NEURO: Neurotransmitter Systems I: Glutamate

Question 1

RECAP: What are the principles of neurotransmission?

Answer 1

Neurotransmission is the fundamental process that drives information transfer between neurons and their targets.

In neuron to neuron communication, this occurs at the synapse between the axon terminal between a pre-synaptic neuron and dendrites of a post-synaptic neuron.

The axon is a neuronal projection that is highly specialised to conduct nerve impulses/action potentials beginning with a region termed the axon hillock, forming the initial segment of the axon. The axon proper is insulated in myelin and functions to speed up the propagation of an action potential down an axon.

There are gaps within the myelin sheath called the nodes of Ranvier which are rich in voltage gated sodium channels, allowing the nodes of Ranvier to propagate the action potentials. The axon terminal is the site at which the axon comes in contact with other neurones and is called the synapse, where neurotransmitters are released across a synaptic cleft by exocytosis.

The dendrites are the highly specialised synaptic projections which contain post synaptic receptors and receive synaptic input from other neurones. Dendrites of a single neurone are a dendritic tree with each branch of this tree, termed a dendritic branch.

Question 2

What is a neurotransmitter?

Answer 2

Neurotransmitters are chemical messengers that transmit signals from a neuron to a target cell across a synapse (i.e. neurotransmission)

Question 3

What is the criteria for a neurotransmitter?

Answer 3

For a chemical to be defined as a neurotransmitter:

1. The molecule must be synthesised and stored in the pre-synaptic neuron.
2. The molecule must be released by the pre-synaptic axon terminal upon stimulation.
3. The molecule must produce a response in the post synaptic cell.

Neurotransmitter is synthesised in cell body or in terminal –> Neurotransmitter is packaged into vesicles –> Neurotransmitter is released when vesicles fuse (exocytosis) –> Neurotransmitter binds to and activates post-synaptic receptors.

Neurons can be classified by the neurotransmitter that they use - these differences arise due to the differential expression of proteins involved in neurotransmitter synthesis, storage and release.
(ACh, GABA, Glutamate, Dopamine, Serotonin, Noradrenaline)

Question 4

What is glutamate?

Answer 4

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS).

Glutamate is at the crossroad of multiple metabolic pathways (e.g. Krebs cycle). The excitatory role of glutamate was discovered in the 1950s and in the late 1970s it became recognised as the principle neurotransmitter in the CNS.

Nearly all excitatory neurons in the CNS are glutamateric and it has been estimated that over half of all brain synapses release glutamate.

Question 5

How is glutamate synthesised and stored prior to its release?

Answer 5

Whilst Glutamate can be synthesised by glucose, its most prevalent precursor is Glutamine.

Glutamine –> catalysed by enzyme Glutaminase (phosphate activated) –> Glutamate

Glutamate is synthesised in the nerve terminals. It is then transported into synaptic vesicles by vesicular glutamate transporters (VGLUT).

Synaptic vesicles are very acidic thus H+ ions move down its concentration gradient. Counter transport with H+ ions allows to drive glutamate entry into vesicles.

Question 6

Describe the glutamate re-uptake and degradation pathways.

Answer 6

Re-Uptake:
Both neurons and glial cells contain Excitatory Amino Acid Transporters (EAAT), these are a family of 5 different Na+ ion dependent glutamate co-transporters which function to transport glutamate from the synaptic cleft back into the neuron or glial cell for subsequent degradation.

Degradation:
Glutamate transported into glial cells via the EAATs are converted into glutamine (catalysed by enzyme glutamine synthetase).

Glutamine is then transported out of the glial cell by a second transporter termed the System N transporter (SN1) where it is the transported into neurones via the System A transporter 2 (SAT2).

This overall sequence of events is termed the glutamate glutamine cycle, and allows the maintenance of an adequate supply of glutamate and the means to terminate its action.

Question 7

What are the 2 broad families of neurotransmitter receptors?

Answer 7

There are 2 broad families of neurotransmitter receptors: the ligand-gated ion channels (ionotropic) and G-protein coupled receptors (metabotropic).

