Insights into their Function and Clinical Applications
Acetylcholine (ACh) is a neurotransmitter in both the peripheral nervous system (PNS) and central nervous system (CNS). In the PNS, ACh activates muscles and is a major neurotransmitter in the autonomic nervous system. In the CNS, ACh tends to cause excitatory actions. ACh receptors are integral membrane proteins that respond to the neurotransmitter acetylcholine. The muscarinic acetylcholine receptors (mAChRs) are one of two subtypes of ACh receptors that respond specifically to muscarine. Muscarine is an organic molecule that is found in the peripheral nervous system and does not affect the central nervous system because it does not cross the blood brain barrier. These mAChRs are present in virtually all organs, tissues, and cell types. Central mAChRs are involved in regulating an extraordinary number of cognitive, behavioral, and sensory motor functions, as well as various autonomic functions. These neurotransmitter receptors have been of great interest to the scientific community due to the discovery that reduced or increased signaling through distinct mAChR subtypes has been found to contribute to the psychophysiology of several major central nervous system diseases such as Alzheimer’s, Parkinson’s, schizophrenia, depression, and epilepsy . There are five subtypes of these receptors, known as M1, M2, M3, M4, and M5. Determining the function of these individual receptors is crucial to the development of novel drugs and treatments for neurological deficits.
There are numerous challenges associated with differentiating the functions of these five receptors. A major problem is the lack of ligands endowed with a high degree of receptor subtype selectivity. Another is that most cell types and tissues exhibit multiple types of mAChRs. These obstacles have made identification of the physiological roles of the individual mAChR subtypes a difficult task. But laboratories have overcome these problems by using gene-targeting technology to generate mutant mice deficient in a specific mAChR subtype by “knocking out” its corresponding gene from the DNA. These individual receptors are knocked out from birth to facilitate experiments that determine the receptor’s specific functions.
M1 receptors were the first mAChR gene to be knocked out in mice. As of now, researchers have collected the most concrete information about this receptor. This receptor is abundant in all major forebrain areas including the cerebral cortex, hippocampus, and striatum. Because of their concentration in these areas, it has been hypothesized that M1 receptors serve important functions in the higher cognitive functions of learning and memory. M1 receptors were not found to be essential for memory formation or initial stability of memory in the hippocampus. They are, however, critical in memory processes involving cerebral cortex and hippocampus interaction (1). There are minor higher cognitive deficits in M1 knockout mice but so far not substantial enough to show that a single subtype is responsible for great deterioration (2).
In 1997 a study found that found that M1 receptors could play a major role in the pathophysiology of some forms of epileptic seizures. Pilocarpine, a nonsubtype-selective muscarinic agonist, was administered to induce epileptic seizures in wild type (WT) mice and M1 receptor deficient mice. Pilocarpine was found to regularly induce seizures in WT mice but not in M1 knockout mice. The lack of the other four receptors did not interfere with these induced seizures (3).
M1 deficient mice also display pronounced increase in locomotor activity. This hyperactivity phenotype of M1 knockout mice is associated with significant increase in extracellular dopamine concentrations in the striatum, most probably owing to dopamine release. This is a great possibility, but the precise mechanism is still being studied. It has been suggested that centrally active M1 mAChR antagonists are potentially useful in the treatment of Parkinson’s disease, a brain disorder characterized by drastically reduced striatal dopamine levels. Studies have also shown that schizophrenia is associated with increased dopaminergic transmission in various forebrain areas, so improper signaling through M1 receptors may also contribute to certain forms of schizophrenia (3). Other reports have shown that M1 knockout mice also have increased response to stimulatory effects of amphetamine. This gives evidence that M1 receptor dysfunction contribute to psychiatric disorders in which dopaminergic transmission is involved (4).
M2 receptors are the most abundant subtype in the peripheral nervous system, especially in smooth muscle tissues. They are involved in smooth muscle contractions, indirectly or by a mechanism involving another contractile receptor, such as M3. M3 receptors mediate most of the contractile responses to cholinergic agents. Lack of both the M2 and M3 receptors results in severe disorders of smooth muscle organs like paralysis ileus (3). Recent research suggests that M2 receptors play an important role in muscarinic agonist-mediated tremors (3). It has been found that administering oxotremorine or other centrally active muscarinic agonists in the nervous system causes akinesia (inability to initiate movement) and whole body tremor. These are two important symptoms of Parkinson’s. The exact mechanisms underlying this disease are unclear but research suggests that striatal mAChRs are involved. This is because during experimentation, the oxotremorine induced tremors were not present in M2 deficient mice but were present in the M1, M3, M4, and M5 deficient mice. This shows that novel Parkinson’s drugs can be created from this oxotremorine induced tremor model. The positive effects of the involvement, or rather the lack of involvement, of the M2 receptor shows that it is a powerful tool for developing drugs with the ability to block this receptor (3).
