Lecture 14: Synaptic Transmission and Vesicle Cycle

Chemical Synapses

• Molecules stored in vesicles are released from one cell onto another to produce an effect

• Molecules diffuse across a “gap” between cell membranes

  • ~20nm distance

• Signalling: relatively slow (~0.5msec)

  • Puts a ‘break’ on one cells ability to affect another

• Unidirectional

  • Post-synaptic cell may release a retrograde transmitter which will affect the pre-synaptic cell

• Majority of synaptic transmission in the nervous system occurs via these synapses

Electrical Synapses

• Small regions of adjacent membranes come into close contact

• Gap junctions present at the site of contacts to bridge the membranes of both cells to allow the movement of ions and small molecules (~500M.W)

  • Holes” in adjoining cell membranes: linked by channels - gap junctions or connexons (hexameric - made up of connexin subunits)

• Signalling: very fast - near instantaneous electrical signalling between cells, what occurs in one cell will happen in another cell due to the transfer of ions

• Bidirectional

• Important for direct electrical coupling between cells - electrical synchronization e.g. in the heart

• Relatively rare in the nervous system – occurs in the inhibitory interneurons or local networks e.g. neocortex, retina

Function of Chemical Synapses

• They allow for flexibility and modification of nervous system function through

  • Neural computation: integration of many input +/- to generate, process, neural signals to produce an output  

  • Exhibit plasticity: supports development, learning and memory by chaining the strength of neural connections involved in the organisation and encoding of information

  • Drug targets: affect neurotransmitter synthesis, release, receptors, uptake, and degradation.

  • Functional Flexibility: Produce complex effects by modifying neural activity.

6 Criteria For Neurotransmitter Classification

• Synthesised and stored in the pre-synaptic neurone (vesicles)

• Released upon stimulation of that neurone in a depolarisation and Ca2+-dependent manner

• Located in the appropriate region at levels sufficient to evoke physiological responses

• Compound must reproduce physiological effects when applied to a postsynaptic neurone

• Transmitter recognition & signal transduction mechanisms (receptors etc.) associated with that postsynaptic neurone

• Transmitter removal mechanisms

Small Molecule Neurotransmitters

• A class of neurotransmitters that includes:

  • Amino acids e.g. Glycine, GABA, glutamate

  • Amine e.g. Noradrenaline, Dopamine and Serotonin

  • Purine e.g . Adenosine and ATP

Peptide Neurotransmitters

• A class of neurotransmitters that consists of short-chained polypeptide sequences and include:

  • Endorphins/ Enkephalins/ Dynorphin

  • Neuropeptide Y

  • Somatostatin

  • Substance P

  • vasoactive intestinal Peptide etc

Dales Principle

• The idea that a neuron releases only one type of neurotransmitter at all of its synapses

• It’s been challenged by the co-existence and release of small molecule neurotransmitters and peptides by interneurons, such as GABA and enkephalins in the striatum, and the release of more than one neurotransmitter in some projection pathways, such as L-glutamate and dopamine in the VTA to nucleus accumbens pathway.

Significance of VTA-Nucleus Accumbens Pathway

• It was thought to be solely dopaminergic, but it is actually both dopaminergic and glutamatergic

Small Synaptic Vesicles (SSVs)

• ‘Clearer synaptic vesicle’ – contains small molecule NT

• Cell type: Neurone

• Diameter: 40nm

• Location: synapse active zones

  • Active zone – pre-synaptic release site opposite the post-synaptic density

• VGCa2+ Channels: Located close by – part of the active zone

• Contents: Small neurotransmitter

• Threshold concentration - [Ca2+] 200uM

  • High concentration threshold explains the close proximity of the channels – a high threshold to initiate the release process

• Physiology: Single Action Potential

• Biogenesis: Constitutive local vesicle, recycling transmitter synthesis and uptake

Large Dense Cored Vesicles (LDCVs)

• Cell type: Neurons and neuroendocrine cells

• Diameter: ~100nm

• Location: non-specific, diffuse i.e all over the cell

  • Originated from the ER and Golgi

  • Transported via the microtubule system and axon transport system to potential release sites

  • Not specifically associated with the active zone

  • distributed relatively diffusely in pre-synaptic terminals

• VGCa2+ Channels: Located relatively distant

• Contents: Peptides, and proteins (sometimes noradrenaline)

• [Ca2+]: 5-10uM – more sensitive to changes in intracellular Ca2+ due to the distance from the channel – to initiate release of more diffuse elevation of [Ca2+]I from the VG Ca2+

• Physiology: Involved in repetitive AP activity  

• Biogenesis: Highly regulated ER-derived vesicles (no recycling) transmitter production and processing under direct genomic control; trafficked to release sites

Classical Vesicle Cycle

1. Exocytosis:

   • Vesicles associate with the plasma membrane and release their contents.

   • The vesicle membrane fuses with the plasma membrane and adds to it.

