Neuro Lecture 4: Synaptic Transmission

What enters the presynaptic terminal during synaptic transmission

Ca2+ (Calcium)

What is CaMKII? What does it do?

CaMKII (Ca2+/Calmodulin-dependent protein kinase, type II) are protein kinases that are activated by incoming calcium. CaMKII phosphorylate Synapsin to detach the vesicles from actin. Then they go to the plasma membrane and dock.

What is the reasoning behind calcium determining which vesicles are released and how many?

Calcium channels are positioned close to the presynaptic membrane, so the local concentration of calcium will be highest there. For example, a low stimulation will result in only a small increase in calcium concentration at the presynaptic membrane. This small release of calcium will only release clear-core vesicles that are pre-made from the readily-releasable pool, because there small-molecule neurotransmitters are easy and ready to go. A high-frequency stimulation will result in a larger and more diffuse calcium concentration, so not only will the clear-core vesicles be released from the readily-releasable pool, but now also the clear-coat vesicles from the reserve pool AND the neuropeptide dense-core vesicles as well.

What is clathrin-mediated endocytosis?

Clathrin is a protein that plays a major role in the formation of coated vesicles. Clathrin triskelia attach to the vesicular membrane and coat the budding vesicle. The molecular shape of clathrin physically forces the membrane it binds to into a ball-like shape.

How was neurotransmitter release studied?

Scientists used the Neuromuscular junction (NMJ) to learn how neurotransmitters are released. (NMJ: A neuromuscular junction is a chemical synapse formed by the contact between a motor neuron and a muscle fiber.) The same principles of synaptic transmission apply whether the postsynaptic cell is muscle or neuron

Summarize the entire process of synaptic transmission

See picture

When does kiss-and run, or the "flicker-fusion" hypothesis occur?

It appears to occur at some synapses under conditions of very high Ca2+ (sustained activity)

If EPC of Na+= gACh(-160) and EPC of K+=gACh(10), how will this current change the membrane potential?

It will depolarize it. When sodium enters it depolarized the cell.

Extra Membrane problem... If vesicles fuse to the plasma membrane, what prevents the terminals from just getting bigger and bigger?

The synaptic vesicle is recycled, or in other words is used again by endocytosing the vesicle back in.

What does a molecular view of a vesicle look like

These are all important for certain functions.

When do MEEPs and EPPs look similar?

They look similar when the calcium level is low. When calcium level is slow it produces a small end plate potential, which looks like the mini end plate potential that happens spontaneously without stimulation/calcium.

What is a co-transmitter?

It's when a neuron releases more than one neurotransmitter at a synapse, the neurotransmitters are called co-transmitters. Many times one neurotransmitter will be a small-molecule neurotransmitter and the other one will be a neuropeptide neurotransmitter.

What do SNARE proteins do?

Promote fusion of vesicles at the membrane by pulling the vesicle close to the plasma membrane. Once synaptobrevin and SNAP-25 wrap around each other the vesicle is then considered "primed" and ready.

What are the steps of vesicle endocytosis?

1. Adaptor proteins connect clathrin to vesicular membrane 2. Clathrin triskelia assemble into coat, curbing membrane 3. Dynamin ring forms and pinches off membrane 4. Coated vesicle translocated by actin filaments 5. Hsc-70 and auxiliary uncut vesicle

How do we figure out what ion or ions carry this current that is traveling through the acetylcholine receptor?

1. First find the reversal potential of the current and see If its close to the equilibrium potentials of any ions. Scientists did this and found the reverse potential of the current was 0mV and that was close to the equilibrium potential of sodium, potassium, and chlorine. This told scientists that the cell must be permeable to more than one ion. 2. Then alter ion concentrations extracellularly and see how it changes the reversal potential Scientists noticed that changing the concentrations of sodium and potassium changed the reverse potential. However changing Chlorine did nothing. This indicated to scientists that the acetylcholine receptor must be permeable to both sodium and potassium ions.

Provide a summary of synaptic transmission?

1. Neurotransmitter release 2. Receptor binding 3. Ion channels open or close 4. Conductance change causes current flow 5. Postsynaptic potential changes 6. Postsynaptic cells excited or inhibited 7. Summation determines whether or not an action potential occurs

What are the main types of neurotransmitter? (Hint: There's 2)

1. Small-molecule neurotransmitter 2. Neuropeptide transmitter

What are the in-depth steps of synaptic transmission?

