Central Muscarinic Receptors Research Articles

Long-term clomipramine treatment upregulates forebrain acetylcholine muscarinic receptors, and reduces behavioural sensitivity to scopolamine in mice.

We have investigated the effects of long-term treatment with clomipramine, a tricyclic antidepressant, on central muscarinic acetylcholine receptors (mAChR) in mice. Repeated clomipramine administration resulted in an increase in the forebrain receptor density value (Bmax) for [3H]quinuclidinyl benzilate, a muscarinic ligand (P < 0.05), that was dependent on dose per administration (saline or 5, 10, or 20 mg kg(-1) once a day for 7 days) and number of days treated (20 mg kg(-1) for 1, 3, 5, or 7 days). No change in apparent affinity (defined as the reciprocal of the dissociation constant) (KD) occurred. Seven daily treatments with clomipramine (saline or 5, 10, or 20 mg kg(-1)) reduced hyperlocomotion induced by scopolamine (0.5 mg kg(-1), s.c.) dose-dependently, and the effect of 20 mg kg(-1) clomipramine was significant (P < 0.05). These results suggest that an upregulation of mAChR is produced by repeated clomipramine administration, and such a change is responsible for the decreased sensitivity to the muscarinic antagonist scopolamine.

The Allocentric Place Discrimination Task Is Selectively and Highly Dependent on the Central Muscarinic System in Rats

The allocentric place discrimination task (APDT) is useful in evaluating working memory separately from and simultaneously with motivation, motor and sensory ability. Muscarinic acetylcholine receptor antagonist scopolamine has been shown to selectively impair the accuracy of APDT without changing swimming speed, distance, and still time. For further evaluation of other neurotransmitters' roles in the APDT, pharmacological manipulations were performed. Neither diazepam 3.0 mg/kg, mecamylamine 10 mg/kg, haloperidol 0.5 mg/kg, nor 8-OH DPAT 1.0 mg/kg affected accuracy of place discrimination. Two kinds of responses were observed following the administration of MK-801 0.3 mg/kg: the accuracy of rats for longer swimming distance tended to decrease, and the accuracy of rats for normal swimming distance did not change. Therefore, NM-801 did not seem to affect the working memory selectively. In addition, neither flumazenil 10 mg/kg, ondansetron 0.3 mg/kg nor R(−)-α-metylhistamine 10 mg/kg attenuated the scopolamine-induced deficits. These results suggest that the central muscarinic receptors are selectively and highly important in the APDT.

Improving the nicotinic pharmacophore with a series of (Isoxazole)methylene-1-azacyclic compounds: synthesis, structure-activity relationship, and molecular modeling.

A series of (isoxazole)methylene-1-azacyclic compounds was prepared. The compounds were tested for affinity to central nicotinic acetylcholine receptors (nAChRs) and central muscarinic receptors. The compounds covered a broad range of affinities for the nAChRs (IC(50) = 0.32 to >1000 nM), with selectivities for the nAChRs over the muscarinic receptors in the range of 3-183. The high-affinity compound (Z)-26 (3-(4-methyl-5-isoxazolyl)methylene-1-azabicyclo[2.2. 2]octane, IC(50) = 3.2 nM) having only one energy minimum was used as the reference structure in a computational study. This ligand has enabled definition of an important distance parameter, and the existence of this parameter was supported by showing that other potent nicotinic ligands (for example, nicotine and epibatidine) fit the model.