2. Endocytosis:

   • Recover membrane added during exocytosis.

   • Vesicles formed from the endosome act as a "holding tank" for membrane and neurotransmitter (NT) storage.

3. Reserve Pool:

   • A ‘backup’ pool of vesicles that are ready to be mobilized and fused with the membrane for NT release.

   • New vesicles filled with NT from the endosome enter the reserve pool,

   • Visualised with electron microscopy

4. Docking and Priming

   • Vesicles under a 2 stage process where they become attached and primed to the membrane ready to undergo function

   • Heavily ATP-dependent process

Electron Microscopy Visualisation of Reserve Pool

• Synapse with pre-synaptic ending  - large .o vesicles associated

• Tissue treated with Antibodies for synapsin

• Depletion of some of the vesicles  - distinction of the reserve pool – disruption of synapsin action allows the vesicles to disperse and disappear

• Vesicles that remain are actively associated with the membrane – can’t dissociate

Slam Freezing

• Technique developed by Heuser and Reese → provided evidence for full vesicle fusion and collapse

• Involved rapidly cooling frog neuromuscular junction on a metal block after electrical stimulation of motor neurone axon fibres to initiate acetylcholine release      

• Freeze fracture electron microscopy is used to visualise the presynaptic membrane    

  • Sections of the presynaptic membrane were visualised at different times after stimulation to follow any changes in presynaptic membrane structure:

• Pits formed at 4ms and persisted after 8ms, indicating sites of vesicle fusion

• Presynaptic Vesicles:

  • 40-50 nm in diameter, clear centres, spherical.

  • Contains thousands of neurotransmitter molecules based on volume.

• Problem: Increased membrane surface area due to fusion.

• Solution: Vesicle recycling maintains membrane area homeostasis.

Vesicle Cycle: Step 1 - Docking

• The close association with plasma membrane

  • Don’t dissociate if the plasma membrane is treated with an Ab

• Synaptic vesicles only dock at the active zone

• This is the presynaptic area adjacent to the signal transduction machinery of the postsynaptic membrane

• Active zones differ between neurones (by vesicle number) - reflects their function and the functions they support

Vesicle Cycle: Step 2 - Priming

• Prepares vesicles for release

  • Synaptic vesicle “maturation” process

  • Vesicles made into a  competent state to release transmitter (in response to Ca2+)

  • Requires ATP w/o won’t occur

  • May be required for a conformational change in proteins that drive the release

• If this step doesn’t occur, the vesicles won’t release

Vesicle Cycle: Step 3 - Fusion/ Exocytosis

• The release of vesicle contents

  • Full fusion of synaptic vesicle and presynaptic terminal membrane

  • Requires Ca2+ (increased)

  • Involves Ca2+ “sensor” protein – detects and initiates release

  • Fusion induces exocytosis - contents discharged (diffusion) – full fusion and vesicle membrane becomes part of the cell

  • Takes about 1msec      

• Results in the continual addition of membrane to the plasma membrane

Vesicle Cycle: Step 4 - Endocytosis

• The recovery and recycling of vesicle fused membrane via endocytosis

  • Vescular membrane recovery is mediated by certain proteins associated with the membrane that will assist in its recovery

  • It is triggered by increased intracellular Ca2+, membrane is recovered as clathrin associates with the protein on the membrane used to form the vesicle to form a clathrin coated pit

  • In the presence of ATP, it will trigger the release of the vesicle from the plasma membrane to be recycled back   

  • Involves cytoskeletal protein lattice formation (from clathrin monomers) to help pinch off the membrane

  • Takes about 5 secs and is ATP-dependent

• Spontaneous pit formation occurs following the vesicular relase

Vesicle Cycle: Step 5: Recycline

• The mechanism used to conserve synaptic vesicle membrane via endosome

• Trafficked back to the endosome to remove clathrin

• Sits in the endosome in a reserve buffer

•   Vesicles refill with transmitter (ATP-dependent again - concentrating neurotransmitter)      

• The entire vesicle cycle (docking to refilling) takes about 1 minute – QUICK/ RAPID TURNOVER as the process is biochemically driven

Kiss and Run

• An alternative vesicle fusion model

• It suggests that full vesicle fusion may not be required for neurotransmitter release

• When the vesicle is activated to release its contents, fusion pores form 

• These are discreet openings between the membrane and the vesicle, which allow neurotransmitters to leak out (down a concentration)

• SSVs are recycled intact from the cell membrane and are not recycled as clathrin-coated vesicles via the endoscope

  • Vesicle structure is maintained

Kiss and Run Model: Electron Microscopy Evidence

• Full vesicle collapse was observed in some areas of the membrane.

• Vescilce integrity is mainated and the formation of narrow conduits/ pores that allow the passage of neurotransmitters are seen

  • This model is harder to confirm with EM since it only provides snapshots; functional studies are more definitive.

