Neuromuscular junction Flashcards
What is a neuromuscular junction
The contact between a motor neuron and a muscle cell- converts electrical energy into mechanical force, specialised for fast and reliable control of muscle contraction
How does the input of skeletal muscles different from most neurons in the CNS
In the CNS, neurons usually receive many small and weak convergent synaptic inputs
Each skeletal muscle cell receives a single synaptic input from one motor axon
Why is the NMJ the prototypical model system for studying synaptic structrue and function
The muscle cell is large and relatively easy to record from, and is innervated by a single motor axon that can be excited by simple electrical stimulation
Who was awarded the Nobel prize for his work on the mechanisms of transmission at the NMJ
Sir Bernard Katz
What 2 observations suggest transmission at the NMJ depends on diffusion of a substance across the synaptic cleft
The synaptic cleft revealed from high-res electron micrographs, and the small delay between the action potential invading the presynaptic terminal and the onset of the electrical response in the muscle cell
What are the 3 established criteria for identifying a chemical as a neurotransmitter
Must be present in a presynaptic neuron, must be released in response to presynaptic stimulation, specific receptors for it must be present on the postsynaptic cell (agnosists of these receptors must mimic synaptic response, antagonists must block synaptic response)
What is the neurotransmitter at the NMJ
Acetylcholine (Ach)
Why does the NMJ use chemical synaptic transmission rather than direct electrical coupling
Chemical transmission allows impedance matching- if the neuron and muscle cell were directly coupled, the small currents flowing across the presynaptic terminal would be too small to charge up the membrane capacitance of the much larger muscle cell and evoke an AP
What is the endplate of the muscle cell
The area that receives synaptic input
What is the resting potential underneath the endplate
If a microelectrode is isnerted underneath the endplate, it records a hyperpolarised resting potential
What is recorded by a microeelctrode inserted under the endplate following stimulation of the efferent motor cell axon
A large synaptic depolarisation called the endplate potential (EPP), which invariably exceeds the threshold the muscle action potential
What happens after the EPP exceeds the threshold for the muscle action potential- MAP vs EPP
The muscle action potential actively propagates to distal regions of the muscle cells
The EPP decreases with distance from the endplate as would be expected with passive propagation
How can we see the EPP waveform experimentally
If we block action potentials in the muscle cell
Example of a study using a microelectrode to record the shape of the EPP and muscle action potential
Von Gersadorff (2008)
What does released acetylcholine bind to to generate the EPP
Acetylcholine binds to nicotinic acetylcholine receptors (nAChR), ligand-gated ion channels
What is the structure of the nicotinic acetylcholine receptors (nAChR)
Composed of 5 subunits (two alpha subunits, a beta, a delta, and epsilon subunit)
What are nAChRs permeable to
Cations, primarily Na+
How is the motorendplate structurally recognisable
Invaginatinos in the postsynaptic membrane called junctional folds- nAChR in high density at the crests, while voltage-gated Na+ channels are at the base of the folds
What is the functional reason for the structural organisation of the junctional folds of the motor endplate
Junctional folds increase the endplate SA, ensuring there is maximal sensitivity to detect ACh release, while keeping lots of voltage Na+ channels close to the synapse- preserves the reliability of the EPP in triggering an AP
What triggers neurotransmitter release at the presynaptic nerve terminal
It opens voltage-gated Ca2+ channels, causing increased presynaptic Ca2+ conc (Ca2+ diffuses down conc gradient), which triggers neurotransmitter release
What is shown if one plots the log of EPP amplitude againsts log of external Ca2+ conc
Reveals a straight line, showing the amount of neurotransmitter release is proportional to external Ca2+ conc
Slope of around 4 suggests the Ca2+ sensor needs to bind 4 Ca2+ ions to trigger release
What spontaneous activity is recorded at the endplate in the absense of presynaptic action potenials
Miniature endplate potentials (mEPPS) caused by a very low rate of neurotransmitter spontaneously released, that have the same shape as EPPs but are several-fold smaller
What is the amplitude of an EPP
50mV
What is the amplitude of basically all mEPPs, and what does this suggest
0.5mV, suggesting mEPPs are a response to a small packet of ACh of fixed size, a quantum
What suggests that ACh shows quantal release
mEPPs all have the same shape, EPP amplitudes when probability of ACh release is very low are all approx multiples corresponding to 1/2/3 quanta
