CS 2015
Functional Aspects of Excitation & Inhibition
Christian StrickerAssociate Professor for Systems Physiology
ANUMS/JCSMR - ANU
[email protected] http://stricker.jcsmr.anu.edu.au/Excitation&Inhibition.pptx
THE AUSTRALIAN NATIONAL UNIVERSITY
CS 2015
CS 2015
Neurophysiology Lectures
• Functional aspects of excitation and inhibition
• Neurotransmitter systems
• Introduction to neuronal networks
• Synaptic plasticity and memory
• Ascending reticular activating system
• Alzheimer disease
• Tutorial on audiometry
CS 2015
Aims
At the end of this lecture students should be able to
• list a variety of neurotransmitters and receptors in the CNS;
• outline the notions of ionotropic and metabotropic receptors;
• discuss the major iono- and metabotropic receptors;
• identify the importance of receptor recycling and the role of
receptor associated proteins;
• recognise the vast molecular heterogeneity of GABAA
receptors; and
• name some glutamate & GABA ant- and agonists.
CS 2015
Contents• Transmitters and
– ANU
– Key concepts: Katz’ postulates
– iono- versus metabotropic receptors
• Glutamate and its receptors– NMDA, Kainate and AMPA receptors
– mGluRs
• GABA and its receptors– GABAA and GABAC receptors
– Pharmacological richness of GABAA receptors
– GABAB receptors
CS 2015
Some “Conceptual” Fathers
Charles Scott Sherrington (1852 - 1952)
John Carew (“Jack”) Eccles (1903 - 1997)
Bernhard Katz (1911 - 2003)
Bert Sakmann (*1942)
CS 2015
Australian Connection
Jeffrey C. Watkins (1929-)David R. Curtis (1927-)
CS 2015
5 Steps of Classical Transmission• Synthesis: presynaptic. Requires specific
enzymes.
• Storage: presynaptic; requires vesicular
transport proteins.
• Release: into synaptic cleft via exocytosis
or a constitutive pathway.
• Binding: concentration dependent; to
iono- or metabotropic receptors.
• Termination: dependent on transmitter
type and extracellular space (tortuosity).
CS 2015
Iono- vs Metabotropic Action
Bor
on &
Bou
lpae
p 20
03
CS 2015
Notion of Excitation & InhibitionExcitability increased if same input current
generates more APs and vice versa.– If the same current causes a larger voltage
change, excitability is increased and vice versa.• Intrinsic properties of ion channels and
membranes.
– Input current is excitatory if it results in an
increase in the rate of action potentials.• Always causes depolarisation
• Erev > Vth
– Input current is inhibitory if it results in a
decrease in the rate of action potentials.• Often causes hyperpolarisation
• Erev < Vthreshold
• Can be depolarising
CS 2015
Excitation
Focus on glutamate (aspartate)
Ionotropic and metabotropic receptors
CS 2015
Glutamate Cycle
• PAG: phosphate-activated glutaminase on mitochondrial membrane.
• Very simple synthesis; no specific degrading enzymes.
• Receptor deactivation via diffusion and re-uptake.
CS 2015
Ionotropic GluRs
• Each branch is pharmacologically distinct; easiest identified by agonists (name).
• RNA editing possible (flip/flop versions).
• Evolved differently to ACh, GABAA/C receptors:– Typically a heteromultimer between 4 subunits.
Bor
on &
Bou
lpae
p 20
03
Boron & Boulpaep 2003
CS 2015
NMDA Receptors• Transmitter: Glutamate• Structure: 4 subunits (NR1-2), 2
glutamate binding sites• Permeable to: Na+, K+, and Ca2+
• Agonists: NMDA• Antagonists: APV, MK801,
ketamine, etc.• Activation/deactivation: slow (~20
ms / >100 ms)• Single channel properties:
P0 ~ 0.05 / 0.3 (?); γ ~ 50
pS• Action: Coincidence detector,
important for synaptic plasticity,
memory and learning.
