Glutamate

What is significant about glutamate?

• Main excitatory neurotransmitter in the brain

Is glutamate excitatory or inhibitory

• Excitatory

What are the uptake transporters for glutamate and where are they expressed

• EEAT1,2 nearby glia

• EEAT3,4 low expression in neurones

• EEAT5, only in retina

• Can be for reuptake into preSN or uptake into astrocytes

Simply, how is glutamate signaling terminated?

• Reuptake into preSN via EEAT

• Uptake into astrocytes via EEAT, use glutamine synthase to form glutamine that can be released via GlnT and uptaken by preSN GlnT to be resynthesized into glutamate via glutaminase

Synthesis of glutamate

• Glutamine released from astrocyte via GlnT

• Uptake into preSN via GlnT

• Glutamine —→ glutamate via glutaminase

What chemicals do glutamate transporters pump in / out?

• Glu, H+, 3Na+ pumped in to preSN / astrocyte

• 1K+ pumped out

• If gradient is disrupted can work in reverse, so glutamate effluxed (neurotoxicity and stroke)

What happens if glutamate uptake is faulty e.g. EAAT1 knockout

• Increases excitation - hyperexcitability

• Seizure and neurotoxicity risk

• EEAT knockout mice have compromised cerebellum function, less motor coordination

What packages glutamate into vesicles in the preSN?

• VGLUT1-3 pumps

Simply, what are the different types of glutamate receptors?

• Ionotropic (LGIC) AMPAR and NDMAR

• Metabotropic (GPCR) mGluR1-8 - not discussed

What type of ion channels are glutamate receptors

• Ligand gated, cation selective

Describe the AMPA receptors

• Na+/K+ permeable

• Subunits made of GluA1-4

• Bind ligand, open and Na+/K+ enters postSN

• Opening in response to ligands measured with patch clamp physiology,

Describe the importance of Glu2 in AMPA neurotransmission

• Subunit of AMPA receptor

• Directs majority of excitatory neurotransmissoin

• 99% of adult GluA2 pre-mRNA is deaminnated so Gln607—→Arg607 in pore loop, change to positive charge prevents Ca2+ influx

• Most adult AMPAR therefore Ca2+ impermeable

Why is it important AMPAR are Ca2+ impermeable?

• Transgenic mice with only Gln607 develop seizures and die early

• Too much Ca2+ influx in response to glutamate signaling triggers seizures and excitotoxicity

What are various ligands of AMPAR

• Glutamate

• AMPA

• Quisqualate

• Domoate

Compare domoate signalling to other AMPAR ligands

• Slower

• Seen on patch clamp EP where peak takes longer to reach

• Glu/AMPA/quisqualate rapidly activate and desensitise to enable sizeable but sharp and brief impulses

• Shellfish poisoning

Describe NMDARs

• Ag: NMDA, glutamate

• Have modulatory sites for glycine and polyamine binding (co-agonists)

What is the NMDAR channel permeable to? What does this mean?

• Ca2+, therefore over-activation can cause cytotoxicity

• A little Na+

How does glutamate mediate synapse learning?

• At positive membrane potentials (at rest), Mg2+ attracted to NMDA channel pore, blocks even if lignd bound

• Moderate glutamate release activates AMPAR, but Mg2+ still binds NMDA

• Increased APs and prolonged depolarisation enables Mg2+ leave pore, Ca2+ influxes postSN which triggers second messenger cascade for synapse remodelling

Compare AMPAR and NMDAR

• AMPAR fast neurotransmission, mostly Na+, some K+

• NDMAR modulatory as only during sustained depolarisation, mainly Ca2+, a little Na+

What are the clinically relevant sites on NMDAR?

• Receptor site for NDMA antagonists

• Modulatory sites for glycine and polyamine antagonists

• Channel blocking drugs

Describe the process of excitotoxicity

• No blood = no O2 = no ATP

• Na+/K+ ATPase antiporter fails

• Synapse depolarizes

• Ca2+v open

• Glutamate released

• Bind AMPAR / NMDAR at high concentrations, depolarisation and Ca2+ influx in postSN

• Ca2+ ATPase antiporter fails, maintains depolarisation and overloads mitochondria, increasing ROS

• Ca2+ also activates proteases, lipases, NOS and ROS, membrane damage

• EEAT inhibited by depolarisation, no glutamate uptake, maintains APs

In general, when is excitotoxicity seen?

• Any condition with excessive glutamate release e.g. brain trauma, some epilepsy

Discuss the problems so far with using ADPAR and NMDAR in stroke treatment?

• AMPAR antagonist reduced post-stroke ischaemic damage in rats, but clinical trials in humans stopped halfway through the trial when interim results suggested unlikely to achieve statistical significance

• NMDAR antagonist nerinetide did not improve outcome of those who had stroke-causing blood clots removed

What other areas could target cytotoxicity in stroke treatment that haven’t yet been successfully pharmacologically targetted?

• Zn2+ neurotoxicity, Zn2+ also stored in vesicles released by neurotransmission

• Acidotoxicity as ischemic areas are acidic, which activates the permeable acid-sensing ion channel 1a which is Ca2+ permeable

Simply, what is a stroke

• Blood clot cuts off circulation to part of the brain

• Decrease O2, decrease ATP

• Neurones die rapidly in ischaemic core, and surrounding neurones in penumbra are damaged by dying cells releasing ros, proteases etc, and this is what rapid response to strokes is trying to minimise