• Otto Loewi studied the heart of the frog, which-like our own hearts- is supplied by two different peripheral nerves. One, the sympathetic nerve, excites the heart and makes it beat more rapidly; the other , the vagus, shows the heart. The problem was to discover the mechanism by which the effects of nerve impulses in either of these nerves are communicated to the heart muscle. Many believed that the electrical nerve impulse spread from the nerve to the muscle as an electrical wave; Loewi thought otherwise.
• Loewi tested two isolated frog hearts, one with the sympathetic and vagus nerves intact, the other with the nerves removed. A small tube containing salt water was placed in the heart with the nerves attached. When he stimulated the vagus nerve, the heartbeat slowed, as expected. Then he took salt solution that had been in the stimulated heart and placed it inside the heart without nerves. It too immediately slowed- exactly as if its own (missing) vagus nerve had been stimulated.
• He repeated the same procedure, stimulating the sympathetic nerve instead. The effect was again as if the nerve of the denervated heart itself were stimulated: the denervated heart began beating faster. These results could not be explained electrically; the nerves must have secreted chemicals into the salt solution that directly affect the muscles of the denervated heart.
• In one simple experiment, Loewi had demonstrated three important findings: (1) that communication at the gap between nerve and heart muscle was chemical, (2) that each nerve released a different transmitter substance, and (3) that it was the characteristics of the different transmitter substances that caused the increase or decrease in heart rate. This was the first direct experimental evidence of the action of chemical neurotransmitters.
• Like the junction between nerve and heart muscle that Loewi studied, nerve cells communicate with each other at special junctions called synapses. Synapses and neurotransmitters are the subject of this chapter.

• A synapse is the point of connection between the endfoot of one neuron and the membrane of another. The word is derived from Greek and means "to fasten together." It is at synapses that one cell influences the activity of another.
• There are two broad categories of synapses: chemical synapses and electrical synapses. In mammals, chemical synapses predominate, but there is evidence of electrical synapses in some parts of our nervous systems, such as the retina of the eye. Electrical synapses are more commonly found in the brains of invertebrates. At a chemical synapse, the arrival of a nerve impulse at the endfoot of an axon triggers the release of a chemical agent, a neurotransmitter or neuromodulator substance, which falls upon the membrane of the postsynaptic cell. There it chemically induces an electrical response in the receiving cell, such as depolarization or hyperpolarization of the cell membrane. In contrast, at an electrical synapse, ionic currents transmit information from one cell to another directly; electrical synapses provide for a continuity of current flow between cells. Because of the relative importance of chemical synapses in the human nervous system, chemical synapses form the focus of this chapter.
• Chemical synapses are not only primary elements in the information-processing functions of the nervous system, they also represent the major point of chemical vulnerability. Most psychopharmacological agents that are therapeutically useful or commonly abused act through chemical synapses. Both substances of abuse, such as the opiates, amphetamines, and LSD, and therapeutic drugs, such as the tranquilizers and antipsychotic agents, exert their effects at chemical synapses. Thus, much of modern psychopharmacology is in fact the investigation of the synaptic effects of psychoactive agents.

• The chemical synapse is composed of the endfoot (or terminal button) of the sending neuron, the membrane of the receiving neuron, the space between them, and some associated intracellular structures on both sides. Figure 5.1 presents an idealized conception of the synapse, illustrating many of its most important features, The transmission of information at the chemical synapse is usually one-directional, from the endfoot, or presynaptic element, across the synaptic cleft, to the receiving membrane, or postsynaptic element of the synapse.
• Perhaps the most striking anatomical feature of the endfoot at a chemical synapse is the presence of vesicles, tiny spheres of membrane that contain neurotransmitter substances. Packaged within a vesicle, the highly active neurotransmitter substance may be transported to and stored within the endfoot.
• Endfeet are also rich in mitochondria. This is an anatomical indication that the biochemical machinery of the endfoot requires an abundant supply of high-energy materials. In fact, a number of complex, energy-utilizing biochemical processes are carried out within the presynaptic element of the synapse.
• The space between the presynaptic and postsynaptic membranes is the synaptic cleft. This gap is about 20-30 manometers wide and contains a large fluid component. The fluid appears to be chemically complex and is poorly understood at present. The synaptic cleft also contains strands, or filaments, that act to hold the presynaptic and postsynaptic membranes in close proximity to each other. This binding, called the synaptic web, may be exceedingly strong; when brain tissue is broken up for chemical analyses, for example, the presynaptic and postsynaptic elements tend to remain together. The entire detached complex- the endfoot, the synaptic web, and the postsynaptic membrane- is called a synaptosome.
• The postsynaptic membrane is the third part of the synapse. This portion of the outer membrane of the receiving neuron is functionally specialized.. The structure of a synapse is clearly illustrated in Figure 5.2, which shows a special type of synapse between the endfoot of a neuron and a muscle fiber.

