Glutamate Flashcards
What is the function of glutamate?
It serves as a neurotransmitter
It is a component of many proteins and has other metabolic roles (but we will focus on its role as an excitatory NT)
Because glutamate is found throughout the brain, it is more difficult to assign specific functional roles to this neurotransmitter
What projection neurons use glutamate?
All pyramidal cells in cerebral cortex
hippocampus, amygdala, thalamus. (limbic system)
Glutamate projection neurons embedded in other subcortical nuclei
How do other neurotransmitters interact with glutamate?
To varying degrees, most other ‘classical’ neurotransmitters modulate effects of glutamate on neural activity
Noradrenaline, serotonin, acetylcholine, etc. usually serve to modulate glutamate activity rather than directly affecting neuron potentials themselves
Glutamate is a form of which amino acid?
Glutamate is ionized form of the amino acid glutamic acid
How is glutamate synthesized? (What reactant/enzyme?)
Synthesized from glutamine by glutaminase.
Why does labeling glutaminase not help us identify which neurons are using glutamate?
While we usually identify neurons that are using a particular NT by labeling the specific enzyme involved in its production, this isn’t useful for glutamate because glutamate is too widely used and many cells express glutaminase despite not necessarily using glutamate.
What are the vesicular transporters associated with glutamate and what do they do? Why might they be of interest to researchers?
Different vesicular transporters move glutamate into synaptic vesicles: VGLUT1, VGLUT2, VGLUT3. Knockout mutation usually fatal.
VGLUT transporters found only in glutamatergic neurons and are thus good markers.
Is VGLUT found only on neurons that use glutamate as a primary NT?
No, it’s also expressed on neurons expressing markers that indciate that they use another neurotransmitter (eg. monoamine NT), which suggests that glutamate can be stored and released as a co-transmitter in addition to the primary NT
Why do we see VGLUT expression in the striatum when the intrinsic medium spiny neurons in that area do not use glutamate as their NT?
VGLUT is found in the TERMINALS of the glutamate-releasing neuron, so the VGLUT expression is coming from neurons that are projecting to and synapsing in the striatum, not the neurons that originate from the striatum
How is glutamate removed from the synapse after release?
After release, glutamate is rapidly removed from synapse by Excitatory Amino Acid Transporters (EAAT1 to EAAT5).
Where are EAAT1, EAAT2 and EAAT3 located?
EAAT1-2 on astrocyte glia (adjacent to the glutamate releasing neuron, rather than on the actual neuron terminal itself)
EAAT3 on presynaptic terminals (eg. the terminal of the glutamate releasing neuron)
Majority of glutamate uptake is done by what? What happens to the glutamate after it gets uptaken?
Majority of glutamate uptake is done by astrocytes that then convert glutamate to glutamine by glutamine synthetase.
Why does glutamate not only get reuptaken, but also converted into glutamine?
Storage of excess glutamate as glutamine may serve to protect brain from excessive excitation
What happens to the glutamine that is being stored in astrocytes (after glutamate is uptaken and converted)?
Glutamine is transported out of astrocytes and back to neurons - it can be transformed back into glutamate and thus recycled
Are metabotropic or ionotropic receptors more common for glutamate?
Ionotropic
What are the three subtypes of ionotropic glutamate receptors? What were they named after?
AMPA- named for the selective agonist (α-Amino-3-hydroxy-5-Methyl-4-isoxazolePropionic Acid)
Kainate-named for the selective agonist kainic acid.
NMDA-named for the agonist N-Methyl-D-Aspartate.
How many subunits are ionotropic receptors composed of?
Four subunit proteins
How do subunits relate to ionotropic receptor subtypes?
The three receptor subtypes have different combinations of subunits, explaining the differences in their pharmacology.
Also, there are subtypes of each subtype (eg. subtypes of NMDA receptor) that are built in different ways using different subunit combinations, causing them to differ in their pharmacology and physical properties.
What are non-NMDA receptors?
