Neuroimmune Pharmacology || Neuronal and Glial Signaling

Keywords Action potential; Calcium; Glia; Ion channel; LTP; Neuron; Neurotransmitter; Potassium; Resting mem-brane potential; Sodium; Structure; Synapse

10.1. Introduction

The major function of the nervous system is to rapidly trans-fer and integrate information with a view to organizing the diverse functions of multicellular organisms. Our present understanding as to how nerve cells communicate originates with studies in the nineteenth century on the anatomy of the nervous system. Work conducted by Camillo Golgi and San-tiago Ramon y Cajal defined the fine structure of neurons for the first time. Golgi favored the idea that all the nerves in the nervous system existed as a sort of reticular net, rather than as separate entities. Cajal, in contrast, concluded that neu-rons were independent entities and that a minute gap existed between the ends of nerve fibers and other nerve cells or the muscles that they innervate. This gap was ultimately named a “synapse” by the great neurologist Sir Charles Sherrington in 1897, from the Greek meaning “to clasp”. Cajal also argued that neurotransmission was basically a unidirectional process, information being received by dendrites and being transmit-ted unidirectionally along axons. The actual existence of the synapse as a structure was not confirmed until the develop-ment of the electron microscope in the 1950s. Around this time a fierce debate took place between two opposing sets of scientists (Valenstein, 2005). One set, primarily electro-physiologists, held the view that information was transmitted between nerves and between nerves and muscles by purely electrical processes. The other group, primarily pharmacolo-gists, suggested that chemical messenger molecules were released by the presynaptic nerve and carried the information across the synapse. The nature of the chemicals that consti-tute neurotransmitters was gradually revealed by the work of several important investigators who characterized the effects of different substances that mimicked or blocked the actions of neurotransmitters on fast skeletal muscles and in the autonomic

nervous system. This finally culminated in the demonstra-tion by Otto Loewi in 1921 that the vagus nerve secreted a chemical (“Vagusstoff”) that mediated the slowing effect of vagal stimulation on the heart. This substance turned out to be acetylcholine.

The term glia, or rather neuroglia (nerve glue), was first introduced by Virchow in 1859 who conceived of these cells as being neutral elements concerned in holding the nervous system together and playing a supportive role. Further studies by Ramon y Cajal and his colleagues divided glia into astrocytes, oligodendrocytes and microglia. We now know that glial cells are far from inactive support systems and are engaged in diverse dynamic interactions with nerve cells. Nissl suggested that microglia had the capacity for migration and phagocytosis and this has indeed proved to be the case. As opposed to other major cell types in the brain, the microglia are of mesenchymal origin and can generally be thought of as resident cells with macrophage like prop-erties. Signaling between neurons and glia is involved in dynamically integrating the functions of the nervous system in health and disease. In general, signaling in the nervous system requires us to understand the properties of the dif-ferent types of neurotransmitters, how they engage receptors and how this impacts the electrical and other properties of neurons and glial cells.

10.2. Electrogenesis and the Action Potential

10.2.1. The Resting Membrane Potential

The most common molecules in the body are water and inor-ganic molecules such as sodium, potassium and chloride ions. A feature that is common among all living cells is that the concentrations of these ions are different in the extracellular and intracellular compartments. The extracellular fluid is high in sodium (Na+) and chloride (Cl−) ions, but low in potassium (K+) ions (Figure 10.1). In contrast, the intracellular solution is

10Neuronal and Glial SignalingMurali Prakriya and Richard J. Miller

T. Ikezu and H.E. Gendelman (eds.), Neuroimmune Pharmacology. 105© Springer 2008

106 Murali Prakriya and Richard J. Miller

low in Na+ and Cl−, but high in K+. This difference is maintained and regulated by control mechanisms residing in the plasma membrane of the cell, a phospholipid bilayer with protein mol-ecules inserted into it. The plasma membrane acts as a selec-tively permeable barrier permitting some molecules to cross while excluding others. When a pathway or “channel” for the movement of these charged molecules opens up across the plasma membrane, a phenomenon known as “gating”, the net movement of ions is governed not only by their concentration gradients, but also by the electrical potential difference across the plasma membrane. As we shall discuss below, neurons express proteins in their plasma membranes that act as chan-nels allowing the selective permeability of different ions.

How does an electrical potential come into this picture? The movement of ions through selective pores in the mem-brane gives rise to a charge separation across the phospholipid bilayer, essentially giving rise to a “capacitor” and a poten-tial difference across the membrane. It takes the movement of only a very small number of charges to give rise to a substan-tial membrane potential difference across the membrane. As a result, ions that are propelled down their concentration gra-dient encounter the electrical force that opposes their move-ment down this concentration gradient. Equilibrium for ion movement across the membrane is reached when the electrical force exactly balances the diffusional force arising from the concentration gradient. A relationship that describes the value of the electrical potential reaches at this equilibrium condition is given by the Nernst equation:

VRT

zF

X

Xou

in

=⎛⎝⎜

⎞⎠⎟

In[ ]

[ ]t

where V is the membrane voltage at equilibrium, [X]out

and [X]

in are the extracellular and intracellular concentrations of

the ion being examined, R is the gas constant, T is the absolute

temperature, z is the valence of the ion, and F is Faraday’s constant. It is important to remember that the Nernst equation applies only to one ion at a time and only to ions that can cross the plasma membrane. For an ion in question, with a certain extracellular and intracellular concentration, the value of the electrical potential given by the Nernst equation is called the equilibrium potential.

For the typical ionic concentrations in mammalian cells (Figure 10.1), the equilibrium potentials for K+ and Na+ can be calculated from the Nernst equation to be −80 mV and +57 mV, respectively. If the plasma membrane is permeable only to K+ and no other ion, the membrane potential would be determined solely by the equilibrium potential for K+ and would be −80 mV. Real cells, however, are permeable to more than one ion at a time, and as a consequence, their resting membrane potential is influenced not only by the move-ments of K+, but also other ions, primarily Na+. For a typical cell that has a large K+ permeability at rest, if we increase the Na+ permeability to the membrane very slightly, the net effect would be to depolarize the membrane potential away from the equilibrium potential of K+ and toward the equilib-rium potential of Na+. There is a struggle between Na+ on the one hand, tending to make the V

m equal to +57 mV, and K+

and Cl− on the other hand, which push to make Vm equal to

−80 mV. An equation that quantitatively relates these factors is the Goldman-Hodgkin-Katz equation (also referred to as the constant field equation because of the assumption made that the membrane field between the intra and extra-cellular compartment varies at a constant rate with distance). For a cell that is permeable to Na+, K+ and Cl−, this equation can be written as:

VRT

zF

p p p

p p pmo o i

i i

=+ ++ +

+ + −

+ +ln[K ] [Na ] [Cl ]

[K ] [Na ] [K Na Cl

K Na Cl CCl ]−

⎛⎝⎜

⎞⎠⎟o

This equation is similar to the Nernst equation except that it simultaneously takes into account the contributions of all three permeant ions. It indicates that the membrane potential is governed by two factors: (1) the ionic concentrations, which determine the equilibrium potentials for the ions, and, (2) their relative permeabilities, which determine the relative importance of a particular ion in governing where V

m lies. For many cells,

including most neurons and immune cells, this equation can be simplified: the chloride term can be dropped altogether because the contribution of chloride to the resting membrane potential is insignificant. In this case, the Goldman equation becomes:

VRT

zF

p p

p pmo o

i i

=++

⎛⎝⎜

⎞⎠⎟

+ +

+ +ln[K ] [Na ]

[K ] [Na ]K Na

K Na

Because it is easier to measure relative ion permeabilities than the absolute permeabilities, this equation can be rewritten in a slightly different form:

Figure 10.1. Composition of major ions in the intracellular and extracellular compartments.