• Several methods of classifying synapses within the central nervous system are commonly employed. One traditional method synapses according to the type of the postsynaptic membrane involved in the synapse. If the synapse has a dendrite as the postsynaptic element, the synapse is said to be axodendritic (from an axon to a dendrite). If the synapse is found on the cell body, it is an axosomatic synapse. If the postsynaptic element is another endfoot or an axon, the synapse is termed axoaxonic. A synapse between dendrites of two neurons is called dendrodendritic. Illustrations of all four types of synapses are shown in Figure 5.3.
• However, synaptic connections are now known to be much more divers than traditional wisdom had indicated. There are also known dendro-dentritic, dendrosomatic, and dendro-axonal synapses. Virtually any conceivable combination of microanatomical connection appears possible, as are more complex synaptic arrangements, such as reciprocal synapses in which both cells affect each other. Perhaps one of the most interesting types of receptors is the autoreceptor. Autoreceptors are located on the presynaptic endfoot and are effected by the release of transmitter from the very same endfoot. Autoreceptors provide a mechanism regulating the future activity of the endfoot on the basis of its previous activity.
• Synapses may also be classified according to the distribution of dense material on the presynaptic and postsynaptic membranes, as seen by electron microscopy with conventional staining methods. This classification is particularly useful in examining synapses on the large, principal neurons cerebral cortex. One type is termed asymmetrical synapse, as the dense material accumulated on the postsynaptic membrane is substantially larger than that on the presynaptic membrane. In these synapses, shown in Figure 5.4, the vesicles have a characteristic spherical shape; these are called round vesicles. In a symmetrical synapse, these two densities are more equal. Here vesicles are elongated or flattened rather than spherical. Figure 5.5 shows a micrograph of symmetrical synapses.
• These differences in the microscopic structure of synapses within the central nervous system have functional significance, not just anatomical interest. It is now clear that the round vesicles of asymmetrical synapses contain an excitatory neurotransmitter, whereas the flattened vesicles of symmetrical synapses contain an inhibitory neurotransmitter. Thus it is possible to learn about the functional properties of synaptic communications by microscopic investigation.
• There is also a relation between types of synapses and their distribution on the large principal neurons of the cerebral cortex. the dendrites of cortical pyramidal cells tend to receive input from asymmetrical synapses with rounded vesicles, whereas on the cell body, synapses tend to be symmetrical, with flattened vesicles. Findings such as these suggest that the cell body many functions as a gate that balances the excitatory input of the dendrites with the inhibitory input to the soma in determining whether or not an action potential will be produced in the initial segment of the axon.

• Just as potassium dominates the resting potential and sodium controls the nerve impulse, it is calcium that regulates the release of a neurotransmitter into the synaptic cleft. However, researchers have just begun to understand the process by which an arriving action potential triggers the release of neurotransmitter and the role that calcium plays in that process.
• Since the action potential is a complex phenomenon, involving the opening and closing of both sodium and potassium channels as well as changes of the membrane potential, more than one aspect of the action potential might control neurotransmitter release. An elegant approach to this problem was provided by Bernard Katz and Miledi (1967), who utilized two ion-specific neurotoxins- tetrodotoxin (TTX) and tetrathylammonium (TEA)- to rule out the effects of ionic movements in governing neurotransmitter release. TTX selectively blocks the voltage-regulated sodium channels of the axon. When it is applied to the presynaptic terminal, the action potential is not propagated, and, for that reason, no neurotransmitter is released by the endfoot. But if the endfoot is electrically depolarized, the chemical synapse continues to function in its normal fashion. This means that the operation of sodium channels is not necessary for neurotransmitter release.
• Conversely, TEA selectively blocks the potassium channels of the axonal membrane. When administered together, TEA and TTX inactivate both the potassium and sodium channels of the membrane. Nevertheless, electrical depolarization of the membrane elicits a perfectly normal release of neurotransmitter substance into the synaptic cleft. This finding indicates that neither potassium nor sodium conductance plays any role in controlling the output of transmitter agent from the presynaptic element; transmitter release is triggered solely by depolarization of the membrane in the vicinity of the synapse.
• In contrast, calcium does have a marked regulatory effect on the neurosecretory activity of the synapse. The amount of neurotransmitter released at a synapse varies directly with the concentration of calcium ions in the extracellular fluid. When extracellular calcium is reduced, the nerve impulse releases only a small amount of transmitter agent. Conversely, when the extracellular fluid is rich in calcium, The secretory output of the synapse is enhanced (Katz & Miledi, 1971).
• It appears that each action potential triggers the opening of calcium-selective channels at the synaptic membrane During the depolarization portion of the action potential, voltage-dependent calcium channels at the synapse are activated. Positively charged calcium ions are electrically attracted to the negatively charged interior of the endfoot as the action potential is initiated. Furthermore, the interior concentration of calcium is very low, approximately 1/1000 of the extracellular concentration. Thus, there is a strong concentration pressure forcing the inward movement of calcium ions through the open calcium channels of the synapse.
• The molecular synapse mechanism by which entering ions facilitate transmitter release are not known I detail, but something like the following probably occurs: A neurotransmitter is packaged in small vesicles of membrane, each containing equivalent amounts of the transmitter substance. In some synapses, the contents of synaptic vesicles have been analyzed and found to contain something on the order of 1000 to 5000 molecules of neurotransmitter (Kuffler & Yoshikami, 1975). These packages are manufactured long before they are actually used, so external calcium levels cannot affect the amount of neurotransmitter within the vesicles.
• The influx of calcium during depolarization of the endfoot appears to activate a system of microtubules within the endfoot. The microtubules exert mechanical force and induce the movement of vesicles toward the presynaptic membrane. The vesicles then eject neurotransmitter into the synaptic cleft. In this way, calcium influx regulates the presynaptic release of neurotransmitter substances.

• The process by which vesicles inside the cell fuse with the cell membrane and release their contents into the synaptic cleft is called exocytosis. There are probably specific neurotransmitter release sites along the presynaptic membrane of the endfoot. The influx of calcium ions in some ways activates these sites, causing the membrane of a vesicle to fuse with the presynaptic membrane, opening the vesicle and spilling its contents into the synaptic cleft. Figure 5.6 illustrates this process.
• The cycle of exocytosis is as follows: First a vesicle approaches the membrane, on its way to an activate site. Second, the vesicle begins to fuse with the membrane at such a site. Third, the vesicle joins the outer membrane of the endfoot and, in so doing, releases its contents into the synaptic cleft. Finally, the vesicle, devoid of most of its neurotransmitter, is reclaimed and returns to the interior of the endfoot for refilling with transmitter substance. Figure 5.7 presents electron microscopic evidence of synaptic vesicles fusing with the membrane of the endfoot and opening their contents to the synaptic cleft. Vesicles at all stages of exocytosis may be seen.

