ACETYLCHOLINE

1
ACETYLCHOLINE

• L.Sivilotti
• Pharmacology Dept.

2

• Agents affecting synthesis or release
• Hemicholinium
• Vesamicol
• Botulinum

Soreq Seidman, Nature Neuroscience, 2001
3
Cholinergic pathways (central)
hippocampus
1
dltn
MS
tpp
NBM
caudal group
DB
3
magnocellular forebrain nuclei
2
interneurones in striatum, accumbens and cortex
4 - lower motoneurones (inc. recurrent
collaterals and autonomic preganglionic)
DB diag. band of Broca MS medial septum NBM, n.
basalis magnocellularis tpp, pedunculopontine
tegmental n. dltn, laterodorsal tegmental n.
IP, interpeduncular nucleus DR, dorsal raphé
Cooper, Bloom Roth- ChAT staining
4
Cholinergic pathways (peripheral)
Spinal cord

• ganglion receptors blocked by hexamethonium
• muscle receptors by decamethonium

5
Muscle nicotinic ACh receptors

• four different subunits

• two copies of a

membrane
Adapted from Nigel Unwin, 1998
6
Topology the nicotinic superfamily
nACh a
nACh non-a
extracellular
intracellular
7
Neuronal nAChR subunits are very numerous
NINE alpha subunits!
THREE beta subunits!
huge potential for diversity in ganglia and
in CNS
8
Neuronal nicotinic ACh receptors assembly rules
Heteromeric
pair
triplet
or b3
9
Torpedo californica, MUSCLE-TYPE RECEPTOR
banded krait, Bungarus multicinctus BUNGAROTOXIN
tobacco, Nicotiana tabacum NICOTINE
10
Nicotinic agents pharmacology
Antagonists Muscle Neuronal aBungarotoxin
only a7 kbungarotoxin - /- Tubocurarine
receptor block channel block Gallamine - Tr
imetaphan - Mecamylamine - Hexamethoniu
m mM mM (trapped block) Dihydroberythroidine
a4b2 Agonists ACh, nicotine, carbachol, DMPP,
cytisine, epibatidine.
11
Neuronal nicotinic receptors - molecular view

• all calcium permeable to some extent, different
  desensitisation time courses and sensitivity to
  bungarotoxins
• ganglia, adrenal medulla
• synaptic a3b4a5(b2) k-Bgt sensitive
• extrasynaptic a7 a-Bgt sensitive
• cochlea a9 or a9/a10 a-Bgt sensitive, nicotine
  insensitive
• CNS pre- and postsynaptic
• a4b2 most abundant
• a7 a-Bgt sensitive, fast desensitisation
• a3b4 at a few locations

12
CNS postsynaptic effects
In the hippocampus, interneurones respond to
nicotinic agonists...
principal neurones dont
fast, sensitive to a-Bgt (a7)
slower, sensitive to k-Bgt or dHBE (a4b2?)
Hippocampus, s.oriens interneurones 1 s 100 pA
(top) 20 pA McQuiston Madison,1999
13
CNS postsynaptic effects
Nicotinic receptors are inward rectifiers Their
activity decreases as the neurone depolarises.
Hippocampus, s.oriens interneurones 100 pA,
1s McQuiston Madison,1999
Discordance detectors?
14
CNS nicotinic synapses chapter 1) 1954!
Nicotinic excitation of Renshaw cells by
recurrent motoneurone collaterals in the spinal
cord (ventral horn) (Eccles et al, 1954 blocked
by dHBE, not MLA, DouradoSargent 2002)
motoneurones
Nicotinic synapse
Renshaw cell
stimulation
15
CNS nicotinic synapses chapter 2) 1998!
Hippocampus, s.oriens interneurones Frazier et
al, 1998
a7 synaptic currents in pyramidal cells (2 of
EPSC? culture)
Hippocampus, CA1, culture or acute slice,
pyramidal cells Hefft et al., 1999
16
CNS presynaptic effects
nAChR agonists increase intracellular Ca2 in
presynaptic terminals
hence the frequency of spontaneous transmitter
release (GABA, Glu, DA, NA, ACh)...
17
CNS more presynaptic effects
Nicotinic agonists can also increase the
amplitude of evoked synaptic currents.
Both a7 and a4b2
Is it physiologically relevant?
18
A physiological presynaptic effect
DA release in striatum depends on tonic
cholinergic transmission via nicotinic receptors
(b2, dHBE -sensitive)
evoked
spontaneous
Zhou, Liang Dani 2001
19
pharmacological relevance...
. . .to strengthen the memory the smoke is
excellent taken by the nostrils, for it is
properly belonging to the brain, and it is easily
conveyed into the cels of it and it cleanseth
that from all the filth (for the brain is the
Metropolis of flegme, as Hippocrates teacheth us
. . .) Dr Giles Everard, 1659
20
Transgenic mice to establish physiology