1. Transmitter is synthesized and then stored in vesicles 2. An action potential invades the presynaptic terminal 3. Depolarization of presynaptic terminal causes opening of voltage-gated Ca2+ channels. 4. Influx of Ca2+ through channels 5. Ca2+ causes vesicle to fuse with presynaptic membrane 6. Transmitter is released into synaptic cleft via exocytosis 7. Transmitter binds to receptor molecules in postsynaptic membrane 8. Opening or closing of postsynaptic channels 9. Postsynaptic current causes excitatory or inhibitory postsynaptic potential that changes the excitability of the postsynaptic cell 10. Removal of neurotransmitter by glial uptake or enzymatic degradation 11. Retrieval of vesicular membrane from plasma membrane

What are the four steps of fusion

1. Vesicle docks 2. SNARE complexes form to pull membranes together 3. Entering Ca2+ binds to synaptotagmin 4. Ca2+ bound synaptotagmin catalyzes membrane fusion

What are the basic steps of synaptic transmission at chemical synapses

1. calcium enters the presynaptic terminal 2. neurotransmitters are released 3a. The vesicles containing the neurotransmitters are endocytose and recycled 3B. Receptors on the postsynaptic membrane open channels and cause signaling postsynaptically

What is a neurotransmitter? (Hint: 3 things)

A neurotransmitter is a stubstance that: 1. A neurotransmitter is present within the presynaptic neuron 2. A neurotransmitter is released in response to presynaptic depolarization 3. A neurotransmitter affects the postsynaptic cell by binding to specific receptors

What is the difference between EPSPs vs IPSPs?

EPSPs: post-synaptic potentials (PSPs) that are excitatory, meaning they increase the likelihood of firing IPSPs: post-synaptic potentials (PSPs) that are inhibitory, meaning they decrease the likelihood of firing.

T or F: Changing the [Cl-] changes the reversal potential of an acetylcholine receptor.

False; Changing [Cl-] does not

How do you calculate the end plate current for each ion?

For each voltage used, we can calculate the end plate current for each ion and then see which ion gives the bigger end plate current. The bigger end plate current indicated it is the ion that contributes more current. Resting membrane potential in a muscle is -90mV and in a muscle, the reversal potential for sodium is +70mV and for potassium it is -100mV. Vm (membrane potential): -90mV E (reversal potential for sodium): +70mV E (reversal potential for potassium: -100mV) Plus these numbers into the equation in the image, and you roughly get the following: EPC of sodium: g*-160 EPC of potassium: g*10 Note: negative charge here means positive charge is leaving the extracellular space, which means it is entering the cell

What is summation of PSPs?

Most neurons receive many inputs from both excitatory and inhibitory synapses. Neurons sum the PSPs to determine their overall membrane potential.

Which ion provides most of the current that will depolarize its membrane?

Most of the current is due to Na+ influx which will depolarize its membrane. However the exact current depends on the membrane potential (Vm) of the muscle cell.

What ion(s) does the AChR let through?

Na+ and K+

What ion or ions carry this current through the AChR?

Na+ and K+ can go through the AChR

EPC total= (EPC of Na) + (EPC of K+) If EPC of Na+= gACh(-160) and EPC of K+=gACh(10), which ion contributes more to the total current?

Na+ because the end plate current of sodium if 160 compared to 10 of potassium.

What are the effects of a neurotransmitters binding to receptors on the postsynaptic cells?

Neurotransmitter binding generates a response in the postsynaptic cell that can be measured with the voltage change: PSP (neuron) or EPP (muscle), or with the current using a voltage-clamp. The inward current that is measured is called the end-plate current (EPC) for muscles and post-synaptic current (PSC) for neurons.

What is the key difference between PSP and EPP

PSP is when the postsynaptic cell is a neuron cell EPP is when the post synaptic cell is a muscle cell

Compare and contrast small-molecular neurotransmitters and neuropeptide transmitter

Small-Molecule neurotransmitter 1. Packaged in SMALL CLEAR-CORE vesicles 2. Synthesized in the TERMINAL 3. Usually RECYCLED by terminal Neuropeptide transmitter 1. Packaged in LARGE DENSE-CORE VESICLES 2. Synthesized in SOMA 3. DEGRADED after release

What is one clue to how neurotransmitter release?

Spontaneous release. Spontaneous release gave the scientists a hint of how neurotransmitters work.

What are spontaneous releases? What are they called?

Spontaneous releases are when you see small EPPs or PSPs without stimulation These mini-events are called MEPPs or mPSPs

What is Synapsin? What does it do?

Synapsin is a protein that regulates neurotransmitters. It binds reversibly to synaptic vesicles. Synapsin cross-links the vesicle to actin to keep them in the reserve pool. Synapsin can be a good marker for presynaptic terminals.

What is synaptotagmin? How is it involved in the fusion process?

Synaptotagmin is a protein on the vesicle that binds to calcium. When it binds to calcium it can then interact with the SNARE proteins. Interacting with the SNARE proteins prompts the vesicle to fuse.

What proteins are involved in fusion?

The SNARE proteins are involved in fusion. Specifically: -Synaptobrevin: It is on the membrane of the vesicle -Syntaxin, SNAP-25: It is on the plasma membrane They all have a motif that can assemble together into a tight bundle of four alpha-helices

What determines which vesicles are released and how many?

The amount of calcium influx determines which vesicles are released and how many.

What is the change in voltage in the postsynaptic cell called?

