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Chapter 3: The synapse – regulating communication and modifying behavior. In the previous chapter we discussed the neuron – the fundamental unit of the nervous system. We learned about its key role in information flow: It transmits an electrical signal from the input site, to the site where the signal will be conveyed onward to its partner cell. We learned the strategies the axon uses to maximize the efficiency with which the electrical signal is conveyed. We examined the transport mechanisms in place to support information flow. The structural proteins the neuron requires are synthesized in the cell body. Hence ensuring that the dendrites, axon and presynaptic terminal are supplied with the right material at the right time requires a complex transport system. We learned how microtubules and ATPase motors collaborate to transfer cargo where and when it is needed. Finally we learned what may go wrong if either the electrical signaling or the support systems are abnormal. We learned how information flow can be disrupted: Disorders of electrical signaling such as those that affect myelin, can directly affect the speed that the electrical signal is conveyed. Disorders of transport mechanisms, such as those that affect the nerve terminal, can directly affect how information is conveyed at the synapse. Disorders of transport mechanisms, such as those that affect axon structure, can indirectly affect the speed that the electrical signal is conveyed, because myelin only remains intact when the axon has a healthy diameter. In this chapter we are going to focus on the region where the electrical signal is transformed into a chemical signal to travel across the gap between a neuron and its partner – and then we will examine two important disorders in which signaling across this gap – the synapse – breaks down. The synapse - where electrical information is converted to a chemical signal. We will begin by describing one of the best-characterized synapses – the vertebrate neuromuscular junction – where spinal motor neurons communicate with skeletal muscle. Once we understand this synapse, we will be better prepared to understand how other neurons have modified this basic structure to meet their specialized requirements. The vertebrate neuromuscular junction: The neurons that innervate skeletal muscle are called motor neurons and their cell bodies are found in the motor (ventral) region of the spinal cord. They send their axons in bundles to contact muscle cells in the periphery. Because the motor neuron axons are long, they need to be heavily myelinated. As a general rule, each muscle fiber receives input (is innervated) from a single motor neuron, but each neuron in the bundle can synapse onto more than one individual muscle fiber. Together the motor neuron and its target muscle fibers create a motor unit that can vary in size from just a few fibers in the small muscles used for fine movements, to up to ~100 fibers in the large muscles used for gross movements. 1 The neuromuscular synapse is excitatory, that is to say, transmitter release from the presynaptic terminal causes the membrane of the muscle fiber to depolarize, eventually causing the muscle to contract. The neuromuscular synapse uses acetylcholine (ACh), as the chemical neurotransmitter to communicate with the muscle. In the presynaptic terminal Ach is found in small organelles called synaptic vesicles. The synaptic vesicles cluster around specialized sites in the presynaptic plasma membrane called active zones, where the vesicles can fuse with the presynaptic plasma membrane. When they do fuse neurotransmitter is released into the synaptic cleft – the extracellular space between the presynaptic site at the axon terminal and the postsynaptic site on the muscle. Directly opposite the active zone, the postsynaptic muscle cell membrane holds the receptors that bind Ach. They are clustered at a very high density (about 10,000 to 20,000 receptors/μm2, which is almost a crystalline array). The synaptic cleft contains a high concentration of acetylcholinesterase, the enzyme that hydrolyzes and inactivates the Ach after it has been released. The cholinesterase is held in place by a collagen matrix on the surface of the muscle. Figure 1: The vertebrate neuromuscular junction. 1A: The motor neurons arise in the motor region of the spinal cord and project out to the skeletal muscles. 1B: Each axon makes many terminal branches, each of which ends in a synapse. The motor neuron uses acetylcholine (Ach) as a neurotransmitter. IC: Ach is synthesized in the terminals and loaded up into synaptic vesicles. When the action potential reaches the terminal it causes vesicles to release Ach into the synaptic cleft. Ach that binds to its receptor on the postsynaptic site depolarizes the postsynaptic membrane, causing a synaptic potential. At healthy synapses, every synaptic potential gives rise to a muscle action potential. 2 The presynaptic side: How neurotransmitter is released. When the action potential moving down the axon of the motor neuron invades the presynaptic terminal, it activates voltage-gated Ca2+ channels localized at the active zones in the terminal membrane. They open, and Ca2+ floods into the terminal, triggering the fusion of synaptic vesicles with the plasma membrane and the release of transmitter into the synaptic cleft. Early investigators, recording electrical signals from the synaptic contacts on muscle, observed that Ach could be released even in the absence of an action potential. But if the nerve is not stimulated, the probability of a vesicle fusing with the presynaptic terminal and releasing transmitter is very low. This is because the release apparatus that controls fusion at the active zone is composed of proteins that depend on Ca2+ to become activated, and in the absence of an action potential the free Ca2+ concentration at the active zones is normally very low. When the action potential enters the presynaptic terminal and causes voltage-dependent Ca2+ channels to open, it increases Ca2+ levels by 1000 fold. Whenever a single vesicle fuses with the plasma membrane and delivers its contents into the synaptic cleft, it releases the same amount of transmitter into the cleft. The amount released is called a quantum. Each quantum contains approximately 3,000 molecules of ACh. A quantum is the minimum amount of Ach able to elicit a single synaptic potential on the post-synaptic side. Many synaptic potentials have to occur to cause a muscle action potential, but in healthy neurons each action potential arriving at the terminal stimulates enough transmitter release to cause a muscle action potential. Figure 2: The Presynaptic side of the neuromuscular junction: a) A diagram of the active zone. The (purple) synaptic vesicles line up next to an area where proteins of the fusion/releasing complex are concentrated (yellow). Once the Ca2+ influx has activated them the vesicles dock and releases Ach into the cleft through a pore (green). b) An actual electron micrograph of a neuromuscular synapse. The synaptic vesicles are arrowed. Notice the fibrous material inside the synaptic