10. Neuronal and Glial Signaling 107

Va

amo o

i i

=++

⎛⎝⎜

⎞⎠⎟

+ +

+ +58 log[K ] [Na ]

[K ] [Na ]

where term a = pK/p

Na is the permeability of K+ relative to Na+,

and the term RT/F has been evaluated at room temperature and converted to log. In most cells at rest, the ratio a is about 50, resulting in a membrane potential of −71 mV for a cell with ionic composition as shown in Figure 10.1.

Because the steady-state membrane potential lies between the equilibrium potentials for Na+ and K+, there is a constant movement of K+ out from the cell and Na+ into the cell. To ensure that this does not lead to a progressive decline in the concentration gradients across the membrane, all cells have a Na–K pump, which uses the hydrolyses of ATP to simultane-ously pump K+ into the cell and push Na+ out. The constant fluxes of K+ and Na+ constitute electrical currents across the cell membrane, and at steady-state, these currents cancel each other out so that the net membrane current is zero.

10.2.2. The Action Potential

As noted above, the membrane potential is governed by the relative permeabilities for K+ and Na+. If the K+ permeabil-ity is greater than the Na+ permeability, the membrane poten-tial is closer to E

K. Conversely, if the Na+ permeability far

exceeds the K+ permeability, the membrane potential should be closer to E

Na than E

K. In excitable cells such as neurons,

ionic permeabilities are not fixed, but can be varied resulting in the occurrence of transient, dramatic changes in the mem-brane potential. It is these transient changes in the membrane potential brought about by changes in ionic permeabilities that underlies the action potential, a fundamental basic signal that sub-serves communication between all brain cells.

The typical profile of membrane potential changes during an action potential is illustrated in Figure 10.2. Following a small depolarization to a “threshold” value, there is a sudden, large jump in the membrane potential during which the poten-tial transiently moves in the positive direction (referred to as a depolarization) and actually reverses in sign for a brief period. After peaking at a positive voltage, the membrane potential begins an equally rapid return toward its resting value, and transiently becomes more negative than its normal resting value. The last part of the action potential is a slow return to its resting value that often lasts several milliseconds. This is called the undershoot of the action potential. This basic elec-trical signal pattern is fundamental to neurons and is the basis of information transfer between neurons of the brain.

The key to understanding the origin of the action poten-tial lies in the factors that influence the membrane potential of the cell as exemplified by the Goldman relationship. Recall that the membrane potential of the cell lies somewhere between E

K and E

Na. At rest, because the relative perme-

ability of the membrane is much higher for K+ than Na+, the

point at which the membrane potential lies is closer to EK.

If the Na+ permeability suddenly increases dramatically, then the membrane potential would correspondingly shift toward E

Na. For example, if p

K/p

Na changed from 50 at rest

to 0.02, then the membrane potential would swing from −71 mV to 53 mV. After a brief delay, if the p

K/p

Na changed

back to 50, the membrane potential would be expected to return to −71 mV. It is these transient changes in Na+ per-meability that are responsible for the swings in membrane potential from near E

K toward E

Na and back during the

action potential.

10.2.3. The Sequence of Activation and Inactivation of Na+ and K+ Channels During an Action Potential

A dramatic increase in Na+ permeability requires a dramatic increase in the number of channels that allow Na+ to enter the cell. Thus, the resting p

Na is only a small fraction of what

it could be because most membrane sodium channels are closed at rest. What stimulus causes the hidden Na+ chan-nels to reveal themselves? It turns out that the activation of these Na+ channels is triggered by membrane depolariza-tion. When V

m is at its usual resting levels around −70 mV,

these Na+ channels are closed and pNa

is low. However, depolarization causes the channels to open. Because the voltage-activated Na+ channels respond to depolarization, the response of the membrane to depolarization is regenerative, and thus explosive (Figure 10.3). A small depolarization of the membrane opens Na+ channels, which causes influx of Na+ into the cell and additional depolarization, which in turn opens more Na+ channels. This explains the all-or-none nature of the action potential: once it is triggered, it runs to completion.

Figure 10.2. Various phases of the action potential. Once a depo-larization reaches a certain threshold, the membrane potential moves rapidly in a regenerative manner toward E

Na. The opening of K+

channels coupled with the inactivation of Na+ channels causes the membrane potential to repolarize. The prolonged hyperpolarization (undershoot) results from slow closing of open K+ channels follow-ing rapid repolarization.


What causes Vm to return to its resting potential following

the regenerative depolarization during the action potential? Two processes cause this: (1) the time-dependent inactiva-tion of the depolarization induced increase in p

Na, and (2) a

delayed increase in the pK initiated by depolarization. A few

milliseconds following the opening of voltage-activated Na+ channels by depolarization, an “inactivation gate” is trig-gered which plugs the ion permeation pathway of Na+ chan-nels and shuts down channel activity, causing a dramatic decrease in p

Na. Almost at the same time as this is happen-

ing, depolarization-activated K+ channels begin to open up, causing the p

K to dramatically increase. The closure of Na+

channels and the opening of K+ channels cause a dramatic increase in the p

K/p

Na ratio, thereby shifting the balance

strongly in favor of EK. Therefore, the repolarizing phase of

the action potential arises from the simultaneous decline in p

Na and an increase in p

K. In fact, the increase in p

K lasts for

several milliseconds after the Na+ channels close, causing the p

K/p

Na ratio to be higher than at rest. This causes the V

m

to be pushed closer to EK, explaining the undershoot that fol-

lows fast repolarization.How does the action potential propagate along the nerve

fiber? This is easily explained by the basis for the generation of the action potential. As we have just seen, the stimulus for an action potential is a depolarization of sufficient strength to open large numbers of Na+ channels to cause a regenerative membrane depolarization. Once such a depolarization occurs in one part of the cell, it brings neighboring regions above the threshold, setting up an action potential in that region. This in turn causes other neighboring regions to reach threshold and triggers the same gating schemes of Na+ and K+ channels to produce action potentials in those regions. The “inactiva-tion” of Na+ channels ensures that once an action potential has occurred in one region, it cannot immediately occur again in that region until Na+ channels have recovered from inactiva-tion. This causes the unidirectional movement of the action potential, away from the region that triggered the initial rise in membrane potential, and typically down the nerve fiber. This is the basis of the fundamental long-distance signal of the nervous system.

10.2.4. Transmission of Signals Between Neurons: Voltage-Activated Ca2+ Channels Mediate Neurotransmitter Release

Propagation of action potentials typically occurs from the somatic regions of neurons, through the axon and into the “ter-minals”—the tiny axon branches that terminate in synapses on neighboring neurons or end organs. As described below, inva-sion of the action potential into nerve terminals causes fusion of pre-packaged neurotransmitter-filled vesicles with the plasma membrane, releasing neurotransmitter across the synapse, and resulting in the activation of receptors on neighboring cells. What process couples the electrical action potential signal in the nerve terminal to the release of neurotransmitter? This is mediated by Ca2+ influx from the extracellular space into the nerve terminal through voltage-activated Ca2+ channels. Com-pared to Na+ and K+, Ca2+ is present in much lower amounts in the extracellular space (1–2 mM) and was therefore ignored in the previous discussions of resting membrane potential and action potentials. However, as a chemical messenger inside the cell, Ca2+ mediates a variety of critical signaling functions. Nature has evolved such that the intracellular concentration of Ca2+ is of the order of only 100 nM. With the Ca2+ concentra-tion in the extracellular space of 1–2 mM, this creates a 10,000-fold concentration gradient across the membrane. In addition, with these ionic concentrations, the Nernst equation indicates that the equilibrium potential for Ca2+ is also very positive. Therefore, near the resting potential, both the concentration and electrical gradients promote the movement of Ca2+ into the cell. When a conduit for Ca2+ entry, such as a voltage-acti-vated Ca2+ channel opens up, Ca2+ rushes into the presynaptic intracellular space, elevating the local Ca2+ concentration, and resulting the fusion of neurotransmitter-filled vesicles through series of very rapid signal transduction events.