AMPA and Kainate receptors (which are both ionotropic glutamate receptors) are often lumped together into non-NMDA receptors
They are more straightforward in their activation - they are activated by molecules of glutamate and open up to allow sodium ions to pass through and depolarize the cell
While some AMPA subtypes allow calcium to flow in along with the sodium ions, this is not the case for all AMPA receptors (so we will generalize and say that non-NMDA receptors usually only allow sodium ions to pass through!)
They are ALWAYS activated when there’s sufficient stimulation by glutamate; though sensitivity to glutamate is tightly regulated and too much stimulation will lead to receptor desensitization
Name and describe a drug we learned in class that acts on both non-NMDA and NMDA receptors
Kynurenic acid: non-selective (‘broad spectrum’) glutamate antagonist for AMPA, Kainate and NMDA receptors
Targets all 3 ionotropic glutamate receptors
Name and describe a drug we learned in class that acts on both non-NMDA receptors but not NMDA receptors. What kind of symptoms might occur when you use it in high doses?
NBQX: competitive antagonist blocks both AMPA and Kainate receptors (not NMDA receptors). It’s more selective than kynurenic acid.
Treatment with high doses of AMPA/Kainate antagonists like NBQX exhibit sedation, reduced locomotor activity, ataxia, and protection against seizures.
What are NMDA receptors?
NMDA receptors are a type of ionotropic glutamate receptor (contrasted with non-NMDA receptors) and have unique characteristics
They allow the flow of both Na+ and Ca2+ into the neuron, which allows for greater depolarization than with non-NMDA receptors (which only allow Na+ to pass through)
In order to be activated, both glutamate and a co-agonist (glycine or D-serine) must bind at the same time to different parts of the receptor (usually, the co-agonist binding site is occupied anyway due to the high amount of glycine in CSF); furthermore, when the neuron is at resting potential, Mg2+ ions are bound to a site in the channel which blocks the channel so depolarization must occur to release the block and allow ions to flow through the channel
Why are NMDA receptors usually situated close to AMPA receptors?
The NMDA receptors are usually situated near AMPA receptors because at a glutamate synapse, the glutamate will activate the AMPA/Kainate receptor which will depolarize the cell and allow the magnesium block to get thrown off, which allows glutamate to activate the NMDA
Repeated AMPA receptor can depolarize neuron enough to remove Mg2+ block and allow NMDA receptor activation
Name and describe a competitive antagonist for NMDA receptors
AP-5(or APV) (amino-5-phosphonovaleric acid) blocks glutamate binding
Name and describe 3 non-competitive antagonists for NMDA receptors. What symptoms do they cause in low doses? High doses?
Non-competitive antagonists: block receptor at site independent of glutamate binding
Phencyclidine(PCP), ketamine, and MK-801(dizocilpine) block NMDA channel when it is open (kinda like what magnesium does normally).
In humans & animals, these drugs can cause schizophrenia-like symptoms at lower doses
At higher doses they cause ataxia (show impairments in movement; fall on their side)and then anesthesia
What kind of drug might we want to give someone who has reduced NMDA receptor activity? (No specific drug name needed)
Agonist that targets the glycine-binding site to enhance the activity of the NMDA receptor
Would an antagonist that targets the glycine-binding site of an NMDA neuron be a competitive or a non-competitive antagonist?
Non-competitive antagonist
What are the differences in the biophysical properties of AMPA vs NMDA receptors?
Activation of AMPA/Kainate vs NMDA receptors can have different excitatory effects on postsynaptic neurons
NMDA receptors can induce larger/ longer-lasting depolarization vs AMPA/Kainate
NMDA receptor activity doesn’t just depolarize neurons; they can also alter their firing patterns (e.g.; promote burst firing)
Explain the experiment that was done to compare the biophysical properties of AMPA vs NMDA receptors
In these studies, presynaptic glutamate inputs are stimulated: AMPA or NMDA antagonists are used to isolate the respective effect of each receptor
They use in-vitro patch clamps to record the depolarizations
To ensure that the NMDA receptors are activated, they bathe the neurons in CSF that is lacking in Mg2+ such that the neurons don’t require depolarization for the NMDA to activate (because the Mg2+ block is not there)
How many metabotropic receptors are there for glutamate?
