Neuro: Neurotransmitters Systems I: Glutamate

Question 1

What are the criteria for a neurotransmitter?

Answer 1

• The molecule must be synthesised and stored in the pre-synaptic neuron
• The molecule must be released by the pre-synaptic axon terminal upon stimulation. This is triggered by the arrival of a action potential in the axons terminal. The depolarisation of the presynaptic membrane causes VGCCs to open, leading to the release of neurotransmitters via exocytosis.
• The molecule must produce a response in the post-synaptic cell.

Question 2

What is glutamate?

Answer 2

• Major excitatory neurotransmitter in the central nervous system (CNS)
• Nearly all excitatory neurons in the CNS are glutamatergic and it has been estimated that over half of all brain synapses release glutamate

Question 3

Describe glutamate synthesis and storage in vesicles.

Answer 3

• Glutamate can be synthesised by glucose.
• However the most prevalent precursor for glutamate synthesis is glutamine. Glutamine is converted to glutamate via the enzyme glutaminase.
• Glutamate is synthesised in the nerve terminals
• It is then transported into synaptic vesicles via a vesicular glutamate transporter (VGLUT)
• The inside of synaptic vesicles is very acidic, maintained by ATP driven hydrogen ion pumps. This is a counter transport process with H+ ions that allows glutamate entry into the vesicles.

Question 4

Describe glutamate re-uptake and degradation.

Answer 4

In terms of glutamate re-uptake both neurones and glial cells contain excitatory amino acid transporters (EAAT). These are a family of five different sodium-ion dependant co-transporters which function to transport glutamate from the synaptic cleft back into the neuronal glial cell for subsequent degradation.

For the subsequent degradation - glutamate transported into the glial cells via the EAATs is converted into glutamine via the action of the enzyme glutamine synthetase. Glutamine is then transported out of the glial cells by a second transporter termed the System N transporter (SN1) where it is then transported into neurones via the System A transporter 2 (SAT2).

Question 5

What are the broad families of neurotransmitter receptors?

Answer 5

Ligand-gated ion channel receptors (iontropic):

• Membrane spanning domain which forms an ion channel
• Neurotransmitter binding allows for ions to pass through the membrane where it can increase or decrease the chance of a nerve impulse or an action potential firing

G protein-coupled receptors (metabotrophic):

• 7-transmembrane domain structure with an extracellular domain for neurotransmitter binding. There is also an intracellular C-terminal domain.
• Neurotransmitter binding activates G proteins which then dissociate from the receptor and interact with ion channels or bind to other effector proteins, activating secondary messenger pathways which can also open/close ion channels.

Question 6

List the different ionotropic receptors.

Answer 6

• AMPA receptors
• NMDA receptors
• Kainate receptors

Question 7

List the different metabotropic receptors.

Answer 7

• Group I (mGlu1 and mGlu5 subtypes)
• Group II (mGlu2 and mGlu3 subtypes)
• Group III (mGlu4, mGlu6, mGlu7, mGlu8 subtypes)

Group I receptors are predominantly postsynaptic whereas group II and group III receptors fit with their inhibitory function as they are predominantly presynaptic.

Question 8

Describe AMPA receptors.

Answer 8

Four subunits types:

• GluA1
• GluA2
• GluA3
• GluA4

These subunits are organised in a hetero-tetrameteric structure ‘dimer of dimers’

Most commonly the structure is composed of:

• 2 GluA2 subunits
• 2 GluA1, 3 or 4
• Two of the four binding sites must be occupied for channel opening
• Current increases (via influx of sodium ions into the channel) as more binding sites are occupied
• Presence of a GluA2 subunit prevents the flow of Ca2+ ions. Important as calcium has a range of intracellular effects that can be excitotoxic. So GluA2 subunit protective against excitotoxicity.

Question 9

Describe NMDA receptors.

Answer 9

Three subunit types:

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

Most commonly this structure is comprised of:

• 2 GluN1 subunits
• 2 GluN2 (or GluN3 less commonly)
• All binding sites need to be occupied for channel opening
• GluN3 subunits are inhibitory to NMDA receptor function
• NMDA receptors are both ligand and voltage gated ion channels.

Ligands:

• Glutamate binds to the GluN2 subunit.
• Glycine or D-serine binds to the GluN1 subunits.

Voltage Gated:
- There is a magnesium ion block in the channel at the resting potential. During depolarisation, the magnesium ion exits the ion channel and enables it to be open. This allows the subsequent influx of ions.

Question 10

Describe Kainate receptors.