Voltage-activated Ca2+ channels are activated by membrane depolarization and represent a large family of related channels with a wide tissue distribution. They are found ubiquitously in neurons, muscles, and endocrine cells as well as in many epithelial and endothelial cells. In addition to neurotransmit-ter release, they mediate a variety of essential functions in the body including muscle contraction, insulin secretion, gene expression, modulation of signal transduction events and in excess can cause cell death.

10.2.5. Membrane Properties of Glial Cells

Intermingled with the neurons in the brain are a variety of other cell types. The most common of these “satellite” cells are glial cells. These make up virtually about one half of the total volume of the brain and exist in several forms such as astrocytes, oligodendrocytes, and Schwann cells. Membrane properties of glial cells exhibit fundamental differences from neurons, the chief difference being their passive nature. Unlike neurons, most glial cells are not excitable and do not fire action potentials. Membrane potential measurements of

Figure 10.3. Behavior of Na+ channels and resulting changes in membrane potential during the rising phase of the action potential.


glia indicate a relatively negative resting membrane poten-tial—around −90 mV in contrast to −70 mV for most neuronal cells. This arises from the fact that, in contrast to neurons, the membranes of glia such as astrocytes and oligodendrocytes are permeable almost exclusively to K+.

Glial cells play several essential roles in neuronal function. Schwann cells form the well-studied myelin sheaths around large axons of peripheral nerves, enabling faster propagation of action potentials along nerve fibers by effectively increas-ing the membrane resistance of the fibers. The end-feet of astrocytic cells helps form the blood-brain barrier, which lim-its what substances cross over from the vasculature into the brain. Glial cells are also involved in guiding axons to their targets during neuronal development and during regeneration of nerve fibers after injury.

One particularly intriguing function of glial cells related to their unique membrane properties is the uptake of excess extracellular K+ by astrocytes. When neurons fire repeatedly, K+ accumulates in the extracellular space. Pumps and trans-porters in the neighboring astrocytes take up the excess K+ and store it to protect neurons from the depolarization that could result from the increase in extracellular K+ concentra-tion. What happens next to the excess K+ taken up? Astrocytes are connected to each other by electrical synapses—essen-tially cytoplasmic bridges between neighboring cells, form-ing sheets of physically connected cells. As a result, the K+ taken up by astrocytes in one area is shuttled to neighboring astrocytes through the cytoplasmic bridges to draw it away from areas of high extracellular K+. It has been discovered that astrocytes lining membranes around the blood vessels have significantly higher K+ channel density than the other cells of this network. The K+ taken up by the astrocytic network is eventually extruded by so called “end-feet” specializations of high K+-channel density directly into the blood vessels. By this mechanism, the high K+ permeability of astrocytes protects neurons from excess depolarization that could result from K+ efflux into the extracellular space.

10.2.6. The Structure of Channel Proteins

A remarkable feature of ion channels is that once open, they promote the diffusion of ions down their concentration gra-dient often with extremely high selectivity and at extremely high rates (tens of millions of ions per second). What essential common structural elements confer ion channels with these properties? As might be expected, a first requirement is the existence of a pore region for passage of ions. The phospho-lipid bilayer is a hostile, low dielectric barrier to the passage of hydrophilic and charged ions. The amino acids of ion chan-nel proteins provide a comfortable conducting hydrophilic pathway across the hydrophobic interior of the membrane. As a result, ion channels are necessarily transmembrane pro-teins with domains that span the membrane. Ion channels are usually constructed from the assembly of several subunits. A second requirement is the existence of a gating mechanism

that regulates the transport of ions across the pore. Channel gating is controlled by external factors (voltage, for voltage-activated channels, ligands for ligand-gated channels). Gat-ing arises from the movements of protein domains that open or occlude the ion permeation pathway. Finally, the channel pores are often ion selective, discriminating between ions of varying sizes and charges to enable the passage of only a sin-gle ionic species.

How nature has evolved to solve these issues can be under-stood by exploring the structure of the K+ channel. The crystal structure of a non-voltage gated, two transmembrane spanning K-channel from the bacteria Streptomyces lividans (KcsA K+ channel) has been solved to 2 Å resolution (Doyle et al., 1998; Zhou et al., 2001). Like its mammalian voltage-gated K+ channel counterparts, the KcsA channel is a tetramer. Each subunit of this tetramer has only two membrane-spanning segments (instead of six for the mammalian channels), but it closely resembles the fifth and sixth transmembrane regions (S5 and S6) of the mammalian voltage-gated K+ channels in its amino acid sequence. The two transmembrane segments in each subunit are α helices, with a peripheral and an inner helix (Figure 10.4D) that run almost in parallel through the membrane. The inner helix, which corresponds to S6 in the well-characterized Shaker potassium channel of Drosophila forms the lining of the inner part of the pore. The four helix pairs are like the support poles for an inverted teepee (with the top inside), widely separated near the outer membrane surface and converging toward a narrow zone near the inner cytoplasmic surface. The inner helices are tilted with respect to the membrane normal by about 25° and are slightly kinked with the wider part facing the outside of the cell allowing the structure to form the pore region near the extracellular sur-face of the membrane. This region contains the K+ channel signature sequence, forming the selectivity filter, which dis-criminates between K+ and Na+ ions. Within the selectivity filter, the orientation of the amino acid side chains preclude their participation in ion coordination, leaving this function to the oxygen atoms of the main chain carbonyls. They form an oxygen ring coordinating dehydrated K+ ions. Roughly in the middle of the membrane is a water-filled cavity, lined by hydrophobic amino acids, and at the bottom of the cavity is another constriction that is likely involved in channel gating (Yellen, 2002).

How does this structure explain the key issues of high ion selectivity, high ion throughput, and gating of K+ channels? Selectivity for K+ over Na+ is explained by the binding of the carbonyl oxygens of the selectivity filter to K+ ions. The spatial geometry of the selectivity filter and its energetics for ion binding is perfectly tuned for K+, the channel’s natural ligand, but not for Na+ and other ions (Yellen, 2001; Zhou et al., 2001). As a consequence, hydrated K+ ions moving into the selectivity filter seamlessly lose their attached water molecules to form bonds with the selectivity filter. For Na+, the pore does not adjust to form good bonding, resulting in poor dehydration and migration of the water-attached Na+


into the selectivity filter. The tuning of the selectivity filter for K+ bonding also allows for its high throughput. New K+ ions entering the selectivity filter from the water-filled cavity expel one or more of the resident ions to the opposite side on the extracellular side and produce net transport. Because the water-filled cavity is hydrophobic, K+ ions in the cavity are less happy and impatient to get into the comfort zone of the selectivity filter, eliciting high throughput rate of ion move-ment. The structural basis for the phenomenon of gating is less well understood and currently the topic of intense discussion. It has been shown that the narrow constriction near the intra-cellular side created by the crossover of the two outer helices and lined by hostile hydrophobic residues, can close the ion permeation pathway and terminate the supply of K+ ions to the selectivity filter (del Camino and Yellen, 2001). This has been proposed to be the primary closing process. Additionally, by adjusting the tuning of K+-bonding and hence the rate of ion throughput, the selectivity filter itself has been proposed to underlie the opening/closing of the channel (Yellen, 2002).

The elucidation of the K+ channel pore structure has pro-vided us with real insight and confirmation of abstract ideas on pores, filters, gates, and ion binding sites. Structures of other channels such as Na+ and Ca2+ channels await elucida-tion. Given the differences in ion selectivity and regulation

between K+ channels and these other channels, many differ-ences are to be expected. However, fundamental similarities between these channels such as their pseudotetrameric sub-unit composition, voltage-dependent gating, and selectivity for specific ions suggest that the lessons gained from examin-ing K+ channel structure are also likely to extend to these and other ion channel families (Hille, 2001).

