Lecture 10 - Neurotransmitters and the Synaptic Vesicle Cycle
A Note on Notes
Most students report that versions of slide notes are often too wordy to manage during lectures; however, these notes become more beneficial when reviewing material afterward to clarify the points of the slides or the definitions of terms.
Learning Objectives
Experimental approaches: Describe at least two common experimental methods for testing synaptic vesicle recycling.
Roles of proteins: Explain the specific roles of clathrin and dynamin in the vesicle recycling process.
Release pools: Describe how vesicles are experimentally categorized into distinct release pools and predict how varying stimulation levels (brief, moderate, prolonged) will affect vesicle release rates.
Naming principles: Recognize and apply common naming principles to analyze neurotransmitter synthesis, packaging, and recycling mechanisms at a synapse.
Comparison of neurotransmitter classes: Compare and contrast the life cycles of small molecule neurotransmitters (e.g., glutamate, acetylcholine) with catecholaminergic/aminergic neuromodulator transmitters (e.g., dopamine, serotonin), particularly regarding recycling processes.
Predicting drug effects: Predict how a drug of known function (e.g., an inhibitor of a specific synthetic enzyme or transporter) will impact neurotransmitter signaling at a synapse.
Key Concepts
Synaptic vesicles are reformed and recycled at the axon terminal.
Clathrin-mediated endocytosis is essential for vesicle recycling.
Vesicles are organized into distinct physiological pools based on their availability for release.
Neurotransmitters comprise a diverse group of over 50 different molecules.
Neurotransmitters are also recycled post-release.
Review of Synaptic Transmission
Neurotransmitter release occurs when synaptic vesicles fuse with the presynaptic cell membrane. Lecture 9 focused on events on this side (Steps 2-6) concerning chemical synaptic transmission. Upcoming lectures (11 and 12) will cover Steps 7-9. This lecture will examine the life cycles of synaptic vesicles and the neurotransmitters contained within them (Steps 1, 10, and 11).
The Synaptic Vesicle Cycle
The synaptic vesicle cycle is a system responsible for the recycling of synaptic vesicles.
Soma Origin: The soma is responsible for the initial synthesis of vesicle proteins. However, used (released) vesicles are reformed at the axon terminal.
Transport Issue: There is slow transport into axon terminals.
Surface Area Issue: Fusing vesicles can increase the surface area of the axon terminal membrane.
To sustain a constant supply of synaptic vesicles, axon terminals possess mechanisms to recover membrane and proteins quickly to form new vesicles. Newly created vesicles are transported to axon terminals at a rate of 50-400 mm per day along microtubules, but this slow rate is inadequate to support the release of multiple quanta per action potential (AP) triggered by an axon.
Evidence for Vesicle Recycling
Electron microscopy has been instrumental in supporting the hypothesis that vesicles are recycled at the axon terminals.
Observations by Heuser and Reese (1973):
High-Frequency Stimulation: When neuromuscular junctions (NMJs) were subjected to high-frequency stimulation before fixation, vesicles appeared to be either undergoing fusion or pinching off from the axon terminal membrane.
Ruffled Membranes: The appearance of ruffled membranes indicates an increased surface area to volume ratio.
Coated Vesicles: Some vesicles appeared to have electron-dense halos, suggesting they were undergoing endocytosis rather than exocytosis.
Experimental Evidence with Horse-Radish Peroxidase (HRP)
To validate whether the observed vesicles were indeed newly formed through recycling:
Experimental Setup: Researchers stimulated neurons in the presence of HRP, a large hydrophilic dye that does not permeate lipid bilayers. An hour later, they fixed the tissue.
Observation: They noted that a fraction of the vesicles were filled with extracellular tracer dye, indicating they had been reformed through the recycling process. Notably, the number of dye-labeled vesicles correlated proportionality to the number of synaptic quanta released.
Key Processes of the Synaptic Vesicle Cycle
Endocytic Budding: Newly formed vesicles are shaped by the assembly of clathrin, a protein coat that influences membrane curvature. Enzymatic cleavage facilitates the vesicle's pinching off from the membrane.
Modern Diagrams: Current diagrams have updated the older textbook versions, reflecting findings from more recent experiments. The intermediate endosomal stage seen in earlier models occurs primarily under intense stimulation conditions.
Physiological Conditions: The budding process can happen faster (less than 1 second instead of 10-20 seconds) under physiological conditions of normal temperature and stimulation.
Clathrin and Dynamin in Vesicle Recycling
Clathrin's Role
Clathrin interacts with adaptor proteins (e.g., AP-2) to bind membranes, forming a complex scaffold that fosters the proper shapes for vesicles.