How can we observe the quantal nature of evoked synaptic transmission
By reducing the probability of release eg reducing external Ca2+
What distribution do evoked EPP amplitudes show when the probability of release is made very low
Multi-peaked distribution- a large no of failures, then peaks at approximate multiples of the mEPP amplitude corresponding to 0/1/2 quanta released per presynaptic action potential
What does quantal release follow
Binomial statistics
How does quantal release follow binomial statistics
If q is quantal size, p is the probability of quantal release and n is the no of release sites, the mean response should be npq, and probability of failure should be (1-p)^n-1
What are the structural correlates of quantal mEPPs
The vesicles of neurotransmitter packed into the presynaptic terminal
How can the vesicles of neurotransmitter be visualised
If the NMJ is frozen just after stimulation, electron microscopy can show these vesicles fusing wit the presynapci membrane (Zucker et al, 2004)
What are the first steps of the vesicle cycle (vesicle cycle summary, before docking)
Neurotransmitters are actively transported into synaptic vesicles that then cluster in front of the active zone
What happens to synaptic vesicles after they are clustered in front of the active zone (vesicle cycle summary)
They dock at the active zone, where they are primed and converted into a state of competence for Ca2+ triggered fusion-pore opening
What are the 3 alternate pathways for synaptic vesicles after fusion-pore opening (vesicle cycle summary)
Synaptic vesicles endocytose and recycle by 3 alternate pathways- kiss-and-stay, kiss-and-run, clathrin-coated pits
3 alternate pathways for synaptic vesicles after fusion-pore opening (vesicle cycle summary)- ‘kiss-and-stay’
Vesicles are reacidified and refilled with neurotransmitters without undocking, thus remaining in the readily releasable pool
3 alternate pathways for synaptic vesicles after fusion-pore opening (vesicle cycle summary)- ‘kiss-and-run’
Vesicles undock and recycle locally to reacidify and refill with neurotransmitters
3 alternate pathways for synaptic vesicles after fusion-pore opening (vesicle cycle summary)- clarithin-coated pits
Vesicles endocytose via clathrin-coated pits and refill with neurotransmitters either directly or after passing through an endosome
How is acetylcholine transported into synaptic vesicles
Vesicular acetylcholine transporter (VAChT) transfers ACh into synaptic vesicles- the vesicles are acidified by accumulation of H+ ions and VAChT exchanges ACH for H+ ions
What are SNARE proteins
Soluble NSF Attachment Protein Receptors- three proteins are the core of the complex mediating synaptic exocytosis
What are the 3 SNARE proteins
Synaptobrevin on synaptic vesicles (v-SNARE), syntaxin 1 and SNAP-25 on the presynaptic plasma membrane (target or t-SNARE)
What is the Ca2+ sensor
Appears to be a synaptotagmin (protein family), Ca2+ binding to it triggers fast exocytosis
How is synaptic transmission terminated (hydrolysis)
Acetylcholinesterase located in the basal lamina between the pre and postsynaptic terminals rapidly hydrolyses ACh into acetate and choline once it dissociates from the receptor
Gives temporal precision
What proportion of ACh molecles are degraded by acetylcholinesterase in the basal lamina before they can bind to nAChR
10%
What happens to the breakdown products of ACh
Reuptake of choline via the choline transporter
Re-synthesis of ACh via chole acetyl transferase (ChAT)
How is the NMJ a high fidelity synapse
It has 1000s of docked vesicles over 700 active zones, hence its large size
This means it has large and reliable responses from trial-to-trial eg mean no of vesicles released per action potential in frog is 350
What does it mean that NMJ is not protected by the blood brain barreir
It is sensitive to blood-born pathogens, including toxins and auto-antibodies
NMJ dysfunction- what is botox
Botukinum toxin cleaves SNARE proteins, preventing exocytosis- causes flaccid paralysis, can lead to cessation of breathing and death
NMJ dysfunction- what is nerve gas
eg sarin blocks acetylcholineresterase, ACh is not broken down and accumulates in synaptic cleft, causing nAChR desensitisation, can cause dizziness, loss of consciousness, convulsive spasms
NMJ dysfunction- myasthenia gravis
An autoimmune disorder, with antibodies targeting nAChR causing muscle weakness and fatigability
NMJ dysfunction- curare-mimetics
eg pancuronium bromide can be used as muscle relaxants during surgery, binds to ACh receptors and prevents muscle contraction
What is synaptic transmission
Process of information transfer at a synapse
What study provides evidence for the concept of chemical synapses
Loewi (1921)- showed that electrical stimulation of axons innervating the heart of a frog causes the release o a chemical that could mimic the effects of neuron stimulation on the heartbeat