10.3. Neurotransmitters and Neurotransmission

10.3.1. Classical Neurotransmitters

What types of molecules act as neurotransmitters and how does the process of neurotransmission proceed? Following the identification of acetylcholine (ACh) as a chemical neurotransmitter by Leowi, noradrenaline (or norepinephrine, NE) was identified through the work of Dale, Cannon and oth-ers as the neurotransmitter at many sympathetic neuroeffector junctions. Although ACh and NE are quite different from each other from the chemical point of view, they do share certain key features in terms of the way they act as neurotransmitters. These features are also shared by numerous other substances

Figure 10.4 (A) Topology of a single subunit of a voltage-gated K+ channels. Hydropathy studies predict the presence of 6 transmembrane α-helices. The sequence that spans the pore region is present between the S5 and S6 transmembrane helices. (B) Four identical copies of the K+ channel subunit shown in A assemble together to form the walls of the K+ channel. (C) Cross-section of an open K+ channel, based on the crystal structure of the bacterial K+ channel, KcsA. The selectivity filter, the wide intracellular cavity, and pore helix dipoles are highlighted. (D) A diagram derived from the high-resolution structure of the KcsA channel, showing the cross-over of the inner helices, corresponding to the S6 transmembrane segment of the mammalian K+ channels. The four inner helices produce a narrow opening that provides access to the water-filled cavity in the middle of the membrane protein. (adapted from Yellen, 2002).


that have subsequently been demonstrated to act as neurotrans-mitters, including other biogenic amines (e.g. dopamine and serotonin), amino acids (e.g. glutamate, GABA, and glycine) as well as many peptides (e.g. Substance P, NPY, CGRP, and the endorphins). Because of these shared features an informal consensus has gradually been reached setting out the “rules” for demonstrating that a substance acted as a neurotransmitter at a particular synapse. These rules are something like this:

(1) The potential neurotransmitter substance should be localized in the presynaptic neuron together with the enzymatic machin-ery for its biosynthesis.

(2) The substance should be released by stimulation of the presynaptic nerve. Because transmitter release has been shown to be dependent on the influx of Ca2+ ions through voltage dependent Ca2+ channels located in the nerve terminal (see above), evoked transmitter release should be Ca2+ dependent.

(3) Drugs that block synaptic transmission at a particular synapse should also block the effects of the substance when it is directly applied to the postsynaptic cell.

(4) A mechanism should exist (in addition to free diffusion) for terminating the action of the proposed neurotransmitter. Inhibition of this mechanism should prolong the time course of action of the proposed neurotransmitter.

It is clear that these criteria are fulfilled in the cases of ACh and NE acting as neurotransmitters at different synapses. Let us consider, for example, the effects of ACh at the synapse made by a motor neuron with fast skeletal muscle (“neuro-muscular junction”).

(1) ACh is found to be stored within the terminals of motor neurons. Detailed analysis has demonstrated that ACh is stored within small packages called synaptic vesicles that are concentrated around “active zones” on the presynaptic membrane. These active zones have been identified as specialized sites for neurotransmitter con-taining vesicle release. The enzyme for synthesizing ACh from choline and acetyl-CoA, choline acetyltrans-ferase, is also found within the presynaptic terminal. Choline acetyltransferase is found in the cytoplasm. When ACh is synthesized it is pumped into synaptic vesicles by means of a specific carrier molecule located in the vesicle membrane. Once released, ACh subse-quently diffuses across the synapse and activates nico-tinic ACh receptors localized on the plasma membrane of the postsynaptic muscle cell producing depolariza-tion of the muscle (see below).

(2) Stimulation of the presynaptic nerve results in the release of ACh in a Ca dependent manner. Release of ACh can be demonstrated using a bioassay of the type originally employed by Loewi or more commonly nowadays by a direct chemical method.

(3) Neurotransmission at these synapses can be inhibited by the drug d-tubocurarine, an antagonist of nicotinic ACh recep-tors. Direct application of ACh mimics the effects of nerve

stimulation, e.g. depolarization of the muscle and muscle con-traction. D-tubocurarine also inhibits both of these effects.

(4) The enzyme acetylcholinesterase (AChE) is localized at the synapse and degrades ACh released by the presynaptic nerve, thus limiting its actions. Inhibition of the effects of AChE (e.g. with a cholinesterase inhibitor such as physo-stigmine) prolongs the time course of action of ACh or of stimulation of the presynaptic nerve.

These observations are clearly consistent with the view that ACh acts as a chemical neurotransmitter at these synapses, indeed, they furnish the necessary “proof” of this proposed hypothesis.

Similarly, if we consider the effects of NE at noradrenergic synapses (Figure 10.5).

(1) As with ACh, NE can be shown to be stored within vesicles localized to the presynaptic terminal. The enzymes responsi-ble for NE biosynthesis are also found within the presynaptic terminal. The synthesis of NE is more complicated than the single step involved in ACh synthesis. In the case of NE an entire biosynthetic pathway exists with the initial step cata-lyzed by the enzyme tyrosine hydroxylase (TH) being rate limiting (Figure 10.5). TH activity can be precisely regulated at several levels (see legend to Figure 10.5). For example, it is subject to feedback inhibition by its products such as the cat-echolamines dopamine and NE. Following its biosynthesis, NE is pumped into synaptic vesicles using a specific pump localized in the vesicle membrane.

(2) Stimulation of NE containing nerves is associated with the Ca2+ dependent release of NE.

(3) Neurotransmission resulting from NE release can be blocked by a variety of drugs that block adrenergic receptors. These may be α or β blockers depending on the situation. Applica-tion of NE mimics the effects of nerve stimulation and these effects can also be inhibited by the same drugs. For exam-ple, vasodilation produced by stimulation of sympathetic innervation of blood vessels can be blocked by a blocker of α adrenergic receptors such as phentolamine.

(4) Rather than being metabolized directly like ACh, the synap-tic terminals of noradrenergic nerves express a high affinity uptake system for NE. Following its postsynaptic actions, NE is retaken up into the nerve terminals from which it was released via this high affinity uptake system and can be repackaged into synaptic vesicles. Drugs that block the high affinity presynaptic uptake system (e.g. cocaine and the tricy-clic antidepressant amitryptaline) enhance the effects of pre-synaptic nerve stimulation or of exogenously applied NE.

(5) The properties of classical neurotransmitters, as described for ACh and NE, can be applied with some variations to numerous other substances which have subsequently been determined to act as neurotransmitters. For exam-ple, in the case of amino acid transmitters such as GABA or glutamate, we would note that much of the released neurotransmitter is cleared from synapses by high affinity uptake into glial cells (astrocytes), in addition to nerve ter-minals. Peptide neurotransmitters, such as one of the endor-


phins, are synthesized in the soma of the neuron as inactive precursor molecules after which they are packaged into synaptic vesicles. These are then transported down the axon to the nerve terminal. The active peptide is cleaved from its precursor by proteases copackaged in the vesicle during its transport from the soma to the terminal. In response to nerve stimulation and Ca2+ influx the contents of the vesicle, including the peptide neurotransmitter are released.

As will be appreciated, in all of these cases neurotransmitters are ultimately packaged into synaptic vesicles. These synaptic vesicles are released from the terminal in response to a rise in intracellular Ca2+. In most instances the source of this Ca2+ is the extracellular medium, and Ca2+ moves into the nerve terminal through voltage sensitive Ca2+ channels. A complex of proteins holds the vesicle in a “primed” state at presynaptic release sites, the active zones discussed above. Some of the proteins involved in this complex are provided by the vesicle (v-SNAREs) and

some by the presynaptic active zone (t-SNAREs). One or more proteins, in this complex senses the rise in Ca2+ and vesicle fusion and transmitter release ensues. The fact that neurotrans-mitters are stored within synaptic vesicles provides an anatomi-cal basis for the properties of neurotransmitter release when recorded electrophysiologically. In particular, in electrophysi-ological recordings, transmitters are observed to be released in discrete packages or “quanta”. The evoked release of these quanta is dependent on Ca2+ influx via voltage dependent Ca2+ channels situated in close juxtaposition to the active zones, exactly where transmitter-containing vesicles are docked.

