Comparative effects of scopolamine and quinpirole on the striatal fos expression induced by stimulation of D(1) dopamine receptors in the rat.

Treatment of intact rats with the full D(1) dopamine agonist A-77636 induced Fos-like immunoreactivity in the medial and, to a lesser extent, the lateral portions of the striatum. Pretreatment with the muscarinic antagonist scopolamine hydrobromide (1.5-6 mg/kg) potentiated the response to A-77636 and eliminated the mediolateral staining gradient seen after A-77636 alone. Similar effects were not produced by scopolamine methylbromide, which fails to cross the blood-brain barrier, demonstrating that the actions of scopolamine were centrally mediated. The effects of scopolamine were further compared to those of the D(2)-like dopamine agonist quinpirole using a factorial design in which subjects were pretreated with either scopolamine, quinpirole, or a combination of the two drugs before receiving A-77636. Pretreatment with either scopolamine or quinpirole increased staining in the lateral striatum, but the combination of the two drugs was no more effective than was quinpirole alone. Pretreatment with quinpirole, but not scopolamine, resulted in a markedly "patchy" pattern of staining and actually suppressed staining in the region between patches in the medial striatum. These findings demonstrate that there are both differences and similarities between the effects of scopolamine and quinpirole on D(1) agonist-induced Fos expression and suggest that although inhibition of cholinergic neurons may be one of the mechanisms through which the effects of quinpirole are produced, other factors must also contribute.

Haloperidol induces Fos expression in the globus pallidus and substantia nigra of cynomolgus monkeys.

Systemic injections of the dopamine antagonist haloperidol (0.1-2.5 mg/kg) induced a dose dependent increase in Fos-like immunoreactivity (FLI) in the internal segment of the globus pallidus (GPi) and in the substantia nigra (SN) of cynomolgus monkeys. These findings are consistent with models of basal ganglia organization which predict that blockade of dopamine receptors should result in a disinhibition of cells in these structures. In the GPi, labeling was most pronounced along the ventral, lateral and medial borders of the nucleus and none of the pallidal cells expressing FLI were immunopositive for choline acetyltransferase. In the SN, immunoreactive nuclei were concentrated in the pars reticulata and the majority of labeled nigral neurons did not display tyrosine hydroxylase-like immunoreactivity. A small number of cells displaying FLI were also observed in the external pallidal segment, but no labeling was seen in the subthalamic nucleus. These findings indicate that blockade of dopamine receptors induces a characteristic pattern of Fos expression in the primate brain which strongly resembles that previously reported in rodents.

Unilateral dopamine depletion paradoxically enhances amphetamine-induced Fos expression in basal ganglia output structures.

The ability of amphetamine to induce expression of the immediate early gene protein, Fos, was examined by immunocytochemistry in animals with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle. Amphetamine induced Fos expression in the globus pallidus (GP) on the intact side of the brain, but this response was greatly attenuated on the dopamine-depleted side. In contrast, amphetamine induced little Fos expression in the entopeduncular nucleus (EPN) and the substantia nigra pars reticulata (SNpr) on the intact side of the brain, but resulted in pronounced expression in these structures on the lesioned side. These findings demonstrate that unilateral dopamine depletion results in a pathophysiological state in which some responses to amphetamine are attenuated while others are paradoxically potentiated. One explanation of these effects is that amphetamine may indirectly activate excitatory inputs to the SNpr and the EPN on both sides of the brain. On the intact side, these effects would be opposed by the simultaneous activation of inhibitory pathways arising in the striatum and the GP, with the result that little Fos expression would be seen. On the dopamine-depleted side, however, engagement of these inhibitory pathways would be attenuated and the unopposed effects of the excitatory inputs mobilized by amphetamine would result in exaggerated Fos synthesis.

Compartmentally specific effects of quinpirole on the striatal Fos expression induced by stimulation of D1-dopamine receptors in intact rats.

