Lecture 10: Glutamatergic Synaptic Transmission and Trafficking of Transmembrane Receptors

Question 1

What is the role of glutamate in the mammalian CNS?

Answer 1

Glutamate serves as a major excitatory neurotransmitter in the mammalian CNS

Question 2

Describe the major components of the tricyclic carbocyclic acid cycle.

Answer 2

Glutamate

Question 3

How many types of ion channels does glutamate have, and what are they?

Answer 3

• Glutamate has two types of ion channels:
  
  1. Ligand-gated ion channels: These channels are fast excitatory channels and include NMDA, AMPA, and kainate receptors.
  2. GPCRs (metabotropic glutamate receptors - mGluR): These are slow modulatory channels that can influence cellular processes such as synaptic transmission. They are categorized into Group I, Group II, and Group III receptors.

Question 4

Which receptors are part of the ligand-gated ion channels for glutamate, and what ions do they bind to?

Answer 4

• NMDA, AMPA, and kainate receptors
• They bind to sodium and calcium ions

Question 5

What is the obligatory receptor for NMDA, among the ligand-gated ion channels of glutamate?

Answer 5

GluN1

Question 6

How are the GPCRs for glutamate categorized, and what are the groups?

Answer 6

1. Group I
2. Group II
3. Group III

Question 7

How is glutamate synthesized and maintained in the brain?

Answer 7

• Uptake of neurotransmitter into glial cells, such as astrocytes, where it is converted to glutamine by glutamine synthetase.
• Glutamine is then taken up by neurons and converted back to glutamate via the enzyme glutaminase. Additionally, glutamate can also be produced through the tricarboxylic acid cycle.

Question 8

Describe the process of maintaining the neurotransmitter pool of glutamate.

Answer 8

The creation of a concentration gradient of hydrogen ions via ATPase supports the uptake of glutamate into synaptic vesicles at high concentrations.

Question 9

How is glutamate released to its receptors?

Answer 9

Glutamate is released to its receptors in the synaptic cleft, with the process influenced by factors like TTX (tetrodotoxin) and DTX (dendrotoxin).

Question 10

How is the termination of glutamate signaling achieved?

Answer 10

Uptake carriers, the location of which depends on the cell type. These carriers recycle glutamate neurotransmitter.

Question 11

Why do astrocytes have glutamate transporter carriers?

Answer 11

• They are closely associated with neuronal synapses, forming part of what is known as a tripartite synapse.
• Astrocytes play an important role in regulating synaptic transmission and have receptors of their own to respond to neurotransmitters.

Question 12

What is meant by a tripartite synapse?

Answer 12

• Presynaptic neuron, the postsynaptic neuron, and an astrocytic process.
• Astrocytes in this structure play a vital role in regulating synaptic transmission and can release their own neurotransmitters, contributing to synaptic modulation.

Question 13

What are the key characteristics of glutamatergic receptors?

Answer 13

• Four subunits, forming a tetrameric structure, unlike acetylcholine receptors which typically have five subunits.
• The transmembrane 2 (TM2) region of the receptor forms the ion channel.
• GluN1 subunit is ubiquitous in the brain, indicating its obligatory role in glutamatergic transmission.

Question 14

Describe the subunits of the NMDA receptor.

Answer 14

The NMDA receptor comprises GluN1 subunits, which are obligatory, and GluN2 subunits.

Question 15

What are the requirements for NMDA receptor activation?

Answer 15

Binding of two agonists: glutamate and either glycine or D-serine, which act as co-agonists.

Question 16

Explain the role of magnesium ions in NMDA receptor function.

Answer 16

• Magnesium ions, present in the extracellular space at concentrations of 1-2 mM, typically block the NMDA receptor channels under normal conditions due to their positive charge and affinity for the intracellular environment of neurons.
• This blockage is dependent on the specific subunit and its variant, and it can be inhibitory.

Question 17

Are there any regulatory sites in the NMDA receptor?

Answer 17

Yes

Question 18

What is the purpose of a cell receptor expression study?

Answer 18

• Expressing receptors, such as NMDA receptors, in a simple cell.
• This allows researchers to investigate the properties of individual ion channels, including their conductance and behavior, based on the subunit composition.

Question 19

How is patch clamp recording used in the Wylie et al 2013 study?

Answer 19

• Patch clamp recording is performed in the outside-out mode, where individual ion channels’ activity is recorded.
• This method allows for the observation of ion flow through individual NMDA receptors and provides insights into their properties.

Question 20

What can be determined by observing individual openings and closings of NMDA receptors?

Answer 20

• By observing individual openings and closings of NMDA receptors, researchers can determine the conductance of the ion channels, which is the number of ions flowing per unit time.
• This information is crucial for understanding the properties of NMDA receptors and can vary based on the subunit composition.

Question 21

Why is studying subunit compositions of NMDA receptors important?

Answer 21

• Studying subunit compositions of NMDA receptors is essential for various purposes, including drug development and medicinal chemistry.
• The properties of NMDA receptors, such as their conductance and activation speed, depend on the subunits they are composed of. Understanding these variations allows for targeted approaches in therapeutic interventions.

