Effect of amygdaloid kindling on rat striatal dopamine D1- and D2-receptors

The effect of kindling on dopaminergic (DA) neurotransmission was assessed by measuring dopamine D₁- and D₂-receptor binding in the dorsal and ventral striatum of rats either 2 hours (short-term) or 3–4 weeks (long-term) after the last kindled seizure. Kindling did not have any significant long-term effect on DA D₂-receptor K_d or B_max values in the dorsal or ventral striatum or on DA D₁-receptor parameters in the dorsal striatum. The short-term effect of kindled seizures was to abolish the asymmetry in DA D₂-receptor density observed in the dorsal striatum of control rats. DA D₁-receptor density was also increased in the dorsal striatum contralateral to the kindled amygdala of short-term rats. The short-term effects support the notion that limbic seizures can modify the lateral imbalance of DA activity in the striatum.     

Regional distribution of monoamines and dopamine D1-and D2-receptors in the striatum of the rat

Dopamine (DA) D₁-and D₂-receptor densities were determined in 18 discrete areas of the caudate-putamen-globus pallidus of male Wistar rats and compared to local DA concentrations. All three parameters were found to decrease caudally. The globus pallidus was distinguished by the low concentration of DA and its receptors and high noradrenaline, (NA) content. While there were no mediolateral differences in DA or DA D₁-receptors, a clear mediolateral gradient was observed for DA D₂-receptors which extended over several sections of the brain. The ratio of DA D₁-to D₂-receptors was significantly higher in the dorsal than in the ventral areas of the mediolateral and caudal striatum. This is the first report of clear dorsoventral differences in parameters relating to DA activity in the striatum. These findings may be of particular significance in understanding the functional dichotomy between the dorsal and ventral striatum.     

Lack of effect of chronic desipramine treatment on dopaminergic activity in the nucleus accumbens of the rat

The binding of [³H]SCH 23390 to dopamine (DA) D₁-receptors was measured in the nucleus accumbens of rats treated chronically with desipramine for 14 days. DA D₁ — and D₂-receptor binding using [³H]SCH 23390 and [³H]spiperone, respectively as ligands, was determined in rats treated for 28 days. NeitherB _max norK _d values were influenced by chronic desipramine treatment. In addition, chronic desipramine treatment (28 days) did not influence the dose dependent, quinpirole (10–1000 nM)-mediated inhibition of the electrically stimulated release of [³H]DA and [¹⁴C]ACh from nucleus accumbens slices or the dose dependent increase in [³H]DA release and decrease in [¹⁴C]ACh release in the presence of 1 and 10 M nomifensine. Therefore, our results suggest that the effect of chronic antidepressant treatment cannot be attributed to changes in either DA D₁₁-or D₂-receptor binding or DA D₂-receptor function in the nucleus accumbens.     

S-(+)-aporphines are not selective for human D3 dopamine receptors

Summary 1. Our aim was to test the hypothesis that selectivity for D₃ dopamine (DA) receptors may contribute to limbic anti-DA selectivity ofS-(+)-aporphine DA partial agonists.2. Affinity was tested with³H-emonapride, using human D₃ receptors in mouse fibroblasts and D₂ receptors in rat striatal tissue.3. D₃ receptors showed a picomolar affinity for³H-emonapride, Na⁺ dependence, and reversible saturability, as well as stereoselectivity. Confirmatory or novel D₃/D₂ pharmacologic selectivity was found with several benzamides, thioxanthenes, buspirone, GBR-12909, and DA agonists including hydroxyaminotetralins [ADTN, (+)-7-OH-DPAT, (–)-PPHT and its fluorescein derivative], (–)-N-propylnorapomorphine, (–)-3-PPP, (–)-quinpirole, and SDZ-205-502, but neither aminoergoline nor (+)-aporphine partial agonists.4. The results extend pharmacologic characterization of D₃-transfected cell membranes but fail to account for the high limbic anti-DA selectivity ofS-(+)-aporphines.     