Injections of the full D1-agonist A-77636 (1.45 mg/kg) were found to induce clear Fos-like immunoreactivity (FLI) in the striatum of neurologically intact rats. Pretreatment with the D2-like agonist quinpirole (3 mg/kg) potentiated staining in the lateral striatum, but actually decreased the number of immunoreactive cells observed in the medial portion of the rostral striatum. Comparison with adjacent sections processed for the calcium binding protein calbindin, indicated that quinpirole pretreatment specifically suppressed staining in the matrix compartment of the striatum while tending to potentiate it in the striosomes, resulting in an extremely patchy pattern of labeling. These results suggest that exogenous stimulation of D2-receptors, although not essential for the induction of FLI, may play an important role in the compartmental patterning of neuronal activity within the striatum.

Substituted hexahydrobenzo[f]thieno[c]quinolines as dopamine D1-selective agonists: synthesis and biological evaluation in vitro and in vivo.

A series of substituted 9,10-dihydroxyhexahydrobenzo[f]thieno[c]quinolines (TB[f]Q), varying with respect to the position of the thiophene relative to the benzo[f]quinoline core and the nature and position of the substituent on the thiophene, were prepared and evaluated for their affinity and selectivity for the dopamine D1-like receptor. The thieno[3,2-c]B[f]Q regioisomers bearing a small alky1 (C1-C3) substituent at the 2 position were potent (Ki < 20 nM) and selective (D2/D1 > 50) D1 agonists with close to full agonist activity (IA > 85%). The compounds were resolved and found to exhibit a high level of enantiospecificity in their interaction with the D1 receptor. Selected compounds were tested in vivo in the 6-OHDA rodent model of Parkinson's disease and for their liability to produce seizure-like activities in mice. (5aR)-trans-2-Propyl-4,5,5a,6,7, 11b-hexahydro-3-thia-5-azacyclopent-1-ena[c]phenanthrene-9,10-diol (5) emerged as the compound with the best overall in vivo profile in terms of potency (ED50 = 0.04 mumol/kg) and safety.

The selective dopamine D1 receptor agonist A-86929 maintains efficacy with repeated treatment in rodent and primate models of Parkinson's disease.

The ability of the selective dopamine D1 receptor agonist (5aR,11bS)-4,5,5a,6,7,11b-hexahydro-2-propyl-3-thia-5-aza cyclopent-1-ena[c]-phenanthrene-9,10-diol (A-86929) to induce contralateral rotation after repeated administration was determined in rodent and primate models of Parkinson's disease. Testing was conducted in rats previously given unilateral 6-hydroxydopamine injections and in macaques previously given unilateral, intracarotid infusions of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Both treatments have been shown to reduce forebrain dopamine levels on the side of the infusion. Such animals rotate contralaterally after injections of direct-acting dopamine receptor agonists. Rats were administered A-86929 (0.11 or 0.22 micromol/kg s.c.) three times daily for 10 days, with injections spaced 3 h apart, and rotation was measured across a 9-h period on various treatment days. Initially, monkeys were given various doses of A-86929 (0.03, 0.10 or 0.30 micromol/kg i.m.), and rotation was monitored for 3 h after each dose. Significant, dose-dependent levels of contralateral rotation were achieved. Monkeys were next treated three times daily at 3-h intervals with A-86929 (0.3 micromol/kg). Analysis of total, daily rotation scores indicated that the magnitude of the behavioral response did not change significantly across the 10-day treatment period in monkeys, although it increased in rats (0.22 micromol/kg). The first daily injection tended to elicit greater and longer-lived responses than the subsequent daily injections in both species. In monkeys, this was particularly true on the first test day and was not seen by the last test. This study suggests that a selective D1 receptor agonist, such as A-86929, with full intrinsic activity relative to dopamine, may be useful for the treatment of Parkinson's disease.

Hyperactivity and behavioral seizures in rodents following treatment with the dopamine D1 receptor agonists A-86929 and ABT-431.

