Lecture 7: Glutamate and Excitotoxicity

What is Glutamate

• A diuretic amino acid widely found in foods with an umami flavour

• This umami flavour occurs due to the presence of modified taste receptors based on mGluRs, which allow for this sensation to be generated

What are the primary neurotransmitters in the human CNS and how do they contribute to brain function?

• Glutamate = Most common neurotransmitter in the CNS (~70% of synapses).

  • Essential for computational power in the brain →drives cognitive processes.

  • Malfunctions → CNS disorders.

  • Number of neurons: 60 x 10⁹.

• GABA = Inhibitory neurotransmitter (~30% of synapses).

  • Number of neurons: 26 x 10⁹.

• Neuromodulators (<0.1% of neurons):

  • Dopamine (VTA, SNpc): 400-600 x 10³ neurons

  • Serotonin (raphe): 300 x 10³ neurons

  • Acetylcholine (nucleus basalis Meynert): 200 x 10³ neurons

  • Noradrenaline (locus coeruleus): 20-50 x 10³ neurons

How is Glutamate Synthesised?

• Glutamate is produced as a side product of major metabolic pathways.

• α-ketoglutarate from the TCA cycle is converted to L-glutamate by aminotransferase → creates a large cellular supply.

• Glutamate is the precursor to GABA via glutamate decarboxylase (GAD).

• It can be generated easily due to abundant precursors and its role in producing the inhibitory neurotransmitter GABA.

• Glutamine–glutamate interconversion is essential for maintaining local glutamate levels for neurotransmission

Why is the Glutamate Synpase Described As Having A Tripartitete Stucture

• It involves three components:

  1. Presynaptic terminal (glutamate-filled vesicles)

  2. Postsynaptic dendritic spine (Glu receptors)

  3. Astrocytic processes

• Astrocytes play key roles in:

  • Regulating glutamate levels

  • Supporting receptor function

  • Maintaining synaptic organisation

• Astrocytic uptake systems are essential for preventing Glu toxicity and shaping synpatic signalling

How is Glutamate Release During Synaptic Transmission

• Neuronal cytosol contains ~10 mM L-glutamate.

• VGLUT loads Glu into vesicles using a proton antiporter → generates high concentrations of Glu (~100 mM) stored in vesicles.

• Upon depolarisation, vesicles fuse with the presynaptic membrane, releasing Glu into the synaptic cleft.

• Glutamate then activates ionotropic or metabotropic receptors on the postsynaptic neuron

• Glutamate actions terminated by Glu uptake processes

How Is Glutamate Cleared From The Synaptic Cleft and Recycled?

• Neuronal EAATs (EAAT1/2): Na⁺/K⁺-dependent uptake back into presynaptic terminals → movement of Glu against its concentration gradient

• Astrocytic transporters:

  • GLT-1 and GLAST (members of the amino acid transporter superfamily) remove Glu from the extracellular space and sequester it in astrocytic processes.

  • Inside astrocytes: Glutamate is converted to glutamine (inactive in excitatory transmission).

  • Glutamine is transported out of the astrocytic terminal, back to presynaptic neurons and reconverted into Glu → glutamate–glutamine cycle.

• System keeps extracellular glutamate at ~1 μM, preventing excitotoxicity

  • Regulates local Glu levels -? provides a source for pre-synaptic vesciles

• α-ketoglutarate from mitochondria replenishes glutamate during high activity.

What Transporters Regulate Glutamate and Glutamine Levels in Neurons and Astrocytes

• EAAT1-5 (Excitatory Amino Acid Transporters) – extracellular glutamate clearance

  • GLu Aspartate Transporter (GLAST/EAAT1) – primarily astrocytes

  • EAAT2 (GLT-1) – astrocytes, major contributor to glutamate uptake

  • EAAT3 (EAAC1) – neuronal glutamate uptake

  • EAAT4/5 – specialised neuronal roles

• 2. System N and A transporters – glutamine transport

  • SN1 (System N) – L-glutamine export from astrocytes

  • SA1 (System A) – L-glutamine import into neurons

• 3. VGLUT1-3 (Vesicular Glutamate Transporters) – intracellular L-glu loading

  • concentrate glutamate into synaptic vesicles for release

• Failure of any of these transporters disrupts glutamate homeostasis, impacting extracellular glutamate levels and synaptic signalling.

