Use of M1-M5 muscarinic receptor knockout mice as novel tools to delineate the physiological roles of the muscarinic cholinergic system.

In this review we report recent findings on the physiological role of the five known muscarinic acetylcholine receptors (mAChRs) as shown by gene targeting technology. Using knockout mice for each mAChRs subtype, the role of mAChRs subtypes in a number of physiological functions was confirmed and new activities were discovered. The M1 mAChRs modulate neurotransmitter signaling in cortex and hippocampus.

Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice.

A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.

The M4 muscarinic receptor-selective effects on keratinocyte crawling locomotion.

We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migratory function of these cells. Several molecular subtypes of muscarinic acetylcholine receptors (mAChRs) have been detected in KC. Early results suggested that M(4) is the predominant mAChR regulating cell motility.

Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review.

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors.

Heterogeneity of presynaptic muscarinic receptors mediating inhibition of sympathetic transmitter release: a study with M2- and M4-receptor-deficient mice.

1 Presynaptic muscarinic receptors modulate sympathetic transmitter release. The goal of the present study was to identify the muscarinic receptor subtype(s) mediating inhibition of sympathetic transmitter release in mouse atria, urinary bladder and vas deferens. To address this question, electrically evoked noradrenaline release was assessed using tissue preparations from NMRI, M(2)- and M(4)-knockout, and the corresponding M(2)- and M(4)-wildtype mice, after preincubation with (3)H-noradrenaline.

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].

Muscarinic agonist-mediated increases in serum corticosterone levels are abolished in m(2) muscarinic acetylcholine receptor knockout mice.

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions in the central and peripheral nervous system. Due to the lack of receptor subtype-selective ligands, however, the physiological roles of individual muscarinic receptor subtypes remain to be determined. In this study, we examined the effects of the muscarinic M(2)/M(4) receptor-preferring agonist [5R-(exo)]-6-[4-butylthio-1,2,5-thiadiazol-3-yl]-1-azabicyclo-[3.

Deletion of the M5 muscarinic acetylcholine receptor attenuates morphine reinforcement and withdrawal but not morphine analgesia.

Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1-M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms.

Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M1-M5 muscarinic receptor knock-out mice.

A proper balance between striatal muscarinic cholinergic and dopaminergic neurotransmission is required for coordinated locomotor control. Activation of striatal muscarinic acetylcholine receptors (mAChRs) is known to modulate striatal dopamine release. To identify the mAChR subtype(s) involved in this activity, we used genetically altered mice that lacked functional M1-M5 mAChRs [knock-out (KO) mice].

M1 muscarinic receptor signaling in mouse hippocampus and cortex.

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands.

Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor.

In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII.

Muscarinic M2 receptors on peripheral nerve endings: a molecular target of antinociception.

We recently described a novel endogenous mechanism of peripheral antinociception, possibly involving activation of muscarinic M2 acetylcholine receptors that are expressed on nociceptive nerve endings and decrease their sensitivity. In the present study, this mechanism was scrutinized in skin taken from mice with targeted deletions of the muscarinic M2 receptor gene and, for control purposes, of the M4 receptor gene. Two different approaches were taken.

M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea.

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases.

M1 muscarinic acetylcholine receptors activate extracellular signal-regulated kinase in CA1 pyramidal neurons in mouse hippocampal slices.

Activation of extracellular signal-regulated kinases (ERK) is crucial for many neural functions, including learning, memory, and synaptic plasticity. As muscarinic acetylcholine receptors (mAChR) modulate many of the same higher brain functions as ERK, we examined mAChR-mediated ERK activation in mouse hippocampal slices. The cholinergic agonist carbachol caused an atropine-sensitive ERK activation in the dendrites and somata CA1 pyramidal neurons.

Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice.

Forebrain muscarinic acetylcholine (ACh) receptors (mAChRs; M1-M5) are predicted to play important roles in many fundamental central functions, including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. Primarily because of the use of ligands with limited receptor subtype selectivity, classical pharmacological studies have led to conflicting results regarding the identity of the mAChR subtypes mediating this activity in different areas of the brain.

Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents.

Oscillatory network activity at gamma frequencies is assumed to be of major importance in cortical information processing. Whereas the synaptic mechanisms of gamma oscillations have been studied in detail, the ionic currents involved at the cellular level remain to be elucidated. Here we show that in vitro gamma oscillations induced by muscarine require activation of M1 receptors on hippocampal CA3 pyramidal neurons and are absent in M1 receptor-deficient mice.

Heterogeneity of release-inhibiting muscarinic autoreceptors in heart atria and urinary bladder: a study with M(2)- and M(4)-receptor-deficient mice.

Release-inhibiting muscarinic autoreceptors were studied in heart atria and the urinary bladder of NMRI mice, M(2)-receptor-deficient mice, M(4)-receptor-deficient mice, and wildtype mice sharing the genetic background of the knockout animals. Segments of the tissues were preincubated with (3)H-choline and then superfused and stimulated electrically. In atrial segments taken from adult mice and stimulated with 120 pulses at 1 Hz, the muscarinic receptor agonist oxotremorine-M reduced the evoked overflow of tritium.

Cholinergic dilation of cerebral blood vessels is abolished in M(5) muscarinic acetylcholine receptor knockout mice.

The M(5) muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M(1)-M(5)) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M(5) receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M(5) receptor-deficient mice (M5R(-/-) mice).

Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy.

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype.

Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor.

Members of the muscarinic acetylcholine receptor family are thought to play key roles in the regulation of a large number of important functions of the CNS. However, the precise roles of the individual muscarinic receptor subtypes in modulating these processes are not well understood at present, primarily because of the lack of ligands with sufficient receptor subtype selectivity. To investigate the behavioral significance of the M(1) muscarinic receptor (M(1)R), which is abundantly expressed in the forebrain, we subjected M(1) receptor-deficient mice (M(1)R(-/-) mice) to a battery of behavioral tests.

Elucidating the role of muscarinic receptors in psychosis.

Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimer's patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses.

Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons.

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons.

Investigations into the physiological role of muscarinic M2 and M4 muscarinic and M4 receptor subtypes using receptor knockout mice.

Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice.

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice.

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia.