5 Chapter 5: Signaling between Neurons

Austin Lim, PhD (DePaul University)Editor: Monica Javidnia, PhD (University of Rochester)

 

 

Previously, we described the electrical properties of a single neuron. A  lone  neuron can send action potentials as a means of communication, but cells become much more interesting when they have partners to talk to. The nervous system of the worm C. elegans is only 300 neurons, and yet it is complex enough to engage in moderately intricate behaviors like responding to repellant or attractant odors, social feeding, and long-term learning. The human brain, with its 86 billion neurons, can engage in these behaviors and so many more – only because of communication between the different neurons in the brain.

In this chapter, we will focus on the molecular-level features of communication between neurons, starting from the    anatomical

Chapter 5 outline
differences between synapses.

A lone neuron can send action potentials as a means of communication, but cells become much more interesting when they have partners to talk to. The nervous system of the worm C. elegans is only 300 neurons, and yet it is complex enough to engage in moderately intricate behaviors like responding to repellant or attractant odors, social feeding, and long-term learning. The human brain, with its 86 billion neurons, can engage in these behaviors and so many more – only because of communication between the different neurons in the brain.

In this chapter, we will focus on the molecular-level features of communication between neurons, starting from the anatomical differences between synapses.

 

 

 

5.1   Electrical vs. chemical synapses

 

 

The synapse is not a part of the structure of a neuron, but rather the site of close proximity between two communicating neurons.  There  are two main types of synapses: electrical and chemical.

Electrical synapses

Electrical synapses are the  simpler  of  the two types of synapses. In an electrical synapse, the main driver of communication between two neurons is a change in potential, and the carrier of charge is almost always an

Connexon Connexin monomer

Cell membrane

 

 

 

 

 

 

 

 

Extracellular space

Channel

Figure 5.1 An electrical synapse exists between two closely-connected neurons. Cytoplasm passes between the two neurons through a protein complex called a connexon.

 

 

ion. The electrical synapse simply means that the cells share their cytoplasm with an adjacent cell. Electrical synapses are what Camillo Golgi imagined when he proposed the reticular theory of nervous system organization. Oftentimes an entire network of many hundreds of neurons is connected by these synapses.

Imagine two neurons that are connected by an electrical synapse. First of all, both of them are complete cells on their own. Each one contains  a  complete  plasma  membrane  surrounding the neuron, a nucleus, and all the individual organelles needed to carry out that cell’s basic life processes. Electrical synapses share the cytoplasm between the two connected cells, so ions, ATP, and larger signaling molecules and proteins are able to move between the two cells. For this to happen, there exists a specialized physical channel between the two that allows for the passage of cytoplasm called a connexon    or hemichannel. Each hemichannel itself is made up of six transmembrane proteins called connexins (you can remember the difference between the channel and the individual protein because proteins often end with the letters   -in).

When two connexons contact each other,     they

interact closely with each other and form the gap junction, which is the structure that connects neurons electrically.

Neurons that are connected by electrical synapses are remarkably close to each other. The synaptic gap between the two electrically connected neurons is about 3.5 nanometers. Logically, the neurons must be close since the hemichannels are like a physical “bridge” between the two cells.

 

Figure 5.2 Electrical synapses are similar to a skybridge that physically connects the cytoplasm between two neurons.

 

 

Electrical synapses are capable of passing information bidirectionally. This means that a signal does not always move sequentially from the presynaptic cell to the postsynaptic cell. Rather, ions and signaling molecules are free to move through the connexons in either direction. Also, each cell within an electrically- coupled network can  receive  inputs at any of the cells, making it able to detect several signals at once – the same way a huge satellite dish can detect more signals than a small dish. Electrical            synapses                     likely            evolved because  of                     evolutionary  pressures            that selected for speed. These synapses can pass signals as fast as electrical charges can move through an electrolyte-rich fluid like cytoplasm, which is almost instantaneous. Therefore, an escape reflex that is made up of communication across electrical synapses is advantageous for animals that need to escape predators. For example, crayfish exhibit a reflexive abdominal flexion response when exposed to threatening stimuli, causing the animal’s body to dart away from a threat within a fraction of a millisecond. Comparing          across                the  phylogenetic   tree, electrical synapses are often found in less complex    organisms,  including                arthropods such as insects and crustaceans, where such

reflexes are likely more critical for survival.

Another     advantage     of     electrical

 

Clinical connection

Charcot-Marie-Tooth (CMT) disease Charcot-Marie-Tooth (CMT) disease is a rare genetic disorder that damages parts of the peripheral nervous system including the motor nerves, resulting in muscle weakness and difficulty with walking, and the sensory nerves, causing some to experience abnormal sensations such as tingling or pain in their extremities. These symptoms are characteristic of signal transduction failure resulting from deficits in myelin. One type of connexin protein, called Cx32, is heavily expressed in Schwann cells, the glia that produce myelin in the PNS. Mutations in the gene that codes for Cx32 are associated with the X-linked form of CMT disease, and knocking- out the gene in experimental mice cause the mice to express similar symptoms as human CMT.

 

 

 

 

 

 

 

 

 

 

 

Figure 5.3 People with CMT disease often have abnormally shaped feet.

synapses is that they can form a large network

of interconnected neurons with  synchronized

activity. For example, neuroendocrine cells  in the hypothalamus are connected by electrical synapses. When the  “go”  signal  arrives,  all  the cells depolarize at once, which can result    in the massive release of hormones into the bloodstream. A network can also cause sudden, powerful inhibition. Like an angry mob of people chanting,   a   network   of   electrical   synapses

connecting inhibitory interneurons allows the network to send an immediate “shut-down” signal under specific circumstances.