What Are Ionotropic Glutamate Receptore (iGluRs)

• Ligand-gated ion channel receptors → Glutamate binds to the extracellular face of the receptor

  • Non-selective cation channels

• Generates an E_rev of 0mV → Non-selective for Na+/K+, with some Ca2+ permeability

  • Ca2+ → important intracellular signal

• Contribute to neuronal excitabilty

What Are Metabotropic Glutamate Receptors (mGluRs)?

• They are G-protein-coupled receptors

  • The receptor binds to the G-protein when activated

  • Hydrolysis of GTP to GDP with the a-protein or B-y dimer component interacting with a secondary messenger system to cause downstream changes and activation of signalling molecules

• 3 Main Groups → Groups I-III

What Are the 3 Main Classes of iGluRs

• Defined by preferential agonist binding

  • AMPA-R

  • NMDA-R

  • Kainate-R

• Located in the postsynaptic density → readily activated by Glu release from the presynaptic terminal, causing depolarisation and AP firing

• Generate an inward current

  • E_rev ~0mV; generate current as membrane is typically hyperpolarised

What are the Key Agonists and Antagonists of NMDA-Rs?

• Agonist: NMDA (N-methyl-D-aspartate) → selectively activates NMDA-Rs only

• Antagonist: D(-)-2-amino-5-phosphonopentanoate (D-AP5) – selectively blocks NMDA-Rs

What are the Key Agonists and Antagonists of AMPA-Rs?

• Agonists: AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)

  • Preferentially activates AMPA over kainate receptors

  • Selective agonists

• Antagonists:

  • NBQX (2,3-Dihydro-6-nitro-7-sulfamoyl-benzo[f]quinoxaline) – competitive antagonist

  • AP5 (competitive antagonist, with NMDA selectivity) shows poor discrimination between AMPA and kainate

What are the Key Agonists and Antagonists of Kainate-Rs?

• Agonists:

  • Kainate (kainic acid)

  • Preferentially activates kainate over AMPA receptors

  • Selective agonists exist, but are not fully exclusive for a single receptor type

• Antagonists:

  • NBQX (2,3-Dihydro-6-nitro-7-sulfamoyl-benzo[f]quinoxaline) – competitive antagonist

  • AP5 – competitive antagonist (primarily NMDA selective)

    • Both NBQX and AP5 bind the glutamate site on ionotropic receptors, competing with endogenous glutamate

      • Poor discrimination between AMPA and kainate receptors

Why are NMDA receptors highly Ca²⁺ permeable, and what is the significance?

• NMDA receptors are highly permeable to Ca²⁺ (≈13× that of Na⁺).

• Changing extracellular Ca²⁺ shifts the equilibrium potential (Erev), highlighting Ca²⁺ contribution and permeability through NDMA.

• Significant contributor to Erev at physiological Ca²⁺ concentrations.

• In CA1 neurons, NMDA-R activation produces large intracellular Ca²⁺ increases (visualised with dyes like Oregon Green BAPTA-1/2).

• Critical for synaptic signalling, excitotoxicity, and stroke pathophysiology.

How are NMDA receptor-mediated synaptic currents isolated in experiments?

• Non-NMDA receptors were blocked with NBQX.

• Membrane potential held at +40 mV to relieve Mg²⁺ voltage-dependent block.

• At -70 mV, Mg²⁺ block prevents NMDA-R activation.

• Depolarisation allows NMDA-Rs to conduct, enabling measurement of Ca²⁺ currents.

• The technique demonstrates voltage-dependent Mg²⁺ block and functional NMDA-R Ca²⁺ signalling.