A-86929 ((-)-trans-9,10-dihydroxy-2-propyl-4,5,5a,6,7,11b-hexahydro-3- thia-5-azacyclopent-1-ena[c]phenanthrene) is a potent and selective full agonist at the dopamine D1 receptor. Both A-86929 and ABT-431 ((-)-trans-9,10-diacetyloxy-2-propyl-4,5,5a,6,7,11b- hexahydro-3-thia-5-azacyclopent-1-ena[c]phenanthrene hydrochloride), the diacetyl prodrug derivative of A-86929, were evaluated for their effects on behavioral excitability in rodents. In rats, A-86929 produced a dose-dependent increase in locomotor activity that was attenuated by the selective dopamine D1 receptor antagonist, SCH 23390, as well as by higher doses of the dopamine D2 receptor antagonist, haloperidol. Repeated administration of A-86929 over 6 days produced hyperactivity which did not change in magnitude across days. Acute administration of A-86929 and ABT-431 to mice produced behavioral seizure activity, with ED50 values of 7.1 and 2.7 mumol/kg, s.c., respectively, that was blocked by SCH 23390. Young rats (35-37 days) exhibited behavioral seizures following A-86929 and ABT-431 treatment (ED50 = 34.2 and 35.6 mumol/kg, s.c., respectively), but at doses higher than those required in mice. Moreover, adult rats (3 months) were less sensitive (ED50 = 345 mumol/kg, s.c.) to A-86929-induced seizures than young rats. Comparison of the ED50 values that produced behavioral seizure activity in rats with those previously established to produce contralateral rotation (ED50 = 0.24 mumol/kg, s.c.) in 6-hydroxydopamine-lesioned rat indicates that a significant dose separation exists between these two properties of A-86929.

MK-801 differentially affects dopamine D1 and D2 receptor agonist-induced behavioral tolerance and desensitization.

In this study we explored the effects of repeated MK-801 (0.10 mg/kg) treatment on rotation in rats with unilateral forebrain dopamine depletions. Daily injections of MK-801 across a 13-day period produced mild ipsilateral rotation which did not change significantly across days compared to daily injections of vehicle. Rats given repeated cotreatment of MK-801 with the selective D1 receptor agonist, A-85653 (0.06 mg/kg), developed response sensitization rather than the behavioral tolerance that was seen in rats given repeated vehicle+A-85653 injections. However, MK-801+A-85653 treated rats did demonstrate behavioral tolerance after an acute vehicle+A-85653 challenge, and the behavioral subsensitivity of rats given repeated vehicle+A-85653 injections reverted to normal in response to an acute MK-801+A-85653 challenge. Thus, MK-801 blocked the expression but not the development of D1-agonist induced behavioral tolerance. MK-801 treatment also enhanced striatal c-fos expression produced by A-85653 but only if MK-801 were given in combination with A-85653 2 h prior to sacrifice; prior daily treatment with MK-801 had no carry-over effect. In contrast to its effects on D1 agonist induced rotation, MK-801 cotreatment inhibited the robust contralateral rotation produced by repeated treatment with the D2/D3 agonist, quinpirole (0.15 mg/kg), and blocked both the development and expression of behavioral supersensitivity compared to rats treated with quinpirole alone. These results demonstrate differential effects of repeated MK-801 treatment on the development and expression of D1 and D2/D3 agonist induced response tolerance and sensitization, respectively.

Amphetamine induces Fos-like immunoreactivity in the striatum of primates.

Injections of D-amphetamine (5 mg/kg) three hours before sacrifice were found to elicit robust Fos-like immunoreactivity in the striatum of cynomolgus monkeys and of the common marmoset. Labeled cells were most frequently observed in the medial portion of the caudate nucleus and were distributed in a patchy fashion. Comparison with adjacent sections stained for the calcium binding protein calbindin indicated that the patches of amphetamine induced labeling corresponded to the calbindin-poor striosomes. These results are consistent with those reported in rats and thus suggest similarities in the basic organization of striatal mechanisms underlying the response to amphetamine in rodents and primates.

Persistent activation of the dopamine D1 receptor contributes to prolonged receptor desensitization: studies with A-77636.

