BIO 3350 Lecture 4 Action Potentials PDF

CHAPTER 4: ACTION POTENTIAL • The ionic basis of action potentials • The sequence of channel openings and closings that generate each action potential • How scientists can study the ionic current and the channel function of neurons Readings • Bear, Chapter 4 1 RECORDING 1 COMMUNICATION IN THE...

CHAPTER 4: ACTION POTENTIAL • The ionic basis of action potentials • The sequence of channel openings and closings that generate each action potential • How scientists can study the ionic current and the channel function of neurons Readings • Bear, Chapter 4 1 RECORDING 1 COMMUNICATION IN THE NERVOUS SYSTEM Sensory stimulus Activation of cutaneous receptors associated with intense deformations Neural code Interpretation/Action Action potential post-synaptic potential Flexor withdrawal reflex ??? 3 INTRODUCTION • Action potentials • Transfers information over long distances … 0.1 mm to 1 m … • Neural Code : frequency and discharge pattern ? • Synonyms: • Spike, nervous impulse, nervous influx, discharge 4 PHASES OF ACTION POTENTIALSS • Phases: • 1. 2. 3. 4. Resting potential Rising phase (depolarisation) Overshoot Falling phase (repolarisation) Hyperpolarisation 1. Absolute Refractory period 2. Relative Refractory period 5 GENERATION OF ACTION POTENTIAL • Caused by a depolarisation of the membrane beyond of a threshold • Electrical, chemical or mechanical stimulation • « All or nothing » • Chain reaction • Feedforward loop OPENING OF CHANNELS PERMEABLE TO NA+ 6 GENERATION OF ACTION POTENTIAL • Artificial injection of current in a neuron by using a micro-electrode • Generation of action potentials 7 GENERATION OF ACTION POTENTIAL • Frequency of discharge depends of the amplitude of injected current 1. stimulation too weak… … does not reach action potential threshold 2. Just above the threshold… … a few action potentials 3. Stronger stimulation … … increase of the frequency of discharge 8 MECHANISM OF GENERATION 9 ACTION POTENTIAL: EXPERIMENT OF HODGKIN + HUXLEY Alan Hodgkin Andrew Huxley 10 ACTION POTENTIAL: EXPERIMENT OF HODGKIN + HUXLEY • Studied the giant axon of the squid • Inserted a metal filament the length of the axon • Determined the importance of the permeabilities of Na+ and K+ • Made a mathematical model 11 SIMPLE MODEL OF AN ACTION POTENTIAL a) Rest: A few K+ channels open Membrane potential gK >> gNa Vm=-65mV EXTRACELLULAR SPACE Na+ K+ 12 + SIMPLE MODEL OF AN ACTION POTENTIAL b) Depolarisation crossing the threshold: Rapid opening of Na+ channels Membrane potential gK << gNa Vm depolarizes EXTRACELLULAR SPACE Na+ K+ 13 + SIMPLE MODEL OF AN ACTION POTENTIAL c) Repolarization Rapid closing of Na+ channels Slow opening of K+ channels Membrane potential gK >> gNa Vm repolarize EXTRACELLULAR SPACE Na+ K+ 14 + SIMPLE MODEL OF AN ACTION POTENTIAL d) Return to rest: Some K+ channels open Membrane potential gK >> gNa Return to rest Vm=-65mV EXTRACELLULAR SPACE Na+ K+ 15 + ACTION POTENTIALS IN REALITY Major problem with this simple model How do Na+ channels close at depolarized Vm and stay close long enough for K+ channels to repolarize the membrane Na+ channels must stay close for K+ channels to repolarize If Na+ channels reopen, this would happen 16 ACTION POTENTIAL IN REALITY • The experiments of Hodgkin and Huxley show that action potentials are more complicated • Require that certain channels inactivated. I.e. they become incapable of passing through current independant of membrane potential. • How can this mechanism of inactivation been verified? 