Austin Lim, PhD (DePaul University)

Editor: Monica Javidnia, PhD (University of Rochester)

Canadian Edition Editor: Drew Mackenzie Neyens (University of Calgary)

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 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 ion. At electrical synapses, 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 2 nanometers. Logically, the neurons must be close since the hemichannels are like a physical “bridge” between the two cells.

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 more critical for survival.

Another advantage of electrical synapses is that they can form a large network of interconnected neurons with synchronized activity. For example, many 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.

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.

Chemical synapses

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

The distance between two cells with chemical synapses can be much larger than that of electrical synapses. On average, a chemical synapse has an intercellular gap of about 20-40 nanometers, 10-20x wider than the space in electrical synapses. Interestingly, this is still roughly a thousand times smaller than the diameter of a human hair.

A single chemical synapse can pass a variety of signals depending on the neurotransmitters and receptors involved. For example, some signals are directly excitatory and allow positively charged cations to enter the postsynaptic neuron, causing depolarization. Other signals are hyperpolarizing, and therefore inhibitory. Still other signals are more complex, inducing changes in protein expression that can modify cellular excitability and connectivity over the course of minutes, hours, or even a lifetime.

Chemical synapses allow for fine-tuning of neural networks that vary in complexity across species, 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.

Most chemical synapses exist between the axon terminal of one neuron, the dendrite or soma of another neuron, or an equivalent receptive feature on non-nervous tissue. 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 lipid spheres with their own lipid bilayer, similar to cell membranes. 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, 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 commonly known and discussed neurotransmitters (i.e., glutamate, GABA, dopamine, norepinephrine, etc.). Small vesicles are almost always exclusively 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 made possible through the joint action of transmembrane proteins called vacuolar-type ATPases and 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 respectively pump protons (H⁺ ions) and molecules of neurotransmitter from the intracellular space of the axon terminal and into vesicles.

Many vesicular transporters are named based on the neurotransmitters that they transport. 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/or serotonin into the vesicles.

Vesicular transporters can function because the interior of the vesicle is highly acidic compared to the interior of the cell. Vesicles have a high concentration of protons and are acidic due to the transmembrane enzyme vacuolar-type ATP-ase, or V-ATP-ase. These membrane-embedded proteins hydrolyze molecules of ATP to concentrate H⁺ ions within the intravesicular space. For each molecule of ATP used, one proton gets pumped into the vesicle. The action of V-ATPases in vesicle membranes creates an electrochemical gradient, where positively-charged protons are highly concentrated within the vesicle, and this store of potential energy is used by vesicular transporters to take up neurotransmitter.

Vesicular transporters pump molecules of neurotransmitter in the vesicle against their concentration gradients, which requires energy. Vesicular transporters use the potential energy from 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 through a vesicular transporter, this causes a conformational change in the vesicular transporter that moves a molecule of neurotransmitter into the vesicle. Because H⁺ ions move opposite of the neurotransmitter molecules, vesicular transporters are also 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 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 have been depleted due to release and are in the process of being refilled with neurotransmitter. They are farther from the cell membrane and the protein machinery is not primed for release, so there is a set time for these vesicles to refill and mobilize to the membrane. If all vesicles of the RRP are depleted due to intense and sustained activity, vesicles in the recycling pool will replenish the RRP at a continuous rate. This set rate of vesicle refilling has been used to study changes in vesicle properties and quantal release of neurotransmitter.
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 vesicles to release, very intense stimulation is often required, and reserve pool vesicles may not be recruited for release under physiological conditions.

Release of vesicles

Neurotransmitter release is tightly regulated at chemical synapses. If there were no mechanisms to control the release of chemicals at the synapse, nerve cells would deplete their entire stock of neurotransmitter. For example, neurotransmitter release that triggers muscle contraction at the NMJ would cause constant muscle tension. In the case of glutamate, over excitation would cause excito-toxicity and cell death.

Regulation of neurotransmitter release depends on several proteins that are often embedded within vesicular and neuronal membranes. These proteins are needed for both “docking” of vesicles at the synapse and for vesicle fusion and neurotransmitter release.

1. V-SNAREs are the proteins expressed on vesicles (v for vesicle). Synaptobrevin and synaptotagmin are two specific v-SNARE proteins that a form one half of the machinery needed for vesicle localization to the membrane (“docking”) and vesicle fusion.
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, are t-SNAREs that function during vesicular fusion.

