Neuromuscular Junction Flashcards by Madison Chu

A synapse is a

specialized area of communication between excitable cells

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Electrical Theory

Cells are continuous, allowing direct current flow
o Allow bi-directional conduction
o Calcium independent
o Very fast

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Evidence against Electrical Theory:

• Evidence against:
o Flow of information was unidirectional
o Existence of excitatory as well as inhibitory synapses
o Existence of a delay

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Electrical Theory now we know:

Some cells are “electrically coupled”
o Gap junctions allow all ions and many small molecules to pass through
( Made up of a pair of hemi-channels, one on eachcell, termed connexons)
Both cells are therefore at the same potential
o Usually between neurons, but important for cardiac AP
propagation

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Chemical Theory

Discrete presynaptic and postsynaptic cells
• Proposed that presynaptic cells released a chemical that initiated
current flow in the postsynaptic cells

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Evidence for Chemical Theory:

Strong evidence for came with Otto Loewi’s 2 heart Experiment (1921):
o Parasympathetic (vagus) and sympathetic nerves
released different transmitter substance
which causes different alterations in fH

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Further evidence for Chemical Theory: came with advent of electron microscope:

§ Pre-synaptic and post-synaptic elements
§ A synaptic cleft
§ Pre-synaptic vesicles

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Neuromuscular Junction

Space of ~50nm separates the nerve
terminal and the plasma membrane of the
muscle fibre

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Neuromuscular Junction Comprised on the

basal lamina:

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§ In muscle, the ____ _______

supports the Sarcomere Length during contraction

basal lamina

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Basal lamina contain

Collagen IV and laminin (primary glycoproteins),

fibronectin, entactin and perlecan (most abundant proteoglycan)

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___ is concentrated (~3,000 per um^2) in the synaptic cleft, and
anchored to the basal lamina

AChE

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At the muscle, the ______ _____ ramifies into branches about 2um thick swellings
called presynaptic boutons, which are covered by a thin layer of Schwann cells

motor axon

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‘motor end plate’ is specific for _____ muscle

skeletal

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The boutons lie over a specialized region of the muscle fibre membrane, the
_______, and are separated from the muscle membrane by a _____ ______

endplate

synaptic cleft.

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§ Boutons contain:

• Hundreds of synaptic vesicles containing the neurotransmitter ACh
• Specialised proteins that regulate the fusion of the vesicles and
the terminal membrane, and release of transmitter from the
active zone

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Post-synaptic membrane:

The membrane of the folds contains two
distinct domains. At the crests, there is a
high concentration of AchRs (green), the
AChR-clustering protein rapsyn and
utrophin.
§ In the depths of the folds there is a high
concentration of VGSCs (red)

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General Mechanism of Synaptic Transmission:

o Presynaptic Events

§ AP in the pre-synaptic cell reaches the nerve terminal
§ Depolarisation opens VGCC (transducers) resulting in calcium influx
§ Exocytosis of vesicles and release of neurotransmitter

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General Mechanism of Synaptic Transmission:

o Postsynaptic events

§ Reaction of transmitter with postsynaptic receptors
§ Activation of synaptic channels to produce a postsynaptic current
§ Change in postsynaptic potential gives rise to a propagated AP in the
post-synaptic cell

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Properties of a Neurotransmitter

o Five minimal criteria:

§ Substance present in the pre-synaptic nerve terminals
§ Substance is released during depolarization of the pre-synaptic nerve
terminal
§ Effect is mimicked by exogenous application to post synaptic membrane
§ Existence of an inactivation mechanism
§ Antagonists affect the response in a similar manner

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Direct-Gated Transmission:
AP in pre-synaptic axon depolarizes the pre-synaptic terminal, opens VGCC.
Ca enters terminal and triggers fusion of vesicles to pre-synaptic active zone
membrane. Opening of the vesicles releases transmitter (via process of
exocytosis) into the synaptic cleft

o At the NMJ, ACh diffuses across cleft and binds to AChRs causing them to
undergo a conformation change which then opens the integral ion channel
o Ion flux through AChRs (synaptic of EPC) depolarizes muscle end plate
o Amplitude of EPP depolarizes muscle fibre membrane beyond threshold to
open voltage gated sodium channels and trigger muscle AP
( The Nicotinic ACh-gated Ion Channel)

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§ Postsynaptic depolarization caused by EPC is known as the

EPP

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Indirect Gated Transmission
o Some transmitter receptors do not have integral ion channels
o Such receptors couple to membrane bound G-proteins (termed transducers) in
the neural membrane which in turn are coupled to one or more membrane bound
enzymes (primary effectors).

o Second messengers in post-synaptic neuron activate effector proteins (e.g.
protein kinase enzyme) which in turn phosphorylate effector proteins to bring
about a change in the post-synaptic neuron function – often resulting in
modification of ion channel function

