Cholinergic neurons play a key role in the functioning of both the peripheral and central nervous systems
Acetylcholine was first identified as a neurotransmitter in the PNS. We have already noted that ACh is the transmitter released at neuromuscular junctions throughout the body. This substance additionally plays a crucial role in both the sympathetic and parasympathetic branches of the autonomic nervous system. To briefly review the relevant features of these branches of the autonomic nervous system, each consists of preganglionic neurons, which are cells located within the CNS that send their axons to the autonomic ganglia, as well as ganglionic neurons located within the ganglia that innervate various target organs throughout the body.
The preganglionic neurons of both branches are cholinergic, as are the ganglionic neurons of the parasympathetic system. The chemical coding of these cells and the synapses they make. The widespread involvement of ACh in both the neuromuscular and autonomic systems explains why drugs interfering with this transmitter exert such powerful physiological effects and are sometimes highly toxic.
Within the brain, the cell bodies of cholinergic neurons are clustered within just a few areas. Some of these nerve cells are interneurons, such as the ones found within the striatum. In the previous post, we saw that the dopaminergic input to the striatum plays a critical role in the regulation of movement. This regulation depends partly on the balance between ACh and dopamine (DA); that is, when DA is low, as in Parkinson’s disease, the resulting neurotransmitter imbalance contributes to the motor symptoms of the disorder. Consequently, anticholinergic drugs such as orphenadrine (Norflex), benztropine mesylate (Cogentin), and trihexyphenidyl (Artane) are sometimes prescribed instead of l-DOPA in the early stages of Parkinsons disease.
Other cholinergic neurons send their axons longer distances to innervate many different brain areas. For example, there is a diffuse collection of cholinergic nerve cells called the basal forebrain cholinergic system (BFCS) that comprises neurons interspersed among several anatomical areas, including the nucleus basalis, substantia innominata, medial septal nucleus, and the diagonal band nuclei. The BFCS is the origin of a dense cholinergic innervation of the cerebral cortex as well as the hippocampus and other limbic system structures.
Research over many years has led to the view that ACh, and more specifically the BFCS, plays an important role in cognitive functioning. Early studies on rats and mice found that anticholinergic drugs such as atropine and scopolamine interfered with the acquisition and maintenance of many different kinds of learning tasks (Spencer and Lai, 1983). Both atropine and scopolamine block one subtype of ACh receptor, the muscarinic receptor, suggesting that this receptor subtype is necessary for the cognition-enhancing effects of ACh. However, this research could not tell us where the key cholinergic synapses are located, since peripherally administered atropine and scopolamine reach all parts of the brain and therefore block all central muscarinic receptors.
More-direct information concerning the role of the BFCS in cognitive functioning has been obtained from studies involving lesions of this system. An important innovation in this research area occurred with the introduction of a cholinergic neurotoxin called 192 IgG-saporin. This odd-sounding substance contains a monoclonal antibody, 192 IgG, that binds specifically to a cell surface protein carried by the basal forebrain cholinergic neurons. The other part of the molecule, saporin, is a cellular toxin obtained from the soapwort plant, Saponaria officinalis. When 192 IgG-saporin is injected into the brain’s ventricular system, the basal forebrain cholinergic neurons take up the toxin due to binding of the antibody part of the molecule. As a result, those neurons are destroyed while neighboring noncholinergic cells are spared. Rats treated with 192 IgG-saporin exhibit disruptions in several different cognitive functions, including learning, memory, and attention (Wrenn and Wiley, 1998). Interestingly, some learning tasks require very large reductions (>75 to 85%) in ACh levels before lesion-induced deficits are observed. It may be that only a relatively small amount of cholinergic input from the BFCS is required for most cognitive functions.
More than 20 years ago, Raymond Bartus and his colleagues proposed that the cognitive decline that often occurs with aging is due, at least in part, to a dysfunction of the BFCS (Bartus et al., 1982). This spurred tremendous interest in the BFCS, not only with respect to normal aging but also regarding a possible role in the age-related disorder,Alzheimer’s disease. As discussed in Box 6.2, while Alzheimer’s disease does involve severe injury to the BFCS, other damaged neural systems undoubtedly contribute to the devastating psychological and behavioral effects of this disorder.
