1. THE AUSTRALIAN NATIONAL UNIVERSITY
Functional Aspects of Excitation & Inhibition Christian Stricker Associate Professor for Systems Physiology ANUMS/JCSMR - ANU

3. 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

4. At the end of this lecture students should be able to
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 antagonists and agonists.

5. Contents Transmitters and Glutamate and its receptors
ANU
Key concepts: Katz’ postulates
iono- versus metabotropic receptors
Glutamate and its receptors
NMDA, AMPA and kainate receptors
mGluRs
GABA and its receptors
GABAA and GABAC receptors
Pharmacological richness of GABAA receptors
GABAB receptors

6. Some “Conceptual” Fathers
Bernhard Katz ( )
Charles Scott Sherrington ( )
Bert Sakmann (*1942)
John Carew (“Jack”) Eccles ( )

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

8. 5 Steps of Classical Transmission
Synthesis: presynaptic. Requires specific enzymes.
Storage: presynaptic; requires vesicular transporter 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).

9. Iono- vs Metabotropic Action
Boron & Boulpaep 2003

10. Notion of Excitation & Inhibition
Excitability 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 > Vthreshold
Input current is inhibitory if it results in a decrease in the rate of action potentials.
Often causes hyperpolarisation
Erev < Vthreshold
Can be depolarising

11. Focus on glutamate (aspartate) Ionotropic and metabotropic receptors
Excitation
Focus on glutamate (aspartate)
Ionotropic and metabotropic receptors

12. Glutamate Cycle
PAG: phosphate-activated glutaminase on mitochondrial membrane.
Very simple synthesis; no specific degrading enzymes.
Receptor deactivation via diffusion and re-uptake.

13. Ionotropic GluRs
Boron & Boulpaep 2003
Boron & Boulpaep 2003
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.

14. NMDA Receptors Transmitter: Glutamate
Structure: 4 subunits (NR1-2), 2 glutamate binding sites
Permeable to: Na+, K+, and Ca2+
Agonists: NMDA
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.

15. 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:

16. 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 / ms)
Agonists: AMPA
Action: Fast CNS signaling; workhorse – most transmission in CNS via AMPAR.
No clinically relevant agonists/antagonists
Finkel & Redman (1983), J. Physiol. 342:

17. Molecular Biology of AMPA-R
At most synapses onto excitatory cells, heteromultimers contain GluR2.
At synapses onto most 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).

18. 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

19. 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
Action:
control of presynaptic release / inhibition: anaesthesia;
kainic acid causes epilepsy (ET).

20. 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.)
Luján et al. (1997), J. Chem. Neuroanat. 13:
Boron & Boulpaep 2003

21. 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).

22. Inhibition Focus on γ-amino-butyric acid (GABA)
Ionotropic & metabotropic GABA
(ionotropic GABA ≈ ionotropic glycine)

23. 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?).

24. 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.

25. 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

26. GABAAR and Disease Angelman syndrome. Alcohol tolerance
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.

27. 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 / 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.
Sadia et al. (2003), Neuron 39: 9-12

28. 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

29. 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.

30. MCQ
Which of the following statements best describes the sedative action of benzodiazepines?
Bind to GABAA and GABAB receptors.
Increase the rate of desensitization at GABAA receptors.
Modulates GABAA mean open time and conductance.
Activate membrane insertion of GABAA receptors.
Slows down GABAA receptor internalisation.

31. That’s it folks…

32. MCQ
Which of the following statements best describes the sedative action of benzodiazepines?
Bind to GABAA and GABAB receptors.
Increase the rate of desensitization at GABAA receptors.
Modulates GABAA mean open time and conductance.
Activate membrane insertion of GABAA receptors.
Slows down GABAA receptor internalisation.