ME.6200

Cholinergic Neuromodulation I

Prof. Gregor Rainer

Discovery of Acetylcholine - Otto Loewi 1921

He is almost as famous for the means by which the idea for his experiment came to him as he is for the experiment itself. On Easter

Saturday 1920, he dreamed of an experiment that would prove once and for all that transmission of nerve impulses was chemical, not

electrical. He woke up, scribbled the experiment onto a scrap of paper on his night-stand, and went back to sleep. The next morning he

arose very excited because he knew this dream had been very important. But he found, to his horror, that he couldn't read his midnight

scribbles. That day, he said, was the longest day of his life, as he could not remember his dream. That night, however, he had the same

dream. This time, he immediately went to his lab to perform the experiment.

Acetylcholine: Metabolism

Acetylcholine is hyrdolyzed by the membrane-associated

Acetylcholineesterase. AChE ranks as one of the most efficient enzymes, since

one molecule of the enzyme can hydrolyze 5000 molecules of acetylcholine per

second.

• Acetylcholine is formed by the combination of an Acetyl group

and Choline in the presence of the ChAT enzyme.

• Acetylcholine is metabolized by the membrane associated

enzyme AChE with very high efficiency.

• Choline is taken up into presynaptic neurons by a high affinity

carrier.

Take Home Message

Acetylcholine: Receptors

Reversal potential Na: ~+40mV

Reversal potential K: ~-75mV

Equilibrium potential typical Neuron: ~-70mV

The Nicotinic ACh Receptor

x5

There are 8 and 4 isoforms.

Note: The nAChR is permeable to Na and K and Ca ions.

The Muscarnic ACh Receptor

Phosphatidylinositoldiphosphate (PIP2)

Inositoltrisphosphate (IP3)

Diacylglycerol (DAG)

Protein Kinase C (PKC)

• There are two types of Acetylcholine receptors (AChR).

• The nicotinic receptor (nAChR) is the best studied of all

receptors. It is a ligand gated ion channel composed of five

subunits. There are many different nAChR varietes that are

specific to particular structures (7 receptor for Cortex and

Hippocampus; 12 for skeletal muscle).

• The muscarinic receptor (mAChR) is coupled to a G-protein

and activates a second messanger cascade. This cascade

triggers protein phosphorylation and Calcium release from

intracellular Calcium stores, and can lead to opening of

Potassium channels. There are five main types of mAChRs.

Take Home Message

Autonomic Nervous System I

Autonomic

Nervous System II

ACh ACh

NE

Adrenaline ACh

ACh ACh

nAChR mAChR

ACh NE, Adrenaline

nAChR adrenergicR

Sympathetic

Parasympathetic

‘rest and digest’

Stimulatory, activational

TARGET

TARGET

stimulates

constricts pupil

salivation

slows heartbeat

constricts lung

stimulates

erection

release NE: Norepinephrine

ACh: Acetylcholine

T, K, S: Thoracic,Lumbar and Sacral segments

• The autonomic nervous system is composed of a sympathetic

and a parasypmpathetic part. Both use (nictonic) Ach in

preganglionic neurotransmission, and the parasympathetic

system also uses (muscarinic) Ach transmission in

postganglionic neurotransmission.

• Sympathic modulation is activational and important for „fight

and flight“; parasympathetic modulation is important for body

maintenance in the resting state („rest and digest“).

• In parasympathetic modulation, ACh: constricts pupils,

stimulates tear glands, induces salivation, constricts lungs,

slows heartbeat, stimulates digestion, causes erections in males

(by vasodilation) and retention of urine.

• Generally, the activation of a particular target is determined by

the relative strength of parasympathetic and sympathetic

pathways.

Take Home Message

Neuromuscular Junction

• When an action potential reaches a neuromuscular synapse,

voltage-gated Ca channels open, and the inflow of Ca triggers

ACh release.

• ACh binds to nAChR on the motor endplate, Na+ moves into

the muscle cell (and less K+ moves out), leading to a local

depolarization of the motor end plate. This depolarization

spreads across the surface of the muscle fiber into transverse

tubules, eliciting the release of calcium and initiating muscle

contraction.

• Myasthenia gravis is an autoimmune neuromuscular disease

characterised by muscle weakness. It is caused by circulating

antibodies that block nAChR receptors at the neuromuscular

junction, and by a reduction in the number of nAChRs.