• The process of using and reusing synaptic vesicles is a fascinating one. Vesicles are manufactured in the cell body, not in the endfoot. Fresh vesicles, filled with neurotransmitter substance and various complexes of enzymes, are shipped from the cell body down the axon to the endfeet of the neuron. This process of axoplasmic transport (axoplasm iis a term for the cytoplasm within an axon) may be demonstrated by tying off the axon between the cell body and endfoot. After some time has passed, a number of vesicles will collect on the cell body side of the obstruction. These are vesicles whose transport to the endfeet was interrupted. Within the endfoot, used vesicles are reclaimed and refilled for further use. This process probably continues for some time, but eventually the vesicles appear to have outlived their usefulness. There is some evidence that these worn-out vesicles are transported back to the cell body, the process of reversed axoplasmic transport, where extensive repairs are undertaken. All of this is especially remarkable in long-axoned neurons, where vesicles may be carried over distances of a meter or more in their movement from cell body to endfoot and perhaps, back again.

• When neurotransmitter substance is released at an excitatory chemical synapse, it acts to depolarize the postsynaptic neuron, sometimes with sufficient strength to induce an action potential in that neuron. The depolarization produced by a single excitatory synapse is usually insufficient to actually trigger a nerve impulse, but its effect is to excite the postsynaptic neuron.
• Figure 5.8 presents examples of typical excitatory postsynaptic potential (EPSP). Although the amplitudes and durations of EPSP’s may differ, all share certain essential features. First, and most important, EPSP’s are depolarizing postsynaptic potentials, moving the membrane potential temporarily toward the cell’s threshold for producing a nerve impulse. Second, the EPSPs are rather long lasting, at least when compared with action potentials; EPSPs typically continue for 5 to 10 msec before their depolarizing effects are completely dissipated, in contrast to the 1-msec duration of a nerve impulse. Third, the size of the EPSP produced by a given amount of neurotransmitter increases with the size of the membrane potential of the postsynaptic cell. EPSPs are larger when the postsynaptic membrane is highly polarized than when it is relatively depolarized. Finally, all EPSPs show a synaptic delay of approximately 1 msec, the time elapsing between the arrival of an action potential at the presynaptic element of the synapse. The presence of synaptic delay indicates conclusively that the EPSP cannot be the result of a spread of current from the presynaptic to the postsynaptic element; current spread is instantaneous. The synaptic delay, instead, reflects the time taken to release packets of neurotransmitter and for the molecules of neurotransmitter to diffuse across the synaptic cleft.
• The ionic basis of the EPSP is now well established, although the details of the molecular mechanisms mediating the EPSP are not yet fully known. The arrival of excitatory transmitter substance at specialized sites on the postsynaptic membrane increases membrane permeability to both Na+ and K+ by opening a nonselective ionic channel. Since the resting potential is established by allowing potassium to cross the membrane while preventing sodium from doing so, allowing both ions to cross freely reduces the membrane potential.
• In an EPSP, there is a breakdown in the selective permeability of the membrane for potassium and sodium. This is demonstrated by measuring the equilibrium potential for the excitatory synapse by observing the effects of transmitter release while artificially varying the resting membrane potential. Such experiments indicate that the equilibrium potential of the EPSP is approximately -10 mV, the "compromise" potential predicted by the Nernst equation for a membrane that is permeable to both potassium and sodium.
• During an EPSP, the sodium - potassium channels are continuously opening and closing: a single sodium - potassium channels remains open for only 1 msec. But during that time, it is estimated that something like 20,000 sodium ions enter the neuron, driven by both the electrical gradient and their own concentration gradient. A much smaller number of potassium ions leave the neuron at the same time. Potassium is driven out by its concentration gradient, but not its electrical gradient, which acts to impede the outward movement of potassium ions. The net dominance of sodium movement produces the depolarizing effect of the EPSP, driving the cell closer to its threshold. In this way, excitatory postsynaptic potentials act to trigger action potentials within the postsynaptic neuron.
• There are important differences between the ion channels opened by an excitatory neurotransmitter and the sodium and potassium channels involved in propagating an action potential. First, in an EPSP, the increase in both sodium and potassium permeability occurs at the same time; this reflects the fact that both ions are using the same channels to cross the membrane. In the action potential, the change in permeabilities occurs in sequence; initially, sodium permeability increases as the sodium channels are opened, and only later does potassium permeability change, reflecting the opening of the potassium channels to restore the membrane potential. Second, the channels opened by an excitatory neurotransmitter are not voltage-sensitive; for this reason , there is no explosive increase in sodium permeability like that characteristic of the nerve impulse. Finally, there are pharmacological differences between the channels involved in the action potential and the channel activated by excitatory neuron transmitter. Neither TTX nor TEA affects the operation of the synaptic sodium - potassium channel, whereas each has a dramatic and specific effect on one of the channels mediating propagation of the action potential.