Central muscarinic cholinergic receptor availability in patients treated with clozapine

Central muscarinic cholinergic receptor availability in patients treated with clozapine

Characterization of the central muscarinic cholinoceptors involved in the cholinergic pressor response in anesthetized dogs

Previous reports have shown that an intracisternal (i.c.) injection of acetylcholine in the dog increases both arterial blood pressure and plasma levels of noradrenaline and vasopressin via central muscarinic receptors. The aim of the present study was to characterize the central muscarinic cholinoceptor subtypes involved in such central cholinergic responses in anesthetized male Beagle–Harrier dogs ( n=12). For this purpose, we studied the relative potency of various muscarinic receptor antagonists to block the acetylcholine-induced pressor responses (30 μg kg −1 i.c.). The acetylcholine-induced pressor response was inhibited in a dose-dependent manner by the i.c. administration of the non-selective muscarinic receptor antagonist atropine (ID 50=0.5 μg kg −1), the muscarinic M 1 receptor antagonist pirenzepine (ID 50=0.45 μg kg −1), the muscarinic M 2 receptor antagonist methoctramine (ID 50=8.5 μg kg −1) and the muscarinic M 3 receptor antagonist para-fluoro-hexahydro-sila-difenidol (ID 50=43.7 μg kg −1). The order of potency of these four muscarinic receptor antagonists was: atropine=pirenzepine>methoctramine≫ para-fluoro-hexahydro-sila-difenidol. In order to confirm the selectivity for muscarinic M 1 receptors of this dose of pirenzepine, we checked that 40- to 50-fold higher concentrations were necessary to block a typical muscarinic M 2 receptor response (bradycardia) and a typical muscarinic M 3 receptor response (endothelial vasodilation) compared with methoctramine and para-fluoro-hexahydro-sila-difenidol, respectively. These results suggest that the pressor response elicited by intracisternal injection of acetylcholine in anesthetized Beagle–Harrier dogs is mediated through the activation of the muscarinic M 1 cholinoceptor subtype.

Antinociceptive activity of a 3(2H)-pyridazinone derivative in mice

The antinociceptive activity of a 3(2H)-pyridazinone derivative (18a) was investigated in mice. 18a administered at doses which did not change either motor coordination or locomotor activity was able to induce antinociceptive effects in four nociceptive tests, the hot plate test, the tail flick test, the writhing test, and the formalin test. In the hot plate and tail flick test, 18a-induced antinociception was observed both after intraperitoneal administration and after intracerebroventricular injection thus indicating 18a has a central site of action. The pretreatment with the opioid antagonist naloxone, the α 2-antagonist yohimbine or the GABA B antagonist CGP 35348 did not change 18a-induced antinociception in the hot plate test and in the tail flick test. Pretreatment with nicotinic antagonist mecamylamine did not change 18a effects either. A reversion of the 18a effects was observed after pretreatment with the muscarinic antagonists atropine and pirenzepine. Binding experiments revealed that 18a binds to muscarinic receptors, suggesting that 18a antinociception is mediated by central muscarinic receptors. The above findings together with the lack of parasympathomimetic cholinergic side effects indicate useful clinical application for this compound.

Synthesis and muscarinic activity of novel aniline derivatives with a 1-azabicyclo[3.3.0]octane moiety.

In order to develop drugs effective against Alzheimer's disease, we synthesized a series of aniline derivatives having a characteristic cyclic amine, 1-azabicyclo[3.3.0]octane ring, and evaluated their binding affinity for the central muscarinic cholinergic receptor. Among these compounds which showed high affinity to the M1 receptor, N-[2-(1-Azabicyclo[3.3.0]octan-5-yl)ethyl]-2-nitroaniline (9f fumarate, SK-946) showed the highest affinity. The ability of this compound to improve cognitive function was assessed using the passive avoidance test in scopolamine-induced dementia mice. Some anilines with a 1-azabicyclo[3.3.1]nonane ring were also synthesized by the ring expansion of the 1-azabicyclo[3.3.0]octane ring, and showed a high affinity for the central muscarinic cholinergic receptor.

Synthesis and biological activity of the metabolites of N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-2-nitroaniline fumarate (SK-946).

Three metabolites of N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-2-nitroaniline fumarate (SK-946), a novel central muscarinic cholinergic receptor agonist, were prepared to confirm their proposed structures, and tested for muscarinic receptor affinity in vitro.

