Decreased prepulse inhibition and increased sensitivity to muscarinic,but not dopaminergic drugs in M<Subscript>5</Subscript> muscarinic acetylcholine receptor knockout mice

Rationale Schizophrenic patients show decreased measures of sensorimotor gating, such as prepulse inhibition of startle (PPI). In preclinical models, these measures may be used to predict antipsychotic activity. While current antipsychotic drugs act largely at dopamine receptors, the muscarinic acetylcholine receptors offer promising novel pharmacotherapy targets. Of these, the M₅ receptor gene was recently implicated in susceptibility to schizophrenia. Due to the lack of selective ligands, muscarinic receptor knockout mice have been generated to elucidate the roles of the five receptor subtypes (M₁–M₅). Objectives Here, we used M₅ receptor knockout (M₅−/−) mice to investigate the involvement of M₅ receptors in behavioral measures pertinent to schizophrenia. We tested the hypothesis that disruption of M₅ receptors affected PPI or the effects of muscarinic or dopaminergic agents in PPI or psychomotor stimulation. Materials and methods We measured PPI in M₅−/−, heterozygous and wild-type mice without drugs, and with clozapine (0.56–3.2 mg/kg) or haloperidol (0.32–3.2 mg/kg) alone, and as pretreatment to d-amphetamine. In addition, we evaluated locomotor stimulation by the muscarinic antagonist trihexyphenidyl (0.56–56 mg/kg) and by cocaine (3.2–56 mg/kg). Results The M₅−/− mice showed decreased PPI relative to wild-type mice, and clozapine appeared to reduce this difference, while haloperidol increased PPI regardless of genotype. The M₅−/− mice also showed more locomotor stimulation by trihexyphenidyl than wild-type mice, while cocaine had similar effects between genotypes. Conclusions These data suggest that disruption of the M₅ receptor gene affected sensorimotor gating mechanisms, increased sensitivity to clozapine and to the psychostimulant effects of muscarinic antagonists without modifying the effect of dopaminergic drugs.     

Locomotor and discriminative-stimulus effects of cocaine in dopamine D<Subscript>5</Subscript> receptor knockout mice

Rationale Dopamine D₁-like antagonists block several effects of cocaine, including its locomotor-stimulant and discriminative-stimulus effects. Because these compounds generally lack selectivity among the dopamine D₁ and D₅ receptors, the specific roles of the subtypes have not been determined. Objectives Dopamine D₅ receptor knockout (DA D₅R KO), heterozygous (HET) and wild-type (WT) mice were used to study the role of D₅ dopamine receptors in the effects of cocaine. In addition, effects of the D₁-like antagonist, SCH 39166 were also studied to further clarify the roles of D₁ and D₅ dopamine receptors in the discriminative-stimulus effects of cocaine. Methods DA D₅R KO, HET and WT mice were treated with cocaine (3–30 mg/kg) or vehicle and their horizontal locomotor activity was assessed. The mice were also trained to discriminate IP injections of saline from cocaine (10 mg/kg) using a two-lever food-reinforcement (FR10) procedure. Doses of cocaine (1.0–10 mg/kg) were administered 5 min before 15-min test-sessions. Results Cocaine dose-dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D₅R WT mice. Both DA D₅R KO and HET mice showed reduced levels of horizontal activity compared to WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine; doses of 1.0–10.0 mg/kg produced dose-related increases in the number of cocaine-appropriate responses. SCH 39166, at inactive to fully active doses (0.01–0.1 mg/kg) produced predominately saline-appropriate responding. SCH 39166 produced a dose-dependent rightward shift in the cocaine dose-effect curve in all genotypes, with similar apparent affinities. Conclusions The present data suggest an involvement of DA D₅R in the locomotor stimulant effects of cocaine. In addition, the data indicate that there is little involvement of the DA D₅R in the discriminative-stimulus effects of cocaine. In addition, the antagonism data suggest a role of the D₁ receptor in the behavioral effects of cocaine.     