• At an inhibitory chemical synapse, the effect of neurotransmitter release is to hyperpolarize the postsynaptic neuron and thereby decrease the probability that the neuron will fire. Like excitation, inhibition plays a critical role in the control of behavior by the brain. Excitatory and inhibitory synapses have opposing effects on the activity of the postsynaptic neuron, the resulting neural activity often depending upon the balance between excitatory and inhibitory influences. Figure 5.9 illustrates inhibitory postsynaptic potentials (IPSP). IPSPs share a number of features with EPSPs. Both are graded potentials; they increase in size as a function of the amount of neurotransmitter released. Both have similar durations, and both show synaptic delay. But one acts to increase and the other to decrease the excitability of the postsynaptic neuron. IPSPs and EPSPs are partners in regulating the activity of neurons.
• The molecular basis of the IPSPs varies at different synapses within the nervous system. In some cases, the inhibitory neurotransmitter acts to increase membrane permeability to potassium; at others, the effect is to increase chloride permeability. In either case, the equilibrium potential for the ion is more negative than the resting potential. Thus, increasing either potassium or chloride permeability will act to hyperpolarize the postsynaptic membrane and drive it away from the critical threshold for triggering a nerve impulse.
• As with the EPSP, the ionic channels opened at the synapse by an inhibitory neurotransmitter are very different from the channels involved in propagating an action potential. For example, the potassium channel at the synapse is not voltage - dependent; a given amount of neurotransmitter opens the same number of channels regardless of the membrane potential of the postsynaptic element. Furthermore, the potassium channels of the synapse differ pharmacologically from those on the axon; TEA blocks axonic, but not synaptic, potassium channels.
• Thus, inhibitory postsynaptic potentials are chemically gated electrochemical events, produced by increases in membrane conductances of potassium, chloride or both types of ions. IPSPs act to hyperpolarize the postsynaptic element, reducing the probability that an action potential will be generated. IPSPs play varying roles in different parts of the nervous system. Often, inhibition serves as a stabilizing influence, preventing neurons from mutually exciting each other and producing a convulsive seizure of electrical firing, such as occurs in epilepsy.

• The neurotransmitters that elicit either EPSPs or IPSPs produce profound effects at the postsynaptic membrane. If these effects were to persist, the synapse would quickly become unresponsive to further synaptic input. Something must be done at the synapse to remove old neurotransmitter molecules and ready the synapse for further input. There are two principal mechanisms by which transmitter substance is disposed of: enzymatic degradation and re-uptake.
• Enzymatic degradation involves the use of specific molecules at the post-synaptic membrane that break down the active transmitter into molecules that do not affect membrane permeability. These inactivated compounds can then be reprocessed by the neuron and used for other purposes. The enzymes involved in inactivating neurotransmitter substance play a critical role in the cycle of synaptic activity and, as we will see later, may be involved in mental illness and its treatment.
• Re-uptake is the mechanism by which neurotransmitter substance is removed from the synaptic cleft. Special high-affinity binding sites for the neurotransmitter are present on the membrane of the presynaptic cell, capturing diffusing molecules of neurotransmitter and returning them safely for repackaging and reuse at the chemical synapse. Both enzymatic degradation and re-uptake appear to be needed to maintain chemical synapses in the required state of readiness for further use.

• In addition to directly affecting ion channels on the postsynaptic membrane, there is yet another synaptic mechanism by which neurons may affect the activity of other neurons.
• In presynaptic inhibition, three neurons are involved. The first (A) synapses upon a second (B) in the conventional manner. But a third neuron (C) can control the effectiveness of the synapse from A to B by its own synapse upon the endfoot of A. Thus, C is an axo-axonic synapse that modulates the primary connection between A and B. Figure 5.10 illustrates these relationships.
• Paradoxically, if C has an excitatory effect on the endfoot A, presynaptic inhibition will occur. Activation of C acts to reduce the efficiency of the voltage-sensitive calcium channels in the endfoot, less transmitter is released by A when it is activated by an action potential. It is in this special sense that the axo-axonic synapse on A reduces the effect of A on B’s postsynaptic membrane.
• It is particularly important that presynaptic inhibition not be confused with postsynaptic inhibition. Presynaptic inhibition always involves a complex of three cells. It modulates the efficiency of a primary synapse between two neurons. No IPSPs are produced; instead, the effectiveness of a normal excitatory synapse is reduced by presynaptic inhibitory input. Presynaptic inhibition is an example of the inventiveness of evolution in producing a method of selectively regulating some information pathways while leaving other unaffected.

• It is important to realize that, in most neurons, a single synapse cannot force the cell to produce an action potential. To trigger a nerve impulse, many synaptic influences must be combined. Synaptic potentials summate; that is, they add with each other in moving the membrane potential closer to or farther from the threshold of the nerve impulse in the process of summation.
• It is often useful to distinguish between spatial and temporal summation of synaptic potentials. Spatial summation refers to the adding together of polarizing and depolarizing effects of different simultaneously active synapses. Temporal summation emphasizes that synaptic potentials linger and therefore can add together over time. Figure 5.11 illustrates the summation of synaptic potentials.
• Spatial summation is particularly important in considering the ways in which neurons and interconnected. Most neurons receive converging information from many other neurons; convergence of information is a major feature of the organization of the nervous system. A large neuron within the human brain may be covered by many tens of thousands of synapses. Spatial summation must be the rule in such cells.
• Temporal summation is an important mechanism by which the rate of firing in one cell affects the size of the postsynaptic response of another. If one nerve impulse arrives at a synapse before the effects of a previous postsynaptic potential have disappeared, the two postsynaptic potentials summate in time, producing a larger change in the membrane potential of the receiving cell. In this way, the synapse converts information coded at the axon by the rate of firing into information coded at the postsynaptic membrane by the size of the summated postsynaptic potential.
• Spatial summation and temporal summation provide a means of integrating information within the postsynaptic neuron. Many influences may be felt, but the neuron must act with one voice; it has but a single axon with which to communicate its decisions. In this way, the effects of the stream of neurochemical released at the many synapses of a cell are integrated.