The LGICRs contain a membrane spanning domain which forms an ion channel. Neurotransmitter binding to the LGICR, allows for ions to pass through the membrane where it can increase (excitatory) or decrease (inhibitory) the chance of an action potential firing.

The GPCRs comprise a characteristic 7 transmembrane domain structure with an extracellular domain for neurotransmitter binding. Neurotransmitter binding to the GPCR activates G proteins (alpha, beta, gamma subunits) which can dissociate from the receptor and interact with ion channels or bind to other effector proteins that active secondary messenger pathways that can also open or close ion channels.

LGICRs take milliseconds whereas GPCRs produce a slower response. LGICRs and GPCRs are found mainly on the dendritic membrane.
There are also voltage-gated ion channels, these are mainly found on the axonal membrane.

Question 8

Glutamate binds to both LGICRs (ionotropic) and GPCRs (metabotropic). List the ionotropic receptors that glutamate binds to.

Answer 8

Ionotropic:

• AMPA receptors
• NMDA receptors
• KAINATE receptors

Ionotropic glutamate receptors are named after the agonists that activate them:

• The AMPA receptor is activated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
• The NMDA receptor is activated by N-methyl-D-aspartate.
• The Kainate receptor is activated by Kainic acid.

The AMPA and NMDA receptors are responsible for the majority of excitatory neurotransmission in the brain.

All 3 receptors are predominantly post-synaptic receptors.

Question 9

What is the response when glutamate activates the different ionotropic receptors?

Answer 9

With AMPA, we get the influx of Na+ and the efflux of K+.

With NMDA, we get the influx of Na+ and Ca2+, and the efflux of K+.

With Kainate, we get the influx of Na+ and the efflux of K+.

Question 10

Describe the AMPA receptors

Answer 10

The AMPA receptors are composed of 4 subunit types (plus alternate splice variants):

• GluA1
• GluA2
• GluA3
• GluA4

The molecule is referred to as hetero-tetrameric, or the ‘dimer of dimers’. This is because there are normally 2 pairs of two types of subunits. The most common orientation is 2 GluA2 subunits, and 2 GluA1/3/4.

There are 4 orthosteric (site at endogenous ligand (glutamate) binds to) binding sites; however, only 2 sites must to be occupied for the channel to be opened. The current increases as more binding sites are occupied.

The presence of GluA2 subunits prevents Ca2+ flow. Thus, they protect the brain against excitotoxicity.

Question 11

Describe NMDA receptors.

Answer 11

There are 3 subunit types (plus alternate splice variants):

• GluN1 (or NR1)
• GluN2 (or NR2)
• GluN3 (or NR3)

It is also hetero-tetrameric. The most common orientation is 2 GluN1 subunits plus 2 GluN2 (or 3) subunits. GluN3 subunits are inhibitory to NMDA receptor function.

NMDA receptors are both LIGAND and VOLTAGE-gated:
- Ligands: Glutamate (binds to GluN2 subunits) and Glycine/D-serine (binds to GluN1 subunits). All binding sites must be occupied for the channel to open.

• Voltage-gated: there is a molecule of Mg2+ blocking the ion channel at the resting membrane potential. Only upon a depolarisation event will the Mg2+ ion exit the NMDA receptor and allow ions to flow. This depolarisation occurs by the initial stimulation of AMPA receptors, which will allow Na+ into the post-synaptic cell, depolarising it in the process.

Question 12

Describe Kainate receptors.

Answer 12

There are 5 subunit types:

• GluK1 (GluR5)
• GluK2 (GluR6)
• GluK3 (GluR7)
• GluK4 (KA1)
• GluK5 (KA2)

They used to be differently named because they were thought to be AMPA receptors.

The receptor is tetrameric and can be made up of homomers or heteromers.
GluK1-3 can form homomers or heteromers. GluK4 and 5 can only form heteromers with GluK1-3 subunits.

It is a ligand-gated ion channel, although we don’t know exactly how many molecules of glutamate are required for the channel to open (since it’s hard to properly crystalise the receptor to study that).

They have a limited distribution in the brain compared to the AMPA/NMDA receptors.