How do NMDA receptors contribute to fast excitatory neurotransmission?

• At hyperpolarised post-synaptic potentials, glutamate preferentially activates AMPA receptors; NMDA-Rs are inactive (voltage-dependent block by Mg²⁺)

• NMDA-Rs contribute when the membrane is significantly depolarised, allowing Ca²⁺ influx.

• Ca²⁺ influx acts as a signal for neurotransmitter release and is critical for synaptic plasticity.

• NMDA-R activation linked to changes in synaptic strength via increased intracellular Ca²⁺.

What are the key functional properties of NMDA receptors?

• Location & Role: Postsynaptic → depolarisation; presynaptic autoreceptors → regulating and increasing neurotransmitter release.

• Ion Permeability: Na⁺, K⁺, high Ca²⁺ permeability

  • generate strong intracellular Ca²⁺ signals.

• Voltage Dependence: Mg²⁺ block at resting potentials; relieved by depolarisation.

• Kinetics: Slow activation and deactivation → produce enduring responses when active.

• Co-agonist Requirement: Glycine + Glutamate needed for full activation

  • Dual agonism receptors

What are the key functional properties of AMPA receptors?

• Location and Role: Postsynaptic → depolarisation; not presynaptic.

• Ion Permeability: Na⁺, K⁺; low Ca²⁺ permeability.

• Kinetics: Fastest on/off → supports the early phase of fast excitatory neurotransmission.

• Equilibrium Potential: ~0 mV.

What are the key functional properties of Kainate Receptors?

• Location: Postsynaptic→ depolarisation; can be presynaptic → modulate NT release,

• Ion Permeability: Na⁺, K⁺; intermediate Ca²⁺ permeability (subunit-dependent).

• Kinetics: Intermediate

• Function: Presynaptic receptors can increase or decrease neurotransmitter release depending on subunit composition.

• Equilibrium Potential: ~0 mV.

What are the functional properties of mGluRs Group I

• Mainly Postsynaptic

• G_q-linked -> hydrolyses PIP

• Depolarisation neurons/increase excitability in post postsynaptic membrane by:

  • Close K⁺ channels

  • Enhance NMDA-R mediated currents

What are the functional properties of mGluRs Group II

• Mainly Presynaptic

• G_i/o-linked

• Inhibits Adenylate cyclase → decrease cAMP

• Decrease NT release by:

  • Downregulate Ca²⁺ channel

What are the functional properties of mGluRs Group III

• Mainly Presynaptic

• G_i/o-linked

• Inhibits Adenylate cyclase → decreases cAMP

• Decrease release

  • Downregulate Ca²⁺ channel

How does activation of Group I mGluRs (e.g., mGluR1) produce excitatory effects?

• Activation by agonist DHPG → slow depolarisation of the postsynaptic neuron.

• TTX blocks action potentials, highlighting the underlying depolarising effect.

• Enhanced NMDA receptor function, seen as an increased outward NMDA current, following DHPG application

• Mechanisms:

  • Closure of K⁺ channels → changes resting membrane potential.

  • Potentiation of NMDA receptors → stronger postsynaptic response.

• Net effect: Excitatory modulation of postsynaptic signalling.

How do Group 2 and Group 3 mGluRs function as inhibitory autoreceptors in the hippocampus?

• Group 3 mGluRs:

  • Regulate synaptic transmission between CA3 and CA1 in the hippocampus. →generate post-synaptic response.

  • Agonist D, L-AP4 depresses postsynaptic potential amplitude → evidence of presynaptic inhibition.

• Group 2 mGluRs:

  • Regulate transmission between dentate gyrus and CA3 → generates postsynaptic response.

  • DCG-IV (group 2 specific inhibitor and agonist) suppresses evoked postsynaptic response → confirms presynaptic modulation.

• Mechanism: Presynaptic mGluR activation reduces Ca²⁺ entry, reducing likelihood of neurotransmitter release

What are the key functional roles of ionotropic and metabotropic glutamate receptors?