• The biochemical processes involved in neurotransmission share several general features, even though the neurons and the effects of synaptic activity may differ markedly in different regions of the nervous system. For every chemical synapse, neurotransmitter substance must be synthesized and stored for future use. The neurotransmitter must be released into the synaptic cleft in response to the arrival of an action potential at the presynaptic element, or endfoot. The neurotransmitter then must bind with a receptor molecule on the outer surface of the postsynaptic membrane to produce its characteristic effects within the receiving neuron. Finally, the neurotransmitter substance must be removed from the synapse, either by returning the molecule to the presynaptic element or by inactivating the molecule through a biochemical transformation. All chemical synapses share these essential features, but the specific mechanisms and molecules vary from synapse to synapse. It is this variation that permits different types of synapse to exert different physiological effects.

• Just as different cells use different neurotransmitters at the presynaptic elements of the synapse, neurons differ in the receptor systems that are present on the post-synaptic membrane. All receptors are now believed to be proteins embedded within the membrane at the synapse. These molecules are known to have an active site that can bind with a specific neurotransmitter molecule in the extracellular space. This site is located on the outer surface of the postsynaptic membrane, facing into the synaptic cleft.
• But receptors are only one part of the postsynaptic machinery that mediates the response to neurotransmitter release; the other part of that molecular machine is an effector molecule. Together, they form a receptor-effector complex.
• Effectors are triggered by the receptor binding to a molecule of neurotransmitter. It is the effector on the inner surface of the membrane - linked to the receptor on the outer surface - that directly produces the postsynaptic response. In many cases, the effector opens an ionic channel through the postsynaptic membrane, thereby producing an EPSP or IPSP, depending upon the properties of the channel. Other effector molecules function in a more complex manner. But since the transmitter-binding site is separate from the response-producing effector, one neurotransmitter substance may have markedly different physiological effects at different receptor complexes. Thus, a single neurotransmitter substance may have excitatory effects at one synapse and inhibitory effects at another. Figure 5.12 illustrates the general properties of the chemical synapse.
• A receptor-effector complex, therefore, may be characterized by the specific neurotransmitter with which it binds. It must also be characterized by the nature of the postsynaptic response that it triggers. Thus, to understand the function of any receptor-effector molecule complex, both the specific binding properties of the receptor molecule and the specific action properties of the effector molecule need to be determined.
• One of the most important methods of studying neurotransmitters and receptors is to examine the effects of different drugs or compounds on synaptic function. Some drugs are capable of binding with a receptor and trigger the response of the effector complex; these drugs, termed agonists, function in the same manner as the naturally occurring neurotransmitter. Heroin, for example, is an agonist of the brain neurotransmitter endorphin. Other drugs, called antagonists, reduce or block the response of the receptor to either the neurotransmitter or its agonist. One way that antagonists produce their effects is to occupy the receptor site without triggering the effector. In this way, action of the neurotransmitter is blocked.
• Still other compounds act at the synapse to potentiate the effects of either neurotransmitter or agonist. One common type of potentiation results from blocking the naturally occurring enzymes that inactivate the neurotransmitter molecules that remain capable of binding with receptors. By determining the types of compounds that act as agonists, antagonists, and potentiators at a particular synapse, the biochemical properties of both neurotransmitter and receptor molecules may be estiablished.

• The process of Neurotransmitter action depends upon a precise sequence of molecular transformations. First, the substance is synthesized within the presynaptic neuron. Upon release, a subset of these molecules successfully crosses the synaptic cleft and binds with a receptor molecule on the postsynaptic membrane, resulting in a second molecular transformation. A third transformation occurs when the neurotransmitter molecule is inactivated. Finally, inactivated molecules are reprocessed and recycled, to enter again into the molecular metabolism of the neuron. Each of these steps involves a biochemical transformation that is guided and controlled by special molecules called enzymes . For each neurotransmitter, the details of these transformations differ, but the same general principles apply.
• Enzymes are specialized molecules that speed and facilitate biochemical reactions but do not themselves enter into those reactions. Enzymes function as biochemical catalysts that act upon a very limited set of very specific molecules. The molecules upon which they act are the substrates of the enzyme and are the precursors that the enzyme chemically transforms. (It is easy to recognize an enzyme when reading about it; the names of most enzymes end in -ase.)
• Enzymes are large protein molecules that are folded into complicated, irregular shapes with grooves, or pockets, into which molecules of the substrate may fit. These pockets form the active sites of the enzyme. They are matched to the conformation of the molecules of the substrate: Where the substrate has a hill, there is a valley in the active site; where the substrate carries a positive charge, there is a negative charge at the active site; and where the substrate is hydrophilic or hydrophobic, the active site is also. The enzyme, by precisely positioning the substrate molecule entering into a reaction, can facilitate the reaction enormously. The active site functions as a complicated molecular lock, with the substrate providing the key. This is the reason that enzymes perform a highly specific manner.

• One of the most fundamental problems in synaptic neurochemistry is to identify the neurotransmitters used in different regions of the brain. Most would agree that any neurotransmitter should pass the following tests:

1. The substance should be present within the nervous system in quantities typical of transmitter agents. Thus, esoteric chemicals that may be produced in the laboratory or extracted from other species may have powerful synaptic effects, but if they are not detectable in the nervous system, they are not likely to be neurotransmitters.
2. The substance must be present in the endfeet of neurons. Neurotransmitters are stored in vesicles within the terminal buttons; therefore, it is in these terminals that the substance should be concentrated.
3. The substance must be synthesized within the neuron. Thus, the specific enzymes responsible for synthesizing the substance from its precursors must be present.
4. There must be evidence of enzymes that inactivate or destroy the substance in the vicinity of the synapse.
5. The substance must act on receptor sites. When applied to the postsynaptic surface as a drug, it should have exactly the same effect as the natural activation of the synapse.