10.3.2. Novel Neurotransmitters

Although these traditional views of neurotransmitter function cover the properties of many neurotransmitters, it is now clear that numerous substances that act as neurotransmitters do not

Figure 10.5. (a) The enzyme choline acetyltransferase catalyzes the synthesis of acetylcholine (ACh) from Acetyl-CoA and choline. (b) Diagram illustrates the different phases of ACh synthesis, release and degradation by a cholinergic neuron. (c) Biosynthetic pathway for catecholamines such as norepinephrine (NE) in a noradrenergic neuron. Note that the rate of biosynthesis of a catecholamine transmitter can be regulated at three levels. First, feedback inhibition of the rate limiting enzyme tyrosine hydroxylase (TH) by NE, adrenaline (E) or dopamine (DA). Second, phosphorylation of TH by second messenger regulated kinases and finally, at the protein level of TH by transcriptional control of mRNA transcription. (d) Diagram illustrates the different phases of NE synthesis, release and uptake in a typical noradrenergic neuron.


fit easily into this description. Consider, for example, the endocannabinoids (Figure 10.6). Neurotransmitters in this class (e.g. anandamide and 2-arachidonoylglycerol, 2-AG) are lipid molecules derived from arachidonic acid which act as endogenous activators of the same receptors as the drug cannabis, the CB1 and CB2 cannabinoid receptors (Piomelli 2003). These transmitters show several features that differ from the above model. First, endocannabinoids are usually synthesized and released from the postsynaptic rather than the presynaptic cell. Secondly, although their synthesis is Ca2+ dependent, endocannabinoids are not stored in synaptic vesicles.

Following their synthesis endocannabinoids leave their cell of origin by a diffusion driven mechanism, rather than as discrete quanta. Endocannabinoids are frequently synthe-sized postsynaptically and then diffuse across the synapse to

produce effects on the presynaptic neuron. Thus, in this case neurotransmission may be viewed as occurring in the reverse direction! Furthermore, consider a molecule like NO, which is a gas and so also diffuses very easily. Here again its syn-thesis is dependent on an increase in Ca2+. Once synthesized, however, NO can leave the cell by diffusion and enter target cells the same way. As in the case of endocannabinoids the information carried by NO may well travel backwards across the synapse, influencing presynaptic functions following its postsynaptic release. Both the endocannabinoids and NO are clearly neurotransmitters. They are responsible for the activity dependent transfer of information across synapses. However, their modus operandi is not at all traditional. Indeed, we could now list of whole host of molecules that can be released from neurons following electrical stimulation and which can then transfer information across synapses in the forward or back-

Figure 10.6. The biosynthesis of endocannabinoids. Biosynthetic pathways for the biosynthesis of the major endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG). Note that the enzymes N-acyltransferase in anandamide biosynthesis. The biosynthesis of 2-AG can proceed via two different routes and is also dependent on an increase in Ca2+. Figures reprinted from Piomelli (2003) with permission from Nature Publishing Group.


ward direction. These include a variety of growth factors and cytokines. Given the wide range of molecules that can appar-ently act as neurotransmitters, it is also true that information transfer across synapses can vary over a vast time scale rang-ing from very rapid electrical events taking a few milliseconds to much longer events taking hours/days and which involve changes in protein synthesis or gene transcription. As we shall now discuss, the type of information involved will depend on the receptor mechanism employed to decode the action of the neurotransmitter. Neurotransmitter receptors are expressed by neurons, glia and microglia and will be involved in decoding information transfer between all of these cell types.

10.4. Neurotransmitter Receptors

Once a neurotransmitter is released it will diffuse across the syn-aptic gap and interact with its target cell. The type of information “transmitted” to the target cell will depend both on the nature of the neurotransmitter and its receptor. As discussed above, this information transfer can be very rapid (perhaps a few millisec-onds) or relatively slow (hundreds of milliseconds or even lon-ger). Sometimes a single neurotransmitter can produce multiple types of signals if it can activate more than one type of receptor. Indeed, more than one receptor for a particular transmitter can be expressed simultaneously by the same target cell. If we our consider our archetypal neurotransmitter ACh for example, its actions can be very rapid due to activation of the nicotinic class of ACh receptors, or its actions can be somewhat slower, due to activation of muscarinic ACh receptors. The effects of ACh typ-ify the two major classes of neurotransmitter receptors-ligand gated ion channels (ionotropic receptors) that mediate rapid syn-aptic transmission, and G-protein coupled receptors (GPCRs, sometimes also called metabotropic receptors) that mediate slower synaptic transmission. Let us consider the structures of these receptors and how they function in neurotransmission.

10.4.1. Ligand-Gated Ion Channels (Ionotropic Receptors)

Ionotropic neurotransmitter receptors are a family of ligand gated ion channels. Normally, these channels exist in the cell membrane in a closed state. However, upon binding the appropri-ate neurotransmitter they open transiently and ions permeate the channel. This results in a redistribution of ions across the plasma membrane of the cell and a change in the membrane potential. This change in potential is the “signal” that can then be propa-gated by the target cell. A change in membrane potential of this type is a very rapid event taking only a few milliseconds.

In the case of the nicotinic ACh receptors at the neuromus-cular junction, the structure of the receptor has been extensively studied and the structural basis for its properties is fairly well established (Figure 10.7). Nicotinic receptors are made up of a pentamer of subunits, which surround a central channel region that spans the membrane. X-ray crystallographic and electron

microscopic studies have revealed the overall structure of the receptor. The majority of its mass exists extracellularly in the form of a “vestibule” in which ions congregate. The receptor then enters the cell membrane as a channel or “pore” region, which narrows to highly restricted area in the middle of the membrane. Less of the mass of the receptor is found on the

Figure 10.7. The structure of nicotinic ACh receptors. Receptors are pentamers of 5 related subunits (a) which form a barrel like array in which the transmembrane region of each subunit (TM2) surrounds a central channel or pore region (b) electron micrographs have demon-strated that most of the mass of the channel is located extracellularly (c) The outside and inside of the channel pore region is surrounded by rings of negatively charged amino acids. These rings of negative charges will repel anions and attract cations resulting is a channel with cationic selectivity.


intracellular (cytoplasmic) side of the membrane. Intracellu-larly, receptors such as nicotinic receptors are often seen to be associated with cytoskeletal proteins that serve to localize them to the appropriate portion of the cell membrane, particu-larly opposite active vesicle release zones in the presynaptic terminal. The 5 subunits that make up the nicotinic receptor are all related transmembrane glycoproteins with molecular weights of around 25 KDa. Each of the subunits is a trans-membrane protein that crosses the membrane 4 times (trans-membrane regions 1–4; TM1-4), so that their N and C termini are both extracellular. The five subunits are arranged like a barrel in which the central ion channel or pore is created by one TM2 region supplied by each subunit. The five subunits that make up the receptor consist of two α subunits, one β subunit, one γ subunit and either a δ or ε subunit. Although these are all proteins of closely related sequence, only the α subunits have binding sites for ACh. ACh molecules bind cooperatively to the receptor producing a conformational change and increasing the probability of channel opening or “gating.” When two ACh molecules bind there is a high prob-ability of channel opening. Once open, the channel is perme-able to cations, which permeate the pore region moving down their electrochemical gradients. As discussed above, at normal resting potentials Na moves into the cell and K moves out. The net result of this ionic flux is depolarization of the cell as the membrane potential moves toward the reversal potential of the nicotinic receptor channel-i.e. 0 mV. Sufficient depolariza-tion of the postsynaptic cell will trigger an action potential. If receptors are exposed to ACh for longer periods of time they change conformation once again to a form a “desensi-tized” state, in which ACh remains bound to the receptor but the channel is closed. The selectivity of nicotinic receptors for cations over anions is engendered by an interesting structural motif. As can be seen in Figure 10.7, both the extra and intra-cellular regions of the receptor just outside the cell membrane are surrounded by a ring of amino acids with a net negative charge. Hence any cation will be attracted into the channel whereas any anion will be repelled. Nicotinic receptors in other parts of the nervous system, including the sympathetic ganglia and the brain, are also thought to be composed of pen-tameric arrays of subunits. These subunits are members of an extended gene family of nicotinic receptor subunits that can associate in multiple different combinations of α and β sub-units (Gotti and Clementi, 2004). For example, in the brain many nicotinic receptors may exist as pentamers of the α4β2 combination. Indeed, some receptors e.g. α7 exist as a homo-meric array of the same subunits. The basic properties of all of these nicotinic receptors are the same but some of the details differ. For example, in addition to being permeable to Na+ and K+, some receptors (e.g. α7 pentamers) are also highly perme-able to Ca2+. Given the importance of Ca2+ as an intracellular second messenger, this may have important signaling conse-quences.