Answer 10

Five subunit types:

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

The Kainate receptors are tetrameric:

• GluK1-3 can form homomers or heteromers
• GluK4 and 5 subunits can only form heteromers with GluK1-3 subunits
• Glutamate binding required for channel opening
• Limited distribution of Kainate receptors in the brain compared to AMPA/NMDA receptors and much less is known about their function.

Question 11

Describe group I metaboreceptors.

Answer 11

• Coupled to the Gq protein.
• PIP2 -> DAG and IP3
• IP3 activates its own receptor - which is a calcium channel, leading to calcium release
• Important roles in synaptic plasticity and long term potentiation (LTP).

Question 12

Describe group II and group III metaboreceptors.

Answer 12

• Coupled to the Gi/o protein.
• Pathway involving the inhibition of the adenylyl cyclase which subsequently inhibits cAMP production.
• Autoreceptors - inhibit neurotransmitter release

Question 13

How can excitatory neurotransmitters e.g. glutamate cause displacement of a membrane potential?

Answer 13

Excitatory neurotransmitters (e.g. glutamate) lead to neuronal membrane depolarisation

• Depolarisation - displacement of a membrane to a more positive value. Required in order to meet the threshold to fire a nerve impulse/action potential.
• Hyper-polarisation - displacement of a membrane potential towards a more negative value. Inhibits action potential firing by increasing the stimulus required to fire that action potential. Can be a result of inhibitory neurotransmission.

Question 14

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

Answer 14

AMPA - influx of Na+ and the efflux of K+.

NMDA - influx of Na+ and Ca2+, and the efflux of K+.

Kainate - influx of Na+ and the efflux of K+.

Question 15

Describe the excitatory post-synaptic current in different ionotropic receptors.

Answer 15

• The excitatory post-synaptic current (EPSC) represents the flow of ions and change in current across a post-synaptic membrane.
• A change in current across a post-synaptic membrane leads to 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. EPSCs generated by AMPA receptors are much larger than the NMDA and Kainate receptors.
• 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 occurs when we have too much excitation. This happens under pathological conditions such as Alzheimer’s disease or a stroke.

One example of this can be seen with damage to vesicular glutamate transporters:

• If these transporters are not functioning correctly glutamate can accumulate in the cytosol in the presynaptic neurones.
• Normally the EAAT take high concentrations of glutamate from the synaptic cleft to lower concentrations in the cytosol (in the neurone). However in this case there is a build up of glutamate in the pre-synaptic neurones cytosol so EAAT reverse their function and begin to pump glutamate out of the neurone into the synaptic cleft.
• Therefore there is glutamate being released without the presence of an action potential.
• This leads to the activation of AMPA receptors and NMDA receptors which leads to the influx of calcium into the post synaptic neurone in an uncontrolled fashion.

Excessive Ca2+ can have multiple implications:

• mitochondrial damage (which can stop cellular respiration taking place)
• oxidative stress (by formation of free radicals)
• apoptosis

Question 17

How is Alzheimer’s disease linked to excitotoxicity and how can it be dealt with?

Answer 17

• Alzheimer’s disease is a neurodegenerative disorder characterised by neuronal cell death in the hippocampus. Also subsequent neuronal cell death throughout the cerebral cortex.
• In Alzheimer’s disease, NMDA receptor over-activation linked with glutamate mediated neurotoxicity contributes to neuronal cell death.
• Memantine is a low-affinity NMDA receptor antagonist. It can be used to treat moderate to severe Alzheimer’s. This drug functions by blocking the excessively open NMDA receptor ion channels to reduce glutamate mediated neurotoxicity. This will therefore reduce neuronal cell death.

Question 18

Describe long term potentiation.

Answer 18

• Refers to the persistent strengthening of a synapse based upon repeated patterns of activity.
• Underlies important processes, including both learning and memory - initial phase involves glutamatergic neurotransmission.

Mechanism of action:

• Glutamate binds to and activates AMPA receptors, resulting in Na+ flowing into the post-synaptic neuron causing post synaptic membrane depolarisation
• Due to depolarisation, NMDA receptors open removing the voltage-gated Mg2+ ion block (allows both sodium and calcium to enter the neurone
• Ca2+ ions that 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 that lead to the insertion of new AMPA receptors into the post-synaptic membrane
• New AMPA receptors into the post-synaptic membrane increases the post-synaptic membranes sensitivity to glutamate and also increases ion channel conductance
• This underlies the initial phase of long-term potentiation (LTP)