CS 2015
NMDA Receptor Properties• Additional features:
- voltage-dependent block by Mg2+
(> -40 mV).- Opening requires for
- subsynaptic receptors: glycine; [gly] in
CSF is sufficiently high.- extrasynaptic receptors: L-serine
• Target of very many modulators or
2nd messengers.- Most cell signalling pathways
modulate this receptor…
• Clinical pharmacology- Antagonist:
- Ketamine: anaesthesia (children)- Memantine: cognitive decline, AD- Stroke: experimental (excitotoxicity)- Phencyclidine: PCP/“angel dust”
- Agonist: experimental
Ascher & Nowak (1988), J. Physiol. 155:247-266
CS 2015
AMPA Receptors• Structure: 4 subunits (iGluR1-4),
2 binding sites occupied with glutamate.• Permeable to Na+, K+; some permeable
for Ca2+ (GluR2-deficient)• Often co-localised with NMDA receptors.• Single channel properties:
P0 ~ 0.8; γ ~ 10 pS
• Activation/deactivation: fast (≤100 µs /
1-10 ms)• Agonists: AMPA• Antagonists: NBQX, CNQX, DNQX,
GYKI53655• Action: Fast CNS signaling; workhorse –
most transmission in CNS via AMPAR.- No clinically relevant agonists/antagonists
Finkel & Redman (1983), J. Physiol. 342:615-632
CS 2015
Molecular Biology of AMPA-R• At synapses onto excitatory cells,
heteromultimers contain GluR2.
• At synapses onto inhibitory cells,
heteromultimers lack GluR2:– inward rectification (polyamine block) and
– significant Ca2+ permeability.
– (Property used to test for AMPA-R cycling).
• AMPA receptor subunits have different roles
at synapse.– GluR1 inserted during synapse formation in an
activity-dependent way: CaMKII and NMDA-R
dependent (source from dendrite).
– GluR2 and it’s tail responsible for constitutive
recycling.
• GluR2 containing are continually recycled:
τ = 40 min.– Changes in recycling rates vary in an activity-
dependent way (synaptic plasticity).
CS 2015
TARPs and AMPA Receptors
• Transmembrane AMPAR regulatory
proteins (TARPs):– “work like” ancillary subunits
(γ subunits on Ca2+ channels, etc.);
– modulate AMPAR activity by direct
interaction with the channel; and
– regulate trafficking of AMPARs.• Can bring extra-synaptic receptors to sub-
synapse.
• TARP phosphorylation stabilises AMPA
receptors in PSD-95.
• Stabilise receptors in postsynaptic density to a
raft size of about 100.
• Likely important in neurodegeneration
and epilepsy.Tomita (2010), Physiol. 25:41-49
CS 2015
Kainate Receptors• Structure: 4 subunits, 2 binding
sites for glutamate• Permeable to: Na+, K+; some
permeable for Ca2+
• Single channel properties:
P0 ~ ??; γ ~ 1.8 pS (?)
• Activation/deactivation: fast
(~100 µs / 1-10 ms)• Agonists: Kainic acid• Antagonists: LY 382884 (GluR5);
not many.• Action:
- control of presynaptic release /
inhibition: anaesthesia;- kainic acid causes epilepsy (ET).
CS 2015
Lujá
n et
al.
(199
7), J
. Che
m. N
euro
anat
. 13:
219-
241
Structure & Function of mGluR1-8
• Location: perisynaptic• Couple to different Gα:– Gi/o (II/III): inhibits adenylyl cyclase
• modulates K+ and Ca2+ channels• inhibitory action on release.
– Gq (I): activates PLC
• can be excitatory in action.
• Role- Group II/III: Autoreceptors (transmitter
release↓).- Group I: postsynaptic (pre?)
• Clinical pharmacology- Experimental (tumours, hypoxic insults,
Parkinson, fragile X syndrome, etc.)
Boron & Boulpaep 2003
CS 2015
Glutamate and Disease
• Jekyll-and-Hyde molecule: essential for normal trans-
mission but with the potential to cause neuronal death.– Ca2+-permeable GluR: excitotoxicity, neurodegeneration
– Involved in epilepsy: overexcitation
– Neurodegeneration: olivopontocerebellar degeneration (glu
dehydrogenase).
• Sources of glutamate– Ingestion: MSG (Chinese restaurant syndrome), plant alkaloids
(chickling pea in India), etc.
– Excitotoxicity: increased glutamate release (positive feedback).
CS 2015
Inhibition
Focus on γ-amino-butyric acid (GABA)
Ionotropic & metabotropic GABA
(ionotropic GABA ≈ ionotropic glycine)
CS 2015
GABA Cycle
• GAD (glutamate decarboxylase)
• Very simple synthesis (depends
on glutamate synthesis); no
specific degrading enzymes.