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 humankind; exposure to concentrations as low as 2ng/kg is lethal. The most common symptoms include muscle weakness or paralysis, especially the muscles of the face or the limbs. In about 5% of untreated botulism cases, death results from paralysis and 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 release.

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 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.

Vesicular fusion

After vesicles are mobilized to and “docked” at the membrane, vesicle fusion with the cell membrane allows for neurotransmitter release. 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. In addition to enabling “docking” at the cell membrane, this protein detects elevated levels of Ca²⁺ in the axon terminal and triggers vesicle fusion.

Neuronal intracellular calcium is generally in the range of 50-100 nM, which 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 open when the surrounding neuronal membrane becomes depolarized, allowing ions to move across the cell membrane. VGCCs selectively pass only Ca²⁺ ions, which flow down their electrochemical gradient into the cell.

When an action potential reaches the axon terminal, it causes a depolarization of the terminal and triggers calcium entry via the VGCCs. Ca²⁺ at the terminal binds to SNARE proteins like 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, resulting in vesicular fusion and the release of neurotransmitter into the synaptic cleft.

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 the contents in their entirety are released 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 enter the synapse via diffusion.

5.3 Receptors

Receptors are membrane proteins that transmit chemical and/or electrical signals 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 specific neurotransmitters to bind to the receptor.

Receptors can be classified into one of two main categories.

1. Ionotropic receptors. Ionotropic receptors are transmembrane proteins with a large-diameter pore through which specific ions can pass. These channels open when the appropriate molecule binds to the active site on the extracellular side of the protein; you can think of this as a “lock-and-key” mechanism. The specific molecules that can bind and open these channels are also called ligands; thus, ionotropic receptors are also called ligand-gated ion channels. Once a neurotransmitter binds and opens the ionotropic receptor, the channel permit ions to l flow down their electrochemical gradient, either into or out of the cell. As a result of ion movement, the cell’s membrane potential will change. For example, nicotinic acetylcholine receptors are ligand-gated sodium channels, so when these receptors are activated by a ligand like acetylcholine, sodium ions enter the cell causing depolarization and excitation. Ionotropic receptors can induce a rapid change in membrane potential, on the order of milliseconds.

Ionotropic recepters can be selective for certain ions depending on the amino acid residues in the channel pore. For example, negatively charged residues lining the inside of the pore repel negatively charged Cl^– (chloride) ions while allowing positively charged cations to pass through the channel.

2. Metabotropic receptors. Rather than passing ions directly to change cellular activity, metabotropic receptors use G proteins to initiate a signaling cascade that changes the activity of other receptors, ion channels, and transporters.

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 can break 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 (α), beta (β), and gamma (γ). The GTP binding sites and catalytic sites are found on the alpha subunit, the largest of the three subunits.

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.

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

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

PKA is a kinase, an enzyme that 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-lasting action of PKA is its ability to change gene transcription, and thus protein synthesis. Some genes downstream of PKA activity include the structural protein actin, important for morphological changes in neuronal structure, or ion channels, which will change neuronal responses to neurotransmitter release.

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

Whereas activation of G_αs increases the action of AC, G_αi proteins decrease AC activity. Therefore, G_αi activation decreases the intracellular concentration of cAMP, in turn decreasing PKA activity. Decreased PKA activity can inhibit cellular activity through multiple mechanisms, including decreased current through glutamate receptors, decreased trafficking of glutamate receptors to the presynaptic neuronal membrane, and decreased transcription of certain genes.

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

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, activating calcium-dependent processes that 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, or 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, 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.

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 unique synapses in the eye, glutamate activates a metabotropic glutamate receptor that causes cellular inhibition.

Glutamate

Glutamate (Glu), also known as glutamic acid, is the main excitatory neurotransmitter used by the nervous system; 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 glutamate receptors. Ionotropic glutamate receptors are all ligand-gated cation channels, which makes them excitatory since they allow cations like Na⁺ to enter the cell. Ionotropic glutamate receptors are generally subdivided into three classes, which are named after exogenous chemicals that can activate the receptor. AMPA receptors are primarily Na⁺-selective 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 (Mg²⁺) 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 G_q, while Group II and Group III are inhibitory via the G_i signal transduction pathway.