cleft where the acetylcholinesterase enzyme is located. c) An electron micrograph that has caught some of the synaptic vesicles in the process of fusion and releasing transmitter into the synaptic cleft. d) Looking up at the active zone of a presynaptic terminal from the presynaptic cleft using a type of electron microscopy that is sensitive to the membrane. The ‘beads’ on the surface are membrane proteins while the ‘dimples’ show pores in the membrane where the synaptic vesicles have fused. After releasing transmitter the pore closes and the vesicles bud off inside the terminal where they can be refilled and reused. The postsynaptic side: How the chemical signal is interpreted. Once ACh is released into the synaptic cleft, two things can happen: It will either bind to nicotinic receptors on the muscle membrane, or it will be inactivated by the cholinesterase in the cleft. The activity of the cholesterase enzyme is one of the highest known for any enzyme; about half of the ACh is broken down even before it reaches its receptor. ACh that does reach 3 the receptors binds (two molecules per receptor) and triggers a conformational change so that the receptor opens a water-filled pore, or channel, that spans the muscle membrane. This receptor channel is different from the voltage-gated channels involved in the action potential because it is permeable to both Na+ and K+. Thus, when the channel opens: Na+ flows into the cell down its electrochemical gradient. K+ flows out of the cell down its gradient. There is more flow of Na+ than K+ ions through the channel, resulting in a potential change across the muscle membrane, called the synaptic potential. It causes the muscle membrane to depolarize. The more Ach that is released by the presynaptic terminal, the more Ach receptor channels are activated, the larger the synaptic current, and the larger the synaptic potential. But unlike action potentials, synaptic potentials are not regenerative. Their amplitude decreases with the distance from the region of synaptic contact. Thus, synaptic potentials are confined to the membrane at or near the synapse. Figure 3: The acetylcholine (Ach) receptor. a, b) Models of the acetylcholine receptor complex a) from the top, b) from the side. It is a pentamer whose subunits surround a central pore, like a donut. When two molecules of Ach bind to the receptor, the ensuing conformational change causes the pore to open. Na+ ions move into the muscle cell and K+ ions move out. c) A model of the receptor from the side, cut away to show the pore. The ‘gate’ is made from negatively charged amino acids that surround the pore and ensure that it is only permeable to cations. The location of the receptor in the membrane is apparent. For muscle contraction to occur the synaptic potential has to be translated into an action potential. This happens when enough ACh is released from the nerve terminal to depolarize the muscle cell past the threshold that will activate the voltage-dependent Na+ channels in the muscle membrane. When these voltage-gated Na+ channels are activated they generate a muscle action potential that is propagated from end to end in the muscle fiber, causing contraction. For this reason, the synaptic potential is excitatory. It is important to note that the AChactivated Ach receptor channels are not themselves directly involved in generating the action potential; they just provide the stimulus current that depolarizes the membrane enough to activate the voltage-dependent Na+ channels in the muscle membrane. At normal, healthy nerve-muscle junctions, neurons always release enough ACh to produce a synaptic potential that is above the threshold that will give rise to a muscle action potential. Thus, every time the nerve is stimulated, the synaptic potential will cause a twitch contraction response in the muscle. Neuropathies in which Ach release is reduced result in decreased synaptic potentials so the muscle membrane doesn’t always reach threshold. In this case, 4 activation of the neuron may not lead to muscle contraction, and synaptic transmission fails. Other Excitatory Synapses Nicotinic Ach receptors produce excitatory synaptic potentials because they cause the postsynaptic membrane to depolarize. This is true whether the membrane the receptors are in skeletal muscle membrane, or another neuron. Nicotinic Ach receptors are also found on autonomic and central nervous system neurons so they cause excitatory synaptic potentials there too. The most widespread receptors involved in excitatory synaptic transmission in the central nervous system are the Glutamate receptors. They exist in many functionally distinct forms and play a critical role in learning and memory. Both Ach and glutamate are known as classical excitatory neurotransmitters because they are small organic molecules. But peptides can also act as excitatory transmitters. For example, the peptide substance P is released from sensory nerve terminals and is a key signal in pain pathways. As with the classical neurotransmitters, each peptide neurotransmitter activates its own unique receptor and gives rise to a characteristic synaptic response. Figure 4: Glutamate receptors. Glutamate synapses are the most common excitatory synapses in the central nervous system. In this picture of a neuron from the hippocampus each fluorescent blue spot marks a glutamate synapse. Note how abundant they are on both the dendritic tree and the neuronal cell body. Central Synapses are Different in Some Ways from the Nerve-Muscle Junction The neuromuscular junction evolved to perform the task of providing a rapid and high fidelity response to activation of motor neurons. Because of this it has many structural features - such as a presynaptic terminal with a high probability of transmitter release and a highly sensitive postsynaptic membrane, specifically designed to carry out such a charge. Synapses in the central nervous system, however, are designed for different functions - not as slaves to a single presynaptic input, but as integrators of information from many inputs that may be both excitatory and inhibitory. As you might anticipate, the structural features of such synapses differ in important ways from the nerve-muscle synapse. Their structure is much more rudimentary, with fewer presynaptic vesicles, less organized active zones, and simpler postsynaptic specializations. In addition, a single postsynaptic cell receives Figure 5: Central Nervous System (CNS) synapse. Many CNS many (hundreds or even synapses are located on protrusions from the dendrite shaft. These protrusions increase the surface area of the neuron. Many CNS neurons have thousands of synapses on their dendrites and cell bodies. 