CS 2015

Functional Aspects of Excitation & Inhibition

Christian StrickerAssociate Professor for Systems Physiology

ANUMS/JCSMR - ANU

Christian.Stricker@anu.edu.au 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.

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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

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Some “Conceptual” Fathers

Charles Scott Sherrington (1852 - 1952)

John Carew (“Jack”) Eccles (1903 - 1997)

Bernhard Katz (1911 - 2003)

Bert Sakmann (*1942)

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Australian Connection

Jeffrey C. Watkins (1929-)David R. Curtis (1927-)

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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).

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Iono- vs Metabotropic Action

Bor

on &

Bou

lpae

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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

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Excitation

Focus on glutamate (aspartate)

Ionotropic and metabotropic receptors

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Glutamate Cycle

• PAG: phosphate-activated glutaminase on mitochondrial membrane.

• Very simple synthesis; no specific degrading enzymes.

• Receptor deactivation via diffusion and re-uptake.

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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

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Boron & Boulpaep 2003

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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.

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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

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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

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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).

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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

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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).

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Lujá

n et

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(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

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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).

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Inhibition

Focus on γ-amino-butyric acid (GABA)

Ionotropic & metabotropic GABA

(ionotropic GABA ≈ ionotropic glycine)

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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?).

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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.

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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

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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.

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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.

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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

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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.

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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.

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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.