Excess signaling by glutamate can lead to neuronal death through a phenomenon called excitotoxicity. Of the 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 main neurotransmitter.

GABA acts as an inhibitory neurotransmitter via three main classes of receptors, called GABA A, B, and C. GABA_A receptors are ligand-gated chloride channels whose activation leads to an influx of negatively-charged Cl^– ions, leading to hyperpolarization and neuronal inhibition. GABA_B and GABA_C receptors are both metabotropic receptors that inhibit neuronal activity through the action of the G_i protein.

Glycine is a neurotransmitter that serves a similar function to GABA. 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. Interestingly, glycine is also a requisite co-agonist at NMDA receptors, meaning both glycine and glutamate are required for NMDARs to be fully activated.

Dopamine

Dopamine (DA) is a biogenic amine and neurotransmitter derived from the amino acid tyrosine through. Tyrosine hydroxylase (TH) is the primary enzyme that converts tyrosine to the dopamine precursor L-DOPA, and 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 clusters of neurons that produce dopamine, most of which are found in the midbrain. Two areas include the ventral tegmental area and the substantia nigra. While dopamine neurons are less abundant than other neuronal phenotypes, these neurons can project widely through the brain and serve essential functions in motor control, cognition, mood and motivation.

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

Since Roy Wise proposed his theory 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 (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 serves a much more complex role. One theory suggests that dopamine elevation serves as a “learning signal” that causes us to pay attention to salient stimuli in the environment, ascribing importance and motivational value to substances, environmental cues, behaviors, and internal states.

DA is also needed for normal motor control. When dopamine-producing neurons 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 dopamine deficiency by introducing an exogenous source of dopamine is our current gold standard of treatment for Parkinson’s disease.

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 the substantia nigra!

For decades, clinicians have been using the biochemical precursor to dopamine, L-DOPA, to treat the symptoms of PD. 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 hyperkinesia, 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 or pedunculopontine tegmental nuclei of the brain. When these brain areas are stimulated, neural circuits are recruited which help restore normal motor control.

Serotonin

Serotonin (5-HT) is a 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 principal source being the raphe nucleus in the brain stem.

Serotonin receptors have a wide variety of actions. We have identified seven major families of 5-HT receptors, which are designated by a number and subclass.

For example, the 5-HT2A receptor is an excitatory metabotropic receptor (G_q-coupled), while the 5-HT5 receptor is an inhibitory metabotropic receptor (G_i-coupled). Most of th denoted in red em are metabotropic receptors; only the 5-HT3 receptor is ionotropic.

in the central nervous system, serotonin has been heavily implicated in the regulation of mood. One of our most effective strategies for treating depression is through administration of drugs like fluoxetine, which act as selective-serotonin reuptake inhibitors (SSRIs). Pharmacologically, fluoxetine increases synaptic levels of serotonin by preventing reuptake, and this has a moderate ability to treat depression in some people. Serotonin signaling is targeted by 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 frog’s vagus nerve decreased heart rate. This chemical was first called Vagusstoff, the German word meaning Vagus substance. Today, we know it as acetylcholine.

ACh acts at ionotropic and metabotropic receptors, and ACh 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. Conversely, 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 G_s or G_i, so they can be either excitatory or inhibitory.

ACh is the main neurotransmitter that the nervous system uses to communicate with the muscles. This communication happens at a special synapse called the neuromuscular junction (NMJ). Here, ACh is released by motor neurons, where it activates nicotinic acetylcholine receptors on muscle cells, causing them to constrict/flex. In contrast, 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 theories to explain the symptoms of Alzheimer’s disease looked at a loss of ACh-producing neurons in the basal forebrain that becomes 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), also called noradrenaline (NA), 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.

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, clusters of nerve cells that run parallel to the spinal cord, on each side of the body. These neurons project out towards the internal organs.

NE receptors 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 beta-agonists are used as bronchodilators for asthma.

Norepinephrine also functions in the brain to modulate alertness, attention, food intake, and metabolic processes.

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.

Neuropeptides

Neuropeptides are a class of large signaling molecules that some neurons synthesize. Neuropeptides are different from the traditional neurotransmitters because of their molecular weight. 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 are packaged in dense-core vesicles close to the site of production near the nucleus, rather than in clear vesicles right at the terminal.