Evaluation of muscarinic agonist-induced analgesia in muscarinic acetylcholine receptor knockout mice

Centrally active muscarinic agonists display pronounced analgesic effects. Identification of the specific muscarinic acetylcholine receptor (mAChR) subtype(s) mediating this activity is of considerable therapeutic interest. To examine the roles of the M(2) and M(4) receptor subtypes, the two G(i)/G(o)-coupled mAChRs, in mediating agonist-dependent antinociception, we generated a mutant mouse line deficient in both M(2) and M(4) mAChRs [M(2)/M(4) double-knockout (KO) mice]. In wild-type mice, systemic, intrathecal, or intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia can be mediated by both spinal and supraspinal mechanisms. Strikingly, muscarinic agonist-induced antinociception was totally abolished in M(2)/M(4) double-KO mice, independent of the route of application. The nonselective muscarinic agonist oxotremorine showed reduced analgesic potency in M(2) receptor single-KO mice, but retained full analgesic activity in M(4) receptor single-KO mice. In contrast, two novel muscarinic agonists chemically derived from epibatidine, CMI-936 and CMI-1145, displayed reduced analgesic activity in both M(2) and M(4) receptor single-KO mice, independent of the route of application. Radioligand binding studies indicated that the two CMI compounds, in contrast to oxotremorine, showed >6-fold higher affinity for M(4) than for M(2) receptors, providing a molecular basis for the observed differences in agonist activity profiles. These data provide unambiguous evidence that muscarinic analgesia is exclusively mediated by a combination of M(2) and M(4) mAChRs at both spinal and supraspinal sites. These findings should be of considerable relevance for the development of receptor subtype-selective muscarinic agonists as novel analgesic drugs.     

Cocaine-induced locomotor activity and cocaine discrimination in dopamine D<Subscript>4</Subscript> receptor mutant mice

Rationale Previous studies have found a role for dopamine D₂-like receptors in many of the behavioral effects of cocaine, including its stimulation of locomotor activity and interoceptive discriminative-stimulus effects. However, given the lack of selectivity of most of the available pharmacological tools among D₂, D₃ and D₄ dopamine receptors, the roles of these specific receptors remain unclear. Objectives The roles of specific dopamine D₄ receptors in the behavioral effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects were investigated using dopamine D₄ receptor knockout (DA D₄R KO) and wild-type (WT) mice. Methods The mice were trained in daily sessions to discriminate IP injections of saline from cocaine (10 mg/kg). Responses on one of two response keys intermittently produced a food pellet; one response was reinforced in sessions following cocaine injection (10 mg/kg), and the other response was reinforced in sessions following saline injection. Each 20th response produced a food pellet (fixed-ratio, or FR20 schedule of reinforcement). The dose-effects of cocaine and its interaction with the D₂-like antagonist, raclopride, were assessed. Horizontal locomotor activity was also assessed in each genotype. Results As previously shown), cocaine was a more potent stimulant of locomotor activity in the DA D₄R KO mice compared to WT littermate mice. In addition, cocaine was more potent in producing discriminative-stimulus effects in DA D₄R KO mice (ED₅₀ value=0.50 mg/kg) compared to their WT littermates (ED₅₀ value=2.6 mg/kg). Raclopride shifted the cocaine dose-effect curve in both DA D₄R KO and WT mice, though the shift was greater for the DA D₄R KO mice. Conclusions The present results on the stimulation of activity and interoceptive/subjective effects of cocaine are consistent with the previously reported disregulation of dopamine synthesis in DA D₄R KO mice, and further suggest a role of the DA D₄R in vulnerability to stimulant abuse.     

Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice

Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic neurotransmission. Overstimulation of cholinergic receptors by excess acetylcholine is known to be lethal. However, AChE knockout mice live to adulthood, although they have weak muscles, do not eat solid food, and die early from seizures. We wanted to know what compensatory factors allowed these mice to survive. We had previously shown that their butyrylcholinesterase activity was normal and had not increased. In this report, we tested the hypothesis that AChE-/- mice adapted to the absence of AChE by downregulating cholinergic receptors. Receptor downregulation is expected to reduce sensitivity to agonists and to increase sensitivity to antagonists. Physiological response to the muscarinic agonists, oxotremorine (OXO) and pilocarpine, showed that AChE-/- mice were resistant to OXO-induced hypothermia, tremor, salivation, and analgesia, and to pilocarpine-induced seizures. AChE+/- mice had an intermediate response. The muscarinic receptor binding sites measured with [3H]quinuclinyl benzilate, as well as the protein levels of M1, M2, and M4 receptors measured with specific antibodies on Western blots, were reduced to be approximately 50% in AChE-/- brain. However, mRNA levels for muscarinic receptors were unchanged. These results indicate that one adaptation to the absence of AChE is downregulation of muscarinic receptors, thus reducing response to cholinergic stimulation.     

Pharmacological analysis of the interaction of antimuscarinic drugs at M<Subscript>2</Subscript> and M<Subscript>3</Subscript> muscarinic receptors in vivo using the pithed rat assay

Muscarinic receptor antagonists form the mainstay of the therapeutic options for airway, bladder, and gastrointestinal smooth muscle disorders. Both M₂ and M₃ muscarinic receptors are involved in mediating smooth muscle contractility, although the relative functional contribution of each subtype, especially in the disease state, is unclear. Because the potency and selectivity of compounds for a given receptor in an in vivo setting can be dissimilar to that observed in an in vitro system, we developed an in vivo assay to simultaneously determine the absolute potency and selectivity of muscarinic receptor antagonists at M₂ and M₃ receptors using the pithed rat. Methacholine (MCh)-induced bradycardia and depressor responses were used as surrogate functional endpoints for M₂ and M₃ receptor activation, respectively. The influence of the muscarinic antagonists, tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, or tiotropium on the MCh-induced responses were studied. The estimated DR₁₀ values (dose producing a tenfold shift in the MCh curve) of tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, and tiotropium for the M₂ muscarinic receptor-mediated bradycardia were 0.22, 1.18, ∼2.6, 0.025, 0.40, and 0.0026 mg/kg, respectively, and 0.14, 0.18, 0.11, 3.0, 0.18, and 0.0017 mg/kg, respectively, for the M₃ muscarinic receptor-mediated depressor response. In a separate set of experiments, a single intravenous dose of tiotropium was administered before a MCh curve at 1, 3, 6, or 9 h to determine if tiotropium exhibited time-dependent selectivity for the M₃ receptor as has been reported from in vitro studies. The results indicate a slight preference of tiotropium for the M₃ receptor at later time points. The pithed rat assay may serve useful for elucidating the functional contribution of M₂ and M₃ receptors to the in vivo pharmacological effects of antagonists in disease animal models.     

Small intestinal motility in soluble guanylate cyclase α<Subscript>1</Subscript> knockout mice

Nitric oxide (NO) activates soluble guanylate cyclase (sGC) to produce guanosine-3′,5′-cyclic-monophosphate (cGMP). The aim of this study was to investigate the nitrergic regulation of jejunal motility in sGCα₁ knockout (KO) mice. Functional responses to nitrergic stimuli and cGMP levels in response to nitrergic stimuli were determined in circular muscle strips. Intestinal transit was determined. Nitrergic relaxations induced by electrical field stimulation and exogenous NO were almost abolished in male KO strips, but only minimally reduced and sensitive to ODQ in female KO strips. Basal cGMP levels were decreased in KO strips, but NO still induced an increase in cGMP levels. Transit was not attenuated in male nor female KO mice. In vitro, sGCα₁β₁ is the most important isoform in nitrergic relaxation of jejunum, but nitrergic relaxation can also occur via sGCα₂β₁ activation. The latter mechanism is more pronounced in female than in male KO mice. In vivo, no important implications on intestinal motility were observed in male and female KO mice.     