Evidence of Kiss and Run Model - Functional Studies

• Measure activity during NT release following an increase in intracellular [Ca²⁺], which triggers membrane depolarisation and VG Ca²⁺ channel activation.

  • Membrane capacitance can be recorded

• Increased capacitance indicates increased membrane surface area during fusion.

• Full fusion shows a step-up in capacitance (more membrane added).

• In kiss and run, capacitance briefly increases and returns to baseline, suggesting a transient increase in surface area (flickering capacitance).

Vesicular Fusion: Capacitance and Surface Area

• Membrane capacitance is proportional to surface area.

• In the full fusion model, membrane capacitance steadily increases, whereas in kiss and run, the change is brief and reversible

Kiss and Run Mechanism of Vesicular Fusion/Release

• Fast recycling

• Low capacity - only a few vesicles over time in the active zone

• Favoured at low frequency stimulation

• 70-80% of glutamate release in the hippocampus mediated via this

Classical Mechanism of Vesciular Fusion/Release

• Slow recycling

• High capacity - many vesicles over time    

• Favoured at high frequency stimulation

Release Machinery Proteins

• Proteins that associate with vesicles or the active zone of the plasma membrane

• They ensure vesicular engagement with the cell membrane and the initiation of the release proteins

  • Involves VAMPs - vesicle assocaited membrane proteins

Vesicle Associated Proteins

• Synapsins

• Synaptobrevins

• Synaptoatagmins

• Rab proteins effectors

• Synaptophyins

• SV2s, SCAMPs, CSPs

• Neurtransmitters transporter

• Vacuolar proton pump

• Amphipysins, AP2 clathrin

• CaMKI and CamKII, pp60src

• Dyamin 1

• Dynein, kinesin

Plasma Associated Proteins

• SNAP-25

• Syntaxins

• Voltage Gated Ca2+

• Complexins

• Munc18s – important in ensuring priming of vesilces

• NSF and SNAPs – important in allowinging vesciles to disengage from the membrane

• Muc13s

• Nerexins

SNARE Proteins

• Proteins that become entangled to allow vesicles to engage in the membrane

• There are 2 types

  • v-SNARE e.g synaptobrevin

  • t-SNARE, e.g. SNAP-25

Synaptobrevin

• A v-SNARE located on the vesicle (VAMP)

• It is 18kDa

• Single transmembrane spanning

Syntaxin

• A t-SNARE located on the target membrane

  • 35kDa

  • Single transmembrane spanning

• Has a regulatory domain important for priming vesicles

SNAP-25 (Synaptosomal-associated protein 25)

• A t-SNARE located on the target membrane

  • 25kDa 

  • Anchored to membrane by S-acylation

S-Palmitolyation

• Post-translational modification that allows the attachment of proteins to the plasma membrane and the cytosolic/ inner leaflet

Docking of Vesicle (Release Machinery)

• Involves Synaptobrevin (on vesicle) and SNAP-25 and Syntaxin (on target membrane)

• Synaptobrevin, Syntaxin, and SNAP-25 form a 1:1:1 trimeric complex, known as the SNARE complex.

• This complex is a coiled-coil structure of α-helices.

  • SNAP-25 has two α-helical regions

• The α-helices of the SNARE proteins wrap tightly around each other, bringing the vesicle into close contact with the target membrane.

• This interaction ensures the vesicle is docked and ready for fusion.

  • how vesicles move from the reserve pool to associate with the cytoskeleton

Priming of Vesicles (Release Machinery)

• The SNAREs form a tighter complex → believed to store energy to facilitate the release process

• SNARE pins form via zippering, where the coiled-coil complex between the 3 SNARE proteins becomes tighter - orchestrated by the regulatory domains of syntaxin

  • SNARE pins trigger vesicular fusion in response to a signal

• Process in ATP dependent - uses MunC18 (ATPase)

Synpatotagmin

• A 65kDa Ca2+ sensor found on vesicles

• It has a cytosolic region where Ca2+ are able to bind and cause a conformation change which then initiates the fusion process

• In the absence of Ca2+ it binds to SNARE pins in priming

• In the presence of Ca2+, it binds to phospholipids and causes the VAMP to vesicle into the membrane for fusion

Release Machinery in Vesicular Fusion

• They are activated through a conformational change in synaptotagmin

• It alters the configuration of the release machinery that pulls the vesicle closer to the membrane and releases the energy stored in the SNARE pin (and SNARE complex) to pull the membranes apart and allow fusion to occurr

NSF

• An ATPase that facilitates the dissociation of SNAREs.

• SNAREs are tightly coupled in coiled-coil structures that keep the vesicular membrane fused with the plasma membrane.

• It uses ATP to "uncoil" the SNAREs, allowing them to dissociate.

• This dissociation is crucial for:

  • Internalisation of empty vesicles.

  • Re-docking of another vesicle with the same T-SNAREs.

• It binds to the SNARE-pin complex to facilitate SNARE release.