foundations in neuroscience i, oct 11...
TRANSCRIPT
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
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
et a
l. N
atur
e 52
5:62
(201
5)
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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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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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