The change in voltage is called: A) PSP (Post synaptic membrane potential) if the postsynaptic cell is a neuron B) EPP (End Plate Potential) if the post synaptic cell is a muscle cell

If EPC of Na+= g(-160) and EPC of K+=g(10), what direction is this current, inward or outward? Note: negative charge here means positive charge is leaving the extracellular space, which means it is entering the cell

The current is entering the cell because sodium makes up the majority of the current, and sodium's end plate current is -160. The negative sign indicated it is leaving the extracellular space and entering the cell.

If the EPC of Na+= gACh(-160) and EPC of K+=gACh(10), which EPC has a higher absolute magnitude?

The end plate current of Na+ has a higher magnitude with 160 compared to the 10 of potassium.

What is the hypothesis to why MEPPs look similar to the EPPs at low CA2+ level

The hypothesis that scientists came up with is maybe neurotransmitters come in "packets". For example Each packet produces one MEPP-sized (mini sized end plate potential) response. An EPP comes from releasing many packets at a time

What is an example from the lecture, of how a neurotransmitter binding to receptors affect the postsynaptic cell?

The lecture focused on the NMJ (neuromuscular junction) as an example of postsynaptic responses to neurotransmitter binding. The NMJ uses acetylcholine (ACh) as a neurotransmitter. The postsynaptic receptor is the acetylcholine receptor (AChR) Scientists were able to determine there is an inward current through an individual acetylcholine receptor

We know that clear-coat vesicles of small-molecule neurotransmitters are synthesized in the terminal. However, some clear-coat vesicles are found closert to the presynaptic membrane and some are not. Why is this?

The ones close to the presynaptic membrane are referred to as the readily-releasable pool because they are ready to be released at any moment. The ones further away form the reserve pool. They are the reserve kept in storage for future use.

How did they test that these quanta of EPPs correspond to vesicles?

They performed a test where they made several EPPs of different amplitudes. For each EPP you can calculate how many quanta must have been involved, by dividing the EPP by the size of a MEPP. Then you can physically count the number of vesicles that fused to the presynaptic membrane using electron microscopy to prove your calculation earlier. The number should match meaning the quanta correspond to vesicles.

T or F: Changing either the [Na+] or the [K+] changes the reversal potential of an acetylcholine receptor.

True

T or F: Neurotransmitters are released from pre-synaptic terminal via help of Ca2+

True

T or F: When membrane potential is made more positive it is called excitatory

True

T or F: Ca2+ influx triggers release of neurotransmitters, which causes a postsynaptic response.

True; Calcium needs to enter the presynaptic membrane in order to release the neurotransmitters into the synapse so they can bind to the receptors on the postsynaptic cell.

T or F: Mechanisms of fusion are similar to those for vesicles fusing with the Golgi

True; Fusing into a membrane is the same regardless if it is a vesicle fusing with the Golgi or a vesicle fusing with the membrane of a neuron

T or F: A neuron can release more than 1 neurotransmitter

True; Many times one neurotransmitter will be a small-molecule neurotransmitter and the other one will be a neuropeptide neurotransmitter.

T or F: Acetylcholine receptor must be permeable to both Na+ and K+ ions

True; This is true because changing either the [Na+] or the [K+] changes the reversal potential.

How are neurotransmitters released from the reserve pool?

Using CaMKII to phosphorylate Synapsin so vesicles can be released from the reserve pool

How do you figure out which ion, Na+ or K+, will contribute more current at a given voltage?

We can figure this out by calculating the end plate current (EPC) for each ion, and then see which ion gives the bigger EPC.

What is a way to measure the amount of neurotransmitter, to prove that Calcium is in fact what releases neurotransmitters?

We can indirectly measure the amount of neurotransmitter release by measuring the resulting change in voltage in the postsynaptic cell.

Is fusion and endocytosis really necessary?

Yes and no. In some special circumstances it isn't necessary, such is the case in the kiss-and-run (or flicker-fusion) hypothesis The kiss and run hypothesis is when Vesicles can release their neurotransmitters without fully fusing with the membrane, therefore avoiding the need for fusion and endocytosis/recyling of vesicles.

Does stimulation cause influx of calcium?

Yes. Stimulation DOES cause an influx of calcium. We can prove it by using Ca2+ sensitive dyes to measure the calcium during a synapse. For ex. we can see Ca2+ signaling at the squid giant synapse (synapse: the junction between two nerve cells) after electrical stimulation. So yes, stimulation causes influx of calcium.

Does the Ca2+ influx cause release of neurotransmitter?

Yes. The influx of calcium does cause release of the neurotransmitter from the presynaptic neuron. How to prove it/How we know: 1. Lowering extracellular calcium changes the amount of neurotransmitter released. 2. Direct injection of Ca2+ presynaptically causes a postsynaptic response 3. Injection of a Ca2+ chelator blocks this response. (chelator: a molecule that binds to something and blocks its action)

Are there voltage-gated Ca2+ channels on the membrane of the presynaptic terminal?

Yes. Voltage-gated calcium channels are found on the membrane of the presynaptic terminal. We can prove it by stimulating the neuron and observing for presynaptic calcium current. If voltage-gated Ca2+ channels sit on the membrane of the presynaptic terminal, we should be able to measure a presynaptic current and block it with Ca2+ channel blockers