 

Chemical synapses

At a chemical synapse, a signaling molecule is released by the presynaptic cell to influence  the  postsynaptic  cell. These signaling

Presynaptic neuron

 

Synaptic vesicle

 

 

Chemical synapse

 

 

Receptor

 

 

Postsynaptic neuron

Figure 5.4  Chemical synapses are the site of close interaction between two neurons (left) or a motor

neuron and a muscle fiber, which is called the neuromuscular junction (right).

 

molecules, generally called neurotransmitters, are synthesized or stored by neurons. After being released, these neurotransmitters diffuse randomly across the synapse, where they are able to affect nearby neurons once the chemical binds to its corresponding receptor.

Since chemical synapses do not rely on a direct physical protein “tunnel” to connect the two neurons, the distance between the two cells can be much larger. On average, a chemical synapse is a distance of about 20-40 nanometers, roughly a thousand times smaller than the diameter of a human hair.

A chemical synapse can pass a variety   of signals, depending on the neurotransmitter and the receptor. For example,  some  signals are directly excitatory and allow positively charged cations to enter the neuron causing depolarization. Other signals are hyperpolarizing, and therefore inhibitory. And yet other signals are much more complex, inducing changes in protein expression  that  can  modify  cellular excitability

over the course of minutes or hours.

Because of the complexity of the signals that chemical synapses can convey, evolutionary development through time has allowed for a tremendous variety of responses. Chemical synapses allow for fine-tuning of neural networks, giving these nervous systems a larger range of possibilities. The nervous systems of “higher” organisms like humans tend to have several chemical synapses since these signals are likely necessary for complex behaviors and cognition.

Many chemical synapses exist between the axon of one neuron and the dendrite of another neuron. One specific type of chemical synapse refers to the space between a motor neuron and muscle tissue, and this is called the neuromuscular junction, or NMJ. When the chemical signaling molecule acetylcholine (ACh) is released by the presynaptic motor neuron, it is detected by receptors that are expressed on the muscle. The release of ACh causes contraction of the muscle.

 

5.2

Properties of vesicles

Types of vesicles

Molecules of neurotransmitters are often stored in synaptic vesicles before being released. Synaptic vesicles are tiny spheres of lipids just like the cell membrane. These vesicles can be roughly characterized into one of two classes:

 

1. Small vesicles. These vesicles have a diameter of 40 nanometers and a volume of about 30 cubic microns. Given the size of  neurotransmitters, we can estimate at somewhere on the order of thousands to tens of thousands of molecules of neurotransmitter can be stored in each vesicle. Small vesicles store most of the neurotransmitters we most often think of, including glutamate, GABA, dopamine, and norepinephrine, for example. Small vesicles are almost always exclusive found in the axon terminals.

 

2.   Large dense-core vesicles. These vesicles are much larger than small vesicles, with a range of diameter from 100 to 250 nanometers. They store peptides such as dynorphin or enkephalin, which have chemical structures much larger than the other neurotransmitters. Since these peptides are packaged into their vesicles near the nucleus, large dense-core vesicles can be found in the cell bodies and all along the axons in addition to the axon terminal.

 

Loading of vesicles

Vesicles need to be filled with molecules of neurotransmitter before release into the synapse. In small vesicles, filling is only made possible through the action of giant transmembrane proteins called vesicular transporters. These  are  protein  complexes  that  span  the vesicular

 

 

 

 

 

membranes, with one side facing the intracellular space and other facing the inside of the vesicle. Their main function is to take molecules of neurotransmitter from the intracellular space of the axon terminal and pump them into vesicles.

Many of the vesicular transporters are named based on the neurotransmitters that they are capable of recognizing and transporting. Some have a single substrate, such as vesicular GABA transporters (VGAT) which move GABA, vesicular glutamate transporters (VGluT) which move glutamate, and vesicular acetylcholine transporter (VAChT) which moves acetylcholine into  vesicles.  Others  recognize  a  broad  class

of neurotransmitters, such as the vesicular monoamine transporters (VMATs), which are responsible for moving monoamines such as dopamine and serotonin into the vesicles.

Vesicular transporters are able to function because the interior of the vesicle is highly acidic compared to the interior of the cell. Vesicles have a high concentration of H+ ions (protons) because of the action of the transmembrane enzyme vesicular ATP-ase, or v-ATP-ase. These membrane-embedded proteins utilize the molecular energy contained in ATP to concentrate H+ ions in the intravesicular space. For each molecule of ATP used, one proton gets pumped into the vesicle.

Vesicular  transporters  pump  molecules of neurotransmitter against their concentration gradients, which is an energetically difficult task. To have enough energy, the vesicular transporters use the high intravesicular concentration of H+ to move molecules of neurotransmitter across the vesicular membrane. When a proton moves from

an area of high concentration to low concentration, energyisgenerated.Thevesiculartransportersuse this energy to push neurotransmitter in. Because H+ ions move opposite of the neurotransmitter molecules, vesicular transporters are called antiporters. Transporters have slightly different stoichiometries, as it requires two protons to move a single molecule  of  dopamine,  while  the energy from a single proton is sufficient to transport GABA or glutamate.

