SYNAPTIC TRANSMISSION I
Tim Murphy NRSC 500, 2011
The definition of synaptic transmission is simply the communication between
two nerve cells. Communication believed to involve specialized structures
termed "synapses".
We will focus on:
1)
The discovery of synaptic transmission
2)
The form of transmission, i.e. chemical or electrical
3)
Criteria for a chemical transmitter
4)
Ionic requirement for release
5)
Quantal aspects of release: vesicle theory
Discovery of synaptic
transmission
• Cajal's golgi staining methods suggested the
presence of contacts between cells that were
used for communication ~1900’s.
• Sherrington proposed the term "synapse"
meaning to clasp to describe the structure,
1890’s.
Cajal’s drawings
of golgi stain.
Shepherd 1997 TINS
Sherrington’s insights 1890s.
Modern golgi staining, YFP mouse
cortical fluorescence, can be bred
to other KO’s, transgenics Feng et al. 2000.
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
Otto Loewi, chemical transmitter.
• 1936 Nobel prize for Medicine
• Showed that vagus nerve
stimulation liberates a diffusible
transmitter.
• Perfusate from one stimulated frog
heart could be transferred to
another and have an effect on beat
frequency.
Debate on synaptic transmission chemical or electrical.
• Otto Loewi showed that acetylcholine could
mimic the effect of vagal nerve stimulation.
• What additional experiment would have been nice
to prove that the vagal nerve released
acetylcholine.
• The results of Loewi's experiments sparked debate
about whether chemical and electrical
transmission was occurring.
• Subsequently shown that both chemical and
electrical transmission exist.
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
Chemical
Electrical
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
Electrical Synapses
• Gap junction-type communication important for rapidly
synchronizing syncytia of cells as is observed in
astrocytes, heart, and developing brains. Present in some
invertebrates to promote rapid defensive secretions.
• Problems with electrical: difficult to modulate gating of
channels (exceptions exist cAMP, pH).
• Can't change sign, i.e. charge always flows "down hill."
• Electrical synaptic transmission requires that the
presynaptic cell or terminal be larger than the
postsynaptic cell for it to inject considerable charge, no
real amplification mechanism.
Electrical synaptic transmission.
Fundamental Neuro.
Chemical transmission inhibitors do not block
transmission in developing Zebrafish.
Saint-Amant and Drapeau
Neuron 2001
Gap junction inhibitors block transmission
in developing Zebrafish.
Saint-Amant and Drapeau
Neuron 2001
Chemical transmission.
• Contrary to electrical transmission multiple steps
are required to release transmitter chemicals and
for them to act on postsynaptic receptors, resulting
in a time delay (can be as short as 0.2 msec, from
Ca2+ entry to secretion).
• Directional, select localization of release
machinery to presynaptic terminals and receptors
to postsynaptic specializations.
• Can change sign by release of inhibitory
transmitter.
• Highly modulatable as it has many steps
presynaptic terminal and at the postsynaptic sites.
Chemical Synaptic Transmission.
• Definition: Communication between cells
which involves the rapid release and
diffusion of a substance to another cell
where it binds to a receptor (at a localized
site) resulting in a change in the
postsynaptic cells properties.
A hall mark of chemical
transmission is a delay
between presynaptic Ca2+
elevation and secretion. The
delay can be as short as 0.2 ms,
but is usually longer due to a
variety of factors.
Fundamental Neuro. 2002
Steps to chemical synaptic
transmission.
• First need to bring the presynaptic neuron to
threshold at axon hillock.
• Conduction down axon, length, R*C
dependent.
• Opening of voltage gated Ca channels.
• Diffusion and action of Ca at release
machinery.
• Exocytosis and diffusion of transmitter in cleft.
• Activation of postsynaptic receptors.
Synaptic delays can be less than 0.2 ms from calcium
entry (Fund. Neurosci. Chap 8) to the beginning of secretion,
but are typically longer when all steps (below) are considered.
From Sudhof 2004
Chemical synapse types.
• Axosomatic, axoaxonic, axodendritic, and
dendrodendritic.
• Excitatory (type I) and inhibitory (type II)
synapses have different structure in CNS neurons.
• CNS synapses usually have one or small number
of release sites while nerve muscle synapses have
up to 300 active zones.
See http://synapses.mcg.edu/atlas
Type I Excitatory
spherical
Type II Inhibitory flattened
See http://synapses.mcg.edu/atlas
Josef Spacek
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
Synapse structure like real estate location, location, location!!
Multiple release sites NMJ
From Squire et al. Fund. Neurosci 2nd ed.
Criteria for a chemical transmitter,
make a case for glutamate.
• The transmitter substance must be synthesized in
the presynaptic neuron. Experiment?
• It must be present in the presynaptic terminal and
released in amounts sufficient to result in the level
of response produced by the endogenous
transmitter. experiment?
• When applied exogenously the substance should
mimic the effect of the endogenous transmitter.
experiment?
• A specific mechanism must exist for removing the
transmitter from the synaptic cleft. experiment?
Ionic requirement for release.
