Session Outline

1. Presentation (~60mins) - Very Brief Introduction to Fundamental concept in neuronal signaling (Membrane potential and Action Potential) - Part 1 (Electrophysiological properties of neurons and Signal processing) - Part 2 (Intrinsic plasticity in learning, aging and CNS disorder) Scenario facts exploration (~40mins) Session Outline

2. Membrane Potential Refer to separation of opposite charges across the cell membrane (unequal distribution of charges establish by active protein pumps present on membrane) Key Ions : Na+ , Cl-, Ca2+ (concentrated outside) K+, anionic proteins (concentrated inside)

3. Membrane Potential All cells possess membrane potential, however neurons and few other cell types could actively manipulate changes in their membrane potential for signaling purpose.

4. Each specify ion possess an equilibrium potential that is determined by their distribution. In general, When Na+ or Ca2+ channel opens, Na+ /Ca2+ rush into the cell producing depolarisation. When K+ channel opens, K+ rush out of the cell producing hyperpolarisation

5. *Changes in membrane potential is brought about by changing the cell permeability to selective ions. Anatomy of an Action Potential

6. *Changes in membrane potential is brought about by changing the cell permeability to selective ions. (Brought about by Neurotransmitter binding resulting in opening of specific ligand gated channels). Iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii ssssssssssssssss

7. Part 1 (Electrophysiological properties of neurons and Signal processing)

8. Neurons exhibit widely varying electrophysiological properties. • Differential patterns of electrical activity are due to the presence of distinct mixture and distribution of different types of voltage gated ion channels with varying kinetics and regulatory properties. • Firing pattern are also affected by input received by neurons (Duration, magnitude and phase of input).

9. Voltage gated ion channel superfamily • Important characteristics/parameter of a voltage gated channel: • Kinetics (mode of activation/inactivation, duration of activation…) • Voltage sensitivity/threshold for activation • Selectivity (Na+,K+,Ca2+..) • Regulatory properties • Types of agonist and antagonist the channel is sensitive to

11. Current trace obtained when membrane depolarised from -100 to -10 mV Voltage dependence and kinetics of different ionic currents mediated by different voltage gated ion channel.

12. Iamm l akmmmbhak General distribution of voltage-gated channels in a typical hippocampus CA1 neuron. Note that different neuronal compartments express characteristic combinations of ion channel subunits. Uikmm,l,kkkm,m Akkkk Kl l

13. Dendritic A-type Current • Key Properties: • Voltage dependent K+ current mediated by Kv4.2 subunits • Activated by depolarisation (activates at subthreshold voltage) • Rapid inactivation at maintained depolarisation • Functions: • Activate by EPSP and increase EPSP attenuation • Attenuates back-propagating action potential (More on this later) • Important regulator of dendritic excitibility (filter off many weak signal)

14. Dendritic H Current (Ih) • Key Properties: • Voltage dependent non-specific monovalent cationic current (both K+ and Na+) • mediated by HCN subunits. • Activated by hyperpolarisation (tonically active at resting potential - *effects mainly • depolarisation) • Range of inactivation kinetics when depolarised (depending on types of subunits) • Ligand gated by cyclic nucleotide • Functions: • Increase EPSP attenuation and reduced summation • Influence resting membrane potential • Pacemaker activity (affecting the rate of neuronal activity oscillation)

15. Iikmmlolmmllma Oppllauuh Opplla ppooka Iikmmlolmmllma Oppllauuh Opplla ppooka Rhythmic pattern during sleep (Thalamic spindles) Iikmmlolmmllma Oppllauuh Opplla ppooka hhb The thalamic neuron spontaneously generates rhythmic bursts of action potentials due to the interaction of the Ca2+ current IT and the inward “pacemaker” current Ih. Depolarization of the neuron changes the firing mode from rhythmic burst firing to tonic action potential generation in which spikes are generated one at a time. Removal of this depolarization during sleep reinstates rhythmic burst firing.

