CJH332

Synapse
Neurobiology
Week 6

Presynaptic transmission
and the role of calcium

Left – A pseudocolored SEM of a synaptic terminal, broken open to show the

synaptic vesicles (blue and orange)
CIL:214, neuron. CIL. Dataset (cellimagelibrary.org)
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© CJH332 Neurobiology of the Synapse 2021
Neurotransmission
• Re-visiting the synapse and why the story of neurotransmission
is so thrilling
• The calcium hypothesis
• Using the NMJ as a specialized synapse and overview of
related techniques (ionophoresis)

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Where have we left off?
• We know that neurons receive
inputs – both excitatory and
inhibitory
• These inputs are summated at
the AIS where an AP can be
initiated
• APs are propagated down axons
through saltatory conduction

• But, what next?

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The Nobel Prize Frenemies?
1871: Camillo Golgi popularizes the
Reticular Theory: everything in the
nervous system is a continuous network

1873: Golgi stain: a silver nitrate stain

to label the anatomy of neurons

1891: Cajal uses the Golgi Stain to

develop the Neuron doctrine: that the
neuron is the basic anatomical and
physiological unit of
the nervous system

1906: Cajal and Golgi share the Nobel

Prize in Physiology or Medicine https://www.the-scientist.com/foundations/the-neuron-doctrine-circa-1894-38476
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Around the same time….
• Sherrington proposed that adjacent cells interact with each
other through a specialized membrane
• He was the first to propose the term “synapse”
• Described the synapse as a functional separation
between cells since he couldn’t visualize synapses
with microscopes at that time….

Sherrington won the Nobel Prize for Medicine

in 1932 for his broad work done in the field
of neuroscience
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How far we’ve come

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 Neurotransmission:
The Chemical
Synapse

• Convert the electrical signal

to a chemical one
• Pre-synaptic terminals are
densely packed with
vesicles
• Membrane expresses
voltage-dependent calcium
channels

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Ca2+ and Excitation
• Ca2+ ions are universal second
messengers in many cell types
• Especially true in neurons and
cells with a neuronal lineage
• Changing Ca2+ ion based
signaling can fundamentally
change properties of neurons

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Ca2+ and Excitation
• Neurons also contain voltage-gated(dependent) Ca2+
channels (VGCCs or VDCCs)
• Ca2+ influx during the AP is critical for the secretion of
neurotransmitters in chemical synapses
• In some neurons the Ca2+ influx is so large it is responsible
for the rising phase of the AP
• gCa2+ is regenerative like gNa+ (positive feedback)
• Ca2+ based APs are most commonly found in cardiac muscle
and invertebrate neurons

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The calcium hypothesis – from the start
• Depolarization by itself did not seem to be able to trigger
post-synaptic responses (see next slide)
• Furthermore, reducing or eliminating calcium in the extracellular
fluid decreased ACh release from NMJ
• Others had shown that irrespective of the neurotransmitter
being studied, that calcium was required for the release of
neurotransmitter (glutamate, GABA etc.)
• Substitution experiments verified that calcium was required
(magnesium, cadmium, nickel, cobalt etc.)
• Release of hormones (adrenaline/salivary secretion)= similar
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Llinas electrophysiology experiments
-18 mV +60 mV

2 separate experiments involving squid giant synapse

(1980s)
Squid giant - Voltage clamp to -18 mV left and +60 mV right
synapse - What else did they have to include in the middle traces?
- Middle traces show the calcium current
- Bottom traces show post-synaptic response

WAIT – did you take a look at this voltage clamp??

Time courses of pre-synaptic action potentials

Opening of voltage gated Ca2+ channels
Release of neurotransmitter
Post-synaptic response

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 Figure 13.4 Microdomains of Calcium within the Presynaptic Terminal at the Squid Giant Synapse

Calcium – where and when

Inject fluorescent dye Aequorin
in the presynaptic neuron

Before activity in (A) observe

nanodomains of calcium

After activity in (B) observe

microdomains of calcium

Suggests spatial regulation

of calcium – it doesn’t go
everywhere

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 Figure 13.4 Microdomains of Calcium within the Presynaptic Terminal at the Squid Giant Synapse

Calcium – where and when

B) Discrete nanodomains: Ca2+ open and Ca2+

open and trigger synaptic vesicle release
- If channels are farther apart, Ca2+ entry
does not summate and there is a linear
relationship between amount of Ca2+ entry
and neurotransmitter release