 

Location of vesicles

Synaptic vesicles can be found in one of three places at the axon terminal.

 

1.  Readily releasable pool (RRP). These vesicles are located close to the cell membrane at the axon terminal. In fact, many of them are already “docked,” meaning that their coat proteins are already interacting closely with the proteins on the inside of the cell membrane. When the depolarizing charge of an action potential reaches

 

 

Fig 5.6 Synaptic vesicles in the axon terminal get filled by the action of two different vesicular transporter proteins. The v-ATP-ase uses energy to pump H+ into the vesicle against it’s concentration gradient (left). Then, a vesicular transporter such as vGluT use the movement of H+ down it’s concentration gradient to increase intravesicular concentration of neurotransmitter (right).

 

 

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the terminal, these vesicles at the RRP are the first ones that fuse with the cell membrane and release their contents.

 

2.    Recycling pool. These vesicles are the ones that have been depleted due to release. They are currently in the process of being refilled or reloaded with neurotransmitter. They are farther from the cell membrane, and the protein machinery is not primed for release, so it requires a more intense stimulation to release the contents of these vesicles.

 

3.   Reserve pool. These vesicles are the farthest from the surface of the cell membrane, and most vesicles are held in this reserve pool. For these neurons to be released, very intense stimulation is required. Reserve pool vesicles may not even be recruited for release under physiological conditions.

Release of vesicles

At a chemical synapse, the process of neurotransmitter release is very tightly regulated. If there were no mechanisms to control the release of chemicals at the synapse, nerve cells would deplete their entire stock of neurotransmitter. The signals that trigger muscle contraction at the NMJ would cause constant muscle tension. All sorts of brain signals would be active, and over-excitation would cause toxicity. Needless to say, regulated control of neurotransmitter release is a normal and essential part of nervous system function.

Regulation of release depends on several proteins that are important parts of the process. These proteins are often embedded within cell membranes of the vesicles or the neuronal membrane.

 

1.  V-SNAREs are the proteins expressed on       vesicles   (v   for   vesicle).   Synaptobrevin

and  synaptotagmin  are  two specific

Fig 5.7 Axon terminals contain hundreds of vesicles roughly divided into three categories.

Axon Terminal Reserve pool

Recycling pool Readily releasable

pool (RRP)

v-SNARE proteins that are involved during synaptic release.

 

2.        T-SNAREs are proteins expressed on the cytoplasmic side of the axon terminal. The inside of the cytoplasm is the “target” for the vesicle (The t in t-SNARE). Syntaxin and synaptosomal nerve-associated protein 25, or SNAP-25 for short, are t-SNAREs that function during vesicular fusion.

 

 

Fig 5.8 v-SNARE proteins interact with t-SNARE proteins to allow for vesicular fusion and release of neurotransmitter.

 

 

 

 

Syntaxin Synaptotagmin SNAP-25

 

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Clinical connection: Botulism

Botulism is a deadly condition that results from exposure to the spores produced by the bacteria Clostridium botulinum. Toxic spores can be found in the soil, contaminated foods, or water. The toxin itself is one of the most potent agents known to man – exposure to concentrations as low as 2ng/kg is lethal. The most common symptoms include  muscle

 

 

Vesicle

 

 

Synaptobrevin Synaptotagmin

Healthy NMJ

 

Acetylcholine

 

 

 

 

 

 

SNAP-25

weakness or paralysis, especially the muscles of the face or the limbs. For about 5% of people who develop botulism, death results from paralysis due to respiratory failure.

Botulinum toxin is known to selectively cleave the proteins that comprise the SNARE complex.  There  are  a  few  specific  types   of botulinum toxin with slightly different intracellular targets, but the result is the same on the molecular level: prevention of vesicular fusion eliminating neurotransmitter signals.

Despite being one of the deadliest toxins so far identified,  millions  of  people  pay to have a preparation of toxin called “Botox” injected into their face. For most, the injection of botox is a cosmetic  procedure  that can reduce the appearance of     wrinkles

Fusing of vesicles

The last step of neurotransmitter release is the fusing of the cell membrane. In order to release their chemical contents into the synapse, vesicles need to fuse with the cell membrane. As the vesicular membrane merges with the interior of the neuronal membrane, the contents of the vesicle become exposed to the extracellular space. Only then are the neurotransmitters capable of activating receptors.

One of the key proteins required for vesicular fusion is the vesicle-embedded V-SNARE    synaptotagmin.   This    protein   is

Botulinum toxin-exposed NMJ

 

 

 

 

 

Botulinum toxin

 

Fig 5.9 Botulinum toxin selectively cleaves vesicular fusion proteins, preventing acetylcholine from being released at the NMJ.

 

by paralyzing the muscles. Botulinum toxin is also used medically for conditions resulting from excessive neurotransmitter release, such as muscle spasms, excessive sweating, or migraine.

 

 

capable of detecting elevated levels of Ca2+ in the axon terminal. As it turns out, an elevation of Ca2+ in the intracellular space is the “go ahead” signal that causes neurotransmitter release.

The concentration of intracellular calcium, generally in the range of 100 nM, is much lower than the concentration outside the cell. Embedded in the cell membrane of  the  axon  terminals  are transmembrane proteins called “voltage- gated calcium channels” or VGCCs. As their name suggests, they function very similarly to the voltage-gated sodium  channels  described in  previous  chapters:  they  are  large    protein

complexes that normally remain closed, but when the surrounding neuronal membrane becomes depolarized, they physically change conformation and open up, allowing ions to move across the cell membrane. These VGCCs selectively pass only Ca2+ ions. The electrochemical gradient causes these Ca2+ ions to enter into the cell.