• Ionotropic Glu-Rs:

  • Activated by Glu, released into the synaptic cleft.

  • Postsynaptic location within PSD: increases neuronal excitability → fast EPSPs)

  • Extrasynaptic location: similar excitatory effect is seen when activated.

  • Presynaptic: can regulate neurotransmitter release.

• mGluRs:

  • Group 1 (postsynaptic): modulate excitability and shape fast/slow EPSPs.

  • Group 2 & 3 (presynaptic): inhibit Glu release (negative feedback).

Describe the Morphology of a Typical Glu Synapse (Grey Type 1)

• Large round vesicles

• Dense asymetrical postsynaptic membrane specialisation

How are Glu-R gene families organised, and how does this contribute to receptor diversity?

• l: Molecular complexity and subunit composition expand functional diversity of Glu receptors.

• Ionotropic Glu receptor diversity arises from multiple gene families and subunit combinations.

  • AMPA-R: 4 subunits → GluA1–GluA4.

  • Kainate-R: 2 subunit families → GluK1–3 + GluK4–5.

  • NMDA-R: Defined by GluN1 + GluN2 (A–D); NR3A/B can form receptors with reduced Glu sensitivity.

• Metabotropic Glu Receptors have homology differnces in the subunits that form Group I, II & IIII receptors,I, each with distinct signalling roles.

What is the Stoichometry of AMPA and Kainate Receptors?

• Tetramers: 4 subunits, each with a glutamate-binding domain.

• 4 Glu molecules needed for full channel activation.

• Can form homomeric or heteromeric receptors.

• Kainate receptors can act as metabotropic receptors due to some subunit combinations (e.g., GluK5) by coupling to G-proteins, influencing secondary pathways and neuronal excitability.

What is the Stoichiometry of NMDA Receptors

• NMDA receptors are tetramers composed of GluN1 + GluN2 subunits.

• GluN1 binds glycine; GluN2 binds glutamate.

• Require dual agonism (Gly + Glu) for full activation.

• Must be heteromers (cannot form functional homomers)

What is the Structure of Metabotropic Glutamate Receptors?

• Class C GPCRs with:

  • Large extracellular N-terminal “Venus flytrap” (VFT) ligand-binding domain

  • Cysteine-rich domain (CRD)

  • 7 transmembrane (7TM) domains per subunit

  • Intracellular C-terminal

  • G-protein binding at intracellular loops 2 and 3

• Function as homodimers, linked by disulfide bonds between VFT domains

  • Glu binds both subunits, but only one subunit must engage with the G-protein for activation

• Structurally related to LIVBP (bacterial leucine/isoleucine/valine-binding protein)

How was excitotoxicity first discovered?

• 1957: Lucas & Newhouse showed that systemic IV administration of MSG in young mice (P2–16) caused inner retinal degeneration within 2 weeks.

  • First report of toxic effects of L-Glu

• 1969: John Olney demonstrated CNS and brain damage in newborn mice & primates after systemic MSG.

  • Led to concern about the use of MSG as a flavour enhancer in baby food, as newborns have vulnerable systems.

• Olney introduced the term “excitotoxicity” to describe amino-acid–induced necrotic neuronal death.

What is excitotoxicity and what causes it?

• Excessive activation of glutamate receptors (iGluRs + mGluRs).

• Driven by overexcitation, often from elevated extracellular L-glutamate.

• Early work showed L-Glu neurotoxicity mimicked by L-aspartate and NMDA, confirming involvement of glutamate receptors.

• The term became widely used in the 1980s as understanding of glutamate neurotransmission expanded.

How is excitotoxicity demonstrated in vitro?

• Embryonic neuronal cell cultures are exposed to iGluR agonists to induce toxicity.