• In practice, no compound has fulfilled all of these criteria for a neurotransmitter within the human brain. Instead, neuroscientists have adopted an attitude of justifiable caution toward this subject. If a reasonable number of these criteria are met, the compound is regarded as a neurotransmitter.

• Loewi had shown that two different neurotransmitter substances are released by the two types of nerves that innervate the heart. Sir Henry Dale, a British pharmacologist who shared the Nobel Prize in physiology with Loewi in 1936, provided the definitive biochemical characterization of the two transmitter substances. His observation that each neuron has its own characteristic neurotransmitter formed the basis of much of Dale’s work. For this reason, many years later, Sir John Eccles, another Nobel laureate, proposed that his observation be considered a fundamental principle of neural functioning. Dale’s Law, as it is now known, holds that any single neuron makes use of the same neurotransmitter at all of its synapses onto other neurons. In other words, each neuron may be characterized by the specific neurotransmitter that it uses at its synapses. Of course, a neuron may receive information from other neurons that use a variety of neurotransmitters; otherwise, it would not be possible to record EPSPs and IPSPs from a single cell.
• Exceptions to Dale’s Law began to appear in recent years, but - at first - they were regarded as unusual anomalies. Many developing neurons are now known to synthesize and release more than one transmitter substance. But now it appears that many neurons - even in the human brain - contain nearly half a dozen different chemical agents, which they release into the synaptic cleft. It is not known whether all endfeet use the same set or subsets of transmitter agents. Thus, it seems a good bet that Dale’s Law certainly does not hold for all neurons. Nonetheless, it is still important to know which neurotransmitters are used in a given set of neural connections.

• Recently, it has become clear that not all substances released by the endfoot behave like traditional neurotransmitters. For this reason, an important - if sometimes imprecise - distinction is made between classical neurotransmitters and what are now called neuromodulators, although the term neurotransmitter is still used to refer to both categories of chemicals when speaking generally. Classical neurotransmitter agents are very quickly to alter the membrane potential of the postsynaptic neuron by controlling chemically gated ion channels. Further, these effects dissipate rapidly.
• In contrast, many newly discovered substances - and some that have been well known for some time - act much more slowly, taking effect many milliseconds after release and continuing their effects for a substantial period of time. These substances are neuromodulators. Although neuromodulators may alter the membrane potential; of the postsynaptic cells, often they do not. Neuromodulators may perform a number of other functions instead.
• Table 5.1 presents the classical view of neuroactive substances. These classical distinctions are being questioned. For example, acetylcholine is now known to function as a neurotransmitter in the peripheral nervous system and as a neuromodulator within the brain.

• One of the two neurotransmitter substances that Sir Henry Dale identified was acetylcholine (ACh). Dale coined the term cholinergic to refer to any neuron that releases acetylcholine at its endfeet. Acetylcholine is widely used within the peripheral nervous system. For example, ACh is the transmitter released by the vagus nerve to the heart as well as the transmitter used to control all of the voluntary skeletal muscles of the body.
• Myasthenia gravis is a disorder of the neuromuscular junction, a synapselike arrangement between the motor nerves and the muscles themselves. The disease results in a progressive weakness that may terminate in death. Electrophysiological studies show that the effect of the release of ACh onto the muscle becomes increasingly diminished as the disease progresses. Either less ACh is released or there are fewer ACh receptors on the muscle fibers. The latter is in fact the case: Myasthenia gravis is an autoimmune disease in which the immune system forms antibodies that attach the ACh receptors on the muscles. As time goes on, the skeletal muscles become increasingly denervated.
• Acetylcholine is synthesized within the cell body; it is then transported down the axon to the endfeet by axoplasmic flow at rates as high as 400 mm per day. The synthesis of ACh is a straightforward chemical reaction involving a single step.
• Acetylcholinesterase is an exceptionally active and powerful enzyme; 1 mg of purified AChE is capable of inactivating up to 150 g of ACh per hour, or 150,000 times its own weight in ACh.
• The distribution of ACh and its related enzymes within the cholenergic neuron and synapse conforms to the pattern required of a neurotransmitter. ACh is found within the endfeet of cholinergic cells, where it is present in a form indicative of a vesicular storage. CAT is also found within the synaptosome. In contrast, AChE - the inactivating enzyme - is more widely distributed and tends to be associated with fragments of cell membrane. That, of course, is the site at which AChE acts.
• Figure 5.13 summarizes the essential features of the cholinergic synapse Acetyl - coenzyme A is produced in the mitochondria and made available for this synthesis of ACh. Apparently, the availability of choline determines the rate of ACh production in the cholinergic neuron; the enzyme CAT is able to rapidly convert any substrate molecule present into free ACh. Once synthesized, ACh is bound into synaptic vesicles and thereby protected from inactivation by AChE. The release of packets of ACh into the synapse is triggered by the arrival of the nerve impulse and accomplished by exocytosis. ACh released into the synaptic cleft may then find its way to a receptor on the postsynaptic membrane, with which it binds.
• There are two types of cholinergic receptors, which differ in their agonists. For one type of cholinergic receptor, nicotine mimics the action of ACh; for the other, the agonist is muscarine, a compound that is derived from the fungus Amanita muscaria. Nicotinic and muscarinic cholinergic receptors have very different properties and mechanisms of action. At a nicotinic receptor, acetylcholine is a classical neurotransmitter; at a muscarinic receptor, it is a neuromodulator.