Apolipoprotein E and Alzheimer???s Disease

Apolipoprotein E (APOE) is a plasma protein that plays an important role in cholesterol transport. The protein exists in 3 different isoforms coded for by alleles e2, e3 and e4. Recent studies have shown that the frequency of the APOE e4 allele is much greater among individuals with Alzheimer’s disease than age-matched healthy controls [an approximate 4-fold increase (36.6 vs 9.4%) in one recent study]. However, due to an insufficient sensitivity of the APOE e4 allele in detecting patients with Alzheimer’s disease and the presence of this allele in demented and nondemented individuals, APOE genotyping should not be used alone as a sole diagnostic test for Alzheimer’s disease. An additional recent findings that central muscarinic receptor binding, as revealed by positron emission tomography (PET) and [11C]benztropine, declines with the progression of Alzheimer’s disease regardless of the presence or absence of APOE e4 allele. These findings suggest that measures of acetylcholine neurotransmission in the living Alzheimer’s disease brain by PET help to visualise altered cholinergic function during the clinical course of Alzheimer’s disease and to identify appropriate individuals who are more likely to respond to emerging cholinergic interventions

Postacquisition scopolamine treatments reveal the time course for the formation of lamb odor recognition memory in parturient ewes.

Institut National de Recherche Agronomique/Centre National de la Recherche Scientifique Within 4 hr after parturition, ewes learn to recognize the odor of their lamb. Whether scopolamine, a muscarinic antagonist, interferes with lamb odor retention was studied. After 4 hr of mother-young contact, ewes were separated from their lambs for 3 hr. During separation, they received intramuscular injections of saline, methylscopolamine (peripheral muscarinic antagonist), or scopolamine. Only scopolamine (100 microg/kg) prevented subsequent lamb recognition. To assess whether this effect depended on the duration of the learning phase, mothers remained with their lambs for 4, 8, or 16 hr before the 3-hr separation period and the scopolamine treatments. Ewes treated after 4 or 8 hr of contact displayed disturbed lamb recognition, whereas those having 16 hr of contact did not. Activation of central muscarinic receptors is therefore important for the formation of lamb odor recognition memory during a critical period of less than 16 hr postpartum.

Central Vagotonic Effects of Atropine Modulate Spectral Oscillations of Sympathetic Nerve Activity

Low-dose atropine causes bradycardia either by acting on the sinoatrial node or by its effects on central muscarinic receptors increasing vagal activity. Any central muscarinic effects of high-dose atropine on RR interval are masked by peripheral muscarinic blockade at the sinoatrial node, which causes tachycardia. Effects of central parasympathetic activation on sympathetic activity are not known. Using power spectral analysis of RR interval, intra-arterial blood pressure, respiration, and muscle sympathetic nerve activity (MSNA), we examined the effects of both low (2 microgram/kg IV) and high (15 microgram/kg IV) doses of atropine. After low-dose atropine, RR increased by 9+/-1% (P<0.0001), the low-frequency (LF) component (in normalized units, NU) of RR variability decreased by -32+/-8%, and the high-frequency (HF)NU component increased (+74+/-19%); hence, LF/HF of RR variability fell by 52+/-10% (all P<0.01). Although overall MSNA did not change, LFNU of MSNA decreased (-15+/-5%), HFNU of MSNA increased (+31+/-3%), and LF/HF of MSNA fell (-41+/-8%) (all P<0.01). After high-dose atropine, LFNU of MSNA decreased (-17+/-12%), HFNU of MSNA increased (+22+/-3%), and LF/HF of MSNA fell (-51+/-21%) (all P<0.02). Increasing central parasympathetic activity with low-dose atropine is associated with an increase in the HF and a decrease in the LF oscillations of both RR interval and MSNA variability. High-dose atropine similarly induces an increase in the HF and a decrease in the LF components of MSNA variability. Thus, central parasympathetic activation is able to modulate the oscillatory characteristics of sympathetic nerve traffic to peripheral blood vessels.