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 via G_i.

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 opioid receptors, like morphine, oxycontin, or fentanyl, are the most effective clinical treatments known for acute pain. Unfortunately, these same drugs also represent a tremendous health risk. Opioid receptors are also present in areas that control respiration, heart rate, and motivation or craving; thus, opioid drugs can be lethal in overdose and have a high risk of substance use disorder.

Endocannabinoids (eCBs)

Endocannabinoids are a class of lipid-based neurotransmitters and 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 are released from postsynaptic sites to communicate with presynaptic receptors. 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 are created and released on demand. The two most well-characterized eCBs in humans are called 2-arachidonoylglycerol (2-AG) and anandamide AEA.

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

Taken together, 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 psychoactive compounds found in plants of the genus Cannabis.

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 ineeded. 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 known as neurotransmitters are released and diffuse across the synapse to transmit chemical signals between neurons. Most classic neurotransmitters are stored in vesicles, tiny spheres that are in the presynaptic axon terminal. Release of neurotransmitter is 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 can undergo a conformational change that allows ions to flow across the membrane (in the case of ionotropic receptors) or initiates intracellular signaling cascades (in the case of metabotropic  receptors). In this way, neurotransmitters can change the excitability of neurons or even affect gene transcription and protein synthesis.

We have so far identified more than 100 neurotransmitters. Many of them are small molecules packaged in vesicles, and these neurotransmitters diffuse from the presynaptic neuron to the postsynaptic neuron upon release (e.g. acetylcholine, glutamate, or GABA). However, there are atypical neurotransmitters such as neuropeptides, endocannabinoids, and nitric oxide that have different mechanisms and directions of communication.

Image Credits

Cover: Image found at https://www.hippopx.com/en/people-letters-envelope-pen-dip-pen-writings-ink-62437

5.1 https://upload.wikimedia.org/wikipedia/commons/7/78/Gap_cell_junction_keys.svg modified by Austin Lim

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5.4 (Left) https://upload.wikimedia.org/wikipedia/commons/4/4c/Synapse_diag1.svg modified by Austin Lim (Right) https://commons.wikimedia.org/wiki/File:Synapse_diag4.png#file modified by Austin Lim

5.5 Tao C-L, Liu Y-T, Zhou ZH, Lau P-M and Bi G-Q (2018) Accumulation of Dense Core Vesicles in Hippocampal Synapses Following Chronic Inactivity. Front. Neuroanat. 12:48. doi: 10.3389/fnana.2018.00048

5.8 https://upload.wikimedia.org/wikipedia/commons/2/28/Opening_of_a_Fusion_Pore_during_Exocytosis.png modified by Austin Lim

5.9 https://upload.wikimedia.org/wikipedia/commons/1/1e/Botulinum_Toxin_Mechanism.png modified by Austin Lim

5.10 https://upload.wikimedia.org/wikipedia/commons/0/0c/0310_Exocytosis_cleaned.png modified by Austin Lim

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5.13 https://upload.wikimedia.org/wikipedia/commons/8/8f/CREB_cAMP_neuron_pathway.svg modified by Austin Lim

5.14 https://upload.wikimedia.org/wikipedia/commons/3/31/Activation_protein_kinase_C.svg modified by Austin Lim

5.17 https://upload.wikimedia.org/wikipedia/commons/d/de/Dopamine_pathways.svg modified by Austin Lim

5.18 Data from K.N. Segovia, M. Correa, J.D. Salamone, Slow phasic changes in nucleus accumbens dopamine release during fixed ratio acquisition: a microdialysis study, Neuroscience, Volume 196, 2011, Pages 178-188, ISSN 0306-4522, https://doi.org/10.1016/j.neuroscience.2011.07.078. Reprinted with permission 2/15/2020

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5.20 https://upload.wikimedia.org/wikipedia/commons/a/aa/Electron_micrograph_of_neuromuscular_junction_%28cross- section%29.jpg

5.21 https://upload.wikimedia.org/wikipedia/commons/4/48/Norepinephrine_Part_1.png modified by Austin Lim

5.23 https://upload.wikimedia.org/wikipedia/commons/2/27/Sistema_endocannabinoide.png modified by Austin Lim

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.