• The nicotinic receptors are one of the least complicated receptor - effector complexes involves in synaptic activity. It is one example of a fast - responding receptor, producing its effect within milliseconds following binding. Four different subtypes of nicotinic receptors have been identified, different subtypes appearing in different parts of the nervous system. The receptor and effector functions are performed by a single molecule, a large protein that is embedded within the postsynaptic membrane. It is thought to have two active sites facing into the synaptic cleft where molecules of ACh may be bound. The molecule is also believed to have a central opening of variable diameter. When two ACh molecules are bound to the outer surface of the protein molecule, the shape of the receptor - effector complex is altered, and the central opening is widened. In this configuration, both sodium and potassium molecules may cross the postsynaptic membrane. But since sodium is driven into the postsynaptic cell by both its concentration gradient and by the electrical gradient of the postsynaptic membrane, the effect of opening the ion channel within the nicotinic receptor is to depolarize the postsynaptic membrane. Thus, the nicotinic cholenergic synapse is excitatory. Figure 5.14 illustrates one model of the receptor.
• The action of ACh at the nicotinic receptor site may be described statistically. Approximately 10,000 molecules of ACh are released from a single vesicle into the synapse. Within about 1/10 msec, these molecules cross the synaptic cleft and reach a receptor site at the postsynaptic membrane. Since both of the active sites on the receptor molecule must be occupied by neurotransmitter molecules to open the channel and re-uptake removes many ACh molecules from the cleft, only about 2,000 receptors are opened by the ACh contained within a single synaptic vesicle. These channels remain open only briefly; ACh molecules are quickly dissociated from the receptor’s binding sites and inactivated by the enzyme AChE. But during the time that the channels are opened, something on the order of 20,000 sodium ions enter the postsynaptic neuron. When more than one vesicle of neurotransmitter is released, more receptor channels are opened, and the magnitude of the resulting excitatory depolarization is increased. The nicotinic receptor is a classic chemically gated ion channel.

• The muscarinic receptor is more complex than the nicotinic receptor, and its physiological effects are more varied. At least five different types have been identified at present. The postsynaptic response to activation of a muscarinic receptor by ACH develops more slowly and is longer lasting than that of the nicotinic receptor.
• Although muscarine receptors are present in the periphery, in the central nervous system they are truly the dominant cholinergic receptor; over 99 percent of all cholinergic synapses within the brain are muscarinic. Some receptor types act directly on ion channels, but others act indirectly. Recently, an understanding has developed of how varied physiological effects may be induced by activation of muscarinic receptors. Central to these hypotheses is the idea that a second messenger system operates within the postsynaptic neuron.
• At any synapse, the neurotransmitter agent functions as the primary messenger, carrying information from one neuron to the next. At some synapses, like the nicotinic synapse, this primary messenger directly induces a change in the permeability of the postsynaptic membrane, producing an excitatory or inhibitory postsynaptic potential. But in a second messenger system, the neurotransmitter does not directly produce a change in permeability in the postsynaptic membrane. Instead, it unleashes other potent biological processes within the postsynaptic neuron that serve as intracellular messengers; these processes may have a wide range of physiological effects. The second messenger could open or close specific ion channels, or it could affect the activity of ionic pumps on the membrane. Muscarinic receptors are able to control potassium, calcium, or chloride gates in different types of cells. Thus, acetylcholine could be either excitatory or inhibitory, depending upon the properties of the postsynaptic muscarinic receptor.
• Further, in a second messenger system, the activation of the receptor could trigger the synthesis of a wide range of proteins and perhaps provide a molecular basis for learning and memory. In short, the second messenger, by initiating the production of specific proteins, not only may control the movement of ions across the membrane but also may affect the internal metabolism of the cell.
• This process occurs at a muscarinic receptor in the following manner. The receptor molecule, located on the outer surface of the cell membrane is coupled with a G-protein (protein guanine nucleotides) located on the interior surface of the cell membrane. A G-protein is composed of three parts, the so-called alpha, beta, and gamma subunits, which are held together by a molecule of cyclic guanosine diphosphate (cGDP). When ACh binds with the receptor, cGDP is converted to cyclic guanosine triphosphate (cGTP), which binds with the alpha subunit. In so doing, the GTP-alpha complex is released into the intercellular fluid, where it can interact with an effector molecule. The effector molecule can then exert its own characteristic response. The muscarinic receptor provides a clear example of a second messenger system.
• It is increasingly clear that the principles developed in the study of cholinergic synapses are of general importance. The ideas of a receptor-effector complex, of chemically gated ionic channels, and of second messenger systems may be equally well applied to other types of synapses using other specific neurotransmitter substances. For this reason, ACh synthesis and utilization have been discussed in some detail. Other systems utilizing a second messenger include adrenergic, serotonergic, dopaminergic, and some opiate receptors, which are summarized below.

