Exocytosis 9
1. Introduction to Synaptic Transmission
Synaptic Plasticity: The brain’s synapses are constantly changing throughout life, which is essential for learning and development.
Model System – NMJ:
Research on the synaptic transmission has often been conducted at the Neuromuscular Junction (NMJ), where motor neurons communicate with muscle fibers.
The NMJ is easier to study due to its simplicity, ease of access, and reliability, which helps form the basis for understanding synaptic transmission in more complex systems such as the Central Nervous System (CNS).
2. Key Anatomical Features of the NMJ
Presynaptic Cell:
The presynaptic motor neuron sends its axon toward the axon terminal at the muscle.
The axon terminal contains voltage-gated ion channels (Na⁺, Ca²⁺) essential for action potential propagation and neurotransmitter release.
Postsynaptic Cell:
The muscle fiber’s membrane, called the sarcolemma, contains acetylcholine (ACh) receptors, which respond to the neurotransmitter ACh.
Synaptic Vesicles:
Neurotransmitter-filled vesicles (primarily ACh) are stored within the presynaptic terminal, poised for release upon activation.
3. Signal Transmission Mechanism at the NMJ
Action Potential Arrival:
An action potential (AP) travels down the motor neuron’s axon, causing depolarization at the axon terminal.
Ion Influx:
Voltage-gated sodium channels open, allowing Na⁺ ions to enter the presynaptic terminal.
Depolarization then triggers voltage-gated calcium channels to open, leading to an influx of Ca²⁺ ions.
Vesicle Fusion & Neurotransmitter Release:
Calcium ions initiate the fusion of synaptic vesicles with the presynaptic membrane.
Neurotransmitter (ACh) is released into the synaptic cleft through the process of exocytosis.
Activation of ACh Receptors:
Released ACh binds to receptors on the postsynaptic membrane (muscle fiber).
This binding opens non-selective cation channels, allowing Na⁺ to enter and K⁺ to exit, generating a graded potential.
Action Potential in Muscle:
If the graded potential reaches threshold, it activates voltage-gated sodium channels in the muscle membrane, triggering its own action potential, leading to muscle contraction.
Vesicle Recycling:
ACh is rapidly broken down by acetylcholinesterase (AChE) into acetate and choline.
Choline is taken back into the presynaptic terminal for repackaging into new vesicles, while vesicle membrane components are recycled via endocytosis.
4. Synaptic Vesicle Recycling & Exocytosis
Vesicle Docking:
Synaptic vesicles are docked at active zones of the presynaptic terminal, ready for rapid release (about 20-100 times per second).
Fusion Mechanism:
Upon Ca²⁺ influx, the vesicle membrane fuses with the presynaptic membrane, releasing its contents.
The vesicle is recaptured after neurotransmitter release via endocytosis, and the membrane is recycled.
5. Evidence for Vesicle Release and Quantal Transmission
Quantal Release:
Early studies by Castillo, Fatt, and Katz (1952-54) showed that neurotransmitter release occurs in discrete packets, or quanta.
Monitoring Endplate Potentials (EPPs) showed that the amplitude of miniature endplate potentials (MEPPs) is about 1 mV, even in the absence of stimulation, suggesting quantal release of ACh.
EPP and MEPP Relationship:
When extracellular Ca²⁺ concentration is reduced, stimulated EPPs show amplitudes similar to MEPPs, confirming the idea that EPPs are a summation of MEPPs.
Electron Microscopy:
Later research using electron microscopy identified that neurotransmitters are stored within membrane-bound vesicles, which fuse with the presynaptic membrane during exocytosis.
This provided direct evidence for the quantal nature of neurotransmitter release.
6. Evidence of Exocytosis & Neurotransmitter Release
Black Widow Spider Venom (BWSV):
BWSV induces excessive, uncontrolled neurotransmitter release at the NMJ.
