Lab report
Abstract: Experiments performed to investigate neurotransmission at the neuromuscular junction.
Acknowledgement: In order to obtain the data for this experiment # collected the data as labpartner.
Introduction: Drugs affecting cholinergic neurotransmission can be divided into drugs that act directly and
drugs that act indirectly. The direct acting binds to the muscarinic receptors (M1,M2,M3,M4 or N) and activates
it (agonistic). While the indirect acting drugs increase the levels/availability of ACh by different target actions.
Another way to categorize the drugs are drugs that causes depolarization and drugs that do not cause
depolarization (non-depolarizing drugs).
Figure of the cholinergic neurotransmission from: Wilkinson, David & Francis, Paul & Schwam, Elias & Payne-Parrish,
Jennifer. (2004). Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between
pharmacological effects and clinical efficacy. Drugs & aging. 21. 453-78.
Illustration depicting the synthesis, degradation and actions of acetylcholine (ACh) on target receptors.
1
,the neuromuscular junction (NMJ) is a chemical synapse between a neuron and a skeletal muscle/smooth
muscle/cardiac muscle. An action potential is initiated by a sufficiently strong enough stimulus which opens
voltage gated sodium (Na+) channels; the depolarization is mainly driven by these channels. The action
potential then travels down the neuron and depolarizes the presynaptic membrane. Next voltage gated calcium
(Ca2+) open and Ca2+ influx on the presynaptic membrane. The SNARE complex then facilitates the vesicles to
fuse with the pre synaptic membrane. The vesicles release acetylcholine (ACh) into the synaptic cleft. On the
postsynaptic membrane (motor end plate) the ACh binds to its receptor (nicotinic) the binding leads to
depolarization of the postsynaptic membrane. Receptors that are voltage sensitive (voltage-sensitive
dihydropyridine receptors DHPR) associate with ryanodine receptors which leads to the release of Ca2+ from
the sarcoplasmic reticulum and through tropomyosin, myosin and actin action a muscle contraction is achieved.
Within the synaptic cleft acetylcholinesterase breakdown ACh to acetate and choline, the presynaptic
membrane then resynthesises ACh by reuptake of choline (Hall, Hall and Hall, 2021) (Boron and Boulpaep,
2003). In the software Twitch you get the option between which tissue you would like to stimulate either nerve
indirectly and muscle directly. The properties of these two tissues are different physiologically and this is
highlighted in the different experiments performed. The role of magnesium in the neuromuscular junction is
that it inhibits the release of ACh presynaptically, less ACh release results in less depolarization post
synaptically. (McLarnon and Quastel, 1983).
Illustration Non depolarizing and depolarizing Neuromuscular blockade drugs.
(Fagerlund and Eriksson, 2009)
Fagerlund, M.J. and Eriksson, L.I. (2009). Current concepts in neuromuscular transmission. British Journal of
Anaesthesia, [online] 103(1), pp.108–114. doi:10.1093/bja/aep150.
2
, Tubocurarine (TUB) is a competitive antagonist that binds to the nicotinic ACh receptor post synaptically. This
blocks depolarization. The drug is not used in clinical practice, it is known for use in arrow poison
(“Neuromuscular Blocking Agents”).
Tetradotoxin (TTX) is a sodium channel inhibitor, it binds to the voltage gated sodium channel and blocks it
physically from letting sodium pass, which prevents depolarization in both muscle and nerve. (Lee and Ruben)
Tetradotoxin is a toxin found in puffer fish with no known antidote (Yıldırım, Y.).
4-aminopyridine (4-AP) is an antagonist that binds to voltage-gated potassium channels. The drug is used in
multiple sclerosis patients in whom the voltage-gated potassium channels have been exposed due to
demyelination and relocated along the neuron. Which would normally be found close to the nodes of Ranvier.
4-AP blocks these exposed mislocated voltage-gated potassium channels and improves signal transduction
(Dietrich et al.).
Neostigmine (NEO) inhibits acetylcholinesterase (AChE) as an antagonist to AChE, this decreases the
breakdown on acetylcholine (ACh), so the levels of ACh increases in the synaptic cleft. It (NEO) stimulates
indirectly nicotinic and muscarinic receptors. The drug is used postoperative in order to reverse the
neuromuscular blockade, urinary retention and neurogenic ileus. In addition it is used in patients with
myasthenia gravis, which is an autoimmune disorder with generalized muscle weakness. (Hristovska et al.).
Atropine (ATR) is a competitive muscarinic antagonist (it blocks muscarinic acetylcholine receptors) in skeletal
muscle exclusively. The drug has several therapeutic uses: it is used as an antidote for anticholinesterase
poisoning. It is used in patients with unstable sinus bradycardia (because atropine increases the heart rate). It is
administered before intubation of patients to decrease secretion of exocrine glands and decrease tone and
motility in smooth muscle. It is used as an antidote for scorpion stings, where a sting would result in
bronchoconstriction and increased salivation!(BRAGA et al., 1994).
Suxamethonium (SUX) is also known as Succinylcholine, which is a depolarizing muscle relaxant. It binds to
ACh receptors (Nm nicotinic receptors at NMJ) as an agonist which causes depolarization in muscle. Increased
influx of Na+ and K+. Which hydrolyses ACh by choline esterase. The drug has no antagonist, AChE can't
break it down and the result is a consistent/prolonged depolarization that leads to a repetitive muscle
contraction and eventually paralysis of the skeletal muscles. Plasma cholinesterase is able to break down
succinylcholine in 5-10 (given that the enzyme is not atypical). (CHURCHILL-DAVIDSON)(Henriquet).
