Glutamate

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Glutamate GABA
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Glutamate

• Glutamate (and aspartate) are two of the major
  excitatory amino acids
• Glutamate is ionized form of the amnio acid
  glutamic acid, which is used by all cells in body
  for construction of proteins
• Only in brain is glutamate used as a
  communication molecule, and ALL neurons in brain
  have glutamate
• However, only glutamatergic neurons release it as
  a NT
• Glutamate can be metabolically derived from
  glucose, or from proteins in diet
• The enzyme glutaminase converts glutamine to
  glutamate

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Glutamate Release/Reuptake

• Glutamate packaged into vesicles by VGLUT1,
  VGLUT2, and VGLUT3
• Good markers for glutamatergic neurons- only
  found in cells that release glutamate
• Most cells have either VGLUT1 or VGLUT2, not both
  (VGLUT3 is rare)

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Glutamate Release/Reuptake

• After release from axon terminal, glutamate is
  rapidly removed from synapse by axon terminals
  and astrocyte transporters
• Excitatory amino acid transporters (EAAT1-5)
• EAAT1 and EAAT2 in astrocytes, EAAT3 most common
  in neurons
• High levels of glutamate can be excitotoxic, and
  reuptake by astrocytes is very important
• Ex amyotrophic lateral sclerosis (ALS or Lou
  Gehrigs Disease) characterized by degeneration
  of motor neurons in cortex and spinal cord have
  abnormalities in EAAT2 transporters
• After astrocytes take up glutamate, it is broken
  down into glutamine by glutamine synthetase
  enzyme
• Glutamine then transported back out of
  astrocytes, taken up by neurons, converted to
  glutamate, and repackaged
• Glutamine does NOT produce neuronal excitation-
  safety mechanism?

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Organization of the Glutamatergic System

• Widely distributed throughout brain- major
  excitatory NT
• Used by pyramidal neurons (motor cortex output)-
  project to thalamus, striatum, limbic system, and
  brain stem
• Also widely found in cerebellar cortex and
  hippocampus
• Heavily involved in synaptic plasticity
  (remodeling synaptic strength and connections),
  learning, and memory
• Glutamate is so widely distributed in the brain,
  it is difficult to map out vital pathways/areas

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Glutamatergic Receptors

• Glutamatergic receptors also apparently used by
  aspartate and other excitatory amino acids
• Ionotropic glutamatergic receptors (each one has
  different types of receptor subunits)
• AMPA receptor (a-amino-3-hydroxy-5-methyl-4-isoxaz
  ole proprionic acid is agonist)- mostly allows
  Na influx
• Kainate receptor (kainic acid is agonist)- mostly
  allows Na influx
• NMDA receptor (N-methyl D-aspartate)- allows Na
  and Ca influx (and increasing Ca can act
  indirectly as a 2nd messenger)
• NBQX (6-nitro-7-sulfamoly-benzo(f)-quinoxaline-2,3
  -dione) is antagonist for AMPA and kainate
  receptors
• NBQX use shows sedation, reduced locomotor
  activity, ataxia (impaired coordination), and
  protection against seizures

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Glutamatergic Receptors

• NMDA receptors are unusual compared to other
  glutamatergic receptors
• NMDA allows Ca through
• Two NTs needed to stimulate glutamate AND
  glycine/D-serine (a co-agonist)
• Co-agonist site is occupied in most situations
  before glutamate
• Mg ion also plugs the channel
• Mg block stays in place at RP, only under
  depolarization AND both co-agonists being present
  is it dislodged (coincidence detector)
• Phencyclidine (PCP) and ketamine binding site
  blocks the channel when the drug is present
  (non-competitive antagonist)

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Glutamatergic Receptors

• Metabotropic receptors
• Classified as mGluR1 to mGluR8
• Typically either inhibit adenylate cyclase
  (decrease cAMP levels) or activate the IP3
  pathway
• Some are located on axon terminals and act as
  presynaptic autoreceptors to inhibit glutamate
  release
• L-AP4 is is a selective agonist at the
  autoreceptors, which suppresses glutamate release
• mGluR1 deletion mutants show reduced locomotion,
  ataxic gait, poor coordination, and learning
  defecits
• Restoring mGluR1 receptors in Purkinje cells of
  cerebellum restored locomotion and normal
  coordination in mice
• Other studies show involvement in pain
  perception, anxiety, and regulating levels of
  brain excitability