There are two acetylcholine receptor subtypes, nicotinic and muscarinic
Like dopamine (DA) and norepinephrine (NE), ACh has many different kinds of receptors. The story can be simplified a little by recognizing that the various cholinergic receptors belong to one of two families: nicotinic receptors and muscarinic receptors. Nicotinic receptors were named because they respond selectively to the agonist nicotine, an alkaloid found in the leaves of the tobacco plant. Muscarinic receptors are selectively stimulated by muscarine, another alkaloid, which was first isolated in 1869 from the fly agaric mushroom, Amanita muscaria.
Nicotinic receptors Nicotinic receptors are highly concentrated on muscle cells at neuromuscular junctions, on ganglionic neurons of both the sympathetic and parasympathetic system, and on certain neurons in the brain. They are ionotropic receptors, which, you will recall from an earlier post, means that they possess an ion channel as an integral part of the receptor complex. When ACh binds to a nicotinic receptor, the channel opens very rapidly and sodium (Na+) and calcium (Ca2+) ions enter the neuron or muscle cell. This flow of ions causes a depolarization of the cell membrane, thereby increasing the cell’s excitability. If the responding cell is a neuron, its likelihood of firing is increased. If it is a muscle cell, it responds by contracting. In this manner, nicotinic receptors mediate fast excitatory responses in both the CNS and PNS.
Another important function of nicotinic receptors within the brain is to enhance the release of neurotransmitters from nerve terminals. In this case, the nicotinic receptors are located presynaptically, right on the terminals. Thus, activation of nicotinic receptors by ACh can stimulate cell firing if the receptors are located postsynaptically on dendrites or cell bodies, or the receptors can stimulate neurotransmitter release without affecting the cell’s firing rate if they are located presynaptically on nerve endings.
The structure of the nicotinic receptor has been known for many years. As members of the family of ionotropic receptors discussed earlier, nicotinic receptors comprise five proteins (subunits) that come together in the cell membrane, forming the ion channel in the center. The subunits are labeled with Greek letters. There are two a-subunits, each of which helps form an ACh binding site on the receptor. Interestingly, both binding sites must be occupied by ACh molecules to open the nicotinic receptor channel.
Even though the nicotinic receptors of neurons and muscles possess five subunits (including two as), the exact proteins making up neuronal and muscle receptors are different. This structural difference leads to significant pharmacological differences between the two types of receptors. For example, muscle nicotinic receptors are not as sensitive to nicotine as are the nicotinic receptors found in the brain and autonomic nervous system. This difference is very important to smokers, because it allows them to obtain the psychological effects of nicotine, which are dependent on activation of brain nicotinic receptors, without experiencing muscle contractions or spasms.
In a living organism, receptors are typically exposed to neurotransmitters in a somewhat sporadic manner. That is, at some moments, there are many transmitter molecules in the vicinity of a particular receptor, whereas at other moments, few transmitter molecules are nearby, because the releasing neuron has slowed its firing or perhaps become completely silent for a brief period of time. We can perform pharmacological experiments, however, in which receptors are continuously exposed to high concentrations of an agonist drug for seconds or even longer intervals of minutes or hours. When nicotinic receptors are subjected to continuous agonist exposure, they become desensitized. Desensitization represents an altered state of the receptor in which the channel remains closed regardless of whether molecules of an agonist such as ACh or nicotine are bound to the receptor. After a short while, desensitized receptors spontaneously resensitize and are then capable of responding again to a nicotinic agonist.
Even if cells are continuously exposed to nicotinic stimulation, the receptors are not all desensitized. Those that remain active produce a persistent depolarization of the cell membrane. If this continues for very long, a process called depolarization block occurs, in which the resting potential of the membrane is lost and the cell cannot be excited until the agonist is removed and the membrane repolarized. A chemical relative of ACh called succinylcholine is a powerful muscle relaxant that is useful in certain surgical procedures where anesthesia alone may not provide sufficient relaxation. Unlike ACh, succinylcholine is resistant to breakdown by AChE, and thus it continuously stimulates the nicotinic receptors and induces a depolarization block of the muscle cells. It is important to note that one of the paralyzed muscles is the diaphragm (the large muscle responsible for inflating and deflating the lungs), so the patient must be maintained on a ventilator until the succinylcholine is finally eliminated and the effect wears off.