28 Do central nicotinic and muscarinic cholinergic receptors play differential roles in attentional and episodic memory mechanisms? A study using a new 5-arm maze protocol

28 Do central nicotinic and muscarinic cholinergic receptors play differential roles in attentional and episodic memory mechanisms? A study using a new 5-arm maze protocol

Muscarinic Receptor Subtype Involvement in Brain Cholinergic Stimulation by Intracerebroventricular Neostigmine in Sinoaortic Denervated Rats

1. The present studies evaluated the participation of central muscarinic receptors in the cardiovascular effects of centrally injected neostigmine, a quaternary anticholinesterase, in conscious, sham-operated rats and in sinoaortic denervated animals. 2. The dose-dependent pressor effect of neostigmine (0.1 to 1 μg ICV) was greater in sinoaortic denervated rats than in sham-operated animals, but only a dose-dependent bradycardic effect was seen in sham-operated rats. 3. Doses of 3.3 nmol (ICV) of both the M 1 muscarinic antagonist, pirenzepine, and the M 3 muscarinic antagonist, 4-DAMP, prevented the pressor response to 1 μg of neostigmine in sham-operated rats and in sinoaortic denervated animals; however, the M 2 muscarinic antagonist, AF-DX116, partially blocked this response in sham-operated rats while failing to do so in sinoaortic denervated rats. In sham rats, doses of 3.3 nmol (ICV) of both pirenzepine and 4-DAMP prevented the bradycardic response to 1 μg (ICV) of neostigmine, whereas AF-DX116 induced a partial blockade. 4. 4-DAMP, at the dose of 0.3 nmol (ICV), but not pirenzepine at the same dose, prevented the pressor effect of neostigmine (0.1 to 1 μg ICV) in both groups of rats. Both muscarinic antagonists at this dose prevented the bradycardia elicited by the anticholinesterase (0.1 to 1 μg ICV), but 4-DAMP showed a greater antagonistic action on this cardiac effect than pirenzepine. In sham-operated rats, ICV injection of 0.3 nmol of AF-DX116 failed to modify the cardiovascular responses to 0.3 μg of neostigmine. 5. Results suggest mainly an involvement of brain M 3-subtype muscarinic receptors in the cardiovascular effect of intracerebroventricular administration of anticholinesterase neostigmine in both groups of rats.

Muscarinic agonists exhibit functional dopamine antagonism in unilaterally 6-OHDA lesioned rats.

(5R,6R) 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]oc tane (PTAC) is a selective muscarinic ligand with high affinity for central muscarinic receptors, agonist mode of action at the muscarinic M2 and M4 receptor subtypes and substantially less or no affinity for central dopamine receptors. In the present study PTAC, as well as the muscarinic agonists oxotremorine, RS86 and pilocarpine, inhibited dopamine D1 and D2 receptor agonist induced contralateral rotation in unilaterally 6-OHDA lesioned rats. The dose of SKF 38393 used to induce contralateral rotation also caused an intense Fos protein immunoreactivity in the rat dorsolateral striatum on the lesioned site which was inhibited by PTAC indicating that the inhibition of rotation by PTAC was not due to non-specific peripheral side effects.

Unexpected antipsychotic-like activity with the muscarinic receptor ligand (5 R,6 R)6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane

(5 R,6 R) 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane (PTAC) is a potent muscarinic receptor ligand with high affinity for central muscarinic receptors and no or substantially less affinity for a large number of other receptors or binding sites including dopamine receptors. The ligand exhibits partial agonist effects at muscarinic M 2 and M 4 receptors and antagonist effects at muscarinic M 1, M 3 and M 5 receptors. PTAC inhibited conditioned avoidance responding, dopamine receptor agonist-induced behavior and d-amphetamine-induced FOS protein M 5 expression in the nucleus accumbens without inducing catalepsy, tremor or salivation at pharmacologically relevant doses. The effect of PTAC on conditioned avoidance responding and dopamine receptor agonist-induced behavior was antagonized by the acetylcholine receptor antagonist scopolamine. The compound selectively inhibited dopamine cell firing (acute administration) as well as the number of spontaneously active dopamine cells (chronic administration) in the limbic ventral tegmental area (A10) relative to the non-limbic substantia nigra, pars compacta (A9). The results demonstrate that PTAC exhibits functional dopamine receptor antagonism despite its lack of affinity for the dopamine receptors and indicate that muscarinic receptor partial agonists may be an important new approach in the medical treatment of schizophrenia.