As the change in membrane potential travels down the length of the axon (the action potential), it causes a depolarization at the terminal, triggering calcium entry via the VGCCs. Ca2+ at the terminal binds with synaptotagmin. The v-SNAREs and the t-SNAREs interact with one another in the presence of Ca2+, forming a molecular structure called a SNARE complex. The SNARE complex looks a lot like two twist ties that are wound tightly together. As they twist

tighter together, it causes the vesicle membrane to approach the inside of the cell membrane, which results in vesicular fusion.

Vesicles are capable of fusing in at   least

two different ways.

1.                Full fusion. A vesicle that undergoes full fusion experiences total exocytosis. The vesicular membrane becomes completely integrated with the cellular membrane, and all of the neurotransmitter spills into the synapse.

2.                                     Kiss-and-run. This method of neurotransmitter release is incomplete fusion. The vesicle only partly connects with the interior surface of the cell membrane, and only a limited number of neurotransmitter molecules are able to enter the synapse via diffusion.

 

 

Fig 5.10 Synaptic vesicles either fuse completely (left) or partially in kiss-and-run (right).

 

5.3   ReceptorsReceptors are proteins that are capable  of sending a signal to change the function or activity of a neuron. Most receptors that function in neurotransmission are large transmembrane proteins. On the extracellular surface is  a specific series of amino acid  residues  called  the active site. The active site, also called the orthosteric site, is shaped to allow molecules of neurotransmitter to bind to the receptor.

 

Fig   5.11   Ionotropic    receptors   (top)   allow   ion movement after receptor binding, while metabotropic

 

 

Receptors are classified into one of    two

main categories.

1.                                Ionotropic    receptors.    Physically, ionotropic receptors are transmembrane proteins with a large-diameter pore through which ions can pass. These channels only open when a molecule of chemical binds to the active site on the extracellular side of the protein. These chemicals are also called ligands, and so ionotropic receptors   are   also   called  ligand-gated

ion channels. Once a neurotransmitter activates the ionotropic receptor, ions    will

receptors (bottom) trigger second messengers to induce signaling.

Neurotransmitter

move based on the electrochemical gradient

for that ion. As a result of ion movement, the cell’s membrane potential will change.   For

Neurotransmitter bound to receptor

 

Ion

released from synapse

example, nicotinic acetylcholine receptors are ligand-gated sodium channels, so when these receptors are activated by a ligand like acetylcholine, sodium ions enter into the cell causing depolarization and excitation.

Ionotropic receptors are able to induce a change in membrane potential very rapidly, on the scale of milliseconds.

Due to the nature of the amino acid residues that make up the pore of ionotropic receptors, they can be very selective for certain ions. For example, negatively charged residues lining the inside of the pore repel negatively charged Cl- ions while allowing positively charged cations to pass through the channel.

2.                  Metabotropic receptors. These receptor complexes cause the cell to change its metabolism in a way that leads to either excitation or inhibition.  Ions  do not pass through these receptors. Instead, metabotropic receptors use the actions of G

proteins, proteins which induce changes in

neuronal excitability through the action of second messenger signaling molecules.

Physically, metabotropic receptors are transmembrane proteins that contain 7 alpha- helix motifs that pass through the cell membrane. The N-terminus of the protein is extracellular, and the protein “weaves” back and forth across the cell membrane, resulting in a protein with three extracellular loops and three intracellular loops. Because of this shape, these receptors are also called seven-transmembrane receptors, or 7-TM receptors.

Another name  for  these  receptors  is  “G protein-coupled receptors”, or GPCRs. Metabotropic receptors are physically linked to proteins  called  G  proteins,  which  exist  on the

inner surface of the cell membrane. Functionally speaking, these G proteins are capable of binding to molecules of guanosine triphosphate (GTP) or guanosine diphosphate (GDP). Chemically similar to ATP, GTP can function as a source of energy. G proteins themselves exhibit catalytic activity of GTP. This means that they are capable of breaking down GTP into the less-energetic GDP. When GTP is bound to the GPCR, the receptor is active. When this molecule is hydrolyzed into GDP, the receptor becomes inactive.

Some G proteins are heterotrimeric, meaning that they are made up of three different subunits, alpha, beta, and gamma. The GTP binding sites and catalytic sites are found on the alpha subunit, the largest of the three   subunits.

 

 

Fig 5.12 GPCRs signal via activation of the G protein attached to the intracellular side of the receptor.

 

 

Usually, the alpha subunit becomes soluble after activation, while the beta / gamma complex stays embedded in the neuronal membrane. The G alpha subunit exists in different varieties.

 

Gαs. When a neurotransmitter activates a GPCR coupled with the Gαs protein, the Gαs protein is excitatory (The “s” stands for stimulatory).

Binding of a ligand to the active site of Galphas-coupled GPCRs results in increased activity of the enzyme adenylate cyclase (AC). AC itself is an enzyme that creates a second messenger, a molecule called cyclic AMP (cAMP). Elevated levels of cAMP activate an enzyme called protein kinase A (PKA).

 

Fig 5.13 GPCRs that are coupled with Gαs are excitatory through adenylate cyclase signaling.