Electrophysiology studies revealed spontaneous release of neurotransmitters, resulting in excessive membrane fusion at the nerve terminal.
This suggests that vesicle fusion and exocytosis add membrane to the terminal, which can be seen as terminal swelling after prolonged neurotransmitter release.
7. Fluorescence Imaging for Real-Time Visualization
FM1-43 Dye:
The dye is hydrophilic and hydrophobic and selectively labels the outer leaflet of the vesicle membrane.
During exocytosis, vesicles move to the surface and become labeled, and the fluorescence intensity decreases as the dye is released into the cleft.
Real-Time Imaging:
Video microscopy shows a 1:1 correlation between dye loss and neurotransmitter release, confirming that the vesicles undergo exocytosis upon action potential stimulation.
8. Spatial Dispersion of Release
Active Zones:
Release occurs at active zones, specialized regions in the presynaptic terminal where vesicles are docked.
In frogs, one vesicle is released per active zone for each action potential.
In mammals, only 1 in 10 active zones are activated by each action potential, reflecting a more complex system of vesicle release.
ACh Breakdown:
Acetylcholinesterase (AChE) rapidly degrades ACh in the synaptic cleft to prevent its spread, ensuring that ACh only affects the local area at the active zone.
9. Calcium’s Role in Vesicle Release
Calcium Dependency:
Calcium ions are essential for vesicle release, with the level of intracellular calcium directly affecting the extent of neurotransmitter release.
Study in rat hippocampal neurones
Depolarisation shows that the calcium goes in, just before the transmitter release from vesicles
FM1-43 – vesicle exocytosis
Fura-2 – internal calcium levels
The more calcium you have, the more release you get ; as the FM1-43 goes down
Studies have shown that about 4 calcium ions are required to trigger the release of a single vesicle, with a 4th power relationship between calcium concentration and vesicle release.
Slater tried to answer how many Ca2+ ions are required for NT release
Depends on a variety of factors
Expected correlation if calcium reqhired and trigfers release - I.e. the more calcium means stronger release of vesicle
Slope = 4 Ca2+ ions/SV
Calcium Channels and Docking Proteins:
Synaptotagmin, a protein on the vesicle membrane, binds calcium, triggering vesicle fusion with the presynaptic membrane.
Proteins like syntaxin, synaptobrevin, and synapsins help organize and tether vesicles at the active zone for efficient release.
10. Vesicle Organisation and Active Zone Structure
Active Zone Morphology:
Electron microscopy reveals the active zone is structured like a network, with filaments connecting vesicles to the presynaptic membrane.
A complex array of proteins links vesicles to active zones and facilitates efficient release during action potentials.
Harlow et al (2001) -> active zone in 3D morphometry
Took a normal EM slice and tilted it by 5 degrees at each interval (±70 degrees at every direction) and image each step
3D reconstruction (cf CAT scan) was created
Imaging of vesicles joined to the membrane – shows that the vesicles are stuck/joined to each other
This filament is a like a string
11. Vesicle Pool Management
Ready Releasable Pool (RRP):
Terminals maintain a ready releasable pool of vesicles that are immediately available for release.
The reserve pool consists of additional vesicles that can be mobilized as needed.
Fatigue Resistance:
Soleus muscle fibers, used for continuous activity, maintain a larger reserve pool of vesicles compared to EDL muscles used for quick bursts of activity, making soleus muscle terminals more fatigue-resistant.
Conclusion
Exocytosis and Neurotransmission: The release of neurotransmitters via exocytosis at the NMJ follows a carefully orchestrated process involving vesicle fusion, calcium influx, and membrane recycling.
Neurotransmitter Release Mechanisms: The quantal nature of neurotransmitter release, supported by techniques like fluorescence imaging and electron microscopy, provides robust evidence for the exocytotic process.
Calcium’s Critical Role: Calcium ions are crucial for vesicle release, and the spatial organization of release sites ensures effective neurotransmission across the synapse.