Neuro Exam 3

Steps of synaptic vesicle cycle

1. NT loading
2. Mobilization and targeting
3. Docking
4. Priming
5. Fusion
6. Endocytosis/Recycling

NT loading machinery

vesicular NT transporter (vTransporter)

* pumps NTs into the vesicle by coupling the pumping of protons out of the vesicle
* V-ATPase- pumps protons into the vesicle so the vTransporter can function. Uses ATP hydrolysis

Mobilization and targeting machinery

Synapsins

* regulates movement of vesicle to the active zone by attaching to vesicle and then to actin to position the vesicle.
* CAMK2 - phosphorylates Synapsin in response to Ca2+ so the vesicle can freely move to the active zone

What would genetic deletion of synapsin cause?

Inhibits NT release by reducing the reserve pool (synapses have less vesicles near the active zones so they cannot sustain high rates of release)

Docking machinery

Rab3A = GTP-binding protein that attaches to the vesicle and helps capture it to the active zone

Munc13/RIM = crosslinks with Rab3A to bring the vesicle to the active zone

What is the consequence of no Rab3A gene?

Enhanced release but have a reduced pool of releasable vesicles

Priming Machinery

SNARE Complex made of one V-SNARE (Synaptobrevin) and two T-SNARES (SNAP 25 and Syntaxin) to create a 4 motif complex

* Active zone membrane contains the T-SNAREs and the vesicle contains the V-SNAREs
* Munc18 is used to stabilize the complex when it comes together at the active zone
* SNARE complex pulls the vesicle and membrane together so they are 2nm apart

Botulinum

A toxin that cuts the T and V-SNAREs so it would block NT release because there would be no priming and fusion of the vesicle and membrane

Tetanus Toxin

Cuts the V-SNARE and would inhibit NT release because there would be no priming and fusion of the vesicle and the plasma membrane.

What facilitates fusion?

Ca2+ dependent

What happens to the SNARE complex after fusion?

NSF and SNAP 25 disassemble the complex

Fusion Machinery

Synaptotagmin = Ca2+ sensor for vesicle-active zone fusion

* transmembrane protein with two Ca2+ binding sites
* when Ca2+ binds and activates the protein, it binds to the SNARE complex and inserts into the active zone
* Dimple forms when the membranes are pulled together and it causes stress that facilitates fusion
* When there is no Ca2+ present, synaptotagmin actually blocks fusion

What does changing the Ca2+ affinity for synaptotagmin do?

Changes the amount of Ca2+ required for both spontaneous and evoked release

Endocytosis/Recycling Machinery

Endosome and plasma membrane transporters (specific for the NT)

What are the three ways to recycle vesicles?

1. Reversible Fusion Pore
2. Clathrin-Mediated
3. Bulk retrieval

Why is it advantageous to have a low affinity calcium sensor for NT release?

A low affinity calcium sensor for NT release gives high signal noise (less background release) and temporal precision (release is restricted to time of peak Ca2+ influx)

NT release is a ___ dependent process

Ca2+ dependent!

What triggers Ca2+ channels to open for NT release?

Ca2+ current follows AP which means membrane depolarization is the trigger to open Ca2+ channels!

* the Ca2+ current peaks after the action potential so VG Na+ channels open faster than Ca2+ channels

What is the speed of Ca2+ inactivation and why?

Slow, to allow more Ca2+ entry

__ triggers the postsynaptic current

NT triggers the postsynaptic current

How much intracellular Ca2+ is needed to trigger the Ca2+ sensor? (threshold?)

about 50 μM

What is the relationship between NT release and Ca2+ flux?

NT release shows steep, non-linear dependence on Ca2+ influx for NT release. Ex: a 2-fold increase in Ca2+ influx could make a 16-fold increase in NT release

What shows evidence that the Ca2+ sensors have low Ca2+ affinity?

most Ca2+ dependent enzymes only need 1 μM for full occupancy of Ca binding

What ceases NT release in regards to the Ca2+ channels?

when the concentration of Ca2+ reaches below the threshold

How are presynaptic Ca2+ channels arranged?

In rows that are localized at active zones so that Ca2+ influx can occur at the site where it is needed

ex: at NMJ, presyn. Ca2+ channels line up with postsyn. ACh receptors

What are some things that can cause a decrease in Ca2+ concentration?

1. greater distance from VG Ca2+ channels means less Ca2+
2. Ca2+ is rapidly buffered by binding proteins, mito uptake and pumps that dump it back outside the terminal

How can sustained Ca2+ entry cause synaptic potentiation?