 

 

PKA is a kinase, an enzyme that is phosphorylates other  proteins.  The addition of a phosphate group onto a protein changes its properties   dramatically.   PKA   phosphorylates protein targets that  increase  cell  excitation. For example, one target of PKA activity is the intracellular side of certain glutamate receptors. Phosphorylation causes these receptors to stay open longer than normal when they are activated by a  molecule  of  glutamate.  This means that a single molecule of glutamate causes more excitation (passes more depolarizing current into the cell) in the presence of increased PKA activity. Targets of PKA activity also include the intracellular store of glutamate receptors. When phosphorylated, these receptors are trafficked to the neuronal membrane. An increase of receptors at the postsynaptic side leads to increased excitatory neurotransmission over a period of minutes  and  hours,  representing  one  form  of

plasticity.

An even longer-term action  of  PKA  is  its ability to change the transcription of various genes, which can trigger the synthesis of proteins. Some genes downstream of PKA activity include the structural protein actin, important for morphological changes in neuronal structure, or ion channels which change neuronal responses to neurotransmitter release.

 

Gαi. A GPCR that is coupled with Gαi causes a decrease in excitability. In many ways, Gαi proteins serve the opposite function as Gαs proteins – the “i” stands for inhibitory.

Whereas  activation  of  Gαs   increases  the action of AC, Gαi proteins decrease AC activity.   Therefore,   Gαi  activation   decreases the intracellular concentration of cAMP, in turn decreasing PKA activity. Given the function of PKA as a kinase that increases cellular excitation   as

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described above, decreased PKA activity inhibits cellular activity through multiple mechanisms, some of which include decreased current through glutamate receptors, decreased trafficking of glutamate receptors to the presynaptic neuronal membrane, and decreased transcription of certain genes.

 

Gαq. Generally, Gαq is an excitatory G protein. Gαq uses a different signaling pathway compared to the PKA pathway that is downstream of Gαs or Gαi. Gαq protein activation leads to activity of the enzyme phospholipase C (PLC).

On  a  biomolecular  level,   PLC   acts   on the phospholipid membrane molecule phosphatidylinositol 4,5-bisphosphate (PIP2). PLC is a hydrolytic enzyme, and it breaks PIP2 into two separate second messenger molecules: the soluble inositol triphosphate (IP3) and the membrane-embedded diacylglycerol (DAG). One of the functions of IP3 is to liberate Ca2+ from intracellular stores, which elevates intracellular Ca2+ levels, depolarizing the cell and activating calcium-dependent processes, which are often excitatory. DAG activates protein kinase C (PKC), an enzyme with substrates that increase neurotransmitter release probability or decrease

potassium channel conductance.

While the alpha subunits carry out a large part of GPCR-mediated changes in cellular excitation, the beta and gamma subunits also affect excitability. The beta and gamma subunits are bound together as a dimer, but they separate from the alpha subunit once the GPCR becomes activated. The beta-gamma complex can also function as a signaling molecule.

Compared to ionotropic receptors, metabotropic receptors affect neuron activity on a slower time scale, on the range of milliseconds to seconds, and possibly even longer depending on the downstream mechanisms that are activated.

 

Presynaptic receptors

In the discussion of receptors, it is common to think of receptors as being expressed at the dendrites, embedded within the postsynaptic membrane. However, not all receptors are located here. Some receptors are presynaptic, meaning they can be found at the axon terminal. Presynaptic receptors are often inhibitory and serve a self-regulatory function. Presynaptically- expressed receptors that respond to the same neurotransmitter that is released are called autoreceptors.

 

 

Fig 5.14 Gαq signals using PLC, which then produces two signaling molecules, IP3 and DAG.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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5.4

Neurotransmitters 

As described previously, neurotransmitters are the substances that are released at chemical synapses, and they are the signaling molecules that allow neurons to communicate with one another. To date,  scientists  have  identified more than 100 neurotransmitters. Here, we will describe six classical neurotransmitters, their receptors, and their actions. Additionally, three atypical neurotransmitters will be introduced.

One important note to keep in mind as you think about neurotransmitters: the  effect  that a neurotransmitter has on a cell depends on the receptor. In other words, a neurotransmitter molecule can either excite or inhibit a neuron depending on the  composition  of  receptors  that are present. For example, glutamate is excitatory at most synapses in the nervous system. Glutamate exerts excitation by activating ionotropic glutamate receptors, which are ligand-gated cation channels. However, at one particular synapse in the eye, glutamate activates a metabotropic glutamate receptor that causes cellular inhibition.

 

Glutamate

Glutamate (Glu) is the main excitatory neurotransmitter used by the nervous system. Glutamate is the same as the amino acid glutamic acid. There is more glutamate per volume of brain tissue than any other neurotransmitter. Glutamatergic neurons are identified by the presence of the vesicular glutamate transporter (vGluT).

Glutamate can activate both ionotropic and metabotropic receptors. Ionotropic glutamate receptors are all ligand-gated cation channels, which  makes  them  excitatory  since  they allow

Na+ to move into the cell. Ionotropic glutamate receptors are generally subdivided into three classes, named after exogenous chemicals that can activate the receptor. AMPA receptors are Na+ channels, but some also allow Ca2+ entry. NMDA receptors allow both Na+ and Ca2+ to pass across the membrane. When the cell is at rest, NMDA receptors also have a large magnesium ion in the pore that blocks ion movement through the channel. The third category of ionotropic glutamate receptors is called kainate receptors, which are similar to AMPA receptors.

