Synaptic Transmission: Communication Between Neurons

Synaptic transmission: communication between neurons

Machinerie d’exocytose

Visit URL hhttp://sites.google.com/site/insermu950/ // i l / i /i 950/

Two principal kinds of synapses: electrical and chemical

Chemical synapses: the predominant means of communication between neurons

Presynaptic Active Zone

An early experiment to support the neurotransmitter hypothesis

Criteria that define a neurotransmitter: 1. Must be p present at p presynaptic y p terminal 2. Must be released by depolarization, Ca++-dependent 3. Specific receptors must be present

Neurotransmitters may be either small molecules or peptides

Neurotransmitter is released in discrete packages, or quanta

Mechanisms and sites of synthesis are different Smallll molecule S l l transmitters are synthesized at terminals, packaged into small clear-core vesicles (often referred to as ‘synaptic synaptic vesicles’ vesicles

Peptides, or Peptides neuropeptides are synthesized in the endoplasmic d l i reticulum and transported to the synapse, sometimes they are p processed along the way. Neuropeptides are packaged in large dense-core vesicles

Failure analysis reveals that neurons release many quanta of neurotransmitter when stimulated stimulated, that all contribute to the response

Quantal content: The number of quanta released q by stimulation of the neuron

From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm

Quantal Q t l size: i How size of the individual quanta

Quanta correspond to release of individual synaptic vesicles EM images and biochemistry suggest that a MEPP could be caused by a single vesicle EM studies revealed correlation between fusion of vesicles with plasma membrane and size of postsynaptic response

From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm

Stimulation mini i i

4X

Evoked amplitudes. 1X 2X

1X

Mini histogram. histogram

3X 4X

2X 1 mV

Squire Fund. Neurosci.

CNS synapses and d quanta. t • A At synapses with i h only l a single i l release l site, i changing the probability of release (changing calcium concentration) does not effect the amplitude of the response (as only zero or one vesicle is released). released) • At synapses with multiple release sites, changing probability can change the response amplitude as more transmitter is released. • At the NMJ a single nerve can elicit a postsynaptic AP given multiquantal release, while at the CNS multiple p synapses y p must cooperate, p , forces a network.

Calcium influx is sufficient for neurotransmitter release

Calcium influx is necessary for neurotransmitter release l

Voltage-gated calcium channels

Synaptic release II The synaptic vesicle release cycle 1. Tools and Pools 2. Molecular biology and biochemistry of vesicle release: 1. Docking 2 Priming 2. 3. Fusion 3. Recovery and recycling of synaptic vesicles

The synaptic vesicle cycle

Synaptic transmission is an adaptatio of normal vesicle trafficking. trafficking

How do we study vesicle dynamics? Morphological techniques Electron microscopy to obtain static pictures of vesicle distribution; TIRFM (total internal reflection fluorescence microscopy) to visualize movement of vesicles close to the membrane

Physiological y g studies Chromaffin cells Neuroendocrine cells derived from adrenal medulla with large dense-core vesicles. p measurements), ), or direct release of Can measure membrane fusion ((capacitance catecholamine transmitters using carbon fiber electrodes (amperometry) Neurons Measure release of neurotransmitter from a presynaptic cell by quantifying the response of a postsynaptic cell

Ge et cs Genetics Delete or overexpress proteins in mice, worms, or flies, and analyze phenotype using the above techniques

Synaptic y p vesicle release consists of three principal steps: 1. Docking Docked vesicles lie close to plasma membrane (within 30 nm)

1. Priming g Primed vesicles can be induced to fuse with the plasma membrane by sustained depolarization, high K+, elevated Ca++, hypertonic yp sucrose treatment

2. Fusion Vesicles fuse with the plasma membrane to release transmitter. Physiologically this occurs near calcium channels, but can be induced experimentally over larger area (see ‘priming’). The ‘active zone’ is the site of physiological release, and can sometimes be recognized as an electrondense structure.