5 thousands) of presynaptic inputs, and some of these can be switched on and off, as circumstances dictate. Figure 6: Central Nervous system (CNS) synapses. Inhibitory as well as excitatory synapses. This is a pseudocolored electron micrograph of two types of CNS synapse. Two presynaptic terminals are shown. The one in pink synapses on a dendritic spine, whereas the one in blue synapses on the dendritic shaft itself. Usually excitatory synapses are on the spines, while inhibitory synapses are on the shaft. The synaptic vesicles are shown in purple. The actual synapse has been colored green – note that the pink terminal makes two excitatory synapses on the dendritic shaft. This will allow summation of the signal. Unlike at the neuromuscular junction the postsynaptic side of the CNS synapse is less specialized. Dark electron dense material indicates where the receptors are located. Excitatory synapses have more electron dense material than inhibitory synapses. The neuromuscular junction and other excitatory synapses depolarize the postsynaptic membrane above the threshold required to elicit a postsynaptic action potential. In contrast an inhibitory synapse suppresses the action potential. As you might imagine, inhibitory neurotransmission is somewhat different than excitatory. Many inhibitory neurotransmitters are known. In the central nervous system the most important is Gamma-aminobutyric acid, more commonly referred to as GABA. GABA regulates many functions including controlling movement by regulating input to the motor neurons in the spinal cord. GABA has many different types of receptors in the nervous system, but the most important is the GABA(A) receptor. Structurally it resembles the nicotinic ACh receptor – when two GABA molecules bind they trigger a conformational change and the receptor opens a water-filled pore, or channel that spans the postsynaptic neuronal membrane. In contrast with the nicotinic Ach receptor, this channel is permeable only to chloride (Cl-) ions. This has several consequences on the response of the postsynaptic membrane: Since Cl– is concentrated outside of cells, opening of GABA-gated channels results in an influx of Cl– into the postsynaptic cell. This causes the postsynaptic membrane to hyperpolarize. As a result of this hyperpolarization, the postsynaptic membrane is driven away from the threshold required to activate voltage-gated sodium channels. The postsynaptic cell is therefore inhibited from firing action potentials. 6 Drugs that target GABA(A) receptors, such as benzodiazepines (e.g., valium) work by increasing the activity of GABAA receptors. This has the result of suppressing excitatory nervous system activity in the brain. The concept that the GABA receptor needs to be more active to produce more inhibition is an important one. Other inhibitory synapses are also important. The glycine receptor is one example. Like the GABA(A) receptor it has a Cl- channel. Glycine has an important role in integrating control of movement. Both GABA and glycine receptors have been implicated in spastic disorders. Slow as well as fast effects Both the excitatory and Figure 7: The GABAA receptor. Like the Ach receptor the GABAA inhibitory receptor types we receptor is a pentameric protein with a pore down the center, like a have discussed discussed donut. Like the Ach receptor, 2 molecules of GABA bind and above are protein complexes trigger a confromational change that opens the pore. Unlike the with integral ion channels – the Ach receptor the GABAA channel is permeable to an anion, Cl-, only differences are the types of which flows into the postsynaptic cell and causes the membrane to hyperpolarize, therefore inhibiting generation of an action ion conveyed through the pore. potential. Many drugs that inhibit nervous system activity bind to As a consequence these receptors the respond extremely quickly when the neurotransmitter binds. GABA receptor. But in other cases the part of the receptor that binds the transmitter is not physically associated with the ion channel. In these cases, when a transmitter binds to its receptor an intervening ‘second messenger module’ conveys information to the channel. These second messengers are familiar from their other functions in cells and include cyclic AMP, cyclic GMP, Ca2+, calmodulin, and GTP-binding proteins. They have the flexibility to exert a number of different effects. For example, they may: Bind directly to a channel, gating it open or closed, or changing its opening probability. Activate another effector molecule (such as a kinase or phosphatase enzyme) that, in turn, alters channel function. Have effects not directly related to channels – such as on gene transcription. Figure 8: A slowly responding receptor. A diagram showing the typical arrangement of a receptor in which the ion channel is not physically associated with the receptor binding protein. The transmitter binding module is usually a 7 transmembrane protein that is coupled to a G protein. The G protein in turn activates a second messenger module. Depending on whether the G protein is inhibitory or stimulatory, and the type of 2nd messenger, the channel may be opened or closed. Then, depending on the type of channel, the membrane may be depolarized or hyperpolarized. Hence the that receptor may have excitatory or An ion channel is directly activated by a neurotransmitter will respond much faster than an inhibitory effects. ion channel that is indirectly activated via a second messenger module. Many transmitters in the CNS have both fast and slow receptors. Because of this the outcome of a synaptic event 7 depends more on which receptor is present than on the transmitter itself. For example, Ach has both fast (nicotinic) and slow (muscarinic) receptors. The muscarinic receptors can have excitatory or inhibitory effects, depending on the second messenger module they are coupled to. Therefore, Ach can have either fast excitatory, slow excitatory, or slow inhibitory effects, depending on location. The combination of excitatory and inhibitory synapses and the differences in speed of response means that the central nervous system has a wide range of possible ways to modulate neuronal behavior, as we shall see below. Presynaptic Inhibition Remember! a single neuron in the central nervous system can receive thousands of inputs, which are often conflicting and overlapping. How does each CNS neuron integrate many inputs into a coherent output? Synaptic connections occur on the dendrites and cell bodies of neurons. The cell body of the neuron integrates all of the synaptic inputs. The goal of input integration is to put the postsynaptic neuron into a final electrical state whereby it can either fire an action potential or not. How synaptic input onto a neuron is integrated. One way is by inhibiting output from a synapse. We have discussed how inhibitory synapses function on the postsynaptic side of the synapse (postsynaptic inhibition). Another critical way that inhibitory synapses influence output is to interfere with transmitter release from the presynaptic nerve terminal. Presynaptic inhibition uses a third modulatory neuron to regulate the behavior of a normally excitatory synapse. In this case a presynaptic inhibitory neuron releases a transmitter onto the presynaptic nerve terminal. The transmitter binds a receptor in the presynaptic terminal that uses a second messenger module to inhibit the voltage-dependent Ca2+ channels that are required to cause vesicle fusion. As a result of this inhibition the amount of Ca2+ entering the presynaptic terminal during the action potential is reduced, and this in turn