The pentameric arrangement of subunits used to construct nicotinic receptors is also utilized to make up other types of

ligand gated ion channels including the GABA-A, glycine and 5-HT3 receptors. Here again the subunits used are mem-bers of the same extended gene family of proteins as those that make up nicotinic receptors, and the basic structure of the receptor as well as its mechanism of action are similar. Interestingly, the GABA-A and glycine receptors are anion (chloride and bicarbonate) rather than cation permeable chan-nels, a property based on the same structural motif that makes the nicotinic receptors cation selective. However, in this case the channels possess rings of positive rather than negative amino acids. GABA-A receptors are highly important as they mediate the rapid effects of GABA at most of the synapses in the brain. Furthermore, these receptors are also the targets for many pharmacologically significant substances includ-ing anxiolytic and hypnotic drugs. As in the case of nicotinic receptors the precise subunit composition and properties of GABA-A receptors vary throughout the neuraxis depending on the precise combination of subunits utilized. The ability to target drugs to these various subclasses or receptors allows for the production of agents with selective anxiolytic or hypnotic properties, for example.

Another important family of ligand gated ion channels is the receptors for the excitatory amino acid neurotransmitters such as glutamate and aspartate (Figure 10.8). These act as neurotransmitters at the vast majority of excitatory synapses in the brain. These receptors are also multisubunit ion channel arrays, in this case consisting of tetramers of related subunits (Wollmuth and Sobolevsky, 2004). The proteins that make up these subunits are all related in structure but are quite different from those that make up the nicotinic and GABA-A receptor family.

In the case of glutamate receptors the basic structure of each subunit is a protein that crosses the membrane 3 times. The TM2 or P region, which again forms the pore of the channel, does not cross the membrane entirely and forms a loop like structure that folds back into the membrane. Indeed, the basic structure of this pore region bears similarities to that described above for K+ channels. The overall structure of each subunit consists of a large N-terminal extracellular region attached to a transmembrane region that forms the channel. It is interesting to note that the large extracellular region is related in structure to ancient bacterial periplasmic amino acid binding proteins. Similar extracellular motifs also make up the N-terminal of metabotropic glutamate receptors, although in this case they are attached to a 7 transmembrane core rather than an ion channel motif (Jingami et al., 2003 Figure 10.8). In the case of the ionotropic glutamate receptors, binding of glutamate to the “clam shell” region in the extra-cellular portion of the receptor closes the clamshell, initiating a conformational change that eventually results in channel gating. There are basically 3 types of glutamate receptors, named for archetypal agonists that activate each class—these being AMPA, kainate and NMDA receptors. The subunits that make up these receptors (GluR subunits) are all related and form an extended gene family. Although their basic structures


and gating mechanisms are similar, there are some interest-ing details that distinguish each type. The AMPA receptors are made up of tetrameric arrays of the GluR 1–4 (or A-D) subunits. These receptors are basically cation selective chan-nels that are relatively impermeable to Ca. However, it was demonstrated that the structure of the channel as coded in the genome was slightly different. The originally coded GluR2 subunit possessed a single glutamine residue in the TM2/P loop region. This is the region of the channel that makes up the cation permeable channel pore. In contrast to the sequence coded in the genome, the subunit normally found to exist in functional AMPA receptors expressed in cells has an arginine residue in exactly the same position. The triplet code for Gln is CAG. It was shown that neurons express a highly specific adenine deaminase that “edits” this triplet to produce C-Ino-sine-G, which is then read by the protein synthetic machinery as CGG or arginine. It was observed that Gln or Arg contain-ing channels differed in their Ca2+ permeabilities, the extra positive charge associated with Arg repelling Ca2+ ions and making the channel relatively Ca2+ impermeable. It is pos-sible, however, that in some cases editing of GluR2 may be incomplete or that some tetrameric AMPA receptors may not include an edited GluR2 subunit and are therefore Ca2+ per-meable. Thus, a single base change produced by RNA editing can completely change an important property of these AMPA receptors, i.e their permeability to Ca2+.

In the case of NMDA receptors Ca2+ permeability is also an important issue and is a key to understanding certain forms of glutamate mediated synaptic plasticity (see below). These properties of NMDA receptors can be readily observed through

a consideration of their electrophysiological properties (Figure 10.9). Let us compare the properties of AMPA and NMDA receptors. When recorded in physiological solutions, currents carried via AMPA receptors display current/ voltage (I/V) relationships that strictly follow Ohm’s law. On the other hand the I/V relationships for NMDA receptors exhibit a region of “negative slope conductance” similar to that displayed by voltage dependent channels, such as voltage sensitive Na+ or Ca2+ channels (see above). An important discovery was that this behavior was not due to the intrinsic gating properties of the NMDA receptor protein itself. It was observed that if Mg2+ was removed from the physiological bathing solution in which the measurements were made, currents carried by NMDA receptors behaved in a strictly Ohmic manner just like the related AMPA receptors. Further investigations revealed that NMDA receptors can be blocked by Mg2+ at hyperpolar-ized membrane potentials. As the cell depolarizes the block by Mg2+ is relieved. This behavior means that glutamate will only activate an NMDA mediated current when the cell is relatively depolarized. This has important implications for the mechanisms underlying some types of synaptic plasticity (see below).

10.4.2. G-Protein-Coupled Receptors/GPCRs

In addition to ligand gated ion channels neurotransmitters often activate GPCRs. These receptors are all based on a structural motif in which the receptor protein crosses the membrane 7 times in a serpentine like manner so that that N-terminal is extracellular and the C-terminal is intracellular. This arrange-

Figure 10.8. The structures of ionotropic and metabotropic glutamate receptors. (a) Family tree of related protein subunits that constitute different types of ionotropic glutamate receptors. (b) Typical domain structure of a glutamate receptor subunit. The extracellular regions show homologies to bacterial periplasmic binding proteins. (c) Structure of a metabotropic glutamate receptor. In this case the extracellular region also bears homology to a bacterial periplasmic binding protein, but this time it is attached to a 7-transmembrane G protein coupled receptor motif. Metabotropic glutamate receptors probably function as dimmers or other higher order arrays.


ment results in the formation of three intracellular loops between the different transmembrane regions of the protein. These loops can be used to transduce signals to the inside of the cell and, in particular, to heterotrimeric G-proteins. GPCRs constitute an extremely large gene family and include many variations on the same basic structural motif (Figure 10.10). However, in virtually every instance the mechanism of action is basically the same. Binding of the agonist produces a con-formational change in the receptor that leads to activation of the G-protein. G-protein activation results in its dissociation into α or βγ subunits-either of which can then mediate sig-nal transduction within the cell. In the classical description of GPCR function the downstream signal is typically produced through the regulation of an enzyme like adenylate cyclase or phospholipase C. Activation of these enzymes produce second messenger molecule such as cAMP, diacylglycerol (DAG) or Ca2+ that then activate protein kinases or other effectors. For example, phosphorylation of ion channels by activated kinases changes their gating properties and so transduces the message to the level of electrical signaling. However, this classical model is only one of many possible alternatives. The subunits of the G-protein may bypass the kinase activation step and interact directly with the channel protein, resulting in direct channel gating. For example, direct interaction of G-protein βγ subunits with K+ channels or N-type Ca2+ channels can

Figure 10.9. Biophysical properties of the N-methyl-D-aspartic acid receptor. The current voltage (i/v) relationship measured in physi-ological solutions shows a region of “negative slope conductance” at hyperpolarized potentials. Little inward current is observed until the cell is substantially depolarized. However, when Mg2+ is removed from the bathing medium the i/v relationship follows Ohm’s law and is represented by a straight line. The reason for this behavior is that Mg2+ block NMDA receptors in a voltage dependent manner (see main text).