16. Processing and regulation of electrical signal at various stages along a neuron Synaptic Input Synaptic weight can undergo bidirectional plasticity in the form of LTP and LTD modulated by AMPA, NMDA 2) Propagation of Synaptic Input to Cell Body Temporal and spatial summation of EPSP and IPSP generated by synaptic input can be modulated by presence of voltage gated channel at dendrites e.g attenuation of EPSP by e.g. A and H Current Apical Dendrites 6) AP Backpropagation In addition, AP can actively propagated back into dendritic region. Magnitude and travel distance can be subjected to modulation by A current and high threshold activated Ca2+ channel (Ca2+ L current) 3) Initiation of Action Potential Once the signal reaches Axon hillocks, initiation of AP can be affected by factors such as AP threshold level, resting membrane potential… Density of Na+ Channel at axon hillocks determine its excitability. Axon 4) Action Potential Properties Properties of AP like duration and amplitude can be affected by variety of voltage gated channel with different kinetics and regulation. E.g. Persistent Na+ current, M current…. 5) Firing Properties Profile of AP in turn determine the overall firing pattern of neurons. E.g. bust firing due to excess charges delivered by persistent Na+ current. Firing Properties can show frequency adaptation due to calcium sensitive K+ Channel Pyramidal neurons

18. Opplakks Aaasd Aaddfdf Addff deffgght Back-Propagating Action Potential • Even though dendrites are not as excitable as axons, they • contain voltage-gated channels that can support action • potential propagation. • Properties and mechanism governing dendritic spike is • fundamentally different from the spikes propagated along axon. • bp-AP primarily supported by relatively high threshold Ca2+ • ion channels (L-type Ca2+) as well as Na+ channels. Opplakks Aaasd Aaddfdf Addff deffgght Opplakks Aaasd Aaddfdf Addff deffgght • Regulation • Degree of attenuation depends : • Density and types of voltage channels. E.g. Kv4.2 (A type current) • and HCN attenuates bp-AP, Na+ and Ca2+ channel supports it. • Amount of Branching (Surface area for current leakage). • Proposed function • Increased intracellular [Ca2+], which could support plasticity role • Important for Spike-Time Dependent Plasticity?

19. Individual Neuron are highly complex processing unit! VS

20. Take Home Messages 1) Different types of neurons have distinct electrophysiological properties that are determined by expression and distribution of different types of voltage gated channels. 2) The processing of electrical signal by neurons are subjected to dynamic modulation. 3) Action Potential once initiated propagate along axon towards axon terminal. Concurrently, the wave of depolarization can actively and passively propagate towards dendrites

21. Part 2 (Intrinsic plasticity in learning, aging and CNS disorder)

22. Learning Neuroplasticity as the substrate for learning and memory establishment Plasticity of brain at cellular level - Changes which modulates efficacy in which electrical signal is transmitted among neurons in the neural circuits. Synaptics connectivity (signal passing from one neurons to another) Intrinsic Properties (Integration and transmission of signal)

23. Learning – Intrinsic Plasticity • In a nutshell, Intrinsic plasticity involves changes to the electrophysiological properties of • neuron. • Plasticity of this nature directly affects excitability of neurons and efficacy of spatial temporal summation…. Both share common induction mechanisms E.g. what induced synaptic potentiation also induce increased in excitability of neurons Synaptic and Intrinsic plasticity operates in functional synergistic manner that enhances reliability of signal transmission.

24. Investigative approach in studying intrinsic plasticity • Measuring number of action potential (frequency) that can • are elicited by certain depolarizing current injected • Measuring threshold of current magnitude needed to elicit • action potential *Coupling between EPSP and Action potential

25. Intrinsic Plasticity can be Local or Global in scale Localized changes – confine to small portion of dendrites. E.g. Downregulation of Kv4.2 that mediate A current -> Increased dendritic excitability Global – functional changes affecting soma, axon or many dendritic regions. E.g. Increased density of Na+ channel at axon hillock -> Lower threshold for AP *Neural Homeostasis – Mechanisms in which each neurons maintain certain balances of excitatory or inhibitory inputs/activites and minimum number of active output in order to survive.