• Three forms of excitotoxicity were observed:

  • Acute: occurs within 1–3 hours

  • Delayed: occurs within 2–12 hours

  • Slow: occurs within 24–72 hours

• The type of excitotoxicity depends on:

  • Receptor subtype involved and mechanism involved

  • Degree of receptor activation

  • Cell type, since different neurons express different iGluRs/mGluRs

• These systems are used as assays to test substances for their ability to block or reduce excitotoxicity.

What is acute L-glutamate excitotoxicity and how is it observed in vitro?

• Seen in embryonic neuronal cultures (somata, neurites, developing axons), with photomicrograph imaging.

• High [Glu] applied for ~30 min causes disruption + death of cell bodies.

  • Rupturing of cell membrane and loss of cellular contents

• Produces rapid necrotic cell death within 1–3 hrs

What is the Mechanism and Pharmacology Behind Acute Swelling Induced L-Glu Excitotoxicity?

• Induced by high extracellular Glu (0.5–1 mM) applied for up to 30 min.

• Blocked by NMDA-R antagonists → necrosis mainly NMDA-R mediated.

• Not Ca²⁺-dependent (still occurs when extracellular Ca²⁺ is removed).

• Prolonged exposure and activation desensitises AMPA/Kainate Receptors→ non-NMDA receptors are not involved due to the duration of Glu application (inactive).

• NMDA-R activation → strong depolarisation → Na⁺ & Ca²⁺ influx (NMDA ligand + VG ion channels) and passive Cl⁻ influx into channel.

• Increased intracellular osmolarity → H₂O enters down the osmotic gradient via aquaporins → swelling (oedema).

• Leads to mitochondrial collapse, membrane rupture, and necrotic lysis.

• Rapid destructive process similar to Olney’s early excitotoxicity observations → traumatic destruction of neruosns due to excesive NMDA-R activation

What is Delayed Excitocity and How Is It Shown In Vitro?

• Described Dennis Choi

• Observed in cultured cortical neurons from 14-17-day-old mice embryos, 5-7 days in vitro

• Brief Glu exposure (5 min) → cells look normal and remain intact at 1 hr.

• After 9 hrs, cell bodies show degeneration and collapse → delayed excitotoxicity.

• Produces delayed cell death 6–12 hrs after exposure (slower than acute).

• Morphology shows a mix of apoptosis + necrosis.

What is the pharmacology of delayed L-Glu excitotoxicity?

• Induced by high levels of extracellular Glu (0.5–1 mM) applied for ≤ 5 min.

• Cells recover from initial acute swelling (receptor activation and recovery from ionic changes)→ no immediate lysis.

• Blocked by BOTH NMDA and non-NMDA antagonists, even when applied 2–8 hrs after L-Glu exposure.

  • Indicates involvement of all iGlu-Rs.

• Delayed antagonist window suggests possible clinical relevance (late therapeutic opportunity).

What is the mechanism of delayed Ca²⁺-dependent excitotoxicity?

• Ca²⁺-dependent with prolonged rise in intracellular Ca²⁺ , mainly via NMDA-Rs and Ca²-permeable AMPA/Kainate-Rs.

  • Blocked by removing extracellular Ca²⁺ from the bathing medium→ less excitatory mechanism

• Involves all iGluRs, not just NMDA-Rs.

• Likely self-propagating and sustaining process: early-dying neurons (1^st wave of cell death) release Glu → triggers more excitotoxic damage → (Release of intracellular content activating excitatory amino acid receptors on other neurons, causing death and further Glu release) → ongoing cell death.

• Similar to progressive neurodegeneration and cell loss as seen in diseases.

What is The Mechanism Behind Slow Excitoxicity

• Described by JA Lamb as cell death (apoptosis) after 1 or more days in vitro

• Likely Ca²-dependent, with a prolonged rise in Ca²⁺ through

  • Ca²⁺-permeable AMPA/Kainate receptors, and/or

  • voltage-gated Ca²⁺ channels.

• Sustained Ca²⁺ elevation → downstream signalling → apoptosis.

• Evidence:AMPA application shows TUNEL-positive staining (DNA fragmentation), confirming apoptosis.