• The catecholamines - dopamine, norepinephrine, and epinephrine - are a set of biochemically related, biologically active compounds that play a variety of roles within the nervous system. They derive their family name from their chemical structures; all are formed by a catechol ring with a tail of amines. Dopamine and norepinephrine certainly serve as neurotransmitters within the central nervous system. Norepinephrine is also the transmitter of the sympathetic nerves of the mammalian heart.
• The case for epinephrine as a central neurotransmitter is much less strong. But outside the brain and spinal cord, epinephrine is the sympathetic transmitter substance. It was epinephrine that Loewi extracted from the frog’s heart following sympathetic stimulation. Dale later suggested that neurons releasing epinephrine, or adrenalin as it was then called, be termed adrenergic.
• Although the proportion of catecholaminergic neurons within the human brain is very small, the influence of these cells upon brain function may be disproportionately large because many of these cells show a great amount of divergence. For example, a dopaminergic neuron in a small area of the rat’s brain (the substantia nigra) sends as many as 500,000 synaptic terminals into its forebrain. In humans, with our large forebrains, the number may be more like five million.
• Catecholaminergic neurons play an important modulating role in human brain function. Their widespread divergence permits them to regulate activity in large regions of the brain. Furthermore, the postsynaptic response to catecholaminergic transmitters is very slow, suggesting the involvement of a second messenger system. Much remains to be learned about the diverse physiological effects produced by second messengers.
• The catecholamines are synthesized from tyrosine, an amino acid that is common in the human diet. This process of synthesis forms a metabolic pathway from tyrosine to epinephrine. The first step is the formation of a precursor for the neurotransmitters, L-dihydroxyphenylalanine (L-dopa), by the enzyme tyrosine hydroxylase:

• The biochemistry of the dopaminergic synapse is shown in Figure 5.16. The synaptic pathway shows the precursor tyrosine transformed first to L-dopa and then to free dopamine. Either this dopamine is then bound into vesicles, where it is stored for synaptic release, or it is destroyed by the intracellular inactivating agent monoamine oxidase (MAO). The arrival of a nerve impulse produces the release of bound dopamine by exocytosis into the synaptic cleft. There, it either binds with a dopaminergic receptor or is inactivated by the synaptic enzyme catechol-O-methyl transferase (COMT).
• There are two types of dopaminergic receptors, D1 and D2. At the post-synaptic receptor complex, dopamine is thought to make its effects felt by regulating cyclic adenosine 3’, 5’ - monophosphate (cAMP). The activating enzyme is adenylate cyclase. Stimulation of a D1 receptor activates adenylate cyclase whereas D2 receptors inhibit the enzyme. This process is analogous to release of cGMP by ACh.

• Figure 5.17 illustrates the biochemistry of the noradrenergic synapse. The biochemical steps involved are exactly those present at the dopaminergic synapse, with the addition of the extra step involved in the synthesis of norepinephrine. In the noradrenergic synapse, free dopamine is first converted to norepinephrine by the enzyme dopa beta-hydroxylase before the neurotransmitter is bound into synaptic vesicles.
• Like dopamine, norepinephrine induces the production of a second messenger within the postsynaptic neuron to produce its biological effects. In both cases, the same releasing enzyme, adenylate cyclase, is employed to activate AMP as a second messenger.

• Serotonin is a central nervous system neurotransmitter and neuromodulator; it is synthesized and inactivated within the brain, it is present at the endfeet of serotonergic neurons, and it meets other tests required of brain neurotransmitters as well. It has been suggested that a disorder of brain serotonin systems is responsible for a number of mental disorders, principally depression.
• Serotonin is synthesized from the dietary amino trytophan. The enzyme trytophan hydroxylase converts trytophan to the 5-hydroxytrytophan (5-HTP), the immediate precursor of serotonin:\

Trytophan hydroxylase

Trytophan 5-HTP

• This serotonin precursor is then converted to serotonin by the enzyme 5-HTP decarboxylase:

5 -HTP decarboxylase

5-HTP Serotonin

• Thus, the synthesis of serotonin very closely parallels the biochemical pathway involved in manufacturing dopamine.
• Chemically, serotonin is considered an indoleamine,meaning that it is composed of an indole ring with an amine tail. This structure is very like that of the catecholamines; thus the indoleamines and catecholamines together are termed monoamines. It is not surprising, therefore, that the similaritites between serotonin and catecholamine metabolism also extend to their inactivating enzyme; like dopamine and noradrenalin, serotonin is inactivated by MAO. MAO is used to inactivate serotonin within both the presynaptic and postsynaptic elements.

• Amino acids are small molecules, each containing an amino group (NH2) and a carboxyl group (COOH). There are only twenty standard amino acids that form building blocks from which proteins are constructed. The idea that amino acids may also serve as neurotransmitters is not particularly new, but until recently, it has been difficult to gather strong evidence on this point. The problem is to distinguish between amino acids functioning as neurotransmitters and those same acids playing other roles in the metabolism of the cell. In contrast, the fact that acetylcholine and monoamines function anly as transmitter substances has made the investigation of these transmitter systems much easier. Nonetheless, it is now apparent that certain amino acids are used as transmitters at chemical synapses; they appear to be the most common neurotransmitters by far within the mammalian brain.
• There are two major categories of amino acid and neurotransmitter: those that depolarize or excite the postsynaptic and thise that hypolarize or inhibit their targets. The excitatory amino acid neurotransmitters include glutamic acid, aspartic acid, cysteic acid, and homocysteic acid. A number of specialized receptors for these amino acids have been demonstrated. Many are simply chemically gated ion channels. Others, like the NMDA receptor (named for its agonist, N-methyl-d-aspartate), appear to play a major role in synaptic pasticity and memory in many species, including humans.
• Amino acids that are thought to be inhibitory neurotransmitters include gamma-aminobutyric (GABA) and glycine. As with other neurotransmitters, some of the amino acid transmitters are known to have multiple receptor types. Further, the postsynaptic effects of these amino acids are not only those of a classicalneurotransmitters, but--in some instances--of neuromodulators as well. Selected amino acids--comparatively simple chemicals--in all probability accomplish much more of the brain’s work than do the more complicated and famous (to neuroscientists) neurotransmitters such as dopamine, norpinephrine, and serotonin.

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