26. Aging • After–Hyperpolarisation (AHP) • Mediated by efflux of K+ • Different type of K+ channel • can mediate AHP : Primary by Ik • but other channels also play • Important role e.g. Ca2+ sensitive • K+ channel, M current, apamin • sensitive K+ channel….

27. Kklolalmm A Sddf Kkjj Addffg d Kklolalmm A Sddf Kkjj Addffg d Enhanced After-Hyperpolarisation current observed in aged mice hippocampus neurons as compared to young mice Kklolalmm A Sddf Kkjj Addffg D ad

28. Gghkak Addff Csss Ffffffffffffffffff aaaaaaaaaaaaaaaa A number of studies have indicated aberrant function of K+ channels as well as intracellular Ca2+ homeostasis that contributes to aged related cognitive decline due to changes in electrophysiological properties of neurons Neurons in aged mice has less capacity in sustaining increased neuronal activity at high frequency

29. CNS Disorder – Epilepsy • In chronic epilepsy, a number of changes in voltage-gated ion channels might act together • to cause output mode changes (E.g. regular firing to busting) • Aberrant downregulation of K+ current and enhance persistent Na+ current, leading to • increase intrinsic excitability of neurons.

30. CNS Disorder – Alzheimer's Disease Chronic neurodegeneration disease characterised by significant loss of neurons and synapses in the cerebral cortex and certain subcortical regions. Impairment in cognitive ability  Profound loss of learning and memory capacity *Changes in electrophysiological properties of neurons in AD patient can be observed. In other neurodegeneration diseased, marked changes in the electrophysiological properties of neurons are also observed

31. In AD, there is a significant loss of forebrain cholinergic projection neurons. Degeneration of Septal nuclei resulted in reduced cholinergic input to hippocampus neurons. In experimental model, cholinergic receptor antagonists impair learning and memory. Normal Cholinergic input to hippocampus 1) Acetylcholine activates muscarinic receptor  2) Activates G proteins leading to chemical Signaling cascade  3) Closure of M current (reduced K+ efflux)  4) Reduced After Hyperpolarisation  5) Busting firing mode that could sustain memory consolidation and LTP Reduced Cholinergic input to hippocampus in AD Increased M current activation at subthreshold voltage depolarisation 2) Enhanced After- hypolarisation following Action potential  3) Fidelity of AP fails at high frequency  4) Suppression of Bust firing  5) Reduced capacity for memory encoding and consolidation Hjjjajj Dsfghhjj Sdgg ssdf

32. Take Home Messages 1) Intrinsic plasticity involves changes to the electrophysiological properties of neuron, and can interact synergistically with synaptic plasticity for producing changes in efficacy of neuronal signal transmission. 2) Intrinsic plasticity can be local or global in scale, and is relevant in maintaining neural homeostasis. 3) Profound changes in intrinsic neuronal properties are observed in many CNS disorders. These changes reflect alterations in functional properties of voltage and chemical messenger sensitive ion channels.

33. Reference 1. Squire B, et al. Fundamental Neuroscience Fourth Edition 2. Zhang W, Linden DJ. The other side of the engra: experience-driven changes in neuronal intrinsic excitability. 3. Heinz B & Yoel Y. Plasticity of intrinsic neuronal properties in CNS disorders. 4. Hausser M, Spruston N & Stuart, G. J. Diversity and dynamics of dendritic signaling. 5. Campanac E & Debanne D. Spike timing-dependent plasticity: a learning rule for dendritic integration in rat CA1 pyramidal neurons. 6. Wang Z., Xu NI, Wu CP, Duan S & Poo MM. Bidirectional changes in spatial dendritic integration accompanying long-term synaptic modifications. 7. Levy WB & Steward O. Temporal contiguity requirements for long-term associative potentiation / depression in the hippocampus. 8. Markram H, Lübke J, Frotscher M & Sakmann, B. Regulation of synaptic efficacy by coincidence of postsynaptic APs and EPSPs.