Eight - mGluR 1 through 8
Which metabotropic glutamate receptors are postsynaptic and which are presynaptic?
mGluR- 1 and -5 mostly postsynaptic, others (mGluR2-4,6-8) are mostly presynaptic
What is the structure and mechanism of metabotropic glutamate receptors?
Resemble other classic metabotropic receptors with 7 transmembrane domains
When activated, they’ll trigger certain G proteins and second messenger systems that can influence neural activity of the cell
What is the connection between metabotropic glutamate receptors and autoreceptors?
Presynaptic mGluRs act as autoreceptors: suppress glutamate release.
Name and describe a drug we learned in class that acts on metabotropic glutamate receptors. Which ones do they act on?
L-AP4:agonist at mGluR 4/6/7/8 auto-receptors. Suppresses glutamate release.
*Note: Don’t confuse this for AP5 which is an NMDA competitive antagonist
What functions are mGluRs involved with?
mGluRs are widely distributed, participate in many functions (locomotion, motor coordination, cognition, mood and pain perception).
What are the potential future applications of mGluR drugs?
mGluR drugs are being developed for treatment of many neuropsychiatric disorders (eg. schizophrenia)
Explain the experimental procedure and findings of the rat study of glutamate and L-AP4
They measured glutamate levels with microdialysis
They collected a bunch of samples to establish a baseline and set this to ‘100%’ (every other measurement thereafter will be normalized to this 100% baseline)
At different time points throughout the experiment, they administered higher and higher concentrations of L-AP4 to the rats, stimulating the metabotropic glutamate autoreceptor
Finding: There was a reduction in baseline levels of glutamate release
Conclusion: Drugs that stimulate the glutamate autoreceptors (eg. L-AP4) can reduce glutamate transmission by reducing glutamate release
What is synaptic strength? How is this relevant to research on synaptic plasticity and learning/memory?
Alterations in the activity of synapses though to underlie learning are measured by changes in synaptic strength
Typically measured by changes in post synaptic potential evoked by an input
Strength = larger EPSP evoked in postsynaptic neuron
Change in synaptic strength doesn’t necessarily mean that the cell is going to fire more all the time, but increases the probability that the particular synapse (just one small part of the larger neuron) will get depolarized enough to exert a greater influence on whether the postsynaptic neuron fires. Remember that neurons usually receive multiple EPSPs and IPSPs. On a larger scale, these changes can add up to affect whole groups of neurons, causing them to fire in the same way that they did when the memory was encoded when presented with the particular stimuli/input. This is thought to underlie certain aspects of long-term memory formation.
How are changes in synaptic strength measured?
Primary way this is measured is with electrophysiological methods
What is LTP?
Most common way that neuroscientists model synaptic plasticity (changes that occur between neurons that regulate their synaptic strengths) is with an electrophysical phenomenon referred to as LTP
Long term potentiation is a model of changes that we can induce between the communication of neurons (primarily glutamate to a neuron)
The cellular and molecular changes that occur in the LTP model may reflect how the brain actually works to help encode memories and alter synaptic strengths that increase the likelihood that you might activate certain groups of neurons in your brain in a manner similar to the way they were activated when you first encoded a memory
Explain the experiment that was done to illustrate LTP
Basic background information: We have a neuron of interest in a dish with some glutamate axons that synapse onto it. We will be measuring the changes in membrane voltage and changes in excitatory postsynaptic potentials (mesaured in mV) using patch clamp electrophysiology.
1. Stimulate the presynaptic glutamate axons at a very low frequency (1/15 sec) to get subthreshold EPSPs. There should be no action potentials. This is to estbalish baseline. We might see a small depolarization of 5mV
2. Stimulate the axons at a very high frequency (eg. 100 Hz) to get lots of action potentials in post synaptic neuron
3. Stimulate axons at low frequency again (same frequency as in #1) - observe a MUCH BIGGER EPSP than before!
Conclusion: The input has become stronger (potentiated). There’s a larger EPSP now and it’s more likely to evoke action potential. These changes can last for days, weeks, months, or even years
Where in the brain has LTP been observed?
LTP has been observed basically anywhere in the brain that has glutaminergic synapses (eg. synapses between cortical pyramidal cells or pyramidal cells in the amygdala or the hippocampus or cortical glutamate synapses that synapse in other types of cells such as in the striatum)
How are NMDA receptors connected to LTP?