The effects of the neurosteroids: pregnenolone,progesterone and dehydroepiandrosterone on muscarinic receptor-induced responses in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes expressing M<Subscript>1</Subscript> and M<Subscript>3</Subscript> receptors

The neurosteroids pregnenolone, progesterone, and dehydroepiandrosterone (DHEA) occur naturally in the nervous system. They act on neural tissues, participate in neuronal signaling, and are reported to alter neuronal excitability via nongenomic mechanisms. Muscarinic receptors have important roles in neuronal functions in the brain and autonomic nervous system. In this study, we investigated the effects of pregnenolone, progesterone, and DHEA on M₁ and M₃ muscarinic receptors using the Xenopus oocyte expression system. Pregnenolone and progesterone inhibited the acetylcholine (ACh)-mediated responses of M₁ and M₃ receptors expressed in Xenopus oocytes, whereas DHEA did not. The half-maximal inhibitory concentrations (IC₅₀) for pregnenolone inhibition of M₁ receptor- and M₃ receptor-mediated currents were 11.4 and 6.0 M respectively; the IC₅₀ values for progesterone inhibition of M₁ receptor- and M₃ receptor-mediated currents were 2.5 and 3.0 M respectively. The selective protein kinase C (PKC) inhibitor GF109203X had little effect on the pregnenolone or progesterone inhibition of the ACh-induced currents in Xenopus oocytes expressing M₁ or M₃ receptors. The inhibitory effects of pregnenolone and progesterone were overcome at higher concentrations of ACh. Pregnenolone and progesterone inhibited the [³H]quinuclidinyl benzilate (QNB) binding to M₁ and M₃ receptor expressed in Xenopus oocytes, and Scatchard plot analysis of [³H]QNB binding revealed that pregnenolone and progesterone altered the K_d value and the B_max, indicating noncompetitive inhibition. In conclusion, pregnenolone and progesterone inhibited M₁ and M₃ receptor functions noncompetitively by the mechanism independent of PKC and by interfering with ACh binding to the receptors.     

Effects of activation of central nervous histamine receptors in cardiovascular regulation; studies in H<Subscript>1</Subscript> and H<Subscript>2</Subscript> receptor gene knockout mice

To elucidate the central roles of histamine receptors in cardiovascular regulatory system, systolic, mean, and diastolic blood pressures (BPs) and heart rate (HR) were examined in conscious H₁ receptor gene knockout (H₁KO) mice, H₂ receptor gene knockout (H₂KO) mice, H₁ and H₂ receptor gene double knockout (DKO) mice, and their respective control mice by the tail-cuff system. Histamine, histamine-trifluoromethyl-toluidine derivative (HTMT, an H₁ agonist), dimaprit (an H₂ agonist), and immepip (an H₃ agonist) were intrathecally administered to these KO mice and control mice. Basal BPs and HR were not different among these three KO mice and their control or wild-type mice. Intrathecal administration of histamine significantly increased BPs and decreased HR in control mice. The increases in BPs were produced by histamine in H₁KO and H₂KO mice and by HTMT and dimaprit in C57BL mice. The pressor responses by HTMT and dimaprit in C57BL mice were greater than those by histamine in H₁KO and H₂KO mice, although the same decreases in HR were induced by histamine in C57BL and H₁KO mice and by dimaprit in C57BL mice. The selective stimulation of H₃ receptors by immepip produced a consistent decrease in BPs in control mice. These results obtained with the exogenous selective agonists of three histamine receptors suggest that the pressor responses to histamine are mediated through the stimulation of both H₁ and H₂ receptors, whereas the atropine-sensitive decrease in heart rate is mainly due to H₂ receptors which activate the vagal output to the heart.     

Role of M<Subscript>3</Subscript> receptor in aconitine/barium-chloride-induced preconditioning against arrhythmias in rats

We demonstrated here that an initial treatment with aconitine- or barium-chloride-induced arrhythmias and resulted in reduced susceptibility of the heart to the induction of arrhythmias by a repeated drug treatment 24 h after the initial one, a delayed preconditioning cardioprotection. This delayed preconditioning was accompanied by enhanced expression of cardiac muscarinic M₃ receptor and abolished by M₃-selective antagonist. We conclude that muscarinic M₃ receptors might play an important role in conferring the pharmacological preconditioning against arrhythmias. This study thus expands our understanding of the cellular function and pathophysiological roles of muscarinic M₃ receptor and reconsolidates our view of cardioprotective effects of muscarinic M₃ receptor on myocardium. Yan Liu and Juan Du made equal contribution to this study.     