High-resolution transmission electron microscopy (TEM) and scanning electron microscopy (SEM) illustrate the cage-like structures formed around vesicle buds, constructed from clathrin.
Dynamin's Role
Dynamin, described as ‘molecular scissors’, is crucial for severing the vesicle from the axon terminal membrane.
Discovery in Drosophila: The temperature-sensitive 'shibire' mutant strain in Drosophila showed paralysis at elevated temperatures due to a depletion of synaptic vesicles. At permissive temperatures below 29ºC, vesicle budding occurred normally. At non-permissive temperatures, long clathrin-coated structures were observed without vesicle release. Upon hydrolyzing GTP to GDP, dynamin tightens around the vesicle neck, severing it from the membrane.
Proteins Involved in Vesicle Reformation
The disassembly of clathrin coats and adaptor protein scaffolds is critical for recycling, alongside the involvement of actin fibers from the cytoskeleton to speed up vesicle transport.
It is essential to recognize that vesicle recycling involves multiple proteins beyond just clathrin and dynamin.
Important Names to Remember
Synapsins: These are vesicle proteins that can tether vesicles, linking them to other non-active zone areas of the axon terminal. Their links are regulated by intracellular signaling.
Organizational Pools of Synaptic Vesicles
Types of Pools
Reserve Pool: These vesicles are filled but tethered by synapsins, remaining relatively immobile, and are typically released only during extreme prolonged stimulation, which inactivates synapsin.
Recycling Pool: Comprising mobile vesicles that can easily reach the active zone to maintain steady release levels during moderate stimulation.
Readily Releasable Pool (RRP): These are docked and primed vesicles available for immediate release upon single Ca^{2+} elevation.
Physiological Locations
In an actual synapse, recycling and reserve pool vesicles do not have fixed distances from the active zone; instead, they are anatomically mixed.
Understanding the Pools of Vesicles
The rate of stored dye release from axon terminals defines three distinct types of vesicle pools:
Dye Loading: Load synaptic vesicles with fluorescent dye while stimulating to assess how different groups of vesicles mobilize for fusion under various stimulation conditions.
Steady Prolonged Stimulation: With steady prolonged stimulation, the slope of fluorescence loss changes over time, showcasing three different groups regarding their availability for fusion.
Methods of Analysis
The fluorescence measurement after stimulating terminals provides insights into vesicle pool categorization based on availability of vesicles for release.
Checkpoint: Proteins in the Vesicle Cycle
Though many proteins participate in the synaptic vesicle cycle, only a few key players need to be remembered for exam purposes:
V-ATPase: A proton pump present in synaptic vesicle membranes responsible for acidifying the vesicle lumen to facilitate neurotransmitter loading.
Vesicular Transporter Proteins: Move neurotransmitters into the vesicle lumen.
Neurotransmitter Communication
Definition
Neurotransmitters are chemicals released at synapses that mediate communication either between two neurons or between a neuron and another excitable cell. This process requires direct mechanical channels rather than slower signaling pathways.
Types of Synthesis
Neurotransmitters can be synthesized from different precursors, including:
Free nucleic acids
Proteins
Phospholipids
Free amino acids (both essential and non-essential)
Dale’s Principle
Dale’s Principle asserts:
Each Neuron Releases Only One Kind of Neurotransmitter: Each neuron typically releases one type of neurotransmitter across all its axon terminals.
Exceptions Exist: There are exceptions (e.g., neurons releasing more than one neurotransmitter) where some neurons have multiple neurotransmitter identities.
The 3 Rs Principle
Non-peptidergic neurotransmitters are recycled effectively, returning them to the axon terminal for reloading.
Recap of Neurotransmitter Use in the Vertebrate Nervous System
A basic road-map for common neurotransmitters in the vertebrate system includes:
Peripheral Nervous System (PNS): Afferents release glutamate; efferents release acetylcholine and noradrenaline (norepinephrine).
Brain:
Excitation: Glutamate
Inhibition: GABA
Modulation: Various others
Spinal Cord:
Excitation: Glutamate
Inhibition: Glycine
Modulation: Various others
Life Cycle of Neurotransmitters
Critical Fact
Each neurotransmitter exhibits a distinctive life cycle involving:
Synthesis
Packaging
Release
Inactivation
Recycling and Repackaging into vesicles.
Similarities in Patterns
While these processes differ across neurotransmitters, the overarching sequence of events follows a similar pattern.
Stepwise Analysis of Neurotransmitter Cycles
Key Questions
How is this molecule synthesized?