Figure 10.10. G-protein coupled receptor (GPCR) structure and function. (a) The basic 7-transmembrane structure of a GPCR is common to all members of the family. (b) The cycle of heterotrimeric G-protein activation. The cycle is initiated by stimulation of GTP/GDP exchange on the G-protein alpha subunit produced by an agonist induced conformational change in the GPCR. (c) Activation of a GPCR also produces interactions with proteins of the β-arrestin family that mediate uncoupling of the GPCR from its G-protein and receptor internalization. (d) Arrestins can also act as scaffold proteins that bring together members of the MAP kinase pathway and so activate MAP kinase signaling.


directly modulate channel function by activating K+ channels or inhibiting N-channels. Such effects can directly modulate neurotransmitter release at nerve terminals. Although such a model is generally applicable to the activation of GPCRs, in the case of certain GPCRs that are important in the nervous system the basis for signal transduction is not entirely clear and may not even involve a traditional G-protein. Thus, for GPCRs of the frizzled or smoothened families, which are involved in the important Wnt and hedgehog signaling path-ways, the precise mechanisms of signal transduction are not known and the role of G-proteins is unclear. Thus, as we shall discuss below, activation of a heterotrimeric G-protein may be only one way in which a GPCR can ultimately transduce information.

How does a GPCR normally work from the molecular point of view? In order to answer this question we should first con-sider how G-proteins produce their effects (Figure 10.10). For heterotrimeric G proteins the α-subunit has intrinsic GTPase activity. Normally GDP is bound to this subunit. In order to initiate a cycle of G-protein mediated signaling this GDP moi-ety must be replaced by a GTP molecule. Binding of GTP results in dissociation of the G-protein into α and β subunits and signaling ensues. Once the GTP has been hydrolyzed the heterotrimer reforms and signaling ceases. In order for these events to be carried out two proteins are important in addition to the G-protein. The first of these is a GEF or “guanyl nucleo-tide exchange factor”. This protein is responsible for stimulat-ing the initial exchange of GTP for GDP. Because the intrinsic GTPase activity of most α-subunits is low a second protein called a GAP (GTPase activating protein) is required. This protein acts upon the GTP bound α–subunit to enhance its GTPase activity and so allow the cycle of G-protein mediated signaling to terminate. In this context the GPCR can be seen to act as a GEF. Under resting conditions the heterotrimeric G-protein is bound to the intracellular loops of the receptor. Binding of the agonist produces a conformational changes such that the receptor now stimulates exchange of GDP by GTP bound to the α-subunit and initiates signaling. Thus, the GPCR is an agonist activated GEF. The arrangement of GPCR and heterotrimeric G-protein is a special case of G-protein mediated signaling. In other cases “small” G-proteins such as Ras, which carry out numerous cellular signaling functions, are not heterotrimers but also function in GTP regulated man-ner. In this case other proteins have GEF like activity to pro-mote guanyl nucleotide exchange.

Signaling mediated by GPCRs has been shown to be a very information rich event which is much more complex than ini-tially supposed. In order to understand this we should consider the entire sequence of events that takes place when a GPCR is activated. Here again there are many variations on the basic theme, but it appears that there are other signaling pathways that are activated in addition to G-proteins. Binding of the agonist to the receptor initiates conformational changes in the intracellular C terminal region. This allows diverse residues in this region to be phosphorylated by kinases of the GRK (GPCR

kinase) family. Phosphorylation of these residues has numerous effects. First, the interaction of the receptor and G-protein is interrupted. Secondly, the phosphorylation of the GPCR allows it to interact with proteins of the β-arrestin family (Lefkowitz and Shenoy, 2005 and Figure 10.10). This interaction was ini-tially shown to allow the GPCR to be relocated to a region of the membrane (coated pit) resulting in receptor endocytosis. When internalized into the cell in this way the receptor may be dephosphorylated and recycled back to the cell surface or else it can be degraded in the lysozomes. As will be obvious all of these actions will result in interference of the basic signaling functions of the receptor by uncoupling it from the G-protein and/or removing it from the surface. This acts as a negative feedback loop controlling the extent of GPCR signaling in the face of continuous receptor stimulation. However, it has subse-quently been demonstrated that β-arrestin like molecules have a very large number of additional receptor related functions. For example, β-arrestins can act as scaffolding proteins for the intermediates of the MAP kinase signaling cascade. Thus, once activated by a GPCRs arrestins might bind MAP kinases allow-ing for their mutual phosphorylation and activation. The overall effect of this is to inhibit the initial signaling of the GPCR and to redirect it down the MAP kinase pathway. Indeed, it has been frequently observed that activation of a GPCR can produce activation of ERK or other MAP kinases (Figure 10.10). Other studies have linked GPCR/β-arrestin interactions to effects on the ubiquitination/proteasome pathway. Thus, the diversity of signaling initiated by a GPCR can potentially be very great and can operate over a wide time course. Rapid signaling events can influence the activity of ion channels, electrical excitability and synaptic communication, whereas longer term effects can influ-ence processes such as neuronal gene transcription possibly leading to changes in neuronal structure or phenotype.

A further recent insight into the mechanism of action of GPCRs is that they often act as dimers or other higher order arrays (Milligan, 2004). Homo and heterodimerization of GPCRs has now been frequently reported. These receptor dimers may have properties, including agonist selectivity and signaling, that are unique and that differ from those of receptor monomers. Thus, the overall impact of signaling by GPCRs may be very diverse.

10.5. Synaptic Plasticity

The strength of synaptic communication is not constant. Moment to moment changes in the strength of synaptic transmission underlie the ongoing requirements of neuronal communication and are probably the basis of long lasting phenomena such as learning and memory. We now understand that there are numerous forms of this synaptic “plasticity”. Some forms of plasticity last for brief periods of time whereas others last for many hours or days-perhaps encompassing the lifetime of the organism. In each case changes in the amount of transmitter released, postsynaptic sensitivity to the trans-mitter or combinations of these processes are involved in


producing these effects. In order to give a general idea as to what these important and widespread neuronal signaling processes involve, we shall discuss two different examples of synaptic plasticity.