A-77636 is a dopamine (DA) D1 receptor-selective agonist that was previously shown to elicit beneficial responses in animal models of Parkinson's disease (PD) (Kebabian et al.: Eur. J. Pharmacol. 229: 203, 1992). However, A-77636 is of limited potential for PD therapy because it induces rapid tolerance in vivo. To understand the basis of rapid onset of tolerance to the compound, we conducted studies to compare the in vitro properties of A-77636 and A-81686; the latter is a structurally related D1 agonist that did not induce significant tolerance in vivo under similar experimental conditions. With SK-N-MC, a neuroblastoma cell line, as an in vitro model for the D1 receptor, significant differences in D1 receptor function were noted after pretreatment with the two compounds. Specifically, 1-hr pretreatment with A-77636 resulted in significant residual cAMP production, even after the drug solution was removed and the cells were washed. The residual cAMP activity was selectively inhibited by SCH 23390, a selective D1 antagonist. The residual cAMP activity declined with pretreatment time, and after 4-hr pretreatment, little residual cAMP production was observed. Cotreatment of SK-N-MC cells with SCH 23390 and A-77636 did not prevent residual cAMP production by A-77636. In contrast, A-81686 did not elicit residual cAMP production is SK-N-MC cells. Although A-77636 treated cells were devoid of agonist response 4 hr after drug removal, A-81686-treated cells exhibited significant cAMP response after drug removal. Preincubation of rat striatal membranes with A-77636 resulted in a large decrease in D1 receptor binding, despite repeated washings, whereas A-81686 pretreatment caused only a small reduction in D1 receptor binding. On the basis of the present data, we conclude that A-77636 dissociates slowly from the D1 receptor. The continued activation of the D1 receptor by A-77636 leads to inability of the receptor to recover its responsivity, which may explain its long duration of action and its ability to induce rapid behavioral tolerance in vivo.

ABT-431: the diacetyl prodrug of A-86929, a potent and selective dopamine D1 receptor agonist: in vitro characterization and effects in animal models of Parkinson's disease.

(-)-Trans 9,10-hydroxy-2-propyl-4,5,5a,6,7,11b-hexahydro-3-thia-5- azacyclopent-1-ena[c]phenanthrene hydrochloride (A-86929) is a potent and selective full agonist at the dopamine (DA) D1-like receptor. Judging by its binding affinities to the D1 and D2 classes of receptors, the compound is approximately 20-fold D1 receptor-selective, whereas relative potencies based on functional in vitro assays indicate that A-86929 is greater than 400-fold D1-selective. A-86929 has moderate to weak (Ki > 1 microM) affinity at other monoaminergic and peptidergic receptors, at ion channels and at monoamine uptake sites. The catechol of A-86929 was bis-acetylated to produce the prodrug, (-)-trans 9,10-acetoxy-2-propyl-4,5,5a,6,7,11-b-hexahydro-3-thia- 5-azacyclopent-1-ena[c]phenanthrene hydrochloride (ABT-431), which is more chemically stable yet is rapidly converted to the parent compound with a half-life of less than 1 min in plasma. Both A-86929 and ABT-431 produced contralateral rotation in rats bearing unilateral 6-hydroxydopamine lesions, with ED50 values of 0.24 mumol/kg s.c. and 0.54 mumol/kg s.c., respectively. A-86929 and ABT-431 improved behavioral disability scores and increased locomotor activity in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned marmoset model of Parkinson's disease in a dose-dependent manner (the minimum effective dose was 0.10 mumol/kg s.c.). When administered three times daily for 30 consecutive days to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned marmosets, A-86929 significantly improved disability scores throughout the duration of the study. Current Parkinson's disease therapy includes L-dopa, which stimulates both classes of DA receptors by virtue of its conversion to DA in vivo, and direct-acting D2-selective agonists. Stimulation of the D2 receptor, which is associated with all current DA agonist-based therapies, may contribute to their dose-limiting side effects. An agent such as A-86929 (or its prodrug ABT-431), which selectively stimulates the D1 receptor, may represent a novel mechanism for Parkinson's disease therapy with the potential for an improved side-effect profile and, consequently, improved patient compliance.

Rotation and striatal c-fos expression after repeated, daily treatment with selective dopamine receptor agonists and levodopa.

