Lecture 3 - Chapter 5: Synaptic Transmission

Question 1

What two classes of synapses are there?

Answer 1

1. Electrical synapse
2. Chemical synapse

Question 2

What is an electrical synapse?

Answer 2

Here, the membranes of two communicating neurons come extremely close at the synapse are are linked together by gap junctions (aligned, paired channels in the membrane of the pre- and postsynaptic neuron).

Question 3

What are characteristics of an electrical synapse?

Answer 3

• Fast transmission
• Bi-directional (allow transmission in both directions)
• No plasticity

Question 4

What is a connexon?

Answer 4

An assembly of six proteins (connexins) that form the pore for a gap junction between the cytoplasm of two adjacent cells.

Question 5

Electrical synapses are characterized by the ability for bidirectional transmission. What function is enhanced by this characteristic?

Answer 5

Synchronization of electrical activity among populations of neurons. As can be seen in the picture.

Question 6

What is a chemical synapse?

Answer 6

Compared to electrical synapses, the space between the pre- and postsynaptic neurons is greater in chemical synapses. This space is called the synaptic cleft. Within the presynaptic terminal, synaptic vesicles are located that are filled with neurotransmitters. Upon presynaptic stimulation, these vesicles fuse with the presynaptic membrane so that neurotransmitters are released in the synaptic cleft.

Question 7

What are characteristics for chemical synapses?

Answer 7

• Slower transmission
• Uni-directional
• Plasticity

Question 8

What are neurotransmitters?

Answer 8

Neurotransmitters are present in synaptic vesicles in the presynaptic neuron. They’re released into the synaptic cleft, upon arrival of an action potential in a calcium-dependent manner. Here, neurotransmitters can bind to postsynaptic (and sometimes also presynaptic) receptors.

Question 9

What two categories of neurotransmitters are there?

Answer 9

• Small-molecule neurotransmitters
• Neuropeptides

Question 10

Neurotransmitters are carried inside vesicles. There are two types of vesicles, name these.

Answer 10

• Synaptic vesicles
• Dense core vesicles

Question 11

Name characteristics of synaptic vesicles.

Answer 11

• 40-60 nm
• Carry small molecule neurotransmitters
• Local recycling of synaptic vesicles
• Found in synapses
• Rapid action
• Can only fuse at the active zone

Question 12

Name characteristics of dense core vesicles.

Answer 12

• 60-120 nm
• Carry (neuro)peptides
• Produced inside golgi
• Found everywhere
• Slow action
• Can fuse anywhere

Question 13

How can the pH be used to visualize secretion?

Answer 13

The intracellular environment of vesicles have a pH of about 5,0-5,5, while the extracellular environment (cerebrospinal fluid, blood, etc.) has a pH of 7,4. This difference in pH can be used to visualize secretion.

This is done through a modified version of GFP, pHluorin. This GFP can either bind to a transmembrane domain of a synaptic vesicle or it can bind to a neuropeptide of a dense core vesicle and via this way can be taken up inside the vesicle.

Note: modified in the sense of that the GFP is more sensitive to pH changes.

Question 14

What fluorescence intensity is expected when pHluorin is taken up inside a vesicle and when the vesicle with pHluorin fuses with the membrane?

Answer 14

The fluorescence intensity of pHluorin increases when the pH increases.

• Since the intracellular environment of a vesicle has a low pH, pHluorin is expected to have a low fluorescence intensity.
• When the vesicle fuses with the membrane, it is exposed to the extracellular environment which has a higher pH (7,4). Thus, upon fusion with the membrane the fluorescence intensity of pHluorin is expected to increase.

Question 15

What’s the relationship between the fusion of a synaptic vesicle and electrical events?

Answer 15

That fusion of one synaptic vesicle corresponds to one spontaneous electrical event.

Question 16

How can synaptic transmission be recorded?

Answer 16

By recording the postsynaptic membrane potential on e.g. a muscle cell when stimulating the axon that is connected to this cell.

Question 17

What three steps are important in the synaptic vesicle cycle?

Answer 17

1. Docking and priming
2. Exocytosis
3. Endocytosis (kiss and run or clathrin-mediated endocytosis)

Question 18

What is clathrin-mediated endocytosis?

Answer 18

Here, vesicles are being reused. So after a neurotransmitter containing vesicle has released its content in the synaptic cleft, the empty vesicle is again taken up by the presynaptic terminal via clathrin-mediated endocytosis. Here, the empty vesicle needs to be refilled again.

(This process will be discussed later in more detail)

Question 19

In this picture you see the protein stoichiometry of some proteins that exist on a synaptic vesicle. Why does synaptotagmin have a high protein stoichiometry?

Answer 19

Because it’s a calcium sensor and calcium is important for the fusion and release of vesicles.