Characterization of dopamine D<Subscript>1</Subscript> and D<Subscript>2</Subscript> receptor function in socially housed cynomolgus monkeys self-administering cocaine

Rationale Social rank has been shown to influence dopamine (DA) D₂ receptor function and vulnerability to cocaine self-administration in cynomolgus monkeys. The present studies were designed to extend these findings to maintenance of cocaine reinforcement and to DA D₁ receptors.Objective Examine the effects of a high-efficacy D₁ agonist on an unconditioned behavior (eyeblinking) and a low-efficacy D₁ agonist on cocaine self-administration, as well as the effects of cocaine exposure on D₂ receptor function across social ranks, as determined by positron emission tomography (PET).Methods Effects of the high-efficacy D₁ agonist SKF 81297 and cocaine (0.3–3.0 mg/kg) on spontaneous blinking were characterized in eight monkeys during 15-min observation periods. Next, the ability of the low-efficacy D₁ agonist SKF 38393 (0.1–17 mg/kg) to decrease cocaine self-administration (0.003–0.1 mg/kg per injection, IV) was assessed in 11 monkeys responding under a fixed-ratio 50 schedule. Finally, D₂ receptor levels in the caudate and putamen were assessed in nineteen monkeys using PET.Results SKF 81297, but not cocaine, significantly increased blinking in all monkeys, with slightly greater potency in dominant monkeys. SKF 38393 dose-dependently decreased cocaine-maintained response rates with similar behavioral potency and efficacy across social rank. After an extensive cocaine self-administration history, D₂ receptor levels did not differ across social ranks.Conclusions These results suggest that D₁ receptor function is not substantially influenced by social rank in monkeys from well-established social groups. While an earlier study showed that dominant monkeys had higher D₂ receptor levels and were less sensitive to the reinforcing effects of cocaine during initial exposure, the present findings indicate that long-term cocaine use changed D₂ receptor levels such that D₂ receptor function and cocaine reinforcement were not different between social ranks. These findings suggest that cocaine exposure attenuated the impact of social housing on DA receptor function.     

Role of the M<Subscript>2</Subscript> muscarinic receptor pathway in lidocaine-induced potentiation of the relaxant response to atrial natriuretic peptide in bovine tracheal smooth muscle

We earlier reported that lidocaine augments the relaxation and accumulation of guanosine 3',5'-cyclic monophosphate produced by atrial natriuretic peptide (ANP) in bovine tracheal smooth muscle contracted with methacholine. However, the mechanism of that augmentation remains to be elucidated.In this study, we examined the role of muscarinic receptor-mediated signalling in the potentiation of ANP-induced relaxation by lidocaine. Lidocaine (100 micro M) augmented the relaxant responses to ANP in methacholine (0.3 microM)-contracted bovine tracheal smooth muscle but had no effect on the relaxant effects of ANP in preparations contracted with 100 micro M histamine. Treatment of tracheal preparations with methoctramine (0.03 microM), an M2 muscarinic receptor antagonist, enhanced ANP-induced relaxation and this treatment abolished the synergistic action of lidocaine on ANP. In radioligand-binding experiments, lidocaine concentration dependently displaced the specific binding of [3H]- N-methyl scopolamine to cloned human M2 and M3 muscarinic receptors expressed in Chinese hamster ovary cells.These results suggest that lidocaine acts as an M2 muscarinic receptor antagonist, thereby potentiating the relaxant responses to ANP in the bovine tracheal smooth muscle contracted with muscarinic receptor agonists.     