What study provides evidence for chemical synapses in the human CNS
Eccles (1951)- used a glass microelectrode to study the physiology of synaptic transmission in the mammalian CNS, found that chemical synapses make up the majority of synapses in the brain
How much of the time does an action potential in the motor axon cause an action potential in the muscle cell it innervates
ALWAYS
What type of neurotransmitter is ACh
Amine
What deposits AChE in the synaptic cleft
Muscle cells
What is the consequence of uninterrupted exposure to high ACh concs after several seconds
Leads to desensitisation where the nAChR channels close, can least many seconds even when the neurotransmitter is removed
Neuromuscular dysfunction- what is bacteria Clostridium botulinum
Produces toxins that block neuromuscular transmission by destroying certain areas of the SNARE proteins in the presynaptic terminals that are critical for transmitter release
What are receptor agonists
Drugs that bind to receptors and mimic the action of the naturally occuring neurotransmitter
What is a receptor agonist of nAChRs
Nicotine- binds to and activates ACh receptors in skeletal muscle
What is synaptic integration
The process by which multiple synaptic potentials combine within one postsynaptic neuron to produce a significant postsynaptic depolarisation/hyperpolarisation
What is quantal analysis
A method of comparing the amplitudes of miniature and evoked PSPs
How many synaptic vesicles are exocytosed following a single action potential in the presynaptic terminal
About 200 synaptic vesicles
How big is the size of the EPSP for the NMJ and why
It generates a huge EPSP because it needs to work every time
What are the two types of EPSP summation
Spatial summation and temporal summation
What is spatial summation
Addnig up EPSPs generated simultaneously at many different synapses on a dendrite
What is temporal summation
Adding up EPSPs generated at the same synapse if they occur in rapid succession ie within about 1msec of each other
What does the motor axon branch to form at the muscle
Synaptic boutons covered by a thin layer of Schwann cells- lie over the end-plate and are where transmitter is released from
What is the basal lamina
The basement membrane, a layer of connective tissue consisting of collagen and glycoproteins that covers the muscle fibres
What does the basal lamina do
Aligns the synaptic boutons with the postsyaptic junctional folds, organising the synapse
Study supporting the role of curare
Fatt and Katz (1950s) isolated the EPP in intracellular voltage recordings using curare, which reduced the amplitude of the EPP below the threshold for the action potential
What is the reversal potential
The membrane potential at which the synaptic current has zero amplitude- there is no net current, as any inward current is balanced by an equal no of outward current
Why is the reversal potential of the endplate potential 0mV
The nAChRs in the end plate are almost equally permeable to Na+ and K+, so during end-plate potential Na+ flows into the cell and K+ flows out, cancelling each other out and creating a weighted average of the equilibium potentials
What does the end-plate potential depolarisation cause to open
Voltage-gated Na+ and K+ channels at the end plate region are activated sequentially- the Na+ channels creating the positive feedback mechanism and eventually an action potential
What is the nature of the behaviour of ion channels during opening/closing
ACh opens individual channels in all-or-non steps, channels conduct a current of fixed amplitude each time they open, the duration of each chanel opening is stochastic but the mean time is around 1ms
What ions move through single ACh-gated channels
About 17,000 Na+ ions flow into the muscle cell, and a smaller no of K+ ions flow out
How do more positive levels of membrane potential affect the magnitude of EPP
The inward driving force on Na+ is less while the outward driving force on K+ is greater at more positive membrane potentials, decreasing the magnitude of endplate currents and thus the size of the EPP
If the synaptic channels at the postsynaptic membrane are equally permeable to Na+ and K+, how come opening them causes depolarisation?
At the usual muscle fibre resting membrane potential of -90mV, there is a small driving force on K+ (outwards) but an even stronger one on Na+ (inwards), so much more Na+ will flow in than K+ out, creating the inward current at the EPC
What is the usual EPSP size of a single action potential
40mV
What is the normal muscle resting membrane potential
-90mV
Why are the nAChR channels not sufficient to generate the action potential on their own
The influx of Na+ through these channels doesn’t cause more ACh channels to open as they can only be opened by ACh, so depolarisation produced by these channels is limited- the Na+ voltage-gated must be recruited for this