• Calcium influx is the trigger for fast evoked
transmitter release
• extracellular calcium (Ca2+) is typically
about 1-2 mM, intracellular calcium at rest is
<0.1 mM.
• An elevation in intracellular calcium
concentration is an absolute requirement for
transmitter release. Na+ and K+ ions not
necessary for release.
• How do you test this hypothesis?
Ion substitution and
pharmacology experiments.
• The influx of calcium is triggered by voltage gated
ion channels.
• Depolarization itself is not needed to stimulate
release, as calcium can be elevated by other means
(caged calcium chelators).
• The relationship between calcium influx and
release is related to the "power (exponent)" of
calcium entry and is highly nonlinear. For
example for a 4th power relationship a doubling of
calcium entry can produce a 16 fold increase in
release.
Slope on a log-log plot indicates power relation, a small
change in calcium produces a large change in release.
Release~[Ca]^3-5
Slope=5.0
Slope=1.6
from Delaney Enc. of Sci.
Neurotransmitter release is triggered by a locally-activated low
affinity sensor since bulk cytosolic [Ca2+]i rarely exceeds 10-6 M,
yet transmitter release requires much higher [Ca2+].
G. Augustine
Curr. Op. in Neurobiol.
Squid giant axon and release
• Due to its large size the squid giant axon has been
used to examine the calcium dependence of
transmitter release.
• Squid studies using different Ca2+ buffers indicate
high concentrations of Ca2+ that are reached for
less than 1 ms trigger release (Adler et al. 1991 J.
Neurosci.).
• Most calcium entry which triggers release occurs
during the falling phase of action potential. Why
is this advantageous?
Rate of calcium binding by buffer
(chelator) provides insight into
release machinery.
• Fast BAPTA (kon 8x108 M -1 *sec -1) buffers block
release whereas,
• slow EGTA (kon 1x107 M-1*sec -1) buffers do not
(Adler et al. 1991 J. Neurosci.).
• Time for equilibration of EGTA with calcium~1000
ms versus 12.5 ms for BAPTA.
• To estimate the buffer equilibration time take
1/(koff +kon*[Ca2+]), use koff of 8x101 sec –1 for
both buffers and 1x10-4 for [Ca2+] at peak.
Temporal requirements.
• Calcium trigger must be able to act within 0.1 ms
of presynaptic stimulation. This requirement
restricts the class of chemical events that may be
involved in the evoked release process.
• Phosphorylation, protein synthesis, gene
expression all out.
• Everything must be ready and localized- diffusion
could not move transmitter vesicles or calcium
very far.
Calcium channels are clustered on the release face
(side with release) of the chick caylx synapse.
Patch config.
Outside
of synapse
Release
face of synapse
Reviewed in EF Stanley TINS 1997
Diffusion time of Ca2+ limits
release latency.
• Buffered calcium diffusion coeff. are on the
order of 200 mm2/sec (D) so calcium could
only diffuse a small distance at the most
(~0.2 mm) during the synaptic delay (0.0001
s, 0.1 ms), so Ca2+ channels need to be very
close to the release machinery.
• distance=Sqrt(2Dt)
D=diffusion coeff., t=time
The diffusion time is dependent on
the square of the distance (d).
• t=d2/(2*D), where t=time, d=distance, and
D=diffusion coefficient.
• For 0.1 mm the time is 0.025 ms.
• However for 1.0 mm the time jumps by the
square to 2.5 ms (100 times longer!), way
longer than the release observed latency.
These equations are for reference, the concept is of interest.
Kd or Km, concentration at ½ max binding or activity.
Kd=Km=kdissoc/kassoc
affinity=1/km, low affinity means a big km which usually
means a large kdissoc
Note koff=kdissoc and kon=kassoc
Low affinity binding
gives rapid off rate.
• Concentration for ½ max activity is ~1/affinity and is
termed the Kd or Km, if a binding site has a low
affinity more ligand is needed to get ½ saturation.
• Therefore Kd=koff/kon, assuming a diffusion limited
kon of 5x108 M –1 s –1 then koff must be 5x104 s-1 if the
Kd is 100 mM.
• To estimate the dissociation time constant take
1/(koff +kon*[Ca]) or 20 ms.
Note after the channels close [Ca2+] is ~ 1x10-7 and the kon*[Ca] is small
compared to koff.
These equations are for reference.
For reference
For a simple reaction.
[E]+[S]
kon*[S]
[ES]
koff
The time constant for Ca2+ dissoc will be:
t=1/(koff+kon*[S])
20 ms=1/(5x104 s-1 + 5x108 M –1 s –1 *1x10-7 M)
Local domains of Ca2+
near channel mouths
control transmitter release.
It is unclear
whether release is always
triggered by a single
channel or whether
multiple ones cooperate.
EF Stanley TINS 1997
Transmitter release microdomains.
Activation
of Ca channels
1.8 mM outside
Resting state
just after channel
closure, bulk Ca
is up only 10%.
To make full use of microdomains the vesicle must
be bound to the calcium channels.
