THE AUSTRALIAN NATIONAL UNIVERSITY

1. THE AUSTRALIAN NATIONAL UNIVERSITY Functional Aspects of Excitation & InhibitionChristian StrickerAssociate Professor for Systems PhysiologyANUMS/JCSMR - ANUChristian.Stricker@anu.edu.auhttp://stricker.jcsmr.anu.edu.au/Excitation&Inhibition.pptx

3. Neurophysiology Lectures • Functional aspects of excitation and inhibition • Neurotransmitter systems • Introduction to neuronal networks • Synaptic plasticity and memory • Alzheimer disease • Tutorial on audiometry

4. 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.

5. 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

6. Some “Conceptual” Fathers Bernhard Katz (1911 - 2003) Charles Scott Sherrington (1852 - 1952) Bert Sakmann (*1942) John Carew (“Jack”) Eccles (1903 - 1997)

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 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).

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 > 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

11. Excitation Focus on glutamate 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/Creceptors: • Typically a heteromultimerbetween 4 subunits.

14. 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.

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 modula-tors or 2nd messengers. • Most cell signalling pathways modulate this receptor… • Clinical pharmacology • Antagonist: • Ketamine: anaesthesia (children) • Memantine: cognitive decline, AD • Stroke: experimental (excitotoxicity) • Agonist: experimental Ascher & Nowak (1988), J. Physiol. 155:247-266

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 / 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

17. 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).

18. TARPs and AMPA Receptors • TransmembraneAMPAR 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 • Antagonists: LY 382884 (GluR5); not many. • 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. 13:219-241 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, bicucul-line, 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. • Similar for BZ (diazepam, etc.); bind to different site: • Endogenous BZ likleydiaze-pam binding inhibitor (DBI) or peptide fragments of it (2013). • Some benzodiazepines can be used to identify specificα2subunits (flunitrazepam). Boron & Boulpaep 2003

26. 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.

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 / 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. 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.