The available evidence suggests that daily injections of selective dopamine (DA) D2 receptor agonists to DA depleted rats typically leads to behavioral sensitization, but the effects of repeated treatment with selective DA D1 receptor agonists are more equivocal. In this study we examined the effects of acute and repeated treatment with DA receptor agonists with various D1/D2 receptor selectivities on rotation and striatal c-fos activation in rats with unilateral DA depletions. Lesioned rats were treated daily for 10 d with either the novel, selective DA D1 receptor agonist, A-85653, the DA D2 receptor agonist, quinpirole, a combination of these compounds, or the indirect D1/D2 receptor agonist levodopa (L-DOPA). Over days, rats given A-85653 alone showed behavioral tolerance, whereas those given either quinpirole or L-DOPA demonstrated behavioral sensitization. Repeated A-85653 + quinpirole treatment s lead to an increase in response magnitude early in the testing sessions and this was accompanied by a reduction in response duration over days. Quantitative analysis of striatal c-fos activation was also conducted in lesioned rats treated acutely or repeatedly with A-85653, A-85653 + quinpirole or L-DOPA. Numbers of Fos-immunoreactive nuclei were sharply reduced after the agonist challenge in all animals given repeated, compared to acute, drug treatment, despite enhanced levels of rotation by rats given quinpirole + A-85653 or L-DOPA repeatedly. These results suggest that desensitization may develop at the DA D1 receptor as a consequence of repeated stimulation, and that the behavioral sensitization seen after repeated L-DOPA treatment may primarily involve the DA D2 receptor.

Dopamine antagonists induce fos-like-immunoreactivity in the substantia nigra and entopeduncular nucleus of the rat.

Injections of the D2 receptor antagonists haloperidol (0.5-8 mg/kg) and metoclopramide (6.25-50 mg/kg) in rats resulted in a dose dependent induction of Fos-like-immunoreactivity in the rostral portion of the entopeduncular nucleus (EPN) and in the medial portion of the pars reticulata of the substantia nigra (SNpr). Nigral staining occurred exclusively in neurons which were not immunoreactive for tyrosine hydroxylase and could be antagonized by pretreatment with the anticholinergic drug scopolamine (3 mg/kg). Effects were much less pronounced following injections of the selective D1 antagonist SCH-23390 (2-8 mg/kg). No staining could be observed following administration of the 5HT3 antagonist MDL-72222 (10 mg/kg) or the 5HT1/5HT2 antagonist metergoline (5 mg/kg), suggesting that the effects observed with dopamine antagonists were not secondary to actions at serotonin receptors. These results are consistant with the hypothesis that blockade of dopamine receptors results in a disinhibition of cells within the SNpr and EPN and further suggest that examination of immediate-early gene expression may provide a useful tool for studying the extrastriatal circuit engaged by manipulations of dopaminergic transmission.

CCK-A-selective tetrapeptides containing lys(N epsilon)-amide residues: favorable in vivo and in vitro effects of N-methylation at the aspartyl residue.

Previous structure-activity studies on a series of CCK-A selective tetrapeptide agonists, typified by A-71623 (Boc-Trp-Lys(CONH-Ph-o-Me)-Asp-(N-Me)Phe-NH2), have shown that replacement of the Lys(N epsilon-carbamoyl) substituent with N epsilon-acyl substituents resulted in partial agonists with moderate to high affinities for the CCK-A receptor and that replacement of the C-terminal dipeptide with either (N-Me)Asp-Phe or (N-Me)Asp-(N-Me)Phe was highly favorable to in vitro and in vivo CCK activity. The present study demonstrates that although analogues in the epsilon-amide series that are N-methylated at the Phe position are weakly active or inactive in an in vivo rat appetite suppression assay, incorporation of (N-Me)Asp or (N-Me)Asp-(N-Me)Phe modifications in this series results in analogues with markedly improved in vivo activity. In in vitro assays, there is minimal effect of N-methylation pattern on binding affinity, whereas there is a trend toward improved functional activity in the phosphatidylinositol hydrolysis assay in analogues containing (N-Me)Asp.

Tetrapeptide CCK-A agonists: effect of backbone N-methylations on in vitro and in vivo CCK activity.