GABA-Dopamine Receptor-Receptor Interactions in Neostriatal Membranes of the Rat

Recent evidence has shown in membrane preparations that the binding of one ligand to its receptor is able to modify the binding parameters of a second receptor (receptor-receptor interactions), allowing the modulation of incoming signals onto a neuron. To further understand the -amino-butyric acid (GABA)-dopamine (DA) interactions in the neostriatum we have carried out experiments to explore whether an activation of the GABA_A receptor could affect the binding characteristics of the D₂ DA receptor in membrane preparations of the rat neostriatum. The results show that GABA (30–100 nM) significantly increases the dissociation constant of the high affinity (K_H) D₂ DA binding site (labelled with the selective D₂ DA receptor antagonist [³H]raclopride and that such an effect is fully counteracted by the GABA_A receptor antagonist bicuculline (1 M). It is suggested that such putative GABA_A/D₂ receptor-receptor interactions may take place in the somato-dendritic membrane of the striato-pallidal GABA neurons and that it may modulate the inhibitory effects of DA on these neurons, mediated via D₂ receptors.     

Differential voltage-sensitivity of D2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D_2L receptor and we now extend our studies to include the other members of the D₂-like receptor subfamily; the D_2S, D₃, and D₄ dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D_2S, D₃, or D₄ receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D_2S and D₄ upon depolarization. In contrast, the concentration-response relationships for D₃-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.     

D1-Like Dopamine Receptor-Mediated Function in Congenic Mutants with D1 vs. D5 Receptor “Knockout”

Current understanding of the functional roles of individual dopamine D₁-like [D₁, D₅] and D₂-like [D_2L/S, D₃, D₄] receptor subtypes remains incomplete. In particular, the lack of pharmacological agonists and antagonists able to distinguish between D₁ and D₅ receptors means that any differential roles in the regulation of behavior are poorly understood. Mutant mice with targeted gene deletion (“knockout”) of individual dopamine receptor subtypes offer an important alternative approach to resolving these functional roles. In congenic D₁ mutants examined ethologically, progressive increases in specific topographies of behavior over wildtypes were considerably greater than those in D₁ mutants on a mixed genetic background; D₁ knockout appears to influence the neuronal substrate(s) of habituation to disrupt sculpture of the changing topography of behavior from initial exploration through to quiescence. Similarly, the D₁ receptor appears to regulate specific topographies of orofacial movement in the mouse as these are “sculpted” in a time-dependent manner. Although the well-recognized role of the D₁-like family in regulating several aspects of behavioral topography has been assumed to involve primarily D₁ receptors, this presumption may require modification to accommodate a subtle but not negligible role for their D₅ counterparts as evidenced in the phenotype of congenic D₅ mutants.     

Dopamine D2 and D4 receptor heteromerization and its allosteric receptor-receptor interactions

Dopamine D₂ and D₄ receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D₂R–D_4.xR (D_4.2R, D_4.4R or D_4.7R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET¹ techniques with the D₂R and D_4.7R receptors being the least effective in forming heteromers. Allosteric receptor–receptor interactions in D₂R–D_4.2R and D₂R–D_4.4 R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor–receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor–receptor interaction interfaces.     

Dopamine D1 receptor activation of adenylyl cyclase, not phospholipase C, in the nucleus accumbens promotes maternal behavior onset in rats

A body of evidence supports the idea that the mesolimbic dopamine (DA) system modulates the natural increase in responsiveness female rats show toward offspring (biological or foster) at birth. In the absence of the full hormonal changes associated with pregnancy and birth, female rats do not show immediate responsiveness toward foster offspring. Activation of the mesolimbic DA system can produce an immediate onset of maternal behavior in these females. For example, female rats that are hysterectomized and ovariectomized on day 15 of pregnancy (15HO) and presented with pups 48 hours later normally show maternal behavior after 2-3 days of pup exposure, but will show maternal behavior on day 0 of testing after microinjection of the DA D₁ receptor agonist, SKF 38393, into the nucleus accumbens (NA) at the time of pup presentation. DA D₁ receptor stimulation is known to activate cAMP intracellular signaling cascades via its stimulation of adenylyl cyclase (AC). However, some DA D₁ receptors are also linked to phospholipase C (PLC) and are capable of activating phosphatidylinositol signaling cascades. SKF 38393 stimulates both types of D₁ receptors. Here we provide evidence that the facilitatory effects of DA D₁ receptor stimulation in the NA on maternal behavior are mediated by AC-linked DA D₁ receptors. By examining the effects of intra-NA application of SKF 83822, a drug which selectively binds DA D₁-AC receptors, or SKF 83959, a drug which selectively activates D₁-PLC-linked receptors, we find that only SKF 83822 facilitates maternal behavior onset.     

Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats

Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D₃ in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D₃ treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D₃ supplementation improved the behavioural deficit. In conclusion, vitamin D₃ shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D₃ modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D₃ supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D₃ and muscarinic M3 receptors in regulating insulin secretion from pancreas.     

Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain

Prenatal stress greatly influences the ability of an individual to manage stressful events in adulthood. Such vulnerability may result from abnormalities in the development and integration of forebrain dopaminergic and glutamatergic projections during the prenatal period. In this study, we assessed the effects of prenatal stress on the expression of selective dopamine and glutamate receptor subtypes in the adult offsprings of rats subjected to repeated restraint stress during the last week of pregnancy. Dopamine D₂-like receptors increased in dorsal frontal cortex (DFC), medial prefrontal cortex (MPC), hippocampal CA₁ region and core region of nucleus accumbens (NAc) of prenatally stressed rats compared to control subjects. Glutamate NMDA receptors increased in MPC, DFC, hippocampal CA₁, medial caudate-putamen, as well as in shell and core regions of NAc. Group III metabotropic glutamate receptors increased in MPC and DFC of prenatally stressed rats, but remained unchanged in all other regions examined. These results indicate that stress suffered during the gestational period has long lasting effects that extend into the adulthood of prenatally stressed offsprings. Changes in dopamine and glutamate receptor subtype levels in different forebrain regions of adult rats suggest that the development and formation of the corticostriatal and corticolimbic pathways may be permanently altered as a result of stress suffered prenatally. Maldevelopment of these pathways may provide a neurobiological substrate for the development of schizophrenia and other idiopathic psychotic disorders.     

Enhanced dopamine D<Subscript>2</Subscript> receptor function in hypothalamus and corpus striatum: their role in liver,plasma and in vitro hepatocyte ALDH regulation in ethahol treated rats

Dopamine D₂ receptors are involved in ethanol self- administration behavior and also suggested to mediate the onset and offset of ethanol drinking. In the present study, we investigated dopamine (DA) content and Dopamine D₂ (DA D₂) receptors in the hypothalamus and corpus striatum of ethanol treated rats and aldehyde dehydrogenase (ALDH) activity in the liver and plasma of ethanol treated rats and in vitro hepatocyte cultures. Hypothalamic and corpus striatal DA content decreased significantly (P < 0.05, P < 0.001 respectively) and homovanillic acid/dopamine (HVA/DA) ratio increased significantly (P < 0.001) in ethanol treated rats when compared to control. Scatchard analysis of [³H] YM-09151-2 binding to DA D₂ receptors in hypothalamus showed a significant increase (P < 0.001) in B_max without any change in K_d in ethanol treated rats compared to control. The K_d of DA D₂ receptors significantly decreased (P < 0.05) in the corpus striatum of ethanol treated rats when compared to control. DA D₂ receptor affinity in the hypothalamus and corpus striatum of control and ethanol treated rats fitted to a single site model with unity as Hill slope value. The in vitro studies on hepatocyte cultures showed that 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in 10% ethanol treated cells to near control level. Sulpiride, an antagonist of DA D₂, reversed the effect of dopamine on 10% ethanol induced ALDH activity in hepatocytes. Our results showed a decreased dopamine concentration with enhanced DA D₂ receptors in the hypothalamus and corpus striatum of ethanol treated rats. Also, increased ALDH was observed in the plasma and liver of ethanol treated rats and in vitro hepatocyte cultures with 10% ethanol as a compensatory mechanism for increased aldehyde production due to increased dopamine metabolism. A decrease in dopamine concentration in major brain regions is coupled with an increase in ALDH activity in liver and plasma, which contributes to the tendency for alcoholism. Since the administration of 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in ethanol treated cells to near control level, this has therapeutic application to correct ethanol addicts from addiction due to allergic reaction observed in aldehyde accumulation.     