During high frequency stimulation, there is so much Ca2+ that flows into the axon terminals that the buffering systems become saturated which leads to the buildup of residual Ca2+.

The residual Ca2+ becomes a second messenger that activates Ca2+ dependent enzymes (ex: CAMKII) which then increase vesicle release in response to subsequence action potentials → thus synaptic potentiation

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Each presynaptic AP will cause a bigger PSP

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Can last seconds to hours

What are the 4 types of VG Ca2+ channels? What is their function?

P/Q - fast release

N - fast release

L - slow release and muscle contraction

T - excitability

ω-agatoxin

Blocks P/Q type Ca2+ channels

ω-conotoxin

blocks N type Ca2+ channels

Dihydropyridines

block L type Ca2+ channels

Mibefradil

Blocks T type Ca2+ channels

Spontaneous NT release is ____

Spontaneous NT release is quantal

Quantal NT release hypothesis

spontaneous fusion of single synaptic vesicle releases a fixed amount of NT which will open a few ligand gated ion channels, which will cause the fixed small depolarization event

Mini/mEPSP

Spontaneous Miniature Postsynaptic Potential

* “The minimum response that a neuron will respond to that produces a depolarization event that is uniform in size”


* A spontaneous event
* One quantum = one vesicle with NT
* Minis occur at all chemical synapses but cannot fire an AP because they are too small
* The total postsynaptic potential is made up of a large number of quantal potentials. The total EPSP looks smooth because there are so many minis

What presynaptic and postsynaptic factors could change the size of a mini?

Presynaptic Factor – increasing the amount of NTs in the vesicle increases quantal size and increases the amplitude of the mEPSP

 

Postsynaptic Factor – increasing receptors in the post synapse can increase the amplitude of the mEPSP

What does increasing Ca2+ concentration do to quantal release?

Vesicles are packaged with a fixed amount of NT (quantum) and increasing or decreasing the Ca2+ influx will not change the size of the quantum, bur rather the rate of quantal release.

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More Ca2+ concentration = more quantal will be released = more vesicles fuse and release NT

What factors can change the amount of NT release?

* # of individual synapses between a pair of pre and post cells
* the number of active zones in the synaptic terminal
* the probability that a presynaptic AP will trigger release of one or more quanta of NT at an active zone

How does capacitance change during NT release?

NT release will cause a brief increase in capacitance of the nerve terminal when it fuses because the surface area of the nerve terminal will increase slightly. Capacitance is directly proportional to surface area, so as surface area increases, so does capacitance.

What are 3 methods to detect NT release?

1. Capacitance
2. Electrochemical detection with Voltammetry
3. Optical Biosensors

Detecting NT release with Voltammetry

a large amount of voltage is applied to the tip of an electrode, which will oxidize certain NTs. The oxidation of the NTs generates free elections. The current of these electrons is proportional to the amount of NT released, so it can be measured and quantified (OLD METHOD)

Detecting NT release with Optical Biosensors

measure NT release with electrochemical detection and light. Using fluorescence to see what and how much NT is in the synapse. KLight is a receptor that only fluoresces when an agonist (the NT) is bound. It is a GPCR and when the NT binds, it will change shape so that it will emit light.

Why is recycling vesicles important?

Helps to prevent the supply of vesicles being rapidly depleted during a fast synaptic transmission

What is the standard release pathway for NT release at active zones?

Complete fusion - clathrin mediated or ultrafast bulk retrieval

State the precursors for the following classical NTs:

* Acetylcholine
* Dopamine
* Norepinephrine
* Epinephrine
* Serotonin
* Histamine
* Glutamate
* Glycine
* GABA

* Choline
* tyrosine
* tyrosine via dopamine
* tyrosine via norepinephrine
* tryptophan
* histidine
* glutamine
* serine
* glutamate and then by glutamic acid decarboxylase (GAD)

What are the biogenic amines?

Catecholamines (dopamine, norepinephrine, epinephrine)

Serotonin

Histamine

What are the amino acid NTs

GABA

Glycine

Glutamate

What makes ACh different than the other classical NTs?