C) IF domains are close, the Ca2+ influx can

summate in microdomains
- supralinear release of neurotransmitter

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 Figure 13.5 Calcium Enters Near the Site of Transmitter Release

Chelators - localization of entry sites

4 mins of recordings in
these panels at top

Over time the injection

of BAPTA causes a decrease
In the post-synaptic response

EGTA injection does not

produce the same effect

BAPTA – very strong and high

affinity for calcium

EGTA – lower affinity and less

speed of binding

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© CJH332 Neurobiology of the Synapse 2021
Nitrophen – calcium responses

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 Figure 13.6 An Increase in Intracellular Calcium Is Sufficient to Trigger Rapid Transmitter Release

Nitrophen – calcium responses

Nitrophen is a “caged” version of calcium

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NMJ Synaptic Potentials

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Spontaneous MEPPs – why important?

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Quantum singularities

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How much ACh in a Quantum?

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Mapping synapses - ionophoresis

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Mapping synapses – immuno-methods

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 Neurotransmitter Release - Exocytosis
• The AP invades the synaptic terminal depolarizing
voltage-gated Ca2+ channels that are highly
localized to the region
• The increase in [Ca2+]i within the synaptic terminal
causes the release of neurotransmitter
• The axon terminal contains vesicles storing
neurotransmitter
• AP induced Ca2+ influx results in the fusion of the
vesicles with the plasma membrane of the axon
terminal. The neurotransmitter is therefore
released by exocytosis.
• The neurotransmitter enters the space between
the neurons called the synaptic cleft. This space
is typically 25 - 40 nm wide, which is actually a little
bit larger than the typical space between adjacent
cells (20 nm). Special synaptic proteins are found
in the axon terminal and in the synaptic cleft.
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 Presynaptic Structures
• Synaptic vesicles –
presynaptic sites for
neurotransmitter storage
• Active zones – presynaptic
regions characterized by
clusters of synaptic vesicles
• Boutons – presynaptic nerve
terminal expansion onto nerve
cells; site of transmitter
release

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Nobel Prize in Physiology or Medicine 2013

Bill Trimble

Shuzo Sugita

Thomas C. Südhof James E. Rothman Randy W. Schekman

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Exocytosis in more detail
• How does AP-induced
Ca2+ entry actually
cause vesicles to
exocytose?
• Vesicles have proteins
associated with them
called v-SNAREs
• These will interact with
other proteins found on
the target pre-synaptic
membrane known as t-
SNAREs

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Complex but necessary interactions

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Vesicle Release – Part 1

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Vesicle Release – Part 2

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Synaptotagmin – a calcium sensor?

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Synaptotagmin – a calcium sensor?

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More complexities pre-synaptically

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 CJH332 Part II
Part I: Fundamental Electrophysiology Part II-A: Advanced techniques
• Channels • Optogenetics of circuits
• Membranes • Calcium imaging
• Action potentials • Chemogenetics
• Myelin
• Calcium

Part II-B: Circuits, mechanisms and

pathologies
• Synaptic plasticity
• Alzheimer
• Stroke
• Synucleinopathies

The goal of part II: is to integrate the fundamentals of synaptic neurobiology to understand neural circuits and pathologies
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Reminders for This Week
• Our next live office hour is Thursday February 25th at 1:10-2:00 PM
• Please let me know which slides and concepts you would like to see
discussed here: https://forms.gle/5nrJz4VYt7Sp44rA6
• QUIZ 4 is due by Friday at 11:59PM (midnight) EST
• TT2 is on March 3rd-4th!

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Term Test 2 Information
• Open book
• Covers weeks 4-6 (everything from week 4 up until this point)
• Open for a 48 hour window beginning on Wednesday, March 3rd
• Wednesday, March 3rd at 12:00AM – Thursday March 4th at 11:59PM EST
• 20 MCQ and 2 short answers
20 MCQ x 1 mark each and
2 short answers worth 20 marks total
= 40 marks
• The MCQ and short answer questions will be available as separate parts
• You will have 1 hour to complete MCQ section once you begin it
• You will have 1 hour to complete the short answer section once you begin it
• Both sections must be completed by the deadline
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Thank you and
good luck!

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