Dopamine D4 Receptors: Beyond Schizophrenia

Dopamine D₄ receptors mediate a wide range of neuronal signal transduction cascades. Malfunctions of these mechanisms may contribute to the pathophysiology of neuropsychiatric disorders, and their modification underlies the actions of many psychotropic drugs. Postmortem neuropathological and genetic studies provide inconclusive associations between D₄ receptors and schizophrenia. Clinical trials of partially selective lead D₄ antagonists have proved them to be ineffective against psychotic symptoms in patients diagnosed with schizophrenia. However, associations are emerging between D₄ receptors and other neuropsychiatric disorders, including attention-deficit hyperactivity disorder as well as specific personality traits such as novelty seeking. Preclinical studies indicate that D₄ receptors play a pivotal role in the cellular mechanisms of hyperactivity, impulsivity, and working memory. Accordingly, D₄ receptors have broader implications for human illnesses than has been suggested by early focus on psychotic illness as a clinical target, and selective D₄ agents may yield clinically useful drugs for several neuropsychiatric disorders that require improved treatments.     

Decreased dopamine D2 receptor function in cerebral cortex and brain stem: their role in hepatic ALDH regulation in ethanol treated rats

Ethanol exerts numerous pharmacological effects through its interaction with various neurotransmitters. The dopaminergic pathway is associated with cognitive, endocrine, and motor functions, and reinforcement of addictive substances or behaviours. Aldehyde dehydrogenase (ALDH) is a vital enzyme involved with alcohol metabolism and detoxification. In the present study, we investigated the role of cerebral cortex and brain stem dopamine D₂ receptors in the functional regulation on ALDH enzyme activity, in ethanol administrated rats. Two groups of rats were selected viz. control and alcoholic. Cerebral cortex, brain stem and the liver dopamine content was decreased significantly (P < 0.05, 0.05, 0.001, respectively) and homovanillic acid/dopamine (HVA/DA) ratio has significantly increased (P < 0.05, 0.001 and 0.001), respectively in ethanol treated rats when compared to control. Scatchard analysis of [³H]YM-09151-2 binding to synaptic membrane preparations of cerebral cortex and brain stem showed a significant decrease (P < 0.001, 0.05, respectively) in B _max in ethanol treated rats compared to control and the K _d also decreased significantly (P < 0.05). The ALDH analysis showed a significant increase (P < 0.05) in V _max in cerebral cortex, plasma and liver of experimental rats when compared with control without having significant change in brain stem but with decreased K _m (P < 0.001). Our results suggest that decreased function of dopamine mediated through DA D₂ receptor in the cerebral cortex and brain stem enhanced the brain, plasma and liver ALDH activity in ethanol treated rats. This ALDH regulation has significance to correct alcoholics from addiction due to allergic reaction observed in aldehyde accumulation.     

Enhanced [<Superscript>3</Superscript>H] Glutamate Binding in the Cerebellum of Insulin-Induced Hypoglycaemic and Streptozotocin-Induced Diabetic Rats