reduces the amount of transmitter released, preventing the postsynaptic membrane the reaching the threshold that will cause a synaptic potential. The axon will fire an action potential if: The excitatory inputs are greater than the inhibitory inputs. It will not fire an action potential if: The inhibitory inputs are greater than the excitatory inputs. Integration of synaptic potentials can be both spatial and temporal Synaptic potentials decay with distance from the synapse. Thus, an input far away from the cell 8 body will be less effective in controlling the fate of the neuron than an input close to the zone where the action potential initiates. Hence, both the amplitude of the synaptic potential and its point of origin relative to the action potential initiating zone are important determinants of whether it will activate the axon. Synaptic potentials are relatively slow events, lasting tens of milliseconds. So if the same excitatory presynaptic input is stimulated more than once within a short period of time it will produce postsynaptic potentials at the same point on the postsynaptic cell that can sum with one another to produce a larger synaptic response than either one alone would produce. Figure 6 has an example of a synapse that could behave in this way. This 'summation in time' is termed temporal summation. If two independent presynaptic inputs are stimulated at exactly the same moment in time, to produce two synchronous postsynaptic potentials at different points on the postsynaptic membrane these potentials will sum to produce an integrated synaptic potential as they spread passively toward the action potential initiation zone at the start of the axon. This summation in space is termed spatial summation. If both synaptic potentials are the same type (both excitatory or both inhibitory) then the summed potential will be larger than that produced by either one alone. On the other hand, if one synaptic potential is excitatory (depolarizing) and the other (from a different presynaptic terminal) is inhibitory (hyperpolarizing), then summation of the two will lead to a synaptic potential whose amplitude is intermediate between the two. These simple principles of synaptic integration are in continuous operation in every neuron in the nervous system. Each cell integrates all of the synaptic information impinging on it and, depending upon the balance of excitation and inhibition, it either fires an action potential or it does not. These are the basic means by which all observable behavior is controlled in an organism. Figure 10: A. Spatial and temporal summation. B. Temporal summation occurs when the same excitatory input fires close together (like the synapse in Fig 6). C. Spatial summation occurs when different excitatory input fires close together. D. Excitatory and inhibitory input can cancel each other out. We will now turn our attention to two types of disorders that exemplify the synaptic behaviors we have discussed above. ____________________________________________________________________________ 9 PAIN Pain is clearly a sensory experience with a crucial protective function; it warns of injury and in its absence the body cannot avoid damage. But there is a key distinction between the neural mechanisms by which we sense pain – called nociception - during which specialized sensory receptors called nociceptors are activated by noxious insults, and pain itself - which is the response to actual (or even perceived) tissue damage. The distinction is clinically and experimentally very important. Nociception does not necessarily lead to the perception of pain. In fact the intensity with which pain is felt depends on the individual and the surrounding conditions almost as much as the sensory stimulus itself. Hence there are no ‘painful’ stimuli that invariably elicit the perception of pain in all individuals. The highly subjective nature of pain is one of the factors that make it difficult to define and treat clinically. There are different categories of pain In order to understand how we perceive and respond to noxious stimuli we must be aware of the distinct categories of pain: 1. The major reason patients usually seek medical attention is because of persistent pain. This is pain with an identifiable cause. It can be divided into two categories: Nociceptive pain is usually caused by inflammation and results from direct activation of sensory nociceptors in the skin or soft tissues. Neuropathic pain is caused by direct injury to nerves and bypasses the nociceptors. 2. The reason patients seek medical attention is because of chronic pain, which is very real even though there is no obvious underlying cause. It can be a significant clinical burden. Pain pathways deliver nociceptive information from the periphery to the brain where it either will or will not be perceived as pain. The ascending pathways headed toward the brain have three important synapses, each of which plays a different role in transmitting the signal to the brain – 1. At the periphery where the nociceptive response is activated. 2. In the spinal cord where nociceptive information is gathered into distinct pathways that ascend towards the brain. 3. In the cortex where nociceptive information is integrated with input from emotional centers, and potential responses are assessed. The brain’s response to nociceptive information has two components: Non-specific motor output that initiates avoidance. Pain-specific outputs that initiate analgesia. 10 We will now take a look at how each of these important synapses function in the pain response. 1. The first pain synapse in the periphery Nociceptive information from the skin and soft tissues: While mechanosensory information from the skin is recognized by a number of specialized receptors, such as Pacinian corpuscles that are responsive to pressure and Merkel’s discs that are responsive to vibration, noxious information is recognized by nociceptors that are simply free nerve endings located in the dermis. Figure 11: Nociceptors in the skin. In contrast with mechanoreceptors that are associated with myelinated Abeta (A fibers, nociceptors are simply free nerve endings. However they can be associated with either myelinated Adelta (Aor non-myelinated C axons. We can identify three types of skin nociceptor based on their distinct functions: Thermal nociceptors are activated by extreme temperatures (>45°C or <5°C). They transmit information quickly because their Adelta (A axons are myelinated. Mechanical nociceptors are activated by intensive pressure. They also transmit information quickly via myelinated A axons. Polymodal nociceptors are activated by high-intensity mechanical, chemical or thermal stimuli. They transmit information more slowly because their C axons are nonmyelinated. Nociceptive information from the internal organs is detected by receptors that are activated by inflammation and chemical insults. They are called ‘silent nociceptors’. We shall see why in a moment. How are nociceptors activated? Nociceptors in the skin are chiefly activated by tissue damage that results in inflammation. The inflamed area releases prostaglandins and bradykinin, which stimulates the