• Nicotinic
• central
• a4 loss of nicotinic analgesia thalamic
  responses
• b2 loss of nicotinic reward analgesia
• a4b2
• a7 loss of nicotinic hippocampal fast currents
• subtle autonomic deficit

• peripheral
• a3 severe autonomic loss (ganglia)- lethal
• b4 mild autonomic deficit
• Combined b2 and b4 severe autonomic loss lethal
• a5 subtle parasympathetic deficit
• Ganglion receptor is a3b2b4 ? Pitfalls
• a9 loss of efferent cochlear responses

21

• Effect of activation of central cholinergic
  pathways
• Important for arousal/attention/sleep cerebral
  cortex
• Short-term memory storage/retrieval
  hippocampus, cortex
• Motor control striatum
• Why?
• Clues central effects of atropine, AChE
  inhibitors, nicotine

• Atropine
• Loss of attention/depression or excitation
• Transient loss of long-term memory
• Cortical/subcortical dissociation (large slow
  waves in EEG deep sleep pattern)
• Anti-emetic (chemoceptor trigger zone)
• Motor control (reduce Parkinsonian tremor
  extrapyramidal side effects of antipsychotics)

22
Muscarinic receptors G-protein linked, 7 TM
domains
membrane
g
b
receptor
a
Wess,1998
23
Fly agaric, Amanita muscaria MYCOATROPINE, MUSCAR
INE
Courtesy of D. Colquhoun
http//www.bryologie.uni-bonn.de/
Deadly nightshade, Atropa belladonna ATROPINE
http//www.botanikus.de/Gift/Toll.html
24
Muscarinic receptors downstream effects
odd-numbered M1, M3, M5 Gq/11 -PLC
IP3/DAG -calcium/PKC -other
mechanisms
even-numbered M2, M4 Gi or Go -inhibit
adenyl cyclase -direct Gbg activation
of potassium channels
Excitation closure of K channels ganglia
IK(M) (M1/M3) hippocampus cortex IK(Ca)
Postsynaptic opening of K channels
heart Kir Inhibition (M2) thalamus
Ch-ergic neurones
Presynaptic opening of K channels
Inhibition closure of Ca channels (M2)
Goodman Gilman, 2001 Caulfield Birdsall, 1998
25
Muscarinic activation can increase membrane
excitability
Firing of a bullfrog sympathetic neurone, Marrion
1997
1)by inhibiting the M-current
a potassium current partially open at rest,
slowly opens further with depolarisation
opposes repetitive firing
26
Muscarinic activation can increase membrane
excitability
AHP
2)by inhibiting spike afterhyperpolarization (AHP
SK channel, hippocampus, calcium entry
unaffected)
27
Muscarinic activation can increase membrane
excitability
3)by inhibiting leak current (TASK channels,
cerebellar granule cells)
M receptors inhibit the native K leak current,
IK(SO) TASK1/TASK3 plus possibly other K2P, in
cerebellar granule neurons A, inhibition of
IK(SO) in rat cerebellar granule neurons
following activation of muscarinic receptors
(from Millar et al. 2000). B, muscarinic
inhibition of IK(SO) enhances the excitability of
cerebellar granule neurons. In current clamp
recordings the depolarizing current injections
give a larger depolarization in the presence of
muscarine and initiate action potential firing
(from Watkins Mathie, 1996).
28
Muscarinic activation can decrease membrane
excitability
by activating a K conductance (KIR-type think
vagus on heart M2 CA1 interneurones, hippocampus)
McQuiston Madison 1999
In cortex M1-calcium release from intracellular
stores and activation of SK channel
5 s
29
Muscarinic receptors location effect of
knockouts
M1 cortex, hippocampus loss of slow epsc
(ganglia, NOT hippoc.) glands loss of
muscarine seizures sympathetic ganglia
M2 basal forebrain less oxotremorine
hypothermia and tremor smooth muscle less M
autoinhib. of ACh release heart loss of
effect of vagus on the heart
M3 cortex, hippocampus peripheral effects,
eye, glands, food intake glands
M4 basal ganglia increased locomotor
activity less M autoinhib. of ACh release
M5 substantia nigra less M increase of DA
release cerebral arteries loss of M dilation
of cerebral arteries
Caulfield Birdsall, 1998 Bymaster et al, 2003
30
Cholinergic transmission and disease

• NMJ ganglia
• autoimmune
• myasthenia gravis (prevalence 1 in 7500)
• Lambert-Eaton syndrome (incidence 1 in 100 000
  per year)
• autoimmune autonomic neuropathy
• congenital myasthenias
• botulism (presynaptic therapeutic uses of BoTox)

• CNS
• schizophrenia?
• Parkinsons disease
• incidence lower in smokers CO or nicotine?
• nicotine has a positive effect in patients more
  DA release? enhance L-dopa?
• M effects benztropine
• Tourette (hyperkinesia, tics motor and verbal
  too much DA)
• nicotine patches reduce tics (contradiction with
  effects in Parkinson desensitisation?)
• Dementia
• Smoking
• Pain
• Autosomal dominant nocturnal frontal lobe
  epilepsy (nicotinic receptor defect)
• Benign familial neonatal convulsions (defect in
  M-current channel)