A well-known nicotinic receptor antagonist is D-tubocurarine. This substance is the main active ingredient of curare, a poison obtained from the tropical plant Chondro- dendron tomentosum. Long before it came to the attention of pharmacologists, curare was being used by South American Indians as an arrow poison for hunting. D-Tubocurarine has a high affinity for muscle nicotinic receptors, thus blocking cholinergic transmission at neuromuscular junctions. Respiratory paralysis is the cause of death in curare poisoning, but this effect can be overcome by treating the victim with an anti-AChE drug such as neostigmine.
Muscarinic receptors As mentioned earlier, muscarinic receptors represent the other family of ACh receptors. Like the receptors for DA and NE, muscarinic receptors are all metabotropic. Five different types of muscarinic receptors (designated to M5) have been characterized, each with specific pharmacological characteristics and coded for by a different gene. Muscarinic receptors operate through several different second-messenger systems. Some activate the phos- phoinositide second-messenger system, while others inhibit the formation of cyclic adenosine monophosphate (cAMP). Another important mechanism of muscarinic receptor action is the stimulation of K+ channel opening. As mentioned in previous posts, this leads to a hyperpolarization of the cell membrane and a reduction in cell firing.
Muscarinic receptors are widely distributed in the brain. Some areas containing high levels of muscarinic receptors are the neocortex, hippocampus, thalamus, striatum, and basal forebrain. The receptors in the neocortex and hippocampus play an important role in the cognitive effects of ACh described earlier, whereas those in the striatum are involved in motor function. There is also recent evidence from Basile and colleagues (2002) that M5 muscarinic receptors are involved in morphine reward and dependence. These investigators compared genetically normal mice to mutant mice in which the M5 receptor gene had been inactivated. In a place-conditioning paradigm, morphine doses that produced a robust place preference in the normal animals had no effect on the mutants.”Loss of M5 receptor function also reduced withdrawal symptoms in mice that were made dependent on morphine, but it had no effect on morphine-induced analgesia. These findings suggest that M5 muscarinic receptors selectively influence the addictive properties of opiate drugs, and they raise the possibility that drugs targeted to these receptors could be useful in treating opiate addicts.
Outside of the brain, muscarinic receptors are found at high densities in the cardiac muscle of the heart and in the smooth muscle associated with many organs, such as the bronchioles, stomach, intestines, bladder, and urogenital organs. These peripheral muscarinic receptors are activated by ACh released from postganglionic fibers of the parasympathetic nervous system. Stimulation of the parasympathetic system has two effects on the heart: a slowing of heart rate and a decrease in the strength of contraction, both of which are mediated by the muscarinic receptors in cardiac muscle. In contrast, smooth muscle cells are typically excited by muscarinic receptor activation, thus causing contraction of the muscle. Muscarinic receptors also mediate various secretory responses of the parasympathetic system, including salivation, sweating, and lacrimation (tearing). Unfortunately, many of the drugs used to treat depression, schizophrenia, and other major psychiatric disorders produce serious side effects due to their blockade of peripheral muscarinic receptors. Patients particularly complain about the so-called dry-mouth effect (technically referred to as xerostomia), which reflects the reduced production of saliva resulting from muscarinic antagonism. For some, the dry-mouth effect is severe enough to cause the patient to stop taking his or her medication. If the medication is continued, the chronic lack of salivation can lead to mouth sores, increased tooth decay, and difficulty in chewing and swallowing food. Later in another post, we will see that pharmaceutical companies have worked to develop newer medications that react less with muscarinic receptors and therefore do not produce the dry-mouth effect.
Several muscarinic receptor agonists occur in nature, including muscarine, from Amanita muscaria; pilocarpine, from the leaves of the South American shrub Pilocarpus jaborandi; and arecoline, which is found in the seeds of the betel nut palm Areca catechu. These substances are sometimes referred to as parasympathomimetic agents, because their ingestion mimics many of the effects of parasympathetic activation. Thus, poisoning due to accidental ingestion of Amanita or any of the other plants leads to exaggerated parasympathetic responses, including lacrimation, salivation, sweating, pinpoint pupils related to constriction of the iris, severe abdominal pain, strong contractions of the smooth muscles of the viscera, and painful diarrhea. High doses can even cause cardiovascular collapse, convulsions, coma, and death.