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Transmitter Inactivation and Recycling
Within the synaptic cleft at the NMJ is a basement membrane composed of
collagen and other ECM proteins

o The enzyme AChE is anchored to the collagen fibrils of the BM
o The enzyme appears to be made by the muscle and deposited in the ECM
o AChE rapidly hydrolyses ACh
Choline then diffuses back to the presynaptic terminal and is reabsorbed
o AChE is the physiological target of insecticides and military nerve gases

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End-Plate Current

At an end-plate, EPC is generated by the near synchronous opening of more than 200,000 individual AChR-ion channels, each permitting around 17,000 Na+ ions to enter the muscle EP (and also a smaller number of K+ ions to leave), so net inward, depolarizing current

o AChE rapidly hydrolyses ACh | Choline then diffuses back to the ________ _________and is reabsorbed

presynaptic terminal

o Equivalent Circuit Representing EPC:

A circuit can be drawn representing the EP region with 3 parallel branches: • 1. A branch representing the flow of synaptic current through transmitter gated channels • 2. A branch representing the return current through resting channels • 3. A branch representing the current flow across the lipid bilayer

Current through synaptic pathway is carried by

both Na+ and K+ ions

EP Conductance depends on the number of channels open, therefore, in the absence of transmitter, the conductance is _____ § Conductance increases to about 5 x 10-6 S when a pre-synaptic AP causes release of transmitter, i.e. about 5 x the conductance in the branch representing the resting, or leakage, channels

§ End-plate conductance is in series with a battery (EEPSP) whose value is given by the __________ for synaptic current flow (0 mV), i.e. the algebraic sum of Na and K equilibrium potentials

reversal potential

End-Plate Potential | The net postsynaptic ........

depolarization produced by the synronised release of Ach | from synaptic vesicles on depolarization of the nerve terminal

o EPP amplitude is determined by

the number of vesicles released by the nerve AP (50-300 in mammalian NMJ) and their quantal content

Characteristics of EPP in skeletal muscle:

§ EPP are always supra-threshold § Like some EPSPs, the EPP itself travels passively along the postsynaptic cell (muscle fibre in this case) for only a short distance (of the order of the muscle length constant, a few mm) before dissipating

Curare

muscle relaxant that blocks the nicotinic acetylcholine receptor (nAChR), one of the two types of acetylcholine (ACh) receptors, at the neuromuscular junction.

Spread of the EPP was electronic (i.e. propagation is passive) • Therefore the EPP can only be recorded at the endplate, since it is conducted with decrement • _____are therefore essential for propagation of the AP and the synchronized contraction of large skeletal muscle fibres, but the EPP relies on ___________caused by the EPC

VGSC | depolarization

The EPC and EPP have different time courses:

EPC and EPP have different time courses: • EPC has time course defined by mean open time of AChR channels (T ms) • EPP has longer time course defined by both AChR channel open time and passive electrical charging/discharging time of muscle membrane (behaves like RC network with characteristic time constant: t > T)

Pharmacological agents used experimentally to study the NMJ | o

Alpha toxins: Specific blockers of nicotinic AChRs o Found in some snake venoms, e.g. alpha-bungarotoxin o Curare, a plant extract used by some South Amerian Indians arrows to paralyse prey (An alternative way to do this would be to restrict the calcium concentration in the solution)

- Quantal Release of Neurotransmitter | o Indications of quantal release of neurotransmitter:

1. Evidence from morphological examination of the synapse | § 2. Evidence from quantal analysis of experimental data

Early experiments observed that when the nerve was stimulated there was a depolarization in the post-synaptic cell of the order of tens of mV (EPP), however they also observed that small depolarisations of ~0.5mV occurred in the absence of nerve stimulation every ~1s

MEPPs

Under normal conditions, MEPPs do not arise from ? | Fatt and Katz showed that their release was random

depolarization of the | presynaptic nerve terminal:

Katz showed that ____arose even in a high [K+]o bathing solution, so that even with the nerve-muscle preparation depolarized you could still record changes in membrane potential caused by ____

MEPPs

Used a fine electrode to deliver a known concentration of ACh into the synapse and record the voltage change, therefore determine how much ACh was required to produce the ____ § They estimated that a single _____was the result of release of about 6000 molecules of ACh

MEPPS

§ Investigation of whether mEPPs and EPPs were related:

• Both EPPs and mEPPs were blocked by curare • Anti-cholinesterase prolonged the duration of both EPPs and mEPPs • The frequency of mEPPs was increased by small depolarisations in the presynaptic cell

Anticholinesterases

are a class of drugs that decrease breakdown of acetylcholine

Katz had previously notes that 1 EPP was equivalent to

~100 simultaneous mEPPs Katz concluded that the EPP was in fact made up of multiple mEPPs