31
Alzheimers disease The cholinergic hypothesis
selective loss of cholinergic neurones (contradic
ted by findings early in disease) Smoking and
Alzheimers risk b-amyloid fragments block
nicotinic receptors
Useful drugs ACh precursors NO Agonists (M
or N) NO
Cholinesterase inhibitors YES tacrine
(obsolete, hepatotoxic) donepezil,
rivastigmine, galantamine AChE or BuChE? N
receptor modulation (galantamine) Modest
benefit in a minority of patients (at best
25) Delay full-time care? Peripheral side
effects (slow titration of dose)
32
Pain nicotinic agonists as analgesics
Nicotine itself, or the more potent epibatidine
are limited by side effects and acute toxicity
(NMJ, autonomic receptors) ABT-594 more
selective for a4b2 receptors (currently phase II
clinical trial) Mechanism Activation of
serotoninergic descending pathways? Nicotinic
receptors on C-fibers (spinal cord and
periphery)?
E. tricolor
33
Smoking
27 of population in 2001 (40-50 in
1970s) Fast delivery to brain redistribution
self-titration Activation then desensitisation
of CNS receptors (a4b2) DA release in limbic
areas (reward areas) chronic increase in
receptor numbers Muscle relaxant, anxiolytic,
stimulant (analgesia? enhancement of
sensory/motor tasks) Peripheral receptors
(tolerance) Cancer (x10), coronary heart
disease, peripheral vascular disease, emphysema,
toxic to foetus Reduces risk of Parkinsons
disease (up to 50) Beneficial in ulcerative
colitis Quitting in US 50 of smokers tried to
stop last year 5-25 success at 1 year nicotine
replacement, bupropion
www.giftpflanzen.com
34
Autosomal dominant frontal lobe nocturnal
epilepsy (ADFLNE) Focal, during sleep,
variable severity- misdiagnosed as
nightmares mutations in a4 or b2 in a small
proportion of cases Incomplete penetrance
(75) loss of function faster desensitisation,
loss of calcium permeability (Kuryatov et al.,
1997 Bertrand et al., 1998) gain of function
use-dependent potentiation (Figl et al., 1998) or
greater sensitivity to ACh (De Fusco et al.,
2001 Phillips et al. 2001) How do these changes
result in epilepsy? Presynaptic a4b2 nicotinic
receptors? Neither a4 nor b2 knockout mice have
epilepsy
35
Is nicotinic epilepsy (ADNFLE) due to a loss
of function or gain of function?
Mutant receptors are more sensitive to ACh
(Phillips et al., 2001)
gain of function?
36
loss of function?
37
benign familial neonatal convulsions
(BFNC) autosomal dominant, 80-90 penetrance
neonatal convulsions, remission by 3
months normal development, increased risk of
later epilepsy (about 15 risk) mutations in
voltage-dependent potassium channel subunits
(KCNQ2 or KCNQ3) causing loss of function, mostly
reduced expression a reduction by 25 in
M-current function is sufficient to give a
phenotype (haploinsufficiency, hence dominant
trait) why in neonate? (mutations in the
related KCNQ1 subunit cause the long QT syndrome).
38
McQuiston Madison 1999
39
(No Transcript)
40
http//www.nigms.nih.gov/news/findings/sept02/snai
ls.html
41
ganglia also have a-Bgt sensitive receptors
(a7)...
42
An unusual synapse the efferent cholinergic
synapse on outer hair cells in the cochlea
ACh produces postsynaptic Ca entry (), which
activates calcium-dependent K channels, which
produce an outward current
nACh receptor here is a9 it is insensitive to
nicotine, blocked by atropine and strychnine!!
43
The a4 ADFLNE mutations are in M2, the
pore-lining region of the nicotinic receptor
out
a
a
K
E
E
outer ring
K
K
S
S? F
S? F
S
S
T
T
T
central ring
T
M2
T
E
E
intermediate ring
E
E
E
D
E
E
cytoplasmic ring
D
D
in
neg. charges
44
(No Transcript)
45
M-channels oppose repetitive firing
Cooper Jan, 2003
46
Congenital myasthenia can be due to lack of nAChR
loss of function
or receptor defects (here slow channel syndrome)
gain of function??
47
Autoimmune reduction in nACh receptors results
in muscle weakness in myasthenia gravis
The edrophonium test (Cogan)
48
The Xenopus oocyte
49
TM2 lines the channel pore and determines open
channel properties (ion permeability,
conductance, binding of channel blockers)
50
The cholinergic synapse
51
Evolutionary tree for nicotinic receptor subunits
millions of years
Comparison of amino acid sequences and estimated
times of divergence of lineages (adapted from Le
Novere Changeux, 1995)
52
what do we get from cloning?
53
McQuiston Madison 1999