foundations in neuroscience i, oct 11...

Synaptic transmission, part 1

Foundations in Neuroscience I, Oct 11 2018

Recap: Resting potential

Recap: Action potential basics

- Rapid, serial increases in conductance

- First Na+ → AP peak is near ENa- Then K+ → AP trough is near EK- Mechanism: voltage-gated

ion-selective channels (next time . . .)

Recap: Propagation and myelin

RmCm

Length constant: longer is faster

Time constant: smaller is faster

Myelin increases AP speed ~50 fold by increasing Rm and decreasing Cm.In myelinated axons, the AP is renewed at the nodes of Ranvier: “saltatory conduction”

Recap: patch clamp and single channel recordings

The “macro” current is the sum of currents provided by many small, voltage-sensitive, transiently and probabilistically active membrane elements: voltage-gated channels

Recap: molecular mechanisms

Voltage sensing: displacement of charged S4 domain

Ion selectivity: pore loop mimics hydration shell

Inactivation: “ball and chain” at cytoplasmic N-terminal

Roadmap

Roadmap

1. Evidence for vesicles

3. Fusion mechanismsActive zone organization

4. Vesicle formation and filling

2. The role of Ca2+

5. Neurotransmitters

3.5 Neurotransmitter degradation/uptake

The neuromuscular junction: a model synapse

Acetylcholine (ACh)

“End plate potential (EPP)”

The neuromuscular junction: a model synapse

Acetylcholine (ACh)

“End plate potential (EPP)”TTXTTX abolishes EPP from axon stimulation, but not ACh application

Evidence for quantal release

Del Castillo & Katz (1954) JPhysiology 124:560

End plate potential (EPP) “mini” EPP

Evidence for quantal release

Extracellular Ca2+ depleted: decreases release of ACh

Stimulatemotor axon

Stimulation-evoked EPP shrinks to mEPP size, but does not disappear.

Spontaneous mEPP(not evoked by stimulation)

Do Poisson distribution here?

The chance of evoking a mEPPis well-fit by a Poisson distribution

Extracellular Ca2+ depleted: decreases release of ACh

Spontaneous mEPPs

Evoked by stimulation

The chance of evoking a mEPPis well-fit by a Poisson distribution

Extracellular Ca2+ depleted: decreases release of ACh

Prediction based onPoisson distribution

Actual data

Spontaneous mEPPs

Evoked by stimulation

Vesicles as seen by electron microscopyFrog NMJ

Vesicle fusion

Vesicles as seen by electron microscopy

Rat cerebellar cortex Human cerebral cortex

The role of Ca2+

2. The role of Ca2+

Ca2+ is necessary for vesicle fusion

Postsynaptic

Presynaptic

Voltage clamp recordings in bothpre- and post-synaptic neurons

with K+ and Na+ channels blocked

Voltage-gated Ca2+ influx

Postsynaptic

Presynaptic

Voltage clamp recordings in bothpre- and post-synaptic neurons

with K+ and Na+ channels blocked

Voltage-gated Ca2+ influx

Postsynaptic

Presynaptic

Voltage clamp recordings in bothpre- and post-synaptic neurons

with K+ and Na+ channels blocked

Ca2+ influx is localized to the synapse

Imaging the SJA synapse using calcium-sensitive dyes(1993)

Roadmap

1. Evidence for vesicles

3. Fusion mechanismsActive zone organization

4. Vesicle formation and filling2. The role of Ca2+

5. Neurotransmitters

3.5 Neurotransmitter degradation/uptake

Synaptic transmission overview

Vesicle fusion mechanisms and other active zone proteins

3. Fusion mechanismsActive zone organization

Mechanisms of vesicle fusion

The molecular mechanisms behind the fusion process are:

- Fast- Reliable- Localized- Effective at getting neurotransmitters to the postsynaptic cell

- Used in other parts of the cell (e.g. internal vesicle trafficking)

Mechanisms of vesicle fusion

Mechanisms of vesicle fusion

SNAREs, SM proteins, synaptotagminSenses Ca2+

Provides mechanical force that initiates fusion

RIM, RIM-BP, Rab3Brings voltage-gated Ca2+ channels close

cadherin, neurexinTies active zone proteins

to the pre- and post-synaptic neurons

SNAREs: providing the fusion force

t-snare

t-snarev-snare

Sec1/Munc18 (SM) proteins: helping(?) the SNAREs

Sec1/Munc18 proteins are essential for proper vesicular fusion

What they do is not precisely known

Four families have been identified

The crystal structure of some SM proteins has been found

Structure of vps33Baker et al. (2013) Plos One, 8:e67409

Synaptotagmin: the calcium sensor

Danko Dimchev Georgiev, M.D.