One thing that many of the different discovered types of LTPs have in common is that they often involve the activation of NMDA glutamate receptor. This is why that higher frequency stimulation is more likely to induce those longer-term changes because the lower frequency stimulation isn’t going to be enough to depolarize the cell enough to activate the NMDA receptor and remove the magnesium dependent voltage block
What is the cellular mechism of LTP? (Explain with reference to the NMDA receptor)
High frequency activation of postsynaptic neurons allows NMDA receptor to be activated = Ca2+ entry into neuron
Once Ca2+ enter the cell, it activates multiple enzyme pathways (kinases)
Ca 2+ activates Calcium-Calmodulin (CAM) kinase which:
1) hits latent AMPA receptor (floating inside cell) and inserts in membrane =more receptors (backup AMPA); glutamate has more opportunities to activator receptor now
2) activates Protein Kinase C and Tyrosine Kinase = short term and long term effects
Leads to formation of retrograde messenger (molecule that goes from postsynaptic to presynaptic neuron)
Messengers such as nitric oxide (NO) promote more transmitter release
Increased synaptic strength by both pre* and postsynaptic mechanisms
What happens to learning/memory when we block the normal function of gluatmate? Explain the evidence for the connections between glutamate and learning/memory with reference to the Morris Water Maze experiment
Blockade of NMDA receptors impairs many forms learning mediated by different systems (hippocampus [spatial memory], amygdala [fear-related memories], striatum [motor memories])
Morris Water Maze (hippocampal dependent): pool filled with opaque water: escape platform hidden under surface
Rat use spatial cues to navigate to platform
Intact rats learn to find platform quickly
NMDA receptor antagonists severely impair learning to find platform efficiently
• NMDA antagonists that impair learning also impair the formation of LTP (usually)
Block NMDA receptors during high-frequency activation of a glutamate pathway= NO LTP
What happens to learning/memory when we enhance the normal function of glutamate? Explain the evidence for the connection between glutamate and learning/memory with reference to a specific drug that can enhance glutamate function.
Enhancing glutamate activity (slightly) can improve learning/memory
Ampakines: Positive allosteric modulators of AMPA receptors
They do not activate AMPA receptors, but prolong open time/reduce desensitization
Ampakines can improve cognition in normal animals (and in animal models of cognitive dysfunction eg. cognitive decline due to aging)
What happens if there’s too much glutamate activity?
High levels of glutamate are toxic to neurons.
Injections of glutamate, kainite or NMDA can lesion any brain nucleus.
Define excitotoxicity
prolonged depolarization of neurons leads to eventual damage or death
In cultured neurons, activating NMDA and non-NMDA receptors with high [glutamate] kills most within a few hours.
Define necrosis
fast death characterized by lysis due to osmotic swelling
When you excessively stimulate with glutamate, a lot of ions flow into the cells, so water will move along with them b/c they’re positively charge, which will lead to swelling and then holes will form in the membrane - can’t maintain membrane potential and cell will die
Define apoptosis/programmed necrosis
a slower death triggered by series of biochemical events. Lysis does not occur.
Can be induced by lower concentration and longer exposure time to glutamate, cell death takes several hours (18-24 hours); depends on NMDA receptor activation.
What is brain ischemia? What happens to the neurons?
Excitotoxic brain damage may occur with brain ischemia (interruption of blood flow from stroke/heart attack etc).
Lack of O2 means Na+/K+ pump stops working, neurons depolarize
Neurons are unable to repolarize so the neurons just end up firing a ton leading to massive glutamate release occurs in the affected area.
Abnormally high Ca2+ levels inside the neuron overloads Ca2+ buffers and they cannot compensate
Super-high Ca2+ levels activates certain enzymes that kill cell
Can we target NMDA receptors to treat stroke? What other alternatives are there?
Tried to treat stroke by targeting the NMDA receptors so that they wouldn’t allow the flow of Ca2+ in, but this didn’t really work (maybe because it’s too late by the time we notice that someone is having a stroke) - try to identify the killer enzymes and target those instead maybe