Question 13

Glutamate binds to both LGICRs (ionotropic) and GPCRs (metabotropic). List the metabotropic receptors that glutamate binds to.

Answer 13

Metabotropic receptors:

• GROUP I
• GROUP II
• GROUP III

The metabotropic glutamate receptors comprise a large extracellular domain for neurotransmitter binding which can be termed a venus flytrap domain. They have characteristic 7 transmembrane domains and an intracellular C terminal domain.

These GPCRs form dimers on the membrane, and there are 3 types of dimers they can make:

• homomers
• heteromers within groups (e.g. mGlu1 and 5)
• heteromers outside of groups (e.g. mGlu2 and 5-HT2A)

There are 8 subtypes of the receptor (mGlu1-8), and they are divided into 3 subgroups (based on their sequence homology).

GROUP 1: mGlu1, mGlu5
GROUP 2: mGlu2, mGlu3
GROUP 3: mGlu4, mGlu6, mGlu7, mGlu8

Group 1 is predominantly found post-synaptically, while Groups 2 and 3 are predominantly pre-synaptically.

They also bind to different G proteins:
- Group 1 binds to αGq/11, ultimately increasing intracellular Ca2+ release (PIP 2 –> DAG and IP3 –> IP3 activation on ER –> increase Ca2+). They contribute to long-term potentiation and therefore plasticity.

• Groups 2 and 3 bind to αGi, ultimately inhibiting adenlyl cyclase and decreasing cAMP formation. They inhibit further neurotransmitter release. (autoreceptors)

Question 14

What can excitatory neurotransmitters like glutamate lead to?

Answer 14

Excitatory neurotransmitters (e.g. glutamate) can lead to neuronal membrane depolarisation – displacement of a membrane potential towards a more positive value.

Hyperpolarisation can be the result of inhibitory neurotransmission. It involves the displacement of a membrane potential towards a more negative value , this inhibits action potential firing by increasing the stimulus required to fire that action potential.

Question 15

What do Excitatory Post-Synaptic Currents (EPSCs) represent and how do the EPSC effects vary in ionotropic receptors?

Answer 15

The excitatory post-synaptic current (EPSC) represents the flow of ions, and change in current, across a post-synaptic membrane.

EPSCs lead to the generation of excitatory post synaptic potentials (EPSPs), which depolarise the cell and increase the likelihood of firing an action potential.

EPSCs produced by the NMDA receptor and kainate receptor are slower and last longer than those produced by AMPA receptors. EPSPs produced by AMPA receptors are much larger.

Accordingly, AMPA receptors are the primary mediators of excitatory neurotransmission in the brain.

Question 16

What is excitotoxicity, and what effect does it have on the neurons?

Answer 16

Excitotoxicity is the pathological process by which excessive excitatory stimulation can led to neuronal damage and death.

Excitotoxicity occurs when we have too much excitation. This happens under pathological conditions.

We could have damage to the vesicular glutamate transporter so that the glutamate stored isn’t released. This build up of glutamate in the vesicle increases its concentration, causing the Glut-H+ transporter to switch and release the glutamate out into the cytosol of the pre-synaptic neuron. This increases the concentration of the glutamate intracellulary, which causes the EAATs to work backwards as well.
These leak out the glutamate into the synaptic cleft without the presence of a stimulus. This leads to the uncontrolled induction of an action potential in the post-synpatic cell. This leads to the activation of the AMPA and NMDA receptors leading to the influx of Ca2+ ions into the post synaptic neuron. (what’s significant about this is the uncontrolled levels of Ca2+).

Excessive Ca2+ can have multiple implications:

• Mitochondrial damage (which can stop cellular respiration taking place)
• Oxidative stress (by formation of free radicals)
• Apoptosis in cells (due to previous reasons)

Excitotoxicity is linked to strokes and Alzheimer’s disease.

Question 17

Describe Glutamate-mediated excitotoxicity in Alzheimer’s disease.