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Long-Term Potentiation

• The coincidence detector aspect of NMDA
  channels thought to be key in learning
• This type of learning is long-term potentiation
  (a specific type of synaptic plasticity)
• Much like classical conditioning, neural basis of
  learning is based on the close timing of two
  events
• In this case, EPSP in cell AND presence of
  glutamate to increase intracellular Ca levels

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Long-Term Potentiation

• If an NMDA antagonist is given (or knockout mice
  for NMDA used) there is impaired learning
• Long-term potentiation is based on NMDA receptor
  activity
• Studies of mice with high-glutamate affinity NMDA
  receptors (Doogie mutation) learn much more
  quickly than control mice
• Doogie mutation also causes enhanced pain
  perception

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Long-Term Potentiation

• LTP is produced by a burst of activity called a
  tetanic stimulus
• LTP can occur in many brain areas, but most
  studied and best understood in hippocampus (which
  use glutamate for LTP)
• Use hippocampal slices kept alive in electolyte
  baths and electrophysiological recordings
• CA1 region recieves glutamate stimulation from
  CA3 region neurons during LTP

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Long-Term Potentiation

• A test pulse is given to CA3 neurons to determine
  synapse strength, and recording is made of
  postsynaptic (CA1) cells
• Get small EPSP in CA1 neurons due to AMPA
  channels
• NMDA do not open since membrane is not
  depolarized enough
• Give tetanic stimulus to CA1 neurons, and get
  much larger CA3 response
• Due to activation of NMDA channels and Ca
  influx, which can act as a 2nd messenger

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Long-Term Potentiation

• Two major phases of LTP
• Induction phase during and immediately after the
  stimulation
• Expression phase enhanced synaptic strength
  measured at a later time
• NMDA receptors are critical for induction phase,
  but not expression phase activity (NMDA blocker
  prevents induction phase, but not expression
  phase)
• AMPA receptors are critical for induction AND
  expression phases

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Long-Term Potentiation

• During LTP, an influx of Ca ions activates a
  Ca/calmodulin protein kinase called CaMKII
• CaMKII phosphorylates AMPA receptors which
  increases AMPA sensitivity to glutamate AND more
  AMPA receptors are inserted into membrane (both
  will increase strength of signaling)
• Ca or CaMKII also phosphorylates NO synthase,
  which leads to increased NO synthesis
• NO can diffuse back presynaptically and increase
  of vesicles of glutamate released per AP
  getting to axon terminal, which increases
  synaptic strength

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Excitotoxicity

• Excessive glutamate release is toxic to neurons
  (experimentally done by injection of MSG)
• Animals showed excessive damage to arcuate
  nucleus of hypothalamus- results in stunted
  skeletal growth, obesity, and reproductive
  abnormalities
• Lesions also occur when other EAAs (aspartate,
  kainate, NMDA) are injected into animals, and
  damage is on postsynaptic cells, but not axons or
  axon terminals

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Excitotoxicity

• Excitotoxicity hypothesis prolonged EAA activity
  holds postsynaptic cell in prolonged
  depolarization leading to cell death
• Excitotoxicity triggered by stimulation of NMDA
  receptors
• If give high levelsEAA for long time- many cells
  die within a few hours due to necrosis (lysis of
  cell due to osmotic swelling)
• If give lower levels of EAA or dont expose as
  long- cells swell, but appear to recover
• However, a few hours later, cells begin to
  disintigrate and undergo apoptosis (can block
  this by NMDA antagonist MK-801)
• Both effects due to excess Ca influx
• Necrosis excess Ca inside causes more water to
  enter cell, bursting it
• Apoptosis excess Ca activates many protein
  kinases, stops ATP production (ATP synthase
  destroyed), release of toxic compounds from
  mitochondria, release of more glutamate and
  aspartate into ECF

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Excitotoxicity

• Can happen from consuming certain foods in humans
• MSG and aspartame? Possibly
• Domoic acid made by marine algae, taken up by
  shellfish. Victims show headache, seizures,
  dizziness, muscle weakness, confusion, and loss
  of short-term memory. Hitchcocks movie The
  Birds based off domoic acid poisoning.
• Also can happen with ischemia (loss of bloodflow
  to areas of the brain). Causes massive release
  of glutamate and EAAs from affected area. An
  NMDA antagonist can reduce excitotoxic effect of
  ischemia.