What is the pharmacology of Slow L-Glu excitotoxicity?

• Induced by prolonged AMPA/Kinate receptor agonists for 24-72 hours at Non-NMDA receptors

• Blocked by CNQX (non-NMDA antagonist) → Non-NMDA-R process

• Not blocked by MK-801 (NMDA R specific antagonists)→ not NMDA-R mediated.

How do glutamate receptors contribute to excitotoxicity?

• Two main processes drive excitotoxicity:

  1. iGluR overactivation (AMPA, Kainate, NMDA).

  2. Group I mGluR activation, which enhances NMDA-R Ca²⁺ entry.

• Ca²⁺ is the central trigger:

  • NMDA-Rs (high Ca²⁺ permeability) + Group I mGluRs trigger a large rise in cytosolic Ca²⁺ .

  • Elevated Ca²⁺ activates signalling pathways causing apoptosis and (to a lesser degree) necrosis.

• Ca²⁺-induced intermediates (e.g., ROS, proteases, lipases, endonucleases) → DNA fragmentation & cell death.

• Ion loading → necrosis:

  • Excess Na⁺ & Ca²⁺ influx, plus passive Cl⁻ entry, increases osmotic pressure.

  • Water influx → neuronal swelling → membrane rupture → necrotic

What morphological profiles occur during excitotoxicity in vivo?

• Excitotoxicity produces an apoptotic–necrotic continuum (shown in vivo after kainate injection in neonatal rat cortex).

• It gives rise to 3 morophological profiles

  • Apoptotic profile

  • Intermediate hybrid profile

  • Necrotic profile

Describe the Apoptotic Profile in Response to Excitotoxicity in Vivo

• Kainate injection into the cerebral cortex neotate the rat in vivo results in

• Dense, round chromatin clumps (condensed).

• Intact nuclear & plasma membranes.

• Condensed cytoplasm.

Describe the Intermediate Hybrid Profile in Response to Excitotoxicity in Vivo

• Kainate injection into the cerebral cortex neotate the rat in vivo results in

• Partially condensed, irregular chromatin.

• Plasma and nuclear membranes are mostly intact.

• Disruption of intracellular organelles (ER, Golgi).

• Many cytoplasmic vacuoles.

Describe the Necrotic Profile in Response to Excitotoxicity in Vivo

• Kainate injection into the cerebral cortex neotate the rat in vivo results in

• Loose, irregular chromatin.

• Membrane dissolution and cytoplasmic breakdown.

• Extensive vacuolation.

What is Olneys Definition of (Neuro)Excitoxicity?

• Cell death caused by over-activation of ionotropic glutamate receptors (iGluRs) → leads to necrosis and/or apoptosis.

• High extracellular L-Glu is not inherently toxic — toxicity occurs when iGluRs are activated persistently and the system loses control.

• mGluRs may contribute:

  • Facilitatory/permissive role → enhance iGluR-mediated effects.

  • Inhibitory role → reduce presynaptic Glu release and limit excitotoxic drive.

What in vivo conditions increase endogenous L-Glu release and cause excitotoxicity?

• Ischaemic stroke / cardiac arrest: restricted blood flow → O₂ deprivation→ depletion of metabolic stores→ loss of ion gradients → mitochondrial failure → uncontrolled Glu release.

• Hypoxia → oxygen depletion (e.g., drowning, asphyxiation):

• Epilepsy: excessive neuronal firing → excessive Glu release → excitotoxic damage; contributes to progressive deterioration .

• Neurodegeneration: necrotic cell death releases Glu → vicious cycle of further excitotoxic damage due to iGluR overactivation → triggers necrosis + apoptosis.

How can exogenous substances trigger excitotoxicity in vivo?

• Exogenous iGluR agonists can mimic glutamate and overstimulate receptors.

• Found in certain environmental or dietary toxins (e.g., some fermented or aged foods such as marmite, parmesan, soy sauce).

• Excessive activation of iGluRs → neuronal damage and potential neurodegeneration.