The metabotropic glutamate receptors (mGluRs) signal using different G proteins. There are a total of 8 of these mGluRs, classified into three groups, called Group I, Group II, and Group

III. Group I are excitatory GPCRs which signal via Gq, while Group II and Group III are inhibitory via the Gi signal transduction pathway.

One theory proposes that excess signaling by glutamate can lead to neuronal death, a phenomenon    called    excitotoxicity.    Of  the

Fig 5.15 Glutamate is the main excitatory neurotransmitter in the nervous system, acting at different categories of receptors, three of which are shown below.

 

 

various glutamate receptors, the NMDA receptor is most strongly implicated in contributing to excitotoxicity,  since  uncontrolled   elevated levels of calcium can be deadly for neurons. Excitotoxicity is observed in a variety of disease states ranging from neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis, but also in injury such as concussion or stroke.

 

GABA + glycine

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. According to one estimate, about 25% of neurons in the brain are GABA-ergic. Chemically speaking, GABA is remarkably similar to glutamate. In  fact, GABA  is  synthesized  from  glutamate  in  a single step by the enzyme glutamic acid decarboxylase (GAD). GAD is often used as a biochemical marker for the presence of GABA- ergic neurons. Many interneurons use GABA as their chemical signaling molecule.

 

Fig 5.16 The inhibitory neurotransmitter GABA is synthesized from glutamate by the action of GAD.

The main action of GABA as an inhibitory neurotransmitter is to activate one of three main classes of receptors, called A, B, and C. GABAA receptors are ligand-gated chloride  channels,  so activating these ion channels causes Cl- flux, which opposes the ability for the cell to reach action potential threshold. GABAB and GABAC receptors are both metabotropic receptors that inhibit neuronal activity through the action of the Gi protein.

A neurotransmitter that is similar to GABA is glycine. Another small amino acid, glycine is mostly used by neurons of the spinal cord and brain stem. Glycine is also inhibitory , and acts at glycine receptors, which are ligand-gated chloride channels.

 

Dopamine

Dopamine (DA) is a biogenic amine derived from the amino acid  tyrosine  through the action of several enzymes. One in particular, tyrosine hydroxylase (TH), is the main marker that is used for identifying dopamine-producing neurons. Unlike glutamate or GABA, dopamine- producing neurons are not widely abundant in the brain. Instead, there are generally only a few patches of neurons that produce dopamine, most of which are found in the midbrain. Two areas include the ventral tegmental area and the substantia nigra.

There are five classes of dopamine receptors, named D1 through D5. All of them are metabotropic receptors. D1 and D5 are generally excitatory receptors, while D2, D3, and D4 are inhibitory receptors.

Since Roy Wise’s  theory  proposed  in the 1960s, DA has been known in pop culture  as the “pleasure neurotransmitter” because of  its involvement in the processing of reward and motivation. For example, if we use  microdialysis

Frontal cortex

 

 

 

 

 

Nucleus accumbens (NAc)

Ventral tegmental area (VTA)

 

Striatum

Substantia nigra (SN)

 

 

 

 

 

 

Hippocampus

in the substantia nigra pars compacta (SNpc) degenerate, as in Parkinson’s disease, a person develops the trademark symptoms: difficulties with motor control, resulting in a resting tremor, postural instability, and  bradykinesia  (slowness  of movement). Reversing the dopamine deficiency by introducing an exogenous source of dopamine is our current gold standard of treatment for PD.

Fig 5.17 Brain dopamine is synthesized in two major midbrain nuclei, the VTA and SN, labeled in green.

 

Serotonin

Serotonin        (5-HT)        is        a

(a technique to measure the concentration of chemicals) in the nucleus accumbens, dopamine levels spike in response to all sorts of pleasurable or rewarding stimuli: food, water, sex, sugar, and exposure to drugs of abuse. However, we now know that dopamine is much more  complex  than once believed. One theory suggests that dopamine elevation serves as a “learning signal” that causes us to pay attention to salient stimuli in the environment.

DA is also needed for normal motor control.               When   dopamine-producing    neurons

neurotransmitter that is derived from the dietary amino acid tryptophan. The enzyme tryptophan hydroxylase is the first step of serotonin biosynthesis and is often used as a marker to identify serotonergic neurons. As with dopamine, there are only a few areas of the brain that synthesize serotonin, the major one being the Raphe nucleus in the brain stem.

Receptors for serotonin have a wide variety of actions. We have identified seven major families of 5-HT receptors, which are designated by the number and subclasses which are designated by

a letter. For example, the 5-HT2A receptor

Fig 5.18 Levels of dopamine in the NAc rise when animals lever press for delicious foods, denoted in red.

 

 

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is metabotropic and excitatory via Gq signaling, while the 5-HT5 receptor is inhibitory via Gi signaling. Most of them are metabotropic receptors; only the 5-HT3 receptor is ionotropic.

Serotonin  is  heavily   implicated in the regulation of mood and complex behavioral conditions. One of our most effective strategies for treating depression is the administration of a drug such as fluoxetine, which acts as a selective- serotonin reuptake inhibitor (SSRI). Pharmacologically,   fluoxetine  increases

Clinical correlation: Parkinson’s disease (PD) and L-DOPA-induced dyskinesia (LID)

Parkinson’s disease is a debilitating neurodegenerative disorder that affects as many as 1% of all people aged 60 or older. Generally, PD is lethal within 16 years. By the time a patient presents to the clinic with motor dysfunction, they have already lost almost 60-80% of dopamine-producing neurons in this area!