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GABA

• Two major fast inhibitory NTs g-aminobutyric
  acid (GABA) and glycine
• GABA is synthesized from glutamate by glutamic
  acid decarboxylase (GAD)
• GAD is blocked by allylglycine,
  thiosemicarbizide, and 3-mercaptoproprionic acid
• Symptoms of GABA block are seizures and
  convulsions (plays role in brain excitability)

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GABA Release/Reuptake

• Vesicular GABA transporters (VGAT) package GABA
  and/or glycine into vesicles in axon terminal
• GABA removed from synapse by GABA transporters
  GAT1, GAT2, and GAT3
• GAT1 and GAT2 in neurons and astrocytes, GAT3 in
  astrocytes only
• GAT1 is selectively inhibitable by tiagabine
  (Gabitril) (increases synaptic GABA)- helps
  prevent seizures
• Enzymatic breakdown of GABA via GABA
  aminotransferase (GABA-T) into succinate
• One molecule of glutamate is also made per
  molecule of GABA broken down
• Vigabatrin (Sabril) is irreversible inhibitor of
  GABA-T (anti-convulsant)

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Organization of the GABAergic System

• Fonnum (1987) found that 10 ro 40 of neurons in
  neocortex, hippocampus, substantia nigra,
  cerebellum, striatum, globus pallidus, and
  olfactory bulbs use GABA
• Many GABAergic neurons are interneurons that
  inhibit other neuronal function
• Some project long distances
• Ex GABA neurons project from striatum to globus
  pallidus and substantia nigra. When DA neurons
  are lost in Parkinsons, abnormal firing of GABA
  neurons in striatum are what cause resting tremor
• Ex Purkinje cells in cerebellum project to deep
  cerebellar nuclei and brainstem, and use GABA for
  fine muscle control and coordination. In Holmes
  disease, purkinje cells degenerate and cause
  walking ataxia, impaired hand movements, tremors,
  and defective speech

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GABA Receptors

• GABAA receptors are ionotropic Cl- channels
  composed of five subunits (which vary depending
  on subtype)
• Agonist for GABAA receptors is muscimol (from fly
  agaric mushroom Amanita muscaria)
• When ingested, causes hallucinations
  (macroscopia), mild stimulatory effects,
  hyperthermia, pupil dilation, mood elevation,
  difficulty in concentration, ataxia, anorexia,
  and catalepsy
• Bicuculine is competivie antagonist (causes
  convusions)
• Pentylenetetrazol (Metrazol) (used to be used as
  convulsant therapy for depression) and picrotoxin
  are non-competitive antagonists (also cause
  convulsions)

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GABA Receptors

• GABAA receptors also respond to drugs with
  anxiolytic, sedative-hypnotic, and anticonvulsant
  effects
• Benzodiazapines (BDZs) and barbiturates
  (agonists)
• These are both thought to potentiate effects of
  GABA on the GABAA receptor (but not bind to GABA
  site)
• BDZs like diazepam (Valium) binds to a secondary
  receptor site, and makes it easier for GABA to
  bind, but cannot activate receptor site by
  themselves
• Some drugs are also inverse agonists they bind
  to receptor and make it harder for GABA to bind
  (doesnt block, just reduces effectiveness)
• These drugs are anxiogenic, arousing, and
  proconvulsant
• Ethanol thought to work by similar effects, but
  exact mechanism not known yet
• Why would the brain express receptors for
  synthetic drugs?
• GABAA receptors also respond to neurosteroids
  like dehydroepiandosterone (DHEA)- also make it
  easier for GABA to bind to receptor
  (sedative-hypnotic, anxiolytic)

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GABA Receptors

• GABAB receptors are metabotropic receptors that
  either open K channels, or inhibit adenylyl
  cyclase (lowers cAMP levels)
• GABAA receptor agonists have no effect on GABAB
  receptors
• Baclofen (Lioresal) is a selective agonist which
  is used as a muscle relaxant and antispastic