It is the only classical NT that is neither an amino acid or derived from one

Life Cycle of Glutamate

1. glial cell reuptakes glutamate by transporter (EAAT/GLT1)
2. Glutamate (Glu) is converted to Glutamine (Gln) in glial cell
3. Neuron converts it back to Glutamate by glutaminase (PAG) in the mitochondria
4. Glu loaded into vesicle by VGluT1-3 and then back out into the synapse

Life Cycle of GABA

1. Glutamate --GAD--→ GABA in neuron and then released into synapse
2. glial cells take up GABA and convert it back to Glu then to Gln
3. Neuron converts Gln back to Glu then back to GABA
4. Packaged into vesicles by transporter (VGAT)

Which of the following are proteins you would need to express in a Glutamatergic neuron to convert it to a GABAergic neuron?

\n A) GAD \n B) Glutaminase \n C) VIAAT + GAD \n D) GABA-T + VIAAT + GAD \n E) VIAAT + GAD + GAT

C

How is acetylcholine made?

Life Cycle of ACh

1. Made by Chat in the neuron
2. packaged into vesicles by choline transporter
3. released into synapse
4. action is terminated by ACh esterase (AChE) that splits ACh into choline and acetic acid
5. Neuron takes up the choline with choline transporter

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GLIAL CELLS NOT USED FOR REUPTAKE

If astrocytes are not needed for recycling Ach, why are they \n present at cholinergic synapses?

\n A) They must process co-released Glu \n B) They still need to metabolically support the neurons \n C) They respond to Ach

B

What are three major functions of Cholinergic neuron projections?

1. Motor gating
2. learning and memory
3. motor output

Explain the pathway of synthesis of Dopamine, Norepinephrine and Epinephrine

How can you use unique proteins to identify neurons that release specific NTs?

Looking at enzymes that make certain NTs can help you distinguish which neuron it is

Ex: Adrenergic neurons (release norepinephrine and epinephrine) will have the dopamine β-hydroxylase enzyme that converts dopamine to norepinephrine

Life Cycle of DA and NE

1. L-tyrosine converted to L-Dopa and then to Dopamine and then if DBH is present, then NE will be made (DBH only in adrenergic neurons)
2. When DA or NE is released they can act on both pre or post neuron receptors
3. Reuptake back into the presynaptic neuron done by either DAT for DA or NET for NE
4. NTs are packaged into vesicles by VMAT/VMAT2 (same for both NE or DA)
5. NTs are degraded by MAO (monoamine oxidase) in the mitochondria

What drives the plasma membrane transporters for reuptake?

Driven by the Na+ electrochemical gradient through a symport mechanism in which Na+ ions and the transmitter move in the same direction (back into the presynaptic neuron from the cleft)

The NE system is a major target for what disorder?

ADHD

Serotonin Synthesis

Life Cycle of Serotonin (5-HT)

1. Synthesized in neuron
2. released into cleft and can act on pre or post receptors
3. Reuptake is with the plasma mem. transporter SERT
4. Packaged back into vesicles with VMAT/VMAT2
5. Degraded by MAO in mitochondria

What type of compound specifically targeting the \n serotonin system might alleviate depression?

\n A) SERT inhibitor \n B) Tryptophan \n C) Serotonin receptor blockers \n D) MAO inhibitor \n E) Tryptophan hydroxylase inhibitor

D

If you wanted to elevate Dopamine in the synaptic cleft without affecting Norepinephrine, which or the following would you do?

\n A) Inhibit MAO \n B) Provide L-Dopa \n C) Stimulate VMAT \n D) Block DAT

D

What type of receptors do neuropeptides bind to?

GPCRs!

What would happen if NT is left in the synaptic cleft for longer than it should?

Leaving NT in the synapse would initially boost the signal but then prevent new signals from getting through because there would be receptor desensitization (decreased responsiveness of the receptor due to repeated exposure to the agonist)

Neuropeptides

Short polymers of amino acids that are work as a signaling mechanism that works in conjunction with NTs

They are made by neurons and processed in the golgi where they are packaged into dense core vesicles

What are the three rules to classify a neuropeptide?

1. made by neurons
2. stored and released on demand
3. modulate neuronal function via receptors (GPCRs)

What are some examples of neuropeptides?

* opioids
* neuropeptide Y (mediates anti-stress response)
* Somatostatin (SST)
* Corticotropin releasing factor (CRF)

Which neurons release neuropeptides?

probably all of them

Co-transmitter

when more than one transmitter is present in a nerve terminal

there is increasing evidence that many neurons actually release more than one type of NT

What are the benefits of co-transmisison?

Having more than one transmitter leads to versatility to synaptic transmission. Different frequencies of stimulation can cause the release of one or both transmitters which affects transmission.