10.5.1. Long-Term Potentiation (LTP)

Long-term potentiation and its flip side, long-term depres-sion (LTD), are probably the most intensively studied of all forms of synaptic plasticity (Collingridge et al., 2004 and Figure 10.11). LTP was first observed in vivo using rabbits by Bliss and Lomo who demonstrated that a brief high frequency stimulation of the perforant path input to the dentate gyrus of the hippocampus from the entorhinal cortex produced a long lasting (many hours) potentiation of the extracellularly recorded field potential. This phenom-enon, or something akin to it, has now been shown to exist at other hippocampal synapses and at many other synapses throughout the brain. Subsequent investigations both in vivo and using hippocampal slice preparations revealed LTP to have some interesting properties. For example, if a neuron receives both a weak and a strong input, in which the weak input is not sufficient to produce potentiation, the weak input can be potentiated if it is paired in time with a tetanic

stimulation of the strong input. This property of “associa-tivity” was seen to be a neurophysiological correlate of the proposal made by Canadian psychologist Donald Hebb that coincident activity between two synaptically coupled neu-rons would cause increases in the synaptic strength between them-a postulate that was made to explain how long-term phenomena such as memory could be represented synapti-cally. From the point of view of the present discussion the mechanism underlying LTP at many excitatory synapses has been shown to depend on the biophysical properties of NMDA receptors as discussed above. Indeed, the induction of LTP at numerous synapses has been shown to depend both on NMDA receptor activation and the associated influx of Ca2+ into the postsynaptic neuron. It will be recalled that the function of NMDA receptors is critically depen-dent on block by physiological concentrations of Mg2+. Accordingly, when synaptic activity is low and the cell maintains a relatively high resting potential NMDA receptors are blocked. At low rates of presynaptic stimulation the synaptic release of glutamate will result in activation of postsynaptic AMPA receptors. Although AMPA receptor acti-vation will result in some postsynaptic depolarization, this will not be sufficient to relieve the Mg2+ block of postsynaptic NMDA receptors, and so no NMDA receptor associated

Figure 10.11. Examples of receptor mediated synaptic plasticity. (a) Long-term potentiation. (LTP). Synaptic transmission at hippocampal synapses (in this example the CA3/CA1 synapse) is potentiated following application of a tetanus (period of rapid stimulation) to the pre-synaptic nerve. This phenomenon is dependent on the activation of synaptic AMPA and NMDA receptors (see main text). (b) Depolarization suppression of inhibition (DSI). Diagram shows how endocannabinoids produce DSI in the hippocampus. Synaptic potentials recorded as hippocampal neurons are depressed following a tetanus.


current will result. Now consider the situation when a tetanus is applied to the presynaptic nerve resulting in increased glutamate release. The degree of postsynaptic depolariza-tion produced by AMPA receptor stimulation may now sufficient to relieve the block of NMDA receptors by Mg2+. Once Mg2+ leaves the receptor and the receptor is unblocked, its activation by glutamate will not only result in the influx of Na+ but also that of Ca2+. Subsequent stud-ies have shown that it is this influx of Ca2+ that acts as a second messenger stimulating the changes responsible for maintained increases in synaptic function, primar-ily the insertion of new AMPA receptor subunits into the postsynaptic cell membrane and hence an increase in the strength of glutamate mediated signaling. There are numer-ous variations on this basic model including observations that, depending on the precise pattern of pairing of synaptic inputs, resulting synaptic transmission may be decreased rather than increased, a phenomenon known as Long-Term Depression (LTD). Thus, synaptic strength at central gluta-mate synapses can be tuned up or down depending on the overall requirements of the synapse in question, a process dependent on glutamate receptor mediated signaling.

10.5.2. Depolarization-Induced Suppression of Inhibition (DSI)

It has been shown that when the pyramidal neurons of the CA1 field of the hippocampus are depolarized, the inhibitory GABA mediated input to these cells is transiently suppressed (Figure 10.11). This phenomenon is known as “depolarization induced suppression of inhibition” (DS1, Diana and Marty, 2004). As with LTP/LTD described above, phenomena of this type occur widely throughout the nervous system. The mecha-nism of this effect and its dependence on receptor signaling is interesting. It has been shown that DSI is dependent on the influx of Ca2+ into the postsynaptic cell (e.g. the pyramidal neuron in this example). However, this results in a reduction of GABA release presynaptically. This means the signal that is responsible for this effect must be transmitted backwards across the synapse. It will be recognized that this is some-thing that applies to endocannabinoid signaling in the brain. Indeed, it has been shown that DS1 can be inhibited by block-ers of CB1 cannabinoid receptors (Piomelli, 2003). Thus, the mechanism involved appears to be as follows. Influx of Ca2+ into the postsynaptic cell as a result of postsynaptic depolar-ization, activates endocannabinoid synthesis. The endocan-nabinoid molecules then leave the cell and diffuse back across the synapse where they bind to CB1 receptors situated on pre-synaptic terminals. Activation of presynaptic CB1 receptors produces activation of heterotrimeric G-proteins. Binding of the G-protein βγ subunits to voltage dependent Ca2+ channels in the nerve terminal, inhibits these channels. Thus, less Ca2+ enters the terminal in response to an action potential and less transmitter is released. The metabolism of the endocannabi-noid results in the transient nature of the phenomenon.

Summary

Neurons communicate with each other, as well as with glial cells, through the propagation of action potentials and the release of chemical neurotransmitters across synapses. In this chapter we discuss the molecular processes that are responsible for the electrical excitability of neurons that allows the genera-tion of action potentials as well as the electrical properties of glia cells. The nature of the resting membrane potential in cells and the structure and function of voltage dependent Na, K and Ca channels are discussed. Many different types of substances can act as neurotransmitters. The first neurotransmitter to be discovered was acetylcholine, but now many other substances including biogenic amines and peptides are also known to be neurotransmitters. Conventional neurotransmitters carry infor-mation across synapses in an anterograde manner, although some recently defined neurotransmitters such as the endocan-nabinoids carry information in a retrograde fashion. Neurotrans-mitters act on different types of receptors. The main classes of receptors are ligand gated ion channels, such as the nicotinic acetylcholine receptor and G-protein coupled receptors, which include the majority of receptors for biogenic amines. Activa-tion of ligand gated ion channels results in a rapid change in the membrane potential, whereas activation of a G-protein coupled receptor can result in numerous changes in ion gradients and second messenger systems. Neurotransmission is not a unvary-ing process and the strength of synaptic communication can vary on a moment to moment basis – a process known as syn-aptic plasticity. Different types of synaptic plasticity exist and may involve the participation of receptors for glutamate or for endocannabinoids for example.

Acknowledgment. The authors thank William Wassom, Graphic Designer at the University of Nebraska Medical Cen-ter for his assistance with the figures.

Review Questions/Problems

1. The undershoot of an action potential occurs from

a. Prolonged opening of voltage-activated Na+ channelsb. Opening of voltage-activated K+ channelsc. Inactivation of Na+ channelsd. All of the above

2. The resting membrane potential is largely influenced by

a. K+ channelsb. Na+ channelsc. none of the aboved. all of the above

3. What is a “selectivity filter” of an ion channel? Do all channels contain a selectivity filter?


4. How many pore-forming K+ channel subunits assem-ble to form a functional channel?

a. One b. Two c. Four d. None of the above

5. Voltage-gated Ca2+ channels mediate

a. Fusion of neurotransmitter-filled vesicles in the pre-synaptic terminals

b. Repolarization of the action potential c. Generation of the resting membrane potential d. All of the above

6. Name four properties that you would expect a chemi-cal substance to possess if it functions as a neurotrans-mitter at a synapse you are investigating.

7. What is an “endocannabinoid”? What evidence exists that these substances act as neurotransmitters?

8. How many protein subunits does a nicotinic acetyl-choline receptor possess?

a. More than one b. Lots c. Five d. Not sure, but definitely around 5 (…..or perhaps 6)

9. How do ionotropic glutamate receptors regulate the movement of Na and Ca ions into neurons? Why is Ca influx through glutamate receptor channels impor-tant?

10. What is the basic structure of a G-protein coupled receptor? How does agonist binding to the receptor lead to the activation of heterotrimeric G-proteins?

11. Which of the following apply to the arrestin class of proteins?

a. They are scaffold proteins that can bind members of the MAP kinase pathway.

b. They can sometimes act as receptors for opioid peptides. c. They are involved in down regulating the effects of

activation of GPCRs d. They act as blockers of ionotropic glutamate receptors,

or something like that.

References

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