Question 22

Which subunits are closely looked at in adult brains, and why?

Answer 22

• In adult brains, there is high expression of 2A and 2B subunits of the NMDA receptor.
• These subunit compositions are closely examined, possibly due to their significance in influencing the properties and functions of NMDA receptors.

Question 23

What are the structural characteristics of glutamate receptors?

Answer 23

Glutamate receptors typically have four transmembrane domains, with the transmembrane region forming the ion channel. The subunits of glutamate receptors are intertwined, forming a corkscrew-like structure through the membrane.

Question 24

Describe the ion path through the AMPA receptor.

Answer 24

In the AMPA receptor, ions flow through the base of the receptor.

Question 25

How do subunits contribute to the function of glutamate receptors?

Answer 25

• Subtypes of glutamate receptors are crucial for their function, providing neurons with the ability to fine-tune activity and behavior.
• Changes in subunit composition, which can occur through development, allow for adjustments in response to different stimuli.
• Post-translational modifications, such as phosphorylation, can also alter the properties of glutamate receptor subunits, influencing the function of the circuit they are embedded in.

Question 26

What are the electrophysiological properties of Ca2+ permeable AMPA receptors?

Answer 26

Ca2+ permeable AMPA receptors, such as GluA1 and GluA3, exhibit a rectifying current-voltage (I/V) curve. This means that the flow of ions through the channel is favored in one direction over the other. GluA2 subunit, which lacks the ability to allow Ca2+ permeation due to the presence of arginine, is most abundant in the adult brain and contributes to a linear I/V curve.

Question 27

How does subunit composition change over time in glutamate receptors?

Answer 27

• During development.
• This change in subunit composition is associated with alterations in the outward response of the receptors.

Question 28

What techniques can be used to observe changes in subunit composition of glutamate receptors?

Answer 28

Immunohistochemistry, immunofluorescence, and Western blotting.

Question 29

What are the roles of glutamate receptors in the mammalian brain?

Answer 29

○ Brain development
§ Subunit switches which require different receptors to be expressed in n out of synapses -> circuits form n established
○ Learning and memory
§ Receptors are expressed in response to stimuli that induce learning
○ Drug addiction
§ Learning n memory circuits may be hijacked during drug taking
§ Becomes an addiction
○ Pain
§ [mis]expression in pain pathways
§ Shift from the cytosol to the membrane of some AMPAR subunits
§ May be part of pain processing / maintenance state
○ Cerebral ischemia (stroke)
§ Loss of GluA2 n increase in Ca2+ influx
○ AD
§ LTD may lead to synapse loss
§ Memantine (NMDAR antagonist) used in AD

Question 30

What is the early evidence of silent synapses in AMPAR trafficking?

Answer 30

• Use of antibodies directed to intracellular (IC) terminals of AMPAR receptors.
• Synapses lacking AMPAR receptors were observed using this approach.

Question 31

How were antibodies used to investigate AMPAR trafficking?

Answer 31

• Antibodies directed to the extracellular (EC) terminal of the AMPAR receptor were compared with antibodies directed to the intracellular (IC) terminal.
• EC antibodies revealed cell surface AMPAR receptors with a spotty distribution, reflecting individual synapses.
• IC antibodies, when allowed to penetrate the cell, revealed a high concentration of receptors within the cell, indicating they were ready to be delivered to synapses.

Question 32

What observations were made from the overlay of EC and IC antibody staining?

Answer 32

• Overlay of EC and IC antibody staining showed yellow spots indicating both IC and EC epitopes targeted.
• However, in spines of contact where AMPAR receptors were expected to be present, they were absent.
• This suggests the presence of synapses lacking AMPAR receptors, indicating silent synapses.

Question 33

What happens to AMPAR receptors when the membrane potential is hyperpolarized?

Answer 33

• Not likely to see a response from AMPAR receptors.
• This is because hyperpolarizing the membrane potential attracts magnesium ions into the channel → blocking the NMDA receptor and preventing activity.

Question 34

What distinguishes active synapses from silent synapses in terms of AMPAR activity?

Answer 34

• Active synapses transmit information at hyperpolarized (resting) membrane potentials through AMPA receptors.
• In contrast, silent synapses lack AMPARs at the synapse and do not transmit information at hyperpolarized membrane potentials due to the magnesium ion block of the NMDA receptors.

Question 35

Where might AMPA receptors be stored in silent synapses?

Answer 35

Intracellular vesicles or at extrasynaptic sites, contributing to the lack of AMPAR activity at the synapse.

Question 36

Describe the process of unsilencing silent synapses as discussed in the Kerncner and Nicoll Nature 2008 paper.