Question 20

Explain the role of calcium in vesicle fusion.

Answer 20

An action potential arrives at the presynaptic terminal of an axon. Depolarization of the presynaptic membrane causes opening of the voltage-gated Ca²⁺ channels. Ca²⁺ flows from the outside of the cell to the inside of the cell. Inside the presynaptic terminal, Ca²⁺ stimulates the fusion of vesicles with the presynaptic membrane. This causes neurotransmitter release into the synaptic cleft.

Question 21

What happens to the fusion process when:

• Calcium is injected
• BAPTA is injected

Answer 21

• Calcium injections triggers fusion
• BAPTA is an intracellular calcium chelator (a binding agent that suppresses chemical activity by forming chelates), which prevents fusion.

Question 22

Synaptotagmin are vesicle-resident calcium sensors. There are several variants of synaptotagmin. There’s one variant that has a high affinity for calcium due to two binding domains for calcium. Name the synaptotagmin variant and its two binding domains.

Answer 22

Synaptotagmin 1 (Syt1) → C₂A and C₂B

Question 23

What happens when calcium binds to synaptotagmin1 with C₂A and C₂B?

Answer 23

After binding of calcium, C₂A and C₂B get a very high affinity for lipid membranes. It then pulls the vesicle closer to the plasma membrane, as depicted in the picture.

Question 24

Synaptotagmin isn’t enough for vesicle (or membrane) fusion. What proteins are imported and how are they important?

Answer 24

SNARE proteins → synaptobrevin (transmembrane vesicle protein), SNAP-25 and syntaxin (transmembrane proteins in plasma membrane).

They form a really tight coil that pulls the synaptic vesicle- and plasma membrane together.

Question 25

What is the function of toxines tetanus and botulinum?

Answer 25

They specifically cleave SNARE-proteins → it prevents fusion

Question 26

Explain the steps of synaptic vesicle fusion.

Answer 26

1. Vesicle docking 2. Entering calcium binds to synaptotagmin 3. SNARE complexes form to pull membranes together 4. Calcium-bound synaptotagmin catalyzes membrane fusion by binding to SNAREs and the plasma membrane

Question 27

Explain the steps of vesicle docking (priming).

Answer 27

1. **Tetherin** protein help recruit the SV to the plasma membrane 2. Priming proteins **RIM**, **Munc13** and **Rac** proteins go into complex with each other. This complex helps align synaptic vesicles with the calcium channels, so that when calcium is released synaptotagmin can rapidly respond to the calcium influx.

Question 28

Synaptic vesicle transport from soma to synapse reaches speeds of 5 to 40 cm per day. What does this say?

Answer 28

That the supply of vesicles via synaptic transport (walking over microtubuli) is not enough to maintain efficient synaptic transmission.

Question 29

What solution is there for the fact that synaptic vesicle transport is not sufficient to maintain efficient synaptic transmission?

Answer 29

Local recycling of synaptic vesicles.

Question 30

What evidence was found for local recycling of synaptic vesicles? (Don't know if you should learn this by heart)

Answer 30

Researchers bathed a neuron in a bath with horseradisch peroxidase (HRP). They then stimulated the presynaptic terminal to release their vesicles. If there's local recycling of synaptic vesicles, there also should be a little HRP inside the recycled vesicles. When this neuron was looked at under a electron microscope, they indeed found that there was local recycling, as why they found HRP inside vesicles.

Question 31

Another way to investigate and visualize synaptic vesicle release and local recycling was with the help of two dyes: FM1-43 and FM4064. What is special about thes dyes?

Answer 31

They only fluorescence when they are inserted into the membrane and thus can also be used to visualize synaptic vesicles.

Question 32

Explain clathrin-mediated endocytosis.

Answer 32

When a vesicle is locally recycled and is about to fuse/has just fused with the presynaptic membrane, clathrin will form a cage around the incoming vesicle. After this, **dynamin** is needed to seperate/pinch off the vesicle from the membrane. Note: the fact that clathrin forms a cage around a vesicle, is also important for the protection of proteins that are attached to the vesicle.

Question 33

After the vesicle has been recycled, by e.g. clathrin-mediated endocytosis, the vesicle is still empty. What is the function of V-ATPase?

Answer 33

V-ATPase pumps protons inside the vesicle and makes the vesicle acidic.

Question 34

After the vesicle has been recycled, by e.g. clathrin-mediated endocytosis, the vesicle is still empty. Why is V-ATPase so important?

Answer 34

Because the only way a vesicle can be refilled is via transporters. Depending on the type of neuron (excitatory or inhibitory), there are either VGLUT (glutamate transporter) or VGAT transporters (GABA transporter) on the vesicle. These transporters make use of protons to transport neurotransmitters into the cell, here protons are pumped out and neurotransmitters are pumped in.