Neurotransmitter Release

E Neher

T Sudhöf

Vesicle release requires many proteins on vesicle and plasma membrane p

P De Camilli R Jahn

SNAREs: targets of clostridial NTs C Montecucco

H Niemann

SNAREs: targets g of clostridial neurotoxins

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

JE Rothman

D Tareste

Vésicule VAMP 2 VAMP-2

d d

Syn1A/SNAP-25

La vésicule s’est arrimée a hemifusionné ou a fusionné

Membrane cible Les SNAREs n’interagissent n interagissent pas encore (d > 8 nm)

Le SNAREpin commence à se former (d ~ 8 nm)

~ 35 kBT

Le SNAREpin est partiellement assemblé (d < 4 nm)

Role of the linker region D Bruns

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

A Brunger

Priming

The SNARE complex

Vesicles in the reserve pool undergo priming to enter the readilyreleasable pool At a molecular level, priming corresponds to the assembly of the SNARE complex

(Sutton et al., Nature 1998)

The SNARE complex

Inhibitory domain, folds back on itself “open” syntaxin doesn’tt fold doesn properly

Synaptotagmin functions as a calcium sensor, promoting vesicle fusion

Calcium & exocytosis

Regulation by calcium: through synaptotagmin? i ?

Mutants off sytt

SNARE & synaptotagmin

Syt accelerates membrane fusion in vitro

Syt acts through SNAREs and lipids ER Chapman

Annuall Reviews i

T Sudhöf

Regulation by complexin & synaptotagmin y p g

A complexin-tagmin complexin tagmin cycle? C Rosenmund

Synaptic vesicles exist in multiple pools within the nerve terminal

Regulation, regulation

(Release stimulated by flash-photolysis of caged calcium)

• Much more is known: (reserve pool)

Munc 13 Munc-13

Munc 18 Munc-18 N Brose

M Verhage

• Much M h more tto come: ?????

B h Becherer, U U, R Rettig, tti J. J Cell C ll Tissue Ti Res R (2006) 326 326:393 393 Morphologically, vesicles are classified as docked or undocked. Docked vesicles g on whether are further subdivided into primed and unprimed pools depending they are competent to fuse when cells are treated with high K+, elevated Ca++, sustained depolarization, or hypertonic sucrose treatment.

Docking: In CNS neurons, vesicles are divided into UNC-18 (or munc-18) is necessary for vesicle docking R Reserve pooll (80 (80-95%) 95%)

(W i (Weimer ett al. l 2003, 2003 Nature N t N Neuroscience i 6 6:1023) 1023)

Recycling pool (5-20%)

1. unc unc-18 18 mutant C. elegans have neurotransmitter release defect

Readily-releasable y pool ((0.1-2%; 5-10 synapses p y p p per active zone))

2. unc-18 mutant C. elegans have reduction of docked vesicles

Rizzoli, Betz (2005). Nature Reviews Neuroscience 6:57-69)

A small fraction of vesicles (the recycling pool) replenishes the RRP upon mild stimulation. Strong stimulation causes the reserve pool to mobilize and be released

Unc-18 mutants are defective for evoked and spontaneous release

Unc-18 mutants are defective for calcium-independent release

primed vesicles occasionally fuse in the absence of calcium; a calcium-independent fusion defect suggests a lack of primed vesicles

UNC-18 (munc18) is required for docking: unc-18 unc 18 mutants have fewer docked vesicles

Summary: Unc-18 mutants are unable to dock vesicles efficiently. Impaired docking leads to fewer primed vesicles; fewer primed vesicles leads to reduced overall neurotransmitter release. release

Synaptic vesicles recycle post-fusion

B Davletov

Modern methods to track recycling membrane

Endocytosis retrieves synaptic vesicle membrane and protein from the plasma p p membrane following g fusion The ATP-ase NSF disassembles the SNARE complex, then clathrindependent endocytosis compensates for exocytosis P De Camilli