Method:
3
Abstract: Experiments performed to investigate neurotransmission at the neuromuscular junction.
Acknowledgement: In order to obtain the data for this experiment # collected the data as labpartner.
Introduction: Drugs affecting cholinergic neurotransmission can be divided into drugs that act directly and
drugs that act indirectly. The direct acting binds to the muscarinic receptors (M1,M2,M3,M4 or N) and activates
it (agonistic). While the indirect acting drugs increase the levels/availability of ACh by different target actions.
Another way to categorize the drugs are drugs that causes depolarization and drugs that do not cause
depolarization (non-depolarizing drugs).
Figure of the cholinergic neurotransmission from: Wilkinson, David & Francis, Paul & Schwam, Elias & Payne-Parrish,
Jennifer. (2004). Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between
pharmacological effects and clinical efficacy. Drugs & aging. 21. 453-78.
Illustration depicting the synthesis, degradation and actions of acetylcholine (ACh) on target receptors.
1
,the neuromuscular junction (NMJ) is a chemical synapse between a neuron and a skeletal muscle/smooth
muscle/cardiac muscle. An action potential is initiated by a sufficiently strong enough stimulus which opens
voltage gated sodium (Na+) channels; the depolarization is mainly driven by these channels. The action
potential then travels down the neuron and depolarizes the presynaptic membrane. Next voltage gated calcium
(Ca2+) open and Ca2+ influx on the presynaptic membrane. The SNARE complex then facilitates the vesicles to
fuse with the pre synaptic membrane. The vesicles release acetylcholine (ACh) into the synaptic cleft. On the
postsynaptic membrane (motor end plate) the ACh binds to its receptor (nicotinic) the binding leads to
depolarization of the postsynaptic membrane. Receptors that are voltage sensitive (voltage-sensitive
dihydropyridine receptors DHPR) associate with ryanodine receptors which leads to the release of Ca2+ from
the sarcoplasmic reticulum and through tropomyosin, myosin and actin action a muscle contraction is achieved.
Within the synaptic cleft acetylcholinesterase breakdown ACh to acetate and choline, the presynaptic
membrane then resynthesises ACh by reuptake of choline (Hall, Hall and Hall, 2021) (Boron and Boulpaep,
2003). In the software Twitch you get the option between which tissue you would like to stimulate either nerve
indirectly and muscle directly. The properties of these two tissues are different physiologically and this is
highlighted in the different experiments performed. The role of magnesium in the neuromuscular junction is
that it inhibits the release of ACh presynaptically, less ACh release results in less depolarization post
synaptically. (McLarnon and Quastel, 1983).
Illustration Non depolarizing and depolarizing Neuromuscular blockade drugs.
(Fagerlund and Eriksson, 2009)
Fagerlund, M.J. and Eriksson, L.I. (2009). Current concepts in neuromuscular transmission. British Journal of
Anaesthesia, [online] 103(1), pp.108–114. doi:10.1093/bja/aep150.
2
, Tubocurarine (TUB) is a competitive antagonist that binds to the nicotinic ACh receptor post synaptically. This
blocks depolarization. The drug is not used in clinical practice, it is known for use in arrow poison
(“Neuromuscular Blocking Agents”).
Tetradotoxin (TTX) is a sodium channel inhibitor, it binds to the voltage gated sodium channel and blocks it
physically from letting sodium pass, which prevents depolarization in both muscle and nerve. (Lee and Ruben)
Tetradotoxin is a toxin found in puffer fish with no known antidote (Yıldırım, Y.).
4-aminopyridine (4-AP) is an antagonist that binds to voltage-gated potassium channels. The drug is used in
multiple sclerosis patients in whom the voltage-gated potassium channels have been exposed due to
demyelination and relocated along the neuron. Which would normally be found close to the nodes of Ranvier.
4-AP blocks these exposed mislocated voltage-gated potassium channels and improves signal transduction
(Dietrich et al.).
Neostigmine (NEO) inhibits acetylcholinesterase (AChE) as an antagonist to AChE, this decreases the
breakdown on acetylcholine (ACh), so the levels of ACh increases in the synaptic cleft. It (NEO) stimulates
indirectly nicotinic and muscarinic receptors. The drug is used postoperative in order to reverse the
neuromuscular blockade, urinary retention and neurogenic ileus. In addition it is used in patients with
myasthenia gravis, which is an autoimmune disorder with generalized muscle weakness. (Hristovska et al.).
Atropine (ATR) is a competitive muscarinic antagonist (it blocks muscarinic acetylcholine receptors) in skeletal
muscle exclusively. The drug has several therapeutic uses: it is used as an antidote for anticholinesterase
poisoning. It is used in patients with unstable sinus bradycardia (because atropine increases the heart rate). It is
administered before intubation of patients to decrease secretion of exocrine glands and decrease tone and
motility in smooth muscle. It is used as an antidote for scorpion stings, where a sting would result in
bronchoconstriction and increased salivation!(BRAGA et al., 1994).
Suxamethonium (SUX) is also known as Succinylcholine, which is a depolarizing muscle relaxant. It binds to
ACh receptors (Nm nicotinic receptors at NMJ) as an agonist which causes depolarization in muscle. Increased
influx of Na+ and K+. Which hydrolyses ACh by choline esterase. The drug has no antagonist, AChE can't
break it down and the result is a consistent/prolonged depolarization that leads to a repetitive muscle
contraction and eventually paralysis of the skeletal muscles. Plasma cholinesterase is able to break down
succinylcholine in 5-10 (given that the enzyme is not atypical). (CHURCHILL-DAVIDSON)(Henriquet).
Method:
3