nociceptors to release substance P. Recall that substance P is a peptide 11 The first pain synapse in the periphery. The free nerve endings are stimulated when the skin is inflamed. This sets up a reflex that amplifies the noxious signal. i.e. the axon does not need to be depolarized for the response to occur. Once the signal is over threshold it is conveyed along the nerve to the next synapse, in the spinal cord. neurotransmitter with an excitatory receptor. The substance P released by the nerve terminal reacts with receptors on mast cells, causing them to degranulate and release histamine. The histamine can excite the nociceptors too, amplifying their response. Histamine also causes blood vessels to dilate and plasma to leak out of capillaries causing swelling (edema). This is known as the triple response of Lewis. Once the axon reflex has amplified the signal above threshold the nociceptive neurons send the noxious information back to the spinal cord. Note that noxious means any painful stuimulus not simply chemical. Nociceptors will transmit noxious information quickly (if myelinated A fibers are activated) or slowly (if non-myleinated C fibers are activated). This impacts how pain is felt: At first, a sharp sensation is due to the A fiber response, then a second burning or aching pain due to the C fibers. Fast pain is due to activation of myelinated Ad fibers, Slow pain is due to activation of unmyelinated C fibers 2. The second pain synapse in the spinal cord. The role of the spinal cord is to convey information to and from the brain. It is divided into two general regions: Gray matter – where the synapses are. White matter – where the ascending and descending pathways are. The gray matter is divided into two main zones. The sensory area is at the back (dorsal) The motor area is at the chest (ventral) Incoming nociceptive fibers synapse in the gray matter of the sensory area (dorsal horn) The gray matter is further divided into a number of layers (laminae). The pain synapses are located in the sensory area, in layers I–VI. Where the myelinated A fibers and non-myelinated C fibers synapse turns out to be crucial for how the initial noxious sensation is controlled. The fast A fibers synapse with projection (output) neurons that move the information up the spinal cord to the brain The slow C fibers synapse with both projection (output) neurons and with interneurons that then synapse on projection (output) neurons. Also: Pressure sensitive Abeta (A fibers (mechanoreceptors) also synapse with the 12 projection (output) neurons. This circuit in the spinal cord is the first way we manage the output of noxious information. 1. Normally the interneurons synapse on projection neurons (that output to the brain) inhibiting them from firing. 2. The C fibers synapse on both the inhibitory interneurons and the projections neurons. 3. When the C fibers are activated by a noxious stimulus they inhibit the inhibitory interneurons. This allows the output neurons to fire when they are activated by A fibers in response to the painful stimulus. 4. The C fibers also stimulate the projection neurons directly – so even more pain information is transmitted up the spinal cord. 5. However, the inhibitory interneurons also receive input from mechanosensitive A fibers. Unlike the C fibers, the A fibers stimulate the inhibitory interneurons. 6. This means that when the A fibers activate the inhibitory interneurons the projection neurons are inhibited - this prevents the noxious stimulus being transmitted to the brain Thus if we can tip the balance toward activating the Ab fibers, we will feel less pain – this is why we rub ourselves after we have experienced a painful stimulus. Mechanosensitive AB fibers regulate nociceptive output at the spinal cord. Chronic pain occurs when C fibers are persistently activated. It is easy to see therefore why chronic activation of C fibers would cause a pain to persist even after the initial stimulus from the periphery has been dealt with. The inhibitory interneurons can no longer work. Persistent changes in C fibers behavior can cause perception of pain even when the initial injury has healed. Referred Pain Nociceptive axons from both the skin and the internal organs project to the same output neurons in the sensory region of the spinal cord. But sensations from the skin normally predominate. Hence, when the nociceptive axons from the internal organs are activated, higher brain centers incorrectly localize the sensations to different areas of the skin. Thus injury to an internal organ is 13 Nociceptive neurons from the skin and the internal organs synapse in the same place in the spinal cord – the brain cannot tell where the stimulus is coming from. experienced on predictable areas of the body surface - when you feel a pain in the arm following a heart attack, this is because the brain is misinterpreting the source of the painful stimulus, not because your arm has been damaged. Why are these areas so predictable? Because pain neurons from the skin and the internal organs are always coupled in the same area. The inability of the brain to directly recognize nociceptive stimuli from the internal organs is why these receptors are called “silent”. Referred pain has a stereotyped distribution The stereotyped distribution of referred pain is used to diagnose damage to internal organs. 3. The third pain synapse - How nociceptive information gets from the second synapse to the cerebral cortex. The pain synapses in the sensory regions of the spinal cord gather neurons together to send nociceptive information to the brain using three important pathways. The most prominent pathway is concerned with where the pain is localized. It ascends from the spinal cord to the thalamus, which is the important way station for all neurons destined for the cortex. From the thalamus the pain pathway ascends to the somatic sensory cortex. Almost as soon as the pathway starts, the axons cross to the opposite side of the spinal cord. Thus the information is represented on the opposite side of the cortex. The second pathway is concerned with the non-specific arousal that occurs after noxious stimulation occurs. It too goes to the cortex, but also branches into the area in the brainstem that organizes arousal. 14 The third pathway ends up in an area of the brain stem that can initiate analgesia. We will look at how this happens shortly. How the cerebral cortex processes nociception The primary nociceptive pathway from the spinal cord ends up in the opposite part of the cortex that recognizes sensation – the parietal cortex. The pink line on the model of the cortical lobes is the location of the primary somatosensory cortex. If we slice down this line and lie the slice flat we can see how the body is mapped in this area of the cortex. It is clear that some areas of the Sensory input from the body maps onto the parietal cortex at the ‘somatosensory body are over- strip’. The homunculus reflects the differences in sensory input from each area. represented on the map, indicating that they send a lot more sensory information to the cortex. Nociceptive information is processed in this area along with other sensory information. It is clear from this map that pain does not simply arise from how nociceptive information is processed in the somatosensory cortex. If it did, the sensation would reflect the small, well-defined, receptive fields of the nociceptors. Instead most clinical pain involves diffuse aches. These reflect the involvement of other brain areas. For example: The insular cortex is found directly underneath the primary somatosensory cortex. It processes information about the internal state of the body and contributes to the emotional response to pain. Patients with lesions in the insular cortex are emotionally unresponsive to pain. The cingulate gyrus is part of the limbic system that is also important in emotional responses. 