Synaptic transmission: communication between neurons
Two principal kinds of synapses: electrical and chemical
Gap junctions are formed where hexameric pores called
connexons connect with one between cells
Electrical synapses are built for speed
Contrast with chemical synapse
Delay of about 1 ms
Electrical coupling is a way to synchronize neurons with one another
Electrical synapses are not presently considered to be the
primary means of communication between neurons in the
mammalian nervous system, but they may prove to be more
important than presently recognized
Rectification and uni-directionality of electrical synapses
…not just simple bidirectional bridges between cells
Conductance through gap junctions may be sensitive to the
junctional potential (i.e the voltage drop between the two
coupled cells), or sensitive to the membrane potential of either
of the coupled cells
Glial cells can also be connected by gap junctions, which allows
synchronous oscillations of intracellular calcium
http://users.umassmed.edu/michael.sanderson/mjslab/MOVIE.HTM
Chemical synapses: the predominant means of
communication between neurons
An early experiment to support the neurotransmitter hypothesis
Criteria that define a neurotransmitter:
1. Must be present at presynaptic terminal
2. Must be released by depolarization, Ca++-dependent
3. Specific receptors must be present
Neurotransmitters may be either small molecules or peptides
Mechanisms and sites of synthesis are different
Small molecule
transmitters are
synthesized at
terminals, packaged
into small clear-core
vesicles (often
referred to as
‘synaptic vesicles’
Peptides, or
neuropeptides are
synthesized in the
endoplasmic
reticulum and
transported to the
synapse, sometimes
they are processed
along the way.
Neuropeptides are
packaged in large
dense-core vesicles
Neurotransmitter is released in discrete packages, or quanta
Failure analysis reveals that neurons release many quanta
of neurotransmitter when stimulated, that all contribute
to the response
Quantal content:
The number of
quanta released by
stimulation of the
neuron
Quantal size:
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
4-AP was used to vary the efficiency of release
Calcium influx is necessary for neurotransmitter
release
Voltage-gated
calcium
channels
Calcium influx is sufficient for neurotransmitter release
Synaptic release II
The synaptic vesicle release cycle
1. Tools and Pools
2. Molecular biology and biochemistry of vesicle release:
1. Docking
2. Priming
3. Fusion
3. Recovery and recycling of synaptic vesicles
The synaptic vesicle cycle
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 studies
Chromaffin cells
Neuroendocrine cells derived from adrenal medulla with large dense-core vesicles. Can
measure membrane fusion (capacitance measurements), or direct release of catecholamine
transmitters using carbon fiber electrodes (amperometry)
Neurons
Measure release of neurotransmitter from a presynaptic cell by quantifying the response of a
postsynaptic cell
Genetics
Delete or overexpress proteins in mice, worms, or flies, and analyze phenotype using the
above techniques
Synaptic vesicle release consists of three principal
steps:
1. Docking
Docked vesicles lie close to plasma membrane (within 30 nm)
1. Priming
Primed vesicles can be induced to fuse with the plasma membrane
by sustained depolarization, high K+, elevated Ca++, hypertonic
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 electron-dense structure.
.
Synaptic vesicles exist in multiple pools within the nerve terminal
(Release stimulated by flash-photolysis of caged calcium)
QuickTime™ and a
(reserve pool) TIFF (Uncompressed)
decompressor
are needed to see this picture.
Becherer, U, Rettig, J. Cell Tissue Res (2006) 326:393
Morphologically, vesicles are classified as docked or undocked. Docked vesicles are
further subdivided into primed and unprimed pools depending on whether they are
competent to fuse when cells are treated with high K+, elevated Ca++, sustained
depolarization, or hypertonic sucrose treatment.
In CNS neurons, vesicles are divided into
Reserve pool (80-95%)
Recycling pool (5-20%)
Readily-releasable pool (0.1-2%; 5-10 synapses per active zone)
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
Vesicle release requires many proteins on vesicle and plasma
membrane
Docking:
UNC-18 (or munc-18) is necessary for vesicle docking
(Weimer et al. 2003, Nature Neuroscience 6:1023)
1. unc-18 mutant C. elegans have neurotransmitter release defect
2. unc-18 mutant C. elegans have reduction of docked vesicles
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 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.
Priming
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
The SNARE complex
UNC-13 is a critical priming factor
Richmond and Jorgensen (1999) Nature Neuroscience 2:959
unc-13 mutants have higher levels of synaptic vesicles than normal
normal
unc-13 mutants
No docking defect was observed
unc-13 mutants have evoked release defect
Calcium-indepenent release is also defective, indicating
that the defect is in priming
Munc-13 function in priming
Inhibitory
domain, folds
back on itself
“open” syntaxin
doesn’t fold
properly
unc-13 defect can be bypassed by providing an “open” form of syntaxin
Model for unc-13, unc-18, syntaxin interaction in priming
Synaptotagmin functions as a calcium sensor, promoting
vesicle fusion
Synaptic vesicles recycle post-fusion
Modern methods to track recycling membrane
Endocytosis retrieves synaptic vesicle membrane and protein
from the plasma membrane following fusion
The ATP-ase NSF disassembles the SNARE
complex