What is co-transmission governed by?

The differential release of co-transmitters is based on the distribution of Ca2+ and vesicles in presynaptic terminals.

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Low firing frequency, the concentration of Ca2+ only causes some opening of local Ca2+ channels so only NT is released

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High firing frequency causes the Ca2+ concentration to be more evenly distributed throughout the presynaptic terminal, thereby inducing the release of neuropeptides as well

List the differences between classical NTs and neuropeptides based on:

* where they are made
* what they are packaged in
* how they are released from the neuron
* reuptake?
* What receptors they activate
* How long/fast their effect is

What are the 5 ways to measure neuropeptides?

1. Slice electrophysiology = use voltage clamp to measure EPSP and apply different drugs to see what it does
2. Optically = stimulate the peptide expressing neuron to measure what is being produced
3. Stimulate the peptide-expressing neuron, show it influences something, and show it is mediated by the peptide receptor system
4. Optogenetically stimulate the peptide neuron, microdialysis in vivo
5. Optical biosensor

What NTs are used to treat ADHD and depression?

biogenic amines (dopamine, norepinephrine, and epinephrine)

How does ADHD treatment work with NTs?

* Target NE
* NE signaling focuses attention
* Increasing NE signaling can increase attention and focus

How does Adderall work?

Acts as VMAT that is designed to increase DA/NE signaling with minimal depletion. To increase attention for patients with ADHD

What is Ritalin and how does it work?

Used to treat ADHD. Directly blocks DAT and NET so there is no reuptake and the NTs stay in the cleft longer

What is the main target for depression treatment?

Serotonin because it signals events to elevate mood and motivation

What are the negative effects of upregulation of dopamine signaling with illegal drugs?

* addiction


* depletion of DA reserves
* increased DA cycling = oxidative stress in neurons which can lead to degeneration

What are the negative effects of upregulation of NE signaling with illegal drugs?

* NE increases blood pressure and heart rate and can lead to heart attack
* oxidative stress and losing ability to focus

What are the negative effects of upregulation of serotonin signaling with illegal drugs?

* depletion leads to profound loss of motivation and can cause depression
* oxidative stress

Oligodendrocytes

Formation of myelin in CNS

Schwann Cells

Formation of myelin in PNS

Astrocytes

Metabolic and structural support in CNS

Satellite Cells

Metabolic and structural support in PNS

Microglia

Support Innate immunity in CNS

What glial cells are derived from the neural tube? the neural crest?

Neural tube - oligodendrocytes and astrocytes

Neural crest - schwann cells and satellite cells

What is myelin?

Made of oligodendrocytes or Schwann cells and wraps around axons to increase the propagation of APs. It acts as an electrical insulator that prevents charge from leaking out of the axon, which prevents the decrease of the AP conduction

What is the ratio of oligodendrocytes and Schwann cells to the axon?

1 oligodendrocyte : 50 axons

1 Schwann cell : 1 axon

How does myelin increase the speed of AP propagation?

Increases the rate of passive spread of depolarization because it acts as an insulator

Nodes of Ranvier

Non-myelinated portions of the axon that contain clusters of Na+ channels (which generate APs)

* The nodes are easily excitable and give the AP a “boost” when they travel down the axon so that propagation can continue and does not decrease
* The signal jumps from node to node (Saltatory Conduction)

Guillain-Barre Syndrome

A rare, autoinflammatory disease involving demyelination of peripheral nerves

* less than 10% mortality
* caused by Zika virus

Multiple Sclerosis

An autoimmune disease involving demyelination in the CNS

* may be triggered by environmental factors like stress
* patients have decreased motor skills because there is no connections between neurons, especially motor regions

Astrocyte Functions

Homeostasis

* store glycogen for energy
* produce growth factors for neuronal survival
* regulate blood flow

Neurotransmission

* NT metabolism
* gliotransmission

Contribute to response to injury and infection

Why is removing glutamate from the synapse with astrocytes important?

Excess glutamate in the synapse can cause epilepsy and Schizophrenia

How do astrocytes help regulate blood flow?

Astrocytes have endfeet on the ends of their processes that bind to the blood vessels

* Ca2+ influx stimulates the astrocyte and they can help with vasodilation and contraction

How do astrocytes respond to injury or disease?

* stimulated by factors that are secreted from microglia or macrophages
* clear dead cells and form glial scar tissue
* secrete cytokines and chemokines

Structure of microglia

Many processes around the cell and the cell moves in circles scanning the environment for injury