17 MINOR(?) ASIDE • CELLULAR ELECTROPHYSIOLOGY • Techniques to observe the electrical activity of a neuron • For exemple, we would like to determine which currents pass the membrane during an action potential 18 ELECTROPHYSIOLOGY Recording electrode Ielectrode Im Rm vm Circuit for measurement electrode in the bath In the lab, we can measure electrical signals. Vm = Rm x Im (Ohm’s law) ELECTROPHYSIOLOGY Recording electrode Ielectrode Im Rm vm Circuit for measurement electrode in the bath On the other hand, we can only measure either of (Vm or Im) at a time Vm = Rm x Im (Ohm's Law) ELECTROPHYSIOLOGY Recording electrode Ielectrode Im Rm vm Circuit for measurement electrode in the bath By using our electrode, we can either 1. Impose a current and measure the Vm that results 2. Impose a voltage and measure the Im that results IMPOSED VOLTAGE VS IMPOSED CURRENT recording clamp Current-clamp V=RxI Voltage-clamp clamp Useful to oberve the behaviour of a neuron by Vm Useful to observe the properties of Ion channel by monitoring their conductances recording 22 CURRENT-CLAMP • Let us measure Vm by injecting currents through an electrode (Ielec) Recording (V) Recording electrode Ielectrode Im Rm vm Circuit for measuremen t electrode in the bath STIMULATION (I) VOLTAGE-CLAMP • The measurement of membrane current (Im) at different membrane potentials (Vm) is more complicated Ex: we would like to know which membrane current (Im) passes at Vm = -55 mV. • Im (-55 mV) = Σ (IK + INa + ICa + ICl) • Problem: IK, INa, ICa and ICl are produced ion channels that can change these currents quickly, which would perturb Vm quickly as well! VOLTAGE-CLAMP • Voltage-clamp • To measure Im at a desired Vm, we use an electronic circuit that can fix (or “clamp”) Vm at a value (Vclamp), and then measure the current that the circuit must inject to maintain Vm at Vclamp. Ielectrode Im Rm vm Voltage-clamp circuit VOLTAGE-CLAMP • The voltage-clamp circuit operates using the following logic 1. Inject a Ielectrode and measure Vm 2. If Vm is different than Vclamp, ajust Ielectrode 3. Repeat steps 1 and 2 iteratively until Vm = Vclamp Ielectrode Voltage-clamp circuit Im Rm vm VOLTAGE-CLAMP • When Vm = Vclamp, Ielectrode = -Im • N.B. the current measured by our circuit (Ielec) has an opposite sign from Im since it is the current that our circuit must inject to maintain Vm at Vclamp Recording (I) of sodium current (note the negative polarity!) 53 mV STIMULATION (V) -86 mV -46 mV VOLTAGE-CLAMP Below is now a voltage-clamp recording of a potassium current (Panels B, C and F) that is activated when the membrane potential is depolarized relative to rest (si the top of panel F for the voltage commands). Dougalis et al Journal of Computational Neuroscience (2017) 28 VOLTAGE-CLAMP • By using the voltage-Clamp, we can measure the Na+ and K+ current activated when Vm goes from rest to the peak of an action potential • The Na+ current (INa) is activated by the depolarisation however it is not sustained throughout the depolarisation. This current inactivates. B. Im (composed of IK and of INa) Im (mA/cm2) • The K+ current (IK) is activated by the depolarisation and it is sustained at throughout the depolarisation A. Voltage-clamp jump of -65 mV to 0 mV C. K+ current (IK) – after block of INa by TTX D. Na+ current (INa) – after block of IK by TEA Can we determine why the Na+ current (INa) seems to inactivate after the depolarisation? 30 PATCH-CLAMPING • Pipette apposed to the membrane • Extract a portion of the membrane • Impose a voltage • Allows you to record from a single ion channel at a time 31 PHASES OF ACTION POTENTIALS • By using patch-clamping, we can measure currents of Na+ channels individually during an action potential • This allows the measurement of the Na+ current that enters • Conclusion: The Na+ current only enters during the Rising phase 32 PHASES OF ACTION POTENTIALS • The recording of K+ currents of individual channels is also possible • This allows us to measure the K+ current entering • Conclusion: The K+ current entres at the end of the Rising phase and peaks at the start of the Falling phase 33 SODIUM CHANNELS • Four domains similar to the four sub-units of K+ channels • Selective pore for Na+ ions • S4 segment charged + • Sensitive to Vm 34 RECORDING OF NA+ CHANNEL • Rapid opening of channels to Na+ • Very short delay (< 1 ms) • Conformational change to open the pore • Stays open for about 1 ms • Unitary conductance • Rapid closing 3 • Mechanism of inactivation • « Ball and chain » • Potential must be repolarised for reactivation4 • Na+ channels must be inactivated after action potential • Absolute refractory period 35 TOXINS TARGETTING NA+ CHANNELS • Tetrodotoxin (TTX) • Blocks the pore of channels to Na+ • Saxitoxin (dinoflagellate) • Lidocaine • Anesthetic • Batrachotoxin (frog) • Blocks inactivation -> channel stays open • Veratridine (lilies) • Lowers the activation threshold • Aconitine (buttercups) • Lowers the activation threshold • Allows to link molecular knowledge to tissue behaviour 36 K+ CHANNELS VS NA+ CHANNELS • Dependance to voltage • The 2: open in response to a depolarisation • K+ channels open more slowly than Na+ channels • Only Na+ channels inactivate • Delayed rectifier • K+ condutance serves to rectify or reset Vm • Necessary to free Na+ channels from their inactivations • Structure • K+ channels: 4 subunits • Na+ channels: 4 domains 37 GENERATION OF AN ACTION POTENTIAL a) At rest: Some K+ channels open. All Na+ channels are closed Membrane potential gK >> gNa Vm=-65mV EXTRACELLULAR SPACE Na+ K+ 38 + GENERATION OF AN ACTION POTENTIAL b) Depolarization crossing the threshold: Opening of a small number of Na+ channels Membrane potential Vm depolarizes EXTRACELLULAR SPACE Na+ K+ 39 + GENERATION OF AN ACTION POTENTIAL c) Rising phase: Rapid opening of all Na+ channels Membrane potential gK << gNa EXTRACELLULAR SPACE Na+ K+ 40 + GENERATION OF AN ACTION POTENTIAL d) Overshoot Rapid inactivation of Na+ channels Slow opening of K+ channels Membrane potential gK >> gNa Vm repolarize EXTRACELLULAR SPACE Na+ K+ 41 + GENERATION OF AN ACTION POTENTIAL e) Falling phase Na+ channels stay inactivated Opening of all K+ channels Membrane potential gK >> gNa Vm repolarize EXTRACELLULAR SPACE Na+ K+ 42 + GENERATION OF AN ACTION POTENTIAL f) Refractory period: Membrane potential Some K+ channels open Na+ channels stay inactivated which prevents the initiation of another action potential EXTRACELLULAR SPACE Na+ K+ 43 + GENERATION OF AN ACTION POTENTIAL g) Return to rest: Membrane potential The inactivation of Na+ channels is terminated The cell can initiate another action potential EXTRACELLULAR SPACE Na+ K+ 44 + PROPAGATION OF ACTION POTENTIALS 45 PROPAGATION OF ACTION POTENTIALS • Orthodromic • Action potential travels towards the nerve terminals (red arrows) • Antidromic • From nerve terminals towards the dendrites (blue arrow) • Duration of an action potential: 2ms • Typical conduction velocity: 10m/s 46 FACTORS INFLUENCING CONDUCTION VELOCITY: SALTATORY CONDUCTION • Myelin • Schwann cells (PNS) • Oligodendrocytes (CNS) • Multiple lipid layers around the axon • Very few Na+ channels • Prevents the leakage of ions • Nodes of Ranvier • Uninsulated • High density of Na+ channels • Allows the passage of ions 47 FACTORS INFLUENCING CONDUCTION VELOCITY • Electrotonic conduction (« passive ») underneath myelin • Passive diffusion of Na+ ions in the longitudinal axis of the axon • Myelin prevents loss of Na+ ions out of the axon • Active conduction (with ion channels) at nodes of Ranvier • Action potentials with Na+ channels 48 ACTION POTENTIALS, AXONS AND DENDRITES • Initiation zone of action potentials • In sensory neurons • Nerve terminals (dendritic extremities) • Other neurones • Axon hillock 49