Atwood & Shanker Karunanithi
DIVERSIFICATION OF SYNAPTIC STRENGTH: PRESYNAPTIC ELEMENTS
Nature Reviews Neuroscience 3, 497 -516 (2002).
BAPTA, a faster buffer than EGTA
more readily blocks synaptic transmission.
Given 100 mM Ca2+, BAPTA equilibrates in <20 ms while
EGTA takes ~1000 ms.
B
Atwood & Shanker Karunanithi
DIVERSIFICATION OF SYNAPTIC STRENGTH: PRESYNAPTIC ELEMENTS
Nature Reviews Neuroscience 3, 497 -516 (2002).
Calcium as a local messenger.
• Fast channel activation, Ca2+ diffuses to
couple excitation to synaptic chemistry.
• Low affinity, rapid off rate, and restricted
microdomains are characteristics of the
calcium flux that evokes release.
• Rapid off rate allows for re-loading of
release mechanism when Ca2+ levels fall.
Affinity=kon/koff
• high off rate = low affinity.
Quantal aspects of release at the
neuromuscular junction.
• At the neuromuscular junction small spontaneous
potentials (depolarizations) termed miniature end plate
potentials are observed. Analogous to CNS miniature
excitatory postsynaptic currents (mEPSCs).
• The amplitude of evoked responses (due to calcium
influx) is always an integer multiple of the unitary
response.
• Shown that calcium increases the probability of
observing a unitary response and not its amplitude.
• These data suggested the existence of transmitter quanta
or packets.
• Evoked transmission mediated by the release of ~ 150
quanta over a 1-3 ms period. Each quantum leads to
about 0.5 mV depolarization.
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
Stimulation
mini
Evoked amplitudes.
1X
Miniature event
histogram.
2X
4X
1X
3X
4X
2X
1 mV
Squire Fund. Neurosci. 2002
CNS synapses and quanta.
• At CNS synapses with only a single release site,
changing the probability of release (i.e. changing
calcium concentration) does not effect the
amplitude of the response (as only zero or one
vesicle is released in theory).
• At CNS synapses with multiple release sites,
changing release probability can change the
postsynaptic response amplitude as more
transmitter is released (graded quantal levels).
• At the NMJ a single nerve can elicit a postsynaptic
AP given multiquantal release, while at the CNS
synapse (with low numbers of release sites)
multiple synapses must cooperate, forces a
network.
CNS synapses and
miniature release.
• Miniature release is produced in the absence
of action potential stimulation.
• Thought to reflect the release of single
vesicles or transmitter quanta.
• Can be stimulated by calcium entry, but may
not necessarily require calcium for release.
• Commonly studied to gain insight into
changes in receptors or release probability
during synaptic plasticity experiments,
although can be difficult to interpret.
Define the number of readily releasable vesicles
a synapse has available. A consequence of having of limited
number is depletion at high stimulus frequency, CNS synapses
may have only a small number of docked vesicles on the
order of 5-10 vesicles for a hippocampal CA1 synapse
(Harris and Sultan, 1995; Schikorski and Stevens, 1997).
From Kristin Harris Lectures.
http://synapses.mcg.edu/lab/harris/lectures.htm
Many vesicles
in the RRP.
Few vesicles
in the RRP, but Pr high
undergoes depression.
Remember depression
over short timescales
can also be caused by other
mechanisms including
desensitization and
autoreceptors.
Fundamental Neuro. 2002
Short term plasticity, history dependent changes in responsiveness,
typically studied over 10-200 ms intervals.
Stim.
Residual Ca in the
terminal
could facilitate
transmission if not all
quanta are released on
the first stimulus (A).
If transmission is
robust on the first
stimulus most readily
releasable vesicles
will be gone and
depression results (B).
Squire Fundamental Neurosci. 2002
Response types at single CNS
synapses with different #s of release sites.
1 release site
2 release sites
2 vesicles
1 vesicle
failure
all or postsyn response?
1 vesicle
failure
Voltage
Time
postsyn amplitude variation?
Electrode
See sum of all
synapses
When multiple synapses (or release sites) are involved facilitation
can reflect an increase in release probability (all or none secretion)
at single synapses that can look like a graded increase in release.
Electrode
sum of all
synapses
Trial 1
Electrode
See sum of all
synapses
Trial 2, 25 ms later
Readings
• Neuroscience 4th Ed. Purves Chapters 4-6 optional
• Fundamental Neuroscience 1st Ed. Chapters 7 and 8
(for Neurochem also), 2nd Ed. Chapters 7 and 8, 3rd Ed.
Chapters 7,8.
• Delaney, Kerry R (March 2000 ) Calcium and
Neurotransmitter Release. In: Encyclopedia of Life
Sciences, London: Nature Publishing Group,
http://www.els.net/doi:10.1038/npg.els.0000027
• Harold L. Atwood & Shanker Karunanithi Diversification
of synaptic strength: presynaptic elements. Nature Reviews
Neuroscience 3, 497 -516 (2002). Advanced review
comprehensive.
• For great EM pictures of synapse see Josef Spacek’s site
http://synapses.mcg.edu/atlas