15 Errors in processing – phantom pain. Phantom pain occurs almost exclusively as a result of amputation. Almost immediately following the amputation of a limb, 90-98% of patients report experiencing a phantom sensation. Nearly 75% of individuals experience the phantom as soon as anesthesia wears off, and the remaining 25% of patients experience phantoms within a few days or weeks. For some people, phantom limb experiences may fade, disappear or change over time, in others they may continue for years or even a lifetime. Phantom limbs are very diverse and individual. The solid lines show the site of amputation, the dotted lines Some describe their phantom where the phantom limbs were experienced. Nearly 95% of limbs as being stuck in a fixed people report a phantom limb experience. position whilst other claim to be able to move their limbs both voluntarily and spontaneously to the extent that they even ‘gesticulate’ when they talk. Amputees often describe that parts of their limb are missing or that the limb is magnified, stretched, or shortened. The posture the phantom ‘adopts’ is often related to the last pose the individual saw their limb in before it was amputated, and this position is often associated with pain. Little is known about the true mechanism causing phantom pains. Historically, phantom pains were thought to originate from nerve scars (neuromas) located at the stump tip, but this does not explain why the sensations appear to emanate from within the space occupied by the missing limb (rather than from the stump). Errors in processing As we saw previously, when a limb is stimulated the corresponding part of the opposite somatosensory cortex is activated. When a limb is removed this part of the brain no longer receives its normal activation. This causes the brain to reorganize so that the part of the brain that used to respond to the missing limb responds to other things instead. For example, it may respond to touch to a different part of the body such as the face. The standard explanation of phantom limb sensation is that the part of the brain that used to respond to the limb is now responding to other things: such as different parts of the body, or maybe to the sight of that part of the body. The pink line shows the area of somatosensory cortex no longer receiving input if the right arm is amputated. Why are some phantom limbs painful? Some people have painless phantom limbs, whereas others experience excruciating pain. We don’t understand why. One factor known to be important is whether the limb was painful prior to amputation. If the real limb was painful prior to amputation then there is a higher chance that the phantom limb will be painful too. One suggestion is that the more that the brain reorganizes itself after amputation, the more pain will be experienced. 16 For most people the intensity of pain and the actual nature of the phantom limb can also change over time. One common phenomenon is called 'telescoping' in which the phantom arm or leg appears to get shorter over time. Mirror box therapy. Many patients experience pain because the phantom limb seems to be clenched; obviously phantom limbs are not under voluntary control, so unclenching is impossible. The theory behind mirror box treatment is that the brain has become accustomed to the fact that a phantom limb is paralyzed because there is no feedback from the phantom back to the brain to inform it otherwise. The neurologists Ramachandran and Rogers-Ramachandran believed that if the brain received visual feedback that the limb had moved, then the phantom limb would become unparalyzed. To create the visual feedback that would allow this illusion, they constructed mirror boxes that have a vertical mirror placed in the center. The intact limb is placed on one side of the mirror, in the patient’s sight, while the amputated limb is placed on the other side, out of sight. The patient sees the intact second limb through the mirror and sends commands to both limbs to make symmetric movements. The movement gives the brain positive feedback that the phantom has moved, and it becomes unparalyzed. In a study of ten patients with upper phantom limb paralysis, nine patients were able to ‘move’ the phantom limb, and eight of those patients had their pain alleviated. Since this pioneering study, multiple additional studies have support the mirror box findings for patients with upper limb phantom pain. The first case of mirror box treatment for lower limb phantoms was reported in 2004. The patient, Alan, experienced a painful crossing of his toes in the morning, and the pain worsened as the day progressed. After three weeks of mirror box treatment twice a day, Alan no longer felt any painful sensations from crossed toes. Stimulating analgesia The pain pathways that ascend the spinal cord to the cortex are mirrored by complementary descending pathways that can stimulate an analgesic response. These pathways release endogenous opioids at the output neurons in the sensory area of the spinal cord. The opioids inhibit the transmission of nociceptive information to the cortex by the ascending pathway. The descending analgesia pathway releases endogenous opioids onto the output neurons in the sensory area of the spinal cord. 17 Endogenous receptors for opioids. Receptors for opioids are found in all areas of the brain important in pain regulation. However they are found in other areas too, explaining why the artificial opioid morphine affects many physiological processes. Because of this, morphine is now commonly administered locally in the spinal cord rather than systemically. This allows the dose to be decreased, which also protects against addiction. In summary It is important to distinguish between a noxious stimulus and the perception of pain. Nociceptive information is sensed in the periphery and then transmitted to the cortex by a multi-synaptic pathway that ascends through the spinal cord. Each ascending synapse is an important site for regulation of the response. A complementary descending pathway modifies the input by stimulating release of analgesic peptides called opioids. ____________________________________________________________ ADDICTION Research has shown us that addiction is a disease that affects brain structure and impacts behavior. Many of the biological and environmental factors that contribute to addiction have been identified, and the search has begun to identify for