Lecture 17- Presynaptic Events

Question 1

What events happen at the presynaptic nerve terminal?

Answer 1

-Action potential travels via axon to reach the presynaptic nerve terminal: Na+ channels (not shown)
-Opens voltage-gated Ca2+ channels
-Ca2+ influx triggers “exocytotic machine”
-Causes release of neurotransmitter (and neuromodulators) from synaptic vesicles

Question 2

What are defining features of the presynaptic nerve terminal as seen in TEM?

Answer 2

-Synaptic vesicles ready for release
-Active zones (where the vesicles fuse with membrane for release, electron dense region)
-Mitochondria (to meet energy requirements)

Question 3

What are the two types of synaptic vesicles?

Answer 3

-Small synaptic vesicles (SSV)
-Large dense core vesicles (LDCV)

Question 4

What are SSV like?

Answer 4

• 50 nm diameter
• Electron-lucent (clear)
• Membrane bound
• Contain classical neurotransmitters
• Glutamate, GABA, glycine

Question 5

What are LDCV like?

Answer 5

• 100 nm diameter
• Electron-dense (dark)
• Membrane bound
• Contain catecholamines neuropeptides, neurotrophins, etc
• Adrenaline, noradrenaline,
• Neuropeptide Y, Brain-derived neurotrophic factor

Question 6

What type of vesicles are primarily in the CNS and what type are primarily in the PNS?

Answer 6

-Some overlap but….
-In CNS typically more: SSV
-In PNS typically more: LDCV

Question 7

What is neurotransmitter release triggered by?

Answer 7

Arrival of an action potential to the nerve terminal

Question 8

What happens at the presynaptic nerve terminal following Depolarisation?

Answer 8

-Voltage-gated calcium channels open= Large inward driving force
-Increased intracellular [Ca2+] in the microdomain around the active zone and
-Signals exocytosis (neurotransmitter
release via fusion of membranes)
-Recycling via endocytosis also occurs
-Note: activation of “reserve pool” of synaptic vesicles occurs also.

Question 9

Where are SSV located?

Answer 9

SVs are attached to the presynaptic plasma membrane and clustered nearby.

Question 10

What are the three ‘types’ of SV?

Answer 10

-Readily releasable pool: SV docked at the active zone
-Reserve pool: distal to active zone, associated with cytoskeleton
-Recycling pool: diffusing

These three types collectively make what is know as the synaptic vesicle cycle

Question 11

Where are small synaptic vesicles made and how do they travel to their location?

Answer 11

Formed in the Golgi Apparatus, and
transported along microtubules to axons

Question 12

What is the vesicle life cycle?

Answer 12

• Formed in the Golgi Apparatus, and transported
  along microtubules to axons
• Enter the “vesicle cycle” (between three types)
• Filled with neurotransmitter in nerve terminal
  -Recycled via endocytosis to endosomes or reserve pool
  -Refilled via transporters

Question 13

What is an example of how SSV are filled with neurotransmitter at the nerve terminal? Is this process fast or slow?

Answer 13

-glutamate via vesicular glutamate transporter (VGLUT)
-Pumps generate H+ gradients across vesicle membrane to power transporters which load neurotransmitters into vesicles

Fast process

Question 14

How is VGLUT used in immunohistochemistry for identification?

Answer 14

VGLUTs are found only in
glutamatergic neurons, and is used to uniquely identify these neurons using
immunochemistry

Question 15

What are LDCV’s role in the CNS?

Answer 15

-Minor players in the CNS: SLOW action
-Comprising 1~2% of vesicles
-May contribute to presynaptic modulation in
addition to postsynaptic effects
-Local diffusion to active synaptic partners: Synchronising or enhancing
-Release can occur at active zone
-Act on G protein-coupled receptors

Question 16

What is one place in the CNS where LDCV are found?

Answer 16

Hippocampus

Question 17

How is the synthesis pathway for LDCV in the CNS different than SSVs

Answer 17

1. Synthesis and modification of
   neuropeptides: RER and Golgi apparatus
2. Packaging: pro-peptide & modifying enzymes
3. Axonal transport (mircotubules)
4. Cleavage of pro-peptide into inactive form
5. Release

SLOW recycling

Question 18

For release of LDCV in the CNS what is needed?