What mechanisms load it into synaptic vesicles?
How is it removed from the synaptic cleft post-release?
What are the necessary precursors and enzymes?
What is the corresponding vesicle transporter?
How is it inactivated – by enzymatic degradation or reuptake?
Case Study 1: Acetylcholine
Acetylcholine Fact Sheet
History: First neurotransmitter discovered associated with synapses in the autonomic nervous system.
Key Role: Sole neurotransmitter at vertebrate neuromuscular junctions, with an exclusively excitatory effect. In the autonomic nervous system, it exhibits both excitatory and inhibitory roles.
Other Roles: Important for attention and memory in select CNS neurons.
Life Cycle of Acetylcholine
Synthesis: Occurs in the cytosol using acetyl CoA (from glucose in mitochondria) and choline (from phospholipid sources) with ChAT as the catalyst.
Loading into Vesicles: Mediated by the vesicular ACh Transporter (VAChT).
Inactivation: Acetylcholine esterase (AChE) catalyzes degradation in the synaptic cleft.
Reuptake of Choline: Conducted via the choline Transporter (ChT), which uses co-transport with Na+.
Case Study 2: Glutamate
Glutamate Fact Sheet
History: First put forth as a neurotransmitter in 1954; became widely accepted by 1980.
Key Role: Abundant neurotransmitter within the vertebrate CNS, used in over 90% of excitatory synapses.
Importance: Acts within at least some synapses in all animals with nervous systems.
Life Cycle of Glutamate
Synthesis: Occurs in the cytosol from glutamine, synthesized locally since it does not cross the blood-brain barrier. The main enzyme is glutaminase.
Loading into Vesicles: Conducted by vesicular glutamate transporter (VGLUT).
Removal from Synaptic Cleft: Executed by excitatory amino acid transporter (EAAT) on astrocytes and less on neurons.
Inactivation: Occurs in astrocytes and recycling of glutamate to neurons by SN1 and SAT2 transporters.
Case Study 3: Biogenic Amines
Overview
History: Noradrenaline was the first biogenic amine neurotransmitter recognized in the 1940s. Dopamine was identified in the 1950s and utilized for Parkinson's Disease treatment.
Key Roles: Biogenic amines, including dopamine and serotonin, act primarily as neuromodulators due to their distribution and effects.
Life Cycle of Dopamine
Synthesis: From tyrosine via L-DOPA with tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC) as key enzymes.
Loading into Vesicles: By vesicular monoamine transporter (VMAT2).
Reuptake: Conducted via the dopamine transporter (DAT) mainly in the presynaptic neuron.
Inactivation: Occurs primarily in astrocytes and presynaptic terminals via monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT).
Life Cycle of Serotonin
Synthesis: From tryptophan, with tryptophan hydroxylase as the key enzyme.
Loading into Vesicles: Conducted by VMAT2.
Reuptake: Via the serotonin transporter (SERT) largely by the presynaptic neuron.
Inactivation: Managed by monoamine oxidase (MAO) in the presynaptic terminal.
Test Your Understanding
Structure Recognition Pattern
In exam situations, similar figures will be presented. Consider recognizing patterns in naming conventions related to neurotransmitter life cycles, particularly regarding transporter proteins and their roles.
Future Lecture Preview
Psychoactive Drugs Examination
Many psychoactive drugs interact with the life cycles of biogenic amines by:
Raising levels of precursor molecules for neurotransmitter synthesis;
Affecting neurotransmitter inactivation or breakdown; and
Inhibiting reuptake by presynaptic neurons, impacting neurotransmitter availability at synapses. Examples include:
L-DOPA for Parkinson's Disease;
MAO inhibitors and COMT inhibitors as antidepressants;
SSRIs, SNRIs in treating anxiety and depression;
DAT inhibitors such as Ritalin, methamphetamine;
MDMA (ecstasy/Molly) impacts SERT;
AChE inhibitors for Alzheimer's Disease.
Summary of Lecture Points
Experiments from the 1970s confirmed local recycling of synaptic vesicles post neurotransmitter release, occurring at the axon terminal.
Clathrin and dynamin serve as dominant mechanisms in synaptic vesicle recycling.
There are more vesicles in presynaptic terminals than can be released, with the process regulated by synapsins and priming proteins crucial for maintaining neuronal signaling during prolonged activity.
The vertebrate system comprises over 50 neurotransmitter types with distinctive pathways for synthesis but similar pathways for packaging and release.
Non-peptidergic neurotransmitters generally undergo recycling at most synapses, providing important targets for medical interventions and psychoactive drug applications.