Answer 36

• The process involves stimulating the tissue at high stimulation strength until a response is observed. Then, the stimulation strength is gradually reduced until no response is recorded.
• If there is no response at -60mV but a response at +30mV, it indicates the presence of NMDA receptors but not AMPA receptors. This suggests the existence of synapses with NMDA but not AMPA receptors.
• To induce AMPA receptor expression in these silent synapses, a pairing protocol is delivered, which involves stimulating the fibers while depolarizing the cell.
• Glutamate is released, and the NMDA receptors are unblocked, potentially recruiting silent synapses to start expressing AMPA receptors.
• This results in the release of glutamate and NMDA receptor activation → AMPA receptor insertion into the synaptic cleft, either through mobilization of intracellular vesicle-bound AMPA receptors or movement of extrasynaptic receptors through the synapse.

Question 37

What evidence supports the trafficking of native AMPARs, as discussed in the study by Pickard et al. in Neuropharmacology 2001?

Answer 37

• The evidence includes using different colored antibodies to label AMPARs and a protocol to induce plasticity or activate NMDA receptors.
• Researchers observed if any new AMPAR receptors were introduced after the protocol.
• In a control experiment, after depolarization, more spots indicating AMPARs were observed.
• However, when an NMDA receptor antagonist was applied, this process of introducing new AMPARs after depolarization was blocked.

Question 38

What are the implications of calcium elevation and receptor immobilization in synaptic plasticity, particularly regarding the mobility and motility of AMPAR receptors along dendrites?

Answer 38

• When calcium levels rise, AMPAR receptors may become immobilized at the synapse, likely due to synaptic plasticity-inducing stimuli.
• This immobilization involves the aggregation of calmodulin and the concentration of CamKII, an important plasticity enzyme, at specific spots.
• These spots aid in anchoring the AMPAR receptors in the synapse. To visualize this trafficking process, AMP receptors labeled with quantum dots can be used. The loss of mobility and motility of AMPAR receptors indicates their immobilization, potentially contributing to synaptic plasticity.

Question 39

What is the process of inducing plasticity in neural tissue through high stimulation, and what are its implications?

Answer 39

• High-frequency stimulation induces a form of plasticity known as long-term potentiation (LTP), resulting in long-lasting synaptic transmission.
• Conversely, long-term depression (LTD) occurs, characterized by a decrease in the size of the response. These processes underpin many aspects of learning and memory within the brain.

Question 40

What is the significance of inducing plasticity in neural tissue through high-frequency stimulation?

Answer 40

Can lead to LTP (process that is fundamental to learning and memory)

Question 41

How does long-term depression (LTD) relate to synaptic plasticity?

Answer 41

• LTD refers to a decrease in the size of synaptic responses and is another form of synaptic plasticity.
• Both LTP and LTD play crucial roles in shaping neural circuits and are involved in learning and memory processes.

Question 42

What role do NMDA receptors (NMDARs) play in synaptic plasticity?

Answer 42

• NMDA receptors are necessary for LTP induction and are crucial for spatial learning.
• Blocking NMDARs with antagonists like AP5/APV prevents the formation of LTP, demonstrating the importance of these receptors in synaptic plasticity and memory formation.

Question 43

How is the involvement of NMDARs in spatial learning demonstrated experimentally?

Answer 43

• Experimental studies, such as the Morris water maze task, show that blocking NMDARs with antagonists like D, L-AP5 impairs spatial learning.
• Rats treated with D, L-AP5 are unable to form a memory of the platform’s location and exhibit aimless swimming behavior, unlike those treated with saline or the inactive isomer of AP5. This indicates that the active isomer of AP5 is necessary for spatial learning.

Question 44

What are the implications of AMPAR trafficking at synapses in brain development?

Answer 44

• Facilitating subunit switches, which involve the expression of different receptor types at synapses.
• This process is crucial for the formation and establishment of neural circuits during development.

Question 45

How does AMPAR trafficking relate to learning and memory?

Answer 45

• Regulating the expression of receptors in response to stimuli that induce learning.
• Changes in AMPAR distribution and abundance contribute to synaptic plasticity, which underlies learning and memory formation.

Question 46

What role does AMPAR trafficking play in drug addiction?

Answer 46

• AMPAR trafficking may be hijacked during drug addiction, as learning and memory circuits involved in addiction may become altered.
• Changes in AMPAR expression and distribution in response to drug-taking behavior can contribute to the development and maintenance of addiction.

Question 47

How is AMPAR trafficking implicated in pain processing?

Answer 47

• AMPAR trafficking, including the shift of some subunits from the cytosol to the membrane, may contribute to pain processing and the maintenance of pain states.
• Dysregulation of AMPAR trafficking in pain pathways could lead to altered synaptic transmission and heightened pain sensitivity.

Question 48

What implications does AMPAR trafficking have in conditions like cerebral ischemia (stroke) and Alzheimer’s disease (AD)?

Answer 48

In conditions such as cerebral ischemia, there may be a loss of GluA2-containing AMPARs and an increase in calcium influx, contributing to neuronal damage. In AD, LTD associated with AMPAR trafficking may lead to synapse loss, while drugs like memantine, an NMDAR antagonist, are used to mitigate excitotoxicity and cognitive decline.