6beta-Acetoxynortropane: a potent muscarinic agonist with apparent selectivity toward M2-receptors.

A series of tropane derivatives, related in structure to baogongteng A (1), an alkaloid from a Chinese herb, were synthesized. 6beta-Acetoxynortropane (5) had weak affinity (Ki 22 microM) for central (M1) muscarinic receptors in a [3H]quinuclidinyl benzilate binding assay but had extremely high affinity (Ki 2.6 nM) and selectivity for M2-muscarinic receptors expressed in CHO cells. It had 13-fold lower affinity for M4-receptors, 260-fold lower affinity for M3-receptors, and 8200-fold lower affinity for M1-receptors expressed in CHO cells. The 6beta-carbomethoxy analogue (14) of baogongteng A had only weak affinity for M2-muscarinic receptors, as did 6beta-carbomethoxynortropane (13) and 6beta-acetoxytropane (4). In transfected CHO cells, 6beta-acetoxynortropane (5) was an agonist at M2-receptors, based on a GTP-elicited decrease in affinity, and a full agonist with an IC50 of 11 nM at M4-receptors, based on inhibition of cyclic AMP accumulation, while being a full agonist at M1-receptors with an EC50 of 23 nM and a partial agonist at M3-receptors with an EC50 of 3.6 nM, based in both cases on stimulation of phosphoinositide breakdown. All of the 16 tropane derivatives had weak affinities for central alpha4beta2-nicotinic receptors with 6beta-carbomethoxynortropane (13) having the highest affinity, which was still 150-fold less than that of nicotine. 6beta-Acetoxynortropane (5) represents a potent muscarinic agonist with apparent selectivity toward M2-receptors.

Choline administration reverses hypotension in spinal cord transected rats: the involvement of vasopressin.

Intracerebroventricular (i.c.v.) choline (50-150 microg) increased blood pressure and decreased heart rate in spinal cord transected, hypotensive rats. Choline administered intraperitoneally (60 mg/kg), also, increased blood pressure, but to a lesser extent. The pressor response to i.c.v. choline was associated with an increase in plasma vasopressin. Mecamylamine pretreatment (50 microg; i.c.v.) blocked the pressor, bradycardic and vasopressin responses to choline (150 microg). Atropine pretreatment (10 microg; i.c.v.) abolished the bradycardia but failed to alter pressor and vasopressin responses. Hemicholinium-3 [HC-3 (20 microg; i.c.v.)] pretreatment attenuated both bradycardia and pressor responses to choline. The vasopressin V1 receptor antagonist, (beta-mercapto-beta,beta-cyclopenta-methylenepropionyl1, O-Me-Tyr2, Arg8)-vasopressin (10 microg/kg) administered intravenously 5 min after choline abolished the pressor response and attenuated the bradycardia-induced by choline. These data show that choline restores hypotension effectively by activating central nicotinic receptors via presynaptic mechanisms, in spinal shock. Choline-induced bradycardia is mediated by central nicotinic and muscarinic receptors. Increase in plasma vasopressin is involved in cardiovascular effects of choline.

Conformationally constrained analogues of the muscarinic agonist 3-(4-(methylthio)-1,2,5-thiadiazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyr idine. Synthesis, receptor affinity, and antinociceptive activity.