Aim Energy deprivation causes neuronal death affecting the cognitive and memory ability of an individual. The kinetic parameters of glutamate dehydrogenase (GDH), the enzyme involved in the production of glutamate, was studied in the cerebellum and liver and the binding parameters of glutamate receptors in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats were studied to reveal the role of glutamate excitotoxicity. Methods A single intrafemoral dose of streptozotocin was administered to induce diabetes. Hypoglycaemia was induced by appropriate doses of insulin subcutaneously in control and diabetic rats. The kinetic parameters V _max and K _m of GDH were studied spectrophotometrically at different substrate concentrations of α-ketoglutarate. Glutamate receptor binding assay was done with different concentrations of [³H] Glutamate. Results The GDH enzyme assay showed a significant increase (P < 0.001) in the V _max of the enzyme in the cerebellum of hypoglycaemic and diabetic rat groups when compared to control. The V _max of hypoglycaemic groups was significantly increased (P < 0.001) when compared to diabetic group. In the liver, the V _max of GDH was significantly increased (P < 0.001) in the diabetic and diabetic hypoglycaemia group when compared to control. The V _max of GDH increased significantly (P < 0.001) in the diabetic hypoglycaemic rats compared to diabetic group, whereas the control hypoglycaemic rats showed a significant decrease in V _max (P < 0.001) when compared to diabetic and diabetic hypoglycaemic rats. The K _m showed no significant change amongst the groups in cerebellum and liver. Scatchard analysis showed a significant increase (P < 0.001) in B _max in the cerebellum of hypoglycaemic and diabetic rats when compared to control. The B _max of hypoglycaemic rats significantly increased (P < 0.001) when compared to diabetic group. In hypoglycaemic groups, B _max of the control hypoglycaemic rats showed a significant increase (P < 0.001) compared to diabetic hypoglycaemic rats. The K _d of the diabetic group decreased significantly (P < 0.01) when compared to control and control hypoglycaemic rats. There was a significant decrease (P < 0.05) in the K _d of diabetic hypoglycaemic group when compared to the control hypoglycaemic rats. Conclusion Our studies demonstrated the increased enzyme activity in the hypoglycaemic rats with increased production of extracellular glutamate. The present study also revealed increased binding parameters of glutamate receptors reflecting an increased receptor number with increase in the affinity. This increased number of receptors and the increased glutamate production will lead to glutamate excitotoxicity and neuronal degeneration which has an impact on the cognitive and memory ability. This has immense clinical significance in the management of diabetes and insulin therapy.     

A solid phase parallel synthesis of diverse amides as dopamine D3 receptor ligands

A solid phase parallel synthesis using SynPhase? technology was used to couple a series of 21 carboxylic with three different 4-(4-arylpiperazinyl)butanamines. The resulting library was evaluated as dopamine D₃ receptor ligands giving rise to several compounds with affinities in the low nanomolar concentration range (9e and 9n with binding affinities at D₃ receptors of 0.10 and 0.35 nM respectively).     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

Discovery of PF-00217830: aryl piperazine napthyridinones as D2 partial agonists for schizophrenia and bipolar disorder

The synthesis and structure-activity relationship (SAR) of a novel series of aryl piperazine napthyridinone D₂ partial agonists is described. Our goal was to optimize the affinities for the D₂, 5-HT_2A and 5-HT_1A receptors, such that the D₂/5-HT_2A ratio was greater than 5 to ensure maximal occupancy of these receptors when the D₂ occupancy reached efficacious levels. This strategy led to identification of PF-00217830 (2) with robust inhibition of sLMA (MED = 0.3 mg/kg) and DOI-induced head twitches in rats (31% and 78% at 0.3 and 1 mg/kg) with no catalepsy observed at the highest dose tested (10 mg/kg).     

Adenylyl Cyclase Interaction with the D2 Dopamine Receptor Family; Differential Coupling to Gi, Gz, and Gs

1.The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D_2S, D_2L, D_3S, D_3L, and D₄) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity.2.COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the subunit of Gz. PTX treatment was employed to inactivate endogenous i, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells.3.D_2S or D_2L receptors can couple to the same extent to Gi and to Gz. The D₄ dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D_3S and D_3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively.4.We have demonstrated for the first time that the two D₃ receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX.5.Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.     

M1 Muscarinic Receptors Contribute to, whereas M4 Receptors Inhibit, Dopamine D1 Receptor-Induced [3H]-Cyclic AMP Accumulation in Rat Striatal Slices

In rat striatal slices labelled with [³H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [³H]-AMP ([³H]-cAMP) accumulation induced by the dopamine D₁ receptor agonist SKF-81297 (1 μM; 177±13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72±3%, IC₅₀ 0.30±0.06 μM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M₁ receptors, reduced the effect of SKF-81297 by 40±7% (IC₅₀ 251±57 pM) and enhanced the inhibitory action of a submaximal (1 μM) concentration of carbachol (69±4% vs. 40±7% inhibition, IC₅₀ 386±105 pM). The toxin MT-1, agonist at M₁ receptors, stimulated [³H]-cAMP accumulation in a modest but significant manner (137±11% of basal at 400 nM), an action additive to that of D₁ receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D₁ receptor-induced [³H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M₄ receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M₁ receptors and PKC stimulation.