N-Methylation of backbone amide bonds was conducted on a tetrapeptide that had been identified previously (Shiosaki, K.; et al. J. Med. Chem. 1991, 34, 2387-2842) as a potent and selective CCK-A agonist. N alpha-Methylation at the position corresponding to Asp32 (CCK-33 numbering) was consistent with high affinity, efficacy, and selectivity for the CCK-A receptor. Combination of this (N-Me)Asp with the (N-Me)Phe modification also provided a highly active analogue. The observation of parallel structure-binding affinity profiles with respect to sites of N-methylation in the C-terminal regions of tetrapeptide vs heptapeptide CCK analogues suggests that the two series interact similarly with the CCK-A receptor.

Repeated D1 receptor agonist treatment blocks cocaine-induced locomotor activity and c-fos expression.

Pretreatment of rats for 4 days with the selective dopamine D1 receptor agonist A-77636 attenuated the ability of cocaine to induce locomotor hyperactivity and to stimulate the expression of the proto-oncogene c-fos in the striatum and nucleus accumbens. Our results suggest that repeated D1 agonist treatment leads to subsensitivity of D1 receptors involved in mediating some of the effects of cocaine on behavior and gene expression.

Interactive effects of stimulation of D1 and D2 dopamine receptors on fos-like immunoreactivity in the normosensitive rat striatum.

Systemic administration of the selective, full, D1 dopamine agonist A-77636 [(1R,3S)3-(1'-adamantyl)-1-aminomethyl-3,4- dihydro-5,6-dihydroxy-1H-2-benzopyran hydrochloride] (0.36-2.9 mg/kg) led to a dose-dependent induction of Fos-like immunoreactivity (FLI) in the striatum. Quantitative analysis of the sections indicated that immunoreactive cells were more numerous in the medial than the lateral striatum and, within these regions, appeared to be randomly distributed. The staining produced by A-77636 could be abolished by pretreatment with the selective D1 antagonist SCH-23390. The selective D2 dopamine agonist quinpirole (3 mg/kg) had no effect on striatal FLI when given by itself, but markedly potentiated the weak striatal staining produced by low doses of A-77636. When combined with the highest dose of A-77636, which produced substantial staining by itself, quinpirole produced an increase in the number of immunoreactive cells seen in the lateral striatum but actually decreased the number present in the medial striatum. Statistical analysis of the distribution of immunoreactive cells demonstrated that, in both regions, quinpirole converted the relatively homogeneous staining seen after A-77636 alone into a markedly patchy pattern. These findings indicate that stimulation of D2 receptors produces both stimulatory and inhibitory effects on the D1-mediated expression of Fos in the striatum and that the interaction between D1 and D2 receptor stimulation must, therefore, be more complex than the simple synergism suggested by previous studies.

Dopamine agonists and stress produce different patterns of Fos-like immunoreactivity in the lateral habenula.

In rats treated systemically with either amphetamine, amfonelic acid or apomorphine, large numbers of cells displaying Fos-like immunoreactivity (FLI) could be seen in the lateral zone of the lateral habenula. The induction of FLI by amphetamine could be blocked either by pretreatment with haloperidol or by 6-hydroxydopamine lesions of ascending dopamine fibers at the level of the lateral hypothalamus. In contrast, a variety of stressors selectively induced FLI in the most medial portion of the lateral habenula. These findings support the concept of a functional differentiation of the medial and lateral regions of the lateral habenula and provide further evidence for involvement of the habenula in the circuitry of the basal ganglia.

Effects of repeated dopamine D1 receptor stimulation on rotation and c-fos expression.

Acute injections of the full dopamine D1 receptor agonist A-77636 ((1R,3S)-3-(1'adamantyl)-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1 H-2-benzopyran hydrochloride) induce ipsilateral Fos-like immunoreactivity and contralateral rotation into rats bearing unilateral dopamine depleting lesions. However, following repeated treatment, A-77636 fails to induce either striatal c-fos or rotation. Our results suggest that striatal c-fos activation mirrors behavioral indices of dopamine D1 receptor sensitivity.