week 6 lecture 2

Synaptic Vesicle Cycle

• Components:

  • Loading

  • Docking

  • Priming

  • Calcium Sensing

  • Fusion

  • Endocytosis

  • Translocation

  • Sorting

  • Mobilization

• Pools of Vesicles:

  • Readily Releasable Pool

  • Reserve Pool

  • Storage

Local Recycling of Synaptic Vesicles in Presynaptic Terminals

• Endometry:

  • Endosome

  • Endocytosis

  • Budding: Takes approximately 1 minute (known to take 10-20 seconds)

  • Docking and Priming followed by Fusion

  • Ca²⁺ involved in 1 millisecond timing

Techniques for Studying Endocytosis

• Horseradish Peroxidase (HRP): An enzyme used for producing dense reaction products visible via electron microscopy (EM)

• Mutations:

  • SNAP-25:

  • SNAP-25 -/+ Heterozygous

  • SNAP-25 -/- Homozygous

Resting Membrane Potential

• Determined by K⁺ Concentration Gradient

  • Equilibrium Potential formula: Ek = $58 mV imes ext{log} rac{[ ext{K}^+]<em>{outside}}{[ ext{K}^+]</em>{inside}}$

  • Calculation Examples:

  • $E_k = 58 mV imes ext{log} rac{20}{400} = -76 mV$

  • $E_k = 58 mV imes ext{log} rac{90}{400} = -13.5 mV$

Synaptic Vesicle Endocytosis Model

• Role of AP2: Binds to synaptotagmin and recruits clathrin

• Dynamin: GTPase that wraps around the neck of the vesicle facilitating vesicle fission

Clathrin-Mediated Endocytosis

• Clathrin proteins form a protective cage around forming vesicles

• Clathrin-mediated endocytosis occurs over the course of tens of seconds, which is relatively slow and inefficient for replenishing the synaptic vesicle pool

Temperature-Sensitive Dynamin Mutant in Flies

• Dynamin function varies with temperature:

  • Permissive Temperature: 20ºC

  • Non-Permissive Temperature: 29ºC

• Dysfunction in dynamin disrupts vesicle recycling

Flash and Freeze Technique

• Modern adaptation of Heuser freezing technique

• Involves channelrhodopsin activation and rapid freeze fixation known as "Flash and Freeze"

  • Chief is a variant of channelrhodopsin activated by blue light

  • Mechanism allows for rapid muscle response recordings in C. elegans

Electron Microscopy Observations

• Flash and freeze EM has shown:

  • Docked synaptic vesicles fuse with the plasma membrane within 20 ms

  • Formation of Omega structures indicating vesicle fusion events

• Rapid endocytosis occurs in response to vesicle fusion, taking 20-100 ms

New Modes of Endocytosis Evidence

• Notable elements:

  • NT transporter

  • NT refilling

  • Reacidification

  • Uncoating

  • Reserve/Resting pool management

  • VATPase

  • Synapsin involvement in vesicle cycling

  • Importance of dynamin, actin, clathrin for endocytosis

• Types of endocytosis recorded: Ultrafast and Kiss and Run modes

Neurological Conditions Related to Synaptic Vesicle Cycle

• Conus Toxins: Known for blocking Ca²⁺ channels

• Congenital Myasthenic Syndromes: Linked to defects in endocytosis, resulting in loss of synaptic vesicles

• Congenital Synaptopathies: Affecting fusion machinery

• Clostridial Toxins: Cause paralysis or muscle spasms

• Lambert-Eaton Myasthenic Syndrome: Autoimmune disorder with antibodies against Ca²⁺ channels

• Familial Infantile Myasthenia: Characterized by smaller vesicles

Lambert-Eaton Myasthenic Syndrome (LEMS)

• Rare autoimmune disorder; immune system targets own tissues

• Symptoms:

  • Muscle weakness, fatigue, pain

  • Difficulty walking, reduced reflexes

  • Weakness in eye, face, and throat muscles

  • Speech and swallowing difficulties

• Patient Groups:

  • Autoimmune (50%) and associated with Small Cell Lung Cancer

  • Diagnosis after age 40

  • Diagnosis in children before age 10

• Symptoms affecting muscle color-coded from most affected (red) to least affected (yellow)

• Estimated LEMS prevalence in the U.S.: Approximately 3,000 patients

LEMS Treatment Options

• 1. Plasma exchange to filter Ca²⁺ channel antibodies

• 2. Immunosuppressants

• 3. Pharmaceuticals such as Firdapse, a K⁺ channel blocker targeting ACh release

The Tripartite Synapse

• Composed of three cell types: presynaptic neuron, postsynaptic neuron, and astrocytes

• Astrocyte Functions:

  • Regulate extracellular environment by uptake of K⁺ and neurotransmitters

  • Respond to neurotransmitters leading to intracellular Ca²⁺ signaling

  • Ca²⁺ waves spread through gap junctions to neighboring astrocytes

  • Trigger release of gliotransmitters (GABA, glutamate, ATP) that modulate neuronal survival and synaptic strength

Neurotransmitter Receptors

• Classifications:

  • Ionotropic receptors

  • Metabotropic receptors

• Time Scale of Action:

  • Ionotropic: milliseconds

  • Metabotropic: hundreds of milliseconds to hours

Cholinergic Neuromuscular Transmission

• Components of the neuromuscular junction:

  • Motor neuron

  • Muscle fiber and synaptic components

  • Presence of ACh receptors and Na+ channels

Passive Properties of EPP

• End-plate potential (EPP) decays with distance from the end-plate

• EPP generates end-plate current (EPC) under voltage clamp

Patch Clamp Techniques for ACh Receptor Currents

• Single-channel patch clamp technique measures ACh receptor current within defined parameters

  • ACh Concentration: Involves 2 µM ACh, observing closed and open channel states over time

Macroscopic Endplate Current (EPC)

• Comprised of numerous ligand-activated ACh receptors

• Levels of channel opening determine postsynaptic potential change (EPP)

• EPC reverses at approximately 0 mV; does not match specific ion reversal but lies between EK and ENa

Changing Ion Reversal Potentials

• Adjusting ENa or EK can cause shifts in EPC reversal potential, suggesting permeability of ACh receptors to both Na⁺ and K⁺

Summary of Ion Movements During EPCs and EPPs

• Net fluxes highlighted includes shifts in net ion flow caused by activation of ACh receptors and the subsequent effects on postsynaptic membrane potential based on concentration and movement patterns.