the underlying genetic variations that predispose and contribute to its development and progression. Every year, abuse of illicit drugs and alcohol contributes to the death of more than 100,000 Americans, while tobacco is linked to an estimated 440,000 deaths per year. People of all ages suffer the harmful consequences of drug abuse and addiction. Babies exposed to legal and illegal drugs in the uterus may be born prematurely and underweight. This drug exposure can slow the child’s intellectual development and affect behavior later in life. Adolescents who abuse drugs often act out, do poorly academically, and drop out of school. They are at risk of unplanned pregnancies, violence, and infectious diseases. Adults who abuse drugs often have problems thinking clearly, remembering, and paying attention. They often develop poor social behaviors, and their work performance and personal relationships suffer. Parents who abuse drugs often live in chaotic, stress-filled homes with child abuse and neglect. Such conditions harm the wellbeing and development of their children in the home and may set the stage for drug abuse in the next generation. Pet scans indicate reduced activity reflecting brain damage in brains of drug abusers. 18 What is drug addiction? Addiction is defined as a chronic, relapsing brain disease that is characterized by compulsive drug seeking and use, despite harmful consequences. It is considered a brain disease because drugs change brain structure and function. These changes can be long lasting, and can lead to the harmful behaviors seen in people who abuse drugs. How do drugs of abuse work in the brain? Drugs of abuse interfere with neurotransmission. For example, the synthetic opioid morphine we discussed above mimics the endogenous opioids involved in the analgesia pathway. Morphine is addictive because its off-target effect on dopamine synapses in the reward pathway. In fact most drugs of abuse target this pathway directly or indirectly. Before we take a look at that pathway in detail, let’s take a look at the characteristics of dopamine that make it such an effective target for drugs of abuse. Dopamine Dopamine is one of three catecholamine neurotransmitters that play important roles in brain function (the others are serotonin and norepinephrine). They are structurally somewhat similar, but each has a distinct receptor. Catecholamine receptors are all coupled to G-protein second messenger modules, which means that, unlike Ach and glutamate, they exert slow modulatory excitatory or inhibitory effects on their target neurons. A second feature of catecholamine neurons affects how they communicate with other axons - their synapses are found along the entire length of the axon, not merely restricted to the axon terminal. These en passant synapses mean that each catecholamine neuron is able to influence a far larger area of target cells than it would if its synapses were restricted to the terminals. Catecholamine neurotransmitters Catecholamine neurons make ‘en passant’ synapses along their entire axons. This means they can influence a larger area of target neurons than if the synapses were restricted to the axon terminals. The structure of the dopaminergic neuron means that even though dopamine pathways within the brain are quite limited, they can nonetheless influence many distinct regions. 19 Dopamine pathways in the brain There are three major dopamine pathways – 1. The nigrostriatal system, which is involved with movement intention, and which is damaged in Parkinson’s disease. 2. The tubero-infundibular system that is involved with hormone secretion and homeostasis. 3. The mesolimbic system or reward pathway, which is the key target for drugs of abuse. The mesolimbic system directly or indirectly regulates emotion, motivation, cognition, movement and feelings of pleasure. Overstimulation of this system, which normally rewards natural behaviors, produces the euphoric effects sought by people who abuse drugs and reinforces the behavior. . The reward pathway in the brain There are three dopamine pathways in the brain. The dopamine reward pathway originates in a subcortical area of the brain near the midline called the Ventral Tegmental Area. Dopamine neurons whose cell bodies are in the VTA end up in the Nucleus Accumbens and the Prefrontal Cortex. The reward pathway connects the ventral tegmental area (VTA) with the nucleus accumbens (NAc). The VTA also connects with the prefrontal cortex. The connections between the VTA and the nucleus accumbens are called the reward pathway because it is activated during pleasurable experiences such as eating, sex or receiving praise, The reward pathway was discovered through the technique of intracranial self-stimulation. An electrode implanted in different areas of the brains of rats could be activated when the rats voluntarily pressed a lever. The rats did not regularly activate the electrode in areas of the brain except the reward pathway: Because of the positive effects felt when this pathway is stimulated, the behavior was reinforced. 20 Dopamine synapses in the reward pathway are critically important for the effects of drugs of abuse. The dopamine synapse is different from the Ach synapse in how it deals with transmitter that has been released into the synaptic cleft. Unlike the Ach synapse that inactivates Ach with cholinesterase, the dopamine synapse clears the dopamine by recapturing into the presynaptic terminal using a specific transporter. This means that any drug inhibiting the reuptake of dopamine from the synaptic cleft The dopamine synapse differs from the nerve – muscle synapse – there is no inactivating enzyme will cause dopamine effects to persist. in the synaptic cleft. Cocaine is one example of a drug that blocks the function of dopamine reuptake transporters. As a result, dopamine levels increase in the synapse, and consequently, the target neuron is continuously stimulated. This constant firing of the neurons leads to a feeling of euphoria. In order to attain a cocaine "high," at least 47% of the binding sites must be blocked. In addicts, cocaine blocks between 60 and 77%. Compare the healthy brain at the top with the brain of a cocaine abuser at the bottom. Why does cocaine reduce brain activity if it increases the activity of dopamine neurons? This is because the output neurons from the nucleus accumbens are inhibitory. That is to say they shut down signaling in the regions they synapse with. When they are stimulated by cocaine the inhibition is even more intense. Amphetamine has effects similar to cocaine. It also prevents dopamine reuptake, but it also stimulates release of dopamine into the synaptic cleft. Cocaine reduces brain activity because output from the nucleus accumbens is largely inhibitory. Drugs of abuse have similar effects All drugs of abuse increase dopamine activity at the synapse between the VTA and the NAc, but they do it in slightly different ways: 21 Nicotine stimulates the VTA, where the dopamine neurons originate, directly, increasing dopamine release. Heroin, other synthetic opiates (like morphine) and ethanol can also affect the VTA synapse but they do so slightly differently: The VTA, is itself normally under significant inhibitory control, to prevent over-activity of the reward pathway. These drugs disrupt that inhibition. The balance between excitation and inhibition is lost and levels of dopamine in the reward pathway surge. Drugs of abuse like cocaine and amphetamine can increase the amount of dopamine in the synaptic cleft between 2-10 fold more than normal. In some cases, this occurs almost immediately (as when drugs are smoked or injected), and the effects can last much longer than Many different drugs of abuse target aspects of the same those produced by natural rewards. pathway For instance the PET scan above was done 10 days after the patient took cocaine. The resulting effects on the brain’s reward circuit dwarfs those produced by naturally rewarding behaviors. This strongly motivates people to take drugs again and again. Tolerance and Dependence Two key consequences of drug abuse – tolerance - increasing doses of a drug are required to obtain the same effect - and dependence, are both particular problems of drugs that affect the reward pathway, because the drive to reinforcement is so strong. Under normal circumstances the Bidirectional interactions between the VTA and the reward pathway exists in homeostasis NAc keep the reward pathway in homeostasis. Drugs because the communication between of abuse increase the steady state level of the pathway. the VTA and the NAc is bidirectional – there are also connections between the NAc and the VTA. The NAc communicates with the VTA by way of an endogenous opioid dynorphin. Recall that nucleus accumbens output is inhibitory. Dynorphin inhibits the VTA, and this in turn reduces dopamine levels in the reward pathway, calming it down. Therefore, anything that increases dopamine release at the VTA/NAc synapse will concurrently increase dynorphin release. This will feed back onto the reward pathway, further depressing its activity. It is not surprising then that the drugs of abuse that increase the activity of the VTA/NAc synapse increase dynorphin as a ‘knock-on’ effect. Down-regulation of the reward pathway means that the same levels of drug no longer produce the same effect, leading 22 to tolerance. But because this is the reward pathway with a significant drive to reinforcement there is pressure to continue the activity – leading to dependence. Drugs can permanently alter the brain’s reward pathways Dopamine’s effects are not limited to transiently depolarizing or hyperpolarizing the postsynaptic cell. Because it acts via a second messenger it can also affect gene transcription in the postsynaptic cell like this: Binding of dopamine to its receptor activates the second messenger cyclic AMP (cAMP). In turn cAMP activates the cAMPdependent kinase. The cAMP-dependent kinase moves into the nucleus and activates the CREB protein. CREB protein binds to the CRE response element in several genes. These genes include dynorphin and many neuropeptide neurotransmitters. Activation of dynorphin causes the The CREB transcription factor is activated via the dopamine receptor pathway. It causes rapid increases in transcription of dynorphin. increases in feedback inhibition of the VTA we saw before. This is a fast response. Binding of dopamine to its receptors can also increase other important transcription factors. One of these is Delta FosB Delta Fos B can activate the neurotrophic factor BDNF (Brain derived neurotrophic factor). BDNF can induce the formation of dendritic spines. Because each spine is the site of a synapse, inducing spines induces synapse formation. This structural response is slow and 23 The delta Fos transcription factor stimulates slow and persistent changes in neuronal structure. Neurons from the methamphetamine abuser brain have more spines than their normal counterpart. sustained. In this way drugs that affect dopamine synapses can have both short-term effects on synapse behavior (like stimulating dynorphin synthesis and release) and long-term effects on neuronal structure like inducing synaptic spines. Genes and addiction While social and environmental factors contribute to the risk of addiction, the finding that several genes are linked to specific addictive behaviors indicates that there is also a genetic susceptibility. Fortunately the reward pathway is located in a part of the brain that is evolutionarily very old, so that all aspects of the pathway are almost identical in mice, rats and humans. This means that mice, which are a valuable genetic tool, are also useful animal models to investigate genetic susceptibility. Mice have the same number of genes as humans (20,000 – 25,000) and each mouse gene is about 85% identical to its human counterpart (or homolog). Among the genes now identified as conferring susceptibility to addictive behaviors are: The Dopamine D2 receptor One particularly interesting gene whose modifications are associated with addictive behavior is the Dopamine D2 receptor. Addictive behaviors are associated with defects to a Binding of dopamine to the D2 number of genes, indicating genetic susceptibilities. receptor inhibits cAMP formation, and this leads to reactions that tend to inhibit addictive behaviors. Reductions in D2 receptor levels in rats are associated with an increase in self-administration of drugs – thought to be a predictor of impulsive, addictive behaviors. Interestingly, when monkeys are raised together under conditions that result in stress in the subordinate monkey, those monkeys have fewer D2 receptors. The subordinate monkeys were more likely to selfadminister drugs than their dominant peers. This is evidence that the environment can also induce molecular changes that impact addictive behaviors. Levels of the important dopamine D2 receptor can be modified by stressful experiences, and then lead to impulsive behaviors. 24 In humans drug abuse is also associated with decrease in levels of the Dopamine D2 receptor, indicating a feed forward inhibition at the level of the postsynaptic cell. Summary: Addiction occurs when the activity of the reward pathway is disturbed. The reward pathway is the mesolimbic branch of the dopamine system, in particular the dopamine synapse between the Ventral Tegmental area and the Nucleus Accumbens. Drugs of abuse act on this pathway in various ways to increase dopamine neurotransmission across this synapse. Dopamine neurotransmission can lead to transient effects on the postsynaptic cell membrane, but also effects on gene transcription. A fast acting effect on gene transcription increases homeostasis within the pathway that may contribute to drug tolerance. Slower effects on gene transcription result in longer acting effects on neuronal structure, including building more synapses on the post-synaptic cell. Finally, a number of genes have been associated with addictive behaviors. Within the dopamine pathway, decreased numbers of the D2 receptor are associated with an increase in impulsive, addictive behaviors. Drug abuse is able to decrease numbers of this receptor. For lesson development This web site has elements of a good high school Addiction curriculum: http://learn.genetics.utah.edu/content/addiction 25