Conformationally constrained analogues of the potent muscarinic agonist 3-(4-methylthio)-1,2,5-thiadiazol-3-yl)-1,2,5,6-tetrahydro-1-methy lpyridine (methylthio-TZTP, 17) were designed and synthesized with the aim of (a) improving the antinociceptive selectivity over salivation and tremor and (b) predicting the active conformation of 17 with respect to the dihedral angle C4-C3-C3'-N2'. Using MOPAC 6.0 tricyclic analogues (7, 15, 16) with C4-C3-C3'-N2' dihedral angles close to 180 degrees and a rotation hindered analogue (9) with a C4-C3-C3'-N2' dihedral angle close to 274 degrees were designed, as these conformations had previously been suggested as being the active conformations. The analogues were tested for central muscarinic receptor binding affinity, for their antinociceptive activity in the mouse grid shock test, and, in the same assay, for their ability to induce tremor and salivation. The data showed that the tricyclic analogues (7, 15, 16) were equipotent with 17 as analgesics, but with no improved side effect profiles. The rotation-hindered analogue 9 had neither muscarinic receptor binding affinity nor antinociceptive activity. These results suggest that the active conformation of 17 has a C3-C4-C3'-N2' dihedral angle close to 180 degrees.

Central activation by CS-932, a functionally relative M1 agonist

1.The present studies evaluated the participation of central muscarinic receptors in the cardiovascular effects of centrally injected neostigmine, a quaternary anticholinesterase, in conscious, sham-operated rats and in sinoaortic denervated animals.2.The dose-dependent pressor effect of neostigmine (0.1 to 1 μg ICV) was greater in sinoaortic denervated rats than in sham-operated animals, but only a dose-dependent bradycardic effect was seen in sham-operated rats.3.Doses of 3.3 nmol (ICV) of both the M1 muscarinic antagonist, pirenzepine, and the M3 muscarinic antagonist, 4-DAMP, prevented the pressor response to 1 μg of neostigmine in sham-operated rats and in sinoaortic denervated animals; however, the M2 muscarinic antagonist, AF-DX116, partially blocked this response in sham-operated rats while failing to do so in sinoaortic denervated rats. In sham rats, doses of 3.3 nmol (ICV) of both pirenzepine and 4-DAMP prevented the bradycardic response to 1 μg (ICV) of neostigmine, whereas AF-DX116 induced a partial blockade.4.4-DAMP, at the dose of 0.3 nmol (ICV), but not pirenzepine at the same dose, prevented the pressor effect of neostigmine (0.1 to 1 μg ICV) in both groups of rats. Both muscarinic antagonists at this dose prevented the bradycardia elicited by the anticholinesterase (0.1 to 1 μg ICV), but 4-DAMP showed a greater antagonistic action on this cardiac effect than pirenzepine. In sham-operated rats, ICV injection of 0.3 nmol of AF-DX116 failed to modify the cardiovascular responses to 0.3 μg of neostigmine.5.Results suggest mainly an involvement of brain M3-subtype muscarinic receptors in the cardiovascular effect of intracerebroventricular administration of anticholinesterase neostigmine in both groups of rats.

Nonhippocampal Muscarinic Receptors Are Required for Nonspatial Working Memory

The effects of scopolamine on nonspatial working memory were examined in rats with hippocampal lesions and sham operations. Performance was examined using a continuous conditional discrimination task in an operant box. Choice accuracy measured nonspatial working memory. Response bias, delay interval responses, and response probability measured response preference, stimulus control, motivation, and sensorimotor ability. Scopolamine (0.05, 0.075, 0.1, and 0.15 mg/kg) or methylscopolamine (0.1 mg/kg) was injected (IP) 15 min prior to behavioral testing. In both control and hippocampal lesioned groups, choice accuracy declined as the delay interval increased. Scopolamine, but not methylscopolamine, produced a dose-dependent impairment of choice accuracy (interaction of Dose × Delay) in both groups. The scopolamine-induced impairment was not different between the control and hippocampally lesioned rats. Response bias, delay interval responses, and response probability were not affected by scopolamine except at the highest dose, which increased delay interval responses. The results suggest that central muscarinic receptors outside the hippocampus are important for working memory of nonspatial stimuli.