Importance of D<Subscript>1</Subscript> receptors for associative components of amphetamine-induced behavioral sensitization and conditioned activity: a study using D<Subscript>1</Subscript> receptor knockout mice

Rationale Repeated exposure to psychostimulant drugs results in conditioned activity and behavioral sensitization. Nonassociative cellular changes are necessary for behavioral sensitization, while associative processes appear to modify the sensitized response.Objective The purpose of the present study was to determine whether the absence of the D₁ receptor would disrupt associative processes modulating sensitization and conditioned activity.Methods Wild-type and D₁ receptor knockout mice (i.e., D₁-deficient mice) were injected with amphetamine (AMPH; 8 mg/kg, IP) before being placed in a previously novel test chamber (AMPH-Test group) or before being returned to the home cage (AMPH-Home group). Separate groups of mice were injected with saline (SAL) at the same time points. Distance traveled was measured 60 min each day, with the preexposure phase lasting 1 or 7 days. Sensitization was subsequently assessed after an injection of AMPH (1 mg/kg, IP), while conditioned activity was assessed after an injection of SAL.Results After a 1-day preexposure phase, wild-type and D₁-deficient mice exhibited similar patterns of sensitization and conditioned activity. After a 7-day preexposure phase, (1) D₁-deficient mice exhibited more robust context-specific sensitization than wild-type mice, (2) only D₁-deficient mice showed context-independent sensitization, and (3) only D₁-deficient mice showed conditioned activity.Conclusions Repeatedly treating D₁-deficient mice with AMPH appears to cause a general increase in responsivity. The reason for this hyper-responsivity is uncertain, but it is possible that cues from the testing environment were unable to inhibit responding (i.e., associative processes were disrupted). Alternatively, compensatory mechanisms (e.g., increases in D₂-like receptors) may affect processes underlying sensitization and conditioned activity.     

Behavioral sensitization to cocaine in rats: evidence for temporal differences in dopamine D<Subscript>3</Subscript> and D<Subscript>2</Subscript> receptor sensitivity

Rationale

Cocaine-induced changes in D₂ receptors have been implicated in the expression of sensitized behavioral responses and addiction-like behaviors; however, the influence of D₃ receptors is less clear.

Objectives

To characterize the effects of repeated cocaine administration on the sensitivity of rats to D₂- and D₃-mediated behaviors, as well as the binding properties of ventral striatal D₂-like and D₃ receptors.

Methods

Pramipexole was used to assess the sensitivity of rats to D₃/D₂ agonist-induced yawning, hypothermia, and locomotor activity, 24 h, 72 h, 10, 21, and 42 days after repeated cocaine or saline administration. The locomotor effects of cocaine (42 day) and the binding properties of ventral striatal D₂-like and D₃ receptors (24 h and 42 days) were also evaluated.

Results

Cocaine-treated rats displayed an enhanced locomotor response to cocaine, as well as a progressive and persistent leftward/upward shift of the ascending limb (72 h–42 day) and leftward shift of the descending limb (42 days) of the pramipexole-induced yawning dose–response curve. Cocaine treatment also decreased B _max and K _d for D₂-like receptors and increased D₃ receptor binding at 42 days. Cocaine treatment did not change pramipexole-induced hypothermia or locomotor activity or yawning induced by cholinergic or serotonergic agonists.

Conclusions

These studies suggest that temporal differences exist in the development of cocaine-induced sensitization of D₃ and D₂ receptors, with enhancements of D₃-mediated behavioral effects observed within 72 h and enhancements of D₂-mediated behavioral effects apparent 42 days after cocaine. These findings highlight the need to consider changes in D₃ receptor function when thinking about the behavioral plasticity that occurs during abstinence from cocaine use.

     

Decreased cocaine self-administration in Kir3 potassium channel subunit knockout mice.