Take Home Message

Cholinergic System Agonists

Direct receptor agonists

Have affinity for nAChR or mAChR, mimic ACh

Act mostly on mAChRs

Exception: Nicotine

AChE inhibitors

Inhibit AChEsterase, increasing Ach

Triggers activation of both nAChR and mAChR

Reversible AChE inhibitors for medical use; irreversible AChE

inhibitors are used as nerve gases and insecticides.

The extremely short half-life of ACh makes it

therapeutically useless.

Carbachol in not hydrolyzed by AChE. Activates

mAChR and nAChR. Does not pass blood brain

barrier.

Bethanecol is not hydrolyzed by AChE. Does not

activate nAChR. Does not pass blood brain

barrier.No cardiovascular effects.Treatment of

urinary retention.

Pilocarpine chief alkaloid in pilocarpus jabordani.

Treatment of glaucoma.

Bethanechol

Cholinergic receptor agonists

Systemic injection of Pilocarpine can

compromise the blood-brain barrier

allowing the pilocarpine to gain

access to the brain. This can lead to

chronic epilepsy.

• Exaggeration of all symptoms of muscarinic agonism

• Significance: High consumption of wild mushrooms (culinary)

• 30-60 minutes, salivation, lacrimation, excessive sweating, nausea, vomiting, diarrhea, bronchospasm, headache, visual disturbances, abdominal colic, bradychardia, hypotension, shock

• ALL SYMPTOMS REVERTED BY ATROPINE (mAChR receptor blocker)

Muscarine chief alkaloid in amanita muscaria.

No therapeutic application.

Cholinergic receptor agonists

• ACh is therapeutically useless due to its extremely efficient

metabolism. Direct ACh receptor agonists mimic ACh at both

nAChR and/or mAChR, but are not rapidly metabolized.

• Carbachol and Bethanecol are choline esters that are more

stable than ACh, and can be administered systemically to

support parasympathetic activation (parasympathomimetics).

• Pilocarpine is a plant alkaloid, used to treat glaucoma by

topical administration to the eye. It constricts iris muscle, and

also ciliary muscle, increasing the flow of acqueous humor from

the eye and reducing intraocular pressure.

• To counteract ACh agonist action, one may deliver an ACh

receptor antagonist such as Atropine.

• All therapeutic AChE inhibitors are reversible; irreversible

AChE inihibitors are used as insecticides and nerve gases.

Take Home Message

Reversible AChE Inhibitors

Physostigmine derived from Calabar bean

(West Africa). Passes blood brain barrier.

Used to treat Alzheimer disease. Can be

used to treat CNS effects of anticholinergic

drug overdoses.

Neostigmine synthesized first 1931.

Does not cross blood brain barrier.

Used to treat the autoimmune

neuromuscular disorder myasthenia gravis.

Edrophonium has short half life (about 5

minutes). Is used to diagnose myasthenia

gravis.

„Horny goat weed“ acts as AChE inhibitor

(active ingredient unknown).

Stimulation of vascular M3 receptors Nitric

oxide release vasodilation. Similar to

Viagra®.

• AChE inhibitors prolong the action of ACh by slowing down it‘s

metabolism, effectively increasing the extracellular

concentration of ACh.

• Physostigmine is a naturally derived AChE inhibitor that is

used to treat CNS disorders such as Alzheimer‘s disease or to

counteract CNS effects of ACh receptor blockers.

• Neostigmine and Edrophonium are synthetic AChE inhibitors,

used rescpectively to treat and diagnose Myasthenia gravis.

• The vasodilation action of AChE causes penile erections by

triggering Nitric Oxide release, and is used to counteract penile

dysfunction.

Take Home Message

Cholinergic System Antagonists

Muscarinic receptor blockers

Competitive antagonists of the mAChR receptor.

Widespread medical applications.

Nicotinic receptor blockers

Ganglion-specific blockers (no medical applications).

Neuromuscular blockers (muscle relaxants).

Atropine extracts from the Egyptian

henbane were used by Cleopatra in

the last century B.C. to dilate her

pupils, in the hope that she would

appear more alluring. In the

Renaissance, women used the juice

of the berries of Atropa belladonna

to enlarge the pupils of their eyes,

for cosmetic reasons

Atropine derived from atropa belladonna.

