Neurotransmitter Release and Synaptic Vesicle Recycling

Neurotransmitter Release

Overview

• A chemical synapse converts an electrical signal to a chemical signal and then back to electrical.

Presynaptic Terminal Organization

• Organization includes:
  • Mitochondria
  • Peri-active zone
  • Active zone
• Exocytosis occurs at the active zone.
• Endocytosis occurs at both the active zone and the peri-active zone.

Visualizing Vesicle Recycling

• Use fluorescent dyes like FM1-43 to load synaptic vesicles.
• Experiment example: Frog NMJ
  • Control: Stimulate with FM1-43 and wash.
  • Observe vesicle dynamics.

Vesicle Usage

• After brief stimuli (30 Hz, 10 sec), only a subset of vesicles are used in frog neuromuscular junctions.
• After intense stimuli (10 Hz, 15 min), only a subset of vesicles are used in frog neuromuscular junctions.
• Not all vesicles are releasable (Heuser and Reese, 1973).

Vesicle Pools

• Readily Releasable Pool:
  • Docked and primed for release.
• Recycling Pool:
  • Maintains release on moderate stimulation.
• Reserve Pool:
  • Intense stimulation.
  • Possibly not during physiological conditions.

Steps in Synaptic Vesicle Recycling and Release

• Clustering/restraint
• Targeting/docking
• Priming
• Exocytosis (fusion)
• Endocytosis
• Vesicle maturation
• The synaptic terminal is a highly specialized compartment.

Vesicle Clustering

• At rest, the movement of synaptic vesicles within nerve terminals is restricted (Takamori et al 2006, Cell).
• Vesicles occur in tight clusters (Henkel et al 1996).
• FRAP (Fluorescence Recovery After Photobleaching) studies show no recovery of fluorescence and no mingling of vesicles.

Mechanisms of Vesicle Clustering

• Synapsin I:
  • Low Ca^{2+}: Synapsin I is unphosphorylated and bound to the cluster.
  • High Ca^{2+}: Synapsin I is phosphorylated and released from the cluster.
  • Phosphorylation by Ca/calmodulin-dependent kinase (CAM Kinase) (Zhang and Augustine, 2021, Cells).
  • Dephosphorylated synapsin can form a distinct “liquid phase” that holds clusters together (Milovanovich and DeCamilli 2017, 2018).
• Phosphorylation dissolves these droplets, releasing vesicles (Boczek and Alberti 2018).

Targeting to the Active Zone

• Scaffolding proteins at the active zone recruit synaptic vesicles and control release.
• Proteins involved: Piccolo, Bassoon, RIM (rab 3 interacting proteins).
• Rab3:
  • A G-protein (GTP bound).
  • Binds to the vesicle membrane and brings it to the presynaptic membrane.
• RIM helps anchor Ca^{++} channels.

SNARE Hypothesis

• vSNAREs (vesicle membrane proteins) bind to tSNAREs (target membrane proteins), leading to fusion of membranes and synaptic vesicle release.
• vSNAREs: VAMP/synaptobrevin
• tSNAREs: Syntaxin, SNAP25
• Ca^{2+} entry and binding to Synaptotagmin brings synaptotagmin into the SNARE complex and triggers fusion.
• Synaptotagmin is the Ca^{++} sensor.
• 4 Ca^{++} needed per vesicle released.

Endocytosis and SNARE Complex

• SNAP and NSF bind to the SNARE complex and unravel it, using ATP.
• Need to ‘untangle’ vSNAREs and tSNAREs.

Coupling of Exocytosis and Endocytosis

• Coupled via a transient fusion pore.
• The rate of endocytosis is tightly coupled to the rate of exocytosis.
• Kiss-and-run (fast)
• Full fusion (slow)

Clathrin-Mediated Endocytosis

• Adaptor protein AP-2 binds to proteins in the “vesicle” membrane (synaptotagmin).
• Clathrin molecules bind to AP2.
• A clathrin lattice forms the coated pit.
• Dynamin (GTPase) pinches off the coated vesicle.

Vesicle Maturation

• Two possible pathways:
  1. After endocytosis, vesicles go straight into the synaptic vesicle pool.
  2. Vesicles go through an intermediate endosomal compartment.
• Steps in vesicle maturation:
  • Acidification: proton (H^+) pump creates an electrochemical gradient.
  • Neurotransmitter uptake by transporters (vGlut).
  • Insertion of vesicle membrane proteins.
  • Vesicle clustering.

Methodologies

• Genetic approaches (shibire mutant).
• Biochemistry (protein-protein interactions).
• Immunocytochemistry.
• Toxins: Botulinum and Tetanus toxins bind to SNAREs.
• Optical methods.

Shibire Mutant

• Mutation in dynamin - no endocytosis.

Optical Methods for Analyzing Vesicle Recycling

• pHFluorins: GFP variant that changes its fluorescence properties with pH. Vesicle proteins tagged with pHFluorin only fluoresce when at the synaptic membrane because the inside of a vesicle is acidic.
• Synaptotagmin-pHluorin (SypHy).

Vesicle Endocytosis Rate

• Vesicle endocytosis proceeds at 1 vesicle/sec per bouton.

Drugs Affecting Vesicle Endocytosis

• Drug A and Drug B can affect SypHy fluorescence, indicating changes in vesicle endocytosis.