Answer 18

-Release requires prolonged stimulation as it is a slower process

It’s slower because LDCV:
-Not restricted to the synaptic specialization
-Not pre-docked (as per SSVs)
-Release occurs via membrane fusion

What is required for release is:
-Requires more widespread increase in [Ca2+] and
therefore stronger/ prolonged stimulation
-Including activation of CaMKII via calcium-induced
calcium release from ER
-Dissociation from the microtubule tracks
-cAMP-dependent protein kinase (PKA; fusion)

Question 19

What are the primary locations of LDCVs?

Answer 19

-neurosecretory cells
-neuroendocrine cells e.g. chromaffin cells
-Sympathetic neurons of the peripheral nervous system
-Neurohypophysis
-Hypothalamus as well as other brain regions

Question 20

What are chromaffin cells and the role of LDCV’s within them?

Answer 20

-Neuroendocrine cells found in the medulla of adrenal glands

-Enriched with LDCVs which create “chromaffin granules”

-Contain and release many substances into
the circulatory system:
* catecholamines (adrenaline, noradrenaline, and dopamine)
* peptides (encephalin and neuropeptide Y)
* proteins (chromogranins, secretogranins)
* microRNA

-Neuromodulators, stress transducers

Question 21

What three components in the membrane of SSV are important for exocytosis to occur?

Answer 21

-Synaptobrevin
-Syntaxin
-Synapsin

Question 22

How does exocytosis occur? How do you
a vesicle

Answer 22

-Exocytosis requires interaction of SNARE proteins
-SNARES are involved in vesicle trafficking throughout the cell
-Specific v-SNARE and t-SNARE pairs mediate synaptic vesicle docking & priming for release
-Translocation and docking also requires “Rab” proteins
-Synaptotagmin allows fusion pore to open
-Dissociation of the SNARE complex is driven by ATPase N-ethylmaleimide-sensitive fusion (NSF)
protein

Question 23

How do the v-SNARE and t-SNARE pairs interact in exocytosis?

Answer 23

-Broadly speaking the proteins wrap around each other with some associated with the presynaptic membrane and some associated with the vesicle membrane. This pulls the vesicle to the membrane allow exocytosis to occur

-The protein associate with the vesicle membrane is Synaptobrevin (v-SNARE)

-SNAP25 and Syntaxin are the two associated with presynaptic membrane (t-SNAREs)

Question 24

How are RAB proteins important in exocytosis?

Answer 24

Translocation and docking requires “Rab”
proteins (small GTP binding proteins)

Question 25

How is Synaptotagmin important in exocytosis?

Answer 25

-Synaptic vesicle protein
-Calcium sensor
-Docking
-Triggers vesicle fusion/ release
-Couples Ca2+ influx through voltage-gated
Ca2+-channels to vesicle fusion and thus
neurotransmitter release
-Ca2+ channels may be part of the docking complex

Basically: Dig into membranes on either side. Twist + bend membrane together in response to calcium influx

Question 26

Why is endocytosis important and what is it mediated by?

Answer 26

-Clathrin
-The process by which vesicular membrane is retrieved back into the cytoplasm
-Ensures that axon terminals continue to function properly even after sustained
neurotransmitter release
-Clathrin assists in the formation of a coated pit on the inner surface of
the plasma membrane of the cell.
-Which buds into the cell to form a coated
vesicle.

Question 27

Structural what is Clathrin like and why is this important for the process of endocytosis?

Answer 27

-Clathrin subunits comprise 3 large and 3 small polypeptide chains that form a triskelion and assemble into basket-like frameworks
-Clathrin provides a scaffold and limiting structure for the vesicle.
-AP2 adaptors link the clathrin lattice to the membrane

Question 28

Why is Dynamin important in endocytosis?

Answer 28

-Dynamin is a GTPase
-Dynamin binds to a forming bud on the membrane
-Forms a helical collar around the neck of the
bud
-GTP hydrolysis is coupled to vesicle scission
-Dynamin spirals undergo a length-wise extension which pinches or pops the vesicle
from the parent membrane.