• Deactivation via diffusion,
uptake into glia and re-uptake
as glutamine.
• Specific transporter at nerve
terminal.
• Also tonically released
(transporter?).
CS 2015
Structure of GABAA Receptor• Structure: 5 subunits, 2 binding sites for
GABA on α subunit- Composed of α (6 genes), β (3) and γ (3) in a
2:2:1 relationship.- γ can be replaced by δ, ε, θ, π and ρ- Large molecular heterogeneity with slightly
different pharmacology.- Mostly, however, only a few dozens are
expressed.
- Most common form is 2α1 2β2 γ2
- Subunit expression varies in different brain
regions (specificity of action …).
• Permeable to Cl-, HCO3-
• Activation/deactivation:
fast (~250 µs / 5-20 ms)• Agonists: muscimol• Antagonists: picrotoxin, bicuculline,
gabazine (potent convulsants)• Action: Fast inhibition in CNS.
CS 2015
GABAA Receptor Modulation• Clinical pharmacology
- Sleep: barbiturates,
benzodiazepines (BZ) – positive
allosteric modulators.- Anaesthesia: volatile interact- Modulated by steroids (θ).
• Increase in single channel open
time and conductance.• BZ (diazepam, etc.) act
similarly; bind to different site:- Endogenous BZ likely diazepam
binding inhibitor (DBI) or peptide
fragments of it (2013).
• Some benzodiazepines can be
used to identify specific α2
subunits (flunitrazepam).
Boron & Boulpaep 2003
CS 2015
GABAAR and Disease• Angelman syndrome.
– Loss of β3 - GABA subunit as part of a partial deletion
of chromosome 15 (classical case of imprinting…).
• Alcohol tolerance– Alcohol in high doses (> 10 mM) increases GABAA
currents via a direct interaction (falling asleep…).
– In mice, a single point mutation in α6 subunit
(cerebellum) renders a benzodiazepine insensitive
into a sensitive channel: increased motor impairment
after Et-OH.
CS 2015
Sadia et al. (2003), Neuron 39: 9-12
Metabotropic GABAB Receptors• Structure: Heterodimer between
GABAB1a/GABAB1b and GABAB2
• Targeting to either dendritic or
axonal compartment• Permeable to: nothing• Activation/deactivation: slow (~50
ms / 100 - 250 ms)
• Coupling: via Gi/oβ/γ to GIRK
channels• Action: Inhibition in CNS
(postsynaptic); presynaptic
inhibition (synaptic triades).• Agonists: Baclofen• Antagonists: saclophen,
phaclofen, CGP 35348, etc.
CS 2015
Clinical Role of GABAB Receptors
• Pre- and postsynaptic inhibition
• Role in absence seizures (thalamic frequency)
• In mice, central role in temporal lobe epilepsy
• Clinical pharmacology– Agonist: used to treat spinal spasticity, dystonia,
some types of neuropathic pain.
also: gastrooesophageal reflux
– Antagonists: experimental• cognitive decline, drug addiction, anxiety
• visceral pain
CS 2015
Take-Home Messages• Ionotropic receptors act fast; metabotropic receptors allow
signal amplification but are much slower.
• Glutamate is the major excitatory transmitter in the brain.
• Some GluR are involved in excitotoxicity, synaptic plasticity,
memory and learning.
• GluRs are continually recycled, the rate depends on subunit
composition.
• GABA is the major inhibitory transmitter.
• GABAA receptors show a large molecular and
pharmacological heterogeneity.
• GABAB receptors provide pre- and postsynaptic inhibition at
the spinal and cortical level.
CS 2015
MCQ
Which of the following statements best describes the sedative
action of benzodiazepines?
A. Bind to GABAA and GABAB receptors.
B. Increase the rate of desensitization at GABAA
receptors.
C. Modulates GABAA mean open time and
conductance.
D. Activate membrane insertion of GABAA receptors.
E. Slows down GABAA receptor internalisation.
CS 2015
That’s it folks…
CS 2015
MCQ
Which of the following statements best describes the sedative
action of benzodiazepines?
A. Bind to GABAA and GABAB receptors.
B. Increase the rate of desensitization at GABAA
receptors.
C. Modulates GABAA mean open time and
conductance.
D. Activate membrane insertion of GABAA receptors.
E. Slows down GABAA receptor internalisation.