Passes blood brain barrier. Deadly in high dose.

Uses: prevents salivation during anesthesia,

treatment for bradycardia, eye examinations,

visual neuroscience experiments.

Muscarinic receptor blockers

Muscarinic receptor blockers

Dose dependence of Atropine action

Scopolamine is main alkaloid in datura

stramonium. Passes blood brain barrier.

Used to treat motion sickness.

Toxin Detector Theory of Seasickness

Vestibular system acts as a toxin detector

Brain has evolved to recognize any changes in expected patterns of vestibular,

visual and kinesthetic information as evidence of CNS malfunction

initiation of vomiting by cholinergic brain vomiting center stimulation as a defense

against possible ingested neurotoxin

Women are more likely to get motion sick than men.

Children are more likely to get motion sick. Peak age around 12.

Prior history of motion sickness makes it more likely to get motion sick.

Bos et al Susceptibility to seasickness. Ergonomics Jun 2007

Muscarinic receptor blockers

Butyl-Scopolamine is a semi-synthetic

derivative of scopolamine that does not pass

the blood brain barrier. Used to treat

menstrual pain.

Glycopyrrolate is a synthetic muscarinic

antagonist that does not pass the blood brain

barrier.

Muscarinic receptor blockers

• Muscarnic antagonist action can be summarized as

“DRY AS BONE, RED AS A BEET, MAD AS HATTER.”

Dry is a consequence of decreased sweating,

salivation and lacrimation - Red is a result of impaired

peripheral vasodilation, problem to dissipate heat

(hyperthermia) - Mad is a result of the CNS effects of

muscarinic inhibition which can lead to sedation,

amnesia (hypersensitivity), or hallucination

• Atropine and Scopolamine are brain-blood-barrier permeable,

naturally derived mAChR blockers (parasympatholytics), with a

wide range of applications including inhibition of salivation

during anesthesia, pupil dilation for eye exams and

neuroscience experiments, treatment of motion sickness.

• Butyl-Scopolamine and Glycopyrrolate are synthetic non brain-

blood-barrier permeable mAChR antagonists with wide

applications. They are used to control for peripheral effects in

CNS studies of BBB-permeable mAChR antagonists.

Take Home Message

Nicotinic receptor blockers

Curare is a plant-derived arrow poison in

South America. Active ingredient is d-

Tubocurarine, it blocks the neuromuscular

nicotinic receptor.

Death occurs by respiratory paralysis.

Was used clinically as muscle relaxant

during surgery. No longer used because

blood pressure drops through histamine

release.

Pancuronium and related compounds are

synthetic neuromuscular nictonic receptor

blockers causing little histamine release, and

are now used for surgical muscle relaxation.

Tubocurarine by itself has no action, it gives neither contraction nor depolarization.

Tubocurarine inhibits muscular contraction induced by stimulation of the motor nerve or by the application of

acetylcholine. It does this by reducing amplitude and duration of the end-plate potential.

The application of an acetylcholine excess or an anticholinesterase agent reduces the effects of tubocurarine.

Tubocurarine has no effect when applied away from the neuromuscular junction, on the nerve or the muscle.

Botulinum toxin (BOTOX):

is produced by anaerobic bacteria called

Clostridium botulinum.

Temporarily blocks the nerve impulses

traveling from the nerve to the muscle

thereby relaxing the muscle.

Botox interferes with vesicle exocitosis, blocking ACh release

• D-tubocurarine, a nAChR blocker specific to the

neuromuscular junction, is the active ingredient in the South

American arrow poison Curare, which kills animals by

respiratory muscle paralysis. Pancuronium and related

compounds are synthetic neuromuscular nAChR

antagonists used for muscle paralysis during surgical

procedures.

• Botox blocks ACh release from presynaptic terminals,

leading to local muscle relaxation. It does this by disrupting

the formation of SNARE complexes that are necessary for

the fusion of vesicles with the cell membrane.

Take Home Message

Botox injection on forehead impairs touch discrimination on hand

• Botox injection on forehead silences muscle acticity for

periods lasting several weeks or months. This leads to

plastic reorganization in somatosensory (touch) cortex, in a

form of maladaptive plasticity that is similar to phantom limb

related cortical reorganization. Subjects’ judgement for

touch temporal order sensitivity is impaired following Botox

administration.

Take Home Message