______is the key molecular trigger for transmitter release

Calcium

Early work was conducted on the NM synapse and squid giant axon o At the squid giant synapse, combined intracellular pre-synaptic and postsynaptic recordings, together with pre-synaptic intracellular electrical stimulation, are possible due to the ?

large size and possibility for visual guidance | of electrode placement

Study of the movement of radioactive Ca2+ into the squid axon and the use of Ca2+ channel blocking agents proved that Ca2+ entry into the pre-synaptic terminal took place via _____________during _______AP and was an absolute requirement for transmitter release

voltage gated Ca2+ channels | pre-synaptic

Del Castillo and Katz showed that reducing [Ca2+]o:

§ Reduced the amplitude of the evoked EPP (from ~70mV to as low as 0.5-2.5mV) § Produced no change in amplitude of unit responses, Reduced the number of quanta released

Voltage clamp + blocking sodium and potassium channels § TEA blocks? § TTX blocks ?

potassium channels | sodium channels

§ Graded depolarisations gave a graded inward calcium current Resulted in graded post-synaptic membrane § Shows that: •

Calcium was essential for transmitter release to take place • Amount of transmitter release was proportional to the amount of calcium entering the nerve terminal (but in a non-linear way)

Location of calcium receptor | § Calcium chelators (small molecules that bind very tightly to metal ions).of different binding kinetics used to show

that the | calcium binding/trigger is close to the release site

_____ _______ ______can be used to record the fusion pore current:

Patch clamp techniques

Steps in Vesicle Cycling

``` 1. Restrain and mobilization Targeting to active zone Docking at the active zone Fusion & exocytosis Retrieval of the fused membrane ```

Rhythmic respiratory activity drive to the diaphragm via the

phrenic nerve

Rhythmic respiratory activity drive to the diaphragm via the

EPP and EPSC associated with the respiratory drive are mediated by

glutamate

Pattern of EPSPs determine the pattern of

AP generation

Pattern of AP generation determines

contractile function

Change in neuronal excitability induced by

neuromodulation | which will change pattern of acitivity

When a nerve impulse arrives at a noradrenaline nerve terminal, noradrenaline is released from synaptic vesicles into the synaptic cleft. Noradrenaline molecules bind to? and the nerve impulse is ?

their receptors on the post-synaptic membrane and the nerve impulse is propagated or inhibited, depending on the specific receptor

Experimental Techniques to study the EPP | {

Normally we are thinking about voltage-gated channels, which we can study by depolarizing them Voltage clamp of the post-synaptic muscle fibre

How are ligand-gated channels studied (AChR is a ligand-gated channel)? § A number of ways agonists can be applied to the end-plate region in vitro

1. A single nerve impulse § 2. Spontaneous quantal release § 3. Agonist applied in bathing solution § 4. Patch of channel with agonist in the patch pipette § 5. Flash photolysis of synthetic agonist molecules

Note: In order to examine the EPP or EPC under voltage clamp, it is necessary to either stimulate the motor axon, or to apply an agonist.. Voltage clamp of the post-synaptic muscle fibre § Controls the voltage across the membrane § Experimental technique

Two microelectrodes inserted at the motor end-plate • (i) Measures membrane potential • (ii) Passes current • Both electrodes are connected to a feedback amplifier that matches the current delivered so that Vm is clamped at the command vol 0mVtage

Unlike the Na+ and K+ channels that underlie the AP, the AChR channel has a reversal potential at

0mV

Individual ion channels open and contribute discrete increments of ___ § EPC rises sharply at start as ___ channels opened in near synchrony by the wave of synaptically released ACh § Channels automatically close as ACh rebinds, and is removed from the EP § Removal of ACh is accelerated bu the enzyme ____ § AChR channels remain open for variable times

EPC AChR AChE

The total EPC is the sum of

``` all individual channel currents: 𝑁 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐ℎ𝑎𝑛𝑛𝑒𝑙𝑠 𝑖𝑛 𝐸𝑃 § 𝑝o = 𝑝𝑟𝑜𝑏𝑎𝑏𝑙𝑖𝑙𝑖𝑡𝑦 𝑜𝑓 𝑐ℎ𝑎𝑛𝑛𝑒𝑙𝑠 𝑏𝑒𝑖𝑛𝑔 𝑜𝑝𝑒𝑛 § 𝛾 = 𝑠𝑖𝑛𝑔𝑙𝑒 𝑐ℎ𝑎𝑛𝑛𝑒𝑙 𝑐𝑜𝑛𝑑𝑢𝑐𝑡𝑎𝑛𝑐𝑒 § 𝑉 = 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒 𝑝𝑜𝑡𝑒𝑛𝑡𝑖𝑎𝑙 § 𝐸 epsp = 𝐶ℎ𝑎𝑛𝑛𝑒𝑙 𝑟𝑒𝑣𝑒𝑟𝑠𝑎𝑙 𝑝𝑜𝑡𝑒𝑛𝑡𝑖𝑎𝑙 ```