For decades, clinicians have been using the biochemical precursor to dopamine, L-DOPA, to treat the symptoms. However, after chronic exposure to L-DOPA, the drug becomes less effective and has a shorter duration of therapeutic action. Worse still, frequent treatment can lead patients to develop hyperkinesias, an abnormal excess of movements. This iatrogenic disorder is called L-DOPA induced dyskinesia (LID).

Biomedical engineers have developed a promising non- drug approach to treating PD called deep brain stimulation. A small stimulating device is surgically implanted into the subthalamic nucleus of the brain. When this brain area is stimulated, neural circuits are recruited which restores normal motor control.

 

Aromatic amino acid decarboxylase (AADC)

 

 

 

L-DOPA                                                  Dopamine

 

 

Fig 5.19 Patients with PD have characteristic changes in gait as a result of low dopamine (top). The current best pharmacological therapy is levodopa administration, which is the biochemical precursor to dopamine (bottom).

 

synaptic levels of serotonin by preventing reuptake, and for some people, this has a moderate ability to reverse depression. Serotonin signaling is also a target for drugs that treat anxiety, post- traumatic stress disorder, obsessive-compulsive disorder, schizophrenia, and more.

Acetylcholine

Acetylcholine  (ACh)  is  a  small  molecule that is made by the enzyme choline acetyltransferase (ChAT), which chemically bonds a molecule of acetyl-CoA with a molecule of choline. The presence of ChAT in a neuron is used as a biochemical marker for neurons that produce acetylcholine.

ACh was the first neurotransmitter discovered and chemically isolated, a feat which earned two researchers the shared Nobel Prize in Physiology or Medicine in 1936. One of the two scientists, a German pharmacologist named Otto Loewi, stimulated the vagus nerve connected to an isolated frog heart, which caused the heart rate to slow down. When he put the surrounding solution on top of another heart, he observed that the second heart also slowed down, despite having no physical connection to the first heart. From this, he concluded that a chemical released by the vagus nerve is able to decrease heart rate. This chemical was first called Vagusstoff, the German word meaning Vagus substance. Today, we know it as acetylcholine.

ACh is able to act at ionotropic and metabotropic receptors, and activity at both receptor classes is essential for normal function. The ionotropic receptors of the nervous system are called nicotinic acetylcholine receptors (nAChRs)  because  they  can  be  activated  by

nicotine in addition to acetylcholine. These ionotropic receptors are ligand-gated sodium channels and are therefore excitatory. On the other hand, the metabotropic receptors are called muscarinic acetylcholine receptors (mAChRs) since they are activated by the chemical muscarine found in some species of mushrooms. MAChRs can be coupled with either Gs or Gi, so they can be either excitatory or inhibitory.

ACh is the main neurotransmitter that the nervous system uses in order to communicate with the muscles at the neuromuscular junction (NMJ). Here, ACh is released by motor neurons, where it activates nicotinic acetylcholine receptors on muscle cells, causing them to constrict, or flex. On the other hand, muscarinic acetylcholine receptors are located in the heart, and their activation causes a decrease in heart rate (as Otto Loewi demonstrated with the isolated frog heart preparation.)

In the central nervous system, ACh is involved in a wide variety of processes, including

                                                                                                                                                                                            attention and learning. One of the first

Fig 5.20 An electron microscope image of the neuromuscular junction showing vesicles (T,  top) forming a synapse with   the muscle cell (M, bottom). Acetylcholine is the main neurotransmitter used in muscle control at the PNS.

theories to explain the symptoms of Alzheimer’s disease looked at a loss of ACh-producing neurons in the basal forebrain that become more  severe as the disease worsens. It has since been demonstrated that Alzheimer’s disease is more complex than this early hypothesis.

 

Norepinephrine

Norepinephrine (NE) is a neurotransmitter that is synthesized from   a   molecule   of    dopamine  by the enzyme dopamine beta- hydroxylase.                                 Norepinephrine- producing cells are localized in the pons  of  the  brain  stem,  a structure

called the locus coeruleus. The locus coeruleus is very small, but these neurons send projections widely throughout the brain.

beta-agonists are used as bronchodilators for asthma.

Norepinephrine  also  functions   in

the brain to modulate behaviors including

Fig 5.21 Norepinephrine in the brain is synthesized by small populations, but these cell bodies project widely across several other areas.

alertness and attention.

 

Atypical neurotransmitters

Although we generally think of neurotransmitters as neurochemicals that function as described above, there are a few atypical neurotransmitters that don’t quite fit the mold of the other chemical signals.

 

 

 

 

 

 

 

Outside of the brain, we think of norepinephrine as being responsible for triggering the sympathetic nervous system response of the body, the “fight-or-flight” reaction that prepares the body for physical activity in times of fear or acute stress. These norepinephrine-producing nerve cells reside in the sympathetic ganglia, a clump of nerve cells that run parallel to the spinal cord, one on each half of the body. These neurons project out towards the internal organs.