Given the autonomic effects of muscarinic agonists, it is understandable that antagonists of these receptors would inhibit the actions of the parasympathetic system. Such compounds, therefore, are called parasympatholytic agents. The major naturally occurring muscarinic antagonists are atropine (also sometimes called hyoscamine) and the closely related drug scopolamine (hyoscine). These alkaloids are found in a group of plants that includes the deadly nightshade (Atropa belladonna) and henbane (Hyoscyamus niger). Extracts of these plants are toxic when taken systemically, a fact that was exploited during the Middle Ages, when the deadly nightshade was used as a lethal agent to settle many political and family intrigues. On the other hand, a cosmetic use of the plant also evolved, in which women instilled the juice of the berries into their eyes to cause pupillary dilation (by blocking the muscarinic receptors on the constrictor muscles of the iris). The effect was considered to make the user more attractive to men. Indeed, the name Atropa belladonna reflects these two facets of the plant, since bella donna means “beautiful woman” in Latin, whereas Atropos was a character in Greek mythology whose duty it was to cut the thread of life at the appropriate time.
Muscarinic antagonists have several current medical applications. Modern ophthalmologists use atropine just as did women of the Middle Ages, except in this case they are dilating the patient’s pupils to obtain a better view of the interior of the eye. Another use is in human or veterinary surgery, where the drug reduces secretions that could clog the patient’s airways. Atropine is also occasionally needed to counteract the effects of poisoning with a cholinergic agonist. Scopolamine in therapeutic doses produces drowsiness, euphoria, amnesia, fatigue, and dreamless sleep. It has sometimes been used along with narcotics as a preanesthetic medication before surgery or alone prior to childbirth to produce “twilight sleep,” a condition characterized by drowsiness and amnesia for events occurring during the duration of drug use.
Despite their therapeutic uses, muscarinic antagonists can themselves be toxic when taken systemically at high doses. The CNS effects of atropine poisoning include restlessness, irritability, disorientation, hallucinations, and delirium. Even higher doses can lead to CNS depression, coma, and eventually death by respiratory paralysis. As in the case of nicotinic drugs, these toxic effects point to the delicate balance of cholinergic activity in both the CNS and PNS that is necessary for normal physiological functioning.
Acetylcholine is an important neurotransmitter in the PNS, where it is released by motor neurons innervating skeletal muscles, by preganglionic neurons of both the parasympathetic and sympathetic branches of the autonomic nervous system, and by ganglionic parasympathetic neurons. In the brain, there are many cholinergic interneurons within the striatum as well as a diffuse system of projection neurons that constitutes the basal forebrain cholinergic system. There is evidence that the BFCS plays an important role in cognitive functioning, and damage to this system may contribute to the dementia observed in Alzheimer’s disease.
Cholinergic receptors are divided into two major families: nicotinic and muscarinic receptors. The nicotinic receptors are ionotropic receptors comprising five subunits. When the receptor channel opens, it produces a fast excitatory response due to an influx of Na+ and Ca2+ ions across the cell membrane. Nicotinic receptors in neurons and muscles possess somewhat different subunits, which leads to significant pharmacological differences between the two types of receptors. With continuous stimulation by an agonist, nicotinic receptors are subject to a phenomenon called desensitization, in which the channel will not open despite the presence of the agonist. These receptors can also lead to a process of depolarization block involving temporary loss of the cell’s resting potential and an inability of the cell to generate action potentials.
There are five kinds of muscarinic receptors, designated M1 to M5, all of which are metabotropic receptors. Muscarinic receptors function through several different signaling mechanisms, including activation of the phosphoinositide second-messenger system, inhibition of cAMP synthesis, and stimulation of K+ channel opening. Muscarinic receptors are widely distributed in the brain, with particularly high densities in various forebrain structures. They are also found in the target organs of the parasympathetic system. Consequently, muscarinic agonists are called parasympathomimetic agents, whereas antagonists are considered parasympatholytic in their actions. Blockade of muscarinic receptors in the salivary glands leads to the dry-mouth effect, which is a serious side effect of many drugs used to treat various psychiatric disorders.