Answer 17

Alzheimer’s disease is a neurodegenerative disorder, characterised by neuronal cell death in the hippocampus (which is associated with memory) and subsequent neuronal cell death throughout the cerebral cortex. In Alzheimer’s disease, NMDA receptor over activation has been shown to be linked with Glutamate-mediated neurotoxicity and has been shown to contribute neuronal cell death.

Memantine is a low-affinity NMDA receptor antagonist that blocks the NMDA receptor ion channel to reduceglutamate mediated neurotoxicity. This is a few select treatment for Alzheimer’s disease (can treat moderate to severe).

Question 18

Describe synaptic plasticity.

Answer 18

Synaptic plasticity - the activity dependent modification of the strength of synaptic transmission at a pre existing synapse.

Question 19

What is Long-term potentiation (LTP)?

Answer 19

Long-term potentiation (LTP) refers to the persistent strengthening of a synapse based upon repeated patterns of activity.

LTP has been extensively studied in the hippocampus as it is involved in the process of learning and memory.

Long-term potentiation (LTP) underlies important processes, including both learning and memory – initial phase involves glutamatergic neurotransmission.

Question 20

How are NMDA receptors dependant on AMDA receptors?

Answer 20

On the post-synaptic cell membrane, the AMPA receptors are activated first. They allow Na+ into the cell, depolarising it, and thus activating the NMDA receptors. The NMDARs allow both Na+ and Ca2+ into the cell, further depolarising the cell.

Furthermore, the Ca2+ traffics more AMPARs to the cell membrane surface, enlarging the signal. It also activates an enzyme called CamKinase II (CamKII) which phosphorylates AMPARs, allowing them to pass more current through. Effectively, it increases their permeability.

Question 21

Describe rhe mechanism of action of how the NMDA and AMPA receptors play a role in long-term potentiation (LTP).

Answer 21

• Glutamate activates AMPA receptors, with Na+ flowing into the post-synaptic neuron and causing post synaptic membrane depolarisation.
• This allows the voltage gated NMDA receptors to open, due to depolarisation removing the voltage-gated Mg2+ ion block allowing both Na+ and Ca2+ to enter.
• Ca2+ ions enter the cell activate post-synaptic protein kinases such as calmodulin kinase II (CaMKII) and protein kinase C (PKC).
• CaMKII and PKC trigger a series of reactions (substrate phosphorylation) that lead to the insertion of new AMPA receptors into the post-synaptic membrane.
• AMPA receptors increase the post-synaptic membranes sensitivity to glutamate and increases ion channel conductance.

This underlies the initial phase of long-term potentiation (LTP).

Changes in gene expression ultimately account for the later phases of LTP, leading to changes in synapse structure.

This potentiation is deemed long-term because we’ve had more proteins inserted into the membrane and phosphorylated. These changes aren’t readily reversible.
This is thought to be a very important mechanism in what is known as synaptic strengthening and also in learning and memory (mostly mediated by calcium influx).

Question 22

Glossary

Answer 22

Glutamate – the major excitatory neurotransmitter in the CNS
Glutamine – Amino acid precursor of glutamate
Phosphate-activated glutaminase – Enzyme that converts glutamine to glutamate
Vesicular glutamate transporter (VGLUT) – Vesicular membrane transporter that mediates glutamate accumulation in presynaptic vesicles (counter-transported with H+)
AMPA receptor – A ligand-gated ionotropic glutamate receptor subtype selective to the synthetic molecule AMPA
NMDA receptor – A ligand- and voltage-gated ionotropic glutamate receptor subtype selective to the synthetic molecule NMDA
Kainate receptor – A ligand-gated ionotropic glutamate receptor subtype selective to the synthetic molecule kainite
Long-term potentiation (LTP) - A persistent increase in synaptic strength following high-frequency stimulation of a chemical synapse. Studies of LTP are often carried out in slices of the hippocampus, an important organ for learning and memory.
Excitatory amino acid transporter (EAAT) – A membrane transporter that mediates glutamate accumulation in the presynaptic bouton from the presynaptic cleft
Excitotoxicity - The pathological process by which neurons are damaged and killed by the overactivations of the NMDA and AMPA receptors.