High-affinity Ca2+ binding sites.Binding is cooperative: binding at one site promotes binding at the others

Effects of deleting synaptotagmin

Synaptotagmin: the calcium sensor

Voltage clamp in synaptically connected hippocampal cells in vitro

Effects of mutations that reduce Ca2+ binding

(single amino acid substitution)

Synaptotagmin binds to the SNARE complex

Zhou

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Synaptotagmin binds to the SNARE complex

Zhou et al. Nature 525:62 (2015)

Two additional notes

Once triggered, release is really, really fast, 0.15ms. (For reference an action potential is around 1.0ms.) This is due to the high binding affinity of Syn for Ca2+

After release, Ca2+ is very quickly removed from the presynaptic terminal by a combination of pumps and binding proteins.

→ High temporal resolution.

Mechanisms of vesicle fusion

RIM, RIM-BP, Rab3Brings voltage-gated Ca2+ channels close

RIM, RIM-BP, Bruchpilot, and Rab3:organizing the active zone

Voltage gated Ca2+channels bind to RIM and RIM-BP

A model of active zone protein organization, derived from super-resolution fluorescence microscopy

“T-Bar” structure with cluster of synaptic vesicles

Giagtzoglou et al. Neuron 64: 595 (2009)

Cadherin and neurexin: synaptic adhesion molecules

homophilic binding

heterophilic binding

Review: cellular cytoskeleton proteins

Universitat Leipzig, Steve Pawlizak, Daniel Koch

actin microtubules

Mechanisms of vesicle fusion

SNAREs, SM proteins, synaptotagminSenses Ca2+

Provides mechanical force that initiates fusion

RIM, RIM-BP, Rab3Brings voltage-gated Ca2+ channels close

cadherin, neurexinTies active zone proteins

to the pre- and post-synaptic neurons

Interlude: What happens to neurotransmitter after it is released

1) Enzymatic degradation within the synapse (e.g. Acetylcholine)

2) Glial uptake (co-transport with Na+)

3) Re-uptake by pre-synaptic neuron, powered by Na+ co-transport (e.g. serotonin, dopamine) → refilling vesicles

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3

Vesicle formation and filling

4. Vesicle formation and filling

Vesicle formation: two mechanisms

1) Endocytosis, mediated by clathrin protein “cage”(same mechanism as all other mammalian cells)

2) “Kiss and run”: vesicle fuses, empties contents, and then quickly re-forms

Vesicle filling: ATP-powered proton gradient

1) Vesicular proton pumps fill the new vesicle with H+ (ATP needed)

2) Vesicular co-transporter allows H+ to exit (flowing down its gradient) while loading neurotransmitter inside

3) The vesicle is now part of the reserve pool, meaning not immediately ready for release

4) After the vesicle moves to active zone and the SNARE/SM complex is assembled, the vesicle is now in the readily releasable pool (not shown)

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Vesicle movie

About neurotransmitters

5. Neurotransmitters

Neurotransmitters

Neurotransmitters: Acetylcholine

Neuromuscular junction (peripheral)

Autonomic nervous system (peripheral)

Cholinergic neurons in brainstem and forebrain project throughout the brain and affect memory, attention, arousal, and more

Cholinergic interneuron neurons in the striatum

(Ask Laszlo, Jim, and Juan!)

Neurotransmitters: Glutamate, GABA, glycine

The major neurotransmitters of the CNS

Most neurons in the brain are glutamatergic, including most long-range projection neurons (excitatory)

GABAergic neurons are typically not long-range projection neurons. Few in number but can have outsize influence in neural circuits. (inhibitory)

Glycine is inhibitory, found in brainstem, retina, spinal cord. Is an amino acid.

← Glutamate and GABA Synthesized from the amino acid glutamine

Neurotransmitters: monoamines

The major neuromodulators of the CNS. Not strictly excitatory or inhibitory: they tend to modulate the responses of neurons to other inputs.

Collectively called “monoamines”

Produced by only a small number of neurons, but have effects throughout the brain: learning, memory, mood, reward, arousal, sleep/wake, drug abuse, etc.

← Dopamine and Norepinephrine are synthesized from tyrosine.

Serotonin is synthesized from tryptophan. →

Histamine is synthesized from histadine.

Neurotransmitters: peptides

Strings of amino acids

Usually co-released with other NTs

Very scarce compared to other NTs, but have broad influence, in particular for regulating hunger, thirst, and other basic drives

Examples: Enkephalin, an endogenous opiate, important for nociception

Leptin, regulates fat metabolism

Oxytocin, involved in pair bonding, parental behavior, labor

Enkephalin by Elaine Meng Leptin by HR Voss

That’s it!

1. Evidence for vesicles

3. Fusion mechanismsActive zone organization

4. Vesicle formation and filling

2. The role of Ca2+

5. Neurotransmitters

3.5 Neurotransmitter degradation/uptake