle for RU 24213 were within the 0– 1 range indicative of normal, non-stereotyped behaviour, and may reflect the heightening of certain spontaneous behaviours in the absence of any treatment. 3 CONCLUSION
The present findings therefore suggest that these topographic behavioural effects do not involve the D1A or indeed any other molecular biologically characterized D1-like receptor having the “defining” characteristic of linkage to the stimulation of adenylyl cyclase, such as D1B; rather, they appear to have their basis in another, functionally prepotent D1-like receptor that is not so linked. Indeed, there are previous data that among a range of partial D1-like agonists, their efficacies both to induce grooming behaviour 25 and to inhibit cell firing in the nucleus accumbens 19 appear unrelated to their efficacies to stimulate adenylyl cyclase. Over recent years, classical pharmacological evidence has emerged to suggest the existence of an additional D1-like receptor linked to activation of phosphoinositide hydrolysis; 9,22,31,32 however, any functional role for this putative receptor at the level of behavioural regulation has remained unclear. We cannot exclude incontrovertibly some involvement of residual D1A receptors or of compensatory mechanisms consequent to the developmental absence of D1A receptors, or that behaviours at issue might involve more than one D1-like receptor or a nonD1-like receptor. However, while the present findings do not in themselves indicate directly the involvement in behavioural regulation of a D1-like receptor linked specifically to phosphoinositide hydrolysis, the action of D1-like agonists to stimulate adenylyl cyclase is lost in D1A-null mice while their action to stimulate phosphoinositide hydrolysis is conserved therein; 14 this parallelism with the present behavioural profile would be in accordance with such a mechanism and with a prominent behavioural role for this novel D1like receptor. Acknowledgements—These studies were supported by the Research Committee of the Royal College of Surgeons in Ireland and the Higher Education Authority. We thank Abbott for A 68930 and Hoechst-Marion-Roussel for RU 24213. SKF 83959 was kindly provided by Research Biochemicals International as part of the Chemical Synthesis Program of the National Institute of Mental Health, contract N01MH30003. John Drago is a Logan Research Fellow of Monash University.
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