Receptors  for  NE  are  classified  into two main categories, alpha or beta. There are subtypes within each category, giving us five major receptors for NE: alpha-1, alpha-2, beta- 1, beta-2, and beta-3. All five of these receptors are metabotropic, and some are excitatory while others are inhibitory. Our internal organs express these noradrenergic receptors. Clinically, the “beta blockers” are a class of drugs that inhibit beta-adrenergic receptors; the resulting action is a decrease in blood pressure. Conversely, some

Neuropeptides

Neuropeptides are a class of large signaling molecules that some neurons synthesize.  Neuropeptides  are   different

from the traditional  neurotransmitters  because of their chemical size. Monoamines like DA, NE, or 5-HT have a molecular weight around 150- 200, while one of the smaller neuropeptides, enkephalin, has a molecular weight of 570. One of the largest, dynorphin, has a molecular weight greater than 2,000. Because of their large size, neuropeptides have to be packaged in dense- core vesicles very close to the site of production near the nucleus rather than in clear vesicles right at the terminal.

 

 

 

 

 

 

 

Fig 5.22 Enkephalin, one of the smaller neuropeptides, is very large compared to other neurotransmitters. Enkephalin is an agonist for both δ and μ opioid receptors.

 

 

Neuropeptides such as enkephalin and dynorphin are agonists at a class of receptors called the opioid receptors. These opioid receptors fall into four main types. The three classical opioid receptors are named using Greek letters: δ (delta), μ (mu), and κ (kappa), and the fourth class is the nociceptin receptor. All of these receptors are inhibitory metabotropic receptors which signal using the Gαi protein.

These receptors are expressed in several brain areas, but expression is particularly heavy in the periaqueductal gray, a midbrain area that functions to inhibit the sensation of pain. Drugs that activate the opioid receptors, like morphine, oxycontin,  or  fentanyl,  are  the  most   effective

clinical treatments that we know of for acute pain.

Unfortunately, these same drugs also represent  a tremendous health risk, as opioid drugs can  be lethal in overdose and have a high risk of substance use disorder.

 

Endocannabinoids (eCBs)

The  eCBs  are  a   class   of   lipid-  based neurotransmitters. They are unusual neurochemicals in a few ways. Instead of sending information from the axon of one neuron to the dendrite of the next neuron (anterograde signaling), eCBs allow the postsynaptic dendritic component to communicate with the presynaptic axon terminal. Since they communicate information in the “opposite” direction of classic neurotransmitter signaling, eCBs are called retrograde signaling molecules. Secondly, eCBs are not packaged into vesicles and released by fusion processes. Instead, eCBs are synthesized de novo, meaning they get created right when they needed and used at that moment. The two most well-characterized eCBs in humans are called 2-AG and AEA.

Fig 5.23 ECBs are synthesized from the postsynaptic cell membrane and signal through presynaptic cannabinoid receptors

 

ECBs activate one of two receptors, CB1 and CB2. Both of them are inhibitory metabotropic receptors that couple with Galphai. Generally, CB1 receptors are found in the nervous system, while the CB2 receptors are found elsewhere in the body, such as in the immune system.

The eCB system is widely used by various systems in the body. It is estimated that eCB receptors are the most abundant GPCRs in the whole body.

These substances were named because they are endogenous chemicals that are functionally similar to compounds found in plants of the genus Cannabis. One reason cannabis is used is because of its ability to interact with our eCB receptors.

 

Nitric oxide

The nervous system is capable of signaling via the gas nitric oxide (NO). This gasotransmitter

is not stored in vesicles but rather is synthesized as it is needed. NO is formed when the amino acid arginine is degraded by the enzyme NO synthase (NOS).

Because NO is a gas, it easily permeates across cell membranes. Therefore, the receptors for NO do not need to be transmembrane proteins expressed  on  the  cell  surface.  Instead,     the

receptor for NO is an intracellular receptor called soluble guanylate cyclase (sGC). SGC works through a signaling pathway that is different from other metabotropic receptors so far described. SGC is linked with the signaling molecule cyclic GMP  (cGMP),  which  activates  protein  kinase

G. PKG can either be excitatory or inhibitory, depending on the intracellular components.

 

 

 

Chapter summary

Neurons communicate with one another in a variety of ways. Anatomically, neurons are separated by a small extracellular  gap  called the synapse. This synapse may directly connect the intracellular cytoplasm, as in an electrical synapse. Alternatively, the gap may be much larger, and chemicals that get released and diffuse across the  synapse  in  order  to  signal to the following neuron. These chemicals, the neurotransmitters, are stored in vesicles, tiny spheres that are located in the presynaptic axon terminal. The release of these chemicals is very closely regulated, and neurons have several mechanisms that regulate vesicular fusion.

Following release, the neurotransmitters diffuse across the synapse  and  can  bind  to  the   active   site   on   transmembrane   proteins

called receptors. Upon binding a molecule of neurotransmitter, these receptors physically change shape,  resulting  in  ion  flow  across  the membrane (ionotropic receptors) or a  change in the activity of intracellular signaling molecules  (metabotropic  receptors).   Binding  of neurotransmitter changes the excitability of neurons.

We have so far  identified  more  than  100 neurotransmitters. Many of them are small molecules that are packaged in vesicles, which then diffuse from the presynaptic neuron to the postsynaptic neuron, such as acetylcholine, glutamate, or GABA. However, there are some atypical neurotransmitters such as neuropeptides, endocannabinoids, and nitric oxide that have different methods of communication.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The Open Neuroscience Initiative is funded by a grant from the Vincentian Endowment Fund of DePaul University.

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.