34. 1. The Glia Revolution 2. Light Activated Mouse Scenario Facts Exploration 3. The Magnetized Brain 4. Death by Fugu

35. 1. The Glial Revolution (more than just glue of the brain) Glia cells outnumber neurons by about 10-1 (depending on source of information) Glia cells especially astrocytes are versatile and highly diverse in function: Remove neurotransmitter, form BBB, couple blood flow with neuronal activity, Immune system of the brain, form myelin sheath, participate in neuronal communication…..

36. Gliotransmission – Tripartite Synapse Astrocytes can sense and integrate synaptic activities. Through release of gliotransmitter, astrocytes can exert feedback signal onto neurons and modulate their activities. • Neurons release Glutamate into synaptic cleft  2) Glutamate binds to mGluR • 3) second messenger activate chemical cascade that opens opens Na+ ion channels • 4) Membrane depolarised (graded potential)  5) Open T type voltage Ca2+ channels • leading to increased Ca2+ concentration  6) Release of gliotransmitters

37. 2. Light Activated Mouse. Neural network is highly complex. In order to understand it better, tool is needed to dissect complexity of brain at high level of specificity – cellular level.

38. 2. Light Activated Mouse. Channelrhodosin Gene identified and Express on neurons Hhhhhhhhhhhhhhhhhhhh Ddfffffffffffffffffffffffffff Wdffffffffffffffffffff tjjjjjjjj Light OFF Light ON Light OFF Chlamydomonasreinhardtii (Unicellular Green Algae) • Light control of aggression in mouse • Gene encoding Channelrhodopsin • was introduced into subpopulation • of hypothalamus neurons. When light • turned on, mouse turned aggressive *Channelrhodosin 2 – non selective cation channel that opens within microsecond following brief exposure to light

39. 3. The Magnetized Brain - Magnetoreception How does some animals sense and use Earth magnetic field to perceive direction and location for orientation and navigation?

40. Magentosensing neurons in C.elegans NATURE REVIEWS NEUROSCIENCE 16, 444 (2015) Using genetic screening procedure, the AFD neuron (previously known to sense CO2 and temperature) was crucial in magnetic field sensing. Similar Magnetosensitive neurons were soon found in migratory pigeons and trouts

41. Ion channel complex that respond to magnetic field found in AFD neurons! Complicated ion channel complex that are associated with oxidase enzyme that catalyst oxidation of Iron to Iron (III) oxide. Iron oxide particle associated with high affinity iron oxide protein domain  2) In response to spatial magnetic field strength protein complex are deform to varying degree  3) open ion channel, that produce depolarisation of graded strength  4) Spatial coordination map of earth magnetic field.

42. 4. Death by Fugu Danger! Tetrodotoxin- potent neurotoxin (20x more toxic than cyanide)

43. 4. Death by Fugu Tetrodotoxin- potent neurotoxin -Binds with high affinity to Na+ channel -Block propagation of action potential leading to failed nerve transmission and respiratory failure

44. Why is Pufferfish resistant to their own toxin? A single amino acid residue substitution in Puffer fish Na+ channel makes them 10000 fold less sensitive to tetradotoxin than Na+ channel in humans.

45. 1. The Glial Revolution False

46. Neurons release Glutamate into synaptic cleft  2) Glutamate binds to mGluR 3) G proteins activates phospholipase that generate IP3  4) IP3 leads to release of Ca2+ from ER  5) Increased Ca2+ concentration  6) Release of gliotransmitters

48. 2. Light Activated Mouse True

49. The field of optogenetics has revolutionized the study of neural circuits -Offers excellent spatial and temporal control. -Frequency of light pulse can control frequency of action potentials. -Can channelrhodopsin can be selectively expressed in neuronal subpopulation. -Complement with synthetic biology produced chimeric proteins from multiple Rhodopsins, each with different kinetics, sensitivity, conductance… Light Activated locomotion of mouse

50. 3. Magnetized Brain False