Multiple G protein-linked neurotransmitter systems have been implicated in the behavioral effects of cocaine. While actions of certain neurotransmitter receptor subtypes and transporters have been identified, the role of individual G protein-regulated enzymes and ion channels in the effects of cocaine remains unclear. Here, we assessed the contribution of G protein-gated, inwardly rectifying potassium (Kir3/GIRK) channels to the locomotor-stimulatory and reinforcing effects of cocaine using knockout mice lacking one or both of the key neuronal channel subunits, Kir3.2 and Kir3.3. Cocaine-stimulated increases in horizontal locomotor activity in wild-type, Kir3.2 knockout, Kir3.3 knockout, and Kir3.2/3.3 double knockout mice, with only minor differences observed between the mouse lines. In contrast, Kir3.2 and Kir3.3 knockout mice exhibited dramatically reduced intravenous self-administration of cocaine relative to wild-type mice over a range of cocaine doses. Paradoxically, Kir3.2/3.3 double knockout mice self-administered cocaine at levels significantly higher than either single knockout alone. These findings suggest that Kir3 channels play significant and complex roles in the reinforcing effect of cocaine.     

Role of dopamine D<Subscript>1</Subscript> receptors in the prefrontal dorsal agranular insular cortex in mediating cocaine self-administration in rats

RATIONALE: Orbital/insular areas of the prefrontal cortex (PFC) are implicated in cocaine addiction. However, the role of dopamine D1 receptors in mediating cocaine self-administration in these sub-regions remains unknown. OBJECTIVES: To define the role of the dorsal agranular insular (AId) sub-region of the PFC, we investigated the effects of D1 receptor manipulation on self-administration behavior maintained by cocaine and cocaine-related stimuli. MATERIALS AND METHODS: Rats were trained to lever press for cocaine (1 mg/kg) under a fixed-interval 5-min (fixed-ratio 5:S) second-order schedule of reinforcement in the presence of conditioned light cues and contextual sound cues. Intra-AId infusions of vehicle, the D1-like receptor agonist SKF 81297 (0.1, 0.2, 0.4 microg/side) or the D1-like receptor antagonist SCH 23390 (1.0, 2.0, 4.0 microg/side), were administered prior to 1-h self-administration test sessions. Food-maintained responding under a second-order schedule was examined in separate rats to determine if pretreatment with D1 ligands produced general impairments in responding. RESULTS: Infusion of SKF 81297 (0.2 and 0.4 microg/side) reduced active lever responses during the first 30 min of 1-h test sessions, but did not influence cocaine intake. Infusion of 4.0 microg/side SCH 23390 reduced active lever responses and cocaine intake throughout the 1-h test sessions. Additionally, this dose of SCH 23390 disrupted food-maintained responding and intake. CONCLUSIONS: D1 receptor agonists and antagonists in the AId have diverse consequences and time courses of action. D1 receptor stimulation in the AId may reduce the motivating influence of cocaine-related stimuli on responding whereas D1 receptor blockade in this PFC sub-region produces global disruptions in behavior.     

Existence of functional M<Subscript>3</Subscript>-muscarinic receptors in the human heart

It has been recently shown that, in adult rat ventricular cardiomyocytes, functional muscarinic receptors (M-receptors) of the M₃-subtype exist that mediate inositol phosphate (IP) formation. The aim of this study was to characterize the M-receptor subtype mediating IP formation in the human heart. For this purpose in [³H]-myo-inositol labeled slices of human right atria, carbachol-induced [³H]-IP formation and its inhibition by several M-receptor antagonists was assessed. Carbachol (0.1 M–100 M) increased [³H]-IP formation; maximal increase at 100 M was 93±16% above basal (n=20); the pEC₅₀-value for carbachol was 5.56. Atropine (1 M) completely suppressed 100 M carbachol-induced [³H]-IP formation. Among the M-receptor subtype selective antagonists himbacine (1 M) and pirenzepine (1 M) only marginally affected carbachol-induced [³H]-IP formation whereas the M₃-receptor antagonist darifenacin (1 nM–1 M) concentration-dependently inhibited carbachol-induced [³H]-IP formation with a pK_i-value of 8.49. We conclude that in human right atrium there exist functional M₃-receptors that couple to IP formation.Abbreviations DAG Diacylglycerol - IP Inositol phosphates - M Muscarinic - PLC Phospholipase C