The M3 deficient mice are leaner, prefer to consume paste-like food, and are hypophagic (undereating). M3 receptors are expressed in high quantities in the hypothalamus which is a key area for regulating appetite. This information of the hypothalamic cholinergic pathway could be pharmacologically manipulated to control food intake. It has also been shown that muscle contraction and salivary secretion is reduced in these M3 knockout mice. This is a likely explanation for the hypophagia. But the involvement of central and peripheral deficits in M3 knockout mice has still yet to be analyzed in further studies (3).
Research has shown that M4 deficient mice have the potential to play a major role in therapeutic treatment of Parkinson’s and forms of psychosis such as schizophrenia. M4 knockout mice have shown a small but statistically significant increase in basal locomotor activity (3). This receptor is mostly expressed in the central nervous system, particularly in the forebrain areas. This increase in activity was greatly enhanced after the administration of a centrally active D1 dopamine receptor agonist. The research found that striatal M4 receptors have an inhibitory effect on D1 receptor-stimulated locomotor activity. This pattern shows that there is a critical interaction between cholinergic and dopaminergic pathways in order to produce proper striatal function, making it a treatment target for Parkinson’s disease.
Behavioral studies have shown that central M4 receptors play a role in attention, specifically modulating prepulse inhibition (PPI) of the startle reflex which is a specific measure of attention. M4 deficient mice particularly exhibit an increase in sensitivity to the PPI disrupting affect of psychomimetic phencyclidine, a noncompetitive NMDA receptor antagonist. This specific disruptive activity has often been used as an animal model of psychosis, leading researchers to believe that M4 receptors are a target for therapeutic treatments of psychosis (3).
Lastly, finding out the various functions of the M5 receptor has been challenging because of its low expression level, coupled with the dearth of selective ligands for subtypes. The gene knockout model has proved to be very useful in this case. The technique has exposed two important roles of M5 receptors. The first involves blood flow regulation and the other involves the reward system. This has prompted research in areas of learning, memory, and addictive disorders. One example of addictive disorder research was done by Harvard Medical School in 2007 revealed that M5 deficient mice had relatively low prepulse inhibition compared to wildtype mice but this difference was reduced with the administration of clozapine (5). It also showed that these M5 knockout mice exhibited an increase in locomotor activity when stimulated by trihexyphenidyl and cocaine. Overall, this showed the M5 receptor gene affected sensorimotor mechanisms due to muscarinic antagonists but not dopaminergic drugs (5).
From all the knowledge gathered from investigating the specific functions of the mAChR subtypes, scientists have found that mAChRs generally have important therapeutic roles in alleviating disease. One of their functions is alleviating cognitive deficits in patients with Alzheimer’s by increasing the amount of ACh levels in the brain. This can be achieved by systemic administration of cholinesterase inhibitors to improve cognitive function through stimulation of both nicotinic and muscarinic ACh. Antimuscarinic agents are used widely in the treatment of Parkinson’s disease, bronchial asthma, peptic ulcer, and overactive bladder. Muscarinic agonists have therapeutic efficacy in facilitating salivation in patients suffering from dry mouth. It has also been hypothesized that an unbalanced autonomic nervous system may be a major cause of the metabolic syndrome. ACh has potential to aide in alleviating the symptoms of these diseases but may also produce undesirable side effects. As mentioned earlier, because of the lack of selectivity in ligands for ACh receptors, it is imperative that more information is collected on the function of these receptors in order to develop novel subtype specific drugs with fewer side effects (2).
Using knockout mice has enabled researchers to perform groundbreaking work investigating specific subtype mAChRs. The next step is to gather even more concrete information about these subtypes as well as investigate how they function in combined forms where multiple mAChR receptors are knocked out in a single strain of mice. With this information, novel therapeutic treatments can be developed to target these subtypes in hopes of treating the many neurological disorders and diseases that plague society.
References
1. J. Wess, Trends in Pharmacological Sciences, 24, 414- 415, (2003).
2. M. Matsui, et al., Life Sciences, 75, 2972- 2974, (2004).
3. J. Wess, Annual Review Pharmacological Toxicology, 44, 423-450, (2004).
4. D. J. Gerber et al., Proc. Natl. Acad. Sci. U.S.A., 98, 15312- 15317, (2001).
5. M. Thomsen et al., Psychopharmacology (Berl)., 192, 97- 110, (2007).